Ecotoxicological assessment of Roundup-ready ...

   

Faculty of Health Sciences UiT

Ecotoxicological assessment of Roundup-ready soybean agriculture investigated in a D. magna model   —   Marek Cuhra A dissertation for the degree of Philosophiae Doctor – March 2015

 

 

          Thomas,    -­  gode  venn,   samvittighetsfulle  veileder     og  dansepartner....    -­  Takk!      

 

 

 

           

 "Writing  a  phd-­thesis  is  like  building  a  house    you  know  you're  not  going  to  live  in"   -­‐    Svein-­‐Anders  Noer  Lie                                                        

 

     

           

 

 

 

          Following  pages  are  an  abridged  and  resubmitted  second  version  of  a  doctoral  thesis,   presenting  the  main  outcome  of  a  2008-­‐2014  scholarship  at  GenØk  Centre  for  Biosafety   Tromsø  Norway.  The  scholarship  is  funded  by  the  Research  Council  of  Norway  through  NFR   Project  184107/S30  LAND:  A  new  model  approach  to  assess  genetically  modified  plants:  their   ecotoxicity  and  potential  interactions  with  environmental  pollutants.   The  text  including  submitted  manuscripts  is  presented  to  the  Faculty  of  Medicine,  University   of  Tromsø,  the  author  thus  aspiring  to  qualify  for  the  doctoral  degree  in  Natural  Sciences  in   compliance  with  regulations  specified  in;  FOR  2012-­10-­25  nr  1150:  Forskrift  for  graden   philosophiae  doctor  (ph.d.)  ved  Universitetet  i  Tromsø  (Lovdata,  2013).     The  text  is  written  by  Marek  Cuhra.  

 

   

 

A"controversial"development"in"industrial"agriculture" Traditional"cultivation"of"landscape"with"deliberate"intent"to"facilitate,"improve"and" increase"production"of"plant"material"for"human"consumption"and"farmed"animal"feed," has"evolved"into"complex"agro:industrial"activities"involving"technological"and" agrochemical"means."It"involves"use"of"machinery,"chemicals,"greenhouses"and" irrigation"to"alter"the"microclimate,"supplementing"nutrients"and"water"and"eradicating" pests."Natural"restrictions"imposed"by"biological"and"climatological"conditions"are" thereby"to"some"extent"manipulated"and"overruled." Taking"this"development"a"step"further,"modification"of"cultivars"by"forced"insertion"of" transgenes"from"other"organisms"has"gradually"been"accepted"in"parts"of"the"world"as"a" legitimate"and"necessary"step"in"the"ongoing"development"of"agriculture."This"genetic" manipulation"has"been"presented"as"potentially"beneficial"for"farmers,"consumers"and" society"in"general,"since"it"is"intended"to"reduce"production"costs"and"create"higher" yields."Genetic"manipulation"has"also"been"heralded"as"allowing"for"more" environmentally"benign"agricultural"practices,"notably"reduced"use"of"pesticides"and" need"for"tillage"(believed"to"be"an"important"factor"for"soil"conservation"in"areas"subject" to"high"rates"of"erosion)."" Plant"varieties"modified"to"tolerate"herbicide"application"were"amongst"the"first" commercially"available"cultivars"to"have"been"genetically"manipulated."These"plants"still" form"the"majority"of"all"genetically"modified"organisms"(GMOs)"produced"worldwide." Herbicide"tolerance"allows"for"reduced"farming"expenses,"use"of"herbicidal"chemicals" substituting"for"generally"more"costly"manual"and"mechanical"eradicative"techniques." Use"of"agrochemicals"will"affect"both"the"quality"of"agricultural"produce"as"well"as"the" surrounding"environment,"which"is"measurable"as"chemical"residue"levels"in" agricultural"commodities"and"impact"on"non:target"organisms." GMOs"have"been"shown"to"facilitate"certain"aspects"of"plant"cultivation"and"bring"down" direct"production"costs."This"not"withstanding,"use"of"GMOs"for"production"of"human" food"and"farm:animal"feed"remains"the"object"of"much"public"scepticism"and"scientific" debate."In"this"regard,"it"might"appear"reassuring"that"agencies"such"as"the"US"Food"and" Drug"Administration"(US"FDA)"as"well"as"the"European"Food"Safety"Authority"(EFSA)," have"both"stated"that"food"produced"from"GMO"plant"material"generally"should"be"

1"

recognized"as"safe."Furthermore,"both"are"claimed"to"have"concluded"that"due"to"the" precision"of"transgenic"techniques,"GMO"plants"have"the"potential"to"produce"safer" foods"than"conventional"(unmodified)"varieties."However,"this"notion"is"contested." Numerous"research"findings"document"that"the"manipulation"methods"are"imprecise" and"unpredictable"and"that"the"cultivation"presents"risk"factors"and"adverse" environmental"impacts"at"production"sites,"in"addition"to"undesirable"effects"on"the" quality"of"produce." The"inherent"complexity"of"assessing"risks"posed"by"genetically"modified"(transgenic)" cultivars"has"been"addressed"by"means"of"traditional"scientific"approaches,"including" visual"characterization,"compositional"analysis"of"constituents"and"animal"feeding"tests." These"are"deemed"sufficient"for"quality:assurance"of"substances"destined"for"human" consumption"or"animal"feed." As"in"numerous"other"instances"of"innovation,"the"professionals"charged"with"testing" and"assessing"are"not"of"one"mind."Scientists"evaluating"transgenic"cultivar"produce" hold"widely"differing"opinions"on"issues"such"as"need"for"precaution,"relevance"of" surveillance"and"essence"of"analysis."This"is"presented"as"scientifically"founded" statements"on"issues"such"as"determination"of"necessary"length"of"exposure"in"feeding" tests,"discussions"on"relevance"of"new"constituents"such"as"novel"proteins"synthesized" by"inserted"transgenes,"labeling"of"consumer"products"and"scope"of"environmental"

monitoring"plans."""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" A"review"of"scientific"literature"and"regulatory"documentation"provides"us"with"an" understanding"of"societal"developments"over"the"last"decades"that"have"led"to"the" present"polarized"situation"in"worldwide"cultivation"and"consumption"of"genetically" modified"material."An"overview"of"this"evolution"should"start"with"analysis"of"regulatory" processes"leading"to"the"1992"US"FDA"policy"brief"on"novel"foods"from"biotech"plants." This"is"vital"to"understanding"the"scientific"and"political"exchanges"leading"to"the" present"deregulation"of"transgenic"produce"in"the"United"States"as"well"as"other" countries"in"North"and"South"America,"including"Canada,"Argentina,"Brazil"and"Uruguay." This"policy"of"deregulation"was"opposed,"based"on"claims"of"perceived"risk,"by"the" majority"of"EU"constituent"nations"and::notably::Norway."

2"

The"scientific"and"political"discussion"on"biotech"food"safety"thus"initially"has"been" characterized"as"a"trans:Atlantic"rift,"with"other"regions"and"individual"countries"joining" in"as"further"evidence"accrued."More"recently,"these"positions"have"changed"somewhat." The"formerly"active"scepticism"regarding"transgenic"plants"in"Europe"has"largely"been" subdued"into"passive"acceptance"as"gradually"biotech"cultivars"are"approved"by"the" EFSA"for"import"and"cultivation."" By"contrast,"in"the"United"States"the"discussion"on"safety"of"food"from"transgenic"plants" has"re:erupted,"generally"focusing"on"mandatory"labeling"of"foods"containing"transgenic" ingredients."With"both"sides"backed"by"their"respective"representatives"in"Congress"and" Senate,"it"led"to"recent"referenda"in"several"states."In"most"of"these"instances"thus"far,"a" narrow"margin"of"voters"rejected"the"proposals"for"labeling." The"safety"issues"concerning"transgenic"plants"are"still"contested."Objective"scientific" evidence"confirming"their"safety"remains"lacking."" In"fact"the"overwhelming"majority"of"data"confirming"safety"of"transgenic"produce"has" been"furnished"by"the"biotech"industry"itself."Critics"have"pointed"out"that"such"industry" tests"are"often"methodologically"flawed,"among"other"things"of"insufficient"duration,"and" thereby"not"useful"in"modelling"the"life:long"exposure"situation"of"consumers."" Independent"research"testing"of"transgenic"produce"has"not"been"welcomed"by"biotech" industry,"which"has"refused"to"supply"material"for"such"testing."To"date,"few"universities" or"research"programs"see"the"importance"of"independent"testing"of"transgenic"foods" substances."Few"independent"researchers"manage"to"obtain"material"for"testing"or" adequate"funding."For"those"who"do,"the"reception"of"findings"disfavourable"to"biotech" products"is"generally"hostile." This"has"created"a"situation"in"which"research"failing"to"document"potentially"adverse" effects"of"transgenic"produce::even"where"exhibiting"obvious"methodical"flaws::has" largely"been"accepted"as"evidence"of"safety,"whereas"research"indicating"the"contrary" tended"to"be"ignored"or"systematically"discredited."

3"

" Contents" A"controversial"development"in"industrial"agriculture .............................................................................................1" Abbreviations"and"key"concepts ..........................................................................................................................................7" Summary"of"key"findings.........................................................................................................................................................9"

BACKGROUND .................................................................................................................13" Soybean........................................................................................................................................................................................ 13" Glyphosate .................................................................................................................................................................................. 13" Risk:assessment"of"transgenic"cultivars ....................................................................................................................... 16" Substantial"equivalence ........................................................................................................................................................ 17" Agriculture"affects"aquatic"ecology.................................................................................................................................. 19" Test:animal"biology ................................................................................................................................................................ 20" Related"work.............................................................................................................................................................................. 21"

AIM .................................................................................................................................25" Research"question ................................................................................................................................................................... 25" Research"hypothesis .............................................................................................................................................................. 25" Form"of"presentation ............................................................................................................................................................. 26"

MATERIALS6AND6METHODS .............................................................................................27" Research"materials.................................................................................................................................................................. 27" Animal"model ............................................................................................................................................................................ 27" Archive"studies ......................................................................................................................................................................... 31"

RESEARCH6MANUSCRIPTS ................................................................................................33" List"of"papers ............................................................................................................................................................................. 35" Paper"I:" Glyphosate"herbicide"ecotoxicity................................................................................................................ 37" Paper"II:" Analysis"of"soybean"constituents .............................................................................................................. 39" Paper"III:" Feeding"study"I. ............................................................................................................................................... 41" Paper"IV:" Feeding"study"II............................................................................................................................................... 43" Paper"V:" Review"of"published"evidence .................................................................................................................... 45"

DISCUSSION .....................................................................................................................47" Glyphosate"ecotoxicity .......................................................................................................................................................... 48" Soy"quality"and"glyphosate"residues............................................................................................................................... 53" Soybean"feeding"studies ....................................................................................................................................................... 57"

4"

Validity"of"results..................................................................................................................................................................... 60" Triangular"model"of"quality ................................................................................................................................................ 62" Regulatory"issues..................................................................................................................................................................... 65" Sustainability"and"societal"context .................................................................................................................................. 67"

CONCLUSION ...................................................................................................................71" Answering"the"research"question..................................................................................................................................... 72"

FORMAL6FRAMEWORK ....................................................................................................73" Mandate ....................................................................................................................................................................................... 73" Declaration"of"independence.............................................................................................................................................. 73" Acknowledgement"of"funding"and"declaration"on"conflicting"interests.......................................................... 74"

ACKNOWLEDGEMENTS ....................................................................................................75" REFERENCES.....................................................................................................................95"

6 6

5"

6"

Abbreviations"and"key"concepts"

6 ADI"

="

Acceptable"daily"intake"(dosage"related"to"bodyweight)"

CAS#"

="

Chemical"abstract"service"of"American"Chemical"Society"

EC50(48)"

="

Concentration"producing"50%"effect"(immobility)"in"test:animal"in"48:hrs"exposure"

FOIA""

="

US"freedom"of"information"act"

GMO"

="

Genetically"modified"organism"

GT"

="

Glyphosate"tolerant"

GTS"

="

Glyphosate"tolerant"soy"

HT"

="

Herbicide"tolerant"

LC50(48)"

="

Concentration"producing"50%"lethal"effect"in"test:animal"in"48:hrs"exposure"

LOEC"

="

Lowest"concentration"of"observed"effect"

MRID"

="

Master"record"identification"number"

MRL"

="

Maximal"residue"level"(limit)"

MSDS"

="

Material"safety"data"sheet"

NOEC"

="

Concentration"of"no"observed"effect"(see"LOEC)"

US"EPA"

="

Environmental"Protection"Agency"of"the"United"States""

US"FDA"

="

Food"and"Drug"Administration"of"the"United"States""

USDA"

="

United"States"Department"of"Agriculture"

Table 1. Abbreviations and key concepts used in introduction, research papers and discussion.

"

7"

8"

Summary"of"key"findings"

"

Despite"two"decades"of"research"into"the"quality"and"biosafety"of"genetically"modified" glyphosate:tolerant"plant"varieties,"these"issues"are"still"contested"by"contradictory" research"findings."In"a"similar"development,"glyphosate"herbicides"such"as"the" commercial"product"Roundup"are"more"controversial"now"than"ever"before,":"despite" four"decades"of"research"into"toxicological"and"ecotoxicological"effects"of"these" chemicals." An"unresolved"issue"seems"to"have"been"largely"ignored"by"scientists"as"well"as" regulators:"glyphosate"tolerant"crops"are"constituents"of"an"industrial"production" system"with"specific"agricultural"practices"and"supplementary"agrochemicals"as" interwoven"additional"elements."Thus"the"transgenic"material"produced"should"not"be" seen"as"an"isolated"product"of"a"specific"modified"genotype"but"rather"as"a"product"of"a" tailored"agriculture"system." The"objective"of"the"research"presented"here"has"been"to"investigate"quality"and"safety" aspects"of"one"such"specific"agriculture"production"system,"that"of"the"glyphosate: tolerant'soybean."This"globally"dominant"crop"has"been"in"open"cultivation"since"1995" and"is"still"predominantly"based"on"hybrid"varieties"of"the"GTS:40:3:2"transgenic"event" soybean"(Roundup:ready"soybean)"in"combination"with"glyphosate"herbicide"co: technology."" The"published"results"were"obtained"via"a"review"of"published"evidence"and"four"quite" large"laboratory"experiments:"a)"an"ecotoxicological"study"of"a"commercial"Roundup" herbicide"and"the"glyphosate"active"ingredient,"b)"a"study"of"biochemical"composition"in" which"genetically"modified"glyphosate:tolerant"Roundup:ready"soybean,"conventional" soybean"and"organic"soybean"are"analyzed"and"compared,"and"c)"two"fully"controlled" laboratory"feeding"studies"with"soybean:meal,"using"water:flea"Daphnia'magna'in"life: long"exposure"as"an"animal"model." Roundup"and"glyphosate:"The"herbicides"were"tested"for"acute"and"chronic"toxicity"in"a" D.'magna"animal"model."It"was"found"that"the"ecotoxicity"is"more"potent"than"what"was" previously"assumed"in"regulatory"assessments"based"on"information"provided"by" chemical"industry."The"tests"in"this"recognized"aquatic"invertebrate"indicator"species'

9"

have"yielded"evidence"of"the"general"effects"these"chemicals"might"have"in"aquatic" ecosystems"that"receive"effluents"from"farmland."Glyphosate"is"found"be"distinctly"more" toxic"than"indicated"in"previously"published"studies."Analysis"of"vital"parameters"from" D.'magna"in"short:term"48:hour"exposure"demonstrate"that"acute:toxicity"of"glyphosate" and"Roundup"herbicide"formulation"are"two"orders"of"magnitude"higher"than"expected" from"previously"published"findings"from"industry"testing."Also,"life:long"exposure"to" low:level"concentration"of"either"glyphosate"or"Roundup"herbicide"formulation" demonstrates"higher"toxicity"than"expected"from"scientific"literature."Notably,"it"was" found"that"low"concentrations"of"Roundup"induce"reproductive"failure"in"D.'magna' (paper"I)."" Through"a"request"for"information:disclosure"under"the"United"States"Freedom"of" Information"Act,"copies"of"the"relevant"industry"tests"from"the"archives"of"United"States" Environmental"Protection"Agency"(US"EPA)"were"obtained."The"material"revealed" evidence"of"systematic"flaws"and"erroneous"interpretation"of"results."These" irregularities"partially"explain"the"discrepancy"between"the"findings"on"glyphosate" ecotoxicity"presented"in"paper"I"and"the"established"assumptions"in"regulatory" assessments"and"some"scientific"literature."" Composition"of"soybean:"Laboratory"analysis"of"selected"minerals,"nutrients"and" pesticide"residues"was"performed"in"transgenic"GTS:40:3:2"soy,"conventional"soy"and" organic"soy"obtained"directly"from"farm:fields"in"Iowa,"USA."The"results"show" systematic"differences"which"characterize"these"products."Substantial"ppm:levels"of" glyphosate"residues"were"found"to"be"systematically"present"in"all"tested"samples"of" Roundup:ready"soybean."In"seven"out"of"ten"samples"these"residue"concentrations"were" unexpectedly"high."Such"residues"were"found"in"neither"conventional"soybean"nor"in" organic"soybean,"although"ppb:"and"ppt:levels"of"other"pesticide"residues"were"present" (paper"II)."" Feeding"studies:"Vital"parameters"of"Daphnia'magna"fed"raw"or"heat:treated"meal"from" either"transgenic"GTS:40:3:2"soybean,"conventional"soybean"or"organic"soybean"were" analyzed."Results"demonstrate"significant"differences"in"test:animal"vital"parameters," reflecting"significant"qualitative"differences"of"feed."The"first"life:long"feeding:study" with"soybean:feed"in"D.'magna"demonstrated"that"overall"animal"performance"

10"

measured"as"growth"and"reproduction"was"significantly"affected"by"soy:meal"diets."The" best"performance"was"found"in"animals"fed"diets"with"organic"soy:meal."By"comparison," animals"fed"transgenic"soybean:meal"showed"significantly"reduced"growth"and" reproduction."The"feeding"studies"documented"that"animals"fed"GTS:40:3:2"soybean" showed"fitness"parameters"inferior"to"animals"fed"conventional"soybean"(paper"III)."" Animal"performance"is"affected"by"glyphosate"residues:"A"subsequent"life:long"feeding: study"using"8"distinct"soybean:meal:diets"from"GTS:40:3:2"Roundup:ready"soybean" demonstrated"that"mortality,"growth"and"reproductive"maturity"of"D.'magna"are" negatively"correlated"with"levels"of"glyphosate"residues"(paper"IV)."" Review"of"evidence:"Genetically"modified"glyphosate:tolerant"crops"have"been" subjected"to"numerous"analyses"and"used"as"feed"in"animal"feeding"studies."Regulatory" review"of"these"studies"in"the"United"States"of"America"and"the"European"Union"has" concluded"that"they"sufficiently"document"that"such"crops"are"essentially"equivalent"to" unmodified"varieties."However,"the"review"indicates"systematic"omission"of"information" on"herbicide"residue"levels"in"the"quality"testing"and"risk"assessment"of"genetically" modified"glyphosate:tolerant"crops."This"finding"is"an"important"background"for" understanding"the"protracted"controversy"on"safety"testing"and"indicates"that"the" current"regulatory"requirements"are"insufficient"to"ensure"identification"of"undesirable" substance"(paper"V)."" Conclusions"from"the"research:"The"findings"in"paper"I"and"the"scrutiny"of"evidence" from"archives"justifies"a"revision"of"industrial"tests"concerning"glyphosate"ecotoxicity"in" aquatic"invertebrates."The"general"indications"from"the"specific"findings"justify"further" measures"for"scrutiny"of"fundamental"documentation"on"ecological"effects,"toxicology" and"health."These"findings"of"faulty"industry"studies"for"assessment"of"glyphosate" ecotoxicity"are"mirrored"by"the"findings"of"paper"V,"which"demonstrates"similar"grave" shortcomings"in"industry"studies"assessing"quality"and"safety"of"glyphosate:tolerant" crops."" The"research"presented"in"papers"II,"II"and"IV"contributes"to"a"body"of"evidence" indicating"that"directly"and"indirectly"measurable"material"differences"between" transgenic"and"conventional"cultivars"are"significant"qualitative"aspects,"which" challenge"the"concept"of"substantial"equivalence."The"traditional"methodology"for"

11"

testing"whether"transgenic"plants"are"substantially"equivalent"to"their"unmodified" origins"should"be"adjusted"based"on"these"findings."" Exposure"to"lowRlevels"of"glyphosate"or"Roundup"in"the"environment"will"impair" D.#magna"growth"and"reproduction."This"indicates"that"adverse"effects"seen"in" feedingRstudies"could"be"caused"by"toxic"effects"from"glyphosate"residues."" The"research"leads"to"reflections"on"issues"such"as"methods"employed"by"biotech" industry"in"production"and"testing"of"transgenic"varieties,"regulatory"authority" oversight"and"wider"socio:political"aspects."" " Primary"research"findings:" •

Glyphosate"and"Roundup"herbicide"are"more"toxic"to"non:target"organism"D.'magna" than"previously"published"evidence"would"suggest"(paper"I)"

•

Glyphosate"tolerant"soybean"accumulates"residues"of"glyphosate"and" aminomethylphosphonic"acid"(AMPA)(paper"II)"

•

Compositional"characteristics"of"biochemical"and"mineral"constituents"in"soybean"is" affected"by"glyphosate"residues"(paper"II)""

•

Feed"quality"of"soybean"meal"from"glyphosate"tolerant"soy"is"not"equivalent"to"that" of"organic"or"conventional"unmodified"comparators"(papers"III"&"IV)""

•

Important"vital"parameters"of"D.'magna"fed"GTS:40:3:2"glyphosate"are"negatively" correlated"with"glyphosate:herbicide"residue"levels"(paper"IV)""

•

Analyses"for"relevant"herbicides"residues"are"systematically"missing"in"industry" tests"of"herbicide:tolerant"transgenic"varieties"(paper"V)"" "

" Table 2. Primary findings from the presented research papers

"

12"

Background Soybean" Soybean"(Glycine'max)"is"predominantly"cultivated"in"North"and"South"America"in" industrial:scale"farming"system"monoculture,"which"is"often"found"to"dominate"the" landscape"(Pengue,"2005)."Total"global"production"of"soybean"was"estimated"to"be"in" the"order"of"287"million"ton"in"the"2013/14"growing:seasons"(USDA,"2014)"and"is" predicted"to"expand."The"majority"of"this"substantial"harvest"continues"to"be"from" glyphosate:tolerant"soybean"(Antoniou"et'al.,"2012;"Binimelis"et'al.,"2009;"Bonny,"2011;" ISAAA,"2014)."This"large"biomass"is"primarily"processed"into"low:cost"ingredients"for" farm:animal"feed"but"also"in"part"for"human"food."The"GTS:40:3:2"variety"of"soybean"is" a"patented"GMO"product"developed"to"withstand"glyphosate"herbicides"in"commercial" formulations"such"as"Roundup."The"plant"is"commonly"known"to"farmers,"agronomists," regulators"and"other"professionals"by"its"trade:name;'Roundup6ready'soybean.""" Roundup:ready"soy"is"widely"adapted"by"farmers"growing"soybean"in"the"USA"and"in" several"countries"in"South"America."As"documented"by"Sylvie"Bonny"(2011),"there"are" several"reasons"explaining"this"success."Even"though"the"Roundup:ready"seed"is" typically"more"expensive"than"conventional"seed"and"farmers"opting"to"use"it"are"bound" by"stewardship"agreement"contracts,"the"benefits"appear"to"outweigh"the"costs." Amongst"these"benefits"we"find"lowered"overall"costs"of"herbicides,"an"initial"decrease" in"number"of"seasonal"herbicide"applications,"potentially"higher"yields"caused"by"better" control"of"weeds,"and"potential"decrease"in"manual"labour"spent"on"weeding"and" plowing."However,"it"is"also"indicated"that"increasing"weed:resistance"necessitates" increase"in"application"rates"of"glyphosate"often"combined"with"use"of"other"herbicides" (in"pre:emergence"or"post:harvest"application),"thus"reducing"overall"efficacy"of"the" Roundup:ready"system." Glyphosate" Just"as"soy"has"become"world"number:one"cultivar,"glyphosate"is"world"number:one" herbicide."Commercial"initiative"combined"these"two"successes"into"genetically" modified"glyphosate:tolerant"soy"thus"creating"an"agroecological"system"which"presents" important"biosafety"challenges.""

13"

It"has"even"been"claimed"that"transgenic"GTS:40:3:2"glyphosate"tolerant"soybean" initially"was"developed"by"chemical"industry"primarily"as"a"strategy"to"increase" consumption"of"glyphosate:herbicide"Roundup"in"agriculture"(Charles,"2001)."Thus,"to" evaluate"the"complexity"still"enshrouding"GTS:40:3:2"glyphosate"tolerant"soybean" nearly"two"decades"after"it"was"released"onto"the"market,"it"is"arguably"essential"to" include"glyphosate"in"the"analysis."Furthermore,"it"has"been"known"for"more"than"a" decade"that"glyphosate:tolerant"soy"can"accumulate"glyphosate"residues,"depending"on" factors"such"as"application"intensity"and"timing"(Duke"et'al.,"2003)."The"herbicidal" properties"of"glyphosate"(N:phosphonomethyl:glycine)"inhibit"biosynthesis"of" chorismate"from"shikimate"(Amrhein"et'al.,"1980),"thereby"lethally"disrupting" photosynthesis"and"plant"cell"metabolism."Transgenic"bypassing"of"this"vital"plant" metabolic"pathway"which"is"specificaly"targeted"by"glyphosate,"allows"for"herbicide" application"onto"growing"crops."Glyphosate"herbicide"main"ingredients"are"usually" isopropylamine:salt"(or"trimesium"salt)"of"N:phosphonomethyl:glycine."These" glyphosates"degrade"into"metabolites,"of"which"Aminomethylphosphonic"Acid"(AMPA)" is"recognized"as"the"most"important."" Glyphosate:tolerance"is"still"the"main"genetic"modification"in"crops"used"in" industrialized"agriculture"(Benbrook,"2012)."Glyphosate"herbicides"such"as"Roundup" have"been"on"the"market"for"four"decades"and"their"use"is"still"increasing,"making"them" the"primary"category"of"pesticides"world:wide."Thus"both"by"volume"and"by"revenue" glyphosate"is"the"globally"dominant"active:ingredient"for"herbicides,"with"recent" estimates"of"global"annual"production"reaching"1.1"million"tonnes"(Székács"&"Darvas," 2012)."" Glyphosate"herbicides"are"controversial."Even"amongst"scientists"these"chemicals"are" perceived"very"differently."Some"describe"them"as"the'ideal'herbicide"(Duke"&"Powles," 2008),"with"little"or"no"impact"on"non:target"species"(Giesy"et'al."2000)"and"presenting" no"danger"to"human"health"(Williams"et'al."2000)."Other"researchers"have"shown"these" chemicals"to"have"considerable"negative"effects"on"non:target"organisms"such"as"aquatic" invertebrates"(Folmar"et'al.,"1979)"and"amphibians"(Relyea,"2005;"Mann"et'al.,"2009)."" In"similar"ways,"scientific"research"into"transgenic"cultivars"also"reaches"opposing" conclusions"regarding"health,"ecology"and"safety"questions."Both"proponents"and"

14"

opponents"present"ostensibly"valid"scientific"evidence"to"support"their"views,"serving" only"to"complicate"the"controversy." Important"societal"challenges"related"to"glyphosate:tolerant"soybean"production" include"ecological"damage"through"deforestation"and"degradation"of"natural"habitats" (Pengue,"2005)"and"glyphosate"pollution"of"environment"(Benbrook,"2012)."The"large: scale"cultivation"of"glyphosate:tolerant"soy"has"also"been"identified"as"a"main"cause"for" emergence"and"widespread"occurrence"of"numerous"glyphosate:resistant"agricultural" weeds"(Duke"&"Powles,"2008)."The"weed:challenges"tend"to"be"met"with"alternative"and" more"potent"mixes"of"herbicides,"whereby"older"and"arguably"more"toxic"herbicides" (notably"atrazine)"are"reintroduced."This"phenomenon"has"shown"positive"correlation" with"increased"occurrence"of"severe"medical"problems"in"farmers"and"farm"village" populations"in"GTS:40:3:2"production"areas"in"Argentina"(Vazquez"&"Nota,"2011)."" Despite"the"challenges"associated"with"both"the"continued"use"of"glyphosate"as"the" principal"herbicide"and"the"continued"cultivation"of"glyphosate"tolerant"crops,"there"are" few"attractive"chemical:biotechnological"alternatives"at"present.""Several"crop"varieties" tolerant"to"herbicidal"chemicals"glufosinate:ammonium,"dicamba"and"2,4:D"are" currently"either"in"development,"awaiting"approval"or"already"on"the"market."But,"it"is" still"an"unresolved"issue"whether"these"crop"varieties"and"agrochemical"systems"(which" are"relying"on""old""herbicide"technology)"are"as"efficient,"cost:effective"or"as" environmentally"benign,"as"the"existing"glyphosate:tolerant"varieties"currently" available."It"should"also"be"noted"that"despite"the"aforementioned"challenges"posed"by" glyphosate:tolerant"GMOs,"several"large"biotech"firms"are"now"releasing"“second: generation”"glyphosate:tolerant"cultivars"touted"as"being"even"more"efficient."Some" offer"“stacked:traits”"biotech:packages;"stacked:traits"cultivars"are"hybrid"varieties" with"a"number"of"transgenes"expressing"herbicide"tolerance"of"several"active" ingredients"or"inducing"additional"production"of"insecticidal"Bt:toxins."This"combining" of"approved"transgenes"into"single"cultivars"is"marketed"in"guise"of"a"partial"solution"to" problems"posed"by"resistant"weed"and"insect"varieties."Sceptics"judge"these"responses" to"constitute"little"more"than"temporary"technological"fixes,"which"are"not"sustainable" for"either"farmers"or"environment"(Antoniou"et'al.,"2010;"Antoniou"et'al.,"2012;" Heinemann"et'al.,"2014)."Developing"a"new"herbicide"and"getting"it"approved"for"use"is" very"costly."According"to"some"estimates,"the"financial"investments"of"industry"can"

15"

amount"to"US"$180"million"and"the"regulatory"approval"can"take"a"decade"(Smith"et'al.," 2008;"Mcdougall,"2010)."Furthermore,"it"is"challenging"for"industry"to"meet"societal" demands"in"such"developments;"new"compounds"are"expected"to"have"high"target" specificity"and"low"general"toxicity"(for"the"environment,"users"and"eventual"consumers" of"agricultural"commodities)."The"biotech:agrochemical"industry"therefore"adheres"to" two"general"strategies:"it"develops"and"registers"new"transgenic"cultivars"and"chemical" compounds"for"the"market"(ISAAA,"2014);"and"it"uses"existing"chemical"compounds"in" new"ways,"notably"through"introduction"of"transgenic"varieties"that"tolerate"higher" doses"of"approved"agrochemicals"such"as"glyphosate"(eg."Cao"et'al.,"2012;"2013)."The" role"of"glyphosate"herbicides"can"therefore"be"expected"to"remain"predominant"in" global"industrial"agriculture,"especially"in"cultivation"of"glyphosate:tolerant"varieties."As" such,"it"is"relevant"to"consider"the"possible"benefits"and"challenges"associated"with" continued"or"increased"glyphosate"use."" RiskRassessment"of"transgenic"cultivars" To"assure"accurate"assessment"of"potential"risk"stemming"from"cultivation"or" consumption"of"transgenic"cultivars,"it"must"be"assumed"that"relevant"qualitative" differences"will"be"detectable"through"existing"methods."This"may"seem"a"trivial"point" but"has"in"fact"led"to"immense"scientific"and"regulatory"debate"involving"societal," political"and"commercial"interests."This"should"not"be"underestimated;"detailed" guidelines"on"analysis"and"risk:assessment"of"transgenic"plants"for"cultivation"and" consumption"have"been"elaborated"by"the"European"Food"Safety"Authority"(EFSA,"2008;" 2010;"Waigmann"et'al.,"2012),"the"United"Nations"Food"and"Agriculture"Organisation" (FAO,"2011)"as"well"as"the"Organisation"for"Economic"Cooperation"and"Development" (OECD,"1993;"1998;"2001;"2002"and"2006)."The"common"recommended"approach"is"to" compare"new"transgenic"cultivars"with"conventional"comparators"(near"isogenic" unmodified"plants)"by"evaluation"of"phenotypical"traits"and"by"analysis"of"molecular" and"biochemical"constituents."Material"from"such"transgenic"plants"is"compared"with" that"from"conventional"varieties"to"ensure"equivalence"of"nutrients,"minerals"and"other" important"content."As"there"will"be"natural"variation"in"levels"of"such"constituents,"it"is" advised"that"both"the"transgenic"variety"and"the"comparator"of"choice"be"cultivated"in" adjacent,"controlled"environments."Complimentary"sprays"applicable"to"transgenic" herbicide"tolerant"varieties"as"well"as"the"complex"genetic"background"of"transgenic"

16"

cultivars"bearing"stacked"events"complicate"such"benchmarking"by"making" representative"agricultural"conditions"more"difficult"to"model"as"well"as"making" representative"comparators"difficult"to"obtain."Thus,"in"assessing"biosafety"aspects"of" transgenic"cultivar"GTS:40:3:2"Roundup:ready"soybean,"I"have"made"certain" assumptions"and"adaptations"in"order"to"perform"relevant"analysis,"ecological" modelling"and"feeding"studies.""One"such"adaptation"has"been"the"decision"to"use"a" selection"of"subsamples"to"produce"representative"test:material"for"diets"of"different" soy:types,"this"is"presented"as"an"alternative"to"using"near:isogenic"comparators."" It"must"be"assumed"that"not"only"genetic"background"of"the"cultivar"in"question,"but"also" abiotic"factors"such"as"soil"quality,"environment"and"cultivating"regime,"will"play"a" significant"role"for"composition"of"the"resulting"produce."Different"varieties"of"the"same" species"of"plant"will"be"more"or"less"suited"for"specific"environments"and"growth" conditions,"thus"the"natural"”normal”"variation"in"nutrients"and"specific"elements"can"be" interpreted"as"significant"qualitative"differences"in"produce"from"the"same"plant"variety." Compositional"differences"will"also"be"seen"in"produce"from"plants"grown"in"the"same" environment,"if"these"plants"happen"to"be"significantly"different"genotypes."Any"living" organism"will"display"phenotypic"variability"and"flexibility,"establishing"visible"or"at" least"somehow"measurable"and"quantifiable"differences,"even"amongst"clonal"or" otherwise"near:isogenic"individuals."" Since"the"transgenic"varieties"of"maize"and"soy"presently"used"in"agriculture"are" primarily"optimized"hybrids"(produced"by"a"mixture"of"transgenic"methods"and" traditional"breeding"techniques),"it"is"very"difficult"to"come"up"with"a"comparator" having"only"the"absence"of"the"specific"modification"as"the"sole"differing"factor."Given" these"facts,"EFSA"has"presented"requirements"for"inclusion"of"several"comparators" when"assessing"a"transgenic"variety."Thus,"it"is"not"seen"as"valid"practice"to"compare" one"specific"transgenic"variety"to"one"specific"unmodified"variety,"rather"the"quality"of" the"specific"transgenic"cultivar"should"be"compared"to"the"variability"of"several" representative"non:transgenic"cultivars." Substantial"equivalence"" The"principle"of"substantial"equivalence"is"at"the"core"of"an"immense"and"complex" discussion"amongst"scientists"and"some"explanation"is"justified;"“substantial"

17"

equivalence"is"a"concept,"developed"by"OECD"in"1991,"that"maintains"that"a"novel"food," for"example,"one"that"derives"from"genetic"modification"or"engineering,"should"be" considered"the"same"as"and"as"safe"as"a"conventional"food"if"it"demonstrates"the"same" characteristics"and"composition"as"the"conventional"food”'(Womach,"2005"p."248).""In" 1997,"the"European"Commission"regulated"its"policy"on"novel"foods"(from"transgenic" plants)"stating"that"food"and"feed"from"such"plants"are"expected"not"to""present"a" danger"for"the"consumer","or""mislead"the"consumer","or""differ"from"foods"or"food" ingredients"which"they"are"intended"to"replace"to"such"an"extent"that"their"normal" consumption"would"be"nutritionally"disadvantageous"for"the"consumer""(EC,"1997"p."5)."" The"regulation"goes"on"to"state"that""[this"policy...]"shall"apply"to"foods"or"food" ingredients"[...]"which,"on"the"basis"of"the"scientific"evidence"available"[...]"are" substantially"equivalent"to"existing"foods"or"food"ingredients"as"regards"their" composition,"nutritional"value,"metabolism,"intended"use"and"the"level"of"undesirable" substances"contained"therein""(EC,"1997"p."5)."This"raises"questions"concerning"the" qualitative"evaluation"of"substances"which"vary"from"benign"to"harmful."It"could"be" argued"that"pesticide"residues"should"simply"be"termed""undesirable"."Such" interpretation"would"define"material"such"as"GTS:40:3:2"as"substantially"different"from" conventional"soybeans"due"to"its"inherent"glyphosate"residue"levels."" Substantial"equivalence"is"difficult"to"confirm"as"it"would"imply"full"analysis"of"all" constituents"in"produce"of"a"specific"new"plant"proving"same"characteristics"and" composition"as"a"conventional"comparator."For"practical"reasons,"such"analysis"is" limited"to"a"spectrum"of"biochemical"and"mineral"constituents."Rhetorically"it"could"be" asked"whether"the"finding"of"no"significant"difference"in"a"rather"limited"analysis"in" itself"confirms"that"the"tested"biological"materials"are"equivalent?"Additional" constituents"can"arguably"be"considered"important"in"specific"cultivars"on"a"case:by: case"basis"supporting"a"demand"that"tests"be"performed"using"supplemental"methods"or" other"approaches."One"such"argument"has"been"presented"as"the"notion"that"it"is"not" enough"to"assess"only"the"potential"for"added"qualities"stemming"from"the"insert,"but" also"to"investigate"possible"effects"of"the"genetic"modification"itself"(Traavik,"1999;" Traavik"&"Lim,"2007)."Opening"this"discussion"would"be"lengthy,"as"it"is"a"fact"that"the" modification"process"itself"has"been"shown"to"have"surprising"results"in"the"host" genome,"often"with"bits"and"pieces"of"the"transgene"scattered"onto"the"receiving"DNA."In"

18"

effect"this"interpretation"can"be"extracted"even"from"the"US"FDA"policy"brief"on"biotech" plants"(US"FDA,"1992)."Thus,"this"concern"is"expressed"not"only"by"independent" scientists."Even"EFSA"has"acknowledged"the"substance"of"this"issue"through"its"recent" adoption"of"guidelines"on"risk"assessment"of"genetically"modified"(GM)"plants,"stating" that"molecular"characterisation"is"a"first"step"to"assess"unintended"effects"of"the" modification"(e.g."to"ensure"that"there"is"no"loss"of"endogenous"gene"function"at"the" insertion"site[s])(EFSA,"2010)."" Published"evidence"on"compositional"analyses"of"glyphosate:tolerant"transgenic"crops" has"been"reviewed"by"biotech"industry"researchers"(Harrigan"et'al.,"2010)"as"well"as" independent"researchers"(Antoniou"et'al.,"2012;"Ricroch"et'al.,"2011)."These"research" groups"reached"differing"conclusions."Based"on"a"total"of"1,840"statistical"comparisons" of"produce"from"genetically"modified"glyphosate"tolerant"cultivars"compared"with" corresponding"conventional"controls,"Harrigan"et'al."found"that"the"majority"of" parameters"(88.5%)"showed"no"significant"differences."They"conclude"that"natural" variation"in"crop"composition"are"the"cause"of"the"remaining"significant"differences," thus"proving"the"transgenic"varieties"to"be"within"the"scope"of"conventional"crop"quality" variation."The"review"by"Ricroch"et'al."supports"the"conclusions"presented"by"Harrigan" et'al."and"concludes"that"the"variation"of"transgenic"crops"compositional"characteristics" are"within"the"range"of"natural"variation"of"unmodified"comparators,"but"goes"on"to" suggest"that"new"profiling"techniques"using"proteomics"and"similar"molecular" fingerprinting"would"be"useful"in"future"evaluations"and"safety"assessment"of"transgenic" varieties."Contrary"to"this,"Antoniou"et'al."mention"several"important"biosafety"issues" and"aspects"of"crop"quality"currently"undercommunicated"in"industry"research,"one" being"glyphosate:tolerant"varieties'"(notably"the"GTS:40:3:2"glyphosate"tolerant" soybean)"physiological"potential"to"absorb"and"accumulate"herbicide"residues,"which" can"then"be"passed"on"to"consumers."" Agriculture"affects"aquatic"ecology" Agriculture"affects"environment"not"only"by"the"fact"that"large"areas"are"under" cultivation"and"thus"denuded"of"native"biosystems,"but"also"through"the"effects"that" agriculture"activities"have"on"surrounding"habitats."Aquatic"habitats"are"affected"by" remains"of"fertilizers"and"pesticides"leaking"from"farmlands"and"also"by"remains"of" plant"material"left"in"the"fields"after"harvest."Such"biomass"is"blown"into"ditches"by"wind"

19"

and"carried"away"by"rainwater"runoff,"often"ending"up"in"the"habitats"of"aquatic"animals" (Rosi:Marshall"et'al.,"2007)."Inconspicuous"plankton"organisms"such"as"the"common" water:flea"Daphnia'magna"perform"important"ecological"functions"in"ponds"and"lakes" globally,"even"in"areas"where"agriculture"is"a"dominant"activity"(Benzie,"2010;" Wesenberg:Lund,"1926)."D.'magna"therefore"constitutes"a"relevant"model"organism"to" test"certain"environmental"effects"of"agriculture."Increasing"use"of"plant:based"feed" ingredients"such"as"soybean:meal"in"feed"formulations"for"aquaculture"of"fish"and" crustaceans"highlights"the"relevance"of"this"animal"model"representative"of"farmed" organisms"in"aquaculture"as"well"as"indicator"of"ecological"effects."It"is"also"relevant"to" further"discuss"the"principal"aspects"of"laboratory:modelling"industrial:scale"feeding"of" various"materials"to"species"of"animal"in"production."Farmers"and"feed:producing" industry"continuously"introduce"unconventional"ingredients"into"the"diets"of"farmed" organisms"which"may"raise"discussions"on"concepts"such"as"natural"food"and"the" possible"intuitive"preconceptions"of"food"and"feed"composition."Established"methods"for" toxicological"testing"of"chemicals"use"different"species"of"microorganisms,"plants"and" animals"as"indicators"of"ecological"effects"on"organism,"biotope"and"ecosystem"level."In" similar"ways,"tests"with"rodents"and"other"mammals,"as"well"as"human"cell:lines,"are" used"to"anticipate"specific"effects"on"human"health."" TestRanimal"biology" Daphnia'magna"is"an"omnivorous"planktonic"filtrator"known"to"feed"on"a"variety"of" organic"material,"such"as"unicellular"algae,"bacteria,"yeast"cells"and"suspended" particulate"material"of"plant"and"animal"origin,"even"benthic"biomass."Under"optimal" conditions"D.'magna"form"large"clonal"populations"by"asexual"parthenogenic" reproduction."The"species"is"a"slow"swimmer"highly"vulnerable"to"predation"from"fish," and"is"thus"found"primarily"in"smaller"bodies"of"stagnant"water."D.'magna"is"abundant"in" Europe"and"is"uncomplicated"to"rear"in"aquaria"and"under"laboratory"condition"due"to" tolerance"to"changes"in"temperature,"water:quality"and"the"overall"phenotypic"plasticity" and"adaptability"of"the"species."D.'magna"is"arguably"the"best"studied"of"the"cladoceran" species"and"subject"of"extensive"research"for"more"than"two:and:a:half"centuries"since" this"species"was"described"by"Schäffer"in"1755"(Fryer,"2008)."Daphnid"breeding"in" captivity"is"relatively"undemanding"and"species"of"water:flies"are"used"in"recreational" aquaristics"as"well"as"intensive"cultivation"of"aquaculture"biomass,"often"serving"as"feed"

20"

for"larger"and"commercially"attractive"species."Daphnid"species"pulex"and"magna"are" globally"recognized"as"indicator"organisms."Published"evidence"shows"D.'magna'to"be"a" useful"indicator"species"for"aquatic"ecotoxicity"testing"of"materials,"substances"and" effluents,"starting"well"before"1930"(Naumann,"1929;"1933;"1934a;"1934b)."Daphnids" have"been"used"for"centuries"in"biological"research"and"for"decades"in"specific" toxicological"testing."National"and"international"institutions,"industrial"organisations," universities"and"laboratories"have"developed"specific"guidelines"and"protocols"for"use"of" daphnids"in"acute"toxicity"testing"and"reproductive"studies"(US:EPA,"1996;"US:EPA," 2002;"OECD,"2004;"OECD,"2008)."At"present,"laboratories"deploy"established"standard" guidelines"for"testing"of"acute"toxicity"(OECD,"2004;"US:EPA,"1996)"or"testing"for" potentially"more"subtle"chronic"effects"in"long:term"exposure"reproduction"studies" (OECD,"2008;"US:EPA,"1996).""" D.'magna"also"has"a"demonstrated"history"as"test:species"in"evaluating"and"comparing" transgenic"plant"material"such"as"Bt:maize"MON"810"(genetically"modified"with"insert"of" toxin:producing"gene"from"Bacillus'thuringiensis)"in"aquatic"suspension"of"powdered" kernel"(Bøhn"et'al.,"2008;"2010)"and"powdered"leaves"(Holderbaum"et'al.,"submitted)." Industry"studies"have"tested"other"varieties"of"transgenic"Bt:maize"in"D.'magna" ecotoxicological"testing"of"whole"pollen"(Collins,"1994;"Privalle,"1997)"or"merely"the"Bt: toxin"itself"in"aquatic"dilution"(Raybould"&"Vlachos,"2011).""" D.'magna"is"very"adaptable"and"its"use"in"testing"is"not"limited"to"that"of"hydrophilic" substances"or"materials"that"can"be"suspended"as"particulate"matter."Interestingly,"in" recent"work"testing"biological"effects"of"electromagnetic"fields,"aquaria"holding"" D.'magna'were"simply"placed"relative"to"physical"location"of"a"source"of"radiation," exposing"replicate"experimental"units"to"gradients"of"the"investigated"factor"(Krylov," 2008)."In"a"recent"testing"of"qualitative"aspects"of"laboratory"plastics,"juvenile"D.'magna" were"reared"in"a"variety"of"plastic"environments,"with"distinct"and"measurable"effects" on"such"parameters"as"growth"and"reproduction"(Cuhra,"in"prep.)." Related"work" Important"evidence"concerning"possible"adverse"effects"of"genetically"modified"plants" emerged"when"it"was"demonstrated"that"transgenic"MON:810"Bt:maize"variety"was"not" substantially"equivalent"to"near:isogenic"conventional"maize"cultivars"grown"in"parallel"

21"

plots"at"the"same"Philippines"farmland"(Bøhn"et'al.,"2008;"2010)."Despite"strong"critique" against"the"2008"study"(EFSA,"2009;"Ricroch"et'al.,"2010,"Romeis"et'al.,"2013)"it"was" considered"important"evidence"of"negative"impact"from"MON:810"towards"non:target" organisms"and"used"for"reassessment"of"industry"application"for"cultivation"of"MON: 810"maize"(Bøhn"et'al.,"2012)."The"study"demonstrated"the"applicability"of"a"well"known" traditional"method"for"substance:testing"as"indicator"of"qualitative"aspects"of"plant" materials."Animals"receiving"feed"produced"from"the"transgenic"variety"had"reduced" reproduction"and"higher"mortality"than"those"fed"conventional"maize."This"indicated" that"either"the"variety"of"Bt:toxin"produced"by"the"transgenic"plant"was"having"adverse" effects"on"organisms"which"are"not"supposed"to"be"affected,"or"some"other"compound"or" mechanism"or"possibly"some"unspecified"consequence"of"the"transgenic"modification" itself,"was"having"significant"negative"impact"on"the"Daphnia."The"evidence"indicated" that"potentially"adverse"ecological"effects"of"MON:810"cultivation"on"non:target" organisms"could"not"be"ruled"out.""The"initial"feeding"studies"in"Bt:maize"were" performed"with"feed"prepared"from"maize"kernels,"which"is"representative"of"the"main" produce"entering"human"food"chain"directly"or"via"animal"feed."These"experiments"were" supplemented"by"series"of"D.'magna"feeding"studies"using"feed"produced"from" lyophilized"leaves"from"MON:810"maize"and"conventional"parent:line"comparators." These"experiments"are"more"representative"in"testing"of"ecological"effects"from" monoculture"waste"biomass"consisting"of"non:harvested"material"such"as"leaves,"stems" and"roots."In"a"review"of"published"evidence,"EFSA"(2009)"concluded"that"the"study"by" Bøhn"et'al."(2008)"was"flawed,"mainly"because"a)"maize"flour"is"not"part"of"the"natural" diet"of"Daphnia"and"the"observed"unusual"delays"in"development"therefore"could"be" caused"by"nutritional"deficiency,"and"b)"internationally"accepted"guidelines"for"toxicity" and"reproduction"testing"in"Daphnia"were"not"followed."This"critique"however,"is"not"a" scientifically"based"rejection"of"the"findings"of"effects"on"non:target"organisms"and"must" be"discussed"further." It"is"also"relevant"in"this"context"to"mention"the"controversial"work"by"Séralini"et'al." (2012),"a"life:long"(two:year)"study"in"which"rats"manifested"adverse"effects"from" Roundup"herbicide"formulation"(in"drinking:water)"and"from"feeding"glyphosate: tolerant"NK:603"maize."It"should"be"noted"that"the"work"by"Séralini"et'al."immediately" provoked"a"storm"of"critical"questioning"from"biotech"industry,"researchers"related"to"

22"

biotech"industry"as"well"as"independent"researchers."The"EFSA"reviewed"the"study"and" in"2013"concluded"that"it"was"methodologically"flawed"and"that"the"results"should"be" disregarded."Subsequently,"in"2013"it"was"retracted"by"the"editor"of"the"journal"that" originally"reviewed"and"published"it."However,"the"following"year"the"work"was" republished"by"a"different"journal"(Séralini"et'al.'2014)." Regardless"of"whether"the"conclusions"drawn"by"Séralini"et'al."were"fully"valid"or"not,"it" is"important"to"acknowledge"that"the"methodology"in"the"mentioned"study"attempted"to" assess"effects"from;"a)"consumption"of"a"glyphosate:tolerant"transgenic"variety"in"feed," b)"ingestion"of"drinking:water"containing"the"relevant"herbicide,"c)"in"life"long"exposure." This"raises"similar"biosafety"questions"as"the"reflections"leading"to"the"work"in"GTS:40: 3:2"soybean"investigated"here."" "The"relevance"of"the"re:assessment"of"fundamental"issues"raised"in"the"retracted"study" was"further"commented"upon"in"an"objection"on"final"rule"to"the"US"EPA"in"2013"(OPP" Docket,"2013)."The"objection"highlighted"aspects"of"a"US"EPA"internal"process"in"1981: 83"on"assessment"of"data"from"a"two:year"rat:feeding"study"submitted"by"industry."The" rats"were"fed"relatively"high"doses"of"glyphosate"but"the"initial"assessment"of"adenoma" and"carcinoma"incidence"was"re:evaluated"by"US"EPA"and"subsequently"the"conclusions" were"changed."The"objection"argued"that"there"were"findings"in"our"studies"presented" here"::"and"the"mentioned"study"by"Séralini"et'al."::"which"indicated"that"the"US:EPA" evaluation"of"the"1981:83"study"was"due"for"revision."However,"this"need"for"regulatory" re:assessment"still"remains"to"be"acknowledged."

23"

24"

Aim Research"question"" Research"question:"The"fundamental"reflections"leading"to"the"research"project"can"be" condensed"in"the"following"research"question:"Is'GTS6406362'genetically'modified' glyphosate6tolerant'soy'substantially'equivalent'to,'or'significantly'different'from,'its' unmodified'counterpart?" Scientific"assumptions:"In"order"to"investigate"the"research"question,"the"following" fundamental"assumption"relating"to"relevance"of"scientific"method"is"stated:''Substantial' equivalence'and/or'significant'differences'are'qualitative'parameters'which'in'GTS6406362' genetically'modified'glyphosate6tolerant'soy'can'be'estimated'by:'1)'analysis'of' composition'of'soybean6meal,'and'2)'through'life6long'feeding"studies"with"soybean:meal" in"test:organism"Daphnia'magna." Since"GTS:40:3:2"soybean"is"designed"to"be"cultivated"in"agriculture"system"dependent" on"glyphosate:herbicide"application,"agroecological"aspects"of"these"chemicals"are" included"in"the"analysis"and"assessment." Research"hypothesis"" Research"hypothesis:"Based"on"the"assumption"of"method"relevance"the"research" question"is"investigated"through"the"following"hypothesis:"Composition'characteristics' and'feed'quality'of'meal'from'GTS6406362'soybean'will'not'differ'significantly'from'meal' from'unmodified'comparators.' Research"methodology:"Following"methods"were"used"to"investigate"the"research" question;"a)"literature"studies"of"relevant"published"evidence,"b)"laboratory"testing"of" soybean"material"c)"feeding"studies"with"soybean"material"in"an"animal"model,"d)" toxicological"and"ecotoxicological"testing"of"glyphosate"chemicals"and"commercial" herbicide"formulations,"and"e)"revision"of"specific"studies"extracted"from"archives"in"the" United"States"of"America"via"a"FOIA:request."" "

25"

" " " Form"of"presentation" The"writing"will"present"research"conducted"to"answer"the"specific"research"question" and"will"further"reflect"on"the"wider"environmental"and"societal"implications."" Thus"the"writing"conforms"to"a"traditional"logical"structure"as"presented"below:" " :"an"introduction"based"on"mandate"and"observations" :"leading"to"a"relevant"specific"research"question" :"which"is"tested"by"scientific"methods" :"producing"results"that"are"discussed" :"leading"to"further"reflections"

" " " " "

:"opening"a"wider"environmental"and"societal"context" "

26"

"

Materials and methods Research"materials" In"2009"I"contacted"biotech"industry"companies"Monsanto"and"Dow"AgroSciences"to" obtain"plant"material"for"testing."Monsanto"replied"to"none"of"my"calls,"emails"or"regular" letters."Dow"AgroSciences"sent"me"an"email"in"response"to"one"of"my"letters,"politely" declining"to"furnish"material"for"my"research."I"thus"had"to"obtain"GTS40:3:2" glyphosate:tolerant"soy"materials"by"other"means."" An"aqueous"solution"of"40"%"b.w."glyphosate"in"the"form"of"N:(p"hosphonomethyl)" glycine:monoisopropylamine"salt"(glyphosate:IPA)"was"obtained"from"Sigma–Aldrich," St."Louis"MO"63103"USA"(Batch"no"10519EJ)."A"typical"commercial"brand"of"Roundup" formulation"was"bought"from"a"US"retailer"a"few"months"prior"to"the"testing."(Lot" I08080/FI/1/5),"containing"18"%"b.w."glyphosate,"0.73"%diquat:dibromide"and," according"to"label,"81.27"%"‘‘other"ingredients’’."The"producer"is"not"required"to"specify" these"other"ingredients"but"they"are"generally"thought"to"consist"of"mainly"water," activator"adjuvants"and"various"surfactants."The"brand"name"of"this"herbicide"is" Roundup"Weed"&"Grass"Killer"Concentrate"Plus"(paper"I)." Animal"model" Experiments"in"the"D.'magna"model"are"initiated"by"preparing"individual"beaker"glasses," typically"pre:fed"(feeding"studies)"or"prepared"with"exposure"gradient"toxins" (toxicological"studies)."A"healthy"stock"of"mother"animals"15"–"40"days"old"are"kept"in"4" :"8"large"4"liter"holding:tanks"(MF:beakers)."The"day"before"experiment"is"to"be" initiated,"all"juvenile"animals"are"removed"from"MF:beakers"by;"a)"transferring"mothers" to"petri"dishes"and"subsequently"b)"filtering"the"holding"medium"through"200µm" plankton"netting."Following"this"cleaning"the"mother"animals"are"transferred"back"into" the"MF:beakers."No"later"than"24"hours"following"this"procedure,"new:born"juveniles" approximately"850µm"in"length"are"carefully"collected"from"MF:beakers"and" transferred"to"a"single"large"petri"dish."Juveniles"are"allowed"to"mix"freely"for"a"few" minutes"and"following"this"homogenization,"individual"experimental"animals"are"taken" with"a"glass"pipette"(figure"1)"and"allocated"to"individual"100ml"beaker"glasses."Each" beaker"glass"contains"one"D.'magna"which"is"daily"monitored"and"fed"according"to""

27"

"

Figure 1. Petri dish with more than 300 juveniles born within 24 hours. The observer selects individual juveniles and allocates these to individual experimental units (glasses) according to randomized setup.

operating"procedures"and"specific"regime"of"individual"experimental"setup"(figures"2" and"4)."Each"beaker"is"an"experimental"unit,"which"is"randomly"allocated"a"position" within"the"experiment."Individual"experimental"units"are"permanently"allocated"to" defined"treatment"groups."Treatment"groups"contain"10,"15,"20"or"30"experimental" units,"depending"on"experimental"setup."Each"beaker"has"a"unique"identifier,"either"as"a" marking"on"the"beaker"itself,"or"as"a"marking"at"the"allocated"position."To"enable" randomization"of"positions,"these"are"affixed"to"plastic"trays"each"containing"6"to"7" treatment"groups,"with"5"experimental"units"in"each"group."According"to"number"of" individual"treatment"groups,"chronic"toxicity"experiments"employ"150:250"individual" animals"and"feeding"studies"employ"100:300"animals."Such"large"quantities"of" experimental"units"are"organized"on"up"to"9"trays"with"35"(30)"beaker"glasses"(figure"4)." Tray"position"(A,"B,"C)"on"individual"tables,"tray"orientation"and"table" position/orientation"is"changed"according"to"outcomes"of"randomization"software"at" defined"intervals,"e.g."days"2,"4,"6"etc."or"days"3,"6,"9"etc."for"the"full"42:day"duration"of" experiments."The"tables"have"wheels,"enabling"rotation"and"relocation."In"order"to" facilitate"handling,"daily"feeding"and"collection"of"data,"the"individual"trays"with"35"(30)" experimental"units"are"transferred"from"tables"in"experimental"area"(figure"4)"to"a" working"area"equipped"with"light:tables,"stereo:loupe"microscopes"and"highly"specific""

28"

Figure 2. Tray with 7 rows, each consisting of 5 experimental units. Row position and tray position and orientation is randomized within experimental setup, and changed at regular intervals according to outcome of randomization software.

tools"designed"and"adapted"for"efficient"and"non:disruptive"handling"and"photography" of"individual"animals."To"describe"these"daily"and"weekly"procedures"in"detail"is"not" possible"here,"however"it"suffices"to"note"that"the"specific"daily"routines"involving" animal"handling,"preparation"of"holding"media,"feeding"of"treatment:groups,"washing" glassware,"harvesting"information"and"registration"of"data"typically"demand"the"full" attention"of"1:2"primary"scientists"and"1:3"assistants"on"a"daily"basis"(weekends" included)"for"a"42:day"period"spanning"the"typical"longevity"of"a"D.'magna"individual." The"operating"costs"for"one"such"42:day"experiment"are"estimated"at"0.25"M"NOK," which"is"relatively"low"when"considering"the"relatively"high"number"of"experimental" units."Experiments"in"D.'magna"are"established"as"an"acknowledged"standard"in" toxicological"and"ecotoxicological"testing."Researchers"employed"by"biotechnological" industries"Monsanto"and"Syngenta"began"using"D.'magna"models"to"assess"toxicological" effects"from"genetically"modified"crops,"initially"as"studies"of"pollen"from"Bt:varieties"of" corn"(Zea'mays)"(Collins"1994;"Privalle"1997)."The"model"developed"by"GenØk"(figure"3)" and"described"here,"was"used"for"obtaing"data"for"Bøhn"et'al.,"2008;"2010"and"papers"III" and"IV."Employed"in"traditional"ecotoxicological"testing"of"herbicides"in"acute"exposure" and"chronic"exposure,"the"GenØk"model"yielded"the"data"reported"in"paper"I."

29"

* Daily registration = survival = ecdysis = reproduction

* Individual longevity = survival rates

* Daily feeding = specific group treatments = base-feed (algae)

* Reproductive outcome = quantification liveborn juveniles = quantification stillborn juveniles = quantification aborted eggs

* Regular renewal holding medium = 2 day cycle = 3 day cycle

* Measurement of carapace length = individual growth

* Animals microphotography = 6 day cycle * Randomization of position

Figure 3. Schematic overview of daily routines and harvesting of endpoints from individual experimental units.

3 2

A

B

1

C

Figure 4. Full experimental setup consisting of 9 trays, each with 7 treatment groups. Individual treatments typically consist of 15-30 experimental units and thus will be distributed as 3-6 rows within the setup. Labels A, B and C denote positions on individual tables 1, 2 and 3.

30"

Archive"studies" In"order"to"investigate"the"research"question,"it"was"found"important"to"access"relevant" archives"to"evaluate"original"toxicological"and"ecotoxicological"studies"which"form"the" basis"of"present"regulation"of"the"herbicides"investigated"through"laboratory"testing." In"November"2011"I"submitted"a"request1"for"disclosure"of"information"to"the"US"EPA," pursuant"to"US"Freedom"of"Information"Act"(FOIA)."The"targeted"information"was" contained"in"21"specific"Master"Record"Identification"Number"(MRID)"study"reports." These"reports"were"originally"submitted"by"industry"applicants"as"evidence"of" ecotoxicological"effects"of"glyphosate,"AMPA,"herbicide"formulation"(Roundup," surfactants"and"inert"ingredients)."The"MRID"is"the"US:EPA"file"containing"the"expert" evaluation"of"the"specific"test"reported"by"the"applicant2."My"request"was"granted"and"I" received"the"materials"in"early"spring"of"2012." There"are"two"ways"that"documentation"is"made"available."The"first"is"through"the" ongoing"scanning"of"archives"and"publishing"of"pdf:files"on"searchable"databases."This" we"may"call"“server:push”;"it"constitutes"the"bulk"of"the"material."The"second"is"by" “client:pull”,"through"specific"requests"to"government"agencies,"in"this"case"the"EPA"and" FDA."The"2011"EPA"report"to"the"US"Department"of"Justice"describes"in"some"detail"how" the"intentions"of"the"FOIA"are"being"implemented"in"the"agency,"with"specific" measurables"on"implementation,"training,"evaluation,"dialogue"with"the"public," proactive"disclosure,"use"of"social"networking"channels"and"reducing"backlog"(Jackson," 2011)."Also,"a"US"EPA/FOIA"Chief"Officer's"report"to"the"US"attorney"general"(US"EPA," 2009)"states"that"proactive"measures"have"been"initiated"regarding"Office"of"Pesticide" Program"(OPP)"documents,"releasing"a"large"number"of"scientific"reviews"as"searchable" documents"in"an"Internet"database"called"“The"Electronic"Reading"Room”."It"is" estimated"that"approximately"13,000"OPP"scientific"reviews"on"300"active"ingredients" are"available"through"this"EPA"website."The"purpose"of"this"proactive"disclosure"is" specifically"stated"as"disclosing"“science"reviews"and"pesticide"registration"files"[...]"vital" """""""""""""""""""""""""""""""""""""""""""""""""""""""" 1"Case"HQ:FOI:00325:12"US"EPA"FOIA"2011." 2"The"Master"Record"Identification"Number"is"defined"as"the"unique"cataloguing"number"assigned"to"an"

individual"pesticide"study"at"the"time"of"its"submission"to"the"EPA."

31"

to"industry,"interest"groups,"state,"local"and"foreign"governments,"and"many"other" stakeholders...”"(US"EPA,"2009)."" The"proactive"disclosure"is"an"ongoing"process."Files"from"the"EPA"archives"are" continuously"being"scanned"and"made"available"to"the"general"public."For"the"purpose" of"assessing"biosafety"of"modern"biotechnology"in"agriculture"and"studying"effects"of" transgenic"plants"and"associated"herbicides,"both"EPA"and"Department"of"Agriculture" (USDA)"and"Food"and"Drug"Administration"(FDA)"documents"are"of"importance."Coming" into"office,"President"Obama"initiated"a"change"in"the"policy"of"his"predecessors:"“In"the" two"years"since"the"President"issued"his"FOIA"Memorandum"directing"agencies"to"apply" a"presumption"of"openness"to"all"decisions"involving"the"FOIA,"agencies"across"the" government"have"taken"steps"to"create"a"more"open"and"accountable"government"' (FOIA,"2011)."" The"administrative"procedures"by"which"different"public"offices"and"parts"of"the"US" administration"incorporate"the"intentions"of"the"FOIA"into"their"daily"routines"vary,"but" there"is"an"underlying"principle"of"openness"which"is"valuable"to"science"as"well"as"to" the"interests"of"the"general"public."“The"U.S."Freedom"of"Information"Act"(FOIA)"is"a"law" ensuring"public"access"to"U.S."government"records."FOIA"carries"a"presumption"of" disclosure;"the"burden"is"on"the"government":"not"the"public":"to"substantiate"why" information"may"not"be"released."Upon"written"request,"agencies"of"the"United"States" government"are"required"to"disclose"those"records,"unless"they"can"be"lawfully"withheld" from"disclosure"under"one"of"nine"specific"exemptions"in"the"FOIA."This"right"of"access"is" ultimately"enforceable"in"federal"court”"(FOIA,"2009)."

32"

Research manuscripts

33"

34"

List"of"papers"

6 Paper"I:"""

Cuhra"M,"Traavik"T,"Bøhn"T"(2013)"" Clone6'and'age6dependent'toxicity'of'a'glyphosate'commercial'formulation'and'its' active'ingredient'in'Daphnia'magna.''

"

"

Ecotoxicology,"22:"251:262."

' Paper"II:""

Bøhn"T,"Cuhra"M,"Traavik"T,"Sanden"M,"Fagan"J,"Primicerio"R"(2014)"" "

"

"

Compositional'differences'in'soybeans'on'the'market:'glyphosate''

'

'

accumulates'in'Roundup'Ready'GM'soybeans.''

'

'

Food"Chemistry,"153:"207:215."

'

" Paper"III:"

Cuhra"M,"Traavik"T,"Bøhn"T"(2014)"" Life'cycle'fitness'differences'in'D.'magna'fed'Roundup'Ready'soybean'conventional' soybean'or'organic'soybean.'' Aquaculture"Nutrition"1365/2095."DOI"10.1111/anu.12199." '

Paper"IV:"

Cuhra"M,"Dando"M,"Traavik"T,"Primicerio"R,"Holderbaum,"D,"Bøhn"T"(2015)" Glyphosate6Residues'in'Roundup6Ready'Soybean'Impair'Daphnia'magna'Life6 Cycle."" Journal"of"Agricultural"Chemistry"and"Environment,"4,"24:36."" "

Paper"V:"

Cuhra"M"(2015)" Analysis'of'herbicide6residues'are'still'missing'in'risk6assessment'of'glyphosate6 tolerant'GMO6cultivars."" Environmental"Sciences"Europe"(manuscript"accepted"for"publication)"

35"

"

36"

Paper"I:"

Glyphosate"herbicide"ecotoxicity"""

" Title:"" "

Clone:"and"age:dependent"toxicity"of"a"glyphosate"commercial" formulation"and"its"active"ingredient"in"Daphnia'magna.""

" Authors:""

Marek"Cuhra,"Terje"Traavik,"Thomas"Bøhn"

Journal:""

Ecotoxicology,"22:"251:262"(2013)."

37"

"

38"

Ecotoxicology (2013) 22:251–262 DOI 10.1007/s10646-012-1021-1

Clone- and age-dependent toxicity of a glyphosate commercial formulation and its active ingredient in Daphnia magna Marek Cuhra • Terje Traavik • Thomas Bøhn

Accepted: 15 November 2012 / Published online: 6 December 2012  The Author(s) 2012. This article is published with open access at Springerlink.com

Abstract Low levels of glyphosate based herbicide induced significant negative effects on the aquatic invertebrate Daphnia magna. Glyphosate herbicides such as brands of Roundup, are known to be toxic to daphnids. However, published findings on acute toxicity show significant discrepancies and variation across several orders of magnitude. To test the acute effects of both glyphosate and a commercial formulation of Roundup (hereafter Roundup), we conducted a series of exposure experiments with different clones and age-classes of D. magna. The results demonstrated EC50 (48) values in the low ppm-range for Roundup as well as for the active ingredient (a.i.) isopropylamine salt of glyphosate (glyphosate IPA) alone. Roundup showed slightly lower acute toxicity than glyphosate IPA alone, i.e. EC50 values of 3.7–10.6 mg a.i./l, as compared to 1.4–7.2 mg a.i./l for glyphosate IPA. However, in chronic toxicity tests spanning the whole life-cycle, Roundup was more toxic. D. magna was exposed to sublethal nominal concentrations of 0.05, 0.15, 0.45, 1.35 and 4.05 mg a.i./l for 55 days. Significant reduction of juvenile size was observed even in the lowest test concentrations of 0.05 mg a.i./l, for both glyphosate and Roundup. At 0.45 mg a.i./l, growth, fecundity and abortion rate was affected, but only in animals exposed to Roundup. At 1.35 and 4.05 mg a.i./l of both glyphosate and Roundup, significant negative effects were seen on most tested parameters, including mortality. D. magna was adversely affected by a near 100 % abortion rate of eggs and M. Cuhra (&)
T. Traavik
T. Bøhn GenØk, Centre for Biosafety, The Science Park, P.O. Box 6418, 9294 Tromsø, Norway e-mail: [email protected] M. Cuhra
T. Traavik
T. Bøhn Faculty of Health Sciences, University of Tromsø, Tromsø, Norway

embryonic stages at 1.35 mg a.i./l of Roundup. The results indicate that aquatic invertebrate ecology can be adversely affected by relevant ambient concentrations of this major herbicide. We conclude that glyphosate and Roundup toxicity to aquatic invertebrates have been underestimated and that current European Commission and US EPA toxicity classification of these chemicals need to be revised. Keywords Cladocera
Glyphosate
Roundup
Aquatic
Chronic toxicity
Toxicology

Introduction The tonnage of glyphosate herbicide application has been constantly increasing since the introduction of this group of chemicals in 1971 (Dill et al. 2010). The 2008 global production was estimated to be 620.000 tonnes, representing a value of 8.3 billion US$, making glyphosate the most widely used herbicide ingredient worldwide (Pollak 2011). The most common herbicide formulations such as the brands of Roundup contain various salts of glyphosate that ensure high water solubility, mainly isopropylamine salt (IPA salt of glyphosate) (Woodburn 2000). Introduction of glyphosate tolerant transgenic crops such as ‘Roundup-Ready’ soy, maize, canola, sugarbeet and cotton, contribute to a further rapid expansion of use (Antoniou et al. 2010, Cerdeira and Duke 2006). Nearly 90 million hectares were planted with herbicide tolerant GM plants in 2010, primarily with glyphosate tolerant traits and primarily in North- and South America (James 2010). Glyphosate has been heralded as an ideal herbicide due to its target specificity and acclaimed low toxicity to non-target organisms (Cerdeira and Duke 2006; Duke and Powles 2008; Giesy et al. 2000). The majority of glyphosate herbicide applications, measured in gross

123

252

tonnage as well as number of herbicide brands, are in agriculture. Some use is also related to forestry, gardening and park management and even specialized applications such as weed management in fresh water lakes and streams (Simenstad et al. 1996). Field studies on effects of prescription dosage application of aquatic glyphosate herbicide on non-target organisms in fresh-water systems have been carried out. Most of these have neither demonstrated shortterm nor long term adverse effects (Gardner and Grue 1996, Siemering et al. 2008), although Pue´rtolas et al. (2010) observed significant toxic effects on the aquatic invertebrate Daphnia magna from established methods for glyphosate control of giant reed in a Spanish River system. Some field studies on effects of glyphosate herbicides in daphnids have not shown adverse effects in modelled instances of herbicide drift from agriculture bordering wetlands (Hessen et al. 1994), even when using dosages much higher (910 and 9100) than prescribed for agriculture use (Hildebrand et al. 1980). However, high levels of glyphosate have been measured in streams draining agricultural fields of transgenic ‘Roundup Ready’ soybeans, with adverse effects on non-target aquatic biodiversity (Ronco et al. 2008). Dynamics of glyphosate in soil, water and sediment have been well studied and its presence has been reported in general surface waters (Scribner et al. 2007; Struger et al. 2008; USGS 2010) as well as in farmland streams (Peruzzo et al. 2008, Ronco et al. 2008). We have reviewed the literature for short- and long-term toxicity studies of glyphosate and glyphosate based herbicides in aquatic organisms. This literature is based mainly on laboratory experiments, with some evidence derived from mesocosmstudies and field-studies. Some studies of effects on nontarget organisms indicate that glyphosate herbicides in fresh-water and marine ecosystems can have significant negative effects on for instance aquatic microbial communities (Pe´rez et al. 2007), macrophytes (Lockhart et al. 1989; Simenstad et al. 1996), cnidaria (Demetrio et al. 2012), sea-urchin embryogenesis (Marc et al. 2004), fish (Servizi et al. 1987), amphibians (Mann et al. 2009; Relyea 2005) and planktonic algae (Peterson et al. 1994; Pe´rez et al. 2007). However, a majority of relevant publications report low toxicity or no adverse effects from prescribed dosage use. This also corresponds to conclusions in published reviews of glyphosate-based herbicide ecotoxicity potential (Giesy et al. 2000; Dill et al. 2010). A recent review of glyphosate herbicide effects in aquatic ecosystems gives a comprehensive overview of individual studies for most investigated taxonomic groups (Pe´rez et al. 2012). Laboratory studies testing ecotoxicological effects of glyphosate and various glyphosate-based herbicides on specific aquatic organisms have been performed for four decades, with varying results even in the same test-species.

123

M. Cuhra et al.

Acute (immediate, short-term) glyphosate toxicity to aquatic invertebrates such as the model organism D. magna is generally considered by regulators to be relatively low (EC 2002; US EPA 1993; Mensink and Janssen 1994). Baseline effect studies and toxicological testing establishing EC50 and LC50 treshold values for glyphosate and glyphosate formulations in D. magna and other species of daphnids have shown highly variable results, ranking these chemicals from practically non-toxic to moderately toxic (FAO 2001; Folmar et al. 1979; McAllister and Forbis 1978; Melnichuk et al. 2007a; Tsui and Chu 2003). To some extent these differences have been attributed to additive or synergistic effects of nonspecified ‘‘inert ingredients’’ in herbicide formulations (Folmar et al. 1979; Melnichuk et al. 2007a) including adjuvants and additives, such as Polyethoxylated tallowamine (POEA) used for dispersal and increased plant uptake (Brausch et al. 2007). A study comparing acute toxicity of 6 different brands of formulated glyphosate-based herbicides in D. magna found LC50 values in the range 4.2–117 mg/l and highlighted the fact that published literature presents EC50 values spanning 3 orders of magnitude (Melnichuk et al. 2007a). Even ecotoxicological testing of the active ingredient glyphosate alone has given highly divergent results in D. magna. Some studies have reported LC50 values of 13–24 mg/l (FAO 2001) whereas others report values of 234 mg/l (Le et al. 2010), 780 mg/l (McAllister and Forbis 1978), 930 mg/l (Forbis and Boudreau 1981) or even above 2000 mg/l (Pereira et al. 2009). Long-term (chronic) exposure studies of glyphosate in D. magna comissioned by the producing industry have shown NOEC (no observed effect concentration) of 50 mg/l for glyphosate-IPA salt (McKee et al. 1982) but later independent studies of formulated glyphosate-based herbicides have shown substantially higher chronic toxicity in D. magna, with effects in concentrations of 2, 0.2 and even 0.02 mg/l glyphosate as active ingredient (a.i.) in a Roundup formulation (Papchenkova 2007). The inconsistent results with regard to toxicity of glyphosate and glyphosate-based herbicides may suggest varying sensitivity between different clones of D. magna. The literature lends some support for clone-specific sensitivity to metals and organic toxins like cadmium, copper, dichloraniline and benzyl sulfonate (Baird et al. 1991). In the present work we tested the following hypotheses: (i) acute toxicity (EC50) of glyphosate and a commercial Roundup formulation differs between clones of D. magna (clone-specific toxicity); (ii) acute toxicity of glyphosate and Roundup decreases with increasing age in D. magna (age-specific toxicity); (iii) chronic exposure to glyphosate and Roundup induces adverse effects on D. magna lifehistory traits (survival, growth, fecundity, abortion rate and juvenile body size) at much lower concentrations than acute EC50-values; (iv) Roundup is more toxic than

Clone- and age-dependent toxicity of a glyphosate commercial formulation

glyphosate at the same concentration of active ingredient since Roundup contains additional potentially toxic chemicals.

Materials and methods Clones of D. magna were obtained from laboratories at the Universities of Oslo (Tromsø-clone, courtesy of Dag Hessen, Dept. of Biology, University of Oslo, P.O. Box 1066 Blindern, NO-0316 Oslo, Norway), Reading (Reading-clone, courtesy of Richard Sibly, Amanda Callaghan, Chris Hill, School of Biological Sciences, The University of Reading, Whiteknights, PO Box 217, READING, Berkshire, RG6 6AH, United Kingdom) and Leuven (Knokke-clones, courtesy of Luc De Meester and Sarah Rousseaux (Katholieke Universiteit Leuven, Laboratory of Aquatic Ecology and Evolutionary Biology, Charles Deberiotstraat 32—box 2439, 3000 Leuven). These clones have very different backgrounds, as some are wild clones collected in Belgium from pristine lakes (Knokke 1 = KNO-15 NF14 and Knokke 2 = KNO-15-F5), from ‘‘extensive agriculture intermediate ponds’’, where some anthropogenic chemicals are expected (Knokke 3 = KNO 208 F1) and from ponds in areas of intensive agriculture (Knokke 4 = KNO-16-F8) (nomenclature of Luc De Meester, Coors et al. 2009). Two clones are from cultures reared for years in laboratories (clones Tromsø and Reading). Taxonomic control of clones (species) was performed according to defined morphological characters (Benzie 2005). Aqueous solution of 40 % b.w. glyphosate in the form of N-(phosphonomethyl) glycine-monoisopropylamine salt (glyphosate-IPA), hereafter ‘glyphosate’ in this work, was obtained from Sigma–Aldrich, St. Louis MO 63103 USA (Batch no 10519EJ). A typical commercial brand of Roundup formulation was bought from a US retailer a few months prior to the testing. The brand name of this herbicide was Roundup Weed & Grass Killer Concentrate Plus, hereafter ‘‘Roundup’’ or ‘‘R’’, (Lot I08080/FI/1/5), containing 18 % b.w. glyphosate, 0.73 % diquat-dibromide and, according to label, 81.27 % ‘‘other ingredients’’. The producer is not required to specify these other ingredients but they are generally thought to consist of mainly water, activator adjuvants and various surfactants (NPIC 2010; PAN 2011; Penner 2000) The Roundup herbicide was produced by Monsanto Company, Marysville, OH, USA. Both chemicals were stored in the dark at room temperature until use. Prior to the experiments, all clones were acclimatized in the laboratory at the University of Tromsø, Norway. Standardized D. magna mother populations brood stock (Benzie 2005) were produced in a fully artificial Aachener Daphnien Medium (ADaM) (modified from Klu¨ttgen et al. 1994) in 4 l glass beakers, fed on a diet of Selenastrum sp. green algae. The ADaM medium was produced in

253

de-ionised water using laboratory grade chemicals and evaporated natural sea-salt. The medium was adjusted to a pH of 7.5 (±0.7) with 0.1 M NaOH solution. Densities of mother-populations brood stock were 20-50 individual mothers in each 4 l beaker. Juveniles less than 24 h old from later than 2nd brood were collected for experiments from these mother populations. All experimental animals were female. Definition of variables; acute toxicity testing For acute toxicity experiments, we used a protocol adapted from ‘‘ISO6341 International Standard (ISO1989), United States Environmental Protection Agency OPPTS 850.1010 Daphnid acute toxicity test’’ (US EPA 1996) and ‘‘OECD202 guidelines for testing of chemicals’’ from the Organisation for Economic Co-operation and Development (OECD 2004). These guidelines are specific for static acute toxicological testing of hydrophilic chemicals in daphnids. Mixtures of fresh chemicals in ADaM were prepared just prior to each exposure. Following range-finding tests, single series, duplicates or triplicates of at least 5 concentrations were tested for each chemical and Daphnia clone. Each experimental unit consisted of 10 juveniles in 100 ml borosilicate glass beakers. Negative controls were established for each experiment and each clone (concentration = 0). Animals were randomly assigned to the different exposure schemes. Subsequent separate testing was performed with juvenile (age \24 h), subadult (age 7 days) and mature (age 19 and 23 days) D. magna in the laboratory house-clone (Tromsø-clone), to assess age-dependent susceptibility to glyphosate and Roundup. Endpoint registered as immobility at 24 and 48 h was recorded by the use of a light-table. Definition of immobility (lack of movement) in guideline OPPTS 850.1010 (US EPA 1996) was used. EC50 values for the individual exposure schemes were calculated using probit analysis in SPSS statistical software. Mortality was not observed in any of the negative control groups. Definition of variables; chronic toxicity testing The chronic testing was performed using an extended and modified protocol based on OECD-211 D. magna reproduction test guideline (OECD 2008). Juvenile daphnids from brood-stock of the D. magna Tromsø-clone were randomly assigned to test regime, kept and reared individually in 100 ml glass beakers. 10 experimental units were set up for each treatment, totalling 110 glasses (animals) for the experiment. Paired test solutions of glyphosate and Roundup were prepared in ADaM medium, as nominal concentrations of 0, 0.05, 0.15, 0.45, 1.35 and 4.05 mg/l of active ingredient (glyphosate). Holding

123

254

Results Clone- and age-specific acute toxicity Acute toxicity testing of juvenile D. magna showed EC50 values of 1.4–7.2 mg/l for glyphosate (6 different clones tested) and 3.7–10.6 for Roundup (3 different clones tested). EC50 values given for both are content of active ingredient glyphosate (Fig. 1). The differences in clonal sensitivity to glyphosate were rather small, the Tromsøclone showing lowest susceptibility with an EC50 value of 7.2 mg/l. Clonal tolerance to Roundup was generally somewhat higher, but still in the same order of magnitude as for glyphosate. The Knokke 4 clone (KNO 16-F8) showed significantly higher tolerance for Roundup than for glyphosate (Fig. 1). By testing different age classes of the Tromsø-clone an age dependent increase in tolerance was

123

Acute toxicity EC50(48h) mg a.i. / l

40

____ Glyphosate ------- Roundup Juvenile

Semiadult

Adult

30

20

10

0

Belgian lakes

English laboratory

Norwegian laboratory

) ks ee w (3 k) sø ee om w Tr (1 sø h) om Tr 24 (< sø om Tr ng di ea R 4 ke ok Kn 3 ke ok Kn 2 ke ok Kn 1 ke ok Kn

medium was replaced with fresh solutions every third day. All daphnids were fed daily from an algae feed consisting of Selenastrum sp. equalling 0.15 mg C/day. Monitoring of survival, reproduction and abortion (counting of visible aborted eggs) was performed every day throughout the 55 day experiment by transferring and observing each glass on a light table. We quantified the following variables: i) Survival (proportion of surviving animals throughout the life-cycle), ii) Growth (i.e. body size at days 6, 12, 24 and 36), iii) Fecundity (number of live offspring/reproductive day, i.e. in the period between first offspring and end of experiment), iv) Abortion rate (number of visible aborted eggs divided by the sum of juveniles born and the aborted eggs) and v) Offspring body size (length of newborns measured within the first 24 h). Experimental animals were photographed every 6 days and 1st instar juveniles from their first and second clutch (brood) were photographed within 24 h after birth in order to measure individual body size of mothers and their offspring. Body size was measured from the top of the head to the base of the caudal spine on photographs using ImageJ software. Animals were handled using glass pipettes. Data were analyzed in SPSS, Systat and R-project softwares. Monitoring of pH, oxygen saturation and laboratory temperature was performed during acclimatization and testing. Oxygen saturation was constantly close to 100 %, pH was within 6.5–8.5 range. Temperature in the laboratory was generally 21 C (±2). Constant 24/7 uniform artificial lighting was provided from standard fluorescent tubes. All results presented refer to nominal concentrations (mg/l) of the active ingredient (a.i.) glyphosate-IPA in glyhosate and Roundup solutions.

M. Cuhra et al.

Clone (age)

Fig. 1 Acute toxicity EC50 (48 h) of glyphosate and Roundup to 6 clones and 3 age classes of D. magna, calculated as nominal concentrations of active ingredient glyphosate. The clones have different backgrounds, some are wild clones collected in Belgium from pristine lakes (Knokke 1 and Knokke 2), from ponds where some anthropogenic chemicals are expected (Knokke 3) and from ponds in areas of intensive agriculture (Knokke 4). Clones ‘Tromsø’ and ‘Reading’ are from cultures reared for years in laboratories. Error bars denote 95 % CI

demonstrated. The EC50 values of adults were 22 and 31 mg/l for Roundup and glyphosate, respectively (Fig. 1). Chronic toxicity Survival, growth, fecundity, abortion rate and juvenile body size in the 5 test concentrations (0.05, 0.15, 0.45, 1.35 and 4.05 mg/l active ingredient glyphosate), for both glyphosate and Roundup, were compared to the performance of the negative control group to determine effects and levels of no observed effect concentration (NOEC). Survival The D. magna survival rates were similar for the control and exposed groups for concentrations up to 1.35 mg/l and 0.45 mg/l of glyphosate and Roundup respectively (Fig. 2). At the 4.05 mg/l glyphosate concentration, there was a significant reduction in survival (p = 0.0035, CoxPH-test), and the median expected longevity was 15 days. Also at the concentrations 1.35 mg/l and 4.05 mg/l for Roundup a significant reduction in survival (p = 0.0055, and p = 0.0022, respectively, CoxPH-test) was recorded. Animals exposed to 1.35 and 4.05 mg/l of Roundup had a median expected longevity of 27 and 8.5 days respectively.

Clone- and age-dependent toxicity of a glyphosate commercial formulation Fig. 2 Survival curves of D. magna exposed to different concentrations of glyphosate (b) and Roundup (c). (a) shows the negative control

a

255

Concentration

Control

0 0.05 0.15 0.45 1.35 4.05

10

Survival

8 6 4 2

b

Concentration

Glyphosate

0 0.05 0.15 0.45 1.35 4.05

Survival

10 8

1.35 6

0.45 4 2

4.05

c

Roundup

Concentration 0 0.05 0.15 0.45 1.35 4.05

Survival

10 8

0.45

6

0.15

4 2

1.35

4.05 0

10

20

30

40

50

60

Time (days)

The NOEC values for glyphosate and Roundup were 1.35 and 0.45 mg/l, respectively. Growth Length measurements at days 6, 12, 24, and 36 demonstrated reduced growth rates for D. magna exposed to glyphosate and Roundup depending on the dose and duration of exposure (Fig. 3). The body sizes of animals exposed to glyphosate concentrations 0.05, 0.15 and 0.45 mg/l as well as to Roundup 0.05, 0.15 and 0.45 mg/l were not significantly different from those of the control group individuals (p [ 0.05). For glyphosate, significant reduction of body size was found in the 4.05 mg/l concentration at day 24, and in the 1.35 mg/l concentration at day 36. For Roundup, exposure to the 4.05 mg/l concentration entailed a significant reduction in body size at day 6 and at all later time points until the animals were

eliminated from analysis by mortality. At 1.35 mg/l animals showed reduced body size at day 36 only. Thus, NOEC levels for growth were 0.45 mg/l for both glyphosate and Roundup.

Reproduction—fecundity and abortion rate Exposures to glyphosate concentrations 1.35 and 4.05 mg/l as well as to Roundup concentration 0.45 mg/l significantly decreased fecundity, as compared to the control group (Fig. 4). Animals exposed to 1.35 mg/l of Roundup reached reproductive age, but almost all eggs and developing embryos were aborted (Fig. 5). (Animals exposed to 4.05 mg/l of Roundup died before reaching maturation). Fecundity in animals exposed to glyphosate concentrations 0.05, 0.15, 0.45 and Roundup concentrations 0.05 and 0.15 were not significantly affected. NOEC

123

256

M. Cuhra et al.

Control Glyphosate Roundup

4.000 3.000

Day 6

2.000

*** 5.000 4.000 Day 12

3.000 2.000

Control glyphosate Roundup

5.00

Fecundity (live offspring/reproductive day)

Body length (mm)

5.000

***

4.00

3.00

* 2.00

1.00

***

*** 0.00

5.000

0

0.05

0.15

0.45

1.35

4.05

Concentration (mg a.i. /l) 4.000

**

3.000

Day 24

2.000

Fig. 4 Fecundity as mean number of live offspring/reproductive day for D. magna exposed to different concentrations of glyphosate and Roundup. Error bars denote 95 % CI and stars denote significant difference to the control group, *p \ 0.05, **p \ 0.01, ***p \ 0.001 (ANOVA, Tukey correction)

5.000 4.000

** 3.000

**

***

Control glyphosate Roundup

1.00

Day 36

0.75

***

0

0.05

0.15

0.45

1.35

4.05

Concentration (mg a.i. /l)

Fig. 3 Mean body size at days 6, 12, 24 and 36 of D. magna exposed to different concentrations of glyphosate and Roundup. Error bars denote 95 % CI and stars denote significant difference to the control group, *p \ 0.05, **p \ 0.01, ***p \ 0.001 (ANOVA, Tukey correction)

levels for fecundity were 0.45 mg/l for glyphosate and 0.15 mg/l for Roundup. The abortion rates for animals exposed to glyphosate as well as Roundup in concentrations of 0.05, 0.15 and 0.45 mg/l, were not significantly different from those of the control group. Abortion rates for animals exposed to 1.35 mg/l of both glyphosate and Roundup were significantly higher than for the control group individuals (Fig. 5), and reached nearly 100 % for animals exposed to Roundup. NOEC levels for abortion were 0.45 mg/l for glyphosate and Roundup.

123

Abortion rate

2.000

0.50

0.25

0.00

0

0.05

0.15

0.45

1.35

4.05

Concentration (mg a.i. /l)

Fig. 5 Mean abortion rate of D. magna exposed to different concentrations of glyphosate and Roundup. Error bars denote 95 % CI and stars denote significant difference to the control group, *p \ 0.05, **p \ 0.01, ***p \ 0.001 (ANOVA, Tukey correction)

Reproduction—size of offspring in first and second clutch First clutch (brood) juveniles born from groups exposed to 0.05 and 1.35 mg/l glyphosate were significantly smaller

Clone- and age-dependent toxicity of a glyphosate commercial formulation 0.975

Treatment

a first clutch

b second clutch

control glyphosate roundup

0.925

Offspring length (mm)

Fig. 6 Mean offspring body size in first (a) and second (b) clutch of D. magna exposed to different concentrations of glyphosate and Roundup. Error bars denote 95 % CI and stars denote significant difference to the control group, *p \ 0.05, **p \ 0.01, ***p \ 0.001 (ANOVA, Tukey correction)

257

0.875

0.825

***

*

***

***

0.775

*

* 0.725 .00

.05

.15

.45

1.35 4.05

.00

.05

.15

.45

1.35 4.05

Concentration (mg a.i. /l)

than those of the control group. This tendency was not significant in the second clutch, but juveniles born in the 4.05 mg/l concentration were significantly smaller. For Roundup, juveniles from the first clutch were smaller, but differences were not significant. In the second clutch the juveniles from animals exposed to 0.05, 0.15 and 0.45 mg/l Roundup and 4.05 mg/l glyphosate were significantly smaller than those of the control group (Fig. 6).

Discussion In the present experiments with D. magna we demonstrate that i) glyphosate and Roundup induce EC50 at concentrations typically below 10 mg/l in 48 h acute experiments, and ii) chronic exposure, particularly to formulated Roundup, causes serious reproduction damage at levels close to (1.35 mg/l) or even below (0.45 mg/l) accepted threshold values for glyphosate in surface waters in the United States in general (0.7 mg/l) and in the state of California specifically (1.0 mg/l)(California EPA 1997). Acute toxicity of glyphosate and Roundup There were only minor differences in tolerance to acute exposure of glyphosate and Roundup between laboratory clones, clones from natural ponds and clones taken from ponds surrounded by intensive agriculture (Fig. 1). A tendency towards higher tolerance to Roundup was observed,

particularly in the Knokke 4-clone. This may be related to this clone’s origin, a pond surrounded by agriculture. Tests of carbaryl pesticide in 10 clones of D. magna, two of which were from the same lakes as two of our tested Knokke clones (Knokke 1 and Knokke 4), indicated an overall correlation between land use intensity (farming) and carbaryl tolerance (as EC50). These findings were attributed to a genetically based resistance, persistent through several generations of toxicant-free laboratory culturing (Coors et al. 2009). Our observed differences in clonal tolerance of glyphosate toxin can be interpreted as response to environmental differences. However, the biology of this response is unclear at present. In this work we have shown a relatively uniform susceptibility to glyphosate and Roundup between clones of D. magna. This is in contrast to the extreme variation seen between published studies. Accordingly, the highly varying EC50 values in D. magna, and other species of daphnids reported in printed reviews (Melnichuk et al. 2007a, Pe´rez et al. 2012, Rico-Martı´nez et al. 2012), and online databases of pesticide exotoxicology such as the Pesticide Alert Network database (PAN 2011), and the US Environmental Protection Agency Ecotox database (US-EPA 2011), should not be primarily attributed to interclonal differences. D. magna toxicity tests are generally considered reproducible and representative, with only small variation between laboratories (Mark and Solbe´ 1998). Still, test conditions and laboratory environments or other contextual factors may cause the discrepant results.

123

258

Furthermore, also the solubility of the different glyphosatebased chemicals may be a decisive factor in glyphosate toxicity testing of aquatic organisms. In the literature the common name ‘‘glyphosate’’ is used somewhat indiscriminately, including chemical compounds that differ substantially from glyphosate-IPA salt (chemical identity CAS# 38641940), e.g. technical grade glyphosate, which has low solubility in water (CAS# 1071836). Toxicological data for technical grade glyphosate are not representative when assessing ecological effects of glyphosate herbicides, which for spraying need to contain a water soluble form of glyphosate, e.g. the IPA-salt, as the active ingredient (Dill et al. 2010). During our review of published studies we contacted the authors of 4 papers from groups that had recently published D. magna toxicity studies with unspecified glyphosate. These studies were performed in Korea (Le et al. 2010), Turkey (Sarigu¨l and Bekcan 2009), Portugal (Pereira et al. 2009) and Mexico (Dominguez-Cortinas et al. 2008). Authors from 3 of these research groups kindly responded to our information request, confirming that the chemical substance tested was technical grade glyphosate, i.e. the non-soluble version of glyphosate. Contrary to this, the glyphosate IPA and Roundup formulation tested in the present study is representative for glyphosate herbicides used in agriculture as active ingredient (glyphosate) and formulated product (Roundup). However, variations in toxicity levels may still be expected due to differences in adjuvants and other ingredients of individual formulations (Gasnier et al. 2009, Melnichuk et al. 2007a). Contrary to the findings of Tsui and Chu (2003) the present work finds acute toxicity of Roundup formulation and active ingredient glyphosate expressed as EC50 (48 h) concentrations, to be in the same order of magnitude. This is in accordance with some published work in other aquatic invertebrates such as Hydra attenuata (Demetrio et al. 2012). We have also shown that the D. magna tolerance for glyphosate and Roundup is enhanced with increasing age of the animals. This has also been shown for Roundup in other freshwater invertebrates, such as the freshwater shrimp Caridina nilotica (Mensah et al. 2011). Both these freshwater invertebrates have relatively low EC50-values as adults (22 and 25.3 mg/l for D. magna and C. nilotica, respectively). Such values are way below previously published results from acute glyphosate toxicity experiments in D. magna, even for juveniles. For example, Mcallister and Forbis (1978) presented an EC50-value of 759.7 mg/l with a sharp 95 % confidence interval (740.8-779.9). The European Commission (EC) working document on glyphosate (EC 2002), which forms the basis for European regulation in the context of health and environment, reports

123

M. Cuhra et al.

the EC50 (48) value of 930 mg/l in D. magna from Forbis and Boudreau (1981). The authors of the EC paper extrapolate this value into a general EC50 value for acute toxicity in aquatic invertebrates. Thus glyphosate is termed ‘‘harmless’’. According to the 2009 WHO guidelines for pesticide classification (WHO 2009), glyphosate is in class 3; slightly hazardous (in relation to human health). The US EPA has defined glyphosate in Toxicity class 4: ‘‘Practically nontoxic’’. For a review see Bates (2000). Furthermore, in 1982 the agrochemicals producer Monsanto presented contrasting data for toxicity of Roundup formulations in Daphnia sp., by simultaneously giving LC50 (96 h) values of 5.3 mg/l for Roundup and 962 mg/l for glyphosate alone (Servizi et al. 1987). Already in 1979 it was pointed out that technical grade glyphosate had properties (notably reduced water solubility) totally different from those of the glyphosate isopropylamine salt (Folmar et al. 1979). This is, however, an important fact that has been commonly overlooked. In contrast to other published toxicity data for formulated glyphosate-based herbicides, our results are comparable to those of Folmar et al. (1979) at 3 mg/l for Roundup in D. magna, and to 4 of the 6 formulations tested by Melnichuk et al. (2007a) at 4.2–10.2 mg/l. The most recent toxicity data presented by the producer, for the specific brand of Roundup that we have tested, is 11 mg/l EC50 (48) for D. magna (Monsanto 2011) and thus in accordance with our findings. A recent publication by Sarigu¨l and Bekcan (2009) reports a much higher toxicity of a 48 % commercial Roundup formulation in D. magna, with LC50 (48 h) values of 0.012 mg/l. We have no explanations for this discrepancy. Some difference in methods may partly explain published experimental test result variations. For example, Servizi et al. (1987) presented the LC50 (96 h)-value for Roundup in D. pulex as 25.5 mg/l, but this referred to the Roundup formulation including water. When the authors assessed only the active ingredient glyphosate IPA and the surfactant MONO818 respectively, LC50 (96 h)-values of 7.8 and 3.8 mg/l were recorded. Chronic toxicity of glyphosate and Roundup When D. magna were exposed to different concentrations in chronic life-cycle experiments, Roundup produced more serious adverse effects than glyphosate alone. This was the case for all tested end-points: survival, growth, fecundity, abortion rates and juvenile body size. Chronic exposure to 0.05 and 0.15 mg/l of Roundup significantly reduced juvenile body size compared to the control group (p \ 0.001). The same was the case for glyphosate at 0.05 mg/l, but to a lower degree (p \ 0.05). This is in accordance with findings of Papchenkova (2007) who

Clone- and age-dependent toxicity of a glyphosate commercial formulation Table 1 Observed significant negative effects caused by chronic exposure to glyphosate IPA salt, administered as glyphosate and Roundup in different concentrations, on D. magna life-history traits mg/l a.i. glyphosate IPA

Glyphosate

Roundup

0.05

Reduced juvenile size

Reduced juvenile size

0.15

No observed effect

Reduced juvenile size

0.45

No observed effect

Reduced fecundity

1.35

Reduced growth

Reduced growth

Reduced fecundity

Increased mortality

Increased abortion

High abortion

Reduced growth

High mortality

High mortality

No reproduction

4.05

Reduced fecundity Increased abortion

found juvenile size significantly reduced (p \ 0.05) by exposure to 2.0 mg/l (6 of 7 generations) and 0.2 mg/l (3 of 7 generations) a.i. concentrations of Roundup. In our present study no other measured end-points were affected at these concentrations (Table 1). No significant effects on fecundity or abortion rates were seen at concentrations 0.05–0.45 mg/l for glyphosate IPA, but exposure to Roundup at 0.45 mg/l concentration significantly reduced fecundity and increased the abortion rate in addition to the reduced juvenile body size. Following exposure to Roundup at the 1.35 mg/l concentration, significantly impaired survival and growth was observed and reproduction failed completely: all eggs were aborted. A summary of the results from the chronic exposure tests is given in Table 1. To put these results and concentrations in context: the US EPA general environmental guideline of 0.7 mg/l and the state specific California EPA environmental guideline limit of 1.0 mg/l glyphosate in surface waters are in between the 0.45 and 1.35 mg/l concentrations we use in our tests. The fact that, in the present study, D. magna subjected to 1.35 mg/l showed complete reproductive failure, aborting all eggs in early to late stages of embryonic development, indicates that the mentioned environmental guidelines may not be sufficiently restrictive to ensure viable populations of D. magna and other aquatic invertebrates. Ronco et al. (2008) investigated pesticide levels in streams draining several sites with transgenic soybean (glyphosate-tolerant) cultivation in Argentina and found the levels to be; ‘‘often below 1 mg glyphosate/l, in Arrecifes tributary, although concentration ranges between 1.8 and 10.9 mg/l (…) were detected’’. The authors concluded that non-target aquatic biodiversity (flora, insects,

259

fish and amphibians) was adversely affected by the pesticide applications. The levels of glyphosate accepted in surface fresh water vary between nations. As far as we have been able to ascertain, the highest tolerated concentrations are found in the earlier mentioned US-EPA guidelines, 0.7–1.0 mg/l, differing strikingly from the EU guideline limit of 0.0001 mg/l (=0.1 ppb), which seems to be the most restrictive. Canada enforces a limit of 0.065 mg/l (Struger et al. 2008), while Ukraine has set the environmental standard to 0.02 mg/l (Melnichuk et al. 2007a, b). The results of the few other published studies on chronic exposure of daphnids to glyphosate or glyphosate-based herbicides are distinctly inconsistent. An industry standard 21-day reproduction test of glyphosate in D. magna, based on test concentrations of 0, 26, 50, 96, 186 and 378 mg/l, was additionally reviewed and extrapolated by dr. Wayne C. Faatz, in a March 1983 report to the US EPA (McKee et al. 1982). Neither significant increase of mortality nor reduction of growth was observed in any of the test concentrations. For reproduction, the same report established 50 mg/l as NOEC, a level 100 times higher than the NOEC determined in our experiments. In contrast, Papchenkova (2007) exposed seven generations of D. magna to 0.02, 0.2 and 2 mg/l a.i. glyphosate in Roundup. Significant reduction of endpoints related to fecundity, length of newborn juveniles and growth in first generation was recorded for D. magna exposed to a concentration of 2 mg/l. Significant effects on the same endpoints were observed also in subsequent generations for concentrations 2.0 and even 0.2 mg/l, but these effects were not consistent in all measured end-points through all of the 7 generations studied. A follow-up generational study of chronic toxicity in D. magna exposed to much higher concentrations of Roundup, i.e. 25 and 50 mg/l a.i. for four generations, showed a significantly reduced fecundity but no adverse effect on the survival of mother animals (Papchenkova et al. 2009). A similar complexity is evident in a chronic effects study of the Fakel herbicide (48 % a.i. glyphosate IPA) in Ceriodaphnia affinis (Melnichuk et al. 2007b). Generational exposures to 10, 5, 2.5, 1 0.1, 0.01 and 0.001 mg/l Fakel established a NOEC of 0.001 mg/l. Even at the very low concentration of 0.01 mg/l, first and second generation fecundity was found to be significantly reduced compared to the control group. Temperature-dependent effects on end-points fecundity and abortion were recorded at test concentrations 1.0–0.1 mg/l. As temperatures were reduced, adverse effects decreased (Melnichuk et al. 2007b). C. affinis was shown to be more sensitive to glyphosate herbicide Fakel in acute LC50 (48) tests than

123

260

D. magna (12.6 mg/l vs. 26.5 mg/l respectively (Melnichuk et al. 2007a). The acidity of the aquatic environment (or laboratory medium) may also be a relevant factor for the toxicity of glyphosate-based herbicides. Chen et al. (2004) exposed the daphnid Simocephalus vetulus to glyphosate herbicide Vision in sublethal concentrations 0.75 and 1.5 mg/l a.i. (acid equvivalent) under two different pH-regimes (pH 5.5 and 7.5). The authors found that survival, fecundity and juvenile maturation time was affected at both concentrations. The effects were more severe at neutral pH 7.5, versus the lower pH 5.5. Thus, the acidity of the experimental or environmental conditions must be taken into account, in particular when the buffering capacity of the artificial holding medium is low and the toxicants tested are acidic. In our experiments, the variation in acidity spanned a range of 2 pH-units. However, they were still within the pH 6–9 range defined as preferred experimental conditions for D. magna testing (OECD 2008). The term ‘‘inert-ingredient’’ for Roundup formulation additives has been used for product labeling. This is problematic when published literature documents that additives may have significant direct or synergistic toxic effects. Numerous studies have demonstrated that surfactants, often called ‘‘adjuvants’’ or ‘‘inert ingredients’’, used in Roundup formulations are the primary toxic agents, with toxicity notably higher than glyphosate (‘‘the active ingredient’’) alone (e.g. Benachour et al. 2007; Folmar et al. 1979; Gasnier et al. 2009; Melnichuk et al. 2007a). Summary and conclusions According to our experimental work and our literature reviews, we find that the previously published EC50 values of 780-930 mg/l for glyphosate (McAllister and Forbis 1978; Forbis and Boudreau 1981) are not representative. The classification of glyphosate as ‘‘practically nontoxic’’ to aquatic invertebrates is based on these non-representative values. The high EC50 values have demonstrated tenacious lives, been extensively referred to in the literature and have also found their ways into regulatory documents. We have found the acute toxicity of glyphosate herbicide active ingredient to be substantially higher, with concentrations below 10 mg/l inducing immobility in D. magna within 48 h. If such more conservative EC50 values were used, glyphosate would be classified as ‘‘toxic’’ or ‘‘moderately toxic’’ to aquatic invertebrates. In our chronic studies covering the whole life-cycle of D. magna, we demonstrated negative and serious effects at very low concentrations (see Table 1 for a summary), i.e. at levels that can be expected with use of the herbicide Roundup at prescribed dosages in agricultural practice.

123

M. Cuhra et al.

The results of our acute and chronic toxicity tests with glyphosate-IPA and Roundup herbicide, in combination with our review of published data, warrant the conclusion that current European Commission and US EPA toxicity classification of these chemicals with regard to effects on D. magna and aquatic invertebrates in general, is based on non-representative evidence and needs to be adjusted. Conflict of interest of interest.

The authors declare that they have no conflict

Funding The work is funded by Forskningsra˚det the Norwegian Research Council, Project No. 184107 Milø2015. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

References Antoniou M, Brack P, Carrasco A, Fagan J, Habib M, Kageyama P, Leifert C, Nodari RO, Pengue W (2010) GM Soy: Sustainable? Responsible?. ARGE Gentechnik-frei, Vienna Baird DJ, Barber I, Bradley M, Soares AMVM, Calow P (1991) A comparative study of genotype sensitivity to acute toxic stress using clones of Daphnia magna straus. Ecotoxical Environ Safe 21(3):257–265 Bates JAR (2000) Health and environmental hazard classification of pesticides. Pestic Outlook 11:109–115 Benachour N, Sipahutar H, Moslerni S, Gasnier C, Travert C, Seralini GE (2007) Time- and dose-dependent effects of roundup on human embryonic and placental cells. Arch Environm Contam Toxicol 53:126–133 Benzie JAH (2005) The Genus Daphnia. Issue 21 In: Dumont HJF (ed) Guides to identification of the Microinvertebrates of the Continental Waters of the World. Backhuys, Leyden Brausch JM, Beall B, Smith PN (2007) Acute and sub-lethal toxicity of three POEA surfactant formulations to Daphnia magna. B Environ Contam Tox 78(6):510–514 California EPA (1997) Public health goal for glyphosate in drinking water. Pesticide and environmental toxicology section, Office of environmental health hazard assessment, California environmental protection agency. http://www.oehha.ca.gov/water/phg/pdf/ glypho_c.pdf. Accessed 8 March 2012 Cerdeira AL, Duke SO (2006) The current status and environmental impacts of glyphosate-resistant crops: a review. J Environ Qual 35(5):1633–1658 Chen CY, Hathaway KM, Folt CL (2004) Multiple stress effects of Vision herbicide, pH, and food on zooplankton and larval amphibian species from forest wetlands. Environ Toxicol Chem 23(4):823–831 Coors A, Vanoverbeke J, De Bie T, De Meester L (2009) Land use, genetic diversity and toxicant tolerance in natural populations of Daphnia magna. Aquat Toxicol 95(1):71–79 Demetrio PM, Bulus Rossini GD, Bonetto CA (2012) Effects of pesticide formulations and active ingredients on the coelenterate Hydra attenuata (Pallas, 1766). Bull Environ Contam Toxicol 88:15–19 Dill GM, Sammons RD, Feng PCC, Kohn F, Kretzmer K, Mehrsheikh A, Bleeke M, Honegger JL, Farmer D, Wright D, Haupfear EA (2010)

Clone- and age-dependent toxicity of a glyphosate commercial formulation Glyphosate: discovery, development, applications, and properties. In: Nandula VK (ed) Glyphosate resistance in crops and weeds: history, development, and management. Wiley, New York, pp 1–33 Dominguez-Cortinas G, Saavedra JM, Santos-Medrano GE, RicoMartinez R (2008) Analysis of the toxicity of glyphosate and Faena using the freshwater invertebrates Daphnia magna and Lecane quadridentata. Toxicol Environ Chem 90(2):377–384 Duke SO, Powles SB (2008) Glyphosate: a once-in-a-century herbicide. Pest Manag Sci 64(4):319–325 EC (2002) European commission working document on glyphosate. Review report for the active substance glyphosate. 6511/VI/99final FAO (2001) Fao specifications and evaluations for plant protection products: Glyphosate N-(phosphonomethyl)glycine. Food and Agriculture Organization of the United Nations. http://www.fao. org/ag/AGP/AGPP/Pesticid/Specs/docs/Pdf/new/glypho01.pdf. Accessed 12 March 2012 Folmar LC, Sanders HO, Julin AM (1979) Toxicity of the herbicide glyphosate and several of its formulations to fish and aquatic invertebrates. Arch Environm Contam Toxicol 8:269–278 Forbis AD, Boudreau P (1981) Acute toxicity of MON0139 (Lot LURT 12011)(AB-81-074) To Daphnia magna: Static acute bioassay report no. 27203. Unpublished study document from US EPA library Gardner SC, Grue CE (1996) Effects of rodeo and garlon3A on nontarget wetland species in central Washington. Environ Toxicol Chem 15(4):441–451 Gasnier C, Dumont C, Benachour N, Clair E, Chagnon MC, Seralini GE (2009) Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines. Toxicology 262:184–191 Giesy JP, Dobson S, Solomon KR (2000) Ecological risk assessment for roundup Herbicide. Rev Environ Contam T 167:35–120 Hessen DO, Ka¨llqvist T, Abdel-Hamid MI, Berge D (1994) Effects of pesticides on different zooplankton taxa in mesocosm experiments. Norw J Agr Sc 13:153–161 Hildebrand LD, Sullivan DS, Sullivan TP (1980) Effects of Roundup Herbicide on Populations of Daphnia magna in a Forest Pond. B Environ Contam Tox 25:353–357 James C (2010) A global overview of biotech (GM) crops: adoption, inpact and future prospects. GM Crops 1(1):8–12 Klu¨ttgen B, Du¨lmer U, Engels M, Ratte HT (1994) ADaM, an artificial freshwater for the culture of zooplankton. Water Res 28:743–746 Le TH, Lim ES, Lee SK, Choi YW, Kim YH, Min J (2010) Effects of glyphosate and methidathion on the expression of the Dhb, Vtg, Arnt, CYP4 and CYP314 in Daphnia magna. Chemosphere 79(1):67–71 Lockhart WL, Billeck BN, Baron CL (1989) Bioassays with a floating aquatic plant (Lemna minor) for effects of sprayed and dissolved glyphosate. Hydrobiologia 188(189):353–359 Mann RM, Hyne RV, Choung CB, Wilson SP (2009) Amphibians and agricultural chemicals: review of the risks in a complex environment. Environ Pollut 157:2903–2927 Marc J, Mulner-Lorillon O, Belle R (2004) Glyphosate-based pesticides affect cell cycle regulation. Biol Cell 96:245–249 Mark U, Solbe´ J (1998) Analysis of the ecetoc aquatic toxicity (EAT) database V—the relevance of Daphnia magna as a representative test species. Chemosphere 36(1):155–166 McAllister W, Forbis A (1978) Acute toxicity of technical glyphosate (AB–78–201) to Daphnia magna. Study reviewed and approved 8–30–85 by EEB/HED US EPA. Unpublished study document from US EPA library McKee MM, McAllister WA, Schofield M (1982) Chronic Toxicity of glyphosate (AB-82-036) to Daphnia magna under flowthrough test conditions, project No. 28742, Analytical biochemistry Laboratories, Submitted by Monsanto Agricultural Project

261

No. on December 27, 1982, for Registration No. 524-308, Accesion No. 249160. Unpublished study document from US EPA library Melnichuk SD, Sherban EP, Lokhanskaya VI (2007a) Estimation of toxicity of glyphosate-based herbicides by biotesting method using cladocera. Hydrobiol J 43(3):80–91 Melnichuk SD, Sherban EP, Lokhanskaya VI (2007b) Effects of Fakel Herbicide on vital activity of Ceriodaphnia affinis in acute and chronic experiments. Hydrobiol J 43(6):83–91 Mensah PK, Muller WJ, Palmer CG (2011) Acute toxicity of Roundup (R) herbicide to three life stages of the freshwater shrimp Caridina nilotica (Decapoda: Atyidae). Phys Chem Earth 36:905–909 Mensink H, Janssen P (1994) Glyphosate—Environmental health criteria. International programme on chemical safety (IPCS– INCHEM), WHO Geneva. http://www.inchem.org/documents/ ehc/ehc/ehc159.htm. Accessed 12 March 2012 Monsanto (2011) MSDS on roundup weed and grass killer pluss. Avalable online at http://www.homedepot.com/buy/outdoors/gardencenter/roundup/1-qt-weed-grass-killer-concentrate-plus-7624.html. Accessed 12 March 2012 NPIC (2010) Glyphosate technical fact sheet. National Pesticide Information Center, Oregon State University, Corvallis OR, USA. www.npic.orst.edu. Accessed 12 March 2012 OECD (2004) OECD-202 Guideline for testing of chemicals: Daphnia sp. acute immobilization test. OECD, Geneva OECD (2008) OECD-211 Guidelines for testing of chemicals: Daphnia magna reproduction test. OECD, Geneva PAN (2011) Online toxicological information at the pesticide action network database. http://www.pesticideinfo.org/Detail_Product. jsp?REG_NR=07199500029&DIST_NR=071995. Accessed 12 March 2012 Papchenkova GA (2007) Study of chronic toxicity of the herbicide roundup in a series of generations of Daphnia magna. Toksikol Vestn 5:14–17 Papchenkova GA, Golovanova IL, Ushakova NV (2009) The parameters of reproduction, sizes, and activities of hydrolases in Daphnia magna Straus of succesive generations afected by roundup Herbicide. Inland Water Biol 2(3):286–291 Penner D (2000) Activator adjuvants. Weed Technol 14(4):785–791 Pereira J, Antunes SC, Castro BB, Marques CR, Goncalves AMM, Goncalves F, Pereira R (2009) Toxicity evaluation of three pesticides on non-target aquatic and soil organisms: commercial formulation versus active ingredient. Ecotoxicology 18:455–463 Pe´rez GL, Torremorell A, Mugni H, Rodrı´guez P, Solange Vera M, Nascimento M, Allende L, Bustingorry J, Escaray R, Ferraro M, Izaguirre I, Pizarro H, Bonetto C, Morris DP, Zagareseet H (2007) Effects of the herbicide Roundup on freshwater microbial communities: a mesocosm study. Ecol Appl 17:2310–2322 Pe´rez GL, Solange Vera M, Miranda LA (2012) Effects of Herbicide Glyphosate and Glyphosate-Based Formulations on Aquatic Ecosystems. In: El-Ghany Hasaneen MNA (ed) Herbicides— Properties, Synthesis and Control of Weeds, pp. 334–368 Peruzzo PJ, Porta AA, Ronco AE (2008) Levels of glyphosate in surface waters, sediments and soils associated with direct sowing soybean cultivation in north pampasic region of Argentina. Environ Pollut 156(1):61–66 Peterson HG, Boutin C, Martin PA, Freemark KE, Ruecker NJ, Moody MJ (1994) Aquatic phyto-toxicity of 23 pesticides applied at expected environmental concentrations. Aquat Toxicol 28:275–292 Pollak P (2011) Fine chemicals: the industry and the business. Wiley, Hoboken Pue´rtolas L, Dama´sio J, Barata C, Soares AMVM, Prat N (2010) Evaluation of side-effects of glyphosate mediated control of giant reed (Arundo donax) on the structure and function of a

123

262 nearby Mediterranean river ecosystem. Environ Res 110: 556–564 Relyea RA (2005) The impact of insecticides and herbicides on the biodiversity and productivity of aquatic communities. Ecol Appl 15(2):618–627 Rico-Martı´nez R, Arias-Almeida JC, Pe´rez-Legaspi IA, Alvarado-Flores J, Retes-Pruneda JL (2012) Adverse Effects of Herbicides on Freshwater Zooplankton. Herbicides - Properties, Synthesis and Control of Weeds. In: El-Ghany Hasaneen MNA (ed) Herbicides— Properties, Synthesis and Control of Weeds, pp. 405–434 Ronco AE, Carriquiriborde P, Natale GS, Martin ML, Mugni H, Bonetto C (2008) Integrated approach for the assessment of biotech soybean pesticides impact on low order stream ecosystems of the Pampasic Region. In: Chen J, Guo C (eds) Ecosystem ecology research trends, pp. 209–239 Sarigu¨l Z, Bekcan S (2009) Acute toxicity of the Herbicide Glyphosate on Daphnia magna. Ankara Universitesi, Tarim Bilimleri Dergisi 15(2): 204–208 Scribner EA, Battaglin WA, Gillom RJ, Meyer MT (2007) concentrations of glyphosate, its degradation product, aminomethylphosphonic acid, and glufosinate in ground- and surface-water, rainfall, and soil samples collected in the United States, 2001–2006. US Geological Survey Scientific Investigations Report 2007–5122, p 111 Servizi JA, Gordon RW, Martens DW (1987) Acute Toxicity of Garlon 4 and Roundup Herbicides to salmon, Daphnia, and Trout. B Environ Contam Tox 39:15–22 Siemering GS, Hayworth JD, Greenfield BK (2008) Assessment of potential aquatic herbicide impacts to California aquatic ecosystems. Arch Environ Con Tox 55:415–431

123

M. Cuhra et al. Simenstad CA, Cordell JR, Tear L, Weitkamp LA, Paveglio FL, Kilbride KM, Fresh KL, Grue CE (1996) Use of rodeo and X-77 spreader to control smooth cordgrass (Spartina alterniflora) in a southwestern Washington estuary: 2. Effects on benthic microflora and invertebrates. Environ Tox Chem 15, 6:969–978 Struger J, Thompson D, Staznik B, Martin P, McDaniel T, Marvin C (2008) Occurrence of Glyphosate in Surface Waters of Southern Ontario. B Environ Contam Tox 80(4):378–384 Tsui MTK, Chu LM (2003) Aquatic toxicity of glyphosate-based formulations: comparison between different organisms and the effects of environmental factors. Chemosphere 52(7):1189–1197 US EPA (1993) EPA 738-R-93-014. Registration Eligibility Decision (RED) Glyphosate. United States Environmental Protection Agency, Washington, DC US EPA (1996) OPPTS 850.1010. Aquatic invertebrate acute toxicity test, freshwater daphnids. EPA Ecological effects Test Guidelines. United States Environmental Protection Agency 712-C-96114 US EPA (2011) Ecotox Database. http://cfpub.epa.gov/ecotox/ Accessed 12 March 2012 USGS (2010) United States Geological Survey at http://water.usgs. gov. Accessed 12 March 2012 WHO (2009) The WHO recommended classification of pesticides by hazard and guidelines to classification. World Health Organization Geneva. http://www.who.int/ipcs/publications/pesticides_hazard_ 2009.pdf. Accessed 12 March 2012 Woodburn AT (2000) Glyphosate: production, pricing and use worldwide. Pest Manag Sci 56:309–312

   

Paper  II:  

Analysis  of  soybean  constituents  

  Title:      

Compositional  differences  in  soybeans  on  the  market:     glyphosate  accumulates  in  Roundup  Ready  GM  soybeans.    

Authors:    

Thomas  Bøhn,  Marek  Cuhra,  Terje  Traavik,  Monica  Sanden,  John  Fagan,   Raul  Primicerio.  

Journal:  

Food  Chemistry,  153:  207-­‐215  (2014).  

39  

40  

Food Chemistry 153 (2014) 207–215

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Compositional differences in soybeans on the market: Glyphosate accumulates in Roundup Ready GM soybeans q T. Bøhn a,b,⇑, M. Cuhra a,b, T. Traavik a,b, M. Sanden c, J. Fagan d, R. Primicerio b a

GenØk, Centre for Biosafety, P.O. Box 6418, 9294 Tromsø, Norway Faculty of Health Sciences, UIT The Arctic University of Norway, 9019 Tromsø, Norway c National Institute of Nutrition and Seafood Research, NIFES, P.O. Box 2029, 5817 Bergen, Norway d Earth Open Source, 2nd Floor 145–157, St. John Street, London EC1V 4PY, United Kingdom b

a r t i c l e

i n f o

Article history: Received 3 July 2013 Received in revised form 7 November 2013 Accepted 11 December 2013 Available online 18 December 2013 Keywords: Agricultural practice GMO Herbicide residues Pesticides Nutrition Substantial equivalence

a b s t r a c t This article describes the nutrient and elemental composition, including residues of herbicides and pesticides, of 31 soybean batches from Iowa, USA. The soy samples were grouped into three different categories: (i) genetically modified, glyphosate-tolerant soy (GM-soy); (ii) unmodified soy cultivated using a conventional ‘‘chemical’’ cultivation regime; and (iii) unmodified soy cultivated using an organic cultivation regime. Organic soybeans showed the healthiest nutritional profile with more sugars, such as glucose, fructose, sucrose and maltose, significantly more total protein, zinc and less fibre than both conventional and GM-soy. Organic soybeans also contained less total saturated fat and total omega-6 fatty acids than both conventional and GM-soy. GM-soy contained high residues of glyphosate and AMPA (mean 3.3 and 5.7 mg/kg, respectively). Conventional and organic soybean batches contained none of these agrochemicals. Using 35 different nutritional and elemental variables to characterise each soy sample, we were able to discriminate GM, conventional and organic soybeans without exception, demonstrating ‘‘substantial non-equivalence’’ in compositional characteristics for ‘ready-to-market’ soybeans. Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

1. Introduction Food and food quality is crucial. Given its significance for human and animal health, we investigate whether plant products from a defined geographical region, produced under different agricultural practices are substantially equivalent or not, in terms of quality indicators like nutritional content, elemental characteristics and herbicide/pesticide residues. By comparing herbicide tolerant (‘‘Roundup Ready’’) GM soybeans directly from farmers’ fields, with extended references to both conventional, i.e., non-GM soybeans cultivated under a conventional ‘‘chemical’’ cultivation regime (pre-plant herbicides and pesticides used), and organic, i.e., non-GM soybeans cultivated under a ‘‘no chemical’’ cultivation regime (no herbicides or pesticides used), a test of real-life samples ‘ready-to-market’ can be performed. Globally, glyphosate-tolerant GM soy is the number one GM crop plant. The herbicide glyphosate is the most widely used herbicide globally, with a production of 620,000 tons in 2008. The world soybean production in 2011 was 251.5 million Metric tons, q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike License, which permits noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⇑ Corresponding author at: GenØk, Centre for Biosafety, P.O. Box 6418, 9294 Tromsø, Norway. Tel.: +47 77644541. E-mail address: [email protected] (T. Bøhn).

with the United States (33%), Brazil (29%), Argentina (19%), China (5%) and India (4%) as the main producing countries. In 2011–2012, soybeans were planted on about 30 million hectares in the USA, with Roundup Ready GM soy contributing 93–94% of the production. Also in the other leading producing countries, this same GM soy dominates the market accounting for 83% and 100% of production, respectively in Brazil and Argentina. Globally, Roundup Ready GM soybeans contributed to 75% of the total soy production in 2011. The first-generation glyphosate-tolerant GM-soy plant (event 40-3-2), produced and patented by Monsanto Company, has been genetically modified to tolerate exposure to glyphosate-based herbicides during the entire growth season. For herbicide-tolerant GM plants, herbicide co-technology is an integral part of the production system and will always be used by the farmer. However, in early studies of the composition of Roundup-Ready GM soy, the researchers did not spray the tested plants with the recommended herbicide (Millstone, Brunner, & Mayer, 1999). This shortcoming was quickly corrected, and also sprayed GM soybeans were claimed to be substantially equivalent to non-GM soybeans (Harrigan et al., 2007). Still, and surprisingly, even in these studies, the residues of herbicides were not measured. The concept of ‘substantial equivalence’ (i.e., close nutritional and elemental similarity between a genetically modified (GM) crop and a non-GM traditional counterpart) has been used to claim that GM crops are substantially equivalent to, and therefore as safe and

0308-8146/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.12.054

208

T. Bøhn et al. / Food Chemistry 153 (2014) 207–215

nutritious as, currently consumed plant-derived foods (Aumaitre, 2002). However, we argue that compositional studies that have overlooked (not measured) pesticide residues contain serious shortcomings. Chemical residues, if present, are important because (i) they are clearly a part of a plants composition, and (ii) they may add toxic properties to the final plant product either by itself or by affecting the plant metabolism. This is particularly relevant for herbicide-tolerant varieties. For the predominantly used GM soy on the market, the 40-3-2 event, herbicide tolerance was achieved by insertion of a transgene construct into the plant genome which constitutively expresses the Agrobacterium strain CP4 analogue of the plant enzyme EPSPS (5enolpyruvylshikimate-3-phosphate synthase). The endogenous plant EPSPS is critically important for the production of certain essential aromatic amino acids. Glyphosate, the active ingredient of Roundup herbicide formulations, is able to bind to all known plant, weed and crop, EPSPS versions. The binding leads to the inactivation of the enzyme and consequently death for the plant. Glyphosate binds the CP4 EPSPS expressed in GM-soy cells in a condensed, non-inhibitory conformation. Hence plants engineered to express the CP4 EPSPS enzyme are tolerant to glyphosate. Accordingly, the farmer may eradicate all kinds of plant weeds by spraying with glyphosate, and not harm the GM crop plants. However, the extensive use of glyphosate over vast land areas may lead to shifts in weed populations and selection of glyphosate-tolerant weeds (Shaner, Lindenmeyer, & Ostlie, 2012). This, in turn, typically triggers the use of higher doses or more applications of glyphosate, which can further accelerate the evolution of glyphosate resistance in weed species (Binimelis, Pengue, & Monterroso, 2009). Such a spiral is clearly not sustainable for farmers, but may also affect the consumer through plant tissue accumulation of glyphosate residues. Evolution of resistance to glyphosate is unfortunately progressing, particularly in the US. System vulnerability to resistance development is enhanced where there is a low diversity in weed management practice coupled with crop and herbicide monoculture. USDA data document dramatic increases in the use of glyphosate-based herbicides and GM soy is a major driver for this development (Benbrook, 2012). US GM soybeans thus represent a system that is influenced by glyphosate exposure and should be an ideal system in which to test whether crop management practices that include spraying with glyphosate might lead to accumulation of chemical residues, or other compositional differences, in the final soy product. Residue analysis is of particular interest, since there are no programmes in the EU, US or Canada designed to monitor the main herbicides used in transgenic crop production. In contrast to real-life samples from the market, transgenic crops intended for scientific studies are often produced in wellcontrolled small experimental plots. In most research studies, application of herbicides has been omitted or has been done at doses lower than those typically used by farmers, giving test materials that are not representative of actual conditions existing in typical agricultural operation, e.g., with regard to glyphosate residues. The knowledge regarding links between glyphosate application rates and soybean nutrient composition is scarce. One study found links between glyphosate application on glyphosate-tolerant soybean and decreased levels of a-linolenic acid (ALA) and iron, and increased levels of oleic acid (Zobiole, Bonini, de Oliveira, Kremer, & Ferrarese, 2010). A 12–14% reduction in phytoestrogen levels in GM soybean strains compared to isogenic conventional strains has been documented (Lappé, Bailey, Childress, & Setchell, 1998). However, Wei et al. showed that GM soybeans may have both a higher and lower content of isoflavones compared to conventional soy (Wei, Jone, & Fang, 2004). Generally, the suggested key food and feed nutrients found in the OECD consensus documents, are considered in safety evalua-

tions of new varieties of soybeans and risk assessment of GM plants has focused on allergenicity and toxicity resulting from the transgenic product itself, or from the possible unintended effects of the transformation process (Podevin & du Jardin, 2012). However, little attention is given to the residues of herbicides and their metabolites that can potentially accumulate in the final product, and also whether exposure to these herbicides, or other functional alterations related to the genetic modification itself (such as alterations in intermediary metabolism of the GM plant), may affect nutrient and elemental composition. In the present study, 31 samples of soybeans grown within a defined area within the state of Iowa in the US, were collected. The influence of agricultural practice on (i) residues of glyphosate, AMPA and other pesticide compounds, and (ii) the nutritional and elemental composition of ‘‘ready-to-market’’ soybeans was analysed. We used methods of multivariate analyses, such as cluster and discriminants analyses, and attempted to track differences (if any), both between individual samples and between the three management systems through which they were produced, namely GM, conventional and organic systems. With H0 as substantial equivalence between the categories of soy, the following hypotheses were tested: H1: The residues of pesticides in soybeans will be influenced by the agricultural practice they have been produced under, specifically: (a). GM-soybeans contain high residue levels of glyphosate and AMPA due to repeated spraying of the plants with glyphosate-based herbicides throughout the production season. Other pesticides may also be present according to use. (b). Conventional soybeans contain low residue levels of glyphosate and AMPA due to pre-planting applications. Other pesticides may also be present according to use. (c). Organic soybeans are expected to represent a control group with zero residues of glyphosate, AMPA and others chemical pesticides. Such pesticides are not allowed in organic farming. H2: The detailed nutritional composition and hence, the nutritional quality (i.e., total fat and protein, main sugars, ash, amino acids, fatty acids and micronutrients/basic elements) of soybean samples will be influenced by the agricultural practices under which they have been produced. 2. Materials and methods 2.1. Soy samples and characterisation Three kg samples of whole soybeans were obtained from n = 31 individual fields/sites in Iowa, USA. Seed type (genetic variety), agricultural practice, i.e. whether samples were ‘GM’ (n = 10), ‘conventional’ (n = 10) or ‘organic’ (n = 11), and pesticide use was noted for all samples (Table 1). All individual soybean samples were analysed for their nutritional content, including total protein, total fat, dry matter, starch, ash, minerals, trace elements, vitamin B6, amino acid and fatty acid composition, in addition to the relevant pesticides. 2.2. Proximate composition of the soybeans Dry matter was analysed by drying at 103 °C for 24 h, ash by weight after burning at 540 °C and lipid after extraction with ethyl-acetate. Nitrogen was measured with a nitrogen determinator (LECO, FP-428, Leco Corporation, St Joseph, MI, USA) according to the Association of Official Agricultural Chemists official methods of analysis and protein calculated as N X 6
25. Glycogen was mea-

209

T. Bøhn et al. / Food Chemistry 153 (2014) 207–215

Table 1 Soy varieties for Roundup Ready (RR) GM, conventional and organic soybeans tested. Farmer information on chemicals used in their soy production is also given. No chemicals were applied to organic soybeans. Type of soy

Variety

RR RR RR RR RR RR RR RR RR RR

Latham 2158 PB 2217VNRR PB 2421 Pioneer 92M76 Stine Stine 2032 Stine 2032 Stine 2062-4 Stine 2538-4 Stine 2602-4

GM GM GM GM GM GM GM GM GM GM

Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional Conventional

Asgrow 2869 Asgrow 2869 Legend 2200 Legend 2375 Legend 2375 Legend 2375 Legend 2932 Legend 2932 Legend 2932 Legend 2932

Organic Organic Organic Organic Organic Organic Organic Organic Organic Organic Organic

ED 4315 ED 4315 Legend 2375 Mark 0427 Mark 0431 PB291N Pioneer 9305 Pioneer 9305 Pioneer 93M52 Stine 2686 US Soy 20333

Seed treatment

Preplant

Cruiser maxx Trifluralin Cruiser extreme

Warden Warden Cruiser maxx

Trust

Cruiser Cruiser Cruiser Cruiser Cruiser Cruiser Cruiser

Treflan Trust Prowl, python Prowl Trust Trust Prowl, python

maxx maxx maxx maxx maxx maxx maxx

sured after enzymatic degradation. Amino acids and Vitamin B6 were determined by high pressure liquid chromatography (HPLC) methods and fatty acids by GLC (gas liquid chromatography). Multielement determination in the soybeans was carried out by inductively coupled plasma MS. Eurofins laboratories GfA, Otto-Hahn-Str. 22, D-48161 Münster (Germany), performed analysis of organochlorine, organophosphorus, pyrethroides, PCBs, glyphosate and AMPA (aminomethylphosponic acid – the major degradation product of glyphosate) based on the list of pesticide brand names used by the farmers (see Table 1). The following Eurofins methods were used; LMBG L00.0034, DFG S19, GC–ECD for organochlorine pesticides, pyrethroides, PCBs and LMBG L00.00-34, DFG S19, GC–FPD for organophosphorus pesticides. DFG 405, HPLC–FLD for glyphosate and AMPA. Three pooled samples (equal amounts of all individual samples) representing each of the soy categories (GM, conventional and organic) were in addition analysed for the average values of monosaccharides, disaccharides and fibre at the Czech Agriculture and Food Inspection Authority (CAFIA), Za Opravnou 300/6, 150 00 Praha 5, (Czech Republic) and for selected organochloride pesticides OCPs (30 active components including their metabolites) at the National Institute of Nutrition and Seafood Research (NIFES), Bergen, Norway. Organochlorine pesticides (OCPs) were determined by GCMS on a Trace GC 2000 series and Trace DSQ single quadrupole (Thermo Fisher Scientific, Waltham, MA, USA). 2.3. Geographic distribution All samples were collected in Iowa (USA) within a 200 km radius. There were examples of GM-soy and organic soy samples collected within the same town/village (the smallest distance between farms was 5 km). Nine out of ten samples from the conventional soy were sampled in a town or village where most of the GM-soy samples (six

Postplant

Insecticide

Touchdown Roundup power max Roundup power max Touchdown Roundup Roundup Roundup Touchdown Roundup (original max), durango Roundup (original max), durango

Warhawk, silencer Warrior, lorsban Warrior Cobalt

Fungicide

Warhawk, silencer Leverage Leverage

Headline Domark Domark

Pursuit plus, select, flexstar Select, flexstar, first rate Pursuit plus, select, flexstar Pursuit plus, flexstar, first rate Flexstar, fusion, first rate Pursuit plus Pursuit, flexstar, fusion Select, flexstar, first rate Flexstar, fusion, first rate Pursuit plus

Lorsban, warrior Lorsban Lorsban, warrior Cobalt Lorsban Cobalt Lorsban Lorsban Lorsban Cobalt

Headline

Apron max

Headline

Headline

Headline

out of ten) were also collected. Organic soy and conventional soy samples were not from the same town/village. 2.4. Soy varieties The ten samples of conventional soybeans were of four different varieties: Legend 2932 (4 samples), Legend 2375 (3 samples), Asgrow 2869 (2 samples) and Legend 2200. The GM samples were from 8 to 9 different varieties: Stine 2032 (2 samples), Stine [unnamed], Stine 2538-4, Stine 2602-4, Stine 2062-4, Latham 2158, PB 2217VNRR, PB 2421, Pioneer 92M76. The organic samples consisted of nine different varieties: Pioneer 9305 and ED 4315 (both 2 samples), Legend 2375, Stine 2686, US Soy 20333, Mark 0427, Mark 0431, PB291N and Pioneer 93M52. The conventional and organic varieties overlapped in the use of ‘‘Legend 2375’’ (n = 3 conventional and n = 1 organic sample). There was no overlap in varieties between the GM and either the conventional or organic varieties. 2.5. Multivariate analyses Characteristics of the soy samples were analysed with the R-project software with library (vegan) for 35 variables: glycogen, all amino acids, sum of unsaturated, mono- and poly-unsaturated fats, omega3, omega6 and trace elements. Glyphosate and AMPA were first taken out of the primary analyses to look for differences beyond/because of these. In later analyses, concentrations of glyphosate or AMPA and soy variety were included to identify co-variation to other variables. GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) and Statistica™ 7 (StatSoft Inc., Tulsa, OK, USA) was used to evaluate correlations between nutrient composition and residue levels of glyphosate and AMPA. Differences in nutrients between the soybean categories were analysed using a

210

T. Bøhn et al. / Food Chemistry 153 (2014) 207–215

one-way ANOVA, and in cases when ANOVA showed significant differences, post hoc tests (Tukey HSD test) were used. 3. Results 3.1. Herbicides and pesticides All individual samples of GM-soy contained residues of both glyphosate and AMPA. In contrast, no sample from the conventional or the organic soybeans showed any residues of these chemicals (Fig. 1). In the GM-soy samples, the concentration of AMPA (mean concentration = 5.74 mg/kg) was on average nearly twice as high as glyphosate (3.26 mg/kg). The minimum maximum values for AMPA and glyphosate were 0.7–10.0 and 0.4–8.8 mg/kg, respectively. Fluazifop-P was found in a concentration of 0.078 mg/kg in one of the GM-soy samples, malathion was found in a concentration of 0.02 mg/kg in one of the conventional soy samples and Dieldrin was found in a concentration of 0.002 mg/kg in one of the organic soy samples. Other residues were not found. The additional testing for pesticide residues in pooled samples of GM, conventional and organic soybeans showed trace-levels of Alpha-endosulfane, Trans-nonachlor and Trans-chlordane, all close to the detection limit of 0.05 lg/kg and in all soy types. Dieldrin was also found in very low levels with 0.51, 0.45 and 0.6 lg/kg in GM, conventional and organic soybeans, respectively. 3.2. Main constituents of the soy – individual samples The organic soybeans differed in nutrient composition compared to the conventional and GM soybeans in several variables (Table 2). The organic samples contained significantly more total protein compared to both the GM-soy and conventional soy (p < 0.01, ANOVA, Tukey correction), which was also reflected with a higher content of the indispensable amino acids (IAAs). There was significantly lower content of 18:2n 6, and sum saturated fats in the organic soybean material. There were no significant differences in the 18:1n 9 (monounsaturated) or the 18:3n 3 (Omega 3) fatty acids between the three groups. The content of Zn was significantly higher in the organic samples compared to the conventional and GM samples (p = 0.001 and p < 0.001, respectively, ANOVA, Tukey correction). Other differences were relatively small (Table 2). There was a significant positive correlation between the AMPA residue levels and iron (p = 0.028, linear regression) and AMPA residue levels and 18:2n 6 content in the GM soybeans (p = 0.016, linear regression).

3.3. Main constituents of the soy – pooled samples Samples representing each of the three production systems, containing equal amounts of all individual samples produced using those production systems were analysed for monosaccharides, disaccharides and fibre. The GM-soy (pooled samples) contained on average less of all the main sugars (glucose, fructose, sucrose and maltose) compared to both the conventional and organic soy (Table 3). The organic soy contained more sugars than both conventional and GM-soy, but less fibre (Table 3). 3.4. Cluster analysis Exploratory cluster analyses were used to group and differentiate the soy samples based on the 35 variables measured. Ten of the organic samples were grouped with 1 of the GM samples, while most of the GM and the conventional samples were intermixed (Fig. 2a). By including the variety name to the samples in the cluster tree (Fig. 2b), the role of the genetic background was highlighted. In some cases, the same agricultural practice in combination with the same soy variety, the outcome was a close grouping (e.g., for conventional Legend 2375). However, a third sample of the same Legend 2375, also grown under a conventional practice showed an intermediate distance to the mentioned samples, but grouped very closely to an organic sample of Legend 2375. For other pairs of varieties grown under the same agricultural practice, samples grouped with an intermediate distance (GM Stine 2032 and conventional Asgrow 2869), yet other pairs showed a great distance between sample characteristics (organic ED4315, organic Pioneer 9305). 3.5. Discriminant analysis Soy from the three different categories, GM, conventional and organic, could be well separated (Fig 3). The first axis of variation mainly separated organic samples from both the GM and conventional, while the second axis differentiated the GM from conventional. 3.6. Redundancy analysis (RDA) GM soybeans were most strongly associated with saturated and mono-unsaturated fatty acids. Organic soybeans were associated with elements and amino acids Zn, Asp, Lys, Ala, Sr, Ba, Glu. Conventional soy were associated with the elements Mo and Cd (Fig. 4). The model accounted for 21.5% of the total variation in the material (PC1 = 19.0%, PC2 = 2.5%).

Glyphosate + AMPA (mg/kg)

20

4. Discussion 15

4.1. General

10

5

0

Organic n=11

Conventional n=10

GM n=10

Fig. 1. Residues of glyphosate and AMPA in individual soybean samples (n = 31).

Our data demonstrate that different agricultural practices lead to markedly different end products, i.e., rejecting the null hypothesis (H0) of substantial equivalence between the three management systems of herbicide tolerant GM, conventional and organic agriculture. Both the H1 and H2 hypotheses were supported due to the key results of high levels of glyphosate/AMPA residues in GM-soybeans, and that all the individual soy samples could be discriminated statistically (without exception) into their respective agricultural practice background – based on their measured compositional characteristics (Fig. 3). Notably, the multivariate analyses of the compositional results was performed excluding the factors glyphosate/AMPA residues, which obviously otherwise

211

T. Bøhn et al. / Food Chemistry 153 (2014) 207–215

Table 2 Composition of nutrients and elements in the different soybean types. Results are given as mean ± SD, based on measurement on individual samples. Significant differences (p < 0.05) between means are indicated by different letters.

A

GM

SD

Conv.

SD

Organic

SD

Anova

Proximate composition Dry matter (%) Protein (%) Fat (%) Ash (%)

89.4 34.6b 19.0 4.6ab

1.4 1.3 0.8 0.2

88.1 34.3b 19.1 4.5b

2.0 1.5 1.3 0.2

88.2 36.3a 18.3 4.7a

2.6 1.1 0.9 0.2

ns p = 0.003 ns p = 0.005

Amino acids (mg/g) Methionine Lysine Histidine Isoleucine Leucine Phenylalanine Threonine Valine Arginine Sum of IAAsA

4.2 22.1b 8.9 15.2 26.3ab 18.0 13.8 15.9 24.0ab 142.3

0.3 1.5 0.3 0.7 0.9 0.6 0.4 0.7 0.9 5.4

4.0 22.2b 8.9 15.0 26.2b 17.7 13.8 15.7 23.4b 140.8

0.3 1.3 0.4 0.7 1.1 0.7 0.5 0.7 1.1 5.2

4.0 24.2a 9.0 15.6 27.4a 18.0 14.3 16.3 24.9a 147.1

0.4 0.9 0.6 0.5 1.0 1.2 0.6 0.6 1.8 5.8

ns p = 0.002 ns ns p = 0.02 ns ns ns p = 0.04 p = 0.037

Vitamins (mg/kg) Vitamin B6

15.7

1.5

14.9

1.2

14.9

1.4

ns

Fatty acids (mg/g) 16:0 (palmitic acid) Sum saturated 18:1n 9 (oleic acid) Sum monounsaturated 18:2n 6 (linoleic acid) 18:3n 3 (linolenic acid)

22.6a 33.0a 41.1 44.4 115.7ab 19.1

1.2 1.4 3.0 3.2 5.2 4.4

21.1ab 31.0ab 38.5 41.5 117.8a 19.6

1.1 1.6 2.9 3.1 5.8 0.8

21.0b 29.7b 38.5 41.5 108.4b 18.0

1.9 2.3 4.3 4.5 9.3 1.6

p = 0.046 p = 0.001 ns ns p = 0.01 ns

Elements (mg/kg) Barium (Ba) Copper (Cu) Iron (Fe) Manganese (Mn) Molybdenum (Mo) Selenium (Se) Zinc (Zn)

6.4b 10.4 86.8 24.1 1.9 0.7b 30.4b

2.2 1.1 7.2 2.8 1.0 0.1 2.4

6.2b 10.8 84.4 22.8 4.5 0.8a 31.7b

1.7 1.1 8.7 1.7 4.0 0.2 2.8

11.0a 11.3 84.7 24.5 2.1 0.2b 37.0a

3.3 1.7 11.3 2.3 1.1 0.2 3.4

p = 0.0005 ns ns ns ns p = 0.0003 p = 0.0002

IAAs, indispensible amino acids (except tryptophan).

Table 3 Composition of sugars and fibre (g/100 g fresh sample) in pooled soybean samples, i.e., mixing of all samples from GM (n = 10), conventional (n = 10), and organic (n = 11) origin.

GM Conv. Organic

Glucose

Fructose

Sucrose

Maltose

Fibre

0,37 0,62 1,04

0,20 0,31 0,62

3,24 4,18 4,82

0,02 0,02 0,54

27,1 28,4 24,7

would have served as a strong grouping variable separating the GM soy from the two non-GM soy types. Since different varieties of soy (different genetic backgrounds) from different fields (environments) grown using different agricultural practices were analysed, we need to acknowledge that variation in composition will come from all three of these sources. However, since 13 samples out of the 31 had at least one ‘sibling’ (same variety) to compare both within and across the different agricultural practices, how the same variety ‘performed’ (i.e., its nutritional and elemental composition) between different environments and agricultural practices could be compared. As some samples of the same variety were highly similar in the cluster analysis, but others were intermediate or even highly different (Fig. 2b), we argue that (i) there was a strong genotype  environment interaction within all three agricultural practices, (ii) the combination of a range of varieties on a range of different farms in a relatively well defined geographical region, and grown in the same climate zone in the same season, give us representative data regarding soy composition from that particular region. To test food products that are not experimentally matched, e.g., for different soil conditions, resembles the situation for a consumer in the store.

4.2. Residues of pesticides in the soy In this study it was found that Roundup Ready GM-soybeans sprayed during the growing season had taken up and accumulated glyphosate and AMPA at concentration levels of 0.4–8.8 and 0.7– 10 mg/kg, respectively. In contrast, conventional and organic soybeans did not contain these chemicals. We thus document what has been considered as a working hypothesis for herbicide tolerant crops, i.e., that: ‘‘there is a theoretical possibility that also the level of residues of the herbicide and its metabolites may have increased’’ (Kleter, Unsworth, & Harris, 2011) is actually happening. Glyphosate is shown to be absorbed and translocated within the entire plant, and has been found in both leaf material and in the beans of glyphosate tolerant GM soy plants. However, FAO have not distinguished GM from non-GM plants in their consideration on glyphosate residues. Monsanto has claimed that residues of glyphosate in GM soy are lower than in conventional soybean, where glyphosate residues have been measured up to 16–17 mg/kg (Monsanto, 1999), which likely must have been due to spraying before harvest (desiccation). Another claim has been that documented maximum residue levels up to 5.6 mg/kg in GM-soy represent ‘‘...extreme levels, and far higher than those typically found’’ (Monsanto, 1999). Seven out of the 10 GM-soy samples tested surpassed this ‘‘extreme level’’ of glyphosate + AMPA residues, indicating a development towards higher residue levels. The increased use of glyphosate on Roundup Ready soybeans in the US (Benbrook, 2012), contributing to selection of glyphosate-tolerant weeds (Shaner et al., 2012) with a response of increased doses and/or more applications used per season, may explain the observed plant tissue accumulation of glyphosate.

T. Bøhn et al. / Food Chemistry 153 (2014) 207–215

10

212

Cluster Dendrogram

5

14

16

a

Conv

12

Conv Conv

0

GM GM GM GM GM GM GM GMGM

Organic Organic Organic Organic Organic GM Organic Organic Organic Organic Organic GM GM Conv Conv Conv Conv Conv GM GM Conv Conv Organic Conv GM GM GM GM GM Conv Conv

Cluster Dendrogram

-5

GM

-5

0

5

10

LD1 Fig. 3. Discriminant analysis for GM, conventional and organic soy samples based on 35 variables. Data were standardised (mean = 0 and SD = 1). Glyphosate/AMPA residues were not included (would have separated the GM soy from non-GM soy).

2.0

12

14

16

b

Organic OrganicOrganic Organ Organic Organic Organic Organic Organic Organic Organic

Conv

Conv

8 2

4

6

Height

10

LD2

Conv Conv Conv Conv Conv

24 27

Asp Lys

Cu

V

glycogen 12 15 His 14

Ala 28 organic Sr Ba Glu

-0.5

25

5 18

30

Zn

Ser Thr 31 Leu Pro Ile Val Arg

Gly Co Phe

29

Mn

7

17

fat_poly omega6

omega3

Se 4

16

0

1.0

Hyp Fe Tyr 13 Met 11 vitb6

9 2

fat_mono 3 6 19 8

10 1

fat_sat

gm

-1

0.5

Mo Cd

22

0.0

RDA2

23

-1.0

Fig. 2. (a) Cluster dendrogram for GM, conventional and organic soy samples, based on 35 variables after standardisation of the data (mean = 0 and SD = 1). Glyphosate/ AMPA residues were not included (would have separated the GM soy from non-GM soy). (b) Same as (a) but including information on the genetic line of soy grown.

1

21

Org_Pioneer9305 Org_ED4315 Org_PB291N Org_ED4315 Org_Pioneer93M52 GM_PB2217VNRR Org_Pioneer9305 Org_USSoy20333 Org_Mark0431 Org_Stine2686 Org_Mark0427 GM_Stine2062 GM_Stine2538 Conv_Legend2375 Conv_Legend2375 Conv_Legend2932 Conv_Asgrow2869 Conv_Legend2200 GM_Pioneer92M76 GM_Stine Conv_Legend2932 Conv_Legend2375 Org_Legend2375 Conv_Asgrow2869 GM_Latham2158 GM_Stine2032 GM_PB2421 GM_Stine2602 GM_Stine2032 Conv_Legend2932 Conv_Legend2932

6 2

4

Height

8

1.5

10

20

26

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

RDA1

A pesticide residue is the combination of the pesticide and its metabolites. According to FAO, the total glyphosate residues should be calculated as the sum of gly + 1.5 AMPA. Using this formula, the data set has on average ‘glyphosate equivalents’ of 11.9 mg/kg for the GM soybeans (max. 20.1 mg/kg). Clear residue definitions are required to establish the compound or compounds of interest, e.g., for estimating dietary intake risks. This issue becomes more complex in the near future as new GM plants may: (i) be tolerant to other/additional herbicides (e.g., 2,4-D and/or dicamba), eventually several stacked in the same plant, (ii) have altered tolerance to glyphosate (likely higher), (iii) metabolise herbicides into new breakdown products having altered toxicity and requiring potentially altered methods of detection. The insertion of GAT-genes into maize and soy for example, makes the plant transform glyphosate into the non-herbicidal N-acetyl-glyphosate, requiring a re-consideration of definitions. Residues of agrochemicals must be expected to increase when repeated applications are carried out and when application takes place later in the growing season. Duke et al. showed that

Fig. 4. Redundancy analysis for the first two axes of variation (RDA1 and RDA2), based on standardised data. The positions of individual variables are indicated and the direction of the different soy types shown in arrows. The red lines indicate increasing levels of glyphosate + AMPA residuals (increasing towards ‘gm’). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

GM-soybeans sprayed at full bloom of the plant contained about 5–10 times more glyphosate and 10–25 times more AMPA than plants sprayed only early in the growing season (Duke, Rimando, Pace, Reddy, & Smeda, 2003). With early spraying, the levels of glyphosate and AMPA were 0.2–0.6 and 0.5–0.9 mg/kg, respectively. Spraying at full bloom gave substantially higher residue levels of glyphosate and AMPA, 2.2–3.1 and 7.3–25 mg/kg, respectively (Duke et al., 2003). The samples in the present study showed residue levels comparable to these (i.e., somewhat higher in glyphosate and lower in AMPA), indicating that spraying later in the season has become common practice in the sampled area. This provides strong support for hypothesis (1a) of high residue levels in GM soy.

T. Bøhn et al. / Food Chemistry 153 (2014) 207–215

Even soybeans grown on areas with no application of glyphosate, have been shown to contain glyphosate and AMPA, e.g., 0.1– 0.2 mg/kg (Duke et al., 2003), possibly due to herbicide drift or indicating plant uptake from a soil reservoir of the herbicide. Our samples from conventional soybean farmers did not contain any glyphosate or AMPA. This was not surprising as the use of preplant herbicides did not include glyphosate-based chemicals. We thus find no support for hypothesis (1b) in our data set. Under all three agricultural practices trace levels of pesticides other than glyphosate were detected (see results), but we consider these pesticide residues of little practical significance for the tested soy materials. Presumably, they are due to residual levels of persistent pesticides in the soil, even in organic fields. 4.3. Nutritional components Soybean nutritional quality is determined by many factors but the protein level, the mineral content and fatty acid (FA) composition are essential components. Our results clearly show that different agricultural practices affect the quality of soybeans. The organic soybeans had significantly higher levels of total protein and lower levels of linoleic acid LA (18:2n 6) and palmitic acid PA (16:0). Soybeans are a major dietary source of LA and although LA is an essential FA, a high and unbalanced intake (high omega 6 and low omega 3) is emerging as a risk factor for developing obesity. We also show that GM-soy had a significantly higher level of PA, a saturated FA, compared to organic soybeans. EFSA has concluded that saturated fatty acids intake should be as low as possible within the context of nutritionally adequate diets. Conventional soybeans were observed to have superior nutrient and dry matter composition compared to glyphosate-treated GMsoybeans (Zobiole et al., 2012). In a review on this topic, however, conflicting results were found, with most studies indicating that mineral nutrition is not affected by glyphosate tolerance trait or application of glyphosate (Duke et al., 2012). 4.4. Direct and indirect effects of glyphosate application on soy nutrition and plant environment Glyphosate has been shown to reduce photosynthesis and nutrient uptake in GM-soy, in greenhouse and field trials, both for first and second generation of glyphosate resistant soy plants. High glyphosate application rates have been shown to reduce alfa-linolenic acid (ALA, 18:3n 3) but increase oleic acid (OL, 18:1n 9) (Bellaloui, Zablotowicz, Reddy, & Abel, 2008), i.e., producing a less healthy profile of fatty acids. Glyphosate may also, depending on soil type, alter micronutrient status, in particular Mn and Zn. Our data showed significantly higher Zn concentrations in organic soy samples (mean 37.0 mg/ kg), but no differences between GM and conventional soy samples (mean 30.4 and 31.7 mg/kg, respectively). This indicates that factors other than glyphosate may be relevant, such as the use of organic versus synthetic fertiliser or long-term accumulated differences in soil treatment and quality. Status of the micronutrient Mn was not affected by the production system in our samples. In general, a healthy microbial community, ‘the plant microbiome’, in the soil of the rhizosphere is an important contributing factor for plant trait characteristics and plant health (Lundberg et al., 2012). Glyphosate has the potential to adversely affect microbial communities present in soils into which plants are rooted, i.e. increased colonisation by Fusarium (Kremer & Means, 2009). AMPA is mildly phytotoxic, and leads to reduced photosynthesis (‘yellowing’) and transpiration rates in soy plants (Ding, Reddy, Zablotowicz, Bellaloui, & Bruns, 2011). Other ingredients of glyphosate-based herbicides have also been described as detrimental

213

to GM-soy. We found a significant positive correlation between AMPA residue levels in the GM soybeans and increasing levels of LA and iron (Fe). 4.5. Maximum residue level (MRL) of glyphosate in food and feed The acceptance level of glyphosate in food and feed, i.e., the maximum residue level (MRL) has been increased by authorities in countries where Roundup-Ready GM crops are produced or where such commodities are imported. In Brazil, the MRL in soybean in 2004 was increased from 0.2 to 10 mg/kg: a 50-fold increase, but only for GM-soy. The MRL for glyphosate in soybeans has also increased in the US and Europe. In Europe, it was raised from 0.1 to 20 mg/kg in 1999, and the same MRL of 20 mg/kg was adopted by the US based on recommendations of the Codex Alimentarius Commission. In all of these cases, MRL values appear to have been adjusted, not based on new evidence indicating glyphosate toxicity was less than previously understood, but pragmatically in response to actual observed increases in the content of residues in glyphosate-tolerant GM soybeans. In the US, in Canada and elsewhere there is a practice of using glyphosate to desiccate crops by spraying the maturing plants, in order to speed up and make the ‘‘maturation’’ of the crop more uniform, thereby facilitating harvest. This may add to the residue levels of glyphosate and AMPA, as shown in field pea, barley and flax seed. Particularly if the plant is still growing, translocation of glyphosate within the plant may result in accumulation of glyphosate residues in the seed, both for GM and unmodified soy. 4.6. Toxicity and health relevance of pesticide/glyphosate residues It is the full, formulated herbicide (typically one of the many Roundup formulations) that is used in the field, and, thus, it is relevant to consider, not only the active ingredient glyphosate and its breakdown product AMPA, but also the other compounds present in the herbicide formulation. For example, herbicide formulations containing glyphosate commonly also contain adjuvants and surfactants to help stabilise the herbicide and to facilitate its penetration into the plant tissue. Polyoxyethylene amine (POEA) and polyethoxylated tallowamine (POE-15) are common ingredients in Roundup formulations, and have been shown to contribute significantly to the toxicity of Roundup formulations (Moore et al., 2012). However, glyphosate alone has been shown to interfere with molecular mechanisms that regulate early development in frogs and chickens, with deformities of embryos as a consequence and the retinoic acid signalling pathway as the affected mediator (Paganelli, Gnazzo, Acosta, Lopez, & Carrasco, 2010). In human cells, Roundup may induce endocrine disturbances at concentrations far below the MRLs cited by authorities in the EU and US (Benachour & Seralini, 2009). A life-cycle feeding study in rats reported negative health effects and found significantly altered blood parameters in animals that were fed Roundup Ready GM maize or were given extremely small amounts of Roundup in the drinking water (Seralini et al., 2012). The authors emphasised the role of pesticide residues in edible herbicide tolerant GM plants and argued that these must be evaluated very carefully to accurately assess potential toxic effects. This study has been criticised for its methods, analysis and reporting by EFSA, which initially rejected the central conclusion of this study, that long term (lifetime) toxicity and carcinogenicity studies are needed. However, EFSA as well as regulatory authorities from multiple EU states are now acknowledging that this study flagged up the need for long term studies. A recent study in the model organism Daphnia magna demonstrated that chronic exposure to glyphosate and a formulation of Roundup resulted in negative effects on several life-history traits, in particular reproductive aberrations like reduced fecundity

214

T. Bøhn et al. / Food Chemistry 153 (2014) 207–215

and increased abortion rate at environmental concentrations of 0.45–1.35 mg/L (active ingredient), i.e., below accepted environmental tolerance limits set in the US (Cuhra, Traavik, & Bøhn, 2013). A reduced body size of juveniles was even observed at an exposure to Roundup at 0.05 mg/L. These results are strikingly different from data reported by a study funded by the European Commission which indicated a NOEC (No Observed Effect Concentration) in D. magna of 455 and 30 mg/l for glyphosateIPA and glyphosate acid, respectively (EC, 2002). The importance of pesticide residuals is recognised by EFSA in feeding studies for risk assessment. For glyphosate-tolerant GM soybeans, EFSA has argued that (i) the levels of glyphosate should be analysed as part of the testing, and (ii) both glyphosate-treated and untreated soybeans should be used in order to separate effects of the plant and the herbicide (van Haver et al., 2008). The toxicity and health relevance of glyphosate and Roundup have been debated widely. Other studies claim that glyphosate is not linked to developmental or reproductive effects in animals and humans, but that surfactants may cause some toxic effects (Williams, Watson, & DeSesso, 2012). This controversy has been reviewed in depth in (Antoniou, Robinson, & Fagan, 2012), with the conclusion that the weight of evidence indicates that glyphosate itself is a teratogen and that adjuvants commonly used in conjunction with glyphosate amplify this effect. 4.7. Organic vs conventional vs GM agriculture Comparisons between organic and conventional agriculture have not reached consistent conclusions on nutritional quality, but a review of 223 compositional studies of nutrients and contaminants found that organic foods have significantly lower levels of pesticide residues (Smith-Spangler et al., 2012). A recent feeding study that compared organic and conventional food products concluded that organic foods may be more nutritionally balanced than conventional foods, or that they contain higher levels of nutrients, since the fruit fly Drosophila melanogaster lived longer and produced more offspring when fed organic soybeans (or potatoes, raisins, bananas) compared to conventional produce (Chhabra, Kolli, & Bauer, 2013). Organic crops may be more variable than industrially produced plant products, but are in general richer in some nutritionally important elements, in antioxidant phytochemicals and lower in pesticide residues. Our data support these conclusions. Organic crops have also been reported to contain a higher content of selenium. This was however not supported by our data, where the selenium content was significantly lower in the organic soybeans compared to the GM and conventional soybeans. 5. Conclusion This study demonstrated that Roundup Ready GM-soy may have high residue levels of glyphosate and AMPA, and also that different agricultural practices may result in a markedly different nutritional composition of soybeans. In the present study organic soybean samples had a more profitable nutritional profile than industrial conventional and GM soybeans. We argue that pesticide residues should have been a part of the compositional analyses of herbicide tolerant GM plants from the beginning. Lack of data on pesticide residues in major crop plants is a serious gap of knowledge with potential consequences for human and animal health. We therefore recommend (i) increased effort on sampling and testing crop material from the market; (ii) testing for possible dose–response effects of chemical residues in long-term feeding studies; (iii) inclusion of pesticide residue measurements and safety testing in the regulatory system for risk-assessment and (iv) further research on the indirect ecological effects of herbicides

and pesticides, i.e., on ecological interactions in the soil community with possible effects on nutrient uptake and plant composition. Acknowledgements We thank the Research Council of Norway for funding under the program ‘‘ENVIRONMENT2015’’ (Project number 184107). References Antoniou, M., Robinson, C., & Fagan, J. (2012). Teratogenic effects of glyphosatebased herbicides: Divergence of regulatory decisions from scientific evidence. Journal of Environmental Analytical Toxicology S4:006. http://www. omicsonline.org/2161-0525/2161-0525-S4-006.php?aid=7453 Aumaitre, L. A. (2002). New feeds from genetically modified plants: Substantial equivalence, nutritional equivalence and safety for animals and animal products. Productions Animales, 15, 97–108. Bellaloui, N., Zablotowicz, R. M., Reddy, K. N., & Abel, C. A. (2008). Nitrogen metabolism and seed composition as influenced by glyphosate application in glyphosate-resistant soybean. Journal of Agricultural and Food Chemistry, 56, 2765–2772. Benachour, N., & Seralini, G. E. (2009). Glyphosate formulations induce apoptosis and necrosis in human umbilical, embryonic, and placental cells. Chemical Research in Toxicology, 22, 97–105. Benbrook, C. M. (2012). Impacts of genetically engineered crops on pesticide use in the U.S. – The first sixteen years. Environmental Science Europe, 24, 24. Binimelis, R., Pengue, W., & Monterroso, I. (2009). ‘‘Transgenic treadmill’’: Responses to the emergence and spread of glyphosate-resistant johnsongrass in Argentina. Geoforum, 40, 623–633. Chhabra, R., Kolli, S., & Bauer, J. H. (2013). Organically grown food provides health benefits to Drosophila melanogaster. PLoS One, 8, 1–8. Cuhra, M., Traavik, T., & Bøhn, T. (2013). Clone- and age-dependent toxicity of a glyphosate commercial formulation and its active ingredient in Daphnia magna. Ecotoxicology, 22, 251–262. http://dx.doi.org/10.1007/s10646-012-1021-1 [open access]. Ding, W., Reddy, K. N., Zablotowicz, R. M., Bellaloui, N., & Bruns, H. A. (2011). Physiological responses of glyphosate-resistant and glyphosate-sensitive soybean to aminomethylphosphonic acid, a metabolite of glyphosate. Chemosphere, 83, 593–598. Duke, S. O., Lydon, J., Koskinen, W. C., Moorman, T. B., Chaney, R. L., & Hammerschmidt, R. (2012). Glyphosate effects on plant mineral nutrition, crop rhizosphere microbiota, and plant disease in glyphosate-resistant crops. Journal of Agricultural and Food Chemistry, 60, 10375–10397. EC. (2002). Review report for the active substance glyphosate (Rep. no. 6511/VI/99final). European Commission. Duke, S. O., Rimando, A. M., Pace, P. F., Reddy, K. N., & Smeda, R. J. (2003). Isoflavone, glyphosate, and aminomethylphosphonic acid levels in seeds of glyphosatetreated, glyphosate-resistant soybean. Journal of Agricultural and Food Chemistry, 51, 340–344. Harrigan, G. G., Ridley, G., Riordan, S. G., Nemeth, M. A., Sorbet, R., Trujillo, W. A., et al. (2007). Chemical composition of glyphosate-tolerant soybean 40-3-2 grown in Europe remains equivalent with that of conventional soybean (Glycine max L.). Journal of Agricultural and Food Chemistry, 55, 6160–6168. Kleter, G. A., Unsworth, J. B., & Harris, C. A. (2011). The impact of altered herbicide residues in transgenic herbicide-resistant crops on standard setting for herbicide residues. Pest Management Science, 67, 1193–1210. Kremer, R. J., & Means, N. E. (2009). Glyphosate and glyphosate-resistant crop interactions with rhizosphere microorganisms. European Journal of Agronomy, 31, 153–161. Lappé, M. A., Bailey, E. B., Childress, M. S., & Setchell, K. D. R. (1998). Alterations in clinically important phytoestrogens in genetically modified, herbicide-tolerant soybeans. Journal of Medicinal Food, 1, 241–245. Lundberg, D. S., Lebeis, S. L., Paredes, S. H., Yourstone, S., Gehring, J., Malfatti, S., et al. (2012). Defining the core Arabidopsis thaliana root microbiome. Nature, 488, 86–90. Millstone, E., Brunner, E., & Mayer, S. (1999). Beyond ‘substantial equivalence’. Nature, 401, 525–526. Monsanto. (1999). Residues in Roundup Ready soya lower than conventional soy. . Ref type: Internet communication. Moore, L. J., Fuentes, L., Rodgers, J. H., Bowerman, W. W., Yarrow, G. K., Chao, W. Y., et al. (2012). Relative toxicity of the components of the original formulation of Roundup (R) to five North American anurans. Ecotoxicology and Environmental Safety, 78, 128–133. Paganelli, A., Gnazzo, V., Acosta, H., Lopez, S. L., & Carrasco, A. E. (2010). Glyphosatebased herbicides produce teratogenic effects on vertebrates by impairing retinoic acid signaling. Chemical Research in Toxicology, 23, 1586–1595. Podevin, N., & du Jardin, P. (2012). Possible consequences of the overlap between the CaMV 35S promoter regions in plant transformation vectors used and the viral gene VI in transgenic plants. GM Crops and Food, 3, 296–300. Seralini, G. E., Clair, E., Mesnage, R., Gress, S., Defarge, N., Malatesta, M., et al. (2012). Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize. Food and Chemical Toxicology, 50, 4221–4231.

T. Bøhn et al. / Food Chemistry 153 (2014) 207–215 Shaner, D. L., Lindenmeyer, R. B., & Ostlie, M. H. (2012). What have the mechanisms of resistance to glyphosate taught us? Pest Management Science, 68, 3–9. Smith-Spangler, C., Brandeau, M. L., Hunter, G. E., Bavinger, C., Pearson, M., Eschbach, P. J., et al. (2012). Are organic foods safer or healthier than conventional alternatives? Annals of Internal Medicine, 157. 348-U112. van Haver, E., Alink, G., Barlow, S., Cockburn, A., Flachowsky, G., Knudsen, I., et al. (2008). Safety and nutritional assessment of GM plants and derived food and feed: The role of animal feeding trials. Food and Chemical Toxicology, 46, S2–S70. Wei, Q. K., Jone, W. W., & Fang, T. J. (2004). Study on isoflavones isomers contents in Taiwan’s soybean and GM soybean. Journal of Food and Drug Analysis, 12, 324–331.

215

Williams, A. L., Watson, R. E., & DeSesso, J. M. (2012). Developmental and reproductive outcomes in humans and animals after glyphosate exposure: A critical analysis. Journal of Toxicology and Environmental Health. Part B. Critical Reviews, 15, 39–96. Zobiole, L. H. S., Bonini, E. A., de Oliveira, R. S., Kremer, R. J., & Ferrarese, O. (2010). Glyphosate affects lignin content and amino acid production in glyphosateresistant soybean. Acta Physiologiae Plantarum, 32, 831–837. Zobiole, L. H. S., Oliveira, R. S., Constantin, J., Oliveira, A., Castro, C., Oliveira, F. A., et al. (2012). Nutrient accumulation in conventional and glyphosateresistant soybean under different types of weed control. Planta Daninha, 30, 75–85.

 

   

Paper  III:  

Feeding  study  I.      

  Title:      

Life  cycle  differences  in  D.  magna  fed  Roundup  Ready  soybean   conventional  soybean  or  organic  soybean.    

Authors:    

Marek  Cuhra,  Terje  Traavik,  Thomas  Bøhn  

Journal:    

Aquaculture  Nutrition  1365/2095.  DOI  10.1111/anu.12199  (2014).  

41  

 

42  

Aquaculture Nutrition 2014

doi: 10.1111/anu.12199

..........................................................................................

1,2 1

1,2

1,2

GenØk – Centre for Biosafety, The Science Park, Tromsø, Norway; Norway, Tromsø, Norway

A lifelong feeding study with soybean from different production systems was carried out in the crustacean Daphnia magna (water flea), an acknowledged model organism for ecotoxicological studies. Experimental diets were prepared with soybean meal from different agriculture production systems: (i) genetically modified Roundup-Ready soy (Glyphosate-Tolerant), (ii) conventional soy and (iii) soy from organic agriculture (agriculture with neither synthetic pesticides nor synthetic fertilizers). Overall, feed produced from organic soybeans resulted in the highest fitness (higher survival, better growth and fecundity) in the model organism. Animals fed Roundup-Ready soybean consistently performed less well compared to animals fed either conventional or organic soybeans. We conclude that accumulation of herbicide residues in Roundup-Ready soy and related nutritional differences between the soy types may have caused the observed fitness differences. The results accentuate the need for further research clarifying qualitative aspects, including potential large-scale consequences for food and feed quality, of this dominant crop. KEY WORDS:

agricultural practice, Daphnia magna ecological indicator organism, glyphosate residues, genetically modified soy, organic soybean, transgenic cultivar risk-assessment

Received 7 October 2013; accepted 6 March 2014 Correspondence: M. Cuhra, GenØk – Centre for Biosafety, The Science Park, PO Box 6418, 9294 Tromsø, Norway. E-mail: [email protected]

Soybean (Glycine max) has become a primary ingredient in feed for farmed domesticated animals and for various

..............................................................................................

ª 2014 John Wiley & Sons Ltd

2

Faculty of Health Sciences UiT, Arctic University of

species of fish and crustacean in aquaculture (Olsen & Hasan 2012). Soybean meal and soy oil are widely used to supplement or substitute fish meal and marine lipids in formulated feeds for aquaculture farming of Atlantic salmon Salmo salar (Sagstad et al. 2008), rainbow trout Oncorhynchus mykiss (Refstie et al. 2000; Yang et al. 2011) and in aquaculture of crustaceans, such as crayfish (Jones & De Silva 1997), prawn and shrimp (Kumaraguru et al. 2005). Methods for soybean meal preparation in aquaculture feed for crustacean species in general have been known and used for more than two decades, confirming soybean meal as an alternative protein sources in crustacean diets (Cuzon et al. 1994). The majority of present global soy production is from genetically modified glyphosate-tolerant Roundup-Ready soybean (GM-soy) (James 2010). The remaining quantity of global non-GM-soy production is largely from ‘conventional’ industrial farming, upheld through national regulation as well as consumer group preferences. To some degree, higher premium on organic soybeans and costs of stewardship agreements motivate farmer transition from GM-soy to organic soybean production (McBride & Greene 2009). Despite this, organic production in the USA is still limited to 0.18; Fig. 5b).

10

15

20

25

30

35

Age (days)

Figure 3 Body size of Daphnia magna at days 7, 13, 19, 25, 31 and 37 after feeding green algae and low doses of raw (dashed lines) or heated (solid lines) soybean meal of organic (green), conventional (black) or GM-soy (red) origin.

High dose 4.0

(a)

3.0 2.5

2.5

15

20

25

30

35

1.5

2.0

2.0 1.5

10

10

15

20

25

30

35

(d)

(c)

1.5

2.0

2.0

2.5

2.5

3.0

Heated soymeal

3.0

3.5

3.5

Raw soymeal

Body size (mm)

(b)

3.5

3.0

3.5

Low dose

Animals fed soybean meal only (unbalanced diets) As expected, animals fed soy only did not perform well compared to groups fed algae and soy combinations. When animals were fed a low dose of raw soybean meal, the organic soy performed significantly better than both

10

15

20

25

30

35

Age (days)

10

15

20

25

30

35

Figure 4 Body size of Daphnia magna at days 7, 13, 19, 25, 31 and 37 after feeding low or high doses, raw or heated soybean meal of organic (green), conventional (black) or GM-soy (red) origin.

..............................................................................................

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

400

Heated soymeal

200

300

(b)

100

100 150 200 250 300 0

0

50

Cumulative fecundity

Raw soymeal

(a)

10

15

20

25

30

35

40

10

15

20

25

30

35

40

Age (days)

Figure 5 Cumulative fecundity for Daphnia magna fed raw (a) or heated (b) soybean meal in addition to green algae. Lines represent regression lines (50% percentiles) for conventional (black), GM-soy (red) and organic (green) soy.

conventional and GM-soy (P < 0.001 for both, quantile regression; Fig. 6a). When animals were fed a high dose of the same raw soybean meal, the organic soy performed better than the conventional and GM-soy (P = 0.013 and P = 0.001, respectively, quantile regression; Fig. 6b). Conventional soy performed better than the GM-soy (P = 0.006, quantile regression). When animals were fed a low dose of heated soybean meal, the GM-soy performed less well than conventional and organic soy (P < 0.001 for both, quantile regression). The conventional and the organic soy performed similarly (Fig. 6c). When animals were fed a high dose of heated soybean meal, the conventional soy performed better than both the GM-soy and the organic soy (P < 0.001 for both, quantile regression; Fig. 6d). Testing across groups For animals fed soybean meal only, with low and high doses pooled, and correcting in the model for the effect of heating, a higher median slope for accumulative fecundity was found for animals fed organic soy than for both conventional and GM-soy (P = 0.003 and P < 0.001, respectively, quantile regression). Conventional soy performed better than GM-soy (P = 0.006, quantile regression).

The life cycle feeding trials in D. magna demonstrate significant differences in fitness measured as survival, growth and fecundity, attributable to soy type. These results were consistent for balanced (with algae) and unbalanced soy

..............................................................................................

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

diets (soy only). Overall, feed produced from organic soybeans resulted in the highest fitness in our model organism, and animals fed Roundup-Ready soy (GM-soy) consistently performed less well, compared to animals fed either conventional or organic soybeans. Our results show that D. magna live, grow and reproduce optimally on full-fat soybean meal diets produced from heat-treated soybeans from all three soy types when given in combination with green alga (balanced diets). Interestingly, when testing raw soy in balanced diets, the organic soy gave significantly improved performance in our test organism as compared to the algae-fed control. In contrast, for the conventional and particularly for the GMsoy, performance was reduced compared to the control, indicating negative effects from toxins or antinutrients. Animals fed unbalanced diets of only soybean meal had increased mortality and reduced growth and fecundity, demonstrating performance well below the control group for all measured variables. This feed composition is not representative of what aquaculture species would receive from commercially produced feed; however, inclusion rates for soybean meal in farm animal feed have increased from 15–40% (OECD 2001), and inclusion rates of 33–40% soybean meal have been validated in feeding trials with Pacific white shrimp (Litopenaeus vannamei) (Amaya et al. 2007). Although diets of soy only are unbalanced for aquaculture purpose, such experimental feed has been validated for D. magna as ecological indicator (Bøhn et al. 2008, 2010). Furthermore, feeding studies with raw and heated soy are complementary. Soy for use as commercial feed is typically heat treated, whereas raw soy represents an environmentally relevant food source. Herbivore species feeding in/ near monoculture fields are exposed to high proportion of

150

High dose

(b)

20

25

Low dose

(a)

100

15

50

10

0

5 0

20

25

30

35

40

100

(c)

10

15

20

25

30

35

40

(d)

0

0

20

10

40

20

60

Heated soymeal

80

40

15

30

Cumulative fecundity

Raw soymeal

10

10

15

20

25

30

35

40

10

15

20

25

Age (days)

specific plant biomass, thus testing protocols for non-target organisms feeding on, for example, transgenic material use 100% of the relevant material (L€ ovei & Arpaia 2005; L€ ovei et al. 2009). In cultivated landscape, streams and ponds allow for diverse aquatic invertebrate species. In such habitats, a majority of allochthonous biomass may come from residuals of adjacent monoculture crops (Rosi-Marshall et al. 2007; Bøhn et al. 2012). Consistent patterns of fitness differences in D. magna are found in balanced and unbalanced diets. For raw soy, organic soy was superior for D. magna reproduction, whereas GM-soy was inferior. Animals fed high doses of GM-soy also showed a markedly increased mortality rate, supporting the observations of toxic or inhibitory effect seen in the balanced diet with algae. Heat treatment of the feed levelled off the large differences in effects from soy types, but the organic soy still gave the highest growth rates. The effects of heating are complex; soybean contains valuable nutritional elements but also major allergens that potentially affect animal health (Lemos et al. 2004; Galbas et al. 2011). Heat and alkali treatment of soy have beneficial effects for nutritional and digestion properties of soy proteins (Arndt et al. 1999), but very high pH and/or temperature should be avoided to maintain nutritional quality (Wu et al. 1999). Both quantity

30

35

40

Figure 6 Cumulative fecundity for Daphnia magna fed soy raw (a,b) or heated soybean meal (c,d), in low (a–c) or high (b–d) doses. Lines represent regression lines (50% percentiles) for conventional (black), GM-soy (red) and organic (green) soy.

and quality of proteins change after heating with a corresponding change in antigenic properties (Galbas et al. 2011). For edible oils, heating may be harmful (Jaarin et al. 2010). Our results indicate positive impact of heating, whereas a study in catfish found no such effect (Evans et al. 2005). As glyphosate is thermally relatively stable, with a degradation point of 199 °C (FAO 2001), heating of soy material is not likely to reduce the levels of glyphosate residues in GM-soy.

Documentation of significant qualitative differences in crops, based on these feeding studies, contributes importantly in discussion of potential effects of farming practice. Here it is important to notice the overall superior performance of animals that had been fed organic soy. A recent review (Smith-Spangler et al. 2012) extracted data from 17 studies in human consumers and 223 compositional studies of nutrients and contaminants and found that there was no strong consistent evidence that organic foods are significantly more nutritious than conventional foods. However, organic foods had significantly lower levels of pesticide residues, which clearly may impact the overall food and feed quality.

..............................................................................................

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

Published studies in general only compare compositional elements or feed quality of either organic versus conventional cultivars (Smith-Spangler et al. 2012) or transgenic versus conventional cultivars (Pryme & Lembcke 2003; Flachowsky et al. 2005).We have not found any other study that covers produce from all three categories of agricultural practices, as presented here. The requirement for animal feeding trials to supplement compositional analyses (EFSA 2008, 2010) highlights obvious shortcomings in assessments of plant material intended for food or feed use. Despite this, stand-alone compositional analysis has been used extensively and is still used (Zhou et al. 2011). As GM-soy now constitutes the majority of soy produced globally (James 2010) and is seen to give an overall inferior fitness in our test animals, it is justified to focus on potential effects from this particular soy type in farm animal and aquaculture feed. Bakke-McKellep et al. (2007) found somewhat higher kidney lysozyme and acid phosphatase levels in Atlantic salmon after feeding Roundup-Ready soybeans, but where not able to conclude if this was caused by the genetic modification or other soy cultivar differences. Residue levels of herbicides were not quantified in the mentioned study, neither in two other feeding studies in Atlantic salmon which have shown minor differences between Roundup-Ready soy and its near-isogenic conventional soy (Sissener et al. 2009a,b). Both these studies used soy material supplied by biotech industry. Sagstad et al. (2008) compared organ development, metabolic markers and general health indicators of Atlantic salmon fed diets from GM-soy with fish fed non-GM-soy of a near-isogenic parental line. The results demonstrated significant differences in investigated endpoint between groups: fish fed GM-soy diets showed increased feed conversion ratios, decreased protein efficiency ratio, decreased apparent digestibility coefficients of lipids and dry matter and decreased plasma triacylglycerol levels. Although these indications of histological and metabolical changes in fish liver, spleen and intestine were attributable to agricultural system (GM versus non-GM), a later review of this evidence was inconclusive (Sissener et al. 2011). Zhu et al. (2004) investigated nutritional qualities of meal from conventional versus Roundup-Ready soybean in a 13-week subchronic rat feeding experiment with high inclusion rates (up to 90% soybean meal). Despite initial significantly reduced feed intake and reduced growth in 90% GM-soy group (in first week of experiment), no subsequent significant differences in feed intake, body weight or mortality were observed. However, it is indicated the

..............................................................................................

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

tested GM-soy was not sprayed with glyphosate herbicide (Huang & Xu 2013). Mice fed Roundup-Ready GM-soy showed altered mitochondrial functions and transcription pathways in hepatocytes (Malatesta et al. 2002, 2008b). Subsequently similar responses were confirmed in cultured liver tissue cells exposed to low concentrations of Roundup (Malatesta et al. 2008a).

A common weakness of most published feeding studies using herbicide-tolerant crops such as Roundup-Ready soy is that the tested material is not sprayed by the relevant herbicide (Millstone et al. 1999; Viljoen 2013), and even if sprayed, measurements of herbicide residue levels are often missing. The causal explanation for the observed fitness differences in our feeding trial is sought in the qualitative aspects of the different types of soy samples used, notably the presence of glyphosate residues as determined in Bøhn et al. (2014). Glyphosate and glyphosate-based herbicides (Roundup) are known to be toxic to D. magna by reducing life expectancy at environmental concentrations of about 1–4 mg L"1, and significantly reducing growth and fecundity at a concentration of 0.45 mg L"1, that is, below threshold acceptance levels in the US (0.7–1.0 mg L"1; Folmar et al. 1979; Cuhra et al. 2013). Obviously, chemical residues of glyphosate cannot explain the significant differences found in our study in fitness of animals fed diets from organic versus conventional soy, as none of these feeds contained such residues. However, Bøhn et al. (2014) showed that the tested organic soybeans contained more total protein, zinc and a healthier fatty acid composition as compared to the conventional soy.

Most feeding studies for risk assessment of transgenic plant produce are planned and executed over a time span which is relatively short, compared to the expected life length of the test animal. However, the actual use of transgenic plants in food and feed represents lifetime chronic exposure with relatively low inclusion rates. Studies of such longtime exposure are relevant and unfortunately the published evidence is scarce. The present study in D. magna represents a life cycle study in a short-lived ecological indicator test organism. Lasting only 42 days, costs are reduced to a

fraction of, for example, a 2-year study in mammals. Although testing in both fish and mammals give numerous richer indications such as histological evidence, biochemical evidence and immunological responses, we argue that indications of toxicity and nutritional quality can be harvested from studies in D. magna and similar model organisms with shorter generation times. Studies in progress further aim to investigate the role of glyphosate residues in Roundup-Ready GM-soy. By feeding diets from individual GM-soy-harvests, it will be investigated whether fitness parameters such as survival, growth and fecundity of D. magna can be correlated to the levels of glyphosate herbicide residues.

The findings demonstrate that soybean meal from different agricultural practices significantly influence D. magna survival, growth and fecundity, with an overall positive effect from organic soybean and an overall negative effect of Roundup-Ready soybean. This indicates these crop types present significantly different quality of feed. This difference may be attributed to residues of glyphosate herbicide determined to be present in the GM-soy or to other nutritional differences attributable to agriculture practice. Such possible effects of herbicide residues have previously been largely ignored in risk assessment procedures for GM plants and should be specifically addressed in future feeding trials.

The work is funded by Forskningsr# adet the Norwegian Research Council, project no. 184107 Milø-2015.

The authors declare that they have no conflict of interest.

Amaya, E.A., Davis, D.A. & Rouse, D.B. (2007) Replacement of fish meal in practical diets for the Pacific white shrimp (Litopenaeus vannamei) reared under pond conditions. Aquaculture, 262, 393–401. Arndt, R.E., Hardy, R.W., Sugiura, S.H. & Dong, F.M. (1999) Effects of heat treatment and substitution level on palatability and nutritional value of soy defatted flour in feeds for Coho Salmon, Oncorhynchus kisutch. Aquaculture, 180, 129–145. Bakke-McKellep, A.M., Koppang, E.O., Gunnes, G., Sanden, M., # (2007) Histological, Hemre, G.I., Landsverk, T. & Krogdahl, A.

digestive, metabolic, hormonal and some immune factor responses in Atlantic salmon, Salmo salar L., fed genetically modified soybeans. J. Fish Dis., 30, 65–79. Bøhn, T., Primicerio, R., Hessen, D.O. & Traavik, T. (2008) Reduced fitness of Daphnia magna Fed a Bt-transgenic maize variety. Arch. Environ. Contam. Toxicol., 55, 584–592. Bøhn, T., Traavik, T. & Primicerio, R. (2010) Demographic responses of Daphnia magna fed transgenic Bt-maize. Ecotoxicology, 19, 419–430. Bøhn, T., Primicerio, R. & Traavik, T. (2012) The German ban on GM maize MON810: scientifically justified or not justified? Environ. Sci. Eur., 24, 22. Bøhn, T., Cuhra, M., Traavik, T., Fagan, J., Primicerio, R. & Sanden, M. (2014) Compositional differences in soybeans on the market: glyphosate accumulates in Roundup Ready GM soybeans. Food Chem., 153, 207–215. Collins, T. (2007) ImageJ for microscopy. Biotechniques, 43, S25– S30. Cuhra, M., Traavik, T. & Bøhn, T.A. (2013) Clone- and age-dependent toxicity of a glyphosate commercial formulation and its active ingredient in Daphnia magna. Ecotocicology, 22, 251–262. Cuzon, G., Guillaume, J. & Cahu, C. (1994) Composition, preparation and utilization of feeds for Crustacea. Aquaculture, 124, 253–267. Dorr, R., Ozu, M. & Parisi, M. (2007) Simple and inexpensive hardware and software method to measure volume changes in Xenopus oocytes expressing aquaporins. J. Neurosci. Methods, 161, 301–305. Duke, S.O., Rimando, A., Pace, P.F., Reddy, K.N. & Smeda, R.J. (2003) Isoflavone, glyphosate, and aminomethylphosphonic acid levels in seeds of glyphosate-treated glyphosate-resistant soybean. J. Agric. Food Chem., 51, 340–344. EFSA (2008) Safety and nutritional assessment of GM plants and derived food and feed: the role of animal feeding trials. Food Chem. Toxicol., 46, S1–S70. EFSA (2010) Guidance on the environmental risk assessment of genetically modified plants. EFSA J., 8, 1–111. Evans, J.J., Pasnik, D.J., Peres, H., Lim, C. & Klesius, P.H. (2005) No apparent differences in intestinal histology of channel catfish (Ictalurus punctatus) fed heat-treated and non-heat-treated raw soybean meal. Aquacult. Nutr., 11, 123–129. FAO (2001) Glyphosate, N(phosphonomethyl)glycine. FAO Specifications and Evaluations for Plant Protection Products, Food and Agriculture Organization of the United Nations, FAO, Rome. Flachowsky, G., Chesson, A. & Aulrich, K. (2005) Animal nutrition with feeds from genetically modified plants. Arch. Anim. Nutr., 59, 1–40. Folmar, L.C., Sanders, H.O. & Julin, A.M. (1979) Toxicity of the herbicide glyphosate and several of its formulations to fish and aquatic invertebrates. Arch. Environ. Con. Tox., 8, 269–278. Galbas, M., Borys, K., Wozniak, A. & Selwet, M. (2011) Impact of globulins derived from genetically modified and conventional soybean on swine lymphocyte proliferation in in vitro cultures. Ann. Anim. Sci., 11, 497–505. Huang, K. & Xu, W. (2013) Reply to letter to the editor. Food Chem. Toxicol., 59, 811–812. Jaarin, K., Hwa, T.C., Umar, N.A., Aishah, M.A.S. & Das, S. (2010) Enzymatic and microstructural changes in the liver of experimental rats fed with fatty diet and fresh or heated soy oil concurrently. Clin. Ter., 161, 429–433. James, C. (2010) A global overview of biotech (GM) crops: adoption, impact and future prospects. GM Crops, 1, 8–12.

..............................................................................................

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

Jones, P.L. & De Silva, S.S. (1997) Apparent nutrient digestibility of formulated diets by the Australian freshwater crayfish Cherax destructor Clark (Decapoda, Parastacidae). Aquacult. Res., 28, 881–891. Kl€ uttgen, B., D€ ulmer, U., Engels, M. & Ratte, H.T. (1994) ADaM, an artificial freshwater for the culture of zooplankton. Water Res., 28, 743–746. Kumaraguru, V.K.P., Ramesh, S. & Balasubramanian, T. (2005) Dietary value of different vegetable oil in black tiger shrimp Penaeus monodon in the presence and absence of soy lecithin supplementation: effect on growth, nutrient digestibility and body composition. Aquaculture, 250, 317–327. Kuminoff, N.V. & Wossink, A. (2010) Why isn’t more US farmland organic? J. Agr. Econ., 61, 240–258. Lemos, D., Navarrete del Toro, A., C! ordova-Murueta, J.H. & Garcia-Carre~ no, F. (2004) Testing feeds and feed ingredients for juvenile pink shrimp Farfantepenaeus paulensis: in vitro determination of protein digestibility and proteinase inhibition. Aquaculture, 239, 307–321. L€ ovei, G.L. & Arpaia, S. (2005) The impact of transgenic plants on natural enemies: a critical review of laboratory studies. Entomol. Exp. Appl., 114, 1–14. L€ ovei, G.L., Andow, D.A. & Arpaia, S. (2009) Transgenic insecticidal crops and natural enemies: a detailed review of laboratory studies. Environ. Entomol., 38, 293–306. Lundry, D.R., Ridley, W.P., Meyer, J.J., Riordan, S.G., Nemeth, M.A., Trujillo, W.A., Breeze, M.L. & Sorbet, R. (2008) Composition of grain, forage, and processed fractions from second-generation glyphosate-tolerant soybean, MON 89788 is equivalent to that of conventional soybean (Glycine max L.). J. Agric. Food Chem., 56, 4611–4622. Malatesta, M., Caporaloni, C., Gavaudan, S., Rocchi, M.B.L., Serafini, S., Tiberi, C. & Gazzanelli, G. (2002) Ultrastructural morphometrical and immunocytochemical analyses of hepatocyte nuclei from mice fed on genetically modified soybean. Cell Struct. Funct., 27, 173–180. Malatesta, M., Perdoni, F., Santin, G., Battistelli, S., Muller, S. & Biggiogera, M. (2008a) Hepatoma tissue culture (HTC) cells as a model for investigating the effects of low concentrations of herbicide on cell structure and function. Toxicol. In Vitro, 22, 1853– 1860. Malatesta, M., Boraldi, F., Annovi, G., Baldelli, B., Battistelli, S., Biggiogera, M. & Quaglino, D. (2008b) A long-term study on female mice fed on a genetically modified soybean: effects on liver ageing. Histochem. Cell Biol., 130, 967–977. McBride, W.D. & Greene, C. (2009) The profitability of organic soybean production. Renewable Agric. Food Syst., 24, 276–284. McCann, M.C., Liu, K., Trujilo, W.A. & Dobert, R.C. (2005) Glyphosate-tolerant soybeans remain compositionally equivalent to conventional soybeans (Glycine max L.) during three years of field testing. J. Agric. Food Chem., 53, 5331–5335. Millstone, E., Brunner, E. & Mayer, S. (1999) Beyond substantial equivalence. Nature, 401, 525–526. OECD (2001) Consensus Document on Compositional Considerations for New Varieties of Soybean: Key Food and Feed Nutrients and Antinutrients. OECD Environmental Health and Safety Publications Series on the Safety of Novel Foods and Feeds, no. 2. Environment Directorate, Organisation for Economic Cooperation and Development, Paris. OECD (2008) Daphnia magna Reproduction Test. OECD Guidelines for the Testing of Chemicals, no. 211. Organisation for Economic Co-operation and Development, Paris.

..............................................................................................

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

Olsen, R.L. & Hasan, M.R. (2012) A limited supply of fishmeal: impact on future increases in global aquaculture production. Trends Food Sci. Tech., 27, 120–128. Place, G.M., Reberg-Horton, S.C., Dunphy, J.E. & Smith, A.N. (2009) Seeding rate effects on weed control and yield for organic soybean production. Weed Technol., 23, 497–502. Podevin, N. & du Jardin, P. (2012) Possible consequences of the overlap between the CaMV 35S promoter regions in plant transformation vectors used and the viral gene VI in transgenic plants. GM Crops Food, 3, 296–300. Pryme, I.F. & Lembcke, R. (2003) In vivo studies of possible health consequences of genetically modified food and feed – with particular regard to ingredients consisting of genetically modified plant materials. Nutr. Health, 17, 1–8. Refstie, S., Korstøen, Ø.J., Stortebakken, T., Baeverfjord, G., Lein, I. & Roem, A.J. (2000) Differing nutritional response to dietary soybean meal in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Aquaculture, 190, 49–63. Rosi-Marshall, E.J., Tank, T., Royer, V., Whiles, M.R., EvansWhite, M., Chambers, C., Griffiths, N.A., Pokelsek, J. & Stephen, M.L. (2007) Toxins in transgenic crop byproducts may affect headwater stream ecosystems. Proc. Natl Acad. Sci. U S A, 104, 16204–16208. # Bakke-McKellep, A.M., Sagstad, A., Sanden, M., Krogdahl, A., Frøystad, M. & Hemre, G.I. (2008) Organs development, gene expression and health of Atlantic salmon (Salmo salar L.) fed genetically modified soybeans compared to the near-isogenic non-modified parental line. Aquacult. Nutr., 14, 556–572. Sissener, N.H., Bakke, A.M., Gu, J., Penn, M.H., Eie, E., Krogdahl, A., Sanden, M. & Hemre, G.I. (2009a) An assessment of organ and intestinal histomorphology and cellular stress response in Atlantic salmon (Salmo salar L.) fed genetically modified Roundup Ready (R) soy. Aquaculture, 298, 101–110. Sissener, N.H., Sanden, M., Bakke, A.M., Krogdahl, A. & Hemre, G.I. (2009b) A long term trial with Atlantic salmon (Salmo salar L.) fed genetically modified soy; focusing general health and performance before, during and after the parr-smolt transformation. Aquaculture, 294, 108–117. # Bakke, A.M., # Sissener, N.H., Sanden, M., Krogdahl, A., Johannessen, L.M. & Hemre, G.I. (2011) Genetically modified plants as fish feed ingredients. Can. J. Fish. Aquat. Sci., 68, 563–574. Smith-Spangler, C., Brandeau, M.L., Hunter, G.E. et al. (2012) Are organic foods safer or healthier than conventional alternatives? A systematic review. Ann. Intern. Med., 157, 348–366. Taylor, N.B., Fuchs, R.L., MacDonald, J., Shariff, A.R. & Padgette, S.R. (1999) Compositional analysis of glyphosate-tolerant soybeans treated with glyphosate. J. Agric. Food Chem., 47, 4469–4473. Trigo, E., Cap, E., Malach, V. & Villareal, F. (2009) The case of zero-tillage technology in Argentina. IFPRI Discussion Paper 00915, International Food Policy Research Institute, Washington, DC, USA. Viljoen, C. (2013) Letter to the editor. Food Chem. Toxicol., 59, 809–810. Wu, W., Hettiarachchy, N.S., Kalapathy, U. & Williams, W.P. (1999) Functional properties and nutritional quality of alkaliand heat-treated soy protein isolate. J. Food Quality, 22, 119– 133. Yang, Y., Wang, Y., Lu, Y. & Li, Q. (2011) Effect of replacing fish meal with soybean meal on growth, feed utilization and nitrogen and phosphorus excretion on rainbow trout (Oncorhynchus mykiss). Aquacult. Int., 19, 405–419.

Zhou, J., Harrigan, G.G., Berman, K.H., Webb, E.G., Klusmeyer, T.H. & Nemeth, M.A. (2011) Stability in the composition equivalence of grain from insect-protected maize and seed from glyphosate-tolerant soybean to conventional counterparts over multiple seasons, locations, and breeding germplasms. J. Agr. Food Chem., 59, 8822–8828. Zhu, Y., Li, D., Wang, F., Yin, J. & Jin, H. (2004) Nutritional assessment and fate of DNA of soybean meal from roundup ready or conventional soybeans using rats. Arch. Anim. Nutr., 58, 295–310. Zobiole, L.H.S., Oliveira, R.S. Jr, Kremer, R.J., Constantin, J., Yamada, T., Castro, C., Oliveira, F.A. & Oliveira, A. Jr (2010a)

Effect of glyphosate on symbiotic N2 fixation and nickel concentration in glyphosate-resistant soybeans. Appl. Soil Ecol., 44, 176–180. Zobiole, L.H.S., Oliveira, R.S., Visentainer, J.V., Kremer, R.J., Bellaloui, N. & Yamada, T. (2010b) Glyphosate affects seed composition in glyphosate-resistant soybean. J. Agric. Food Chem., 58, 4517–4522. Zobiole, L.H.S., Kremer, R.J., Oliveira, R.S. Jr & Constantin, J. (2011) Glyphosate affects chlorophyll, nodulation and nutrient accumulation of “second generation” glyphosate-resistant soybean (Glycine max L.). Pestic. Biochem. Phys., 99, 53–60.

..............................................................................................

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

   

Paper  IV:  

Feeding  study  II.      

  Title:      

Glyphosate-­‐Residues  in  Roundup-­‐Ready  Soybean     Impair  Daphnia  magna  Life-­‐Cycle.    

Authors:    

Marek  Cuhra,  Mickaël  Dando,  Terje  Traavik,  Raul  Primicerio,  Daniel   Holderbaum,  Thomas  Bøhn.  

Journal:    

Journal  of  Agricultural  Chemistry  and  Environment,  4,  24-­‐36  (2015).  

43  

44  

Journal of Agricultural Chemistry and Environment, 2015, 4, 24-36 Published Online February 2015 in SciRes. http://www.scirp.org/journal/jacen http://dx.doi.org/10.4236/jacen.2015.41003

Glyphosate-Residues in Roundup-Ready Soybean Impair Daphnia magna Life-Cycle Marek Cuhra1*, Terje Traavik1,2, Mickaël Dando1,2, Raul Primicerio3, Daniel Ferreira Holderbaum4, Thomas Bøhn1,2 1

Faculty of Health Sciences, The Arctic University of Norway, Tromsø, Norway GenØk—Centre for Biosafety, The Science Park, Tromsø, Norway 3 Faculty of Biosciences, Fisheries and Economics, The Arctic University of Norway, Tromsø, Norway 4 Graduate Program in Plant Genetic Resources, Federal University of Santa Catarina (UFSC), Florianópolis, Brazil Email: *[email protected] 2

Received 5 January 2015; accepted 24 January 2015; published 30 January 2015 Copyright © 2015 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

Abstract Herbicide tolerant plants such as Roundup-Ready soybean contain residues of glyphosate herbicide. These residues are considered safe and previous animal-feeding-studies have failed to find negative effects related to such chemical residues. The present study tests 8 experimental soymeal diets as feed in groups (each containing 20 individuals) of test-animals (D. magna). The diets have different levels of glyphosate residues and we show that animal growth, reproductive maturity and number of offspring are correlated with these chemicals. The tested soybeans are from ordinary agriculture in Iowa USA and the residues are below the regulatory limits. Despite this, clear negative effects are seen in life-long feeding. The work enhances the need for including analysis of herbicide residues in future assessment of GMO.

Keywords Transgenic GTS 40-3-2 Roundup-Ready Soybean, Glyphosate Residues, Life-Long Animal Feeding Study, GMO Risk-Assessment, Herbicide-Tolerant Cultivar Quality, Aquatic Invertebrate Ecotoxicology

1. Introduction Transgenic glyphosate-tolerant soy (Roundup-Ready soybean = RR-soybean) is the most commonly cultivated genetically modified crop, contributing approximately 80% of global annual soy production of 283 million metric *

Corresponding author.

How to cite this paper: Cuhra, M., Traavik, T., Dando, M., Primicerio, R., Holderbaum, D.F. and Bøhn, T. (2015) GlyphosateResidues in Roundup-Ready Soybean Impair Daphnia magna Life-Cycle. Journal of Agricultural Chemistry and Environment, 4, 24-36. http://dx.doi.org/10.4236/jacen.2015.41003

M. Cuhra et al.

tons in the 2013/14 growing season [1]. Previous studies found compositional differences between organic, conventional and RR-soybean and demonstrated that these differences were correlated with cultivating regime, notably the use of pesticides and resulting residue levels in plant material [2]. Subsequent feeding studies with the same soy materials demonstrated that D. magna vital parameters survival, growth and fecundity were significantly affected by soy type, with animals fed diets made from genetically modified soybean demonstrating significantly reduced fitness. The observed differences were attributed to differences in soybean composition, notably content of glyphosate residues [3]. Ecotoxicological testing of Roundup herbicide and its glyphosate active ingredient indicated that D. magna sensitivity to these toxins is considerably higher than previously reported. Cuhra et al. [4] found that concentrations of 1.35 - 4.05 mg/l of glyphosate or Roundup herbicide in aqueous medium, negatively affected survival, and 0.05 - 0.45 mg/l reduced growth, reproduction and offspring birth size. Through their systemic mode of action, glyphosate-herbicides such as Roundup enter plant tissue and affect vegetative parts of plants by interfering with important metabolic pathways [5]. Transgenic crops such as GTS40-3-2 RR-soybean have been modified to have alternative and non-susceptible metabolic pathways and thus to withstand the toxic action. However, such modified plants are found to accumulate glyphosate residues from applied glyphosate-herbicide formulations [2] [6]. In this study we define “glyphosate residues” as the sum of glyphosate and its main degradation product aminomethyl-phosphonic acid (AMPA). Regrettably, information concerning glyphosate-herbicide residues in soybean is sparse [6] [7], but recent studies have disclosed surprisingly high levels of glyphosate residues in RR-soybean (Figure 1), even in excess of regulatory guidelines limits [8]. Furthermore, it was found that the presence of glyphosate residues was significantly correlated with distinct differences in nutritional components of soybean, indicating a possible influence of such residues on soy nutritional constituents [2]. Glyphosate residues in glyphosate-tolerant cultivars are found to impact plant metabolism affecting grain composition [9]-[11]. Globally, protein-meal and oil from soybean (Glycine max) have become important ingredients in feed formulations for domestic mammals and are increasingly used as an affordable substitute for exhausted and costly traditional ingredients such as marine proteins and lipids for formulated feeds in aquaculture farming of fish such as Atlantic salmon Salmo salar [12] [13], rainbow trout Oncorhynchus mykiss [14] [15], and in aquaculture of crustaceans, such as crayfish [16] [17], prawn and shrimp [18]. Accordingly, the consequences of glyphosate accumulation in RR-soybean and the influence of such residues on in-plantae metabolic processes and soybean quality should be further investigated. In the present study we fed 8 defined experimental diets to D. magna in order to reveal possible impacts of soybean glyphosate. The diets were produced from individual harvests of RR-soy, representing a range of glyphosate residues, from 1.1 mg/kg up to 15.1 mg/kg in the highest concentration, well below current USA maximum-residue-limits (MRLs) of 40 mg/kg.

Experimental Hypothesis H0: Soybean-meal diets with varying content of glyphosate residues will not induce differences in measurable end-points for life-cycle traits survival, growth and reproduction in D. magna life-long feeding studies.

2. Results 2.1. Survival Three out of the four groups of D. magna with the highest mortality were the groups fed soy with the highest glyphosate residues (Figure 1). And the group with lowest mortality (exempting the control group) was the group fed soy with the lowest residue level of glyphosate (Figure 2). However, there was not a full consistency in the results to support a simple dose-response relationship between glyphosate residues and mortality. Mortality was significantly higher in the groups fed soy with high glyphosate (13.6 - 15.1 mg/kg) residues as compared to medium (9.1 - 10.7 mg/kg) or low (1.1 and 2.5 mg/kg) levels (p = 0.0066 and 0.0068 respectively, Cox-PH test). No difference was found between animals fed soy that contained low and medium levels of glyphosate (p = 0.75).

2.2. Growth Measurement of carapace length of D. magna at days 12, 21, 30 and 42 showed that growth was negatively affected by levels of glyphosate residues at the ages of 21 and 30 days (p = 0.02, R2 = 4.5% and p = 0.031, R2 =

25

M. Cuhra et al.

Figure 1. Soy glyphosate and AMPA levels (mg/kg) in relation to relevant regulatory limit (MRL). Data from [2].

Figure 2. D. magna survival rates in relation to levels of glyphosate/AMPA residues.

26

M. Cuhra et al.

5.3%, respectively) (Figure 3). At ages 12 and 42 days no significant correlation was found between body size and residue content of the diet. By day 42, the few surviving animals displayed a high variation in body size, visibly influencing the width of the confidence bands.

2.3. Reproduction Age at maturity increased with the glyphosate residues in the soy feed (p = 0.005, R2 = 5.3%) (Figure 4). Cumulative fecundity of D. magna was significantly, negatively correlated with the herbicide residue level at the ages of 6 days and 9 days (p = 0.006, R2 = 4.2%, and p = 0.019, R2 = 2.0%, Pearson-correlation, respectively). However, analyses of data on fecundity at ages 12, 15, 18, 21, 27, 30, 33 and 36 days showed that the negative effect on fecundity disappeared over time. At the age of 42 days there was a significant positive correlation (p = 0.019, R2 = 17.5%), based on small sample sizes due to high mortality (Figure 5). Analysis of abortion rates (Figure S1) showed no correlation with feed glyphosate residue content (p = 0.407). The total cumulated fecundity for the whole life-span (Figure S2) was negatively correlated with the level of glyphosate residues in feed (p = 0.024, R2 = 3.4%, Pearson correlation).

Figure 3. Body length (length of carapace) measured at age 12, 21, 30 and 42 days for D. magna fed experimental diets with varying levels of glyphosate residues (Error-bands: 95% CI).

27

M. Cuhra et al.

Figure 4. Mean age at maturation (time of first live birth) of D. magna fed experimental diets with varying levels of glyphosate residues (Error bars: 95% CI).

3. Discussion The life-cycle feeding studies with Roundup-Ready soybean (RR-soybean) in D. magna demonstrated several interesting findings: high levels of glyphosate residues in the soybean feed tested correlated with 1) higher mortality, 2) reduced growth and fecundity in some parts of the life-cycle, 3) delayed reproduction, and a reduced total number of offspring. None of the statistical associations were particularly strong with R2 (variation explained) typically below 5% - 6%. However, the consistent and significantly negative effect of glyphosate on a range of life-history traits can arguably be used as a proof of concept that glyphosate residues in Roundup Ready soybean have adverse biological effects at “normally occurring levels” (i.e. as found in “ready to market” soybeans) in the investigated dominant feed-ingredient. These results accentuate the demand for further studies on potential effects of herbicide residues present in genetically modified herbicide tolerant crops such as RR-soybean. Whether the observed negative impacts are due to, e.g., low-grade toxicity, endocrine disruption or antinutritional effects, can only be clarified by further well-designed feeding experiments. Importantly, life-cycle ecotoxicology testing of Roundup herbicide and glyphosate in living-environment of D. magna [4] has demonstrated reduced survival, growth and fecundity attributable to low environmental levels of these chemicals. It is thus not unreasonable to relate the effects we see in animals given progressive concentrations of these chemicals in feed, to effects observed from progressive concentrations of the same chemicals in the aqueous living environment. Despite nearly two decades of commercial cultivation and use, environmental impact of and health risk assessment of transgenic plants is a contested issue [19]-[21]. Also the interpretation of published evidence for risk assessment is contested [22]. It is thus appropriate to discuss how initial studies by chemical industry and biotech industry found transgenic RR-soybean as nutritious and safe as conventional soybean varieties. Compositional analyses of RR-soybean [23] led to the conclusion that the composition was essentially similar (substantially equivalent) to unmodified soy. Feeding studies [24] gave no indications of negative health impacts on rat, chicken, catfish or cattle. However, subsequent reviews by independent scientists highlighted that these initial studies were conducted with soybean material produced under non-representative agricultural conditions, since the tested Roundup-Ready-soy was not exposed to Roundup in the field [25]. Following feeding studies with RR-soybean were performed in swine [26] and mouse [27]. In addition, several composition studies assessing substantial equivalence of nutrient profiles [28] [29] were conducted. It was reported that the plants used in these studies had been exposed to the prescribed amounts of Roundup. Regrettably, measurement of residue levels was not provided in these studies. Recently Viljoen [30] reported that evidence in risk-assessment of RRcrops is still based on analysis of biological material which has not been subjected to application of glyphosate and thus can not be accepted as relevant safety assurance representative for the material actually entering feed production and human food.

28

M. Cuhra et al.

Figure 5. Fecundity (cumulated live births) of surviving D. magna fed experimental diets with varying levels of glyphosate residues, at ages 9, 12, 15, 27, 39 and 42 days (Error bands: 95% CI).

29

M. Cuhra et al.

Independent researchers attempting to replicate industry studies have experienced various difficulties, mainly represented by prohibitive formalities enforced through patent laws and intellectual property rights which limit access to research materials [31]. Despite such difficulties, several independent researchers have obtained transgenic plant material, such as RR-soybean, and assessed its quality in compositional studies and animal feeding trials. Mice fed RR-soybean demonstrated significant morphological changes in liver cells [32]. The data suggested that RR-soybean intake was influencing liver cell nuclear features in both young and adult mice, but the mechanisms responsible for the alterations could not be identified by the experimental design of these studies. Malatesta and co-workers showed that mice fed RR-soybean developed altered mitochondrial functions and transcription pathways in hepatocytes [32] [33] and confirmed similar responses in hepatome-derived cell cultures by exposing them to low concentrations of Roundup [34]. Even without measurements of the herbicide residues levels, this work indicates that Roundup may have adverse toxicological effects. Zhu et al. [35] compared nutritional qualities of meal from conventional and RR-soybean in a 13-week sub-chronic rat feeding experiment with high inclusion rates (up to 90% soy). Despite initial significant reduced feed intake and growth in the RR-soybean group (during the first week of experiment), no subsequent significant differences in feed intake, body weight nor mortality was seen. Neither post mortem histology nor haematological and urinary analyses showed significant effects attributable to feed type. As RR-soybean now accounts for the majority of globally produced soybean, concern has been raised in relation to its increasing use in aquaculture feed. Several studies have tested effects of soybean as ingredients in aquaculture feed formulations. Sagstad et al. [13] compared organ development, metabolic markers and general health indicators of Atlantic salmon fed diets with RR-soybean to fish fed non-modified soy of a near-isogenic parental line. The results showed significant differences in investigated end-points between groups. Fish having been fed RR-soybean diets had significantly increased feed conversion ratios, significantly decreased protein efficiency ratio, significantly decreased apparent digestibility-coefficients of lipids and dry matter, and significantly decreased plasma triacylglycerol levels. These indications of functional and metabolic changes in fish liver, spleen and intestines were attributable to soy type (RR-soy versus unmodified isoline) and thus could be related to possible residues of glyphosate herbicide or other characteristics of RR-soybean. Later investigations provided new data that were inconclusive [36]. However, neither presence nor quantity of glyphosate residues was investigated. Bakke-Mckellep et al. [37] detected somewhat higher kidney lysozyme and acid phosphatase levels in Atlantic salmon after feeding RR-soybeans, but were not able to conclude whether this was caused by the genetic modification or other factors representing soy-cultivar difference. The authors did not mention herbicide residue levels. Other long-term feeding studies in Atlantic salmon have shown minor differences between RR-soybean and a near-isogenic conventional soybean, but not related to the genetic modification per se [38] [39]. Both these studies used soy material supplied by biotech industry and content of glyphosate residues were not analyzed. Previous experimental protocols for testing transgenic (GM) food and feed in D. magna [40] [41] have been criticized for 1) high inclusion rates of plant material [42] [43] and 2) longer duration than standard OECD D. magna 21-day reproduction testing [42]. These are important aspects to discuss. EFSA guidelines for animal feeding studies in nutritional and safety assessment of GM plants and derived feed specify that; “When testing whole foods, it is desirable to obtain the highest concentration possible of the GM food and feed in laboratory animal diet without causing nutritional imbalance” [44]. Thus, in order to investigate potential quality differences in the soybean meal, the present study was designed with multiple treatment groups, to combine the rationale for obtaining the highest possible concentration of the feed under testing, while keeping a balanced nutritional composition (i.e. standard green algae diet in addition to soy). We chose to use experimental diets consisting of 80% biomass (organic carbon) from soymeal and 20% biomass from unicellular green algae. The study is thus not representative of what consumer organisms would receive from commercially produced feed. Normal inclusion rates for soybean-meal are 15% - 40% in terrestrial farm animal feed [45] and 20% - 30% in aquaculture feed for species such as salmon and omnivorous fish [46] [47]. However, experimental protocols have been developed and validated for non-target organisms like D. magna for testing plant quality as food over the full life-cycle with 100% of the diet being the tested material [42] [43]. Herbivore species that live in proximity to monoculture fields will be exposed to large quantities/a high proportion of that crop, both in the terrestrial and in the soil ecosystem. Most testing protocols for non-target organisms feeding on transgenic material use 100% of the relevant material for testing (see e.g. reviews [48] [49]). Also in the aquatic run-off system, where other non-target biodiversity is found, including Daphnia spp., close to 100% of the incoming (allochthonous) plant material may come from crop residuals, debris and by-products [50]. Furthermore, most published

30

M. Cuhra et al.

feeding studies represent short-term exposure, compared to the life expectancy of the test organisms. However, the intended use of plant ingredients in food and feed for animals, including humans, impose lifetime chronic exposure for the consumer organism. Life-long feeding studies are time-consuming, expensive and hence rare. The first life-long study of a genetically modified plant and its co-technology herbicide in a rodent species tested the cp4 epsps-transgenic NK 603 maize (RR-maize) and Roundup during two years of continuous feeding in rats ([51], retracted and republished). The study indicated that rats fed RR-maize (with and without the application of Roundup), or given Roundup separately in the drinking water, developed problems in detoxification organs and developed significantly changed blood parameters. The retracted study by Seralini et al. highlights the role and relevance of life-cycle studies for herbicide tolerant GM crops, despite high costs. This has also been recognized by the European Commission and EFSA that agree to the need for two-year GMO feeding studies [52]. Our study in D. magna also represents a life-cycle study, but lasting 42 days only and with much lower costs than a two-year study in mammals. Although vertebrates have numerous responses that are unique to those organisms, e.g. due to their immune systems, we argue that many qualitative aspects of toxicity and nutrition can be tested with valuable information obtained in D. magna or similar invertebrate model organisms with relatively short life span and short generation times. The revised EFSA guidelines for animal feeding studies in risk assessments also specify that it is not enough to only assess the added qualities stemming from expression of the inserted gene in a GM-plant, but also investigate possible effects of the genetic modification itself [53]. We argue that feeding studies with herbicide tolerant genetically modified plants also have to deal with and, ideally, should have the ability to differentiate effects stemming from the plant and those derived from the herbicides. Due to increasing occurrence of glyphosate-resistant weeds in agricultural systems, particularly in fields of glyphosate tolerant RR-crops [20], functionality of cultivars such as GTS 40-3-2 RR-soybean has decreased. To combat resistant weeds new varieties of genetically modified soybeans are developed in order to 1) have higher tolerance to herbicides such as glyphosate and 2) be tolerant to additional active herbicide ingredients such as glufosinate-ammonium, dicamba and 2,4-D [54]. Even glyphosate resistant weeds can be eradicated with glyphosate (Roundup) alone, if the dosage is sufficiently high. New varieties of glyphosate-tolerant soybean, such as MON 89788 are designed to provide tolerance to higher doses than first-generation GTS 40-3-2 soybean and may thus allow for intensive eradication of semi-resistant weeds. This may lead to increased application rates and thus also increased residue levels in the end products. To our knowledge, no previous feeding studies have addressed and tested dose-response relationship in plant samples with different amounts of pesticide residues. This becomes more and more relevant as the regulatory systems allow progressively higher maximum residue levels of glyphosate and other herbicides present in biomass produced from herbicide-tolerant crops.

4. Materials and Methods 4.1. Materials The tested soybeans were grown on a number of agricultural farm fields in the State of Iowa, USA. All samples were from agriculture of glyphosate tolerant (Roundup-Ready) soybean. Soy material for testing was obtained from bags of 3kg samples of whole beans, representing harvests from 8 individual fields. Seed type, farming history including pesticide use and nutritional composition was known for all samples [2]. All fields were treated with normal application rates of either Roundup glyphosate herbicide (Monsanto) or Touchdown glyphosate herbicide (Syngenta). D. magna mother-populations were reared for several generations in fully synthetic Elendt M7 artificial lake water [55] fed diets of Selenastrum sp. unicellular algae. Test animals consisted of juvenile D. magna less than 24 hrs old, taken from mother population 3 - 5 clutch.

4.2. Methods The feeding experiment setup included 8 individual feed-types, with 20 animals in each feeding group, plus an integrated control group fed only algae. Soy feed was given in a Daphnia magna long-term reproduction study for a duration of 42 days, which covers the full life-cycle of the animal (life expectancy is 30 - 45 days). The experiment was conducted in the laboratories of GenØk-Centre for Biosafety, Tromsø, Norway. Upon arrival in the laboratory, soybean samples were cleaned manually, i.e. impurities and other plant material were removed. Standardized subsamples for analysis and feeding tests were prepared by homogenization after weighing and

31

M. Cuhra et al.

grinding. Material was kept cool during grinding, to avoid degradation of essential components. Ground soy samples were analyzed for pesticide residues at Eurofins laboratories GfA, Otto-Hahn-Str. 22, D-48161 Münster (Germany). Details on soy-meal composition including multivariate analyses are presented in Bøhn et al., 2014. The raw feed from Roundup Ready GM soy contained both glyphosate and the degradation product aminomethylphosphonic acid (AMPA) (Figure 1). Soy diets for the main experiments were produced from finely ground and dried soy-bean meal, homogenized and subsequently filtered through 200 μm Retsch metal filters. Subsamples of 0.800 g (±0.5 mg) were taken from each category, each homogenized into 1000 ml ultraclean screw-cork flasks containing 200.0 ml of ultrapure water by manual shaking for 60 seconds, settling and subsequent 5 minute shaking. Flasks with the feed solutions were blinded, and individual 8 ml aliquots were prepared for freezing in colour-coded 14 ml PP tubes (Falcon). Feed was kept frozen at −18 C until use. Daily feeding dose of 100 μl containing 0.2 mg organic carbon soy biomass (80% of feed), was precisely administered using micropipette combined with supplementary 0.05 mg C/d as Selenastrum sp. algal feed in all diets (20% of feed). 160 juvenile D. magna test animals were reared and kept as individual experimental units in 100 ml glasses for the duration of the experiment. 13 animals fed only standard laboratory diets of 0.15 mg C/d as Selenastrum algae feed were integrated as control. At day 21 of the experimental period, average fecundity of the 12 surviving individuals in this control was 79 (SE = 5). Holding medium for experiment was Elendt-M7 artificial lake water [55]. Physical parameters temperature, dissolved oxygen, conductivity and pH were measured regularly. The feed identity was blinded in the experiment and allocation of animals to feed treatment and position of experimental units on lab bench were randomized. Experimental setup was on 6 trays each having 6 treatments with 5 experimental units consisting of colour coded and numbered individual glasses of each treatment, in laboratory environment. Feeding and registration of mortality, reproductive maturity and fecundity endpoints were performed on a daily basis for the 42-day duration of the experiment. Juveniles were quantified and removed every 3 days. On days 12, 21, 30 and 42 all surviving animals were photographed through a Leitz fixed magnification loupe fitted with a Nikon D300 high-resolution digital camera, for subsequent measurement of carapace length (anterior extreme of head-shield to base of caudal spine) according to standardized procedure using Wayne-Rasband Image-J software [56] [57] calibrated to an Agar-L4078 scale. The holding-medium was renewed every 3d day. Experimental units were randomly re-positioned following medium renewal. Survival, growth, age at maturation, fecundity and abortion rate, were compared across treatments and correlated to feed characteristic pesticide content, defined as total glyphosate residue levels calculated on the basis of analytic results for glyphosate + aminomethylphosphonic acid (AMPA). The performance of the control group (exclusively fed algae diet) validated overall quality of experimental conditions. Collected data were analyzed with the SPSS and R statistical softwares. Mortality of individual animals in different feeding groups was analyzed by Cox Proportional Hazard (Cox-PH-test). The main measured outcomes with respect to growth, age at maturity and fecundity (number of living young as well as abortions) were analyzed in relation to magnitude of glyphosate residues by Pearson correlation analysis.

5. Conclusion Published studies for regulatory assessment of herbicide tolerant crops largely lack information on herbicide residue presence. This is a serious shortcoming on a key factor that may impair the quality of such crops. We show that glyphosate residues in “ready to market” RR-soybean affected D. magna life-history traits negatively in a dose-response model. This indicates that “normally occurring levels” of glyphosate residues in soy-meal feed can have negative biological effects. The investigated residue levels are well within current MRLs of 40 mg/kg for soybean in USA. The tested RR-soybean material was harvested from commercial farm fields and is representative of the soybean biomass which is dominantly used for feed purposes world-wide. Our results warrant further research related to potential health consequences in consumer organisms. Pesticide residues in food and feed produced from herbicide-tolerant plants needs to be routinely monitored and we urge for further testing of potential effects on farmed animals at realistic chronic exposure rates. Finally, we recommend revisions of guidelines for risk-assessment of genetically modified herbicide tolerant cultivars and for animal feeding-trials to specifically address herbicide residues in food and feed.

References [1]

USDA (2013) United States Department of Agriculture. Foreign Agricultural Service, Table 07: Soybeans: World

32

Supply and Distribution. (Online Statistics) www.fas.usda.gov/psdonline

M. Cuhra et al.

[2]

Bøhn, T., et al. (2014) Compositional Differences in Soybeans on the Market: Glyphosate Accumulates in Roundup Ready GM Soybeans. Food Chemistry, 153, 207-215. http://dx.doi.org/10.1016/j.foodchem.2013.12.054

[3]

Cuhra, M., Traavik, T. and Bøhn, T. (2014) Growth and Fecundity of Crustacean D. magna in Life-Cycle Feeding with Roundup Ready, Conventional or Organic Soybean (accepted 7. March 2014: Aquaculture Nutrition).

[4]

Cuhra, M., Traavik, T. and Bøhn, T. (2013) Clone- and Age-Dependent Toxicity of a Glyphosate Commercial Formulation and Its Active Ingredient in Daphnia magna. Ecotocicology, 22, 251-262. http://dx.doi.org/10.1007/s10646-012-1021-1

[5]

Amrhein, N., Schab, J. and Steinrücken, H.C. (1980) The Mode of Action of the Herbicide Glyphosate. Naturwissenschaften, 67, 356-357. http://dx.doi.org/10.1007/BF01106593

[6]

Duke, S.O., Rimando, A., Pace, P.F., Reddy, K.N. and Smeda, R.J. (2003) Isoflavone, Glyphosate, and Aminomethylphosphonic Acid Levels in Seeds of Glyphosate-Treated, Glyphosate-Resistant Soybean. Journal of Agricultural and Food Chemistry, 51, 340-344. http://dx.doi.org/10.1021/jf025908i

[7]

Arregui, M.C., et al. (2004) Monitoring Glyphosate Residues in Transgenic Glyphosate-Resistant Soybean. Pest Management Science, 60, 163-166. http://dx.doi.org/10.1002/ps.775

[8]

Then, C. (2013) High Levels of Residues from Spraying with Glyphosate Found in Soybeans in Argentina. Testbiotech Institute for Independent Impact Assessment in Biotechnology Report. http://www.testbiotech.de/en/node/926

[9]

Zobiole, L.H.S., et al. (2010) Effect of Glyphosate on Symbiotic N2 Fixation and Nickel Concentration in Glyphosate-Resistant Soybeans. Applied Soil Ecology, 44, 176-180. http://dx.doi.org/10.1016/j.apsoil.2009.12.003

[10] Zobiole, L.H.S., et al. (2010) Glyphosate Affects Seed Composition in Glyphosate-Resistant Soybean. Journal of Agricultural and Food Chemistry, 58, 4517-4522. http://dx.doi.org/10.1021/jf904342t [11] Zobiole, L.H.S., Kremer, R.J., Oliveira Jr., R.S. and Constantin, J. (2011) Glyphosate Affects Chlorophyll, Nodulation and Nutrient Accumulateion of “Second Generation” Glyphosate-Resistant Soybean (Glycine max L.). Pesticide Biochemistry and Physiology, 99, 53-60. http://dx.doi.org/10.1016/j.pestbp.2010.10.005 [12] Gillund, F. and Myhr, A.I. (2010) Perspectives on Salmon Feed: A Deliberative Assessment of Several Alternative Feed Resources. Journal of Agricultural and Environmental Ethics, 23, 527-550. http://dx.doi.org/10.1007/s10806-010-9237-7 [13] Sagstad, A., Sanden, M., Krogdahl, Å., Bakke-McKellep, A.M., Frøystad, M. and Hemre, G.-I. (2008) Organs Development, Gene Expression and Health of Atlantic Salmon (Salmo salar L.) Fed Genetically Modified Soybeans Compared to the Near-Isogenic Non-Modified Parental Line. Aquaculture Nutrition, 14, 556-572. http://dx.doi.org/10.1111/j.1365-2095.2008.00630.x [14] Refstie, S., Korsøen, Ø.J., Storebakken, T., Baeverfjord, G., Lein, I., et al. (2000) Differing Nutritional Response to Dietary Soybean Meal in Rainbow Trout (Oncorhynchus mykiss) and Atlantic Salmon (Salmo salar). Aquaculture, 190, 49-63. http://dx.doi.org/10.1016/S0044-8486(00)00382-3 [15] Yang, Y., Wang, Y., Lu, Y. and Li, Q.-Z. (2011) Effect of Replacing Fish Meal with Soybean Meal on Growth, Feed Utilization and Nitrogen and Phosphorus Excretion on Rainbow Trout (Oncorhynchus mykiss). Aquaculture International, 19, 405-419. http://dx.doi.org/10.1007/s10499-010-9359-y [16] Cuzon, G., Guillaume, J. and Cahu, C. (1994) Composition, Preparation and Utilization of Feeds for Crustacea. Aquaculture, 124, 253-267. http://dx.doi.org/10.1016/0044-8486(94)90387-5 [17] Jones, P.L. and De Silva, S.S. (1997) Apparent Nutrient Digestibility of Formulated Diets by the Australian Freshwater Crayfish Cherax destructor Clark (Decapoda, Parastacidae). Aquaculture Research, 28, 881-891. [18] Kumaraguru Vasagam, K.P., Ramesh, S. and Balasubramanian, T. (2005) Dietary Value of Different Vegetable Oil in Black Tiger Shrimp Penaeus monodon in the Presence and Absence of Soy Lecithin Supplementation: Effect on Growth, Nutrient Digestibility and Body Composition. Aquaculture, 250, 317-327. http://dx.doi.org/10.1016/j.aquaculture.2005.02.035 [19] Antoniou, M., Robinson, C. and Fagan, J. (2012) Teratogenic Effects of Glyphosate-Based Herbicides: Divergence of Regulatory Decisions from Scientific Evidence. Journal of Environmental and Analytical Toxicology, 4, 006. [20] Benbrook, C.M. (2012) Impacts of Genetically Engineered Crops on Pesticide Use in the U.S.—The First Sixteen Years. Environmental Science Europe, 24, 24. http://dx.doi.org/10.1186/2190-4715-24-24 [21] Jennings, R.C. (2013) Social and Ethical Issues Raised by NGOs and How They Can Be Understood. In: Bennet, D.J. and Jennings, R.C., Eds., Successful Agricultural Innovation in Emerging Economies, Cambridge University Press, Cambridge, 386-399. http://dx.doi.org/10.1017/CBO9781139208475.028

33

M. Cuhra et al.

[22] Wickson, F., Bøhn, T., Wynne, B., Hilbeck, A. and Funtowicz, S. (2013) Science-Based Risk Assessment Requires Careful Evaluation of All Studies. Nature Biotechnology, 31, 1077-1078. http://dx.doi.org/10.1038/nbt.2761

[23] Padgette, S.R., Taylor, N.B., Nida, D.L., Bailey, M.R., MacDonald, J., Holden, L.R. and Fuchs, R.L. (1996) The Composition of Glyphosate-Tolerant Soybeans Is Equivalent to That of Conventional Soybeans. Journal of Nutrition, 126, 702-716. [24] Hammond, B.G., Vicini, J.L., Hartnell, G.F., Naylor, M.W., Knight, C.D., Robinson, E.H., et al. (1996) The Feeding Value of Soybeans Fed to Rats, Chickens, Catfish and Dairy Cattle Is Not Altered by Incorporation of Glyphosate Tolerance. Journal of Nutrition, 126, 717-727. [25] Millstone, E., Brunner, E. and Mayer, S. (1999) Beyond Substantial Equivalence. Nature, 401, 525-526. http://dx.doi.org/10.1038/44006 [26] Cromwell, G.L., Lindemann, M.D., Randolph, J.H., Parker, G.R., Coffey, R.D., Laurent, K.M., et al. (2002) Soybean Meal from Roundup Ready or Conventional Soybeans in Diets for Growing-Finishing Swine. Journal of Animal Science, 80, 708-715. [27] Brake, D.G. and Evenson, D.P. (2004) A Generational Study of Glyphosate-Tolerant Soybeans on Mouse Fetal, Postnatal, Pubertal and Adult Testicular Development. Food and Chemical Toxicology, 42, 29-36. http://dx.doi.org/10.1016/j.fct.2003.08.003 [28] Harrigan, G.G., Ridley, W.P., Riordan, S.G., Nemeth, M.A., Sorbet, R., Trujillo, W.A., et al. (2007) Chemical Composition of Glyphosate-Tolerant Soybean 40-3-2 Grown in Europe Remains Equivalent with That of Conventional Soybean (Glycine max L.). Journal of Agricultural and Food Chemistry, 55, 6160-6168. http://dx.doi.org/10.1021/jf0704920 [29] Lundry, D.R., Ridley, W.P., Meyer, J.J., Riordan, S.G., Nemeth, M.A., et al. (2008) Composition of Grain, Forage, and Processed Fractions from Second-Generation Glyphosate-Tolerant Soybean, MON 89788 Is Equivalent to That of Conventional Soybean (Glycine max L.). Journal of Agricultural and Food Chemistry, 56, 4611-4622. http://dx.doi.org/10.1021/jf073087h [30] Viljoen, C. (2013) Letter to the Editor. Food and Chemical Toxicology, 59, 809-810. http://dx.doi.org/10.1016/j.fct.2013.07.003 [31] Nielsen, K.M. (2013) Biosafety Data as Confidential Business Information. PLoS Biology, 11, e1001499. http://dx.doi.org/10.1371/journal.pbio.1001499 [32] Malatesta, M., Caporaloni, C., Gavaudan, S., Rocchi, M.B.L., Serafini, S., Tiberi, C., et al. (2002) Ultrastructural Morphometrical and Immunecytochemical Analysis of Hepatocyte Nuclei from Mice Fed on Genetically Modified Soy Bean. Cell Structure and Function, 27, 173-180. http://dx.doi.org/10.1247/csf.27.173 [33] Malatesta, M., Boraldi, F., Annovi, G., Baldelli, B., Battistelli, S., Biggiogera, M., et al. (2008b) A Long-Term Study on Female Mice Fed on a Genetically Modified Soybean: Effects on Liver Ageing. Histochemistry and Cell Biology, 130, 967-977. http://dx.doi.org/10.1007/s00418-008-0476-x [34] Malatesta, M., Perdoni, F., Santin, G., Battistelli, S., Muller, S., Biggioger, M., et al. (2008a) Hepatoma Tissue Culture (HTC) Cells as a Model for Investigating the Effects of Low Concentrations of Herbicide on Cell Structure and Function. Toxicology in Vitro, 22, 1853-1860. http://dx.doi.org/10.1016/j.tiv.2008.09.006 [35] Zhu, Y., Li, D., Wang, F., Yin, J. and Jin, H. (2004) Nutritional Assessment and Fate of Dna of Soybean Meal from Roundup Ready or Conventional Soybeans Using Rats. Archives of Animal Nutrition, 58, 295-310. http://dx.doi.org/10.1080/00039420412331273277 [36] Sissener, N.H., Sanden, M., Krogdahl, Å., Bakke, A.-M., Johannessen, L.E. and Hemre, G.-I. (2011) Genetically Modified Plants as Fish Feed Ingredients. Canadian Journal of Fisheries and Aquatic Sciences, 68, 563-574. http://dx.doi.org/10.1139/F10-154 [37] Bakke-McKellep, A.M., Koppang, E.O., Gunnes, G., Sanden, M., Hemre, G-I., Landsverk, T. and Krogdahl, Å. (2007) Histological, Digestive, Metabolic, Hormonal and Some Immune Factor Responses in Atlantic Salmon, Salmo salar L., Fed Genetically Modified Soybeans. Journal of Fish Diseases, 30, 65-79. http://dx.doi.org/10.1111/j.1365-2761.2007.00782.x [38] Sissener, N.H., Bakke, A.M., Gu, J., Penn, M.H., Eie, E., Krogdahl, Å., et al. (2009) An Assessment of Organ and Intestinal Histomorphology and Cellular Stress Response in Atlantic Salmon (Salmo salar L.) Fed Genetically Modified Roundup Ready ® Soy. Aquaculture, 298, 101-110. http://dx.doi.org/10.1016/j.aquaculture.2009.10.011 [39] Sissener, N.H., Sanden, M., Bakke, A.M., Krogdahl, A. and Hemre, G.I. (2009) A Long Term Trial with Atlantic Salmon (Salmo salar L.) Fed Genetically Modified Soy; Focusing General Health and Performance Before, during and after the Parr-Smolt Transformation. Aquaculture, 294, 108-117. http://dx.doi.org/10.1016/j.aquaculture.2009.05.002 [40] Bøhn, T., Primicerio, R., Hessen, D.O. and Traavik, T. (2008) Reduced Fitness of Daphnia magna Fed a Bt-Transgenic Maize Variety. Archives of Environmental Contamination and Toxicology, 55, 584-592. http://dx.doi.org/10.1007/s00244-008-9150-5

34

M. Cuhra et al.

[41] Bøhn, T., Traavik, T. and Primicerio, R. (2010) Demographic Responses of Daphnia magna Fed Transgenic Bt-Maize. Ecotoxicology, 19, 419-430. http://dx.doi.org/10.1007/s10646-009-0427-x [42] EFSA (2009) Scientific Opinion on Applications for Renewal of Authorization for the Continued Marketing of Maize MON 81 and Existing Derived Food and Feed Products. EFSA Journal, 1149, 38-85. [43] Ricroch, A., Bergé, J.B. and Kuntz, M. (2010) Is the German Suspension of MON810 Maize Cultivation Scientifically Justified? Transgenic Research, 19, 1-12. http://dx.doi.org/10.1007/s11248-009-9297-5 [44] EFSA (2008) Safety and Nutritional Assessment of GM Plants and Derived Food and Feed: The Role of Animal Feeding Trials. Food and Chemical Toxicology, 46, S1-S70. http://dx.doi.org/10.1016/j.fct.2008.02.013 [45] OECD (2001) Consensus Document on Compositional Considerations for New Varieties of Soybean: Key Food and Feed Nutrients and Antinutrients. OECD Environmental Health and Safety Publications Series on the Safety of Novel Foods and Feeds, No. 2. Environment Directorate, Organization for Economic Co-Operation and Development, Paris. [46] Ellingsen, H., Olaussen, J.O. and Utne, I.B. (2009) Environmental Analysis of the Norwegian Fishery and Aquaculture Industry—A Preliminary Study Focusing on Farmed Salmon. Marine Policy, 33, 479-488. http://dx.doi.org/10.1016/j.marpol.2008.11.003 [47] Olsen, R.L. and Hasan, M.R. (2012) A Limited Supply of Fishmeal: Impact on Future Increases in Global Aquaculture Production. Trends in Food Science & Technology, 27, 120-128. [48] Lövei, G.L. and Arpaia, S. (2005) The Impact of Transgenic Plants on Natural Enemies: A Critical Review of Laboratory Studies. Entomologia Experimentalis et Applicata, 114, 1-14. [49] Lövei, G.L., Andow, D.A. and Arpaia, S. (2009) Transgenic Insecticidal Crops and Natural Enemies: A Detailed Review of Laboratory Studies. Environmental Entomology, 38, 293-306. http://dx.doi.org/10.1603/022.038.0201 [50] Rosi-Marshall, E.J., Tank, J.L., Royer, T.V., Whiles, M.R., Evans-White, M., Chambers, C., et al. (2007) Toxins in Transgenic Crop Byproducts May Affect Headwater Stream Ecosystems. Proceedings of the National Academy of Sciences of the United States of America, 104, 16204-16208. http://dx.doi.org/10.1073/pnas.0707177104 [51] Séralini, G.E., Clair, E., Mesnage, R., Gress, S., Defarge, N., Malatesta, M., et al. (2014) Long Term Toxicity of a Roundup Herbicide and a Roundup-Tolerant Genetically Modified Maize. Environmental Sciences Europe, 26, 14. [52] EFSA (2013) Considerations on the Applicability of OECD TG 453 to Whole Food/Feed Testing. EFSA Journal, 11, 3347. [53] EFSA (2010) Guidance on the Environmental Risk Assessment of Genetically Modified Plants. EFSA Journal, 8, 1111. [54] ISAAA (2014) GM Approval Database. International Service for the Acquisition of Agri-Biotech Applications (ISAAA). http://www.isaaa.org/gmapprovaldatabase/ [55] OECD (2008) OECD-211 Guidelines for Testing of Chemicals: Daphnia magna Reproduction Test. OECD, Geneva. [56] Collins, T. (2007) ImageJ for Microscopy. BioTechniques, 43, S25-S30. http://dx.doi.org/10.2144/000112517 [57] Dorr, R., Ozu, M. and Parisi, M. (2007) Simple and Inexpensive Hardware and Software Method to Measure Volume Changes in Xenopus Oocytes Expressing Aquaporins. Journal of Neuroscience Methods, 161, 301-305. http://dx.doi.org/10.1016/j.jneumeth.2006.11.005

35

M. Cuhra et al.

Supporting Information

Figure S1. Abortion rates for each individual D. magna, related to level of glyphosate residues in feed (Error bands: 95% CI).

Figure S2. Total D. magna fecundity (cumulated live births) related to levels of glyphosate residues (Error bands: 95% CI).

36

   

Paper  V:  

Review  of  published  evidence  

  Title:      

Analysis  of  herbicide-­‐residues  is  essentially  missing     in  risk-­‐assessment  of  herbicide-­‐tolerant  genetically  modified  cultivars.    

Author:    

Marek  Cuhra.  

Journal:    

Environmental  Sciences  Europe  (accepted  manuscript).

45  

 

!

1! 2!

3!

Analyses(of(herbicide1residues(are(still(missing((

4!

in(risk1assessment(of(glyphosate1tolerant(GMO1cultivars(

5!

!

6!

Keywords:!Glyphosate!tolerant!GMO,!substantial!equivalence,!herbicide!tolerance,!

7!

RoundupBready!cultivars,!herbicide!residues,!industrial!agriculture!chemistry!

8!

!

9!

Author:!Marek!Cuhra!

10!

!

11!

Authors!affiliation:!!

12!

GenØk!–!Centre!for!Biosafety,!The!Science!Park,!P.O.Box!6418,!9294!Tromsø,!Norway!

13!

Faculty!of!Health!Sciences!B!UiT!Arctic!University!of!Norway!

14!

!

15!

Corresponding!author:!!

16!

eBmail:[email protected]!

17!

telephone:!+47!–!99585427!

18!

telefax:!+47!B!77646100!

19!

!

20!

!

1!

!

20!

Abstract!

21!

Background:!Genetically!modified!glyphosateBtolerant!cultivar!varieties!(GMBcrops)!

22!

have!been!a!commercial!success!widely!known!as!RoundupBready!plants.!As!new!

23!

glyphosateBtolerant!varieties!are!introduced!to!satisfy!agriculture!demand,!it!is!relevant!

24!

to!review!the!scientific!evidence!that!documents!the!quality!and!safety!of!such!

25!

biotechnology.!!Assessment!of!genetically!modified!glyphosateBtolerant!plants!is!partly!

26!

based!on!reports!from!laboratory!comparisons!with!nonBmodified!plants!(near!isogenic!

27!

relatives).!Such!comparative!testing!is!typically!performed!as!analysis!of!plantBmaterial!

28!

composition!and!in!animal!feeding!studies.!The!material!for!testing!is!typically!

29!

produced!in!testBfields!set!up!as!modelBenvironments.!Researchers!employed!by!biotech!

30!

industry!companies!plan,!perform!and!report!most!of!this!research.!!

31!

Perspective:!The!present!paper!aims!to;!i)!review!15!reports!on!compositional!analyses!

32!

of!glyphosateBtolerant!cultivars!and!15!reports!from!animalBfeedingBstudies,!ii)!discuss!

33!

recent!data!indicating!glyphosate!residue!in!RoundupBready!soybean,!iii)!outline!recent!

34!

developments!of!cultivars!with!increased!tolerance!to!glyphosate.!!

35!

Findings:!The!reviewed!industry!studies!show!methodological!flaws:!GlyphosateB

36!

tolerant!GMBcrops!are!designed!for!use!with!glyphosate!herbicide.!However,!glyphosate!

37!

herbicides!are!often!not!applied!in!testBstudy!cultivation.!In!the!studies!where!

38!

glyphosate!herbicides!were!applied!to!growing!plants,!the!produced!plant!material!was!

39!

not!analysed!for!glyphosate!residues.!This!review!has!failed!to!identify!industry!studies!

40!

that!mention!glyphosate!residues!in!glyphosateBtolerant!plants.!This!indicates!that!

41!

questions!and!evidence!of!importance!for!regulatory!assessment!have!been!

42!

systematically!ignored.!Independent!research!has!investigated!this!issue!and!found!that!

43!

glyphosateBtolerant!plants!accumulate!glyphosate!residues!at!unexpected!high!levels.!

44!

Glyphosate!residues!are!found!to!have!potential!to!affect!plant!material!composition.!

45!

Furthermore,!these!residues!are!passed!on!to!consumers.!

46!

Conclusions:!Industry!studies!are!not!sufficient!for!regulation.!Despite!decades!of!risk!

47!

assessments!and!research!in!this!field,!specific!unanswered!questions!relating!to!safety!

48!

and!quality!aspects!of!food!and!feed!from!transgenic!cultivars!need!to!be!addressed!by!

49!

regulators.!Independent!research!gives!important!supplementary!insight.!

!

2!

!

50!

Introduction(

51!

Recent!changes!in!the!Europen!Union!(EU)!legislative!framework!for!assessments!and!

52!

approvals!of!genetically!modified!agriculture!cultivars!(GMBcrops)!have!led!to!a!

53!

delegation!of!responsibility!to!regulatory!authorities!in!individual!EU!member!states.!

54!

The!increased!challenges!of!evaluating!applications!for!import!or!cultivation!of!GMB

55!

crops!accentuate!the!need!for!reliable!and!transparent!evidence!on!GMBcrop!quality!and!

56!

safety!issues.!

57!

In!a!2013!review!of!two!decades!of!research!on!possible!unintended!compositional!

58!

changes!in!GMBcrops,!two!senior!scientists!conclude!that!such!GMBcrops!have!been!

59!

subjected!to!a!large!number!of!analytical!studies!which!confirm!compositional!

60!

equivalence.!They!conclude!that!GMBcrops!are!safe!and!rhetorically!ask;!"How!much!

61!

uncertainty!remains!after!20!years!of!research?"![1].!

62!

I!see!that!the!authors!have!concluded!that!compositional!equivalence!is!sufficiently!

63!

established!and!I!hear!their!argument!stating!that!further!safety!studies!of!GMBcrops!

64!

thus!no!longer!are!necessary.!However,!I!still!propose!to!answer!the!rhetorical!question!

65!

presented!by!these!senior!scientist!authors,!of!whom!one!is!representing!a!major!

66!

industrial!producer!of!GMBcrops!and!the!other!is!retired!from!the!Food!and!Drug!

67!

Administration!of!the!United!States.!

68!

Unresolved!important!uncertainties!remain!in!relation!to!genetically!modified!crop!

69!

quality!and!safety.!One!such!specific!issue!will!be!reviewed!here:!the!somewhat!

70!

neglected!fact!that!GMBcultivars!designed!and!modified!to!be!tolerant!to!herbicides!such!

71!

as!glyphosate,!will!be!subjected!to!application!of!such!chemicals!in!the!field!and!

72!

therefore!must!be!expected!to!have!biological!interaction!with!these!herbicidal!sprays.!!

73!

Background(

74!

Herbicides!such!as!glyphosate!disrupt!plant!metabolism!by!having!chemical!and!

75!

physical!qualities!that!facilitate!penetration!into!the!plant!tissue!and!transportation!

76!

within!the!plant,!killing!the!recipient!by!systemic!action![2].!!Glyphosate!is!an!important!

77!

chemical;!it!is!a!bestBselling!herbicide!with!an!annual!application!in!the!order!of!0.6B1.2!

78!

million!tonnes!globally![3,!4].!Glyphosate!is!used!in!farming,!parks,!gardening,!forestry! !

3!

!

79!

and!wetland!management![5].!Glyphosate!is!widely!used!as!a!desiccant!to!induce!

80!

ripening!in!semi!mature!crops![6]!and!it!has!been!found!to!have!antibiotic!qualities![7].!

81!

In!the!context!of!this!review!it!must!be!noted!that!the!advent!of!glyphosateBtolerant!

82!

crops!has!contributed!to!a!sharp!increase!in!global!dispersal!of!this!chemical![8].!

83!

Early!findings!justified!that!glyphosate!has!been!widely!recognized!as!having!relatively!

84!

low!environmental!impact![9],!low!toxicity!for!field!workers!handling!the!chemical,!and!

85!

low!toxicity!for!consumers!ingesting!residues!of!it!through!food![10].!However,!in!recent!

86!

years!such!established!indications!of!safety!have!come!under!revision,!as!glyphosate!is!

87!

found!to!have!more!subtle!and!complex!effects!than!what!has!previously!been!

88!

acknowledged![11,!86].!Furthermore,!recent!pesticide!screenings!of!fruits,!vegetables!

89!

and!other!food!in!the!EU!have!shown!that!a!majority!of!the!samples!(97%)!contain!trace!

90!

quantities!of!pesticide!residues.!Glyphosate!stands!out!as!the!most!common!detected!

91!

chemical!in!European!food,!present!in!approximately!half!of!all!samples![12].!However,!

92!

the!vast!majority!of!samples!show!concentrations!well!below!the!existing!acceptance!

93!

levels.!It!must!be!noted!that!although!acceptance!levels!of!most!pesticides!are!defined!in!

94!

ng/kg!(ppt)!or!μg/kg!(ppb)!orders!of!magnitude,!the!acceptance!levels!for!glyphosate!in!

95!

numerous!foods!and!agriculture!products!are!defined!in!mg/kg!(ppm).!Despite!the!

96!

detected!wideBspread!occurrence!of!glyphosate!residues!in!food,!animal!feed,!water![5],!

97!

air![13],!human!blood![14],!human!milk![14]!and!human!urine![14,!15],!it!is!not!within!

98!

the!mandate!of!this!review!to!evaluate!whether!the!relatively!high!acceptance!levels!for!

99!

glyphosate!residues!in!food!and!feed!are!scientifically!justified,!however!relevant!the!

100!

question!may!seem.!

101!

HerbicideBtolerant!cultivars!dominate!agriculture!

102!

It!has!been!estimated!that!an!overwhelming!81%!majority!of!transgenic!crops!in!

103!

cultivation,!are!herbicideBtolerant!varieties![16].!The!majority!of!those!herbicideB

104!

tolerant!crops!are!Roundup'Ready!plant!cultivars!(RRBcrops)!genetically!modified!to!

105!

tolerate!glyphosate!herbicides!such!as!the!commercial!product!Roundup.!The!first!such!

106!

varieties!were!introduced!in!1996!and!rapidly!gained!popularity!amongst!farmers.!

107!

HerbicideBtolerance!allows!for!postBemergence!application!and!in!principle!eliminates!

108!

the!need!for!preBplant!tillage!and!manual!weeding.!!This!is!an!advantage!which!

109!

contributes!to!reduced!soil!erosion!and!reduced!production!expenses![16,!17,!18].!

!

4!

!

110!

Despite!challenges!from!increasing!numbers!of!agriculture!weeds!that!are!resistant!to!

111!

glyphosate!herbicide,!glyphosate!tolerant!cultivars!such!as!RRBsoy,!RRBcorn,!RRBcanola!

112!

and!RRBcotton!are!still!the!most!popular!and!widely!grown!genetically!modified!plant!

113!

varieties![8].!Additional!glyphosateBtolerant!cultivars!such!as!RRBsugar!beet,!RRBwheat!

114!

and!RRBalfalfa!are!introduced!as!promising!and!potentially!important!crops![4].!

115!

Glyphosate!tolerant!plants!thus!form!a!dominant!and!increasing!proportion!of!the!

116!

biomass!produced!globally!from!industrial!agriculture.!This!biomass!is!used!for!farmB

117!

animal!feed!purposes!and!for!important!constituents!in!human!food!products.!

118!

Industry!provides!most!data!for!riskBassessment!of!GMBcrops!

119!

Regulatory!assessments!of!applications!for!import!and!use!of!products!from!GM!

120!

cultivars!into!the!European!Union/EEC!area,!and!applications!for!open!cultivation!of!

121!

such!plants!in!Europe,!have!until!recently!been!centrally!processed!by!the!European!

122!

Food!Safety!Authority+(EFSA)!based!on!documentation!submitted!by!applicants![19,!21,!

123!

22,!24].!Recent!changes!in!regulation!of!GMBcrops!within!the!European!Union!delegate!

124!

this!responsibility,!as!individual!member!states!now!have!the!obligation!to!

125!

independently!approve!or!reject!specific!applications!on!a!national!level.!

126!

In!a!standing!controversy!over!GMO!safety,!the!EU!approval!process!as!conducted!by!

127!

EFSA!has!been!claimed!to!be!unsupportive!of!independent!research!findings![19,!20,!

128!

87].!

129!

Typically!industry!applications!for!import!and/or!cultivation!contain!3!main!categories!

130!

of!information;!1)!molecular!information!on!the!actual!event!including!the!structure!and!

131!

origin!of!transgenic!construct,!2)!information!from!compositional!analysis!where!the!

132!

transgenic!cultivar!in!question!is!compared!to!nearBisogenic!motherBlines!or!other!

133!

comparators!representative!of!unmodified!varieties!grown!under!similar!conditions,!

134!

and!3)!results!from!feeding!studies!in!testBanimals!such!as!rodents!or!farmBanimals!such!

135!

as!pigs!and!poultry.!Implicitly,!the!transgenic!material!in!such!tests!should!be!

136!

representative!of!the!actual!material!intended!for!consumption.!

137!

European!regulation!defines!guidelines!for!animal!feeding!studies!and!compositional!

138!

analysis!to!determine!whether!food!and!feed!from!transgenic!cultivars!reliably!has!

139!

qualitative!equivalence!to!that!of!conventional!nonBmodified!cultivars![21,!22].!!

!

5!

!

140!

The!concept!of!substantial+equivalence![23]!is!used!by!regulators!and!industry!scientists!

141!

to!validate!transgenic!crop!quality.!Comparative!analysis!of!composition!and!

142!

comparative!testing!in!animal!feedingBtrials!are!still!the!two!fundamental!methods!in!

143!

use!for!assessment!of!substantial!equivalence!of!produce!from!herbicideBtolerant!crops!

144!

and!other!genetically!modified!biomass!intended!for!consumption.!Guidelines!for!such!

145!

analysis!and!testing!aim!to!ensure!that!the!new!varieties!are!as!safe!and!nutritious!as!

146!

conventional!plants.!Therefore!such!testing!includes!riskBassessments!which!anticipate!

147!

potentially!adverse!effects!stemming!from!qualitative!differences!or!undesirable!

148!

constituents![23,!24].!The!Food!and!Agriculture!Organisation!(FAO)!and!World!Health!

149!

Organisation!(WHO)!established!the!Codex!Alimentarius!commission!in!1963!to!

150!

develop!harmonised!international!food!standards,!guidelines!and!codes!of!practice!to!

151!

protect!the!health!of!the!consumers.!The!aim!of!the!Codex!regulation!is!to!anticipate!not!

152!

only!direct!risk,!but!also!indirect/unaticipated+risk![25].!Thus!it!is!interesting!to!note!

153!

that!the!Codex!Alimentarius!commission!in!1999B2001!had!protracted!evaluations!on!

154!

the!possibility!of!establishing!specific!and!unique!standards!for!herbicideBresidueBlevels!

155!

in!herbicideBtolerant!transgenic!crops![26].!The!reports!of!this!regulatory!process!

156!

document!that!this!question!was!seen!relevant!at!that!time.!However,!the!result!of!this!

157!

process!was!a!decision!not!to!establish!separate!residue!limits!for!transgenic!herbicideB

158!

tolerant!cultivars.!!

159!

Although!generally!recognized!as!safe!by!regulators!in!the!United!States!Food!and!Drug!

160!

Administration![27],!safety!assessment!of!produce!from!transgenic!cultivars!is!a!

161!

contested!issue!in!Europe![19]!and!numerous!other!countries!worldBwide.!Safety!

162!

assessment!is!mostly!based!on!testing!performed!by!industry!companies!or!by!

163!

researchers!working!for!such!companies.!Complex!legal!and!commercial!aspects!of!

164!

patented!biotechnology!products!restrict!independent!researcher!access!to!both!such!

165!

transgenic!material!(patented!property!of!industry)!and!to!data!from!development!and!

166!

testing,!which!is!regarded!as!intellectual!property!and!thus!confidential![28,!29].!!

167!

!

168!

!

6!

!

168! 169! 170! 171! 172! 173!

! Figure61.6Recent6data6on6glyphosate6residues6in6glyphosate6tolerant6soybean.!! Data!from!analysis!of!samples!from!fields!in!Iowa,!USA![30]!and!province!of!Salta,!Argentina![90].! Residues!are!shown!as!detections!of!glyphosate!and!the!primary!metabolite,!AMPA.!Reference!lines! indicate!maximum!residue!limit!(MRL).!Former!European!MRL!of!0.1!mg/kg!was!raised!200Bfold!in!1999! to!20!mg/kg.!US!MRL!at!20!mg/kg!was!raised!to!40!mg/kg!in!2014.!

!

7!

!

174!

!

175!

!

176!

Review(of(published(evidence(

177!

15!published!reports!from!compositional!analyses!of!plant!material!grown!from!

178!

glyphosateBtolerant!cultivars!and!15!published!reports!from!tests!of!such!material!in!

179!

animalBfeedingBstudies!were!extracted!from!peerBreviewed!scientific!journals!(tables!1!

180!

&!2).!The!majority!of!these!studies!were!designed!and!performed!by!researchers!

181!

affiliated!with!biotech!industry!companies.!These!companies!are!also!seen!to!have!

182!

funded!the!research.!Although!the!numbers!are!relatively!low!and!not!suited!for!

183!

statistical!analysis,!a!graphic!visualisation!and!percentual!representation!is!appropriate.!!

184!

Analyses!of!composition:!Reviewing!15!publications!on!results!from!comparative!

185!

analyses!in!which!specific!transgenic!glyphosateBtolerant!plant!material!was!compared!

186!

to!nearBisogenic!unmodified!material!or!other!relevant!conventional!plant!material,!it!

187!

was!found!that!14!of!the!15!published!analyses!were!industry!studies!(Table!1).!Of!

188!

these,!only!7!(50%)!reported!to!have!applied!glyphosate!herbicide!during!cultivation.!

189!

None!of!these!14!industry!studies!presented!data!from!quantification!of!pesticide!

190!

residues!or!gave!indications!that!such!analysis!had!been!performed.!Only!the!one!

191!

independent!study!reported!glyphosate!residues![30]!(Figure!1).!

192!

All!14!studies!in!which!glyphosate!had!not!been!applied!(the!industry!studies)!found!the!

193!

various!glyphosateBtolerant!GMBcrops!(soybean,!corn!and!canola)!to!be!compositionally!

194!

equivalent!to!nonBmodified!comparators.!The!one!study!in!which!glyphosate!had!been!

195!

applied!found!significant!differences!in!composition!of!glyphosateBtolerant!soybean!

196!

compared!to!nonBmodified!varieties!of!soybean.!

197!

Animal!feeding!studies:!Reviewing!published!reports!from!feedingBstudies!in!which!

198!

animals!are!fed!feed!from!specific!glyphosateBtolerant!plant!material!and!compared!to!

199!

animals!fed!nearBisogenic!unmodified!material!(or!other!relevant!conventional!

200!

produce),!are!also!seen!to!lack!information!on!herbicideBresidues!(Table!2).!Through!

201!

literature!searches,!15!studies!were!identified.!6!of!these!studies!were!performed!by!

202!

researchers!with!industry!affiliation.!The!remaining!9!studies!were!independent!studies!

203!

performed!by!researchers!affiliated!with!government!agencies,!universities!or!other! !

8!

!

204!

institutions!recognized!to!be!independent!of!the!implicit!financial!issues!associated!with!

205!

GMBcrop!commercialisation!and!production.!!

206!

Of!the!6!studies!performed!by!industry,!plant!material!for!feed!had!been!produced!with!

207!

application!of!glyphosate!herbicide!in!3!studies!(50%).!!In!the!9!independent!studies,!

208!

plant!material!for!feed!had!been!produced!with!application!of!glyphosate!herbicide!in!5!

209!

studies!(56%).!

210!

3!of!the!9!independent!studies!reported!that!pesticide!analysis!had!been!performed!

211!

(33%).!Of!the!6!industry!studies,!only!one!(17%)!reported!that!pesticide!analysis!had!

212!

been!performed.!However,!although!glyphosate!herbicide!was!the!only!

213!

pesticide!applied!in!the!mentioned!study,!the!subsequent!analysis!for!pesticide!residues!

214!

included!numerous!chemical!compounds!known!as!active!ingredients!in!various!other!

215!

commercial!pesticide!formulations.!!Paradoxically,!the!analysis!did!not!include!

216!

glyphosate!or!the!main!metabolite!of!this!chemical.!Due!to!those!obvious!shortcomings!

217!

of!the!pesticide!analysis!in!the!mentioned!study,!it!is!concluded!that!none!of!the!6!

218!

industry!studies!include!analyses!for!relevant!pesticides!(0%).!

219!

Based!on!data!from!testBanimal!performance!and!histology,!7!studies!found!that!there!

220!

were!no!significant!effects!from!the!GMBfeed!produced!from!glyphosateBtolerant!plant!

221!

material.!These!7!studies!consisted!of!one!of!the!independent!studies!and!all!6!industry!

222!

studies.!The!remaining!8!independent!studies!found!significant!effects!attributable!to!

223!

GMBfeed!produced!from!glyphosateBtolerant!plant!material!(Table!2).!

224!

In!the!8!studies!reporting!significant!effects!from!GMBfeed,!2!studies!related!these!

225!

effects!to!residues!of!glyphosate.!One!of!the!studies!found!that!testBanimal!fitness!

226!

decreased!in!correlation!with!increasing!levels!of!glyphosate!residues![32].!

227!

Discussion(

228!

30!Published!reports!from!studies!of!compostitional!analysis!glyphosateBtolerant!GMB

229!

plant!varieties!and!from!feeding!studies!using!glyphosateBtolerant!GMBplant!varieties!

230!

have!been!reviewed.!These!studies!were!performed!in!the!years!1996B2015.!A!simple!

231!

synthesis!of!available!information!on!study!design,!methods!and!results!shows!that:!

!

9!

!

232!

•

233!

14!of!15!studies!on!composition!and!6!of!15!animal!feeding!studies!were! performed!by!biotech!industry!companies.!

234!

•

235!

16!of!30!studies!(53%),!used!material!actually!sprayed!with!glyphosate! herbicide!during!cultivation.!

236!

•

237!

4!of!30!studies!(13%)!address!the!issue!of!glyphosate!residues.!None!of! these!4!studies!were!funded!by!industry.!!

238!

The!findings!of!this!review!fundamentally!challenge!the!basis!for!regulatory!assumption!

239!

of!substantial!equivalence!between!glyphosateBtolerant!GMBvarieties!and!unmodified!

240!

comparators.!The!findings!are!a!strong!argument!for!mandatory!inclusion!of!pesticide!

241!

analysis!data!in!regulatory!assessment!of!GMBcrop,!notably!in!assessments!of!herbicideB

242!

tolerant!crops.!Two!of!the!animal!feeding!studies!performed!by!independent!

243!

researchers![87,!88]!used!GMBcrop!material!as!well!as!unmodified!comparators!

244!

supplied!by!industry.!No!analysis!was!performed!to!control!the!compositional!quality!of!

245!

this!material.!

246!

The!literature!review!indicates!that!there!are!relatively!few!studies!available!for!

247!

regulatory!evaluation!of!scientific!evidence!on!herbicideBtolerant!crop!quality!and!

248!

safety.!Furthermore,!it!is!found!that!the!majority!of!such!studies!are!presented!as!

249!

reports!from!compositional!analyses!and!animal!feedingBstudies,!and!predominantly!

250!

performed!either!by!biotech!industry!companies!with!potentially!conflicting!interests!in!

251!

research!outcome!or!by!subcontractors!working!for!the!biotech!industry!companies!

252!

(Tables!1!&!2).!Society!should!expect!the!biotech!industry!companies!to!continue!to!

253!

conduct!such!studies!to!peerBreview!standard!and!the!industry!should!continue!to!bear!

254!

associated!costs.!However,!it!is!evident!that!appropriate!revisions!of!standards!are!

255!

needed,!and!supplementary!studies!by!independent!researchers!should!be!encouraged.!

256!

Published!evidence!on!safety!testing!presented!by!the!industry!has!generally!been!

257!

recognized!by!EFSA!as!sufficient!for!regulatory!purpose,!despite!the!fact!that!several!

258!

potentially!conflicting!issues!have!been!continuously!raised!by!independent!researchers!

259!

[19,!20,!28,!33,!34,!87].!Such!critique!has!also!questioned!both!the!principle!of!delegated!

260!

selfBcontrol!and!the!validity!of!industry!methods.!Some!of!this!critique!has!led!to!

261!

temporary!adjustments!of!protocols!and!changes!in!methodology.!A!review!by!

262!

independent!scientists!in!1999![33]!examined!results!of!three!initial!industry!tests!that! !

10!

!

263!

were!published!in!1996.!These!first!industry!tests!claimed!substantial!equivalence!of!

264!

transgenic!glyphosateBtolerant!GTS!40B3B2!soybean![34,!35]!and!seed!from!glyphosateB

265!

tolerant!cotton![36].!However,!the!review!noted!that!the!industry!reports!were!based!on!

266!

tests!of!glyphosateBtolerant!material!grown!in!artificial!conditions!without!application!

267!

of!complimentary!glyphosate!herbicides.!The!GMBcrop!thus!produced!was!claimed!to!be!

268!

not!representative!of!the!crops!actually!produced!in!agriculture![33].!Several!industry!

269!

researchers!immediately!acknowledged!the!necessity!to!change!these!specific!

270!

approaches!and!subsequent!industry!publications!on!quality!of!glyphosateBtolerant!

271!

varieties!of!soy![37,!38,!39,!40],!maize![41],!alfalfa![42]!and!cotton![43]!specified!that!

272!

normal!cultivation!practice!had!been!used!in!production,!including!prescription!rate!

273!

application!of!glyphosate!via!commercial!glyphosate!herbicides!such!as!Roundup.!One!

274!

paper!published!immediately!following!the!1999!criticism!even!specifies!in!its!title!that!

275!

glyphosate!herbicides!have!been!applied![44].!Despite!this!change!of!practice!and!the!

276!

acknowledged!need!for!realistic!field!conditions!to!produce!material!for!evaluation,!

277!

numerous!subsequent!tests!have!been!published!where!again!produce!of!glyphosateB

278!

tolerant!cultivars!is!used!for!comparison!despite!having!been!grown!in!artificial!

279!

conditions!without!application!of!complimentary!herbicides![34].!!Recently10!studies!

280!

presented!by!industry!applicants!as!evidence!for!regulatory!approval!of!glyphosateB

281!

tolerant!cultivars!were!reviewed!and!the!author!concludes!that!lack!of!relevant!

282!

herbicide!application!is!still!a!discrediting!flaw!in!such!studies![34].!However,!although!

283!

this!highlights!one!systematic!flaw!in!studies!currently!accepted!for!regulatory!purpose!

284!

documentation,!the!role!of!herbicideBresidues!must!be!recognized!as!a!subsequent!and!

285!

not!least!important!aspect.!

286!

The!relevance!of!testing!for!herbicide!residues!is!highlighted!by!the!findings!of!a!recent!

287!

study!on!the!composition!of!plantBmaterial![30]!performed!by!independent!researchers.!

288!

The!study!reports!high!levels!of!glyphosate!residues!(Figure!1)!in!glyphosateBtolerant!

289!

soybean!(RoundupBready!soy!GTS!40B3B2).!The!study!also!found!that!residues!of!

290!

glyphosate!and!the!primary!metabolite!aminomethyphosphonicBacid!(AMPA)!are!

291!

correlated!to!differences!in!crop!composition.!In!2003!and!2004!independent!research!

292!

demonstrated!that!residues!of!glyphosate!herbicides!will!accumulate!in!glyphosateB

293!

tolerant!plant!material![45,!46]!but!showed!lower!quantities!than!the!subsequent!

294!

findings!reported!in!2014![30].!!Another!recent!report!from!tests!performed!in! !

11!

!

295!

Argentina!by!independent!scientists!working!for!the!German!NGO!TestBBiotech!have!

296!

reported!findings!of!even!higher!levels!of!glyphosate!residues!in!harvests!of!glyphosateB

297!

tolerant!soy,![65]!(Figure!1).!These!results!indicate!very!high!glyphosate!residue!levels!

298!

up!to!100!mg/kg!in!soybean.!The!tests!were!performed!at!the!University!of!Buenos!

299!

Aires!and!although!it!is!unclear!whether!this!laboratory!is!formally!accredited!for!the!

300!

analytical!methods!used!in!quantification,!the!results!stand!as!an!important!indication!

301!

that!justifies!further!sampling!and!analysis.!!

302!

The!results!indicate!a!rise!in!glyphosate!residue!levels!in!recent!decades.!In!1999!a!

303!

major!producer!of!glyphosate!and!GMBcrops!declared!that!glyphosate!residue!levels!of!

304!

5.6!mg/kg!in!glyphosateBtolerant!soybean!were!considered!to!be!extreme!high!values!

305!

[30].!It!seems!apparent!from!figure!1!that!such!levels!at!present!would!be!considered!

306!

moderate!or!even!low.!To!explain!tendencies!of!rising!residue!levels!it!would!be!

307!

relevant!to!investigate!actual!application!rates.!Global!production!figures!support!the!

308!

notion!that!very!large!quantities!of!glyphosate!are!being!sold!and!dispersed.!

309!

It!is!interesting!to!note!that!several!independent!researchers!have!mentioned!the!

310!

specific!question!of!glyphosateBresidues!in!glyphosateBtolerant!crops,!demonstrating!

311!

the!need!for!more!data!to!clarify!this!issue![45,!46,!61].!The!question!has!also!been!

312!

addressed!in!a!review!of!concepts!and!controversies!in!EFSA!environmental!risk!

313!

assessment!of!GMBcrops;!it!was!found!that!also!in!the!environmental!context!more!data!

314!

on!glyphosate!residues!is!needed,!as!postBharvest!biomass!is!potentially!affecting!both!

315!

soil!and!adjacent!environments![19].!

316!

Studies!of!glyphosateBtolerant!cultivar!composition!have!identified!differences!in!

317!

essential!plant!constituents,!which!have!been!attributed!to!in'plantae!metabolic!effects!

318!

of!glyphosate!residues![47,!48,!49].!This!research!indicates,!that!glyphosateBresidues!

319!

have!negative!effects!on!composition.!Contrary!to!this,!a!recent!review!by!authors!from!

320!

the!United!States!Department!of!Agriculture![50]!conclude!that!there!is!not!sufficient!

321!

evidence!for!claiming!that!glyphosate!in!glyphosateBtolerant!crops!a)!significantly!

322!

affects!mineral!composition!or!b)!changes!rhizosphere!microbial!community!or!c)!

323!

induces!susceptibility!to!disease.!

324!

As!a!direct!critique!of!the!regulatory!policies!enforced!of!the!European!Food!safety!

325!

Authority!EFSA,!independent!scientists!have!claimed!that!the!present!regime!of! !

12!

!

326!

industry!selfBcontrol!(autoregulation)!is!insufficient!to!provide!necessary!evidence!and!

327!

ensure!the!longBterm!interests!of!society.!Industry!studies!therefore!must!be!

328!

supplemented!with!additional,!independent,!research![19,!20].!This!however!is!not!a!

329!

view!shared!by!researchers!representing!interests!of!biotech!companies,!who!often!

330!

participate!in!systematic!opposition!to!any!results!questioning!industryBstudies.!It!has!

331!

been!described!as!highly!regrettable!that!independent!scientific!work!is!often!attacked!

332!

and!discredited!by!concerted!efforts!of!industry!proponents!and!journal!editors!loyal!to!

333!

biotech!sector!interests![28].!A!recent!study![51]!found!clear!evidence!of!doubleB

334!

standards!in!criteria!for!evaluation!of!safetyBstudies!on!GMO!cultivars!such!as!herbicideB

335!

resistant!plants.!The!authors!document!that!evidence!confirming!safety!is!not!exposed!

336!

to!same!the!intensive!scrutiny!as!evidence!indicating!possible!harm.!This!is!paradoxical,!

337!

as!it!should!be!evident!that!faulty!findings!in!the!first!of!these!categories!has!potential!

338!

for!inflicting!negative!effects!on!consumer!health.!Faulty!findings!in!the!second!category!

339!

will!not!have!the!same!implications,!but!may!lead!to!exaggerated!precaution,!which!can!

340!

be!conflicting!in!relation!to!commercial!interests.!

341!

Evidence!has!emerged!during!the!compilation!of!this!review,!which!to!a!certain!degree!

342!

confirms!the!claims!of!doubleBstandards:!One!of!the!industry!studies!reviewed!here!

343!

serves!as!a!noteworthy!example!of!malpractice.!The!study![31]!was!published!by!journal!

344!

Food!and!Chemical!Toxicology.!The!scientists!authoring!the!study!were!employees!of!

345!

commercial!companies!Pioneer+Hi'bred!and!DuPont.!They!conducted!a!safety!study!on!

346!

DPB356Ø43B5!glyphosateBtolerant!soybean!by!testing!cultivated!material!in!a!feedingB

347!

study!using!rats.!According!to!the!methods!chapter!of!the!study!the!tested!DPB356Ø43B5!

348!

glyphosateBtolerant!soybean!was!sprayed!with!glyphosate!herbicide.!Glyphosate!

349!

herbicide!was!the!only!pesticide!used!in!the!strictly!controlled!production!on!parallel!

350!

fields!of;!a)!glyphosateBtolerant!soy!(sprayed)!and!b)!unmodified!soy!(not!sprayed).!The!

351!

irregular!aspect!relates!to!the!fact!that!a!wide!array!of!subsequent!tests!for!pesticides!

352!

was!performed!on!the!produced!soy!materials,!screening!these!for!a!variety!of!active!

353!

ingredient!chemicals.!And,!although!glyphosateBherbicide!was!specified!to!be!the!only!

354!

pesticide!applied!in!the!strictly!controlled!testBplot!cultivation,!an!analysis!for!

355!

glyphosate!residues!was!omitted.!Instead!the!cultivated!material!was!analysed!for!

356!

numerous!herbicide!ingredients!that!were!fundamentally!irrelevant.!This!published!

357!

study!should!be!seen!as!an!example!supporting!the!arguments!demanding!revision!of!

!

13!

!

358!

the!regulatory!framework!mandating!selfBcontrol!of!biotech!industry!products.!

359!

Furthermore,!given!the!recent!heightening!of!qualitative!requirements!for!such!studies,!

360!

which!in!its!utmost!consequence!is!seen!as!retractions!of!publications,!I!nominate!the!

361!

mentioned!study![31]!as!a!prime!candidate!for!editorial!reBevaluation.!

362!

Other!reviews!of!published!testing:!

363!

Four!recent!reviews!of!produce!from!transgenic!plants!in!agriculture![52,!53,!54,!55]!

364!

present!evidence!confirming!transgenic!herbicideBtolerant!cultivar!equivalence,!as!

365!

compared!to!nonBmodified!comparators.!None!of!these!reviews!mention!herbicide!

366!

residues!or!their!potentially!conflicting!nature!in!relation!to!concept!of!substantial!

367!

equivalence.!Contrary!to!this,!three!reviews!by!independent!scientists!approach!the!role!

368!

of!herbicide!residues!in!transgenic!cultivars!or!present!indications!of!toxicity.!In!one!of!

369!

these![56]!the!authors!review!available!safety!assessment!and!speculate!whether!

370!

adverse!effects!reported!in!results!of!animalBtesting!published!in!2002![57],!2004![58]!

371!

and!2009![59]!could!be!attributable!to!pesticides!contained!in!the!tested!transgenic!

372!

material.!Another!recent!review![60]!concludes!that!parts!of!published!evidence!in!

373!

assessments!of!health!risks!of!GMO!foods!are!indications!of!general!toxicity.!!

374!

The!regulatory!developments!of!standards!for!defining!herbicide!residues!in!herbicide!

375!

resistant!crops!have!been!reviewed!recently!and!important!recommendations!have!

376!

been!presented![61].!The!recommendations!include!specific!measures,!such!as!the!

377!

concept!of!supervised+field+trials,!which!is!seen!as!important!potential!improvement!of!

378!

the!current!system!of!industry!selfBcontrol!and!scientific!autonomy.!

379!

The!future!of!herbicideBtolerant!crops:!

380!

Undoubtedly!transgenic!herbicide!tolerant!cultivars!are!popular!amongst!farmers!in!

381!

dominantly!important!agricultural!sectors!such!as!production!of!maize!and!soybean!in!

382!

countries!of!NorthB!and!South!America.!From!a!database!listing!transgenic!crop!

383!

varieties!pending!regulatory!approval![62]!it!seems!that!a!majority!of!these!GMBcrops!

384!

are!either!herbicide!tolerant!varieties!or!varieties!with!stacked!events!which!include!

385!

herbicide!tolerance.!!

386!

Some!new!varieties!have!herbicideBtolerance!traits!which!are!selected!from!

387!

microorganisms!systematically!bred!in!environments!with!high!glyphosate!

388!

concentrations![63].!Traditional!firstBgeneration!glyphosateBtolerant!crops,!such!as!the! !

14!

!

389!

GTSB40B3B2!soybean!which!still!dominates!global!production,!are!only!45B50!times!more!

390!

tolerant!than!unmodified!varieties!(the!glyphosate!dose!inducing!LC50Boutcome!in!GTSB

391!

40B3B2!is!about!50x!that!of!unmodified!soy).!!

392!

By!using!new!sources!of!transgenes!and!geneBstacks!with!combinations!of!several!

393!

transgenes!conveying!multiBpathway!tolerance!to!specific!active!ingredients,!secondB

394!

generation!cultivars!are!seen!as!having!significantly!improved!tolerance!to!specific!

395!

herbicides!or!combinations!of!herbicides.!This!development!should!be!seen!primarily!as!

396!

a!method!allowing!for!increased!herbicide!application.!!It!seems!that!in!the!onBgoing!

397!

struggle!to!eradicate!resistant!weeds,!the!agriculture!environments!rely!heavily!on!

398!

solutions!offered!by!commercial!producers!of!herbicides.!A!main!strategy!seems!to!be!

399!

developments!that!allow!for!higher!dosage!of!herbicides!such!as!glyphosate.!!

400!

It!is!recommended!that!such!developments!should!be!met!by!regulatory!initiative!to!

401!

ensure!necessary!oversight!of!secondary!consequences,!such!as!compositional!changes.!

402!

These!potential!changes!must!be!monitored!in!analysis!of!representative!material,!

403!

which!can!be!taken!as!samples!from!the!actual!agroBecological!production!systems.!

404!

The!present!maximal!residue!limits!(MRLs)!allow!for!relative!high!concentrations!of!

405!

herbicide!residues.!In!Brazil!in!2004!the!MRL!in!soybean!was!increased!from!0.2!mg/kg!

406!

to!10!mg/kg:!a!50Bfold!increase,!but!only!for!glyphosate!tolerant!soy![64].!In!Europe,!the!

407!

MRL!for!glyphosate!in!soybean!was!raised!by!a!factor!200!from!0.1!mg/kg!to!20!mg/kg!

408!

in!1999![66]!and!the!same!MRL!of!20!mg/kg!was!adopted!by!the!US!based!on!

409!

recommendations!of!the!Codex!Alimentarius!Commission.!In!2013!the!MRL!tolerance!

410!

levels!for!glyphosate!residues!in!US!soybean!were!raised!from!20!mg/kg!to!40!mg/kg!

411!

(Figure!1).!The!increases!coincide!with!industry!development!of!new!transgenic!

412!

varieties!with!stronger!tolerance!to!glyphosate.!In!these!cases!the!MRL!values!appear!to!

413!

have!been!adjusted!in!response!to!actual!observed,!or!expected,!increases!in!the!content!

414!

of!residues!in!glyphosateBtolerant!soybeans.!In!this!context!it!would!be!appropriate!to!

415!

collect!and!review!more!of!the!existing!data!on!glyphosate!residues!in!glyphosateB

416!

tolerant!crops.!However!relevant!such!a!question!may!be,!it!cannot!be!satisfactorily!

417!

answered!here!due!to!the!fact!that!only!sparse!published!information!exists!on!this!

418!

issue.!!

!

15!

!

419!

Despite!the!limited!number!of!analyses!for!glyphosate!residues!in!glyphosate!tolerant!

420!

crops,!the!few!tests!reported![30,!90]!indicate!surprisingly!high!levels!of!glyphosate!

421!

residues.!Such!findings!fundamentally!challenge!regulatory!assumption!of!substantial!

422!

equivalence!between!glyphosateBtolerant!varieties!and!their!unmodified!comparators.!!

423!

Substantial!equivalence:!

424!

The!principle!of!substantial!equivalence!is!fundamental!for!assessment!of!genetically!

425!

modified!plants,!so!some!explanation!is!justified!here.!Substantial!equivalence!is!a!

426!

concept!developed!by!OECD!in!1991B93![23],!establishing!that!a!novel!food,!for!example,!

427!

one!derived!from!genetic!modification!or!engineering,!should!be!considered!the!same!as!

428!

and!as!safe!as!a!conventional!food,!if!it!demonstrates!the!same!characteristics!and!

429!

composition!as!the!conventional!food![67].!In!1997,!the!European!Commission!

430!

regulated!its!policy!on!novel!foods!(from!transgenic!plants)!stating!that!food!and!feed!

431!

from!such!plants!are!expected!not!to!"present!a!danger!for!the!consumer",!or!"mislead!

432!

the!consumer",!or!"differ!from!foods!or!food!ingredients!which!they!are!intended!to!

433!

replace!to!such!an!extent!that!their!normal!consumption!would!be!nutritionally!

434!

disadvantageous!for!the!consumer"![68]!The!regulation!goes!on!to!state!that!"[this!

435!

policy...]!shall!apply!to!foods!or!food!ingredients![...]!which,!on!the!basis!of!the!scientific!

436!

evidence!available![...]!are!substantially!equivalent!to!existing!foods!or!food!ingredients!

437!

as!regards!their!composition,!nutritional!value,!metabolism,!intended!use!and!the!level!

438!

of!undesirable!substances!contained!therein"![68].!This!allows!a!discussion!on!the!

439!

qualitative!evaluation!of!substances!which!vary!from!benign!to!harmful.!It!seems!

440!

evident!that!pesticide!residues!belong!in!the!category!"undesirable!substances".!

441!

PostBmarket!monitoring:!

442!

European!Union!legislation![69]!specifies!framework!for!postBmarket!monitoring!of!

443!

transgenic!produce,!to!ensure!traceability!of!individual!feedBlots!entering!the!European!

444!

common!market.!This!is!important,!as!only!such!traceability!through!proper!labelling!

445!

will!ensure!that!possible!adverse!effects!from!specific!harvests!or!specific!batches!of!

446!

feed!can!be!identified.!At!present!the!USA,!which!is!the!largest!market!for!transgenic!

447!

consumer!products!entering!food!for!human!consumption,!has!a!lack!of!such!

448!

traceability.!In!the!USA,!this!situation!has!been!established!through!commercial!and!

449!

political!influence.!Contrary!to!this,!European!legislation!accommodates!traceability!of!

450!

feed!for!industrial!scale!production!of!farmed!animals.!Such!as!pigs,!poultry!and!cattle.! !

16!

!

451!

This!traceability!however,!is!not!enforced!at!present.!It!has!been!claimed!that!such!a!

452!

deductive!approach!to!material!quality!would!be!unfeasible![22].!Contrary!to!this!it!can!

453!

be!argued,!that!labelling!and!traceability!should!be!used!systematically!in!enforced!

454!

postBmarket!monitoring.!Especially!as!this!systematic!approach!allows!for!efficient!

455!

identification!of!possible!adverse!effects!from!novel!feed!ingredients!following!largeB

456!

scale!introduction.!In!guidance!documents!for!riskBassessment!of!food!and!feed!from!

457!

transgenic!plants,!EFSA!has!specifically!stated!the!need!for!post!market!monitoring!of!

458!

"undesirable!substances"![70].!This!is!a!clearly!defined!regulatory!intension.!Based!on!

459!

the!findings!on!potentially!high!residue!levels!reported!here,!it!is!recommended!that!

460!

EFSA!gives!priority!to!implementation!of!the!existing!regulation.!

461!

!

17!

!

461!

Conclusion(

462!

Of!30!reviewed!studies!on!composition!and!feedBquality!of!glyphosateBtolerant!GMBcrop!

463!

material,!only!half!of!the!studies!use!material!produced!with!application!of!glyphosate!

464!

herbicide.!Only!one!of!the!30!studies!has!analysed!the!material!for!glyphosate!residues.!!

465!

Application!of!representative!dosage!of!herbicides!as!well!as!subsequent!analysis!of!

466!

herbicide!residues!is!missing!in!industry!testing!of!glyphosateBtolerant!GMBcrops.!This!

467!

implies!that!central!data!from!compositional!analysis,!animal!feeding!studies!and!

468!

overall!riskBassessment!performed!by!industry!and!submitted!to!national!and!

469!

international!regulatory!bodies!as!evidence!as!safety,!is!not!representative!of!the!

470!

materials!actually!delivered!onto!the!commercial!market.!In!part!the!scientific!evidence!

471!

produced!by!industry!is!found!to!have!unacceptable!standard!for!regulatory!purpose.!

472!

Such!evidence!should!be!disregarded!and!demands!for!new!evidence!should!be!brought!

473!

forward.!

474!

Published!data!on!glyphosate!residues!in!glyphosateBtolerant!crops!are!sparse.!The!

475!

findings!presented!here!suggest!that!this!could!be!an!issue!with!important!implications.!

476!

Scientific!evidence!produced!by!biotechBindustry!companies!should!be!supplemented!

477!

with!data!from!independent!research.!Alternatively,!the!riskBassessments!and!analyses!

478!

performed!by!industry!should!be!competently!supervised!to!ensure!transparency!and!

479!

an!overall!satisfactory!standard!of!testing.!!

480!

This!leads!to!a!recommendation!to!regulatory!authorities!such!as!the!European!Food!

481!

Safety!Authority!(EFSA)!and!the!Organisation!for!Economic!Cooperation!and!

482!

Development!(OECD)!to!apply!necessary!measures!and!enforce!routines.!!

483!

Regular!revision!of!regulatory!framework,!routines!and!operating!procedures!is!needed!

484!

to!secure!future!quality!of!important!food!and!feed!material.!Such!action!is!fundamental!

485!

for!safeguarding!coherence,!relevance!and!public!trust.!

486!

!

18!

!

486!

!

487!

!

488!

Competing!interest:!The!author!declares!that!he!has!no!competing!interests.!

489!

!

490!

Acknowledgements:!The!work!is!funded!by!the!Norwegian!Research!Council,!project!

491!

84107!MiljøB2015.!

492!

!

!

19!

!

Reference

Year

Author

published [35]#

1996

Author

Crop studied

Subject

Main finding

Padgette et al.

Industry

1996

Nida et al.

Industry

GTS 40-3-2 soybean

Compositional

Substantial equivalence assumed

GT-cotton

Compositional

1999

Taylor et al.

Industry

GTS 40-3-2 soybean

Compositional

Substantial equivalence assumed

2000

Sidhu et al.

Industry

GT-corn

Compositional

Not relevant

Glyphosate herbicide not

Not relevant

applied Substantial equivalence assumed

analysis [71]#

Glyphosate herbicide not applied

analysis [44]#

Relevant analysis for herbicide residues

analysis [36]#

Relevant co-technology

affiliation

Glyphosate herbicide applied

No analysis

at prescribed rate Substantial equivalence assumed

Unclear

No analysis

Substantial equivalence assumed

Glyphosate herbicide applied

No analysis

analysis [43]#

2002

Ridley et al.

Industry

GT-maize NK603

Compositional analysis

[72]#

2004

Sidhu et al.

Industry

GT-corn

Compositional

at prescribed rate Substantial equivalence assumed

Unclear

No analysis

Substantial equivalence assumed

Glyphosate herbicide not

Not relevant

analysis [73]#

2004

Obert et al.

Industry

GT-wheat MON71800

Compositional analysis

[40]#

2005

McCann et al.

Industry

GTS 40-3-2 soybean

Compositional

applied Substantial equivalence assumed

analysis [42]#

2006

McCann et al.

Industry

GT-alfalfa

Compositional

2007

Harrigan et al

Industry

GTS 40-3-2 soybean

Compositional

Substantial equivalence assumed

2007

McCann et al.

Industry

GT-corn MON88017

Compositional

Glyphosate herbicide applied

No analysis

at prescribed rate Substantial equivalence assumed

analysis [41]#

No analysis

at prescribed rate

analysis [38]#

Glyphosate herbicide applied

Glyphosate herbicide applied

No analysis

at prescribed rate Substantial equivalence assumed

Unclear

No analysis

Substantial equivalence assumed

Glyphosate herbicide applied

No analysis

analysis [39]#

2008

Lundry et al.

Industry

GT-soybean MON89788

Compositional analysis

[74]#

2010

Berman et al.

Industry

GT-soybean MON89788

Compositional

at prescribed rate Substantial equivalence assumed

analysis [30]#

2014

Bøhn et al.

Public

GTS 40-3-2 soybean

(university)

Compositional

Glyphosate herbicide not

No analysis

applied Significant differences found

analysis

Material from farm-fields:

Pesticiode analysis

Glyphosate herbicides

performed and

applied at realistic

reported

representative rate [75]#

2014

Delaney et al.

Industry

GT-canola DP-Ø73496-4

Compositional analysis

Substantial equivalence assumed

Glyphosate herbicide applied at prescribed rate

Table 1. Compositional analysis. 15 Published studies comparing compositional quality of transgenic glyphosate-tolerant crop varieties with unmodified conventional comparators.

!

1!

No analysis

!

Reference

Year

Author

published [34]#

1996

Author

Crop studied

Subject

Main finding

Relevant co-technology

affiliation Hammond et al.

Industry

Relevant analysis for herbicide residues

GTS 40-3-2 soybean

Feeding study in rat,

No significant effects found.

Glyphosate herbicide not

chicken, catfish and

Equivalence assumed

applied

Not relevant

No significant effects found.

Glyphosate herbicide applied

Equivalence assumed

at prescribed rate

No significant effects found.

Glyphosate herbicide applied

Equivalence assumed

at prescribed rate

Reduced feeding and weight gain significant

Unclear

No analysis

Unclear

No analysis

cattle [37]#

[76]#

2002

2004

Cromwell et al.

Brake et al.

Industry

Public

GTS 40-3-2 soybean

GTS 40-3-2 soybean

Feeding study in swine

Feeding study in mouse

(university) [77]#

2004

Zhu et al.

Public

GTS 40-3-2 soybean

Feeding study in rat

(university)

No analysis

No analysis

in young rat treatment group. Not significant in adult rat

[84]#

2007

Taylor et al.

Industry

GT-soy MON 89788

Feeding study in broiler

No significant effects found. Equivalence assumed

[88]#

[31]#

[78]#

2007

2008

2008

Bakke-McKellet et

Public

al.

(university)

Appenzeller et al.

Industry

Healy et al.

Industry

GTS 40-3-2 soybean

Feeding study in salmon

Significant effects found

Unclear

No analysis

GT-soy DP-356Ø43-5

Feeding study in rat

No significant effects found.

Glyphosate herbicide applied

Irrelevant analysis

Equivalence assumed

at prescribed rate

presented

No significant effects found.

Glyphosate herbicide applied

Pesticiode analysis

Equivalence assumed

at prescribed rate

performed and

GT-maize MON88017

Feeding study in rat

reported [79,#80]# [89]#

2008

Malatesta et al.

University

GTS 40-3-2 soybean

Feeding study in mouse

Significant effects found

Unclear

No analysis

2009

Sissener et al.

Public

GTS 40-3-2 soybean

Life-long feeding in

Significant effects found

Unclear

No analysis

Significant effects found

Glyphosate herbicide applied

No analysis

(university) [81]#

2012

Seralini et al.

Public

salmon GT-maize NK603

(university) [82]#

2013

Carman et al.

Public

Life-long feeding study in rat

Mixed diets with NK603

at prescribed rate

Long-term study in pig

Significant effects found

GT-maize

Material from farm-fields:

No analysis

Glyphosate herbicides applied at realistic representative rate

[83]#

2014

Cuhra et al.

Public

GTS 40-3-2 soybean

(university)

Life-long feeding study

Significant effects found

in crustacean D. magna

Material from farm-fields:

Pesticiode analysis

Glyphosate herbicides

performed and

applied at realistic

reported

representative rate [75]#

2014

Delaney et al.

Industry

HT-canola DP-Ø73496-

Rodent

4 [32]#

2015

Cuhra et al.

Public (university)

GTS 40-3-2 soybean

No significant effects found.

Unclear

No analysis

Material from farm-fields:

Pesticiode analysis

Glyphosate herbicides

performed and

applied at realistic

reported

Equivalence assumed Life-long feeding study in crustacean D. magna

Significant effects found

representative rate

Table!2.!Animal#feeding#studies.!15!Published!studies!comparing!transgenic!glyphosate:tolerant!crop!varieties!with!unmodified!conventional!comparators!as!feed!in!animal!feeding! studies.

!

2!

1" Herman'R,'Price''WD'(2013)'Unintended'Compositional'Changes'in'Genetically'

Modified'(GM)'Crops:'20'Years'of'Research.'J.'Agric.'Food''Chem.,'dx.doi.org/10.1021.'

2" Amrhei'et'al'1980' 3" the'620'kilotonn'reference'on'Gly'productioin' 4" Szeḱács'A,'Darvas'B'(2012)'Forty'Years'with'Glyphosate.'In;'Herbicides'Z'Properties,' 5" 6" 7" 8" 9" 10" 11" 12" 13" 14" 15" 16" 17" 18" 19" 20" 21" 22" 23" 24" 25" 26" 27" 28" 29" 30" 31"

Synthesis'and'Control'of'Weeds,'Dr.'Mohammed'Nagib'Hasaneen'(Ed.),'InTech' (www.intechopen.com'accessed'March'2014).' Cuhra'2013'glyphosate'ecotox' Halcke'&'reinecen'dessiccation' reference'on'anbtibiotic'patented'qualities' Benbrook'CM'(2012)'Impacts'of'genetically'engineered'crops'on'pesticide'use'in'the' U.S.'ZZ'the'first'sixteen'years.'Env.'Sci.'Eu.,'24(24):'1Z13.' Giesy'2000' Williams'2000' Senef,'on'the'subtle'effects'of'glyphosate' EFSA'2014'on'pesticide'residues' ref'on'gly'in'air' ref'on'gly'in'human'blood,'milk'and'urine' human'urine'scepticism' Bonny'S'(2011)'HerbicideZtolerant'Transgenic'Soybean'over'15'Years'of'Cultivation:' Pesticide'Use,'Weed'Resistance,'and'Some'Economic'Issues.'The'Case'of'the'USA.' Sustainability'3(9):'1302Z1322.' Duke'&'powles'2008,'gly'the'ideal'herbicide' James'C'(2010)'A'global'overview'of'biotech'(GM)'crops:'Adoption,'impact'and'future' prospects.'GM'Crops'1,'8–12.'doi:10.4161/gmcr.1.1.9756.' Hilbeck,'A'et'al.,'2011.'Environmental'risk'assessment'of'genetically'modified'plantsZ concepts'and'controversies.'Environ.'Sci.'Eur.'Z'Env.'Sci'Eur'23.'doi:10.1186/2190Z 4715Z23Z13' Dolezel'and'Hilbeck'2013' Kuiper'HA,'Kleter'GA,'Noteborn'HPJM,'Kok'EJ'(2001)'Assessment'of'the'food'safety' issues'related'to'genetically'modified'foods.'The'Plant'Journal,'27:'503–528.' Kok'EJ,'Keijer'J,'Kleter'GA,'Kuiper'HA'(2008)'Comparative'safety'assessment'of'plantZ derived'foods.'Regul.'Toxicol.'Pharm.,'50:'98–113.' OECD'(1993)'Safety'Evaluation'of'Foods'Derived'by'Modern'Biotechnology.'Concepts' and'Principles.'Organisation'for'Economic'CoZoperation'and'Development'OECD'Paris,' France.'' EFSA'(2010)'Guidance'on'the'environmental'risk'assessment'of'genetically'modified' plants.'EFSA'journal'8(11):'1Z111'(1879).' Haslberger'A'(2003)'Codex'guidelines'for'GM'foods'include'the'analysis'of'unintended' effects.'Nature'Biotechnol.,'21(7):'739Z740.' FAO'(2001)'Feasibility'of'establishing'MRLs'for'genetically'modified'crops'and' metabolite'residues.'Codex'Alimentarius'commission.'Food'and'Agriculture' Organization'of'the'United'Nations,'The'Hague,'Netherlands.' US'FDA'biotech'policy'the'1992'brief' Waltz'E'(2009)'Battlefield.'Nature,'461:'27Z32.' Nielsen'K'(2013)'Biosafety'Data'as'Confidential'Business'Information.'PLOS'Biology,' 11(3)'e1001499.' Bøhn'T,'Cuhra'M,'Traavik'T,'Sanden'M,'Fagan'J,'Primicerio'R'(2014)'Compositional' differences'in'soybeans'on'the'market:'Glyphosate'accumulates'in'Roundup'Ready'GM' soybeans.'Food'Chem.,'153,'207–215.' Appenzeller'LM,'Munley'SM,'Hoban'D,'Sykes'GP,'Malley'LA,'Delaney'B'(2008)'

32" 33" 34"

34" 35" 36" 37"

38"

39"

40" 41"

42" 43" 44" 45" 46" 47"

Subchronic'feeding'study'of'herbicide–tolerant'soybean'DPZ356Ø43Z5'in'Sprague– Dawley'rats.'Food'Chem.'Toxicol.,'46:'2201–2213.' Cuhra'2015'JACEN' Millstone'E,'Brunner'E,'Mayer'S'(1999)'Beyond'substantial'equivalence.'Nature,'401,' 525Z526.' Hammond'BG,'Vicini'JL,'Hartnell'GF,'Naylor'MW,'Knight'CD,'Robinson'EH,'Fuchs'RL,' Padgette'SR'(1996)'The'feeding'value'of'soybeans'fed'to'rats,'chickens,'catfish'and' dairy'cattle'is'not'altered'by'incorporation'of'glyphosate'tolerance.'J.'Nutr.,'126:'717Z 727.'' Viljoen'C'(2013)'Letter'to'the'Editor.'Food'Chem.'Toxicol.,'59:'809–810.' Padgette'SR,'Taylor'NB,'Nida'DL,'Bailey'MR,'MacDonald'J,'Holden'LR,'Fuchs'RL'(1996)' The'composition'of'glyphosateZtolerant'soybean'seeds'is'equivalent'to'that'of' conventional'soybeans.'J.'Nutr.,'126:'702–716.' Nida'DL,'Patzer'S,'Harvey'P,'Stipanovic'R,'Wood'R,'Fuchs'RL'(1996)'GlyphosateZ tolerant'cotton:'the'composition'of'the'cottonseed'is'equivalent'to'that'of'conventional' cottonseed.'J.'Agric.'Food'Chem.,'44:'1967–1974.' Cromwell'GL,'Lindemann'MD,'Randolph'JH,'Parker'GR,'Coffey'RD,'Laurent'KM,' Armstrong'CL,'Mikel'WB,'Stanisiewski'EP,'Hartnell'GF'(2002)'Soybean'meal'from' roundup'ready'or'conventional'soybeans'in'diets'for'growingZfinishing'swine.'J.'Anim.' Sci.,'80(3):'708Z715.' Harrigan'GG,'Ridley'WP,'Riordan'SG,'Nemeth'MA,'Sorbet'R,'Trujillo'WA,'Breeze'ML,' Schneider'RW'(2007)'Chemical'composition'of'glyphosateZtolerant'soybean'40–3Z2' grown'in'Europe'remains'equivalent'with'that'of'conventional'soybean'(Glycine'max' L.).'J.'Agric.'Food'Chem.,'55:'6160–6168.' Lundry'DR,'Ridley'WP,'Meyer'JJ,'Riordan'SG,'Nemeth'MA,'Trujillo'WA,'Breeze'ML,' Sorbet'R'(2008)'Composition'of'grain,'forage,'and'processed'fractions'from'secondZ generation'glyphosateZtolerant'soybean,'MON'89788'Is'equivalent'to'that'of' conventional'soybean'(Glycine'max'L.).'J.'Agric.'Food'Chem.,'56:'4611–4622.' McCann'MC,'Liu'K,'Trujillo'WA,'Dobert'RC'(2005)'GlyphosateZtolerant'soybeans'remain' compositionally'equivalent'to'conventional'soybeans'(Glycine'max'L.)'during'three' years'of'field'testing.'J.'Agric.'Food'Chem.,'53:'5331–5335.' McCann'MC,'Trujillo'WA,'Riodan'SG,'Sorbet'R,'Bogdanova'NN,'Sidhu'RS'(2007)' Comparison'of'the'forage'and'grain'composition'from'insectZprotected'and'glyphosateZ tolerant'MON'88017'corn'to'conventional'corn'(Zea'mays'L.).'J.'Agric.'Food'Chem.,'55:' 4034–4042.' McCann'MC,'Rogan'GJ,'Fitzpatrick'S,'Trujillo'WA,'Sorbet'R,'Hartnell'GF,'Riodan'SG,' Nemeth'MA'(2006)'GlyphosateZtolerant'alfalfa'is'compositionally'equivalent'to' conventional'alfalfa'(Medicago'sativa'L.).'J.'Agric.'Food'Chem.,'54:'7187–7192.' Ridley'WP,'Sidhu'RS,'Pyla'PD,'Nemeth'MA,'Breeze'ML,'Astwood'JD'(2002)'Comparison' of'the'nutritional'profile'of'glyphosateZtolerant'corn'event'NK603'with'that'of' conventional'corn'(Zea'mays'L.).'J.'Agric.'Food'Chem.,'50:'7235–7243.' Taylor'NB,'Fuchs'RL,'MacDonald'J,'Shariff'AR,'Padgette'SR'(1999)'Compositional' analysis'of'glyphosateZtolerant'soybeans'treated'with'glyphosate.'J.'Agric.'Food'Chem.,' 47:'4469–4473.'' Arregui'MC,'Lenardon'A,'Sanchez'D,'Maitre'MI,'Scotta'R,'Enrique'S'(2004)'Monitoring' glyphosate'residues'in'transgenic'glyphosateZresistant'soybean.'Pest'Man.'Sci.,'60:'163Z 166.' Duke'SO,'Rimando'AM,'Pace'PF,'Reddy'KN,'Smeda'RJ'(2003)'Isoflavone,'glyphosate,' and'aminomethylphosphonic'acid'levels'in'seeds'of'glyphosateZtreated,'glyphosateZ resistant'soybean.'Z'J.'Agric.'Food'Chem.,'51:'340Z344.' Zobiole'LHS,'Oliveira'Jr.'RS,'Kremer'RJ,'Constantin'J,'Yamada'T,'Castro'C,'Oliveira'FA,' Oliveira'Jr.'A'(2010a)'Effect'of'glyphosate'on'symbiotic'N2'fixation'and'nickel' concentration'in'glyphosateZresistant'soybeans.'Appl.'Soil'Ecol.,'44'(2):'176–180.'

48" Zobiole'LHS,'Oliveira'RS,'Visentainer'JV,'Kremer'RJ'Bellaloui'N,'Yamada'T'(2010b)' 49" 50" 51" 52" 53" 54" 55"

56" 57"

58" 59" 60" 61" 62" 63" 64" 65" 66" 67" 68" 69"

Glyphosate'Affects'Seed'Composition'in'GlyphosateZResistant'Soybean.'J.'Agric.'Food' Chem.'58:'4517–4522.' Zobiole'LHS,'Kremer,'RJ,'Oliveira'Jr.'RS,'Constantin'J'(2011)'Glyphosate'affects' chlorophyll,'nodulation'and'nutrient'accumulation'of'“second'generation”'glyphosateZ resistant'soybean'(Glycine'max'L.).'Pestic.'Biochem.'Physiol.,'99(1):'53–60.' Duke'SO,'Lydon'J,'Koskinen'WC,'Moorman'TB,'Chaney'RL,'Hammerschmidt'R'(2012)' Glyphosate'Effects'on'Plant'Mineral'Nutrition,'Crop'Rhizosphere'Microbiota,'and'Plant' Disease'in'GlyphosateZResistant'Crops.'J.'Agric.'Food'Chem.,'60:'10375Z10397.' Wickson'F,'Bøhn'T,'Wynne'B,'Hilbeck'A,'Funtowicz'S'(2013)'ScienceZBased'Risk' Assessment'Requires'Careful'Evaluation'of'All'Studies.'Nature'Biotechnol.,'31(12):' 1077–1078.' Delaney'B'(2007)'Strategies'to'evaluate'the'safety'of'bioengineered'foods.'Int.'J.' Toxicol.,'26:'389–399.'' Harrigan'GG,'Lundry'D,'Drury'S,'Berman'K,'Riordan'SG,'Nemeth'MA,'Ridley'WP,'Glenn' KC'(2010)'Natural'variation'in'crop'composition'and'the'impact'of'transgenesis.' Nature'Biotechnol.,'28:'402–404.' Ricroch'A,'Bergé'JB,'Kuntz'M'(2011)'Evaluation'of'Genetically'Engineered'Crops'Using' Transcriptomic,'Proteomic,'and'Metabolomic'Profiling'Techniques.'Plant'Physiol' 155(4):'1752Z1761.' Snell'C,'Berheim'A,'Bergé'JB,'Kuntz'M,'Pascal'G,'Paris'A,'&'Ricroch'A.'(2011)' Assessment'of'the'Health'Impact'of'GM'Plant'Diets'in'Long'Term'and'Multigenerational' Animal'Feeding'Trials:'a'Literature'Review.'Food'Chem.'Tox.' doi:10.1016/j.fct.2011.11.048' Domingo'JL,'Bordonaba'JG'(2011)'A'literature'review'on'the'safety'assessment'of' genetically'modified'plants.'Environment'International,'37(4):'734Z742.' Malatesta'M,'Caporaloni'C,'Gavaudan'S,'Rocchi'MBL,'Serafini'S,'Tiberi'C,'Gazzanelli'G' (2002)'Ultrastructural'morphometrical'and'immunocytochemical'analyses'of' hepatocyte'nuclei'from'mice'fed'on'genetically'modified'soybean.'Cell'Struct.'Funct.,'27,' 173Z180.' Vecchio''L,'Cisterna'B,'Malatesta'M,'Martin'TE,'Biggiogera'M'(2004)'Ultrastructural' analysis'of'testes'from'mice'fed'genetically'modified'soybean.'Eur.'J.'Histochem.,'48:' 448Z54.' Vendemois'JS,'Roullier'F,'Cellier'D,'Seralini'GE'(2009)'A'comparison'of'the'effects'of'GM' corn'varieties'on'mammalian'health.'Int.'J.'Biol.'Sci.,'5:'706Z26.' Dona'A,'Arvanitoyannis'IS'(2009)'Health'Risks'of'Genetically'Modified'Foods.'Crit.'rev.' Food'Sci.'Nutr.,'49:'164Z175.' Kleter'GA,'Unsworth'JB,'Harris'CA'(2011)'The'impact'of'altered'herbicide'residues'in' transgenic'herbicideZresistant'crops'on'standard'setting'for'herbicide'residues.'Pest' Manag.'Sci.,'67:'1193–1210.' ISAAA'(2014)'GM'Approval'Database.'International'Service'for'the'Acquisition'of'AgriZ biotech'Applications'(ISAAA)'(www.isaaa.org/gmapprovaldatabase/'Z'accessed'March' 2014)' Cao'G,'Liu'Y,'Liu'G,'Wang'J,'Wang'G'(2013)'Draft'Genome'Sequence'of'Pseudomonas' Strain'P818,'Isolated'from'GlyphosateZPolluted'Soil.'Genome'Announc.,'1,'e01079–13.' agencia'nacional'de'vigilancia'sanitaria'Brazil'2003'&'2004' testbiotech'argentina'soy'report' reference'for'European'MRL'soybean'Z'in'Samsel'and'seneff'2013' Womach'2005'Z'intro'the'thesis' EC'1997'Z'see'thesis'intro' EC'(2003)'Regulation'1830/2003'of'the'European'Parliament'and'of'the'Council'of'22' September'2003'concerning'the'traceability'and'labelling'of'genetically'modified' organisms'and'the'traceability'of'food'and'feed'products'produced'from'genetically'

70" 71"

72" 73"

74"

75" 76" 77" 78" 79" 80" 81"

82" 83" 84"

85" 86"

modified'organisms'and'amending'Directive'2001/18/EC.' http://europa.eu/legislation_summaries/environment/nature_and_biodiversity/l2117 0_en.htm' EFSA'(2011)'Guidance'on'the'PostZMarket'Environmental'Monitoring'(PMEM)'of' genetically'modified'plants.'EFSA'Journal,'9(8):'2316.' Sidhu'RS,'Hammond'BG,'Fuchs'RL,'Mutz'JN,'Holden'LR,'George'B,'Olson'T'(2000)' GlyphosateZtolerant'corn:'the'composition'and'feeding'value'of'grain'from'glyphosateZ tolerant'corn'is'equivalent'to'that'of'conventional'corn'(Zea'mays'L.).'J.'Agric.'Food' Chem.,'48:'2305–2312.' Sidhu'RS,'Brown'S'(2004)'Petition'for'Determination'of'Nonregulated'Status'for'MON' 88017'Corn'(www.aphis.usda.gov/brs/aphisdocs/04Z12501p.pdf'Z'accessed'March' 2014).' Obert'JC,'Ridley'WP,'Schneider'RW,'Riordan'SG,'Nemeth'MA,'Trujillo'WA,'Breeze'ML,' Sorbet'R,'Astwood'JD'(2004)'The'composition'of'grain'and'forage'from'glyphosate' tolerant'wheat'MON'71800'is'equivalent'to'that'of'conventional'wheat'(Triticum' aestivum'L.).'J.'Agric.'Food'Chem.,'52'(2004),'pp.'1375–1384.' Berman'KH,'Harrigan'GG,'Riordan'SG,'Nemeth'MA,'Hanson'C,'Smith'M,'Sorbet'R,'Zhu'E,' Ridley'WP'(2010)'Compositions'of'forage'and'seed'from'secondZgeneration' glyphosateZtolerant'soybean'MON'89788'and'insectZprotected'soybean'MON'87701' from'Brazil'are'equivalent'to'those'of'conventional'soy'bean'(Glycine'max).'J.'Agric.' Food'Chem.,'58:'6270–6276.' Delaney'B,'Appenzeller'LM,'Roper'JM,'Mukerji'P,'Hoban'D,'Sykes'GP'(2014)'Thirteen' Week'Rodent'Feeding'Study'with'Processed'Fractions'from'Herbicide'Tolerant'(DPZ Ø73496Z4)'Canola.'Food'Chem.'Toxicol.'' brake'2004' Zhu'Y,'Li'D,'Wang'F,'Yin'J,'Jin'H'(2004)'Nutritional'assessment'and'fate'of'DNA'of' soybean'meal'from'Roundup'Ready'or'conventional'soybeans'using'rats.'Arch.'Anim.' Nutr.,'58(4):'295Z310.' Healy'C,'Hammond'B,'Kirkpatrick'J'(2008)'Results'of'a'13Zweek'safety'assurance'study' with'rats'fed'grain'from'corn'rootwormZprotected,'glyphosateZtolerant'MON'88017' corn,'Food'Chem.'Toxicol.,'46(7):'2517Z2524.' Malatesta'M,'Boraldi'F,'Annovi'G,'Baldelli'B,'Battistelli'S,'Biggiogera'M,'Quaglino'D' (2008)'A'longZterm'study'on'female'mice'fed'on'a'genetically'modified'soybean:'effects' on'liver'ageing.'Histochem.'Cell'Biol.,'130,'967Z977.' Malatesta'M,'Boraldi'F,'Annovi'G,'Baldelli'B,'Battistelli'S,'Biggiogera'M,'Quaglino'D' (2008b)'A'longZterm'study'on'female'mice'fed'on'a'genetically'modified'soybean:' effects'on'liver'ageing.'Histochem.'Cell'Biol.,'130:'967Z977.' Séralini'GE,'Clair'E,'Mesnage'R,'Gress'S,'Defarge'N,'Malatesta'M,'Hennequin'D,'de' Vendômois'JS'(2014)'Long'term'toxicity'of'a'Roundup'herbicide'and'a'RoundupZ tolerant'genetically'modified'maize.'Environmental'Sciences'Europe,'26'(1):'14.'doi:' 10.1186/s12302Z014Z0014Z5.' Carman'JA,'Vlieger'HR,'Ver'Steeg'LJ,'Sneller'VE,'Robinson'GW,'ClinchZJones'CA,'Haynes' JI,'Edwards'JW'(2013)'A'longZterm'toxicology'study'on'pigs'fed'a'combined'genetically' modified'(GM)'soy'and'GM'maize'diet.'J.'Organic'Syst.,'8(1):38Z54.' Cuhra'M,''Traavik'T,'Bøhn'T'(2014)'Life'cycle'fitness'differences'in'Daphnia'magna'fed' RoundupZReady'soybean'or'conventional'soybean'or'organic'soybean.'DOI:' 10.1111/anu.12199.' Taylor'M,'Hartnell'G,'Lucas'D,'Davis'S,'Nemeth'M'(2007)'Comparison'of'broiler' performance'and'carcass'parameters'when'fed'diets'containing'soybean'meal' produced'from'glyphosateZtolerant'(MON'89788),'control,'or'conventional'reference' soybeans.'Poult.'Sci.'86,'2608–2614.' antoniou,'robinson'and'fagan' Teratogenic'effects'gly/R'Robinson'et'al'

87" robinson,'holland,'leloup,'muilerman'2013'conflicts'of'interest'at'EFSA' 88" bakke'mckellep'et'al'2007'salmon'fed'gtZsoy' 89" sissener'et'al'2009'histomorphology'of'organs'in'longterm'feeding'trials'w'salmon'fed' gt'soy'

90" Then,'C'(2013)'High'levels'of'residues'from'spraying'with'glyphosate'found'in' soybeans'in'Argentina.'TestBiotech'background'report'22Z10Z2013'

" ' " Codex'(2003)'Guideline'for'the'Conduct'of'Food'Safety'Assessment'of'Foods'Derived' from'RecombinantZDNA'Plants'(CAC/GL'45Z2003).'

" EC'1997' " OECD'(1998)'Report'of'the'OECD'Workshop'on'the'Toxicological'and'Nutritional' "

" "

" " " " " " " " " " " " " " " " " " " " " " " " "

Testing'of'Novel'Foods.'Organisation'for'Economic'Cooperation'and'Development,' Paris,'France.' OECD'(2001)'Consensus'Document'on'Compositional'Considerations'for'New'Varieties' of'Soybean:'Key'Food'and'Feed'Nutrients'and'Antinutrients.'OECD'Environmental' Health'and'Safety'Publications'Series'on'the'Safety'of'Novel'Foods'and'Feeds,'No.'2.' Environment'Directorate,'Organisation'for'Economic'CoZoperation'and'Development,' Paris,'France.' OECD'(2006)'An'Introduction'to'the'Food/Feed'Safety'Consensus'Documents'of'the' Task'Force.'Organization'for'Economic'Cooperation'and'Development,'Paris,'France.' Taylor'ML,'Y'Hyun'GF,'Hartnell'MA,'Nemeth'M,'Karunanandaa'K,'George'B,'Glenn'KC' (2005)'Comparison'of'broiler'performance'when'fed'diets'containing'MON'88017,' MON'88017'x'MON'810,'control,'or'commercial'corn.'Poultry'Sci.,'83(S1):'322'(Abst.' W39)' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' '

" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " "

' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' '

" " " " " " " " " " " " " " " " " " " " " '

' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' '

!

Discussion This!work!began!with!a!research!question!regarding!the!Roundup9ready!soybean!at!the! centre!of!attention!here,!asking!whether!GTS9409392!genetically!modified!glyphosate9 tolerant!soy!is!substantially!equivalent!to,!or!significantly!different!from,!its!unmodified! origin.!From!the!published!papers!I!bring!several!conclusions!forward!for!further! discussion,!primarily!the!findings!of!glyphosate!toxicity!and!the!finding!of!glyphosate! residues!accumulated!in!Roundup9ready!soybean.!A!key!issue!is!whether!such!residues! contribute!to!making!Roundup9ready!soybean!substantially!different!from!conventional! and!organic!varieties.!The!documented!compositional!differences!go!beyond!the!narrow! question!of!residue!presence!and!are!also!seen!as!differences!in!concentrations!of! specific!minerals!and!nutrients.!It!has!not!been!possible!to!deduce!whether!those! compositional!differences!primarily!are!consequences!of!seed!genotype,!of!agriculture! system!in!general!or!of!glyphosate!herbicide!application!in!particular.! Life9long!feeding!studies!in!the!animal!model!indicate!that!the!systematic!compositional! differences!seen!in!the!3!categories!of!soybean,!are!also!systematically!and!significantly! affecting!test!animal!performance.!In!D.#magna,!organic!soybeans!allow!for!better! longevity!and!reproductive!success,!compared!to!the!two!industrial!varieties.!Of!these,! the!transgenic!soybeans!systematically!give!the!poorest!performance!in!the!test9 animals.!Subsequent!life9long!feeding!studies!with!8!distinct!diets!produced!from! transgenic!soy!indicate!a!correlation!between!magnitude!of!residue!and!negative!effect! on!animal!performance,!measured!as!longevity,!growth!and!reproduction.! The!results!of!the!ecotoxicological!testing!indicate!that!glyphosate!is!more!toxic!to!D.# magna!than!previous!published!results!have!stated.!Low!levels!of!Roundup!herbicide! and!glyphosate!in!the!living9environment!of!D.magna!is!shown!to!induce!reduced! survival,!growth!and!fecundity.!It!is!thus!not!unreasonable!to!relate!the!effects!we!see!in! animals!given!progressive!concentrations!of!these!chemicals!in!feed,!to!effects! previously!observed!from!progressive!concentrations!of!the!same!chemicals!in!the! aqueous!living!environment.! From!the!laboratory!studies!in!the!animal!model!it!cannot!be!excluded,!that!glyphosate! residues!have!a!significant!negative!effect,!even!at!levels!well!below!the!limits!currently! !

47! !

!

accepted!by!society.!A!review!of!30!scientific!publications!assessing!quality!of! glyphosate9tolerant!transgenic!plants!demonstrates!that!the!issue!of!accumulation!of! herbicide!residues!in!glyphosate!tolerant!varieties,!still!is!an!unanswered!question.! There!is!little!published!evidence!available!for!researchers!and!regulators!attempting!to! assess!the!issue.!! The#work#thus#not#only#indicates#that#glyphosate#residues#in#soybean#feed#can#have# negative#effect#on#animal#consumers#in#life;long#feeding.#The#work#also#indicates,#that# this#question#has#not#been#sufficiently#addressed#previously.## The#overall#indication#from#this#research,#is#a#ladder#of#scientific#evidence#suggesting# that#herbicide#residues#in#glyphosate;tolerant#cultivars#such#as#soybean#should#be# investigated#further,#with#the#aim#of#determining#whether#these#residues#are#truly#as# harmless#as#previously#thought.# The!following!discussion!describes!these!points!in!some!detail,!based!on!the!findings!of! individual!papers!and!other!evidence.! Glyphosate+ecotoxicity+ It!is!found!that!glyphosate!and!Roundup!induce!mortality!in!D.#magna!at!EC50(48h)! concentrations!below!10!mg/l!in!48!h!acute!experiments.!Chronic!exposure!to! glyphosate,!particularly!when!given!as!formulated!herbicide!Roundup,!causes! significant!biological!effects!at!levels!of!0.0590.15!mg/l!and!serious!reproduction! damage!at!levels!of!1.35!mg/l,!which!is!close!to!or!even!below!accepted!threshold!values! for!glyphosate!in!surface!waters!in!the!United!States!(paper+I).!! There!were!only!minor!differences!in!tolerance!to!acute!exposure!of!glyphosate!and! Roundup!between!laboratory!clones,!clones!from!natural!ponds!and!clones!taken!from! ponds!surrounded!by!intensive!agriculture.! The!work!shows!a!relatively!uniform!susceptibility!to!glyphosate!and!Roundup!between! clones!of!D.#magna.!This!is!in!contrast!to!the!extreme!variation!seen!between!published! studies.!Accordingly,!the!highly!varying!EC50!values!in!D.#magna,!and!other!species!of! daphnids!reported!in!printed!reviews!should!not!be!primarily!attributed!to!interclonal! differences.!! D.#magna!!toxicity!tests!are!generally!considered!reproducible!and!representative,!with! ! 48! !

!

only!small!variation!between!laboratories.!Still,!test!conditions!such!as!neutralizing! capacity!of!holding!medium,!laboratory!environments!or!other!contextual!factors!may! cause!the!discrepant!results.!Furthermore,!the!solubility!of!the!different!glyphosate! based!chemicals!may!be!a!decisive!factor!in!glyphosate!toxicity!testing!of!aquatic! organisms.!As!shown,!the!common!name!‘‘glyphosate’’!is!used!somewhat! indiscriminately!in!published!literature,!including!chemical!compounds!that!differ! substantially!from!glyphosate9IPA!salt!(chemical!identity!CAS#!38641940),!e.g.! technical!grade!glyphosate,!which!has!low!solubility!in!water!(CAS#!1071836).! Toxicological!data!for!technical!grade!glyphosate!are!not!representative!when!assessing! ecological!effects!of!glyphosate!herbicides,!which!for!spraying!need!to!contain!a!water! soluble!form!of!glyphosate,!e.g.!the!IPA9salt,!as!the!active!ingredient.!! Contrary!to!previous!published!findings!from!studies!in!daphnids,!the!present!work! finds!acute!toxicity!of!Roundup!formulation!and!active!ingredient!glyphosate!expressed! as!EC50(48h)!concentrations,!to!be!in!the!same!order!of!magnitude.!This!is!in!accordance! with!some!published!work!in!other!aquatic!invertebrates!such!as!Hydra#attenuata.!! D.#magna#tolerance!for!glyphosate!and!Roundup!is!found!to!increase!with!increasing!age! of!the!animals.!This!has!also!been!shown!for!Roundup!in!other!freshwater! invertebrates,!such!as!the!freshwater!shrimp!Caridina#nilotica.!Still,!adult!individuals!of! both!these!freshwater!invertebrates!show!relatively!low!EC50!9values!(22!and!25.3!mg/l! for!D.#magna##and!C.#nilotica,!respectively).!Such!values!are!way!below!previously! published!results!from!acute!glyphosate!toxicity!experiments!in!D.#magna,!even!for! juveniles.!Notably!the!original!work!submitted!by!chemical!producer!Monsanto! company!(Mcallister!and!Forbis,!1978)!presented!an!EC50(48h)!value!of!759.7!mg/l!with! a!sharp!95!%!confidence!interval!(740.89779.9!mg/l).!The!European!Commission!(EC)! working!document!on!glyphosate!(EC,!2002),!which!forms!the!basis!for!European! regulation!in!the!context!of!health!and!environment,!reports!the!EC50(48)!value!of!930! mg/l!in!D.#magna!!from!Forbis!and!Boudreau,!1981.!The!authors!of!the!EC!paper! extrapolate!this!value!into!a!general!EC50!value!for!acute!toxicity!in!aquatic! invertebrates.!Thus!glyphosate!is!termed!‘‘harmless’’!for!aquatic!invertebrates.! According!to!the!2009!WHO!guidelines!for!pesticide!classification!(WHO,!2009),! glyphosate!is!in!class93;!slightly!hazardous!(in!relation!to!human!health).!The!US9EPA! has!defined!glyphosate!in!Toxicity!class!4:!‘‘Practically!nontoxic’’.!Paradoxically,!in!1982! ! 49! !

!

the!agrochemicals!producer!Monsanto!presented!contrasting!data!for!toxicity!of! Roundup!formulations!in!Daphnia#sp,!by!simultaneously!giving!LC50(96h)!values!of!5.3! mg/l!for!Roundup!and!962!mg/l!for!glyphosate!alone!(Servizi!et!al.,!1987).!Already!in! 1979!it!was!pointed!out!that!technical!grade!glyphosate!had!properties!(notably! reduced!water!solubility)!importantly!different!from!those!of!the!glyphosate! isopropylamine!salt!(Folmar!et!al.,!1979).!This!is,!however,!an!important!fact!that!has! been!commonly!overlooked.!! During!our!review!of!published!studies!I!contacted!the!authors!of!4!papers!from!groups! that!had!recently!published!D.#magna#toxicity!studies!with!unspecified!glyphosate.! Authors!from!3!of!these!research!groups!confirmed!that!the!chemical!substance!tested! was!technical!grade!glyphosate,!i.e.!the!non9soluble!version!of!glyphosate.!Contrary!to! this,!the!chemicals!tested!in!the!present!study!are!representative!for!glyphosate! herbicides!used!in!agriculture!as!active!ingredient!(glyphosate)!and!formulated!product! (Roundup).!However,!variations!in!toxicity!levels!may!still!be!expected!due!to! differences!in!adjuvants!and!other!ingredients!of!individual!formulations.! In!contrast!to!other!published!toxicity!data!for!formulated!glyphosate9based!herbicides,! our!results!are!comparable!to!those!of!Folmar!et#al.,!(1979)!at!3!mg/l!for!Roundup!in!D.# magna,!and!to!4!of!the!6!formulations!tested!by!Melnichuk!et#al.,!(2007)!at!4.2–10.2! mg/l.!The!most!recent!toxicity!data!presented!by!the!producer,!for!the!specific!brand!of! Roundup!that!we!have!tested,!is!11!mg/l!EC50(48)!for!D.#magna!and!thus!importantly!is! in!accordance!with!our!findings.!! Importantly,!a!review!of!documentation!obtained!from!the!US9EPA!through!the!FOIA! request!has!demonstrated!that!the!original!research!reports!produced!by!ABC! laboratories!(McAllister!and!Forbis,!1978;!Forbis!and!Boudreau,!1981)!and!the! subsequent!approval!processes!at!the!US!EPA,!are!flawed.!There!are!numerous!mistakes! and!erroneous!interpretations.!Although!not!conclusive,!the!research!protocols!also! indicate!that!test!animals!have!succumbed!to!conspicuous!mortality!patterns,!which! produce!a!symmetry!not!seen!in!other!studies.!The!EPA!reviewers!have!made!hand9 written!changes!onto!the!evaluation!sheets,!in!effect!changing!conclusions!and!in!some! cases!downplaying!indications!of!toxicity.!This!evidence!contributes!to!a!mosaic!of! faults!in!data!computations,!in!written!assessments!and!in!extraction!of!conclusions!and! !

50! !

!

adds!up!to!an!overall!reduced!credibility!of!the!results.! From!the!experimental!work,!the!literature!reviews!and!the!revision!of!US9EPA!archive! material,!it!is!found!that!these!widely!quoted!EC50(48)!values!of!7809930!mg/l!for! glyphosate!(McAllister!and!Forbis,!1978;!Forbis!and!Boudreau,!1981)!are!not! representative.!The!classification!of!glyphosate!as!‘‘practically!nontoxic’’!to!aquatic! invertebrates!is!based!on!these!non9representative!values.!The!high!EC50!values!have! demonstrated!tenacious!lives,!been!extensively!referred!to!in!the!literature!and!have! also!found!their!ways!into!regulatory!documents.!! We!have!found!the!acute!toxicity!of!glyphosate!herbicide!active!ingredient!to!be! substantially!higher,!with!concentrations!below!10!mg/l!inducing!immobility!in!D.# magna!within!48!h.!If!such!more!conservative!EC50!values!were!used,!glyphosate!would! be!classified!as!‘‘toxic’’!or!‘‘moderately!toxic’’!to!aquatic!invertebrates.!In!our!chronic! studies!covering!the!whole!life9cycle!of!D.#magna,!we!demonstrated!negative!and! serious!effects!at!very!low!concentrations,!i.e.!at!levels!that!can!be!expected!with!use!of! the!herbicide!Roundup!at!prescribed!dosages!in!agricultural!practice.!The!results!of!our! acute!and!chronic!toxicity!tests!with!glyphosate9IPA!and!Roundup!herbicide,!in! combination!with!our!review!of!published!data,!warrant!the!conclusion!that!current! European!Commission!and!US!EPA!toxicity!classification!of!these!chemicals!with!regard! to!effects!on!D.!magna!and!aquatic!invertebrates!in!general,!is!based!on!non9 representative!evidence!and!needs!to!be!adjusted.! During!the!progression!of!this!analysis!it!was!realised!that!the!flawed!results!from!the! 1978982!tests!at!ABC!9laboratories!have!penetrated!into!regulation!of!pesticides! worldwide.!Thus!it!is!not!surprising!that!we!find!it!even!in!our!Norwegian!regulation!of! glyphosate,!as!seen!in!a!policy!document!review!elaborated!by!Mattilsynet!(Tveit!&! Holten,!2004)(Norwegian!Food!Control!Authority).!This!review!is!(at!time!of!writing)! the!most!recent!revision!of!the!Norwegian!guidance!document!on!glyphosate!herbicides! ecotoxicity.!It!presents!large!amounts!of!data!for!various!organisms,!but!few!references! to!the!origin!of!the!data.!For!D.#magna!the!review!quotes!4!studies!on!toxicity!of! glyphosate9isopropylamine!salt,!of!these!one!is!a!219day!chronic!exposure!study!with!a! NOEC!of!455mg/l!(endpoints!reproduction!and!mortality).!The!remaining!3!studies!are! acute!toxicity!tests,!with!the!following!results:!LC50(24)!>!1000!mg/l,!LC50(48)!930!mg/l! !

51! !

!

and!LC50(48)!>!1000!mg/l.!For!the!study!finding!a!LC50(48)!value!of!930mg/l!there!is!a! corresponding!NOEC!value!of!320!mg/l,!so!presumably!this!is!data!from!the!test!ABC! laboratories!(Forbis!&!Boudreau,!1981),!but!again,!no!reference.!In!a!subsequent! chapter!the!authors!compile!studies!on!formulated!glyphosate!herbicides.!Of!11!studies! in!daphnids,!7!are!in!D.#magna.!One!study!is!on!MON90139!and!presents!the!values!930! mg/l!(LC50/48)!and!320!mg/l!(NOEC).!! Again,!presumably!these!are!results!from!the!Forbis!&!Boudreau!1981!test.!The!authors! present!a!code!reference,!but!no!further!information!on!the!origin!of!the!data.!Further,! the!authors!mistake!MON90139!for!a!formulated!product!while!actually!it!is!only!an! aqueous!solution!of!the!Monsanto!brand!of!glyphosate9isopropylamine!salt.!The! remaining!6!studies!on!formulation!toxicity!show!LC50(48)!values!of!105,!317,!676,!105,! >1000!and!>1000!mg/l!respectively.!Corresponding!NOEC!levels!are!given!for!the!first! four!tests!as;!26,!33,!356!and!26!mg/l!respectively.!It!is!possible!that!test!#1!and!#4! refers!to!the!same!data.!It!seems!these!data!are!for!complete!formulations!and!not! recalculated!in!relation!to!percentage!of!active!ingredient.!! The!authors!of!the!review!conclude!that!all!the!mentioned!studies!are!of!”acceptable! quality”.!This!would!indicate!that!the!authors!have!reviewed!the!original!studies!and!are! not!merely!quoting!other!reviews!or!regulatory!compilations.!Never!the!less,!I!find!the! imprecisions!important!to!clarify!in!future!revisions.!Furthermore,!it!is!surprising!that! the!authors!do!not!acknowledge!the!widely!quoted!results!published!by!Folmar#et#al.,! (1979),!showing!LC50(48)!values!of!3!mg/l!for!Roundup!formulations,!in!other!words! much!more!toxic!than!the!mentioned!LC50(48)!values!of!10591000+!mg/l.!! It!is!important!to!address!whether!the!confusion!we!have!described!regarding!the!”true”! toxicity!of!glyphosate!and!glyphosate!herbicides!in!D.#magna,!could!also!have!parallels! when!it!comes!to!assessments!of!toxicity!of!these!chemicals!in!other!invertebrate!taxa! or!in!vertebrates!such!as!fish!and!birds.!Or,!in!mammals.!We!do!not!possess!the! experimental!facilities!necessary!to!conduct!such!wide!arrays!of!specialized!tests,!but!

!

52! !

!

from!brief!literature!surveys!it!seems!there!may!be!some!such!flaws,!at!least!concerning! assessment!of!glyphosate!acute!toxicity!in!some!species!of!fish3.! Also!importantly,!I!have!shown!that!several!studies!assessing!glyphosate!toxicity!have! used!chemical!substance!which!only!with!partial!justification!could!be!claimed!to!be! "glyphosate",!as!it!is!parent!compound!glyphosate9techical!or!glyphosate9acid.!As!I!have! pointed!out,!this!type!of!parent!compound!is!not!relevant!in!toxicological!and! ecotoxicological!testing!of!glyphosate!herbicides,!since!it!will!be!different!salts!of! glyphosate,!notably!the!isopropylammonium!salts!or!the!trimesium!salts!which!will!be! the!actual!ingredients!in!such!formulations.!! Again!I!must!underline!the!importance!of!testing!chemicals!representative!of! compounds!which!will!be!dispersed!into!environment!and!pass!into!consumer!food! chain.!Suspecting!other!parts!of!the!regulatory!assumptions!as!flawed!as!the!regulatory! interpretations!of!evidence!for!aquatic!toxicity,!the!entity!of!regulatory!background! material!on!glyphosate!might!benefit!from!a!thorough!revision.!! Soy+quality+and+glyphosate+residues+ Organic!soybeans!showed!a!distinct!nutritional!profile!with!more!sugars,!such!as! glucose,!fructose,!sucrose!and!maltose,!significantly!more!total!protein,!zinc!and!less! fibre!than!both!conventional!and!Roundup9ready!soy.!Organic!soybeans!also!contained! less!total!saturated!fat!and!total!omega96!fatty!acids!than!both!conventional!and! Roundup9ready!soy.!Roundup9ready!soy!contained!high!residues!of!glyphosate!and! AMPA!(mean!3.3!and!5.7!mg/kg,!respectively).!Conventional!and!organic!soybean! batches!contained!none!of!these!agrochemicals!(paper+II).!! Using!35!different!nutritional!and!elemental!variables!(not!including!pesticide!residues)! to!characterize!each!soy!sample,!we!were!able!to!discriminate!Roundup9ready,! conventional!and!organic!soybeans!without!exception,!demonstrating!‘‘substantial!non9 equivalence’’!of!compositional!characteristics!for!these!types!of!commercial!soybeans.! All!individual!samples!of!Roundup9ready!soy!contained!residues!of!both!glyphosate!and! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 3!Comparing!data!from!toxicity!testing!in!fish!as!presented!by!Folmar#et#al.,!(1979)!and!by!Tveit#&#Holten,!

(2004),!it!seems!that!again!the!latter!present!data!showing!low!toxicity!(as!found!in!Giesy#et#al.,!2000).!

!

53! !

!

AMPA.!In!contrast,!no!sample!from!the!conventional!or!the!organic!soybeans!showed! any!residues!of!these!chemicals.!In!the!Roundup9ready!samples,!the!concentration!of! AMPA!(mean!concentration!=!5.74!mg/kg)!was!on!average!nearly!twice!as!high!as! glyphosate!(3.26!mg/kg).!The!minimum!/!maximum!values!for!AMPA!and!glyphosate! were!0.7–10.0!and!0.4–8.8!mg/kg,!respectively.!Fluazifop9P!was!found!in!a! concentration!of!0.078!mg/kg!in!one!of!the!GM9soy!samples,!malathion!was!found!in!a! concentration!of!0.02!mg/kg!in!one!of!the!conventional!soy!samples!and!dieldrin!was! found!in!a!concentration!of!0.002!mg/kg!in!one!of!the!organic!soy!samples.!Other! residues!were!not!found.!The!additional!testing!for!pesticide!residues!in!pooled!samples! of!Roundup9ready,!conventional!and!organic!soybeans!showed!trace9levels!of!alpha9 endosulfane,!trans9nonachlor!and!trans9chlordane,!all!close!to!the!detection!limit!of!0.05! µg/kg!and!in!all!soy!types.!Dieldrin!was!also!found!in!low!levels!with!0.51,!0.45!and!0.6! µg/kg!in!GM,!conventional!and!organic!soybeans,!respectively.! The!lack!of!published!data!on!glyphosate!residues!in!glyphosate9tolerant!crops!such!as! Roundup9ready!soybean!is!obviously!unfortunate!when!considering!the!aspects!which! have!emerged!during!this!research.!In!this!situation!estimates!must!be!based!on!the!few! existing!data,!the!data!from!Duke!et#al.,!(2003),!our!data!from!USA!(paper+II)!and!the! Testbiotech!report!from!Argentina!(Then,!2013).!The!data!shown!in!figure!1.!of!the!final! article!in!this!compilation!(paper+V),!is!recalculated!from!the!data!in!paper+II#and! shows!AMPA!as!glyphosate!equivalents!conforming!to!the!FAO!standards!used!in!the! data!presented!by!Testbiotech.!Average!glyphosate9equivalent!residue!concentrations! are!11.87!mg/kg!in!the!tests!of!soybeans!from!USA!and!39.87!mg/kg!in!the!tests!of! soybeans!from!Argentina.!It!is!evident!that!these!average!concentrations!are!in! compliance!with!the!maximum!residue!levels!defined!by!the!US!FDA!(40!mg/kg)!and!the! results!from!USA!are!also!in!compliance!with!the!EU!MRL!of!20!mg/kg.!However,! individual!samples!are!seen!to!exceed!MRL!limits.!Given!the!very!large!quantities!of! material!produced,!it!is!relevant!to!reflect!upon!the!total!amount!of!residues!thus! transported!and!mediated!to!consumers!(mainly!farm!animals).!According!to!recent! estimates!79%!of!global!soybean!production!stems!from!glyphosate!tolerant!soy!(James,! 2013).!The!total!global!production!in!the!2013/14!growing!season,!was!estimated!to!be! 284!Mt!(USDA,!2014).!Simple!calculation!thus!gives!us!an!estimate!of!the!total!global! quantity!of!soybean!from!glyphosate!tolerant!soy,!of!approximately!224.4!Mt!per!annum! !

54! !

!

at!present.!If!the!residue!concentrations!from!paper+II.!are!representative,!the!total! amount!of!glyphosate!residues!accumulated,!translocated!and!consumed!via!glyphosate! tolerant!soy,!is!approximately!270!000!kg.!Recalculating!by!using!the!data!from! Testbiotech,!this!figure!could!be!as!high!as!897!000!kg.!However!large!it!may!seem,!this! quantity!is!just!a!fraction!of!the!total!load!of!glyphosate!herbicides!applied!in!soybean! cultivation.!Exact!figures!are!difficult!to!obtain,!not!least!since!the!cultivation!of! transgenic!soybean!is!continuously!expanding!and!application!rates!of!glyphosate!active! ingredient!are!increasing!due!to!such!factors!as!have!been!discussed!in!papers+II+and+V.! Based!on!data!from!the!US!Soybean!association,!the!USDA,!the!Penn!State!University! online!Agronomy9Guide!and!similar!readily!accessible!sources,!it!is!not!unreasonable!to! use!production!figures!of!2.592.9!ton/ha!for!present!soybean!yield!and!estimates!of!809 90!Mha!for!the!total!area!currently!in!global!cultivation!with!glyphosate!tolerant! soybean.!Based!on!the!same!sources,!realistic!application!rates!for!glyphosate! herbicides!are!not!significantly!less!than!1.692!kg!active!ingredient!per!hectare.!This!is! partially!estimated!from!the!USDA!maximal!application!rate!of!1.5!lb/acre,!which!is!not! an!unrealistic!seasonal!figure.!Basing!an!estimate!on!total!area!in!cultivation!and!one! seasonal!pass!of!maximum!allowed!application,!the!total!quantity!of!glyphosate!active! ingredient!applied!on!glyphosate!tolerant!soybean!globally,!would!be!in!the!magnitude! of!135!Mkg.!Assessing!the!application!figures!via!the!available!production!data!for! soybean!yield,!the!estimates!are!nearly!identical,!approximately!140!Mkg.!Again,!these! figures!are!rather!rough!estimates,!but!would!indicate!that!0.290.7!%!of!the!applied! glyphosate!active!ingredient!is!accumulated!in!the!soybean!commodity.!It!would! undeniably!be!relevant!to!investigate!the!dynamics!of!the!uptake,!translocation!and! accumulation!further.!Due!to!the!lack!of!data!it!is!not!possible!to!assess!whether!there!is! a!linear!relation!between!application!rate!and!accumulation.!Duke!et#al.,!(2003)! attempted!to!address!the!dynamics!of!uptake!and!degradation!by!measuring! glyphosate/AMPA!ratios!in!soybean!following!different!application!rates,!and!b)! applications!in!different!times!during!the!growth!season!of!the!soybean.!Not! surprisingly,!one!main!conclusion!from!that!work!was!the!finding,!that!applications!late! in!the!season!99!eg.!at!full!flowering!of!the!soy!plants!99!would!result!in!relatively!high! levels!of!residues.!Other!findings!are!less!obvious!to!interpret,!amongst!those!the! finding!of!very!low!glyphosate/AMPA!ratios!in!soybean!following!late!application.!Thus! !

55! !

!

it!seems!that!glyphosate!degradation!into!metabolite!AMPA!is!not!a!linear!process!and! may!proceed!faster!when!the!herbicide!is!applied!late!in!growing!season.!We!see!similar! indications!in!our!data!from!Iowa!(paper+II)!as!there!is!no!predictable!ratio!between! glyphosate!active!ingredient,!and!the!main!metabolite.!This!99!along!with!the!fact!that! glyphosate!is!known!to!actively!interplay!with!biochemical!processes!in!plant! metabolism!99!suggests!that!the!dynamics!of!glyphosate!degradation!and!transformation! in!growing!plants!should!be!investigated!further.! Roundup!and!similar!formulated!glyphosate!herbicides!contain!various!adjuvants!and! inert!ingredients!(paper+I).!I!have!described!some!of!the!confusion!that!enshrouds! ecotoxicological!and!toxicological!assessments!of!these!compounds,!which!are!seen!as! significantly!contributing!to!toxicological!properties!of!formulated!herbicides.! Recognizing!the!inherent!complexity!of!assessing!compounds!which!are!partially! unknown!to!scientists!and!regulators!(paper+I)!it!is!my!heartfelt!recommendation!that! such!ingredients!be!regulated!and!labeling!be!enforced.!As!discussed,!the!present! regulation!allows!producers!of!formulations!to!simply!declare!these!additives!and! adjuvants!as!“inert!ingrediets”,!although!they!are!widely!recognized!to!have!biological! and!toxicological!effects!in!non9target!organisms.!It!should!be!mentioned!that!during! this!work!I!have!not!seen!a!single!published!paper!which!deals!with!this!issue!in!any! systematic!way,!other!than!mentioning!it!as!a!possible!issue!of!concern!(e.g.!Séralini!et# al.,!2014;!Duke!et#al.,!2003).! The!global!annual!soy!production!equals!approximately!40!kg!for!each!inhabitant!on!the! planet.!Of!this!quantum,!approximately!32!kg!would!be!from!glyphosate!tolerant! varieties.!Direct!human!consumption!of!soy!is!minimal!as!the!majority!of!the!global! production!at!present!is!utilized!in!production!of!feed!for!farmed!animals.!Many!species! of!farmed!animals!(cattle,!poultry,!pigs,!fish,!crustacea!etc)!are!fed!diets!with!a! considerable!proportion!of!soy.!Such!feeding!is!life9long,!accentuating!the!relevance!of! adequate!testing!for!effects!from!glyphosate!residues!(paper+V). The!feeding!tests!found!different!types!of!soybean!to!have!different!qualities!as!feed!for! D.#magna.!Primarily!it!was!found!that!organic!soybean!was!superior!to!the!conventional! and!transgenic!types.!It!was!also!evident!that!balanced!diets!with!a!soy9meal!in! combination!with!the!unicellular!algae!(which!are!the!staple!food!for!our!test!animal)! !

56! !

!

were!optimally!suitable!for!D.#magna!growth,!development!and!reproduction!(paper+ III).!This!good!performance!of!test9animals!is!seen!as!a!primary!validation!of!our!model! and!I!find!no!reason!to!hesitate!interpreting!data!from!this!and!similar!feeding!studies.!! When!testing!soy9feed!with!varying!concentrations!of!glyphosate!residues,!it!was!found! that!several!vital!parameters!were!correlated!to!residue!magnitude.!From!this!it!is! concluded!that!Roundup!residues!at!normal!concentrations!found!in!Roundup9ready! soybean!farm9harvests,!have!adverse!biological!effects.!We!observe!that!regulatory! threshold!of!accepted!levels!of!such!glyphosate!residues!are!continuously!being!raised.! At!present!the!maximal!residue!levels!(MRLs)!of!glyphosate!in!soybean!in!the!USA!has! been!increased!from!20!mg/kg!up!to!40!mg/kg!in!the!fall!of!2013.!I!must!accentuate!the! fact!that!such!milligram9levels!are!high!when!compared!to!limitations!on!other!pesticide! active!ingredients,!which!are!typically!tolerated!at!merely!microgram!or!nanogram! levels.!Furthermore!I!see!that!raised!acceptance!levels!will!allow!for!higher! concentrations!of!residues!than!found!in!the!material!which!we!have!tested,!allowing! for!even!higher!exposure!in!consumers.!! Following!these!results!from!experimental!work,!I!must!also!highlight!the!conclusions! from!my!review!of!the!safety!data!which!are!at!the!foundations!of!basic!safety9 assumptions!regarding!the!Roundup9ready!soy!and!other!herbicide!tolerant!cultivars! (paper+V),!stressing!the!importance!of!a!requirement!for!ensuring!use!of!truly! representative!materials!for!regulatory!purpose!testing.!Also,!based!on!these!initial! reviews!and!tests!I!find!that!there!are!serious!flaws!and!systematic!mistakes!that!could! have!general!implication!for!validity!of!current!foundations!for!regulation!of!genetically! modified!cultivars!and!associated!agrochemicals.!! Soybean+feeding+studies++ A!life9long!feeding!study!was!performed!in!D.#magna!with!the!same!soybean!types!from! different!production!systems!as!analyzed!in!paper+II.!! 3!experimental!diets!were!prepared!with!soybean!meal!from!the!different!agriculture! production!systems.!The!diets!each!contained!one!distinct!type!of!soy,!produced!as!a! representative!mixture!of!10(11)!sub9samples.!These!3!diets!were!each!administered!as! 6!different!treatments;!raw!high!dose,!heated!high!dose,!raw!low!dose,!heated!low!dose,! !

57! !

!

raw!with!algae!and!heated!with!algae.!Thus!the!experimental!setup!included!a!total!of! 18!experimental!diets!and!a!control!treatment!given!only!green!algae.!Heat9treated!feed! formulations!were!produced!by!boiling!aqueous!solutions!of!soy9meal!(paper+III).! Overall,!feed!produced!from!organic!soybeans!resulted!in!the!highest!fitness!(higher! survival,!better!growth!and!fecundity)!in!the!model!organism.!Animals!fed!Roundup9 ready!soybean!consistently!performed!less!well!compared!to!animals!fed!either! conventional!or!organic!soybeans.!It!is!concluded!that!accumulation!of!herbicide! residues!in!Roundup9ready!soy!and!related!nutritional!differences!between!the!soy! types!may!have!caused!the!observed!fitness!differences.!The!life!cycle!feeding!trials!in!D.# magna!demonstrate!significant!differences!in!fitness!measured!as!survival,!growth!and! fecundity,!attributable!to!soy!type.!These!results!were!consistent!for!balanced!(with! algae)!and!unbalanced!soy!diets!(soy!only).!! The!results!show!that!D.#magna!live,!grow!and!reproduce!optimally!on!full9fat!soybean! meal!diets!produced!from!heat9treated!soybeans!from!either!soy!type,!when!given!in! combination!with!green!alga!(balanced!diets).!Interestingly,!when!testing!raw!soy!in! balanced!diets,!the!organic!soy!gave!significantly!improved!performance!in!our!test! organism!as!compared!to!the!algae9fed!control.!In!contrast,!for!the!conventional!and! particularly!for!the!Roundup9ready!soy,!performance!was!reduced!compared!to!the! control,!indicating!negative!effects!from!toxins!or!antinutrients.!Animals!fed!unbalanced! diets!of!only!soybean!meal!had!increased!mortality!and!reduced!growth!and!fecundity,! demonstrating!performance!well!below!the!control!group!for!all!measured!variables.!! This!extreme!feed!composition!is!not!representative!of!what!aquaculture!species!would! receive!from!commercially!produced!feed;!however,!inclusion!rates!for!soybean!meal!in! farm!animal!feed!have!increased!from15–40%,!and!inclusion!rates!of!33–40%! soybeanmeal!have!been!validated!in!feeding!trials!with!Pacific!white!shrimp! (Litopenaeus#vannamei).!Although!diets!of!soy!only!are!unbalanced!for!aquaculture! purpose,!such!experimental!feed!has!previously!been!validated!for!D.#magna!as! ecological!indicator!(Bøhn!et!al.!2008,!2010).!Furthermore,!feeding!studies!with!raw! and!heated!soy!are!complementary.!Soy!for!use!as!commercial!feed!is!typically!heat9 treated,!whereas!raw!soy!represents!an!environmentally!relevant!food!source.! Herbivore!species!feeding!in/near!monoculture!fields!are!exposed!to!high!proportion!of! !

58! !

!

specific!plant!biomass,!thus!testing!protocols!for!non9target!organisms!feeding!on,!for! example,!transgenic!material!use!100%!of!the!relevant!material.!In!cultivated! landscapes,!streams!and!ponds!allow!for!diverse!aquatic!invertebrate!species.!In!such! habitats,!a!majority!of!allochthonous!biomass!may!come!from!residuals!of!adjacent! monoculture!crops.!! Consistent!patterns!of!fitness!differences!in!D.#magna!are!found!in!balanced!and! unbalanced!diets.!For!raw!soy,!organic!soy!was!superior!for!D.#magna!reproduction,! whereas!Roundup9ready!soy!was!inferior.!Animals!fed!high!doses!of!Roundup9ready!soy! also!showed!a!markedly!increased!mortality!rate,!supporting!the!observations!of!toxic! or!inhibitory!effect!seen!in!the!balanced!diet!with!algae.!Heat!treatment!of!the!feed! leveled!off!the!large!differences!in!effects!from!soy!types,!but!the!organic!soy!still!gave! the!highest!growth!rates.!The!effects!of!heating!are!complex;!soybean!contains!valuable! nutritional!elements!but!also!major!allergens!that!potentially!affect!animal!health.!Heat! and!alkali!treatment!of!soy!have!beneficial!effects!for!nutritional!and!digestion! properties!of!soy!proteins,!but!very!high!pH!and/or!temperature!should!be!avoided!to! maintain!nutritional!quality.!Both!quantity!and!quality!of!proteins!change!after!heating! with!a!corresponding!change!in!antigenic!properties.!For!edible!oils,!heating!may!be! harmful.!! The!results!from!testing!in!D.#magna!indicate!positive!impact!of!heating,!whereas! previous!study!in!catfish!found!no!such!effect.!As!glyphosate!is!thermally!relatively! stable,!with!a!degradation!point!of!199!°C,!boiling!of!soy!material!is!not!likely!to!reduce! the!levels!of!glyphosate!residues!in!GM9soy.! Subsequent!life9cycle!feeding!studies!with!Roundup9Ready!soybean!in!D.#magna! demonstrated!several!interesting!findings:!high!levels!of!glyphosate!residues!in!the! soybean!feed!tested!correlated!with!1)!higher!mortality,!2)!reduced!growth!and! fecundity!in!some!parts!of!the!life9cycle,!3)!delayed!reproduction,!and!a!reduced!total! number!of!offspring.!None!of!the!statistical!associations!were!particularly!strong!with! R2!!(variation!explained)!typically!below!5%!9!6%!(paper+IV)! However,!the!consistent!and!significantly!negative!effect!of!glyphosate!on!a!range!of!life9 history!traits!can!arguably!be!used!as!a!proof!of!concept!that!glyphosate!residues!in! Roundup!Ready!soybean!have!adverse!biological!effects!at!“normally!occurring!levels”! !

59! !

!

(i.e.!as!found!in!commercial!soybeans)!in!the!investigated!dominant!feed9ingredient.! These!results!accentuate!the!demand!for!further!studies!on!potential!effects!of!herbicide! residues!present!in!genetically!modified!herbicide!tolerant!crops!such!as!Roundup9 Ready!soybean.!Whether!the!observed!negative!impacts!are!due!to,!e.g.,!low9grade! toxicity,!endocrine!disruption!or!antinutritional!effects,!can!only!be!clarified!by!further! well9designed!feeding!experiments.! Validity+of+results+ It!is!important!to!reflect!upon!the!previously!mentioned!critique!(EFSA,!2009;!Ricroch! et#al.,!2010;!Romeis!et#al.,!2013)!against!the!laboratory!tests!of!MON810!maize,!given!as! feed!in!the!D.#magna!model!(Bøhn!et#al.,!2008;!2010).!The!critique!more!than!suggests! that!the!results!should!be!disregarded.!The!rejection!of!the!evidence!is!partially!based! on!a!conception!of!the!employed!methodology,!which!is!deemed!unsuitable!for!testing! of!such!plant9material!(EFSA,!2009).!Also,!it!is!claimed!that!the!results!do!not! specifically!address!causality!of!the!Cry1ab!toxin!in!the!maize!but!merely!shows!adverse! effect!of!the!Bt9maize!itself!(Romeis!et#al.,!2013).!The!critique!by!Ricroch!et#al.,!(2010)!is! more!detailed!and!discusses!several!issues!which!are!presented!as!weaknesses!of!the! study.!The!critique!was!refuted!in!a!detailed!response!by!Bøhn!et#al.,!(2012).! Furthermore,!numerous!of!these!issues!are!specific!points!which!justifiably!have! parallel!shortcomings!which!could!be!raised!against!the!majority!of!the!industrial! studies!that!I!have!reviewed!(paper+V).!Several!of!the!specific!questions!are!addressed! here,!as!they!obviously!have!implications!for!the!present!research.!First!of!all,!it!must!be! acknowledged!that!in!natural!environments!species!of!Daphnia!are!not!exposed!to! homogenized!and!heat9treated!plant!material!such!as!powdered!maize!kernel!or! powdered!soybean.!Thus,!in!an!ecotoxicological!model!testing!potential!effects!of! allochthonous!material!from!agriculture,!the!raw!material!(not!heat!treated)!which!we! also!have!used!in!our!testing,!is!more!representative.!However,!the!heat!treated! material!is!by!no!means!irrelevant,!since!this!will!be!representative!of!material!given!in! feed!formulations!in!farm!animal!feed,!including!aquaculture!feed!for!fish!and! crustacean!species.!Furthermore,!it!is!a!fact!that!existing!guidelines!for!toxicological! testing!such!as!the!OECD9211!protocol!for!D.#magna!reproduction!testing!(OECD,!2008)! specify!a!shorter!period!for!testing!(21!days)!than!the!life9long!exposure!employed!in! !

60! !

!

our!adapted!model!(42!days).!This!critique!is!valid!in!relation!to!exposure!time!and!I! will!acknowledge!that!our!regime!of!testing!is!an!adaptation!of!the!OECD9211!protocol,! but!this!in!no!way!invalidates!the!results!of!the!first!21!days.!If!our!protocols!had! specified!a!shorter!exposure!time!than!21!days,!the!situation!would!be!different!and!the! critique!would!have!some!obvious!justification.!In!the!given!situation!most!of!our! presentation!of!data!allows!for!differentiation!of!results!according!to!age!and!thus! allowing!for!extraction!of!results!from!the!first!21!days,!if!so!desired.!Furthermore,!it!is! important!to!test!such!material!over!the!whole!life9cycle!of!animals,!as!effects!from!life9 long!exposure!can!be!expected!to!be!found!at!different!stages!of!the!life!cycle.!This!in! effect!is!the!underlying!rationale!for!expanding!the!909day!tests!in!rat!typically! performed!by!industry!company!researchers!as!safety!assessment!of!such!materials,! making!the!exposure!life9long!(Séralini!et!al.!2014)!and!I!find!it!a!valid!argument.!I!must! also!accentuate!the!findings!from!our!first!soy9feeding!study!(paper+III)!showing!that! animals!given!organic!soybean!in!addition!to!green!algae!(balanced!diets)!performed! better!than!the!control!animals!fed!only!green!algae.!Thus!it!could!be!suggested,!that! organic!soybean!meal!given!in!addition!to!green!algae!could!be!a!better!standard!diet! for!rearing!healthy!and!vital!experimental!animals!in!any!laboratory!working!in!D.# magna.! Review+of+published+evidence+ Of!30!reviewed!studies!on!composition!and!feed9quality!of!glyphosate9tolerant!GM9crop! material,!only!half!of!the!studies!used!material!produced!with!application!of!glyphosate! herbicide!(paper+V).!Furthermore,!only!one!of!the!30!studies!has!analyzed!the!material! for!glyphosate!residues.!Application!of!representative!dosage!of!herbicides!as!well!as! subsequent!analysis!of!herbicide!residues!is!missing!in!industry!testing!of!glyphosate9 tolerant!GM9crops.!This!implies!that!central!data!from!compositional!analysis,!animal! feeding!studies!and!overall!risk9assessment!performed!by!industry!and!submitted!to! national!and!international!regulatory!bodies!as!evidence!as!safety,!is!not!representative! of!the!materials!actually!delivered!onto!the!commercial!market.!! In!part!the!scientific!evidence!produced!by!industry!is!found!to!have!unacceptable! standard!for!regulatory!purpose!(papers+I+and+V).!Such!evidence!should!be!disregarded! and!demands!for!new!evidence!should!be!brought!forward.!! !

61! !

!

Triangular+model+of+quality++ During!this!work,!some!thought!has!been!given!to!a!concept!of!an!“agroecological! system!model”,!aiming!at!visualizing!interplay!and!relativity!of!factors!found!to!be! dominant!aspects!in!soybean!agriculture.!From!a!model!system!consisting!of!three! denominating!factors,!the!figures!on!agroecological!balance!attempt!such!visualization.! Industrial!agriculture!is!taking!place!within!a!dynamic!frame!illustrated!by!the!triangle! in!figure!1.!The!production!will!have!specific!goals,!which!often!are!condensed!into!a! strategy!of!producing!as!much!of!a!certain!commodity!as!possible!on!a!give!area!of!land! (yield,!or#quantity)!with!lowest!possible!expense.!! Also,!society!has!certain!qualitative!requirements!for!agriculture!and!specific!types!of! food!and!feed!which!partially!define!this!dynamic!frame,!e.g.!recommendation!on! specific!practice!and!regulation!of!pesticide!use.!Additionally!the!products!are!primarily! delivered!onto!an!established!consumer!market!regulated!by!specific!demands!for! quality!and!price.!Thus!we!see!that!producers!of!specific!agriculture!commodities!such! as!soybean!will!balance!the!production!within!the!framework!of!opposing!factors.!Less! use!of!fertilizer!will!attractively!lower!nominal!value!of!denominator!“expense”!but!may! probably!also!lower!nominal!value!of!denominator!“quantity”,!a!development!which! typically!is!considered!unattractive.!! In!the!same!model!a!priority!of!denominator!“quality”!e.g.!through!strict!limits!on!use!of! fertilizers!or!pesticides!as!seen!in!organic!agroecological!production!systems,!may!shift! the!commodity!towards!“quality”!but!thus!also!away!from!optimal!status!for! denominators!“quantity”!and!“expense”.!! The!model!of!agroeclogical!balance!implies!that!it!is!not!feasible!to!obtain!optimal! outcomes!for!all!3!denominators!in!a!given!production!system,!9!the!farmer!cannot!have! high!yields!of!high!quality!produce!at!low!costs.!Typically!the!model!will!allow!for! optimizing!two!factors!at!the!expense!of!the!third.!It!thus!would!be!possible!to!produce! optimal!quality!at!optimal!costs,!but!then!the!quantity!may!not!be!expected!to!reach! optimal!level.!Shifting!towards!a!quantitative!focus,!commodities!can!be!produced!in! optimal!quantity!at!optimal!expense.!In!which!case!the!quality!can!be!expected!to!be! less!than!optimal.! !

62! !

!

expense

quality

quantity

! Figure 1. Agroecological balance of conventional soybean in relation to factors expense (cost of production), quality (measured as quality of consumer product) and quantity (measured as harvested biomass).

! ! optimal cost

B optimal quality

A

C optimal quantity

! Figure 2. Relative agroecological balance of 3 different production systems for soybean; A – conventional industrial agriculture, B – organic agriculture and C – agriculture of glyphosate-tolerant soy.

The!analysis!of!soybean!from!three!distinct!agroecological!systems!demonstrates!that! these!have!distinct!descriptors,!characteristically!separating!soybean!crops!from! transgenic,!conventional!and!organic!agricultures.!Soybean!is!an!example!of!a! commodity!from!an!agroecological!system,!which!can!be!defined!by!keywords! !

63! !

!

"quantity",!"quality"!and!"expense"!("expense"!inverted!into!positive!denominator;! “optimal!cost”),!as!illustrated!in!figure!2.!! Conventional!soybean!(A)!stands!in!equilibrium!as!comparator!in!the!model!and!we!see! the!two!other!types!relative!to!these!conventional!varieties.!Based!on!results!from! pesticide!analysis!and!feeding!studies!as!indicative!measurables!for!denominator! "quality"!organic!soybean!(B)!occupies!a!distinct!niche!of!the!agroecological!model,!with! a!stronger!affinity!towards!denominator!"quality"!and!lower!affinity!towards! denominators!"quantity"!and!"cost".!! Contrary!to!this,!the!transgenic!soy!(C)!is!seen!transgressing!those!model!boundaries! defined!by!the!normal!comparator!(A).!The!niche!occupied!by!transgenic!soybean!is! defined!less!by!affinity!towards!denominators!"quantity"!and!"cost",!as!by!an! antagonistic!relationship!towards!denominator!"quality".!The!strong!affinity!towards! fulfilment!of!both!denominators!“optimal!cost”!and!“optimal!quantity”!forces!the! transgenic!soybean!out!of!equilibrium!and!places!it!at!a!substantial!distance!from! denominator!“quality”,!in!effect!breaking!out!from!the!normality!confinement!defined! by!the!model!framework.!! The!model!also!illustrates!the!relativity!of!soybean!agriculture!area!in!the!USA,! proportionally!divided.!Organic!soy!is!planted!on!smaller!parts!of!the!available!total! area,!compared!to!conventional!soy.!Also,!compared!to!conventional!soy,!transgenic!soy! is!planted!on!dominating!larger!parts!of!the!available!area!(papers+II,+III).! A!review!of!agroecological!systems!(Heinemann!et#al.,!2014)!partially!extending!the! work!of!the!International#Assessment#on#Agricultural#Knowledge,#Science#and#Technology# for#Development!(IAASTD,!2009;!UNEP,!2014)!may!be!interpreted!as!supportive!for!such! a!triangular!model!of!agroecological!balance.!A!recent!review!(Smith9Spangler!et#al.,! 2012)!of!studies!comparing!produce!from!organic!agroecosystems!and!conventional! agroecosystems!finds!qualitative!differences!notably!in!detections!and!levels!of! pesticide!residues,!and!thus!arguably!also!can!be!seen!to!support!a!model!involving! more!than!two!denominators.!On!the!contrary,!reviews!of!produce!from!conventional! agroecosystem!versus!that!of!transgenic!agroecosystems!(Delaney,!2007;!Harrigan!et# al.,!2010)!can!be!interpreted!to!support!mainly!a!bilateral!model!encompassing! denominators!quantity!and!cost.!! !

64! !

!

The!triangular!model!of!agroecological!balance!has!at!least!one!major!shortcoming,! which!could!be!discussed!and!possibly!improved:!Two!of!the!three!denominators! ("quantity"!and!"cost")!are!scale!variables!to!which!numerical!values!can!be!attributed,! alas!measurables.!However,!the!third!denominator;!"quality"!is!not!a!scale!variable!but! rather!a!categorical!denominator!based!on!criteria!which!should!be!defined4.!In!the! current!model!the!denominator!"quality"!is!implicitly!representative!of;!a)! concentrations!of!pesticide!residues!(compositional!analysis),!and!b)!quality!as!feed! (animal!feeding!studies).!In!a!revised!model!all!denominators!should!ideally!be! quantifiable!or!at!least!less!imprecise!than!subjective!terms!such!as!denominator! quality.!An!adjustment!that!could!be!explored!would!be!to!use!a!numerical!ecological! indicator!as!a!scale9variable!for!denominator!quality.!The!proposed!adaptation!could! draw!inspiration!from!the!Environmental#Impact#Quotient!for!pesticides!(Kovach,!1993),! which!is!a!model!encompassing!qualitative!data!from!a!wide!array!of!toxicological!and! ecotoxicological!testing.! Regulatory+issues+ It!is!relevant!to!discuss!regulatory!issues!arising!from!the!findings!of!high!residue!levels! of!glyphosate!in!the!Roundup9ready!soybean!(papers+II+and+V).!Given!the!context!of! presented!justification!for!reassessment!of!glyphosate!toxicity,!presence!of!such! residues!must!be!seen!as!both!challenging!and!undesirable.!Again,!such!regulatory! changes!would!depend!on!new!understandings!of!the!causal!relationships!of!singular! indications!from!toxicology!and!ecotoxicology,!summed!into!re9evaluations!of!concepts! such!as!Environmental!Impact!Quotient!of!specific!compounds!and!subsequently! revisions!of!Allowable!Daily!Intake!(ADI)!and!adjustments!of!Maximal!Residue!Levels! (MRLs)5!to!implement!such!precautionary!revision!and!achieve!reduction!in!consumer! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 4!Furthermore,!from!a!puritan!view!of!graphic!communication!as!convincingly!presented!by!Tufte!(2001)!

it!could!be!argued!that!denominator!"quantity"!should!be!at!the!top!of!the!triangle!and!denominator! "cost"!at!the!bottom,!as!visual!display!of!information!usually!employs!vertical!progression!to!imply! correlating!increases!in!numerical!values!(Tufte!has!produced!several!noteworthy!books!on!the!subject).! 5!One!main!arguments,!which!I!only!have!lightly!touched!upon!in!this!text,!is!the!paradoxical!discrepancy!

in!scientific!findings!on!glyphosate!toxicity!and!parallel!developments!in!regulatory!measures.!These!two! aspects!would!be!expected!to!be!interdependent,!so!as!the!regulatory!measures!would!directly!relate!to!

!

65! !

!

total!exposure.!It!must!also!be!relevant!to!assess!regulatory!framework!in!relation!to! recent!evidence!on!environmental!persistence!of!glyphosate,!as!these!chemicals!seem!to! be!less!biodegradable!than!initially!assumed!and!contrary!to!expectations!are!also! found!in!ground9water!and!other!reservoirs!recognized!as!protectable!for!drinking9 water!purpose.!Glyphosate!is!also!found!in!air!in!agricultural!areas!and!is!diffusely! present!in!even!urban!environments.!This!is!not!surprising,!given!the!large!quantity! produced!and!dispersed!annually.!! Turning!to!regulatory!authorities,!a!recommendation!from!this!may!be!that!since!overall! exposure!is!increasing!through!additional!channels!of!consumption,!inhalation!and! other,!a!continuing!policy!of!regularly!allowing!for!increasing!MRLs!in!individual! sources!such!as!soybean,!may!be!conflicting!with!the!general!intensions!of!preserving! acceptable!lowest!possible!levels!of!consumer!intake!in!relation!to!ADI!ambitions.!It!is! also!appropriate!to!mention!the!issue!of!post9market9monitoring,!as!this!is!seen!to!have! importance!in!assessing!possible!adverse!effects!in!consumer!organisms!at!farm9level.! Anecdotal!evidence!from!farmers!raising!swine!and!poultry!on!GMO9feedstuffs!presents! claims!of!perceived!improved!fecundity,!growth!or!survival,!even!behaviour,!as!a! consequence!of!having!discontinued!use!of!feed9formulations!known!to!contain!GMO! ingredients.!Such!postulated!observations!have!limited!value!when!it!cannot!be! documented!which!ingredients!actually!were!present!in!the!feed!batches!in!question.! Extending!this!perspective!upwards!to!the!level!of!human!consumers!we!realize!that!the! present!regime!of!non9labeling!of!genetically!modified!ingredients!in!the!globally!largest! consumer!market!for!such!products,!is!unwise!from!a!precautionary!health!perspective! (paper+V).!The!non9labeling!should!not!be!seen!mainly!to!serve!the!interests!of! producers!cautious!of!possible!litigation.! Published!data!on!glyphosate!residues!in!glyphosate9tolerant!crops!are!sparse!(papers+ II+and+V).!The!findings!presented!here!suggest!that!this!could!be!an!issue!with! important!implications.!Scientific!evidence!produced!by!biotech9industry!companies! should!be!supplemented!with!data!from!independent!research.!Alternatively,!the!risk9 !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! aforementioned!findings.!Remarkably,!this!seems!not!to!be!a!valid!assumption!when!discussing! glyphosate!regulation.!

!

66! !

!

assessments!and!analyses!performed!by!industry!should!be!competently!supervised!to! ensure!transparency!and!an!overall!satisfactory!standard!of!testing.!This!leads!to!a! recommendation!to!regulatory!authorities!such!as!the!European!Food!Safety!Authority! (EFSA)!and!the!Organisation!for!Economic!Cooperation!and!Development!(OECD)!to! apply!necessary!measures!and!enforce!routines.!Regular!revision!of!regulatory! framework,!routines!and!operating!procedures!is!needed!to!secure!future!quality!of! important!food!and!feed!material.!Such!action!is!fundamental!for!safeguarding! coherence,!relevance!and!public!trust.! Sustainability+and+societal+context++ We!must!acknowledge!the!importance!of!soybean!as!bulk!ingredient!in!feed!for!farmed! animals.!Soybean!generally!has!a!composition!of!proteins!and!lipids!which!is!favorable! for!feed!production!purposes!and!the!known!potentially!problematic!compounds!such! as!antinutrients!can!be!neutralized!through!heating!or!other!industrial!processes!during! the!manufacturing!of!feed!formulations.!Even!more!important!than!the!mentioned! compositional!qualities!is!the!fact!that!soybean!can!be!obtained!at!low!costs!compared! to!other!sources!of!proteins!and!lipids.!As!we!have!discussed!in!our!papers!on! compositional!equivalence!and!in!our!feeding!studies;!soybean!meal,!soybean!oils!and! other!soybean!ingredients!are!increasingly!penetrating!into!all!levels!of!food!supply!in! industrialized!societies,!either!as!direct!ingredients!in!food!or!as!indirect!ingredients!via! the!important!role!as!prime!constituents!of!farm9animal!feed.!At!the!present!rate!of! development!even!feed!formulations!for!crustaceans!and!fish!in!aquaculture!are!found! to!substantially!consist!of!soybean!meal!and!soybean!oil.!These!farmed!ingredients!are! used!to!substitute!increasingly!scarce!and!costly!marine!oils!and!marine!proteins!such! as!traditional!fishmeal!(papers+I,+II+and+III).!! This!is!also!very!relevant!in!a!wider!Norwegian!context,!as!the!Norwegian!farmed! salmon!is!given!feed!with!high!proportions!of!vegetal!proteins!and!oils.!Norwegian! domestic!production!of!oilseed!is!in!the!order!of!11.000!metric!ton!annually!(primarily! seed!of!Brassica),!while!the!annual!import!of!soybean!to!Norway!is!approximately! 413.000!metric!ton!(SSB,!2014).!Thus!an!overwhelming!majority!of!oilseed!used!in! domestic!feed!production,!is!imported!soybean.!So!far!these!imports!have!been!non9 GMO.!Thus!Norwegian!feed9formulations!for!farm9animals!and!for!aquaculture!do!not! !

67! !

!

contain!the!glyphosate9tolerant!soy!which!dominates!other!parts!of!the!world!market.! This!is!a!consequence!of!a!deliberate!policy!which!is!justifiable!from!a!number!of! reasons;!first!of!all!the!Norwegian!legislation!(The#Gene#Technology#Act),!which!states! requirements!for!sustainability!and!societal!benefit!of!biotechnological!products.!! Also,!we!see!new!indications!of!important!qualitative!differences!attributable!to! agriculture!system!and,!as!we!have!shown!throughout!the!present!work,!important! indications!have!emerged!substantiating!claims!stating!that!several!previous!studies! upon!which!the!assumption!of!qualitative!equivalence!was!based,!are!flawed.! Furthermore,!the!Norwegian!farmed9salmon!industry!is!an!important!financial!sector!in! society,!creating!jobs!in!sparsely!populated!parts!of!coastal!regions!and!fetching! substantial!export!revenues!for!the!Norwegian!state!economy.!! The!vulnerability!of!this!sector,!the!product!of!which!is!marketed!as!originating!from! pristine!crystal9clear!fiords!and!an!unspoilt!natural!environment,!is!evident.!I!expect!the! image!of!Norwegian!aquaculture!not!to!be!irreparably!marred!by!a!public!outcry!over! feed!composition!issues,!but!I!do!not!recommend!any!further!testing!of!this!hypothesis.!! Thus!even!when!keeping!a!narrow!focus!on!optimizing!revenues,!the!potential!long9 term!consequences!of!relatively!minor!savings!on!feed!ingredients!can!be!fully!realized.!! Paradoxically!it!should!be!notice!that!certain!researchers!uphold,!that!since!only!plants! (and!some!lichens!and!microorganisms)!have!the!59enolpyruvylshikimic!acid939 phosphate!synthase!metabolic!pathway!defined!as!glyphosate!target9site,!only!such! organisms!can!be!expected!to!be!targeted!by!toxic!effects!of!this!chemical!(Duke!et#al.,! 2012).!Obviously!this!deduction!of!safety!towards!non9target!organisms!is!scientifically! unconventional!and!it!is!remarkable!that!it!has!passed!into!publication.!I!do!find!it! spectacular!that!parts!of!the!researcher!community!have!managed!to!convince! themselves!and!possibly!parts!of!their!target#audience!into!assuming!that!agrochemicals! such!as!herbicides,!may!be!expected!to!have!restricted!singular!modes!of!action.!! Based!on!the!present!findings!(papers+I,+II,+III,+IV)!it!can!be!concluded!that!the! agroecological!system!of!GTS!409392!cultivation!is!seen!to!have!adverse!effects!on! aquatic!invertebrate!D.#magna#indicative!of!recipient!biota.!However,!based!on!present! findings!it!is!not!valid!to!extrapolate!and!speculate!that!similar!effects!could!occur!in! !

68! !

!

related!or!unrelated!ecosystems.!In!a!similar!way,!based!on!present!findings!it!is!not! possible!to!dismiss!such!speculation.! Likewise,!it!is!not!scientifically!valid!to!extrapolate!and!speculate!that!Roundup9ready! soybean!agroecology!has!a!potential!to!cause!adverse!effects!in!mammalian!consumer! organisms.!And,!based!on!the!present!findings!it!is!not!justified!to!dismiss!such! questions!as!irrelevant.! The!work!presented!here!(paper+V)!should!also!be!acknowledged!to!support!the! concerns!raised!by!reviewers!who!have!highlighted!a!general!need!for!transparency!in! regard!to!biosafety!evidence!from!biotech!industry,!which!is!perceived!as!uncooperative! and!antagonistic!towards!both!scientifically!based!criticism!and!independent!revision!of! information!(Nielsen,!2013;!Waltz,!2009).!! From!my!work!I!can!not!claim!to!have!proven!a!fundamental!irregularity!at!the!basis!of! current!regulatory!systems!for!genetically!modified!herbicide!tolerant!cultivars,! involving!commercial!and!political!interests!engaging!in!strategies!to!divert!regulatory! focus!from!obvious!biosafety!challenges!related!to!existing!and!emerging! biotechnological!agriculture!patents.!Neither!can!it!be!concluded!that!biotech!industry! withholds!essential!information!which!under!the!intensions!of!US!regulation!should! have!been!voluntarily!presented!to!regulators.!! But!on!the!other!hand,!based!on!the!available!evidence!I!unfortunately!cannot!state!that! the!mentioned!wording!on!irregularity!is!not!a!relevant!description.!

!

69! !

!

!

70! !

!

Conclusion The!analysis!of!soybean!meal!composition!show!that!transgensgenic!GTS!409392! soybean,!conventional!soybean!and!organic!soybean!are!distinct!and!proves!that! products!of!these!3!farming!practices!is!substantially!different!from!each!of!the!others.! One!main!result!concerns!the!glyphosate!residues,!which!are!present!at!ppm9levels!in! all!10!transgenic!samples.!It!is!relevant!to!consider!the!wording!high,!very#high!and! extreme,!when!describing!ppm!concentrations!of!chemical!residues!otherwise! quantified!at!ppb!levels6.!This!further!relates!to!scientific!and!social!definitions!such!as! “Maximum!Residue!Levels”!(MRLs),!as!acceptance!levels!of!pesticide!residue!toxins!in! food!and!feed.!These!acceptance!levels!in!turn!are!based!on!expert!opinion,!which!is! based!on!scientific!studies.!Those!studies!are!mainly!performed,!interpreted!and! published!by!researchers!who!are!employees!of!the!applicant!industry.!! The!feeding!experiments!further!confirms!that!the!3!soy9meal!types!are!not!equivalent,! having!distinctly!different!qualities!deduced!from!feeding!in!D.#magna.!Growth!and! fecundity!was!best!in!animals!fed!diets!made!from!organic!soy,!whereas!the!data! indicate!that!animals!fed!diets!made!from!transgenic!GTS!409392!soybean!had!poorest! growth!and!poorest!overall!fecundity.!! Furthermore,!the!testing!of!toxicity!of!Roundup!in!D.#magna!proves!that!the!herbicide! formulation!is!as!toxic!to!this!species!as!previously!indicated!by!A):!independent! research!reaching!back!to!the!work!of!Folmar#et#al.!(1979)!and!B):!manufacturer! information!material!data!sheet.!However,!the!testing!of!the!glyphosate!active! ingredient!alone!showed!surprisingly!higher!toxicity!in!D.#magna,!compared!to!data! referenced!in!important!published!literature.!The!acute!toxicity!as!well!as!the!chronic! long9term!toxicity!was!up!to!1009fold!higher!in!our!testing,!then!stated!in!regulatory! documents!based!on!such!experimental!evidence.!Not!surprisingly,!these!conclusions! immediately!lead!to!new!questions.!!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 6!ppb!is!”parts!per!billion”!and!ppm!is!”parts!per!million”,!commonly!expressed!as!ng/g!or!ug/g!

respectively.!

!

71! !

!

A!review!of!published!literature!on!several!specific!issues!studied!here,!such!as!toxicity! of!glyphosate!and!biosafety!of!transgenic!cultivars,!shows!surprisingly!divergent! results:!I!see!that!numerous!publications!contribute!to!a!"body!of!scientific!evidence"! supporting!the!following!conclusions;!1)!substantial!equivalence!of!transgenic!cultivars! as!compared!to!conventional!varieties!of!the!same!plants,!and!2)!no9effect!of!transgenic! feed!ingredients!along!with!3)!evidence!of!glyphosate!low9toxicity!towards!aquatic! invertebrates,!vertebrates!and!mammals.!However,!another!body!of!scientific!evidence! is!presenting!opposite!conclusions;!1)!the!concept!of!substantial!equivalence!is!not! supported,!and!2)!transgenic!feed!ingredients!do!have!the!potential!to!induce!adverse! effects!in!animals,!and!also!that!3)!glyphosate!has!adverse!toxicological!effects!in! aquatic!invertebrates,!mammals!and!other!vertebrates.!! These!“bodies!of!evidence”!are!incompatible,!as!they!indicate,!substantiate!and!prove,! completely!opposite!conclusions.!It!cannot!be!concluded!whether!one!or!the!other!part! of!these!scientific!communities!in!general!is!more!or!less!correct!in!method,! interpretation!and!conclusion.!However,!this!need!for!clarification!is!found!to!justify! further!investigation.!! Answering+the+research+question+ The!presented!findings!and!the!outcome!from!the!model!of!agroecological!balance!all! contribute!to!a!falsification!of!the!research!hypothesis!stemming!from!the!initial! research!question.!! Thus,!the!glyphosate9tolerant!GTS9409392!variety!of!soy!cannot!be!claimed!to!be! substantially!equivalent!to!soybean!from!conventional!or!organic!agriculture.!! It!is!concluded!that!the!GTS9409392!variety!of!soy!is!significantly!different!from!the!two! mentioned!alternative!categories!of!soy.!! It!will!be!appropriate!to!further!discuss!whether!this!difference!has!physiological,! ecological!or!societal!importance,!or!whether!it!is!merely!an!acceptable!variation.!!

!

72! !

!

Formal framework Mandate+ The!research!was!conducted!within!the!framework!of!Norwegian!Research!Council! project!184107/S30#LAND:#A#new#model#approach#to#assess#genetically#modified#plants:# their#ecotoxicity#and#potential#interactions#with#environmental#pollutants.!! Declaration+of+independence++ According!to!§19!(3)!in!the!regulation!of!doctoral!studies!at!University!of!Tromsø! (Lovdata,!2013)!a!work!such!as!the!present!thesis,!which!is!based!on!contributions!of! several!authors!including!supervisors!and!co9authors!of!manuscripts,!must!have!a! written!and!signed!declaration!of!independence!and!interdependence!specifying!the! contributions!by!the!doctoral!candidate.!In!the!present!version!of!thesis!the! introduction!(sub9chapters:!Preface,#Summary,#List#of#papers,#Key#findings#related#to# individual#papers,#Abbreviations,#Research#question#and#hypothesis,#Mandate)!and!the! chapter!on!Background!(sub9chapters:!Soybean,#Glyphosate,#Risk;assessment#of# transgenic#cultivars,#Substantial#equivalence,#Regulatory#framework,#Information#access),# the!chapter!on#Model+system!(sub9chapters:!Research#materials,#Test;animal#ecology,# Related#work)!and!the!chapters!Discussion!and!Postscript,!are!written!by!the!doctoral! candidate!Marek!Cuhra.!Research+papers+I,!II,!III!and!IV!were!written!with!co9authors! as!stated!in!"List#of#papers".!Research!paper!V!is+written!by!Marek!Cuhra,!who! furthermore!is!principal!author!of!manuscripts!I,!III!and!IV.!Thomas!Bøhn!is!principal! author!of!manuscript!II.!Marek!Cuhra!performed!data+collection!for!manuscripts!II,!III! and!V.!Thomas!Bøhn!and!Marek!Cuhra!performed!data!collection!for!manuscript!I.! Michaël!Dando!and!Marek!Cuhra!performed!data!collection!for!manuscript!IV.!Marek! Cuhra,!Raul!Primicerio!and!Thomas!Bøhn!performed!statistical+computations!for! manuscripts!II,!III,!IV!and!V.!Marek!Cuhra!was!principal+scientist!in!laboratory!work!on! manuscript!II,!III.!Thomas!Bøhn!was!principal!scientist!in!laboratory!work!on! manuscript!I.!Michaël!Dando!and!Marek!Cuhra!shared!the!role!of!principal!scientist!in! laboratory!work!for!manuscript!IV.!! !

!

73! !

!

Acknowledgement+of+funding+and+declaration+on+conflicting+interests+ The!research!was!fully!funded!by!the!Norwegian!Research!Council!project!184107/S30# LAND:#A#new#model#approach#to#assess#genetically#modified#plants:#their#ecotoxicity#and# potential#interactions#with#environmental#pollutants.!! The!author!declares!he!has!no!conflicting!interests!relating!to!the!presented!work.! ! Tromsø,!February!2015!

! Marek!Cuhra! ! !

!

74! !

!

!

Acknowledgements+ Recognizing!the!complexity!of!the!investigated!subject!and!the!collective!effort,!a!proper! acknowledgement!is!undoubtedly!not!only!appropriate,!but!may!even!be!claimed!to!be! scientifically!relevant.!Although!discussing!the!principal!implications!of!my!findings!in!a! societal!context!is!somewhat!beyond!what!is!usually!expected!in!work!such!as!this! qualitative!assessment!of!the!specific!constituents!in!industrial!agriculture!systems!for! production!of!soybean,!I!see!that!these!aspects!are!important.!Arguably!a!further!broad! analysis!of!such!and!related!issues!could!have!relevance,!validating!subsequent! inclusion!into!a!treatise!otherwise!expected!to!have!a!rather!narrow!ecotoxicological! focus.!I!will!not!discuss!principal!aspects!of!the!doctoral!thesis!as!product!of! socialization!here,!but!I!must!reflect!upon!whether!the!work!leading!to!this!discussion! has!produced!new!knowledge,!or!if!it!has!been!mainly!a!researchers!educative! evolution.!Evaluating!the!research!findings!it!is!concluded!that!the!educational! progression!should!not!be!considered!the!main!outcome.!! The!previous!version!of!this!thesis!99!which!was!submitted!on!March!17th!2014!and! rejected!by!the!University!of!Tromsø!on!October!1st!the!same!year!99!consisted!of!eight! manuscripts!and!an!enclosed!100!page!monography!in!the!form!of!a!postscript.!The! committee!evaluating!the!work!decided!that!three!specific!manuscripts!should!be! eliminated!from!the!thesis,!along!with!the!postscript.!The!committee!argued!that!my! work!was!controversial,!political,!not!in!compliance!with!formal!requirements!and!as!if! that!was!not!bad!enough,!9!it!was!found!to!be!transdisciplinary.!Furthermore,!the! committee!had!specific!recommendations!relating!to!aspects!such!as!experimental! design,!experimental!animal!biology!and!data!statistics.!I!did!not!accept!that!part!of!the! argumentation,!but!I!agreed!to!revise!the!thesis!and!remove!the!three!controversial! manuscripts!and!the!postscript.!! When!a!doctoral!thesis!is!rejected,!the!candidate!is!usually!given!3!months!to!present!a! new!thesis,!which!should!be!in!compliance!with!the!expert!recommendations!of!the! committee!and!the!formal!rulings!of!the!faculty!leadership.!Thus,!the!present!version!is! a!revised,!albeit!sadly!anaemic!and!fundamentally!reduced!version!of!my!work.!!

!

75! !

!

Literacy!at!the!receiving!end!is!not!an!absolute!criterion!for!successful!writing,!9!but!it! helps.!Cultivation!is!a!nice!word,!it!has!several!layers!of!meaning!and!its!use!in!language! spans!bridges!from!classrooms!to!cornfields.!Our!parents!had!large!collections!of!books! and!partially!spent!evenings!reading!and!writing.!Family!holidays!involved!mandatory! admiration!of!historical!evidence!from!former!European!civilization;!the!gothic! cathedrals,!the!baroque!churches,!the!frescoes!by!Signorelli,!the!aquaduct!at!Gard,!the! pillars!of!Paestum!and!visits!at!countless!other!marvels!scattered!in!European!and! Mediterranean!landscapes.!Despite!our!initial!reluctance!when!facing!things!we!did!not! understand!or!appreciate!at!the!time,!each!summer,!Mother!dragged!us!through!a!fair! bit!of!such!heritage!before!we!were!allowed!to!search!for!shells!at!the!beaches.!I! remember!how!she!showed!us!the!lead!linings!of!the!aquaducts!and!explained!how!the! widespread!use!of!this!toxic!metal!had!affected!the!water!supply!and!brought!the! Roman!Empire!to!its!knees!through!a!degeneration!of!intellect!and!general!public! health.!I!still!do!not!know!if!this!was!just!an!opinion!or!if!her!theories!were!supported! by!scientific!evidence!and!biochemical!analysis!of!dusty!bones,!but!it!filled!my!head!with! reflections!and!made!me!wonder!how!the!consecutive!generations!of!the!dominating! culture!gradually!were!poisoned!and!incapacitated.!Initially!I!thought!the!Romans! probably!could!not!envisage!possible!consequences!arising!from!this!specific!use!of! technology;!lead!plumbing!to!contain!water!for!consumption.!This!is!all!in!the!past!and! the!main!point!is!whether!we!have!learned!anything!from!the!unfortunate!use!of!lead! sealings!in!aquaducts.!Surprisingly,!when!contemplating!certain!aspects!of!our!modern! modus!vivendi,!such!as!the!nature!of!materials!which!our!dentist!use!to!seal!cavities!in! our!teeth,!it!could!seem!that!our!societies!are!not!too!preoccupied!with!lessons!possibly! learned!from!earlier!exposure!to!minute!concentrations!of!toxic!metals.!! In!our!Danish!childhood,!Father!used!to!take!us!for!long!walks!along!the!white!stretches! of!sand!on!the!western!coast!of!Jutland.!We!would!discuss!the!purpose!of!it!all!and!often! have!wonderful!enlightening!moments.!One!evening,!in!an!autumn!storm!far!south!of! our!childhood!home,!the!thundering!sea!and!waves!dug!away!the!sands!at!Harboøre! Tange!and!consequently!washed!out!the!hidden!toxic9waste!dump!of!Cheminova! industries.!It!took!us!some!time!to!realize!the!extent!of!the!pollution!disaster,!as!well!as! the!implication!that!Cheminova!was!a!major!producer!of!glyphosate!herbicides!and!a! main!European!affiliate!of!US!chemical!giant!Monsanto.!And,!it!was!interesting!to!realize! !

76! !

!

the!fact!that!the!major!stockholder!owning!and!operating!Cheminova!was!the!University! of!Århus,!where!I!was!later!to!study!biology.! In!countries!of!the!Western!World,!agriculture!is!regulated!by!policy!and!in!principle! such!policy!is!based!on!factual!evidence,!scientific!findings!and!democratic!involvement! of!all!major!interests.!However,!the!reality!of!agriculture!policy!can!be!quite!different,! with!the!USA!no!better!nor!worse!than!the!rest!of!the!mechanized!and!industrialized! world.!This!is!a!world!where!farms!have!become!increasing!larger,!cost9effective,! dependent!on!unfathomable!amounts!of!agrochemicals!and!fossil!fuel!to!produce!food! that!may!possibly!satisfy!the!demands!of!society!for!cheap!commodities,!but!which! often!is!of!a!quality!that!leaves!a!lot!to!be!wished!for.!In!many!countries!the! consequences!of!industrial!agriculture!are!devastating,!be!it!countries!like!Denmark,!the! Netherlands!or!Germany,!where!intensive!farming!covers!the!majority!of!unpaved!area! and!open!landscape,!where!natural!habitats!have!been!erased!and!wildlife!has!largely! disappeared.!In!such!European!countries!the!farming!history!goes!back!two!thousand! years,!to!the!ages!when!settlers!slashed!and!burned!the!forest!and!began!claiming!the! land.!This!has!been!a!gradual!ecological!change!of!landscape,!but!especially!this!last! century!has!seen!dramatic!increase!in!farming!intensity,!with!reclamation!of!wetlands! and!utilization!of!every!potential!surface!for!production!purposes7.!The!latest!decades! have!seen!new!types!of!farming!grasping!hold!of!the!landscapes,!such!as!the!“seas!of! greenhouses”!in!Spain!and!the!Netherlands,!where!vegetables!are!partially!grown!in! artificial!soils!drenched!in!chemical!fertilizers.!Or!take!the!example!of!Denmark,!which! gradually!has!become!one!of!the!worlds!largest!industrial!producers!of!low9cost!animal! protein8,!mainly!swine!that!are!held!in!huge!production!facilities!which!no!longer!have! any!connection!to!the!surrounding!farmland9.!The!primary!production!of!the!Danish! farmlands!is!nowhere!near!the!capacity!to!supply!the!demands!for!fodder,!which!is! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 7!Hansen,!2003;!2008.! 8!In!2011!Denmark!produced!30!million!pigs!and!continues!to!be!the!world’s!leading!pork!exporter.!The!

production!volume!and!total!financial!revenue!parallels!the!Norwegian!production!and!export!of!farmed! salmon.!Furthermore,!the!qualitative!aspects!of!feed!ingredients!are!contested!for!both!production! systems.!Soy!is!an!important!ingredient!in!feed!for!Danish!pigs!and!Norwegian!salmon.! 9!For!an!analysis!of!this!development!see!Hansen,!2003!p.!3129328.!

!

77! !

!

largely!imported!cheap!sources!of!protein,!fats!and!carbohydrates,!arriving!in!huge! quantities!as!soy,!maize,!rape9seed!and!other!cash9crops!from!South!America,!Canada! and!the!USA.!Instead!of!being!productive!areas,!the!Danish!landscape!has!gained!new! importance!as!a!receiving!environment!for!the!putrid!waste!from!these!farm!industries.! An!interesting!aspect!here!is!the!fact!that!the!development!has!been!gradual,!with!no! singular!factor!responsible!for!the!resulting!situation.!A!different!picture!emerges!if!we! cross!the!Atlantic!Ocean,!to!the!Argentinean!pampas!region!where!much!of!the!fodder! for!the!Danish!swine!is!produced.!Here!the!changes!in!overall!landscape!have!been!very! dramatic.!In!just!a!few!decades!Argentina!has!gone!from!extensive!cattle!grazing!to!a! new!agriculture,!with!huge!monocultures!of!mainly!transgenic!soybean10.!This! development!has!brought!enormous!consequences!there!as!well.!Not!mentioning!the! loss!of!habitat!and!depletion!of!species!richness,!we!can!focus!narrowly!on!the! consequences!for!farmland!communities,!small!towns!where!incidence!of!grave!medical! problems!have!risen!sharply!and!where!this!is!documented!as!mainly!a!consequence!of! increased!use!of!pesticides!in!cultivation!of!glyphosate9tolerant!soybean11.!! So!this!is!based!on!logic,!rationale!and!common!sense?!It!just!does!not!seem!evident,! that!societies!should!deliberately!strive!for!development!of!such!agricultures!that!go! against!and!destroy!the!natural!environment.!But,!somehow!this!very!situation!has! evolved!into!existence,!possibly!primarily!driven!by!financial!incitements12.!In!the!world! of!today,!agriculture!is!a!main!polluter!and!a!main!threat!to!the!remaining!fragments!of! diversity!and!terrestrial!life!on!the!planet.!In!this!situation!we!could!expect!mankind!to! demand!high!quality!agriculture!produce,!healthier!and!more!nutritious!foods!and!a! reduction!of!the!negative!impact!on!surrounding!nature.!But!my!claim!is!that!although! we!as!human!societies!definitely!could!afford!such!priorities,!still!we!choose!differently.! Our!dominant!agriculture!strategies!rely!on!intensive!industrial!farming,!producing! commodities!which!bring!chemical!residues!onto!dinner!plates!of!our!children!and! where!ethical!standards!seem!not!to!be!an!issue!of!concern.!This!is!a!deliberate!choice,! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 10!Pengue,!2005! 11!Vazquez#&#Nota,!2010# 12!Hansen,!2003;!2008!

!

78! !

!

maximizing!output!quantity!at!a!lowest!possible!price13.! Thus!we!are!back!to!the!important!role!of!scientific!research,!9!the!only!way!that!facts! can!be!presented!and!policy!documents!can!be!developed,!is!through!systematic!and! meticulous!work!of!scientists!in!universities,!in!administration!and!in!research!and! development!of!private!and!public!sectors.!Documentation,!development!of!alternative! solutions!and!remediation!of!consequence!are!important!outcomes,!but!unfortunately,! often!the!workings!of!scientists!are!at!the!roots!of!the!problems!themselves,!9!many!of! the!chemicals!that!have!proven!to!be!persistently!toxic!and!which!accumulate!in!the! natural!food!chains,!are!the!results!of!concentrated!meticulous!scientific!research14.! Research!laboratories!have!developed!technical!inventions!that!were!supposed!to!make! life!easier,!reduce!labour!and!reduce!costs,!but!which!have!proved!to!also!have!adverse! consequence!that!we!could!not!envisage.!Just!as!we!get!surprised!when!we!discover!that! flocks!of!geese!contribute!to!the!dispersal!of!transgenic!seed,!we!also!get!surprised! when!we!realize!that!the!plasticizers!added!to!beverage9bottles!or!children!toys,!and! flame9retardants!added!to!clothes!and!furniture,!may!actually!have!adverse! consequences!for!health.!Sometimes!it!is!a!matter!of!calculated#risk,!as!when!our! advisors!set!maximum#residue#limits!for!substances!that!our!researchers!are!relatively! sure!about,!but!sometimes!negative!effects!emerge!as!complete!surprise!for!authorities,! regulators!and!most!members!of!the!scientific!communities.! And,!sometimes!it!seems!that!society!as!a!collective!is!somehow!just!not!aware!of! actually!having!the!particular!knowledge!which!could!prevent!adverse!consequence.!To! some!part!it!can!be!explained!by!the!complexity!of!it!all;!the!risk9factors!in!our!world!of! today!are!often!investigated!and!described!in!great!detail!and!the!body!of!evidence!is!so! overwhelming!and!contradictory,!that!it!is!quite!understandable!that!even!experts! ignore!important!aspects,!loose!the!grand!perspective!or!get!absorbed!in!detail.!Finally,! it!is!no!secret!that!decisions!which!potentially!have!profound!consequence!for!health! and!environment,!are!often!products!of!compromise!between!competing!interests.! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 13!Cuhra,!2009.! 14!A!quote!attributed!to!Georges#Pompidou!can!be!translated!into!something!like!this;!there#are#three#ways#

to#ruin;#women,#gambling#and#experts.#The#fastest#one#is#gambling,#the#most#pleasurable#with#women.#But# the#most#reliable#one#is#with#experts.!

!

79! !

!

These!are!deliberations!where!advice!based!in!science!and!research!plays!an!important! part,!but!where!politics!and!corporate!finances!also!have!strong!influence.15! Approaching+the+heritage++ During!the!years!of!work!preparing!results!and!studying!literature!I!have!willingly! stumbled!onto!ideas!and!evidence,!even!such!which!was!not!always!directly!associated! with!my!main!subjects,!but!which!never!the!less!seemed!important!and!thus!to!some! degree!was!included!in!this!writing.!The!decision!to!do!so!was!based!on!several!lines!of! argumentation.!First!I!will!try!to!substantiate!the!conviction,!that!these!other!aspects! and!larger!overarching!issues!do!have!direct!relevance!to!the!work!performed!and!thus! must!be!seen!as!valid!parts!of!the!reflections!leading!to!analysis.!The!other!important! realisation!that!came!to!me!during!this!work,!is!the!fact!that!certain!parts!of!the! scientific!heritage!seem!to!have!been!if!not!forgotten,!then!at!least!overlooked.!As!I!feel! that!some!of!this!work!has!relevance!in!the!core!discussion!of!the!central!issues!such!as! the!nature!and!function!of!the!germ9plasm,!the!role!of!epigenetics!and!the!phenomena! termed!as!phenotypic!plasticity,!I!have!tried!to!mention!some!of!this.!I!see!that!some!of! the!thinkers!who!have!inspired!me,!have!ventured!out!into!ambitious!attempts!at! obtaining!broad!transdisciplinary!hold!of!these!hugely!important!questions16.!+ I!began!this!work!in!the!spring!of!2008,!having!been!away!from!academia!for!the! previous!14!years.!Thus!my!research,!studies,!staggering!and!writing!has!also! necessitated!an!abrupt!reorienting!and!updating!of!my!scientific!background,!9!as!my! former!specialist!knowledge!of!bottom9dwelling!invertebrate!communities,!hard9coral! diversity!and!other!marine!ecology,!has!not!been!of!much!direct!use!when!evaluating! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 15!A!collection!of!case9stories!substantiating!the!hypothesis!that!society!repeats!such!mistakes!is!

presented!in!”Late#lessons#from#early#warnings:#the#precautionary#principle#1896;2000”!(Harremoës#et#al.,! 2001).!Available!online!and!from!the!European!Environment!Agency.!As!there!unfortunately!are!plenty!of! such!cases!throughout!recent!history,!a!second!volume!was!published!in!2013).! 16!I!will!not!dare!to!think,!let!alone!communicate!to!the!reader,!that!I!have!approached!these!issues!with!

necessary!depth!and!stamina.!I!humbly!realise!that!the!work!of!Lamarck,!Kammerer,!Haeckel!and!other! great!scientist!that!I!quote!and!discuss,!have!effort!and!understanding!belayered!into!it!and!not!readily! extracted.!Thus,!in!those!parts!of!the!work!where!I!have!felt!my!own!limited!understanding!of!the!matter! as!restricting!true!wide!reflection,!I!have!touched!only!onto!the!main!principles!and!abstained!from!any! attempt!at!analysis,!reducing!my!own!part!to!that!of!a!mere!presenter.!

!

80! !

!

qualitative!aspects!of!transgenic!plants,!for!analyzing!modern!agriculture!nor!for! perfecting!and!running!the!laboratory!for!ecotoxicological!testing!in!D.#Magna.!I!believe! that!during!this!development!I!have!touched!upon!some!very!interesting!questions!and,! even!more!importantly,!9!some!very!interesting!answers.!The!expansion!of!laboratory! methods,!using!canteen!trays!and!light!tables,!subsequently!realising!the!importance!of! offspring!sex!ratio!and!reproductive!failure,!could!be!seen!mainly!as!development!of! methods,!which!has!contributed!to!the!quality,!efficacy!and!productivity!of!the!GenØk# Daphnia#laboratory.!These!qualitative!improvements!along!with!some!of!my!research! findings,!give!me!reason!to!feel!that!I!have!contributed!not!only!via!the!scientific!results! but!also!in!the!overall!purpose!and!development!of!the!research!facility.!! The!wide!span!of!this!thesis!brings!certain!disadvantages,!mainly!as!a!possibly! perceived!lack!of!overall!coherence!as!well!as!limited!depth!of!penetration!in!some! individual!subjects.!Often,!a!plural!form!of!“we”!has!been!used!when!describing! experimental!work!and!the!mental!processes!leading!to!the!research!results!presented,! recognizing!contributions!and!collective!efforts!of!colleagues,!co9authors!of! manuscripts,!discussion!partners,!supervisors!and!laboratory!co9workers.!However,! although!the!plural!form!is!used!extensively,!the!wording!of!the!thesis!and!the!aspects!of! its!quality!99!or!even!a!regrettable!lack!of!such!quality!99!is!solely!the!responsibility!of!the! singular!author17.! Our+scientific+heritage+should+not+be+ignored+ ”We!will!never!cease!our!critique!of!those!persons!who!distort!the!past,!rewrite!it,! falsify!it,!who!exaggerate!the!importance!of!one!event!and!fail!to!mention!some!other;! such!a!critique!is!proper![...]!but!it!does!not!count!for!much!unless!a!more!basic!critique! precedes!it:!a!critique!of!human!memory!as!such”!18+ I!start!this!last!line!of!thought!by!outlining!western!thinking!as!mainly!dualistic,!and!we! have!seen!there!is!an!unfortunate!systematic!division!between!matters!physical,!which! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 17!With!the!notable!exception!of!the!essays!on#genesis#of#scientific#facts#and#researcher#misbehaviour!which!

both!have!been!discussed!with!Brian#Wynne,#Anne#Myhr!and!Fern#Wickson,!and!the!interpretations!of! primary!research!results!which!have!been!discussed!with!Thomas#Bøhn!and!Terje#Traavik.! 18!Milan#Kundera!(2002)!9!In!a!translation!from!the!original!French!text!by!Linda!Asher.!

!

81! !

!

are!perceived!as!being!in!the!hands!of!scientists,!and!the!metaphysical!or!spiritual!sides! of!this!world.!This!leads!to!a!misconceived!scientific!legitimating!of!such!practice!that! does!not!respect!nature!nor!the!integrity!of!the!living!organism.!Again,!it!is!both! relevant!and!interesting!to!go!back!into!history.!Somehow!it!seems!that!many! researchers!aspire!to!use!the!most!recent!publications!in!their!field,!as!references!and! support!for!their!own!work.!Although!this!approach!undoubtedly!is!very!justifiable!in! the!research!of!specific!issues!where!modern!technological!means!have!recently! advanced!our!understanding,!it!is!not!entirely!advisable!in!subjects!of!natural!history!or! in!many!of!the!fields!outside!of!biological!sciences.!Along!the!same!line!of!thought!it!can! be!argued!that!for!a!deeper!understanding!of!policy!it!is!still!relevant!to!read!the!studies! on!autonomy!of!administration!by!former!US!president!Woodrow#Wilson,!published!in! 1887!and!important!in!evaluating!the!recent!interference!into!the!regulation!of!food! produced!from!transgenic!plants.! Naturally,!for!most!of!us!it!is!most!convenient!to!use!Scholar!or!Science#direct!or!some! other!provider!of!readily!available!factual!knowledge,!mostly!online.!Thus!it!seems!that! evidence!not!accessible!online,!may!not!be!used!as!much!and!as!often.!And!definitely! this!must!be!the!situation!for!many!of!the!publications!that!were!published!before!the! age!of!the!Internet.!Luckily,!many!pdf9copies!of!evidence!dating!as!far!back!as!1980'!or! even#1970'!are!surfacing!and!made!available,!but!if!one!searches!for!work!much!older! than!that,!one!has!to!be!able!to!use!traditional!methods,!such!as!those!mastered!by! research!librarians.!One!treasure!which!surfaced!through!such!traditional!search!for! knowledge,!was!the!1926!monograph!by!Carl#Wesenberg#Lund,!entitled!Contributions#to# the#Biology#and#Morphology#of#the#genus#Daphnia,#with#some#remarks#on#Heredity.!The! complex!question!of!heredity!and!the!biological!aspects!of!dynamic!evolution!and! adaptation!of!life9forms!as!well!as!simply!physical!heritage!of!knowledge!as!dusty!pages! in!forgotten!volumes!are!both!aspects!that!can!be!read!into!that!last!part!of!the!title,! which!leaves!us!hoping!for!a!heritage!that!we!can!expand!further.!! The!quote!at!the!start!of!this!small!chapter!is!by!French!author!Milan#Kundera!who! shares!my!distant!origin.!I!find!it!appropriate!as!introduction!to!a!small!excursion!into! history!of!science,!the!purpose!of!which!will!be!to!substantiate!that!we!as!scientists!can! not!expect!to!understand!the!perplexing!questions!of!heredity!and!the!principles! !

82! !

!

underlying!the!phenotypic!expressions!of!biodiversity,!unless!we!accept!that!there!are! fundamental!issues!that!are!unknown!to!us,!incomprehensible!to!us.!Some!of!these! questions!have!been!left!unanswered!through!centuries!of!wonder,!as!we!are! contemplating!or!rather!mostly!avoiding!eternal!scientific!paradoxes!that!form! unbridgeable!oceans!dividing!and!fragmenting!our!understanding!of!the!world!in!which! we!live.!In!order!not!to!make!this!excursion!life9long,!we!have!to!march!directly!to!the! shores!of!the!nearest!of!these!Seas#of#Ignorance!and!set!sail!in!the!dusty!vessels!our! predecessors!left!at!the!beaches.!To!understand!biological!heredity,!the!role!of!the! biochemical!structures!which!we!have!come!to!know!as!the!genetic!code!and!that!our! predecessors!knew!as!germ9plasm,!we!must!try!and!accept!that!despite!centuries!of! research,!we!still!are!on!the!outside!of!the!understandings!we!have!hoped!to!achieve.! The!scientist!who!perform!manipulations!of!hereditary!material,!translocating!pieces!of! biochemistry!between!species!and!constructing!transgenic!plants,!microbes!and! animals,!9!those!scientists!might!claim!to!have!sufficient!understanding!of!what!they!are! doing,!but!there!are!many!other!scientist!amongst!us!who!think!differently.!! Throughout!the!previous!chapters!I!have!tried!to!present!some!of!the!facts!and! reflections!which!should!cast!light!on!the!problematic!aspects!of!the!present! developments.!We!have!performed!research!showing!that!there!are!fundamental!faults! in!the!present!regulation!of!pesticides!and!transgenic!cultivars!and!the!astonishing!fact! of!that!matter!is,!that!we!have!only!but!poked!a!little#bit!beneath!the!polished!surfaces! visible!to!the!outside!world.!And!what!have!we!found?!That!the!worlds!most!commonly! used!chemical!pesticide!has!been!accepted!by!global!societies!on!scientifically!based! premises,!which!must!be!termed!as!misleading?!That!the!most!fundamental! assumptions!of!safety!and!predictability!of!transgenic!plants!are!not!based!on!scientific! evidence!but!have!arisen!as!political!decisions?!That!the!transgenic!plants!are!useless! for!feeding!the!world,!for!adapting!to!climate!change!or!for!ensuring!better!food?!! Yes,!and!not!only!that,!we!have!found!that!a!main!quality!of!the!genetically!manipulated! transgenic!plants!is!a!surprising!ability!to!accumulate!pesticide!residues!and!pass!these! toxins!on!to!the!consumers.!It!is!a!fact!that!our!small!research!institution!far!north!of!the! Arctic!Circle!has!only!limited!resources!and!few!heads!employed!in!analysis.!We!still!do! not!have!access!to!crucial!parts!of!industry!information!nor!are!we!even!allowed!to! !

83! !

!

independently!analyze!the!plant!materials!developed!by!this!industry.!Still,!by!opening! just!a!small!PhD9project!into!a!few!aspects!of!modern!biotechnology!we!have!discovered! such!fundamental!flaws?!Is!this!just!imagination!or!could!it!possible!be!indicative!of!a! wider!complexity,!suggesting!that!the!gleaming!ivory!towers!of!giant!biotech!industry! have!surprisingly!weak!foundations,!9!as!to!allow!for!such!immediate!dismantling?!! Václav!Havel!claimed!that!sooner!or!later!truth!will!prevail,!in!his!famous!words;!láska#a# pravda#zvítezí#nad#lží#a#nenávistí,!but!sometimes!truth!emerges!only!after!such!a!long! time,!that!the!lies!and!falsehood!have!solidified!and!intoxicated!the!landscape.!! I!will!not!venture!further!down!that!path!but!ask!you!to!follow!me!back!to!the!Seas#of# Ignorance,!where!we!now!will!look!at!other!means!left!by!our!predecessors!for!our! disposal;!the!scientific!heritage!which!will!help!us!gain!understanding!of!biological! heredity.!This!is!not!a!crime!novel!and!there!is!no!need!to!keep!the!suspense!by!saving! essential!information!for!the!last!pages,!9!what!I!aim!to!do!here,!despite!the!somewhat! lengthy!introduction,!is!to!show!that!there!is!good!and!credible!evidence!for!biological! phenomena!which!we!due!to!a!lack!of!better!words!at!present,!will!term!as!epigenetics! and!activation!of!dormant!characteristics!inherent!in!the!germplasm.!I!will!take!you!to! the!work!of!Paul!Kammerer.!Kammerer!was!working!with!something!that!even!today!is! difficult!to!comprehend;!he!was!investigating!the!phenotypic!plasticity!of!organisms!and! how!this!sleeping!potentiality!can!be!released!as!adaptations!to!new!environments19.! There!is!a!fundamental!difference!between!this!type!of!thinking,!and!the!traditional! misinterpretation!of!those!who!claim!to!have!read!Lamarck.!The!fundamental!difference! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 19!Despite!the!fact!that!recently!research!(Vargas,!2009)!has!focused!on!the!work!which!Kammerer!did!in!

Aleytes,!speculating!that!this!is!the!main!evidence!of!epigenetics,!9!I!tend!to!disagree.!For!those!not!familiar! with!the!work!of!Paul#Kammerer,!we!will!introduce!the!Aleytes!experiment!here;!in!short!Kammerer!used! two!closely!related!by!ecologically!distinct!species!of!toad,!who!have!the!same!apparent!morphological! features!but!which!have!rather!different!ecological!adaptation,!one!species!has!an!aquatic!form!of!life,! mating!in!water,!depositing!its!eggs!in!water!and!living!most!of!its!life!in!water.!The!other!species,!which! resembles!the!first!one!down!to!a!few!important!anatomical!differences!such!as!the!nupital!pads!which! were!the!focus!of!the!mentioned!experimental!series!that!Kammerer!performed,!lives!a!more!terrestrial! existence,!even!mating!on!land!and!depositing!its!eggs!in!moist!vegetation.!This!species!does!not!have!the! nupital!pads!seen!in!the!former.!

!

84! !

!

is!in!the!understanding!of!how!potentiality!is!inherited,!whether!the!seeds!of!certain! characteristics!are!omnipresent!and!dormant,!or!if!the!potentiality!is!acquired!and! somehow!made!heritable20.!The!work!in!Aleytes!became!the!Achilles!heel!of!Kammerer,! his!adversaries!led!by!English!scientist!William!Bateson!found!that!histological!samples! had!been!forged!and!shortly!afterwards!Kammerer!committed!suicide.!This!unfortunate! development,!along!with!the!political!turmoil!which!absorbed!Europe!at!that!time,! meant!that!the!scientific!communities!nearly!forgot!Kammerer,!his!research!and!his! publications.!Reviving!his!work!is!not!difficult!however,!as!he!wrote!a!synthesis!of!his! main!experiments!and!the!underlying!principles,!which!was!translated!into!English!and! has!recently!been!reprinted.!Reflecting!on!this!and!the!work!of!Arthur#Koestler,!who! discovered!Kammerer!in!the!1960'!and!compiled!a!fascinating!biography!of!his!life!as! scientist,!we!see!that!there!are!some!very!important!findings!supporting!the!mentioned! theories.!Although!the!recent!focus!on!Kammerer!in!the!context!of!the!scientific! buzzword!epigenetics!has!the!right!direction,!still!in!my!opinion!it!manages!to!arrive!at! the!wrong!target.!The!work!which!should!be!in!focus!when!discussing!epigenetics!is!not! the!experiments!in!Aleytes,!nor!do!I!agree!with!Koestler!that!repeating!the!experiments! done!in!Ciona!will!produce!the!necessary!evidence!of!dormancy.!However,!one! experiment!which!in!my!opinion!is!truly!spectacular!and!should!be!repeated,!is!the! work!Kammerer!did!in!Proteus.!However,!it!would!be!necessary!to!carefully!replicate! the!methods!used.!Kammerer!was!an!excellent!laboratory!professional!and!he!was! partially!mistrusted!by!adversaries!who!possibly!failed!to!repeat!his!experiments!on!the! grounds!of!using!somewhat!less!sophisticated!means.!In!the!Proteus!experiments!this! importantly!implies!that!the!light!used!should!be!similar!to!that!used!in!the!darkroom!of! photographers.! The!life!of!Carl#Wesenberg;Lund,!the!founding!father!of!Danish!limnological!research,! was!not!as!dramatic!as!that!of!Paul!Kammerer,!but!Wesenberg9Lund!is!another!scientist! who!produced!work!which!also!has!been!partially!forgotten!by!time.!When!working!in! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 20!A!complimentary!theory!is!presented!by!Lie!(2009)!as!the!concept!of!"categorical!base"!for!a!specific!

"functional!property".!Conventional!discipline!would!use!the!terminology!genotype!and!phenotype,!but!Lie! opens!another!complexity!in!suggesting!that!a!strive!for!a!specific!disposition!(function)!can!have!its! origin!in!distinctly!different!hereditary!bases.!I!interpret!this!view!as!approaching!coevolving!analogues.!

!

85! !

!

waterflea!as!we!have,!one!might!feel!overwhelmed!by!the!enormous!published!evidence! from!centuries!of!work!in!cladoceran!species!such!as!Daphnia#pulex!and!Daphnia# magna.!But!most!of!the!work!which!researchers!use!today!as!reference!material!seems! to!be!recent!publications.!A!century!ago!Wesenberg9Lund!published!detailed!work21,! which!gives!a!thorough!description!of!the!natural!history!of!waterfleas,!with!a!special! focus!on!Daphnia#magna.!Wesenberg9Lund!spent!decades!of!his!life!meticulously! studying!fresh!water!invertebrate!plankton!and!his!writings!are!interesting.!Besides!the! scientific!publications!he!also!authored!popular!books!on!the!rich!diversity!of!critters! and!creatures!found!in!ponds!and!lakes.!Reading!these!enthusiastic!depictions!of! natural!beauty!fills!a!modern!reader!with!some!nostalgia,!as!these!small!windows!of! diversity!have!all!but!disappeared!from!the!Danish!rural!landscape.!Most!ponds!have! been!filled!and!erased!to!pave!way!for!expanding!agriculture!surface!and!the!remaining! few!are!mostly!too!polluted!to!support!anything!worth!admiring.!However,!back!in!the! days!when!Wesenberg9Lund!was!gathering!inspiration,!a!century!ago,!things!were! different!and!species!such!as!Daphnia#magna!were!present!in!any!pool,!pond!or!lake22.!! Returning!to!the!observations!leading!to!the!remarks!on!heredity!that!Wesenberg9Lund! presented,!9!in!effect!his!observations!were!much!the!same!as!those!presented!by! Kammerer!9,!species!of!Daphnia!showed!morphological!variations,!which!we!could!term! phenotypic!plasticity,!manifesting!dependent!on!environment.!Thus!Wesenberg9Lund! observed!that!certain!species!in!one!specific!environment!would!have!anatomical! features!just!a!little!bit!different!from!the!individuals!of!the!same!species!found!in!other! environments.!But!in!generational!studies!on!such!distinct!morphological!structures!the! characteristic!were!befittingly!revived!when!clones!were!moved!between! environments.!These!observations!of!adaptations!to!specific!confined!environments! were!facilitated!by!the!nature!of!the!investigated!habitats,!which!were!separate!ponds! in!a!varied!landscape.!! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 21!Wesenberg9Lund,!1926.! 22!As!described!by!Wesenberg9Lund,!even!in!pools!consisting!of!little!more!than!manure.!Those!were!

times!when!such!effluents!were!mainly!of!organic!nature,!whereas!today!municipal!wastewaters!are!too! polluted!by!true!contaminants!such!as!pharmaceuticals!and!heavy!metals!from!human!excretions!to! sustain!any!forms!of!life!other!than!a!few!species!of!hardened!microbes.!

!

86! !

!

Such!lengthy!introductions!are!merely!meant!as!windows!into!a!past!in!which!scientist! may!be!seen!as!progressing!with!manners!which!were!possibly!more!cautious,! respectfully!humble!and!driven!mainly!by!curiosity.!Again,!those!predecessors!were! dealing!with!questions!with!which!we!still!struggle!today,!such!as!the!enigma!of! heredity.!Thus!it!could!be!claimed!that!we!still!have!no!true!understanding!of!the!germ9 plasm,!but!still!we!manipulate!biochemistry!and!forcefully!mix!apples!and!pears!and! jellyfish,!9!and!sometimes!this!does!produce!viable!results,!although!I!have!a!suspicion! that!we!as!scientists!often!are!taken!by!surprise!from!this,!just!as!the!layman.!! I!will!in!no!way!attempt!to!claim!that!we!have!moral!obligations!to!abstain!from!messing! with!those!essentials!which!we!do!not!fully!understand,!but!I!do!feel!that!it!would!be! appropriate!that!we!as!academic!professionals!confess,!that!we!do!not!understand!how! hereditary!material!functions.!Further!this!naturally!leads!to!a!discussion!of!the! precision!of!genetic!manipulation!methods!and!the!predictability!of!inserts!and! consequentially!the!biosafety!of!genetically!modified!organisms.!The!existing!claims!as! worded!in!US9biotech!regulation!quoted!several!times!in!this!work!are!simply!not! scientifically!justified!and!this!fact!alone!gives!me!all!necessary!freedom!to!venture!into! dissection!and!reflection!which!may!seem!far9fetched.!Never!the!less,!my!speculations! could!never!arrive!at!a!conclusion!as!far9fetched!as!the!horrendous!accusation!that!US! chemical!industry!is!dictating!global!regulation!of!biotechnology!through!simple! financial!and!political!means,!which!in!turn!overrule!scientifically!based!conclusions.! It!is!tempting!to!continue!this!into!reflections!on!texts!such!as!Freedom#in#Science#and# Teaching!(1879)!by!brilliant!German!zoologist!and!artist!Ernst#Haeckel#(182091917).! Haeckel!was!an!enthusiastic!founder!of!Deutches#Monistenbund;!the!German!association! of!monists.!Monism!is!the!basic!belief!that!everything!is!inseparably!connected,!in! opposition!to!the!dualistic!views!that!somehow!came!to!dominate!amongst!scientists! and!in!Western!Society!in!general.!And,!I!confess,!my!holistic!approach!is!flavoured!by!a! bit!of!underlying!monism23.!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 23!It!must!be!stated!that!the!historical!monism!interpreted!from!the!mentioned!literature,!may!be!seen!as!

somewhat!different!than!the!reinterpretation!of!monism!presented!in!more!recent!writing,!e.g.!Lie,!2009.!

!

87! !

!

As!any!scientific!researcher!might!acknowledge,!the!fact!of!scientific!publishing!is!that! we!all!will!be!judged!by!our!contributions.!Most!of!us!will!simply!be!forgotten,!some!of! us!even!while!we!are!still!walking!the!Earth.!A!few!selected!minds!will!have!produced! work!of!a!lasting!nature,!due!to!brilliant!discovery,!strength!of!argument!or!simply! beauty.!But,!as!I!have!argued!when!describing!the!misrepresentations!of!Darwin,!even! that!of!our!work!which!will!be!read!and!remembered!will!be!misinterpreted,! misrepresented,!quoted!out!of!context!and!misunderstood.!Unfortunately!simplification! seems!to!be!a!legitimate!method!for!condensing!interpretations!before!adding!them!as! bricks!in!the!pyramids.!Such!is!the!nature!of!that!architecture;!there!simply!is!less!space! available!as!we!gradually!progress.!Again,!regarding!Haeckel!I!have!decided!to!try!and! keep!most!of!my!reflections!to!myself,!as!his!breathtaking!excellence!in!both!science!and! arts!leaves!me!somewhat!stupified.!Our!university!library!has!a!copy!of!his!monograph! on!medusae!and!my!own!library!has!a!few!volumes!with!reprints!of!his!numerous! plates,!which!serve!as!visually!strong!appetizers!for!further!exploration.!When! contemplating!the!wording!of!his!books!and!lectures!it!is!interesting!to!notice!that!there! are!strange!inconsistencies!between!different!editions!and!translations.!These! inconsistencies!relate!to!aspects!expanding!beyond!material!biology!and!mechanistic! function,!where!metaphysical!aspects!opened!and!discussed!in!the!original!German! texts!partially!are!missing!in!the!English!translations.!This!can!also!provide!some! explanation!for!the!misunderstandings!of!key!concept!monism,!which!subsequently!has! been!embraced!by!authors!such!as!Richard#Dawkins,!who!arguably!has!infused!it!with! somewhat!different!meaning!than!that!what!should!be!read!from!the!original!work.!! God+in+his+room+trying+to+make+a+chicken++ A!recent!presentation!of!the!Large#Hadron#Collider!in!journal!New#Scientist24!made!me! remember!the!wonderful!cartoon!by!Gary#Larson,!depicting!God!as!a!young!boy,!9!with!a! surprised!look,!blackened!by!soot!and!with!chicken9feathers!flying!around.!The!title!of! the!article!was!”Large!Hadron!Collider!hints!at!why!we!exist”.!! I!was!thinking!that!Large#Hadron#Collider#hints#at#why#we#may#cease#to#exist,!might!be!an! interesting!twist!of!the!title.!I!have!often!reflected!on!the!ways!that!some!scientist!seem! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 24!Shiga,!2011.!!

!

88! !

!

to!pretend!to!have!full!oversight,!ignore!precaution!and!let!arrogance!and!disrespectful! curiosity!lead!their!work.!I!would!not!be!the!least!surprised!if!a!survey!of!opinion!in!the! scientific!communities!would!even!reveal!that!some!researchers!are!annoyed!by!the! insecurity!or!precaution!or!scepticism!that!others!might!find!appropriate!when!engaging! in!fields!of!science!that!may!have!unexpected!consequence.!For!the!future!of!the!Large! Hadron!Collider!project!and!the!rest!of!us,!I!hope!for!a!group!of!international!advisors!to! include!such!opinion!as!the!grandmother9common9sense!of!non9scientists!and!NGOs.! Such!common!sense!could!seem!needed!when!dealing!with!biotechnology!as!well,!9!just! imagine!something!so!obviously!challenging!as!to!developing!a!herbicide9resistant!plant! and!after!drenching!it!with!herbicide!toxins!simply!straight!away!eating#it,!9!without! testing!for!herbicide!residues.!The!human!being!is!a!creature!in!which!creative!intellect! and!roaring!stupidity!go!hand9in9hand.! I!have!used!the!word!finally!at!the!forefront!of!numerous!sentences!and!for!introducing! new!paragraphs!and!reflections,!but!now,!running!out!of!pages,!I!find!it!highly! appropriate.!Not!always!does!a!text!have!meaning,!but!here,!at!this!very!point!in!space! and!time,!I!will!try!to!put!down!the!words!which!will!be!the!concluding!remarks! regarding!the!scientific!explorations!of!my!thesis.!The!investigations!into!what!we!might! denominate!as!agnotology#of#risk;assessment#of#herbicide;tolerant#transgenic#cultivars! lead!us!to!the!reflections!on!fundamentally!agnotological!aspects!of!heredity,!which!set! the!stage!for!a!discussion!on!biotechnological!precision!versus!curiosity,!trial9and9error.! Hopefully!the!main!reflection!on!vulnerability!of!dormant!potentiality!subjected!to! biolistic!showers!of!tungsten!meteorites,!99!the!relative!size!of!which!must!be! astronomical!compared!to!those!fragile!hairs!of!coiled!biochemistry!99,!may!be!inspiring.!+ Since!a!doctoral!thesis!must!somewhat!be!seen!as!an!ultimately!individual!statement,!it! was!considered!appropriate!and!necessary!to!present!not!only!results!of!research! performed!and!scientific!evidence!extracted,!but!also!associated!topics!and!opinion!held! by!the!author.!I!realize!this!is!a!risky!approach!as!one!of!the!first!things!I!learned!in! university!was!at!a!lecture!in!cytology,!where!our!witty!and!inspiring!professor!told!the! auditorium!how!important!it!is!to!keep!scientific!communication!at!a!high!level!of! complexity.!Through!experience,!he!had!realized!that!it!was!necessary!to!use!a!plethora! of!scientific!terms,!complicated!figures!and!an!overall!language!which!would!ensure!that! !

89! !

!

the!audience!did!not!fully!comprehend!all!that!was!being!said;!to#enhance#and# substantiate#ones#professional#credibility,!as!he!put!it.!If!not,!he!concluded;!they#will#not# believe#you#are#truly#an#academic.!+ Additional!support!for!my!approach!is!lent!from!Melville!and!his!essay!on!Hawthorne,! where!he!reflects!on!the!art!of!writing;!But#it#is#better#to#fail#in#originality,#than#to# succeed#in#imitation25.!He!then!added!that!he!who!never!failed!somewhere,!that!man!can! not!be!great.!I!will!add!a!few!words!to!that!addition!myself,!as!I!humbly!realize!that!too! much!of!a!good!thing!does!not!necessarily!add!up!to!something!better.!Thus,!after!some! trial!and!error!one!might!be!motivated!to!avoid!the!gravest!risks!of!failure,!maybe!even! at!the!expense!of!personal!and!professional!integrity26.!! When!Thomas!once!asked!me!what!I!would!envisage!as!a!possible!subject!for!a!doctoral! thesis,!I!told!him!from!the!top!of!my!head!that!I!would!like!to!investigate!decomposing# whales.!Whales!are!abundant!in!our!part!of!the!world,!we!swim!with!them,!we!eat!them! and!we!use!their!bones!to!decorate!our!homes.!We!love!whales,!whales!are!great! wonderful!exciting!intelligent!creatures.!Whales!are!unbelievably!big!and!they!dive! down!into!the!abyss!of!black!cold!death!to!fight!and!devour!giant!octopi.!! I!wanted!to!write!about!whales.! But,!in!the!end!I!had!to!write!about!the!flea,!9!the!waterflea.! Melville!had!his!time,!he!wrote!of!whales,!creatures!too!intelligent!to!eat!just!anything.! Melville!penned!his!reflections!in!an!explosion!of!colour,!which!makes!any!attempt!I! may!give!seem!simple;!One#often#hears#of#writers#that#rise#and#swell#with#their#subject,# though#it#may#seem#but#an#ordinary#one.#How,#then,#with#me,#writing#of#this#Leviathan?# Unconsciously#my#chirography#expands#into#placard#capitals.#Give#me#a#condor's#quill!# Give#me#Vesuvius'#crater#for#an#inkstand!#Friends,#hold#my#arms!#For#in#the#mere#act#of# !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 25!Melville,!1850.! 26!How!very!true!these!words!came!to!be.!This!acknowledgement!is!taken!as!it!is,!from!the!first!version!of!

the!thesis!(March!2014),!which!was!subsequently!rejected!by!the!University!of!Tromsø!in!October!2014.! This!footnote!is!a!brief!addition!to!the!second!version,!9!the!2015!resubmission!of!the!somewhat!castrated! original!work.!

!

90! !

!

penning#my#thoughts#of#this#Leviathan,#they#weary#me,#and#make#me#faint#with#their# outreaching#comprehensiveness#of#sweep,#as#if#to#include#the#whole#circle#of#the#sciences,# and#all#the#generations#of#whales,#and#men,#and#mastodons,#past,#present,#and#to#come,# with#all#the#revolving#panoramas#of#empire#on#earth,#and#throughout#the#whole#universe,# not#excluding#its#suburbs.#Such,#and#so#magnifying,#is#the#virtue#of#a#large#and#liberal# theme!#We#expand#to#its#bulk.#To#produce#a#mighty#book,#you#must#choose#a#mighty#theme.# No#great#and#enduring#volume#can#ever#be#written#on#the#flea,#though#many#there#be#who# have#tried#it.!27! None!of!this!had!been!possible!alone.!I’m!standing!on!the!shoulders!of!predecessors,! harvesting!fruits!of!their!efforts.!I!have!tried!as!best!as!I!can,!to!credit!these!sources!of! facts,!inspiration!and!general!direction,!by!referring!to!them!and!their!work.!I!also! extend!my!gratitude!to!Petr,#Svein;Anders,#Brian,#Daniel,#Mickaël,#John,#Suzan,#Fern,#Jan,# Arinze,#Earl,#Odd;Gunnar,#Anne,#Raul,#Walter,#Angelika,#Jack,#Julia,#Lise,#Kaare,#Kåre,# Conny,#Inger,#Monica,#Luc,#Sarah,#Vlado,#Trond,#Idun,#Elisabeth,#Berit,#Ignacio,#David,# Frode#and!those!many!others!who!have!helped!in!the!laboratory,!to!my!lecturers,!my! students!and!to!the!other!good!people!at!the!US9EPA.!! And,!Steve!showed!me!how!to!turn!a!few!sequences!of!words!into!writing.! Probably!I!forgot!important!names,!which!should!be!in!this!list.!The!previous!time!I! wrote!something!like!this!it!was!about!marinelife#on#shipwrecks,!with!reflections!on! biological!phenomena!determining!what!will!grow!on!your!foot!if!you!stand!in!the!water! for!too!long28,!9!and!in!my!acknowledgements!I!listed!a!lot!of!people!but!forgot!to!credit! Marianne#Pereira!of!Hempel!industries!in!Copehagen,!despite!the!fact!that!she!was!a! main!source!of!inspiration.!So,!if!you!are!one!of!the!people!who!I!forgot!to!mention!this! time,!I'll!simply!get!back!to!you!next!time!I!write!a!thesis.!! And,!while!we!are!back!in!the!past,!I!should!mention!that!two!decades!ago!my!fellow! undergraduate!student!Marina!in!a!few!sentences!taught!me!more!on!scientific!method! than!all!my!teachers!combined,!still!a!pivotal!point!in!my!understanding!of!science.!! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 27!The!Whale,!chapter!104.#Melville,!1850.! 28!Cuhra,#1994.!

!

91! !

!

I!have!been!very!fortunate!to!happen!upon!understanding!and!interested!editors,!thus! my!few!submitted!manuscripts!have!been!thoroughly!scrutinized!by!anonymous! reviewers!who!have!contributed!importantly!and!helpfully!to!the!overall!result.!Even! when!major!revisions!were!necessary!(they!sometimes!truly!were)!the!reviewer! comments!gave!me!constructive!guidelines.!I!am!immensely!grateful!for!those! anonymous!reviews!and!I!could!not!have!wished!for!any!other!method!of!publishing!my! scientific!results.!And!in!the!same!breath!I!must!excuse!the!obvious!lack#of#review! affecting!this!remaining!part!of!my!writing.! The!inspiring!and!heated!discussions!on!scientific!methods!and!statistics!have!been!an! important!part!of!the!overall!progression!of!the!scientist!and!I!thank!all!my!supervisors! and!discussion!partners,!especially!Einar,#Thomas!and!Terje.!The!Norwegian#Research# Council!and!GenØk!generously!funded!my!salary!and!made!it!possible!for!me!to!quit!my! job!and!concentrate!on!research.!! My#kids!have!shown!an!amazing!understanding!of!the!importance!of!having!copies!of! scientific!articles!floating!about!everywhere!in!the!house,!especially!on!the!dinner!table.! Hanna,#Kristin,#Jakob!and!Mathilde!have!cooked!and!baked,!shopped!groceries,!cleaned! the!house!and!allowed!me!to!concentrate!on!less!essential!tasks,!such!as!statistical! analysis!and!writing.!Their!mother!Ingvil!has!been!supportive!and!understanding!to!an! extent!which!should!make!other!men!envious,!9!takk.!! And!speaking!of!earthly!existence!as!opportunity!for!pleasurable!pursuit!of!beauty,!9! just!as!Daphne!the!water9nymph!was!pursued!by!Apollon,!immortalized!in!mythology! and!more!recently!even!in!biological!sciences!through!intervention!of!taxonomists!9,!her! daughters!have!given!me!hundreds!of!enlightening!as!well!as!pleasurable!moments! these!past!years,!although!i!have!subjected!them!to!so!much!cruelty,!intoxicating!them! with!herbicides!and!force9feeding!them!with!genetically!modified!material.!Terrible.! But,!without!the!aid!of!these!innumerable!female!creatures!I!could!not!have!completed! my!laboratory!experiments,!harvested!data,!nor!performed!the!calculations!and! analyses!necessary!to!reach!the!scientific!conclusions!presented.!So,!excuse!my! wrongdoing!and!the!tortuous!time!we!spent!together,!it!was!all!in!the!name!of!science.!

!

92! !

!

I!wish!to!especially!mention!journalist!Marie#Monique#Robin.!Her!film!Le#Monde#selon# Monsanto!and!her!book!bearing!the!same!title!has!contributed!more!to!my! understanding!of!the!deeper!complexity!undermining!this!scientific!field!than!most! research!papers.!I!will!also!thank!the!librarians!at!UB!UiT!who!have!been!most!helpful,! as!have!the!colleagues!at!the!Institute!of!Farmacology.!I!fear!that!probably!only!few! people!will!ever!read!these!words,!as!doctoral!theses!seldom!are!amongst!the!literary! bestsellers.!Thus!I’m!realizing!that!I’m!mainly!writing!this!for!my!supervisors,!for!the! committee!who!will!evaluate!the!work!and!for!myself.!I!have!an!astonishing!ability!to! forget!(which!can!be!useful!in!other!aspects!of!life)!and!in!a!few!years!all!of!this! transgene;conspiracy;puzzlework,!which!is!sort!of!a!diary!of!six!years!of!life,!again!may! seem!new!and!interesting.!! During!this!work!I!have!submerged!into!heaps!of!paper,!innumerable!books!and!articles,! of!which!selected!relevant!material!is!included!and!listed!in!the!references!of! manuscripts!and!chapters.!I!extend!my!gratitude!to!the!many!authors!who!have! produced!work!that!often!turned!out!to!be!interesting!and!constructive!as!either! concrete!foundations!or!as!trees!to!climb,!inspiring!me!and!giving!me!a!wealth!of!new! directions!to!explore29.!These!thinkers,!researchers!and!authors,!of!whom!some!are! unfortunately!rather!forgotten!or!even!misunderstood,!left!a!heritage!which!I!have!tried! to!approach!in!a!scientific!way!and!with!an!open!mind.!I!will!not!list!names!of!the!many! scientists,!colleagues!and!adversaries!who!have!triggered!sparks!of!creativity!and! enjoyable!research!moments,!but!I!must!repeat!the!words!I!said!to!my!colleagues!when!I! started!this!job;!it#is#an#honour#and#a#privilege#to#be#allowed#to#work#amongst#people#who# are#more#intelligent#than#one#self.!Be!that!in!person!or!on!paper.!! I’ve!done!my!best!to!be!as!truthful!as!possible!and!as!precise!as!needed,!while!also! attempting!to!use!a!bit!of!common!sense.!I!hope!my!ideas!will!be!seen!as!contributing!to! the!expanding!foundation!for!further!research.!You!are!welcome!to!stand!on!these! shoulders!and!enjoy!the!view.!! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 29!Traditionally!a!doctoral!thesis!submerges!sharply,!vertically!and!narrowly!into!an!exact!subject,!which!

is!then!thoroughly!analyzed!and!exhausted.!The!scientific!issues!covered!in!this!work!are!somewhat! broader!in!an!attempt!at!presenting!a!horizontal!approach!via!the!discipline!of!lateral!thinking.!

!

93! !

!

!

94! !

!

!

References ! Amrhein N, Deus B, Gehrke P, Steinrücken HC (1980) The Site of the Inhibition of the Shikimate Pathway by Glyphosate. Plant Phys 66(5): 830-834. Antoniou M, Brack P, Carrasco A, Fagan J, Habib M, Kageyama P, Leifert C, Nodari RO, Pengue W (2010) GM Soy: Sustainable? Responsible? Report from ARGE Gentechnik-frei, Vienna, Austria. Antoniou M, Robinson C, Fagan J (2012) GMO Myths and Truths: An evidence-based examination of the claims made for the safety and efficacy of genetically modified crops. Earth Open Source, London UK (www.earthopensource.org. Accessed March 2014). Benbrook CM (2012) Impacts of genetically engineered crops on pesticide use in the U.S. -- the first sixteen years. Envir Sci Europe 24(24): 1-13. Benzie JAH (2005) The Genus Daphnia. Issue 21 in: Dumont HJF (ed) Guides to Identification of the Microinvertebrates of the Continental Waters of the World. Backhuys, Leyden. Binimelis R, Pengue W, Monterroso I (2009) Transgenic treadmill: Responses to the emergence and spread of glyphosate-resistant johnsongrass in Argentina. Geoforum 40(4): 623-633. Bonny S (2011) Herbicide-tolerant Transgenic Soybean over 15 Years of Cultivation: Pesticide Use, Weed Resistance, and Some Economic Issues. The Case of the USA. Sustainability 3(9): 13021322. Bøhn T, Primicerio R, Hessen DO, Traavik T (2008) Reduced Fitness of Daphnia magna Fed a BtTransgenic Maize Variety. Arch Environ Contam Toxicol 55: 584–592. Bøhn T, Primicerio R, Traavik T (2012) The German ban on GM maize MON810: scientifically justified or unjustified? Envir Sci Europe 24: 22. Bøhn T, Traavik T, Primicerio R (2010) Demographic responses of Daphnia magna fed transgenic Bt-maize. Ecotoxicology 19: 419–430. Cao G et al. (2012) A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase Shows High Glyphosate Tolerance in Escherichia coli and Tobacco Plants. PLoS ONE 7(6): e38718. Cao G, Liu Y, Liu G, Wang J, Wang G (2013) Draft Genome Sequence of Pseudomonas Strain P818, Isolated from Glyphosate-Polluted Soil. Genome Announc 1, e01079–13. Charles D (2001) Lords of the harvest. Perseus publishers, New York, USA. Collins M (1994) Bt Maize Pollen (PHO176-0194): Acute Toxicity to Daphnids (Daphnia magna) under Static-Renewal Conditions: Lab Project Number: 94/3/5217: 1781/0394/6419/110. Unpublished study prepared for Monsanto Company by Springborn Laboratories Inc, 59 p.

!

95! !

!

Connor S (2010) Is the Catholic Church in favour of GM crops? The independent 14/12/2010 (www.independent.co.uk). Cuhra M (1994) Epifauna. Dissertation M. Sc. biology, University of Copenhagen. Cuhra M (2009) Du blir det du spiser. Kronikk, Nordlys 30. september 2009 (www.nordlys.no/debatt/kronikk/article3817826.ece). Cuhra M (in prep.) In vitro or in plastico: fitness differences in D. magna raised in various holding medium container materials. Cummings JL, Handley LW, Macbryde B, Tupper SK, Werner SJ, Byram ZJ (2008) Dispersal of viable row-crop seeds of commercial agriculture by farmland birds: Implication for genetically modified crops. Environ Biosafety Res 7(4): 241-52. Delaney B (2007) Strategies to evaluate the safety of bioengineered foods. Int J Toxicol 26: 389– 399. Duke SO, Lydon J, Koskinen WC, Moorman TB, Chaney RL, Hammerschmidt R (2012) Glyphosate Effects on Plant Mineral Nutrition, Crop Rhizosphere Microbiota, and Plant Disease in Glyphosate-Resistant Crops. J Agric Food Chem 60: 10375-10397. Duke SO, Powles SB (2008) Glyphosate: a once-in-a-century herbicide. Pest Manag Sci 64(4): 319325. Duke SO, Rimando A, Pace PF, Reddy KN, Smeda RJ (2003) Isoflavone, Glyphosate, and Aminomethylphosphonic Acid Levels in Seeds of Glyphosate-Treated, Glyphosate-Resistant Soybean. J Agric Food Chem 51: 340−344. EC (1997) Regulation (EC) No 258/97 concerning novel foods and novel food ingredients. European Commission, Brussels. EC (2002) European commission working document on glyphosate. Review report for the active substance glyphosate. 6511/VI/99-final. EFSA (2008) Safety and nutritional assessment of GM plants and derived food and feed: the role of animal feeding trials. Food Chem Toxicol 46: S1-S70. EFSA (2009) Scientific opinion on applications for renewal of authorisation for the continued marketing of maize MON810 and existing food and feed products. EFSA Journal 1149: 39-85. EFSA (2010) Guidance on the environmental risk assessment of genetically modified plants. EFSA journal 8(11): 1-111 (1879). FAO (2011) Biosafety Resource Book. Food and Agriculture Organization of the United Nations, Rome. FOIA (2009) The Freedom of Information Act. The National security Archive (www.gwu.edu/~nsarchiv/nsa/foia.html). FOIA (2011) 2010-2011 Agency FOIA Success Stories: Creating a Culture of Transparency (www.foia.gov/iso/opa/foia/oip-1103111.html).

!

96! !

!

Folmar LC, Sanders HO, Julin AM (1979) Toxicity of the herbicide glyphosate and several of its formulations to fish and aquatic invertebrates. Arch Environm Contam Toxicol 8: 269-278. Forbis AD, Boudreau P (1981) Acute toxicity of MON0139 (Lot LURT 12011)(AB-81-074) To Daphnia magna: Static acute bio-assay report no. 27203. Unpublished study document from US EPA library (accessed through FOIA). Fryer G (2008) Jacob Christian Schäffer FRS, a versatile eighteenth-century naturalist, and his remarkable pioneering researches on microscopic crustaceans. Notes Rec R Soc 62: 167–185. Giesy J. Dobson S, Solomon KR (2000) Ecotoxicological risk assessment for Roundup herbicide. Rev Environ Contam Toxicol 167: 35-120. Haeckel E (1879) Freedom in Science and Teaching. As translated from the German original, - Freie Wissenschaft und freie Lehre, 1877. Hansen K (2003) Det er et yndigt land. En fattig natur i et rigt samfund – status over Danmarks natur. Gads Forlag, Copenhagen, Denmark. Hansen K (2008) Det tabte land. Den store fortælling om magten over det danske landskab. Gads Forlag, Copenhagen, Denmark. Harremoës P (Ed.)(2001) Late Lessons from Early Warnings. European Environmental Agency, Copehagen, Denmark. Harrigan GG, Lundry D, Drury S, Berman K, Riordan SG, Nemeth MA, Ridley WP, Glenn KC (2010) Natural variation in crop composition and the impact of transgenesis. Nature Biotechnol 28: 402–404. Heinemann J (2009) Hope not Hype. The future of agriculture guided by the IAASTD. TWN Third World Network, Penang, Malaysia. Heinemann JA, Massaro M, Coray DS, Agapito-Tenfen SZ, Wen JD (2014) Sustainability and innovation in staple crop production in the US Midwest. Int J Agric Sustain 12: 71–88. Holderbaum DF, Cuhra M, Wickson F, Orth AI, Nodari RO, Bøhn T: Transgenic maize (MON810) affects growth and reproduction of the aquatic non-target organism Daphnia magna: a full life-cycle feeding bioassay with maize leaf diets (submitted). IAASTD (2009) International Assessment of Agricultural Knowledge, Science and Technology for Development (www.unep.org/dewa/assessments/ecosystems/iaastd/tabid/105853/default.aspx). ISAAA (2014) On-line database and report on status of transgenic cultivars. (http://www.isaaa.org/gmapprovaldatabase). Jackson MD (2011) Report of the Chief FOIA officer to the US Department of Justice. US EPA, Washington, USA. James C (2013) Global Status of Commercialized Biotech/GM Crops: 2013. ISAAA, Ithaca NY, USA. Kammerer P (1924) Environmental Vitalism: The Inheritance of Acquired Characteristics. 2005 reprint, High Sierra Books, New York NY, USA. Kershen DL (2004) Of Straying Crops and Patent Rights. Washburn L J 43: 575-610.

!

97! !

!

Koestler A (1971) The Case of the Midwife Toad. Hutchinson, London, Great Britain. Kovach (1993) Measuring the Environmental Impact of Pesticides. HortScience 28(5): 469. Krylov VV (2008) Impact of alternating electromagnetic field of ultralow and low frequencies upon survival, development, and production parameters in Daphnia magna straus (Crustacea, Cladocera). Inland Water Biol 1: 134–140. Kundera M (2002) Ignorance (translated by Linda Asher). Faber and Faber, London, Great Britain. Kuntz M, Ricroch AE (2012) Is it Time to Adjust the Current Regulatory Risk Assessment for GM Food and Feed? ISB News Report. Lie SA (2009) Naturalness reconcidered: Ontlogy, ethics and dispositions. Doctoral thesis, Department of Philosophy, University of Tromsø, Norway. Lovdata (2013) FOR 2012-10-25 nr 1150: Forskrift for graden philosophiae doctor (ph.d.) ved Universitetet i Tromsø (www.lovdata.no/cgi-wift/ldles?doc=/sf/sf/sf-201210251150.html#map004). Mann RM, Hyne RV, Choung CB, Wilson SP (2009) Amphibians and agricultural chemicals: Review of the risks in a complex environment. Envir Poll 157: 2903–2927. McAllister W, Forbis A (1978) Acute toxicity of technical glyphosate (AB–78–201) to Daphnia magna. Study reviewed and approved 8–30–85 by EEB/HED US EPA. Unpublished study document from US EPA library (accessed through FOIA) McDougall P (2010) The cost of new agrochemical product discovery, development and registration in 1995, 2000 and 2005-8. Report for European Crop Protection association and Crop Life America. Melnichuk SD, Sherban EP, Lokhanskaya VI (2007) Estimation of toxicity of glyphosate-based herbicides by biotesting method using cladocera. Hydrobiol J 43(3): 80–91. Melville H (1850) Hawthorne and His Mosses. The Literary World, August 17-24. Melville H (1851) The Whale (Moby Dick). 1937 Reprint by Garden City publishing co (Random house), New York, USA. Naumann E (1929) Grundlinien der experimentellen Planktonforschung. Sammlung Binnengewässer 6. Stuttgart, Germany. Naumann E (1933) Daphnia magna als Versuchstier. D. magna probe prüfung Materialien für Wasserbäuten, Luftanalysen und Toxicologischen Untersuchungen. Spezialarbeit von Kungl Fysiografiska Sällskapets i Lund Förhandlinger, Lund, Sweden. Naumann E (1934a) Über die Anwendung von Daphnia magna Straus als Versuchstier zur experimentellen Klarlegung der Lebensverhältnisse im Wasser. Int Revue ges Hydrobiol Hydrogr 31: 421–431. Naumann E (1934b) Über die Beeinflussung von D. magna durch einige Saltze und saltzmischungen, pH werte, freies CO2, toxicität des Kupfersaltz, Zinksulafts und Bleichlorids . Spezialarbeit von Kungl Fysiografiska Sällskapets i Lund Förhandlinger, Lund, Sweden. Nielsen KM (2013) Biosafety Data as Confidential Business Information. PLoS Biol 11, e1001499.

!

98! !

!

OECD (1993) Safety Evaluation of Foods Derived by Modern Biotechnology. Concepts and Principles. Organisation for Economic Co-operation and Development, Paris, France. OECD (1998) Report of the OECD Workshop on the Toxicological and Nutritional Testing of Novel Foods. Organisation for Economic Cooperation and Development, Paris, France. OECD (2001) Consensus Document on Compositional Considerations for New Varieties of Soybean: Key Food and Feed Nutrients and Antinutrients. OECD Environmental Health and Safety Publications Series on the Safety of Novel Foods and Feeds, No. 2. Environment Directorate, Organisation for Economic Co-operation and Development, Paris, France. OECD (2002) Consensus Document on Compositional Considerations for New Varieties of Maize (Zea Mays): Key Food and Feed Nutrients, Anti-nutrients and Secondary Plant Metabolites. OECD Environmental Health and Safety Publications Series on the Safety of Novel Foods and Feeds, No. 6. Environment Directorate, Organisation for Economic Co-operation and Development, Paris, France. OECD (2004) OECD-202 Guideline for testing of chemicals: Daphnia sp. acute immobilization test. OECD, Geneva, Switzerland. OECD (2006) An Introduction to the Food/Feed Safety Consensus Documents of the Task Force. Organization for Economic Cooperation and Development, Paris, France. OECD (2008) OECD-211 Guidelines for testing of chemicals: Daphnia magna Reproduction test. OECD, Geneva, Switzerland. OPP Docket (2013) Objection on US-EPA Final Rule under FFDCA section 408(g), 21 U.S.C. 346a and request for hearing on objections. (http://www.torpedotext.net/USEPA_OPP_DOCKET_June_15_2013.pdf). Pengue W (2005) Transgenic Crops in Argentina: The Ecological and Social Debt. Bull Sci Technol Society 25(4): 314-322. Privalle L (1997) Comparison of CryIA(b) Levels in Transgenic BT11-Derived Maize (Corn) Pollen and Event 176-Derived Maize Pollen and Justification for Citation of Daphnia magna Toxicity Study of Event 176-Derived Pollen in Support of BT11 Daphnia magna Data Requirement: Lab Project Number: NSB-001-97. Unpublished study prepared by Novartis Seeds, 11p. Raybould A, Vlachos D (2011) Non-target organism effects tests on Vip3A and their application to the ecological risk assessment for cultivation of MIR162 maize. Transgenic Res 20: 599–611. Relyea RA (2005). The impact of insecticides and herbicides on the biodiversity and productivity of aquatic communities. Ecol Appl 15(2): 618–627. Ricroch A, Bergé JB, Kuntz M (2010) Is the German suspension of MON810 maize cultivation scientifically justified? Transgenic Res 19: 1-12. Ricroch A, Bergé JB, Kuntz M (2011) Evaluation of Genetically Engineered Crops Using Transcriptomic, Proteomic, and Metabolomic Profiling Techniques. Plant Physiol 155(4): 17521761. Robin MM (2008) Le Monde selon Monsanto. Arte film production, Paris, France. Romeis J, McLean MA, Shelton AM (2013) When bad science makes good headlines: Bt maize and regulatory bans. Nature Biotechnology 31(5): 386-387.

!

99! !

!

Rosi-Marshall EJ, Tank JL, Royer TV, Whiles MR, Evans-White M, Chambers C, Griffiths NA, Pokelsek J, Stephen ML (2007) Toxins in transgenic crop byproducts may affect headwater stream ecosystems. PNAS 104(41): 16204-16208. Séralini GE, Clair E, Mesnage R, Gress S, Defarge N, Malatesta M, Hennequin D, de Vendômois JS (2012) Retracted study: Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize. Food Chem Tox, retracted by editor. Séralini GE, Clair E, Mesnage R, Gress S, Defarge N, Malatesta M, Hennequin D, de Vendômois JS (2014) Republished study: Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize. Environmental Sciences Europe 2014, 26:14 Servizi JA, Gordon RW, Martens DW (1987) Acute Toxicity of Garlon 4 and Roundup Herbicides to salmon, Daphnia, and Trout. B Environ Contam Tox 39:15–22. Shiga D (2011) LHC hints at why we exist. New Scientist 2840: 6-7. Smith K, Evans DA, El-Hiti GA (2008) Role of modern chemistry in sustainable arable crop protection. Phil Trans R Soc B 363: 623-637. Smith-Spangler C, Brandeau ML, Hunter GE, Bavinger JC, Pearson M, Eschbach PJ, Sundaram V, Liu H, Schirmer P, Stave C, Olkin I, Bravata DM (2012) Are Organic Foods Safer or Healthier Than Conventional Alternatives? A Systematic Review. Annals of Internal Medicine 157(5): 348366. SSB (2014) Online database at Statistics Norway (https://www.ssb.no/statistikkbanken - Accessed March 2014) Székács A, Darvas B (2012) Forty Years with Glyphosate. In; Herbicides - Properties, Synthesis and Control of Weeds, Hasaneen MN (Ed.), InTech (http://www.intechopen.com). Then C (2013) High levels of residues from spraying with glyphosate found in soybeans in Argentina. TESTBIOTECH Background report. (www.testbiotech.de/sites/default/files/TBT_Background_Glyphosate_Argentina_0). Traavik T (1995) For tidlig kan være for sent. DN Rapportserie, 95/5. DN Trondheim. Traavik T (1999) Too early may be too late. Ecological risks associated with the use of naked DNA as a biological tool for research, production and therapy. Research report 1999-1, Directorate for Nature Management, Trondheim Norway. Traavik T, Lim LC (2007) Biosafety first: hollistic approaches to risk and uncertainty in genetic engineering and genetically modified organisms. Tapir Academic Press, Trondheim. Traavik T, Nielsen K, Quist D (2009) Genetically Engineered Cells and Organisms: Substantially Equivalentor Different? TWN Biosafety series, TWN Penang, Malaysia. Tufte E (2001) The visual display of quantitative information. Graphics Press, Cheshire CT USA. Tveit CW, Holten R (2004) Økotoksikologisk vurdering av glyfosat (isopropylamin-saltet) Mattilsynet – statens tilsyn for planter, fisk, dyr og næringsmidler. Norwegian national regulatory document on glyphosate herbicide ecotoxicity (http://english.vkm.no/dav/65af99d9a0.pdf).

!

100! !

!

UNEP (2014) International Assessment of Agricultural Knowledge, Science and Technology for Development (www.unep.org/dewa/assessments/ecosystems/iaastd/tabid/105853/default.aspx). US FDA (1992) Policy brief in the Federal Register of May 29, 1992 (57 FR 22984), FDA Statement of Policy: Foods Derived from New Plant Varieties (www.fda.gov/food/biotechnology/default.html). US-EPA (1996) OPPTS 850.1010. Aquatic invertebrate acute toxicity test, freshwater daphnids. EPA Ecological effects Test Guidelines. EPA-712-C-96-114. US-EPA (2002) Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Fourth Edition. United States Environmental Protection agency EPA-821-R-02-013. US-EPA (2009) US Environmental Protection Agency Chief Freedom of Information Act Officer´s Report To The attorney General (www.epa.gov/foia/docs/Chief_FOIA_Report.pdf). USDA (2014) United States Department of Agriculture. Foreign Agricultural Service, Table 07: Soybeans: world supply and distribution (www.fas.usda.gov/psdonline). Vargas AO (2009) Did Paul Kammerer discover epigenetic inheritance? a modern look at the controversial midwife toad experiments. J Exp Zool (Mol Dev Evol) 312B: 667–678. Vazquez MA, Nota C (2011) Report from the 1st national meeting of physicians in the crop-sprayed towns, Faculty of Medical Sciences, National University of Cordoba August 2010. Cordoba, Argentina. Waigmann E, Paoletti C, Davies H, Perry J, Kärenlampi S, Kuiper H; Special issue: Risk assessment of Genetically Modified Organisms (GMOs). EFSA Journal 10(10): s1008 Waltz E (2009) Battlefield. Nature 461: 27-32. Wesenberg-Lund, C (1926) Contributions to the Biology and Morphology of the genus Daphnia, with some remarks on Heredity. Det Kongelige Danske videnskabernes selskabs skrifter, 126 p. Høst & Søn Copenhagen. WHO (2009) The WHO recommended classification of pesticides by hazard and guidelines to classification. World Health Organization Geneva. (www.who.int/ipcs/publications/pesticides_hazard_2009.pdf). Wickson F, Bøhn T, Wynne B, Hilbeck A, Funtowicz S (2013) Science-Based Risk Assessment Requires Careful Evaluation of All Studies. Nature Biotechnol 31(12): 1077–1078. Williams GM, Kroes R, Munro IC (2000) Safety Evaluation and Risk Assessment of the Herbicide Roundup and Its Active Ingredient, Glyphosate, for Humans. Regul Toxicol Pharmacol 31: 117– 165. Womach J (2005) Agriculture: A Glossary of Terms, Programs, and Laws, 2005 Edition. Coordinator Agricultural Policy Specialist Resources, Science, and Industry Division. CRS Report for Congress (www.cnie.org/NLE/CRSreports/05jun/97-905.pdf).

!

101! !

!

!

102! !