Proceedings of the 13th International Conference of Environmental Science and Technology Athens, Greece, 5-7 September 2013
THE EFFECTS OF GLYPHOSATE HERBICIDE ON SOME SOIL ORGANISMS IN SELECTED AREAS OF OKPOKWU, BENUE STATE, NIGERIA. ELLA, A. B1, ELLA, F. A.2 and EFFIONG, M. U.3 1
Department of Science Laboratory Technology, Benue State Polytechnic, Ugbokolo,.Box 01, Ugbokolo,Benue State, Nigeria;
[email protected] 2 Department of Biology Education, Federal College of Education (Technical), Umunze, P.O.Box 0189 Anambra State, Nigeria. 3 Department of Aquaculture and Fisheries Technology, Federal Polytechnic, Oko, Anambra State, Nigeria ABSTRACT Glyphosate herbicide is among the most commonly used herbicides worldwide. Despite their value to agriculture, they have in some circumstances posed direct or indirect threats to the health of humans and beneficial organisms in the delicate web of nature. The effects of glyphosate on some soil organisms were investigated in Ugbokolo, Ogene and Opialu in Okpokwu LGA of Benue State. Culturable bacteria were isolated using tryptic soy agar and identification made on the basis of classification schemes published in Bergey’s manual of systematic bacteriology. Fungi were identified based on their cultural morphology and microscopic characteristics of their spores and haphae using the simplified key to the classes of fungi. Invertebrates were enumerated using simplified key to invertebrate identification. The soil samples were collected randomly from selected plots located in three towns in Okpokwu LGA of Benue state. The plots had no previous history of any herbicidal application. The soil samples were collected from the upper 10 cm of the soil profile. Results showed that composition of soil in the three locations did not differ significantly at P = 0.05 according to Tukey – Karamer Multiple Comparisons Tes. Ugbokolo had the highest percentage of organic carbon of 0.96, while Ogene had the least percentage of organic carbon of 0.73 before the application of glyphosate. The number of bacteria isolated at the control concentration (0 kg\ha) was less than that isolated at the concentration of 0.84 kg\ha and 840 kg\ha in all the three locations. Opialu had the highest number of bacteria colonies of 1.7 x 107 cfu\ml followed by Ugbokolo with 9.8 x 106 cfu\ml while Ogene had the least number of 7.6 x 106 cfu\ml. The results also showed that bacterial colonies (especially, Agrobacterium sp.,Bacillus sp. and Pseudomonas sp.) increased with increased concentration of glyphosate; while fungal colonies were not affected significantly (at P = 0.05) by the herbicide. Invertebrates (especially, Caterpillars and earth worms were affected adversely by the glyphosate herbicide. The effects of the herbicide on bacteria and invertebrates were more severe in the month of November than in the month of July. Though, there was no significant adverse effect of glyphosate on soil bacteria and fungi, but the unintended effect on soil invertebrates and other non – target organisms is enough reasons to justify its replacement by the use of non – chemical biological turn over activities. KEYWORDS: Effects, Glyphosate, Herbicide, Soil, Organisms. INTRODUCTION Glyphosate is one of the most commonly used, herbicides worldwide. The use of herbicides have increased 50 fold since 1950 and about 2.5 million tons of industrial herbicides are now used each year to secure food supply for the growing global population (Miller and Tyler, 2002). In the tropical regions of the world, agricultural intensification has led to higher
pesticide utilization (Racke et al., 1997; Shipitalo et al., 2008). Glyphosate is a broad spectrum, non selective herbicide used in the control and or killing of grasses, herbaceous plants, including deep rooted perennial weeds, some broad – leaf trees and some shrubs (United States Department of Agriculture (USDA, 2000). Primarily applied to agricultural lands, glyphosate is also popular in the production forestry because of its effectiveness in controlling many under story plant species, and its benign effects on conifers (Powers and Reynolds, 1999; 2000).in addition, it has a low mammalian toxicity (Williams et al , 2000), and rapid inactivation in soil (Veiga et al., 2001; Ramwell et al., 2004). However, it has been found to pollute virtually every lake, pond, river and stream in areas of use due to run off and leaching (Andrea, 2003). The potential non-target effects of glyphosate on soil microorganisms and their processes, such as nutrient recycling and maintenance of soil structure and synthesis via the shikimic acid pathway in bacteria and fungi (Bentley, 1990; Franz, et al., 1997), and one of its surfactants, polyxy-ethyleneamine, is toxic to species of bacteria and protozoa (Tsui and Chu, 2003). In Nigeria, most of the rural settlers are farmers and Okpokwu Local Government Area is a typical example in Benue State where over 80% of the adult population engages in farming. From preliminary studies, it was observed that herbicides are extensively used for farming and the farmers due to illiteracy, and ignorance use above the stipulated quantity of herbicides per application. These may persist relatively for long periods of time in the environment and may affect non-target organisms. Without increased professional and public understanding of the fate of herbicides in the environment, current concern about contamination of the environment will not be quitted and merits of herbicides will continue to constitute a subject of controversy. Since information must be accumulated before understanding is possible, the objective of this work was to determine whether glyphosate application results in short-term changes that are either deleterious or beneficial to soil microorganisms in some grassland soil of Okpokwu L.G.A. BASIC CHEMISTRY OF GLYPHOSATE Glyphosate is an aminophosphoric analogue of the natural amino acid glycine and the name is a contraction of glycine, phospho, and ate. Glyphosate was first discovered to have herbicidal activity in 1970 by John Frank, while working for Monsanto (Murtaza and William, 2001). Glyphosate kills plants by inhibiting the enzymes 5-enolpyruvyl shikimate – 3phosphate synthase (EPSPS) which catalyzes the reaction of Shikimate – 3 – phosphate (S3p) and phosphoenolpyruvate to form 5 – Enolpyruvyl – Shikimate – 3- phosphate (ESP). ESP is subsequently dephosphorylated to chorismate an essential precursor in plants for the aromatic amino acides: phenylalanine, tyrosine and tryptophan biosynthesis is inhibited (Zablotowiez and Reddy, 2004; USEPA, 2006). TOXICITY OF GLYPHOSATE Glyphosate is less toxic than a number of other herbicides such as those from the organochlorine family (Williams et al., 2000, USEPA, 2006). Williams et al., (2000) consider the likely effects experienced by the two groups most likely to have high exposures namely, herbicide applicators and children aged 1 – 6 years, noted that exposure in those subpopulations was a health concern. Glyphosate has been found by ingestion (one or several mouthfuls) to cause fatalities in people, despite immediate and intensive treatment (Sorensen and Gregersen, 1999; Stella and Ryan, 2004). Acute toxicity induced by glyphosate is enhanced when ingestion (100 ml or more) is accompanied by esophageal injuries (Chang et al., 1999). Despite industrial claims to the contrary, a recent study indicated lymphatic cancer increases that was associated with glyphosate exposure (Hardell and Erickson, 1999). Glyphosate has
also been found to cause growth abnormalities in human neurons (Axelrade et al., 2003). In addition, the herbicide has been found to cause mutations in human lymphocytes as well as DNA damage in the livers and kidneys of rats exposed to the chemical (Bologneis, 1997). Due to the similarities in physiology between humans and rats, rats are often used as a proxy indicator of human toxicity, avoiding the need for human tests. Leuken et al. (2003) reported damage to DNA in human fibroblast cells in the presence of low amounts of glyphosate and mild oxidative stress (Manthey et al., 2004). Glyphosate has been shown to drastically alter the balance of soil microbial populations and metabolites, by enhancing mycobacteria and fungi while decreasing bacteria and photosynthetic microorganisms (Araujo et al., 2003). By altering the balances of soil ecology in these ways, glyphosate poses a hazard to one or more foundational branches of the microbial food chain. The authors also showed evidence of near complete biogenic breakdown of glyphosate after 32 days. Accinelli et al., (2002) revealed that though glyphosate applied in agricultural and commercial doses can increase bulk microbial activity in agricultural soils, but did not indicate the various species that were affected. BIODEGRADATION OF GLYPHOSATE Glyphosate breaks down most quickly in coarse – textured gravel or sand, and most slowly in clay-like soil (Strange – hansen et al., 2004). The authors also found that glyphosate becomes much more stable in soils below 10oc, indicating that cold – season applications or situations where glyphosate is allowed to penetrate deep into soil may allow for more persistence and contamination. Glyphosate has also been found to bind with copper in soil (Undabeytia et al., 1996; Morllio et al., 2002.) Glyphosate selectively binds with iron and aluminum oxides in the soil, but is released when phosphates are heavily introduced (Gimsing and Borggard, 2002a ; 2002b). The authors also noted that glyphosate adsorption is highest in low – pH system (pH of 6 or below), and that alkaline soils (pH > 7) tend to poorly adsorb glyphosate, allowing for greater contamination of water. Glyphosate is mainly degraded biologically by soil microorganisms (including Pseudomonas putida, Arthrobacter ramosus, Arthrobacter viscosus and Bacilus circulans and has a minimal effect on soil microflora (Smith et al., 1997). In the soil environment, glyphosate is resistant to chemical degradation such as hydrolysis and is stable to sunlight (Smith et al., 1997). The primary metabolite of glyphosate is amino-methyl phosphonic acid (AMPA) which has a slower degradation rate than glyphosate. The persistence of AMPA is reported to be longer than glyphosate, possibly due to tighter binding to soil (Gimsing et al., 2004). MATERIALS AND METHODS COLLECTION OF SOIL SAMPLES The soil samples were collected randomly from selected plots located in three towns in Okpokwu LGA of Benue state. The plots had no previous history of any herbicidal application. The soil samples were collected from the upper 10 cm of the soil profile as proposed by Lal and Saxens (1982). PRE-TREATMENT OF SOIL SAMPLES After removal of living materials (such as mosses, roots etc) and objects > 2 cm, about 500g of fresh soil was obtained using hand trowel and cutlass. The samples were taking to the laboratory in polythene bags. The samples where air dried for 72 hours and kept in polyethylene bags at 4oC for subsequent analysis (Haney et al., 2000).
Table 1. Experimental design for the treatment combinations. Factor A (Days of sampling) b1 b3 b7 b30
TREATMENT CONDITIONS Factor B (concentration of glyphosate- kg ha-1) a0 a0,84 b1a0 b1a0,84 b2a0 b3a0,84 b3a0 b7a0,84 b30a0 b30a0,84
a840 b1a840 b3a840 b7a840 b3a840
PROCEDURE FOR FIELD SAMPLING Treatment included three levels of glyphosate application which were 0, 0.84 and 840 kg a.i./ha and four sampling days (1, 3, 7 and 30) after application. The glyphosate was applied to plots at the rates expressed above using Carbon dioxide pressurized knapsack sprayer. The sprayer had two nozzles (TT110.02), spaced 1.0m with pressure of 200 kpa and spray flow of 100L ha-1. During the spraying the sky was clear and soil moist (Parions et al., 2003). The layout of the experiment was completely randomized design as shown in Table 1. Intermediate glyphosate concentration used was based on the recommended rate of glyphosate being 0.84 kg ha-1 (Haney et al, 2000). The highest concentration used (100-fold) was selected to test effects since farmers sometimes use this or higher concentration other than the recommended field rate. A control treatment with no glyphosate was included for each soil; therefore, comparison with glyphosate addition to each soil would measure the positive or negative influences on bacterial growth for each soil. (Haney et al., 2002). ISOLATION AND IDENTIFICATION OF BACTERIA The bacterial isolates were identified on the basis of classification schemes published in Bergey’s Manual of Systemic Bacteriology (Holt et al., 1994), using the following characteristics :Gram reaction, Cell shape, Motility in liquid medium, colony pigmentation, Oxidase and Catalase test, and Indole production test. CULTURING AND ISOLATION OF FUNGAL ISOLATES The method adopted in this work is in line with that outlined by Rataliff et al. (2006). Fungi were extracted by agitating a 3g sub-sample of soil (oven dry equivalent) in 10 fold dilution of 0.15m sodium chloride (NaCl) with 3mm glass beads for 10 minutes. Serial dilutions were plated in duplicate on malt extract agar for fungal propagules. Fungi were counted after 14 days incubation at 28oC The fungi were identified based on their cultural morphology and microscopic characteristics of their spores and hyphae using the simplified key to the classes of fungi (Division: Mycota) and fungus like organisms devoid of chlorophyll (Axelrade et al.,2003). INVERTEBRATE ENUMERATION The method adopted in this work is in line with manual of Cox (1996), using Key to Invertebrate Identification.
Simplified
RESULTS Table 2: Number of microorganisms detected in the different locations after glyphosate treatment in the month of July Microorganisms Day1 Day3 Day7 Day30 And soil locations _____________________________________________________________________________ 0kgha-1 0.84kgha-1 840kgha-1
0kgha-10.84kgha-1 840kgha-1
0kgha-1 0.84kgha-1 840kgha-1
0kgha-1 0.84kgha-1 840kgha-1
Bacteria (cfu/ml) Ugbokolo Ogene Opialo
9.8x106 1.2x107 1.7x107 7.6x106 9.4x106 1.6x107 1.7x107 6.1x107 8.8x107
8.0x106 7.2x108 9.0x108 8.2x106 1.2x108 1.8x109 7.5x107 6.6x109 5.4x109
6.9x106 9.4x108 1.4x109 6.2x108 6.8x108 8.8x108 1.4x107 1.8x109 8.6x109
8.8x106 6.3x107 8.2x107 5.8x106 1.1x107 1.3x107 1.6x107 6.2x107 1.7x108
Fungi (cfu/ml) Ugbokolo Ogene Opialu
4.2x104 4.4x104 4.6x104 4.8x104 4.4x104 4.6x104 4.8x104 4.4x104 5.0x103
4.4x104 4.6x104 4.1x104 5.1x104 5.0x104 4.9x104 4.5x104 4.6x104 4.5x104
4.3x104 6.0x104 4.5x104 4.9x104 4.6x104 5.1x104 4.7x104 4.3x104 4.8x104
4.5x104 4.8x104 4.8x104 4.7x104 4.2x104 4.7x104 4.9x104 4.8x104 4.7x104
Invertebrates (No./m2) Ugbokolo 600 418 221 583 246 140 608 212 Ogene 564 352 197 581 303 164 573 208 Opialu 816 548 382 822 201 304 799 427 ______________________________________________________________________________ *values for each locations are mean of triplicates sample per plots
120 146 260
614 592 831
200 244 438
100 128 248
Table 3: Number of microorganisms detected in the different locations after glyphosate treatment in the month of November Microorganisms Day1 Day3 Day7 Day30 And soil locations________________________________________________________________________________________________ 0kgha-1 0.84kgha-1 840kgha-1 Bacteria (cfu/ml) Ugbokolo 2.4x106 9.6x106 8.8x106 Ogene 7.6x105 9.8x105 9.2x106 Opialo 8.9x106 6.3x107 8.5x107 Fungi (cfu/ml) Ugbokolo Ogene Opialu
2.6x104 3.0x104 2.8x104 3.3x104 3.0x104 2.8x104 2.1x104 2.2x104 2.4x103
0kgha-10.84kgha-1 840kgha-1
0kgha-1 0.84kgha-1 840kgha-1 0kgha-1 0.84kgha-1 840kgha-1
2.6x106 1.8x107 2.3x107 9.3x105 7.1x106 7.8x107 6.4x106 9.0x107 2.7x108
1.8x106 3.4x107 5.4x107 5.0x105 8.4x107 9.6x107 8.4x106 2.2x108 6.1x108
3.2x106 5.7x107 7.2x107 7.2x105 5.1x107 1.4x108 7.8x106 6.6x108 8.7x108
2.8x104 2.4x104 3.4x104 3.1x104 3.2x104 3.0x104 2.3x104 2.4x104 2.2x104
3.1x104 2.8x104 2.7x104 2.9x104 3.1x104 3.2x104 2.0x104 2.0x104 2.1x104
2.7x104 3.3x104 3.2x104 3.0x104 3.2x104 3.1x104 2.2x104 2.3x104 2.3x104
Invertebrates (No./m2) Ugbokolo 412 216 163 386 164 111 420 158 Ogene 394 201 156 403 170 121 418 149 Opialu 608 401 321 618 342 236 585 303 ______________________________________________________________________________ *values for each locations are mean of triplicates sample per plots
104 96 188
400 380 601
142 132 264
86 66 128
Table: 4
Differential characteristics of culturable soil bacterial isolated
rods
rods
+
-
-
rods
rods
rods
rods
chains
chains single filamentous single clumps single
single
chains single
Motility in liquid medium
+
+
+
+
-
+
+
+
Yellow colonies
-
-
-
-
-
Red colonies
-
-
-
-
-
-
Violet colonies
-
Oxidase
+
-
-
-
-
-
-
-
-
+ -
-
-
-
-
-
-
-
-
-
-
-
+
-
+
+
+
-
-
+
+
-
Catalase
+
+
rods single
+
+
+
-
+
+
+
+
-
+
-
Production of acid from glucose
+
-
+
+
-
-
+
+
+
-
Indole production
-
-
-
-
-
-
-
+
-
-
Endospore production
-
-
+
-
+
Strict aerobes
+
+
-
-
+
-
-
-
-
+
Facultative anaerobes
-
-
+
+
-
+
+
+
-
-
-
Obligate anaerobes
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
____________________________________________________________________________________
-
-
Rhizobacter daucus
Azotobacter sp. -
Pseudomonas sp .
ovoid
rods
-
Bacillus sp.
-
Arthrobacter sp.
-
Arrangement of cells
+
coccoid
+
Clostridium sp .
rods
-
Citrobacter sp .
Cell shape
-
Agromyces sp.
-
Agrobacterium sp .
Acetobacter sp.
Characteristics ______________________________________ Gram reaction
Acinetobacter sp.
Genera of soil bacterial isolated ______________________________________________________
Table:5 The mean of bacterial Colonies isolated from the three locations. Bacteria Number of colonies {(cfu/ml)x104} ____________________________________________________________ Before glyphosate application After glyphosate appliation Acetobacter sp.
10
16
Acintobacter sp.
17
22
Agrobacterium sp.
15
35
Agromyces ramosus
21
Arthrobacter sp.
38
16
19
Azotobacter sp.
08
12
Bacillus sp.
11
34
Citrobacter sp.
10
18
Clostridium sp.
13
20
Pseudomonas sp.
11
45
Rhizobacter daucus
13
15
Table:6 Fungal colonies isolated from the three loctions Number of colonies (cfu/ml)x102 _________________________________________________________________________________
Fungi Before glyphosate application After glyphosate application ________________________________________________________________________ Aspergillus niger
37
34
Aspergillus flavus
22
26
Geomyces pannorum
11
14
Penicillium sp.
28
31
Cladosporium cladosporiodex
07
06
Rhizopus stolonifer
12
08
Rhizopus oryzae
17
18
Mucor hiemalis
21
17
Fusaium soloni
02
06
Table:7 The mean number of invertebrates observed from the three locations Number of invertebrates per square metre ______________________________________________ Invertebrates Before glyphosate application After glyphosate application ________________________________________________________________________ Ants
182
179
Beetle
36
30
Caterpillar
18
09
Centipede
28
21
Earth worm
132
62
Millipede
48
41
Spider
26
24
________________________________________________________________________
DISCUSSION The result of the viable bacterial cells isolated from the three locations – Ugbokolo, Ogene and Opialu in the month of July showed very little or no change in number of viable bacterial cells in plots with 0kg ha-1 application of glyphosate. This could stem from the fact that the soil had no appliction of glyphosate. However at the concentration of 0.84 ad 840 kgha-1, the viable bacterial cells isolated increased which could be that the glyphosate herbicide according to Haney et al.(2000) and Heney et al.(2002) can increase soil microbial biomass, respiration and carbon and nitrogen mineralization. This report was supported by Barrett and McBride (2005) that both biotic and abiotic oxidative degradation of glyphosate caused breakage of both the CP and C-N bond thereby releasing carbon, phosphorous and Nitrogen to the soil. On day 30 after glyphosate treatment, the viable microbial counts declined compared to day 7. The reason could be attributed to the decline in the available nutrients such as carbon, phophorus and nitrogen due to rapid minieralization as suggested by Mamy et al. (2005) that glyphosate availability in soil have shown to decline between avarage of 25 to 35 days). The result was statistically tested using Dunette Multiple Comparisons Test (p > 0.05) and Bartlette Test (p < 0.0001) which suggest that the differences among the Standard Deviations (S Ds) is extremely significant. The result of fungal cells isolated from Ugbokolo, Ogene and Opialu in the month of July showed no significant differences among the control, 0.84 kgha-1 and 840kgha-1 levels of the herbicide treatment. The result obtained in this investigation is in line with the work of Lal and Saxena (1982) which showed that no significant main effect were found for fungal cells isolated in whitmore soil. A semilar result was also obtained by Bahig et al., (2008).The work of Wardle and Parkinson(1990), however, suggested an increase in the number of fungal cells directly or indirectly after glyphosate application, which is contrally to our result. The result is tested using One-way Analysis of Variance (ANOVA) (P = 0.4589) and Tukey-Kramer Multiple Comparisons Test (P > 0.05). These tests suggest that variations among column means is not significantly greater than expected. The result of the number of invertebrates observed from soil smaples in Ugbokolo, Ogene and Opialu in the month of July showed a higher number of invertebrates at the control rate (0 kgha 1 ), while the number of invertebrates where low and lowest at the concentration of 0.840 kgha-1 and 840 kgha-1 of the glyphosate herbicide respectively. The results have shown that glyphosate herbicide affects the soil invertebrate adversely, Krutz et al., (2009) statal that an indirect effect on weeed species compositions and densities may likely affect ivestebrate population than the direct effect of the herbicide. CONCLUSIONS In this investigation, glyphosate herbicide was found to increase the bacterial cell count with minimal effect on fungal cells. Invertebrates where adversely affected by the glyphosate herbicide in this study. Since invertebrates plays vital role in the overall soil fertility, continous and repeated use of glyphosate might have detrimental effects on the soil fertility over time. Although, the effects of glyphosate herbicide on soil bacteria and fungi in the study was small and short lived, but the effects on structural diversity of microbial communities is enough reason to shift to the use of non-chemical herbicide alternatives such as biologicl turn over activity like mulching and compost applications. RECOMMENDATION The recommended field rate for glyphosate herbicide application to most farm land is 0.84kg-ha. This rate should be maintained to reduce detrimental effects of the herbicide on invertebrate populations.
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