and cylindrospermopsin. The State of California's Office of Environmental Health Hazard. Assessment (OEHHA) 2012 criteria provides public health protection ...
Region 1: Cyanobacteria Monitoring Program Development Following a report of 3 dogs that appeared ill after swimming in the Russian River in early August 2015, the North Coast Regional Water Quality Control Board (Regional Water Board) and Sonoma County Department of Public Health began sampling the Russian River for various cyanotoxins. The Department of Public Health focused their efforts on water column sampling, believing that the most likely exposure route to the public would be the inadvertent consumption of surface water during recreational activities, while the focus of this program was on the complimentary sampling of algal mats, identifying the sources of the cyanotoxins and better understanding the risks to dogs and children.
Initial water column results were predominantly non-detect, while reach-integrated shoreline algal mats documented the presence of the cyanotoxins; microcystin, anatoxin, and cylindrospermopsin. The State of California’s Office of Environmental Health Hazard Assessment (OEHHA) 2012 criteria provides public health protection advisory posting guidance for water column samples, but the guidance is mute on the subject of cyanotoxin concentrations in cyanobacteria mats. Without existing cyanobacteria mat criteria there is no guidance on whether advisory signs should be posted on the river based upon mat concentrations. Water column results and observed mat concentrations in relation to the available water column criteria (OEHHA 2102) were only elevated for microcystin, so the Public Health Department and Regional Water Board focused all of our efforts on microcystin sampling.
In addition to testing for cyanotoxins in the algal mats, a taxonomic investigation was initiated in collaboration with the California State University at San Marcos (CSU San Marcos) to document the various cyanobacteria species that were present in the Russian River. An extensive literature search to identify the cyanotoxins that might be produced by the various cyanobacteria found in the Russian River determined that the species identified in taxonomic samples were dominated by microcystin producers.
However, on August 29, 2015 a dog died on the Russian River to anatoxin poisoning. At that time, the Public Health Department and Regional Water Board added anatoxin analysis to the sampling program. Through the remainder of September, anatoxin was detected in water column (48.9 ug/L) and algal mat (21.2 ug/L) samples but never exceeded the 2012 OEHHA guidance criteria of 90 ug/L.
Dog deaths due to cyanobacteria (anatoxin) have now been suspected or documented (in 9 of 11 cases) in the mainstem Eel River (2015), the Russian River (2015), the Van Duzen River (2009), and the South Fork Eel River (2002, 2004, and 2009). In 2016, the Regional Water Board began investigating the spatial and temporal extent of cyanobacteria in the Eel, South Fork Eel and Russian Rivers, the species common to these rivers, as well as identifying the cyanotoxins that they produce. To that end, this program focused on the collection of water grab samples (testing for 6 toxins), integrated and eventually singlespecies dominant algal mat samples (testing for 4 toxins and taxonomic identification), and deployed Solid Phase Adsorbing Toxin Tracking (SPATT) passive samplers (testing for 6
toxins). Through this work we identified the presence of between 3-5 cyanotoxins in SPATT samplers at most locations (unquantifiable concentrations), 2-3 cyanotoxins in water column grab samples (low concentrations), and 2-4 cyanotoxins in algal mats (extremely elevated concentrations in some cases). To definitively determine which cyanobacteria species were responsible for the cyanotoxins documented in our rivers, CSU San Marcos monocultured various cyanobacteria which were collected as part of this program to document cyanotoxin production.
During the 2016 monitoring program, we documented multiple locations with multiple cyanotoxins in algal mats and limited low-level detections in water column sampling. After observing floating chunks/mats of cyanobacteria, observing children playing in locations completely covered by algae, and observing dogs swimming/running/drinking from areas where algae was being churned into the water column, it became apparent that more information was needed to determine toxic the mats were should some be ingested. This led me analyze algal mat samples for Ash Free Dry Mass (AFDM) analysis to determine the relative toxicity of each species or mat.
To determine the relative (normalized) toxicity of each mat, an integrated sample from a single species dominated mat is homogenized, with 50ml pipetted into a bottle for toxin analysis and 5-25 ml (depending on species) filtered and frozen for AFDM analysis (drying and combusting), providing a measure of mg of cyanotoxin per kg of cyanobacteria organic material. Though the results obtained vary by species and time of year, some extremely high toxin concentrations have been documented, leading us to convene an international benthic cyanobacteria workgroup to assist with the development of spatial benthic criteria based upon genera and benthic cover for which public health posting could be developed. 2016
Waterbody Russian River Russian River Russian River Russian River Eel River Eel River Eel River Eel River
ANALYTE Result (ug/L) AFDM (mg/L) (mg/kg dry wt) Dominant Species Anatoxin >15750 9030 >1744.19 Phormidium Anatoxin 2631.00 1940 1356.2 Phormidium Anatoxin 8115.00 6670 1216.6 Phormidium Anatoxin 3396.00 7290 465.8 Phormidium Anatoxin 45.30 800 56.6 Anatoxin 15.60 1850 8.4 Cylindrospermum Anatoxin 38.0 4950 7.7 Oscillatoria Anatoxin 11.5 2370 4.8 Geitlerinema
2017
Waterbody SF Eel River SF Eel River Eel River Eel River Eel River Russian River Garcia River Russian River SF Eel River SF Eel River SF Eel River Russian River Russian River Eel River Russian River Eel River Russian River SF Eel River Russian River Garcia River
ANALYTE Result (ug/L) AFDM Results (mg/L) (mg/Kg dry wt) Dominant Species Anatoxins 26.1 5160 5.1 Anabaena Anatoxins 8.05 1570.00 5.1 Nostoc Anatoxins 49.7 8460.00 5.9 Phormidium Anatoxins 54.8 8390 6.5 Phormidium Anatoxins 20.55 2940.00 7.0 Phormidium Anatoxins 91.9 6020 15.3 Phormidium Microcystins 0.23 13.60 16.9 Nostoc Microcystins 0.8 36.20 22.1 Anabaena Anatoxins 123.2 5460 22.6 Phormidium Anatoxins 41.25 1420.00 29.0 Phormidium Anatoxins 175.3 4980.00 35.2 Anabaena Anatoxins 220.6 4410.00 50.0 Phormidium Anatoxins 369.3 6500 56.8 Phormidium Anatoxins 616.4 10000.00 61.6 Phormidium Anatoxins 565.8 8340 67.8 Phormidium Anatoxins 819.4 10300.00 79.6 Phormidium Anatoxins 1195.8 9910 120.7 Phormidium Anatoxins 179.2 170 1,054.1 Anabaena Anatoxins 46.5 36.20 1,284.5 Anabaena Anatoxins 158.4 37.40 4,235.3 Phormidium
DATA RESULTS (rough) •
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We believe that the results collected to date under report the level of toxicity that may have been reported in our water column and SPATT bag samples. Our use of LCMS analysis for the Anatoxin analysis for may not truly represent the conditions as they existed in the water column. The laboratory used for Anatoxin analysis did not have standards for any Anatoxin variants except Anatoxin-A. Depending on the site, or perhaps the dominant cyanobacteria species, toxicity increased, decreased, or seemed to remain consistent throughout the season.
The highest concentrations in algal mats were predominantly Anatoxin while the SPATT bags documented elevated Microcystin and Nodularin concentrations.
LCMS analysis and genomic evaluation of some species collected from the Russian River suggest that di-hydro Anatoxin may be produced and not Anatoxin-a providing additional evidence that LCMS analysis under reports the total Anatoxin water-column or algal mat concentrations.
LESSON LEARNED / QUESTIONS TO ASK?
We have initiated a cyanobacteria monitoring plan designed to provide information for the protection of public health. We are investigating the magnitude and extent of cyanobacteria in river reaches which have experienced dog deaths related to toxic cyanobacteria. In addition to public protection, we have collaborated with various researchers to further our knowledge of benthic cyanobacteria and impacts to the environment. We have begun to evaluate the data and find that the we are being led to ask more questions rather than answering the questions that we have.
Do the rivers of Region 1 play host to a novel Nodularin producer? • Our water column and SPATT bag samples demonstrated the presence of Nodularin. To date, the only known Nodularin producer is Nodularia and one species of arctic Nostoc. The toxicity of Nodularin has not been quantified and currently no water quality objectives exist. Are benthic macroinvertebrate populations (biomass and species composition) affected by cyanobacteria growth (including algal speciation and toxicity) through the season? • The results from our culture study demonstrated that low levels of cyanotoxins can have a deleterious effect on benthic macroinvertebrates in the laboratory setting. Does cyanotoxins affect benthic communities in a natural setting? Are we seeing impacts in our rivers and streams as demonstrated by impaired benthic macroinvertebrate communities?
What are the dominant cyanobacteria species in the watersheds of Region 1 and what are the microhabitats that they inhabit? • We deployed SPATT bags, and collected water and cyanobacteria mat grabs for toxin analysis. In addition, we performed in-house genera identification and laboratory species identification from a subset of our complete sample set. We photo documented some cyanobacteria mats in-situ and collected “general” habitat information for the reach as a whole. We did not emphasize the documentation of microhabitat conditions for the individual genera or species that exist in the various rivers. This additional information would be extremely helpful as we put together a field guide. What is the relative toxicity of the various species of cyanobacteria? What are the drivers that dictate when the cyanobacteria become toxic and to what degree? • Our 2016 cyanobacteria monitoring effort initially collected composite cyanobacteria samples for cyanotoxins and as the genera/species composition simplified (less diversity) later in the season, we collected single-genera mats for the analysis of cyanotoxins. We found that some single-genera mats can be more toxic than others, but the samples were not collected in a manner that lent itself to the quantification of toxicity per cyanobacteria mat mass. • Posting guidelines for recreation and water use focus on water column concentrations in either absolute toxicity or cell counts. Current posting guidelines do not take into consideration toxicity of benthic mats. We want to continue to
document the toxicity of single species mats and demonstrate the need for posting based upon intracellular concentrations.
What if any are the water quality response conditions that suggest algal blooms including cyanobacteria may be occurring? • Algae, including cyanobacteria, are always present within all waterbodies. Impacts to water quality are considered adverse when algal and cyanobacteria populations become excessive and are considered an aesthetic nuisance, affect the chemistry of the waterbody (dissolved oxygen and pH concentrations), or produce illnesses or death in animals and humans. The physio-chemical response to excess algae in a waterbody is generally the documentation of exaggerated diurnal swings in dissolved oxygen and pH (biostimulatory response). In 2016 we relied upon a small set of datasondes documenting biostimulatory conditions in the Russian River. Biostimulatory conditions were only noted on one datasonde throughout the season, yet algae and cyanobacteria mats were quite prevalent throughout much of the river.
Are flows the major determining factor in the development of algal blooms including cyanobacteria? • Of great concern to many is the relationship between flow and algal growth, especially cyanobacteria. Many hypothesize that the benthic cyanobacterial blooms we are experiencing are directly related to lower flows (warmer water, greater light penetration/intensity) associated with the prolonged drought or water management (Russian River). This is particularly important as water use continues to increase and drought events become more frequent. In 2016, did we under report Anatoxin toxicity in water column and SPATT bags analysis due to our reliance upon LCMS analysis that did not include dh-ATX? • The analysis of the cultures collected from the Russian River suggest that some of the (more toxic) species may actually be producing dh-ATX rather than ATX. The SPATT bags and water column grabs were analyzed using LCMS and an Anatoxin-A standard. They were not analyzed for any other variants at the time. The lab did not have a dh-ATX standard at the time of analysis. ELISA analysis of cyanobacteria mats demonstrated toxic concentrations in most mat samples.
What are the toxic benthic cyanobacteria species in these systems? • We collected several single species cyanobacteria mats throughout these watersheds for toxin analysis. With additional sampling, can we begin to develop an understanding of the various species which are toxic? Can we create a guide to share with recreaters, the public, and regulators so that determinations regarding public health can be made without the high cost of toxin analysis? What is the extent of benthic cyanobacteria blooms in these systems? • Our sampling effort has been focused on 9 sites encompassing no more than two miles of river length in a combined 300 miles of river and does not take into
consideration any tributary contributions. Are the cyanobacteria isolated in certain reaches or are the located throughout these river systems?
How do cyanobacteria respond to issues of turbidity? • Our site “Russian River upstream of Hopland” was somewhat turbid throughout most of the season with limited amounts of algae growing except along the margins. As it began to clear in late September, benthic cyanobacteria began to colonize and bloom.
What are the nutrient conditions under which the cyanobacteria grow and bloom in the Russian and Eel River watersheds? • Very little ancillary data has been collected during our sampling efforts. We have been so focused on public health monitoring that we have not had an opportunity to expand the program and include nutrients. How do cyanobacteria respond to issues of turbidity? • One sample site experienced higher turbidity throughout most of the season with limited amounts of algae growing except along the margins. As the water clarity improved in late September, benthic cyanobacteria began to colonize and bloom.
TAKE AWAY MESSAGE?
The ongoing development and refinement of this monitoring program is designed to provide the Regional Water Board with insight and understanding of the benthic cyanobacteria communities in the Region and the impact that they have on public health and recreation. We first and foremost utilize the data from this effort to inform the local health departments and water agencies to conditions that may warrant health postings or waterbody closures as well as providing information to the various water purveyors on the river. Our secondary driver is to provide additional information to policy makers and encourage them to adopt additional measures for public health postings of affected waterbodies. Though much of the study has been conducted on planktonic cyanobacteria, academic researchers in New Zealand have been leading the way in identifying the presence, distribution, and toxicity of benthic species. Our efforts pale in comparison to those of the New Zealand researchers, however we are no less committed to working on this matter and providing as much information as we can to interested parties with the hope of expanding the collective knowledge base. We believe that the publishing our efforts and results coupled with that of our partners in academia and laboratories both here and abroad will provide a peer reviewed source of information that can then be used for multiple purposes including; •
updating the California guidance for benthic bloom posting guidance,
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providing additional information to the State of California’s water quality programs, the Governor’s office, and the State Legislature to develop a formal program to address the threats of benthic blooms, and triggering additional interest in studying benthic blooms which will in turn benefit our ability to protect public health, domestic animals, and wildlife.
In 2018, this porgram will once again collect water column grab samples, single species dominated algal mats, SPATT bags, nutrient samples, and time series data (dissolved oxygen, ph, conductivity, temperature, and turbidity).
