Concerned Individuals and Belize Organizations
Bibliography of Published Peer-Reviewed Scientific Documents
Belize Pesticide Control Board
Registration Review Committee
30 August 2016 10 a.m.
Pesticide Control Board, Central Farm, Cayo, Belize
Bibliography of Peer-Reviewed Documents Supporting Petition to Ban Glyphosate
1. Glyphosate Information, Testing, and Use
Impact on Plant Physiology
1. Noctor, G.; Foyer, C.H. “Perturbations of amino acid metabolism associated with glyphosate-dependentinhibition of shikimic acid metabolism affect cellular redox homeostasis and alter the abundanceof proteins involved in photosynthesis and photorespiration”. Plant Physiol. 2011, 157, 256–268. Abstract: The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway.
2. de María, N.; Becerril J.M.; García-Plazaola, J.I.;Ndez, A.H.; de Felipe, M.R.; Fernández-Pascual, M.“New insights on glyphosate mode of action in nodular metabolism: Role of shikimate Accumulation”. J. Agric. Food Chem. 2006, 54, 2621–2628. Abstract: The short-term effects of the herbicide glyphosate (1.25-10 mM) on the growth, nitrogen fixation, carbohydrate metabolism, and shikimate pathway were investigated in leaves and nodules of nodulated lupine plants. All glyphosate treatments decreased nitrogenase activity rapidly (24 h) after application, even at the lowest and sublethal dose used (1.25 mM). This early effect on nitrogenase could not be related to either damage to nitrogenase components (I and II) or limitation of carbohydrates supplied by the host plant. In fact, further exposure to increasing glyphosate concentrations (5 mM) and greater time after exposure (5 days) decreased nodule starch content and sucrose synthase (SS; EC 188.8.131.52) activity but increased sucrose content within the nodule. These effects were accompanied by a great inhibition of the activity of phosphoenolpyruvate carboxylase (PEPC; EC 184.108.40.206). There were remarkable and rapid effects on the increase of shikimic and protocatechuic (PCA) acids in nodules and leaves after herbicide application. On the basis of the role of shikimic acid and PCA in the regulation of PEPC, as potent competitive inhibitors, this additional effect provoked by glyphosate on 5-enolpyruvylshikimic-3-phosphate synthase enzyme (EPSPS; EC 220.127.116.11) inhibition would divert most PEP into the shikimate pathway, depriving energy substrates to bacteroids to maintain nitrogen fixation. These findings provide a new explanation for the effectiveness of glyphosate as a herbicide in other plant tissues, for the observed differences in tolerance among species or cultivars, and for the transitory effects on glyphosate-resistant transgenic crops under several environmental conditions.
Ingredients: Active and “Inert”
3. Mesnage, Robin, et al., “Major Pesticides Are More Toxic to Human Cells than Their Declared Active Principles,” BioMed Research International (December 2013). Abstract: Pesticides are used throughout the world as mixtures called formulations. They contain adjuvants, which are often kept confidential and are called inerts by the manufacturing companies, plus a declared active principle, which is usually tested alone. We tested the toxicity of 9 pesticides, comparing active principles and their formulations, on three human cell lines (HepG2, HEK293, and JEG3). Glyphosate, isoproturon, fluroxypyr, pirimicarb, imidacloprid, acetamiprid, tebuconazole, epoxiconazole, and prochloraz constitute, respectively, the active principles of 3 major herbicides, 3 insecticides, and 3 fungicides. We measured mitochondrial activities, membrane degradations, and caspases 3/7 activities. Fungicides were the most toxic from concentrations 300–600 times lower than agricultural dilutions, followed by herbicides and then insecticides, with very similar profiles in all cell types. Despite its relatively benign reputation, Roundup was among the most toxic herbicides and insecticides tested. Most importantly, 8 formulations out of 9 were up to one thousand times more toxic than their active principles. Our results challenge the relevance of the acceptable daily intake for pesticides because this norm is calculated from the toxicity of the active principle alone. Chronic tests on pesticides may not reflect relevant environmental exposures if only one ingredient of these mixtures is tested alone.
4. Mesnage, Robin, Bernay, Benoit, and Seralini, Gilles-Eric, “Ethoxylated Adjuvants of Glyphosate-Based Herbicides are Active Principals of Human Cell Toxicity,” Toxicology 313, nos. 2 – 3 (November 2013). Published online September21, 2012 Abstract: Pesticides are always used in formulations as mixtures of an active principle with adjuvants. Glyphosate, the active ingredient of the major pesticide in the world, is an herbicide supposed to be specific on plant metabolism. Its adjuvants are generally considered as inert diluents. Since side effects for all these compounds have been claimed, we studied potential active principles for toxicity on human cells for 9 glyphosate-based formulations. For this we detailed their compositions and toxicities, and as controls we used a major adjuvant (the polyethoxylatedtallowamine POE-15), glyphosate alone, and a total formulation without glyphosate. This was performed after 24h exposures on hepatic (HepG2), embryonic (HEK293) and placental (JEG3) cell lines. We measured mitochondrial activities, membrane degradations, and caspases 3/7 activities. The compositions in adjuvants were analyzed by mass spectrometry. Here we demonstrate that all formulations are more toxic than glyphosate, and we separated experimentally three groups of formulations differentially toxic according to their concentrations in ethoxylated adjuvants. Among them, POE-15 clearly appears to be the most toxic principle against human cells, even if others are not excluded. It begins to be active with negative dose-dependent effects on cellular respiration and membrane integrity between 1 and 3ppm, at environmental/occupational doses. We demonstrate in addition that POE-15 induces necrosis when its first micellization process occurs, by contrast to glyphosate which is known to promote endocrine disrupting effects after entering cells. Altogether, these results challenge the establishment of guidance values such as the acceptable daily intake of glyphosate, when these are mostly based on a long term in vivo test of glyphosate alone. Since pesticides are always used with adjuvants that could change their toxicity, the necessity to assess their whole formulations as mixtures becomes obvious. This challenges the concept of active principle of pesticides for non-target species.
5. Bonnet, J.L., F. Bonnemoy, M. Dusser, and J. Bohatier(2007). “Assessment of the potential toxicity of herbicides and their degradation products to nontarget cells using two microorganisms, the bacteria Vibrio fischeri and the ciliate Tetrahymena pyriformis.” Abstract: The potential toxicity of several herbicides-alachlor, diuron and its photo and biotransformation products, glyphosate and its metabolite aminomethyl phosphonic acid (AMPA)-to nontarget cells was assessed using two microorganisms frequently used in ecotoxicology, Vibrio fischeri and Tetrahymena pyriformis. Toxicity assays involved the Microtox test, the T. pyriformis population growth impairment test employing three different processes (flasks, tubes, microplates), and the T. pyriformis nonspecific esterase activities test. Several IC(50) or EC(50) values are reported for each molecule. Alachlor exerted a toxic effect on the two nontarget cells used. The results for diuron and its photo and biotransformation products indicated that most of the metabolites presented nontarget toxicity higher than that of diuron. Glyphosate and AMPA had a less negative effect on T. pyriformis than on V. fischeri. Nevertheless, in all cases, glyphosate was found to be more toxic than AMPA. Comparison analysis of the sensitivity of the different tests showed that, in general, tests using the eukaryotic cell (T. pyriformis) were more sensitive than test using the prokaryotic cell (V. fischeri), and that a population growth criterion is more sensitive than an enzymatic criterion. The three different processes that could be used to evaluate effects on population growth rate were equally sensitive for the herbicides tested. A significant correlation between toxicity data and the hydrophobicity of the chemicals could only be established with the growth population test. This study demonstrates that it is essential to assess the toxicity of the metabolites formed to complete a more comprehensive study of the environmental impact of a polluting agent. Environmental Toxicology, 22(1): 78–91.
6. Vandenberg, Laura N. et al., “Hormones and Endocrine-Disrupting Chemicals: Low-Dose Effects and Nonmonotonic Dose Responses,” Endocrine Reviews 33, no. 3 (June 2012):378-455. Abstract: For decades, studies of endocrine-disrupting chemicals (EDCs) have challenged traditional concepts in toxicology, in particular the dogma of "the dose makes the poison," because EDCs can have effects at low doses that are not predicted by effects at higher doses. Here, we review two major concepts in EDC studies: low dose and nonmonotonicity. Low-dose effects were defined by the National Toxicology Program as those that occur in the range of human exposures or effects observed at doses below those used for traditional toxicological studies. We review the mechanistic data for low-dose effects and use a weight-of-evidence approach to analyze five examples from the EDC literature. Additionally, we explore nonmonotonic dose-response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. We provide a detailed discussion of the mechanisms responsible for generating these phenomena, plus hundreds of examples from the cell culture, animal, and epidemiology literature. We illustrate that nonmonotonic responses and low-dose effects are remarkably common in studies of natural hormones and EDCs. Whether low doses of EDCs influence certain human disorders is no longer conjecture, because epidemiological studies show that environmental exposures to EDCs are associated with human diseases and disabilities. We conclude that when nonmonotonic dose-response curves occur, the effects of low doses cannot be predicted by the effects observed at high doses. Thus, fundamental changes in chemical testing and safety determination are needed to protect human health.
7. Bergman,Ake,Heinde, Jerrold J., Jobling , Susan, Kidd, Karen A., Zoeller, R. Thomas, “State of the Science of Endocrine Disrupting Chemicals”, Inter-Organisation Programme for the Sound Management of Chemicals (IOMC), with The Participating Organisations FAO, ILO, UNDP, UNEP, UNIDO, UNITAR, WHO, World Bank and OECD, 2012, Excerpt: Since there are data from epidemiological studies showing associations between human disease end-points and EDC exposures, it is likely that endocrine diseases and disorders are occurring at current exposure levels. Put another way, this means that there are situations in which individually safe exposures of EDCs have reached a collectively harmful level or in which levels thought to be safe are not so. When chemicals are tested for endocrine disrupting activity under specific validated Guideline studies, it is customary to examine three doses to determine a level not apparently associated with observable effects. This level, termed the no-observed-adverse-effect level, is then divided by a so-called safety or uncertainty factor (of 100, for example) to extrapolate to levels expected to be safe for humans or wildlife. The doses declared safe are not actually tested, nor are the mixtures. These studies also assume that there is a threshold for EDC effects, that there will be no effects at low doses and that the dose–response curve rises with increasing dose. As noted above, there is no threshold for EDC effects due to the presence of active hormone pathways, and EDCs are likely to have effects at low doses. Consequently, their dose–response curves will not necessarily rise in proportion to dose. Regulatory guideline studies also focus on histopathology and organ and body weights as the end-points. As noted above, EDCs can cause many diseases and affect many disease end-points that are not currently assessed in regulatory studies. Also, risk assessment approaches do not always assess toxicity during development, which is the most sensitive window for EDC action, and also do not follow the animals for their lifetime, which is needed to assess resulting diseases.
2. Toxicity Impact on Animal/Human Health
Oxidative Stress / Cell Damage
8. Cattani, Daiane et al., “Mechanisms Underlying the Neurotoxicity Induced by Glyphosate-Based Herbicide in Immature Rat Hippocampus: Involvement of Glutamate Excitotoxicity,” Toxicology 320 (March 2014): 34-45. Abstract: Previous studies demonstrate that glyphosate exposure is associated with oxidative damage and neurotoxicity. Therefore, the mechanism of glyphosate-induced neurotoxic effects needs to be determined. The aim of this study was to investigate whether Roundup(®) (a glyphosate-based herbicide) leads to neurotoxicity in hippocampus of immature rats following acute (30min) and chronic (pregnancy and lactation) pesticide exposure. Maternal exposure to pesticide was undertaken by treating dams orally with 1% Roundup(®) (0.38% glyphosate) during pregnancy and lactation (till 15-day-old). Hippocampal slices from 15 day old rats were acutely exposed to Roundup(®) (0.00005 to 0.1%) during 30min and experiments were carried out to determine whether glyphosate affects (45)Ca(2+) influx and cell viability. Moreover, we investigated the pesticide effects on oxidative stress parameters, (14)C-α-methyl-amino-isobutyric acid ((14)C-MeAIB) accumulation, as well as glutamate uptake, release and metabolism. Results showed that acute exposure to Roundup(®) (30min) increases (45)Ca(2+) influx by activating NMDA receptors and voltage-dependent Ca(2+) channels, leading to oxidative stress and neural cell death. The mechanisms underlying Roundup(®)-induced neurotoxicity also involve the activation of CaMKII and ERK. Moreover, acute exposure to Roundup(®) increased (3)H-glutamate released into the synaptic cleft, decreased GSH content and increased the lipoperoxidation, characterizing excitotoxicity and oxidative damage. We also observed that both acute and chronic exposure to Roundup(®) decreased (3)H-glutamate uptake and metabolism, while induced (45)Ca(2+) uptake and (14)C-MeAIB accumulation in immature rat hippocampus. Taken together, these results demonstrated that Roundup(®) might lead to excessive extracellular glutamate levels and consequently to glutamate excitotoxicity and oxidative stress in rat hippocampus.
9. Lushchak, Oleh V. et al., “Low Toxic Herbicide Roundup Induces Mild Oxidative Stress in Goldfish Tissues” Chemosphere 76 no.7 (2009): 932-37 Abstract: The formulation of Roundup consists of the herbicide glyphosate as the active ingredient with polyethoxylene amine added as a surfactant. The acute toxicity of Roundup (particularly of glyphosate) to animals is considered to be low according to the World Health Organization, but the extensive use of Roundup may still cause environmental problems with negative impact on wildlife, particularly in an aquatic environment where chemicals may persist for a long time. Therefore, we studied the effects of Roundup on markers of oxidative stress and antioxidant defense in goldfish, Carassiusauratus.
10. El-Shenawy, Nahia S., “Oxidative Stress Responses of Rats Exposed to Roundup and Its Active Ingredient Glyphosate,” Environmental Toxicology and Pharmacology 28, no.3 (November 2009): 379-85 Abstract: Glyphosate is the active ingredient and polyoxyethyleneamine, the major component, is the surfactant present in the herbicide Roundup formulation. The objective of this study was to analyze potential cytotoxicity of the Roundup and its fundamental substance (glyphosate). Albino male rats were intraperitoneally treated with sub-lethal concentration of Roundup (269.9mg/kg) or glyphosate (134.95mg/kg) each 2 days, during 2 weeks. Hepatotoxicity was monitored by quantitative analysis of the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities, total protein, albumin, triglyceride and cholesterol. Creatinine and urea were used as the biochemical markers of kidney damages. The second aim of this study to investigate how glyphosate alone or included in herbicide Roundup affected hepatic reduced glutathione (GSH) and lipid peroxidation (LPO) levels of animals as an index of antioxidant status and oxidative stress, respectively, as well as the serum nitric oxide (NO) and alpha tumour necrosis factor (TNF-α) were measured. Treatment of animals with Roundup induced the leakage of hepatic intracellular enzymes, ALT, AST and ALP suggesting irreversible damage in hepatocytes starting from the first week. It was found that the effects were different on the enzymes in Roundup and glyphosate-treated groups. Significant time-dependent depletion of GSH levels and induction of oxidative stress in liver by the elevated levels of LPO, further confirmed the potential of Roundup to induce oxidative stress in hepatic tissue. However, glyphosate caused significant increases in NO levels more than Roundup after 2 weeks of treatment. Both treatments increased the level of TNF-α by the same manner. The results suggest that excessive antioxidant disruptor and oxidative stress is induced with Roundup than glyphosate.
11. Richard, S., Moslemi, S. et al. 2005. Differential effects of glyphosate and roundup on human placental cells and aromatase. Environ Health Perspect 113(6): 716–720. Abstract: Roundup is a glyphosate-based herbicide used worldwide, including on most genetically modified plants that have been designed to tolerate it. Its residues may thus enter the food chain, and glyphosate is found as a contaminant in rivers. Some agricultural workers using glyphosate have pregnancy problems, but its mechanism of action in mammals is questioned. Here we show that glyphosate is toxic to human placental JEG3 cells within 18 hr with concentrations lower than those found with agricultural use, and this effect increases with concentration and time or in the presence of Roundup adjuvants. Surprisingly, Roundup is always more toxic than its active ingredient. We tested the effects of glyphosate and Roundup at lower nontoxic concentrations on aromatase, the enzyme responsible for estrogen synthesis. The glyphosate-based herbicide disrupts aromatase activity and mRNA levels and interacts with the active site of the purified enzyme, but the effects of glyphosate are facilitated by the Roundup formulation in microsomes or in cell culture. We conclude that endocrine and toxic effects of Roundup, not just glyphosate, can be observed in mammals. We suggest that the presence of Roundup adjuvants enhances glyphosate bioavailability and/or bioaccumulation.
12. Mesnage, Robin, Bernay, Benoit, and Seralini, Gilles-Eric, “Ethoxylated Adjuvants of Glyphosate-Based Herbicides are Active Principals of Human Cell Toxicity,” Toxicology 313, nos. 2 – 3 (November 2013). Published online September21, 2012 Abstract: Pesticides are always used in formulations as mixtures of an active principle with adjuvants. Glyphosate, the active ingredient of the major pesticide in the world, is an herbicide supposed to be specific on plant metabolism. Its adjuvants are generally considered as inert diluents. Since side effects for all these compounds have been claimed, we studied potential active principles for toxicity on human cells for 9 glyphosate-based formulations. For this we detailed their compositions and toxicities, and as controls we used a major adjuvant (the polyethoxylatedtallowamine POE-15), glyphosate alone, and a total formulation without glyphosate. This was performed after 24h exposures on hepatic (HepG2), embryonic (HEK293) and placental (JEG3) cell lines. We measured mitochondrial activities, membrane degradations, and caspases 3/7 activities. The compositions in adjuvants were analyzed by mass spectrometry. Here we demonstrate that all formulations are more toxic than glyphosate, and we separated experimentally three groups of formulations differentially toxic according to their concentrations in ethoxylated adjuvants. Among them, POE-15 clearly appears to be the most toxic principle against human cells, even if others are not excluded. It begins to be active with negative dose-dependent effects on cellular respiration and membrane integrity between 1 and 3ppm, at environmental/occupational doses. We demonstrate in addition that POE-15 induces necrosis when its first micellization process occurs, by contrast to glyphosate which is known to promote endocrine disrupting effects after entering cells. Altogether, these results challenge the establishment of guidance values such as the acceptable daily intake of glyphosate, when these are mostly based on a long term in vivo test of glyphosate alone. Since pesticides are always used with adjuvants that could change their toxicity, the necessity to assess their whole formulations as mixtures becomes obvious. This challenges the concept of active principle of pesticides for non-target species.
13. Marc, Julie.,Mulner-Lorillon, O., Bellé, R. 2004. “Glyphosate-based pesticides affect cell cycle regulation”. Biology of the Cell 96: 245–249. Abstract: Cell-cycle dysregulation is a hallmark of tumor cells and human cancers. Failure in the cell-cycle checkpoints leads to genomic instability and subsequent development of cancers from the initial affected cell. A worldwide used product Roundup 3plus, based on glyphosate as the active herbicide, was suggested to be of human health concern since it induced cell cycle dysfunction as judged from analysis of the first cell division of sea urchin embryos, a recognized model for cell cycle studies. Several glyphosate-based pesticides from different manufacturers were assayed in comparison with Roundup 3plus for their ability to interfere with the cell cycle regulation. All the tested products, Amega, Cargly, Cosmic, and Roundup Biovert induced cell cycle dysfunction. The threshold concentration for induction of cell cycle dysfunction was evaluated for each product and suggests high risk by inhalation for people in the vicinity of the pesticide handling sprayed at 500 to 4000 times higher dose than the cell-cycle adverse concentration.
14. Chaufan, G. et al 2014, “Glyphosate commercial formulation causes cytotoxicty, oxidative effects, and apoptosis on human cells: differences with its active ingredient”, Int. J. Toxicol 33(1) 29-38. Abstract: In the present study, the effects on oxidative balance and cellular end points of glyphosate, aminomethylphosphonic acid (AMPA), and a glyphosate formulation (G formulation) were examined in HepG2 cell line, at dilution levels far below agricultural recommendations. Our results show that G formulation had toxic effects while no effects were found with acid glyphosate and AMPA treatments. Glyphosate formulation exposure produced an increase in reactive oxygen species, nitrotyrosine formation, superoxide dismutase activity, and glutathione (GSH) levels, while no effects were observed for catalase and GSH-S-transferase activities. Also, G formulation triggered caspase 3/7 activation and hence induced apoptosis pathway in this cell line. Aminomethylphosphonic acid exposure produced an increase in GSH levels while no differences were observed in other antioxidant parameters. No effects were observed when the cells were exposed to acid glyphosate. These results confirm that G formulations have adjuvants working together with the active ingredient and causing toxic effects that are not seen with acid glyphosate.
15. Vivancos, P.D.; Driscoll, S.P.; Bulman, C.A.; Ying, L.; Emami, K.; Treumann, A.; Mauve, C.; Noctor, G.; Foyer, C.H. “Perturbations of amino acid metabolism associated with glyphosate-dependent inhibition of shikimic acid metabolism affect cellular redox homeostasis and alter the abundance of proteins involved in photosynthesis and photorespiration.” Plant Physiol. 2011, 157, 256–268. Abstract: The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway.
16. Belle, R, Le Bouffant R, Morales J, Cosson B, Cormier P, Mulner-Lorillon O., “Sea urchin embryo, DNA-damaged cell cycle checkpoint and the mechanisms initiating cancer development” J. Soc. Biology 2007;201(3):317-27. Abstract: Cell division is an essential process for heredity, maintenance and evolution of the whole living kingdom. Sea urchin early development represents an excellent experimental model for the analysis of cell cycle checkpoint mechanisms since embryonic cells contain a functional DNA-damage checkpoint and since the whole sea urchin genome is sequenced. The DNA-damaged checkpoint is responsible for an arrest in the cell cycle when DNA is damaged or incorrectly replicated, for activation of the DNA repair mechanism, and for commitment to cell death by apoptosis in the case of failure to repair. New insights in cancer biology lead to two fundamental concepts about the very first origin of cancerogenesis. Cancers result from dysfunction of DNA-damaged checkpoints and cancers appear as a result of normal stem cell (NCS) transformation into a cancer stem cell (CSC). The second aspect suggests a new definition of "cancer", since CSC can be detected well before any clinical evidence. Since early development starts from the zygote, which is a primary stem cell, sea urchin early development allows analysis of the early steps of the cancerization process. Although sea urchins do not develop cancers, the model is alternative and complementary to stem cells which are not easy to isolate, do not divide in a short time and do not divide synchronously. In the field of toxicology and incidence on human health, the sea urchin experimental model allows assessment of cancer risk from single or combined molecules long before any epidemiologic evidence is available. Sea urchin embryos were used to test the worldwide used pesticide Roundup that contains glyphosate as the active herbicide agent; it was shown to activate the DNA-damage checkpoint of the first cell cycle of development. The model therefore allows considerable increase in risk evaluation of new products in the field of cancer and offers a tool for the discovery of molecular markers for early diagnostic in cancer biology. Prevention and early diagnosis are two decisive elements of human cancer therapy.
17. Hedberg, D., and M. Wallin(2010). “Effects of Roundup and glyphosate formulations on intracellular transport, microtubules and actin filaments in Xenopus laevis melanophores.” Abstract: Glyphosate containing herbicides, such as Roundup, are commonly used and generally considered to be safe. However, some toxic effects are found on amphibians in vivo and human and mouse cells in vitro. In this study the effects of Roundup, glyphosate, glyphosateisopropylamine and isopropylamine were studied on intracellular transport by measuring aggregation capacity in Xenopus laevis melanophores. The chemicals inhibited retrograde transport of melanosomes in the range of 0.5-5mM. Cellular morphology and localization of microtubules and actin filaments were affected as determined by immunocytochemistry. Both glyphosate and Roundup decreased pH in the media. Acidic pH inhibited melanosome transport and altered microtubule and actin morphology in the absence of chemicals, while transport inhibiting concentrations of glyphosate, Roundup and glyphosateisopropylamine disassembled both microtubules and actin filaments. At physiological pH the effects of Roundup decreased whereas glyphosate failed to inhibit transport. Physiological pH decreases glyphosate lipophilicity and its diffusion into the cytoplasm. The Roundup formulation contains surfactants, such as POEA (polyetylated tallow amine) that increases membrane permeability allowing cellular uptake at physiological pH. Our results show that the effects of glyphosate containing compounds are pH-dependent and that they inhibit intracellular transport through disassembly of the cytoskeleton possibly by interfering with intracellular Ca(2+)-balance. Toxicology.in Vitro 24: 795–802.
Breaks in DNA Strands
18. Gasnier, C.; Dumont, C.; Benachour, N.; Clair, E.; Chagnon, M.C.; Séralini, G.E. “Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines”. Toxicology 2009, 262, 184–191. Abstract: Glyphosate-based herbicides are the most widely used across the world; they are commercialized in different formulations. Their residues are frequent pollutants in the environment. In addition, these herbicides are spread on most eaten transgenic plants, modified to tolerate high levels of these compounds in their cells. Up to 400 ppm of their residues are accepted in some feed. We exposed human liver HepG2 cells, a well-known model to study xenobiotic toxicity, to four different formulations and to glyphosate, which is usually tested alone in chronic in vivo regulatory studies. We measured cytotoxicity with three assays (Alamar Blue, MTT, ToxiLight), plus genotoxicity (comet assay), anti-estrogenic (on ERalpha, ERbeta) and anti-androgenic effects (on AR) using gene reporter tests. We also checked androgen to estrogen conversion by aromatase activity and mRNA. All parameters were disrupted at sub-agricultural doses with all formulations within 24h. These effects were more dependent on the formulation than on the glyphosate concentration. First, we observed a human cell endocrine disruption from 0.5 ppm on the androgen receptor in MDA-MB453-kb2 cells for the most active formulation (R400), then from 2 ppm the transcriptional activities on both estrogen receptors were also inhibited on HepG2. Aromatase transcription and activity were disrupted from 10 ppm. Cytotoxic effects started at 10 ppm with Alamar Blue assay (the most sensitive), and DNA damages at 5 ppm. A real cell impact of glyphosate-based herbicides residues in food, feed or in the environment has thus to be considered, and their classifications as carcinogens/mutagens/reprotoxics is discussed.
19. Bagchi D, M. Bagchi, E.A. Hassoun, and S.J. Stohs(1995). “In vitro and in vivo generation of reactive oxygen species, DNA damage and lactate dehydrogenase leakage by selected pesticides.” Abstract: Reactive oxygen species may be involved in the toxicity of various pesticides and we have, therefore, examined the in vivo effects of structurally dissimilar polyhalogenated cyclic hydrocarbons (PCH), such as endrin and chlordane, chlorinated acetamide herbicides (CAH), such as alachlor, and organophosphate pesticides (OPS), such as chlorpyrifos and fenthion, on the production of hepatic and brain lipid peroxidation and DNA-single strand breaks (SSB), two indices of oxidative stress and oxidative tissue damage. The selected pesticides were administered p.o. to female Sprague-Dawley rats in two 0.25 LD50 doses at 0 h and 21 h and killed at 24 h. In a parallel set of experiments, we have determined the in vitro effects of these pesticides on the DNA-SSB and enhanced lactate dehydrogenase leakage (LDH) from neuroactive PC-12 cells in culture. In vitro production of reactive oxygen species by these pesticides was also assessed by determining the enhanced chemiluminescence responses of hepatic and brain homogenates. Following treatment of rats with endrin, chlordane, alachlor, chlorpyrifos and fenthion, increases of 2.8-, 3.0-, 4.2-, 4.3- and 4.8-fold were observed in hepatic lipid peroxidation, respectively, while at these same doses, increases in lipid peroxidation of 2.4-, 2.1-, 3.6-, 4.6- and 5.3-fold, respectively, were observed in brain homogenates. Increases of 4.4-, 3.9-, 1.6-, 3.0- and 3.5-fold were observed in hepatic DNA-SSB following treatment of the rats with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively, while at these same doses, increases of 1.9-, 1.7-, 2.2-, 1.4-, 1.4-fold, respectively, were observed in brain nuclear DNA-SSB. Following in vitro incubation of hepatic and brain tissues with 1 nmol/ml of each of the five pesticides, maximum increases in chemiluminescence occurred within 4-7 min of incubation and persisted for over 10 min. Increases of 3.0-, 2.7-, 3.6-, 4.9- and 4.4-fold were observed in chemiluminescence following in vitro incubation of the liver homogenates with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively, while increases of 1.7-, 1.8-, 2.0-, 3.4- and 3.7-fold, respectively, were observed in the brain homogenates. Increases of 2.2-, 2.3-, 2.9-, 2.9- and 3.4-fold were observed in the chemiluminescence responses in the liver homogenates of the animals treated with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively, while increases of 1.8-, 2.0-, 3.2-, 2.9- and 2.4-fold, respectively, were observed in the brain homogenates. Cultured neuroactive PC-12 cells were incubated with the pesticides and the release of the enzyme lactate dehydrogenase (LDH) into the media as an indicator of cellular damage and cytotoxicity was examined. Maximal release of LDH from cultured PC-12 cells was observed at 100 nM concentrations of the pesticides. Increases of 2.3-, 2.5-, 2.8-, 3.1 and 3.4-fold were observed in LDH leakage following incubation of the PC-12 cells with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively. Following incubation of the cultured PC-12 cells with 100 nM concentrations of these same pesticides, increases in DNA-SSB of 2.5-, 2.2-, 2.1-, 2.4- and 2.5-fold, respectively, were observed. The results clearly demonstrate that these different classes of pesticides induce production of reactive oxygen species and oxidative tissue damage which may contribute to the toxic manifestations of these xenobiotics. Reactive oxygen species may serve as common mediators of programmed cell death (apoptosis) in response to many toxicants and pathological conditions. Toxicology, 104(1-3):129–140.
20. Anthony Samsel and Stephanie Seneff, “Glyphosate, Pathways to Modern Diseases II: Celiac Sprue and Gluten Intolerance,” Interdisciplinary Toxicology 6, no.4 (2013): 159 – 84. Abstract: Celiac disease, and, more generally, gluten intolerance, is a growing problem worldwide, but especially in North America and Europe, where an estimated 5% of the population now suffers from it. Symptoms include nausea, diarrhea, skin rashes, macrocytic anemia and depression. It is a multifactorial disease associated with numerous nutritional deficiencies as well as reproductive issues and increased risk to thyroid disease, kidney failure and cancer. Here, we propose that glyphosate, the active ingredient in the herbicide, Roundup(®), is the most important causal factor in this epidemic. Fish exposed to glyphosate develop digestive problems that are reminiscent of celiac disease. Celiac disease is associated with imbalances in gut bacteria that can be fully explained by the known effects of glyphosate on gut bacteria. Characteristics of celiac disease point to impairment in many cytochrome P450 enzymes, which are involved with detoxifying environmental toxins, activating vitamin D3, catabolizing vitamin A, and maintaining bile acid production and sulfate supplies to the gut. Glyphosate is known to inhibit cytochrome P450 enzymes. Deficiencies in iron, cobalt, molybdenum, copper and other rare metals associated with celiac disease can be attributed to glyphosate's strong ability to chelate these elements. Deficiencies in tryptophan, tyrosine, methionine and selenomethionine associated with celiac disease match glyphosate's known depletion of these amino acids. Celiac disease patients have an increased risk to non-Hodgkin's lymphoma, which has also been implicated in glyphosate exposure. Reproductive issues associated with celiac disease, such as infertility, miscarriages, and birth defects, can also be explained by glyphosate. Glyphosate residues in wheat and other crops are likely increasing recently due to the growing practice of crop desiccation just prior to the harvest. We argue that the practice of "ripening" sugar cane with glyphosate may explain the recent surge in kidney failure among agricultural workers in Central America. We conclude with a plea to governments to reconsider policies regarding the safety of glyphosate residues in foods.
21. Shehata, Awad Ali, Schrodl, Wieland, Aldin, Alaa A. Hafex, Hafex M. and Kruger, Monika, “The Effect of Glyphosate on Potential Pathogens and Beneficial Members of Poultry Microbiota in Vitro,” Current Microbiology 66, no 4 (2013): 350-58. Abstract: The use of glyphosate modifies the environment which stresses the living microorganisms. The aim of the present study was to determine the real impact of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro. The presented results evidence that the highly pathogenic bacteria as Salmonella Entritidis, Salmonella Gallinarum, Salmonella Typhimurium, Clostridium perfringens and Clostridium botulinum are highly resistant to glyphosate. However, most of beneficial bacteria as Enterococcus faecalis, Enterococcus faecium, Bacillus badius, Bifidobacteriumadolescentis and Lactobacillus spp. were found to be moderate to highly susceptible. Also Campylobacter spp. were found to be susceptible to glyphosate. A reduction of beneficial bacteria in the gastrointestinal tract microbiota by ingestion of glyphosate could disturb the normal gut bacterial community. Also, the toxicity of glyphosate to the most prevalent Enterococcus spp. could be a significant predisposing factor that is associated with the increase in C. botulinum-mediated diseases by suppressing the antagonistic effect of these bacteria on clostridia.
22. Samsel, Anthony and Seneff, Stepanie, “Glyphosate’s Suppression of Cytochrome P450 Enzymes and Amino Acid Biosynthesis by the Gut Microbiome: Pathways to Modern Diseases,” Entropy 15, no. 4 (2013): 1416-63. Abstract: Glyphosate, the active ingredient in Roundup®, is the most popular herbicide used worldwide. The industry asserts it is minimally toxic to humans, but here we argue otherwise. Residues are found in the main foods of the Western diet, comprised primarily of sugar, corn, soy and wheat. Glyphosate's inhibition of cytochrome P450 (CYP) enzymes is an overlooked component of its toxicity to mammals. CYP enzymes play crucial roles in biology, one of which is to detoxify xenobiotics. Thus, glyphosate enhances the damaging effects of other food borne chemical residues and environmental toxins. Negative impact on the body is insidious and manifests slowly over time as inflammation damages cellular systems throughout the body. Here, we show how interference with CYP enzymes acts synergistically with disruption of the biosynthesis of aromatic amino acids by gut bacteria, as well as impairment in serum sulfate transport. Consequences are most of the diseases and conditions associated with a Western diet, which include gastrointestinal disorders, obesity, diabetes, heart disease, depression, autism, infertility, cancer and Alzheimer’s disease. We explain the documented effects of glyphosate and its ability to induce disease, and we show that glyphosate is the “textbook example” of exogenous semiotic entropy: the disruption of homeostasis by environmental toxins.
23. Seneff, S., Swanson, N. and Li, C. (2015) “Aluminum and Glyphosate Can Synergistically Induce Pineal Gland Pathology: Connection to Gut Dysbiosis and Neurological Disease”. Agricultural Sciences, 6, 42-70. Abstract: Many neurological diseases, including autism, depression, dementia, anxiety disorder and Parkinson’s disease, are associated with abnormal sleep patterns, which are directly linked to pineal gland dysfunction. The pineal gland is highly susceptible to environmental toxicants. Two pervasive substances in modern industrialized nations are aluminum and glyphosate, the active ingredient in the herbicide, Roundup?. In this paper, we show how these two toxicants work synergistically to induce neurological damage. Glyphosate disrupts gut bacteria, leading to an overgrowth of Clostridium difficile. Its toxic product, p-cresol, is linked to autism in both human and mouse models. p-Cresol enhances uptake of aluminum via transferrin. Anemia, a result of both aluminum disruption of heme and impaired heme synthesis by glyphosate, leads to hypoxia, which induces increased pineal gland transferrin synthesis. Premature birth is associated with hypoxic stress and with substantial increased risk to the subsequent development of autism, linking hypoxia to autism. Glyphosate chelates aluminum, allowing ingested aluminum to bypass the gut barrier. This leads to anemia-induced hypoxia, promoting neurotoxicity and damaging the pineal gland. Both glyphosate and aluminum disrupt cytochrome P450 enzymes, which are involved in melatonin metabolism. Furthermore, melatonin is derived from tryptophan, whose synthesis in plants and microbes is blocked by glyphosate. We also demonstrate a plausible role for vitamin D3 dysbiosis in impaired gut function and impaired serotonin synthesis. This paper proposes that impaired sulfate supply to the brain mediates the damage induced by the synergistic action of aluminum and glyphosate on the pineal gland and related midbrain nuclei.
Metabolic Pathway-Endocrine Disruption
24. Vandenberg, Laura N. et al., “Hormones and Endocrine-Disrupting Chemicals: Low-Dose Effects and Nonmonotonic Dose Responses,” Endocrine Reviews 33, no. 3 (June 2012):378-455. Abstract: For decades, studies of endocrine-disrupting chemicals (EDCs) have challenged traditional concepts in toxicology, in particular the dogma of "the dose makes the poison," because EDCs can have effects at low doses that are not predicted by effects at higher doses. Here, we review two major concepts in EDC studies: low dose and nonmonotonicity. Low-dose effects were defined by the National Toxicology Program as those that occur in the range of human exposures or effects observed at doses below those used for traditional toxicological studies. We review the mechanistic data for low-dose effects and use a weight-of-evidence approach to analyze five examples from the EDC literature. Additionally, we explore nonmonotonic dose-response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. We provide a detailed discussion of the mechanisms responsible for generating these phenomena, plus hundreds of examples from the cell culture, animal, and epidemiology literature. We illustrate that nonmonotonic responses and low-dose effects are remarkably common in studies of natural hormones and EDCs. Whether low doses of EDCs influence certain human disorders is no longer conjecture, because epidemiological studies show that environmental exposures to EDCs are associated with human diseases and disabilities. We conclude that when nonmonotonic dose-response curves occur, the effects of low doses cannot be predicted by the effects observed at high doses. Thus, fundamental changes in chemical testing and safety determination are needed to protect human health.
25. Peraldi, P.; Hotamisligil, G.S.; Buurman, W.A.; White, M.F.; Spiegelman, B.M.“Tumor necrosis factor (TNF)-alpha inhibits insulin signaling through stimulation of the p55 TNF receptor and activation of sphingomyelinase”. J. Biol. Chem. 1996, 271, 13018–13022. Abstract: Tumor necrosis factor (TNF)-alpha plays a central role in the state of insulin resistance associated with obesity. It has previously been shown that one important mechanism by which TNF-alpha interferes with insulin signaling is through the serine phosphorylation of insulin receptor substrate-1 (IRS-1), which can then function as an inhibitor of the tyrosine kinase activity of the insulin receptor (IR). However, the receptors and the signaling pathway used by TNF-alpha that mediate the inhibition of IR activity are unknown. We show here that human TNF-alpha, which binds only to the murine p55 TNF receptor (TNFR), is as effective at inhibiting insulin-dependent tyrosine phosphorylation of IR and IRS-1 in adipocytes and myeloid 32D cells as murine TNF-alpha, which binds to both p55 TNFR and p75 TNFR. Likewise, antibodies that are specific agonists for p55 TNFR or p75 TNFR demonstrate that stimulation of p55 TNFR is sufficient to inhibit insulin signaling, though a small effect can also be seen with antibodies to p75 TNFR. Exogenous sphingomyelinase and ceramides, known to be formed by activation of p55 TNFR, inhibit IR and IRS-1 tyrosine phosphorylation and convert IRS-1 into an inhibitor of IR tyrosine kinase in vitro. Myeloid 32D cells expressing IR and IRS-1 are sensitive to this inhibition, but cells expressing IR and IRS-2 are resistant, pointing to an important difference in the biological function between IRS-1 and IRS-2. These data strongly suggest that TNF-alpha inhibits insulin signaling via stimulation of p55 TNFR and sphingomyelinase activity, which results in the production of an inhibitory form of IRS-1.
Declining Sperm Count - Testicular Issues (DNA)
26. Romano, R. M., Romano, M. A. et al. 2010. “Prepubertal exposure to commercial formulation of the herbicide Glyphosate alters testosterone levels and testicular morphology”. Archives of Toxicology 84(4): 309–317. Abstract: Glyphosate is a herbicide widely used to kill weeds both in agricultural and non-agricultural landscapes. Its reproductive toxicity is related to the inhibition of a StAR protein and an aromatase enzyme, which causes an in vitro reduction in testosterone and estradiol synthesis. Studies in vivo about this herbicide effects in prepubertalWistar rats reproductive development were not performed at this moment. Evaluations included the progression of puberty, body development, the hormonal production of testosterone, estradiol and corticosterone, and the morphology of the testis. Results showed that the herbicide (1) significantly changed the progression of puberty in a dose-dependent manner; (2) reduced the testosterone production, in semineferous tubules' morphology, decreased significantly the epithelium height (P < 0.001; control = 85.8 +/- 2.8 microm; 5 mg/kg = 71.9 +/- 5.3 microm; 50 mg/kg = 69.1 +/- 1.7 microm; 250 mg/kg = 65.2 +/- 1.3 microm) and increased the luminal diameter (P < 0.01; control = 94.0 +/- 5.7 microm; 5 mg/kg = 116.6 +/- 6.6 microm; 50 mg/kg = 114.3 +/- 3.1 microm; 250 mg/kg = 130.3 +/- 4.8 microm); (4) no difference in tubular diameter was observed; and (5) relative to the controls, no differences in serum corticosterone or estradiol levels were detected, but the concentrations of testosterone serum were lower in all treated groups (P < 0.001; control = 154.5 +/- 12.9 ng/dL; 5 mg/kg = 108.6 +/- 19.6 ng/dL; 50 mg/dL = 84.5 +/- 12.2 ng/dL; 250 mg/kg = 76.9 +/- 14.2 ng/dL). These results suggest that commercial formulation of glyphosate is a potent endocrine disruptor in vivo, causing disturbances in the reproductive development of rats when the exposure was performed during the puberty period.
27. Clair, E.; Mesnage, R.; Travert, C.; Séralini, G.E. “A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and testosterone decrease at lower levels”. Toxicol.In Vitro 2012, 26, 269–279. Abstract: The major herbicide used worldwide, Roundup, is a glyphosate-based pesticide with adjuvants. Glyphosate, its active ingredient in plants and its main metabolite (AMPA) are among the first contaminants of surface waters. Roundup is being used increasingly in particular on genetically modified plants grown for food and feed that contain its residues. Here we tested glyphosate and its formulation on mature rat fresh testicular cells from 1 to 10000ppm, thus from the range in some human urine and in environment to agricultural levels. We show that from 1 to 48h of Roundup exposure Leydig cells are damaged. Within 24-48h this formulation is also toxic on the other cells, mainly by necrosis, by contrast to glyphosate alone which is essentially toxic on Sertoli cells. Later, it also induces apoptosis at higher doses in germ cells and in Sertoli/germ cells co-cultures. At lower non toxic concentrations of Roundup and glyphosate (1ppm), the main endocrine disruption is a testosterone decrease by 35%. The pesticide has thus an endocrine impact at very low environmental doses, but only a high contamination appears to provoke an acute rat testicular toxicity. This does not anticipate the chronic toxicity which is insufficiently tested, and only with glyphosate in regulatory tests.
28. Walsh, L.P.; McCormick, C.; Martin, C.; Stocco, D.M. “Roundup inhibits steroidogenesis by disrupting steroidogenic acute regulatory (StAR) protein expression”. Environ. Health Persp. 2000, 108, 769–776. Abstract: Recent reports demonstrate that many currently used pesticides have the capacity to disrupt reproductive function in animals. Although this reproductive dysfunction is typically characterized by alterations in serum steroid hormone levels, disruptions in spermatogenesis, and loss of fertility, the mechanisms involved in pesticide-induced infertility remain unclear. Because testicular Leydig cells play a crucial role in male reproductive function by producing testosterone, we used the mouse MA-10 Leydig tumor cell line to study the molecular events involved in pesticide-induced alterations in steroid hormone biosynthesis. We previously showed that the organochlorine insecticide lindane and the organophosphate insecticide Dimethoate directly inhibit steroidogenesis in Leydig cells by disrupting expression of the steroidogenic acute regulatory (StAR) protein. StAR protein mediates the rate-limiting and acutely regulated step in steroidogenesis, the transfer of cholesterol from the outer to the inner mitochondrial membrane where the cytochrome P450 side chain cleavage (P450scc) enzyme initiates the synthesis of all steroid hormones. In the present study, we screened eight currently used pesticide formulations for their ability to inhibit steroidogenesis, concentrating on their effects on StAR expression in MA-10 cells. In addition, we determined the effects of these compounds on the levels and activities of the P450scc enzyme (which converts cholesterol to pregnenolone) and the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzyme (which converts pregnenolone to progesterone). Of the pesticides screened, only the pesticide Roundup inhibited dibutyryl [(Bu)(2)]cAMP-stimulated progesterone production in MA-10 cells without causing cellular toxicity. Roundup inhibited steroidogenesis by disrupting StAR protein expression, further demonstrating the susceptibility of StAR to environmental pollutants.
29. Cavalli, V.L., et al. “Roundup disrupts male reproductive functions by triggering calcium-mediated cell death in rat testis and Sertoli cells”. Free Radical Biology and Medicine 65 (2013)335–346. Abstract: Glyphosate is the primary active constituent of the commercial pesticide Roundup. The present results show that acute Roundup exposure at low doses (36 ppm, 0.036 g/L) for 30 min induces oxidative stress and activates multiple stress-response pathways leading to Sertoli cell death in prepubertal rat testis. The pesticide increased intracellular Ca(2+) concentration by opening L-type voltage-dependent Ca(2+) channels as well as endoplasmic reticulum IP3 and ryanodine receptors, leading to Ca(2+) overload within the cells, which set off oxidative stress and necrotic cell death. Similarly, 30 min incubation of testis with glyphosate alone (36 ppm) also increased (45)Ca(2+) uptake. These events were prevented by the antioxidants Trolox and ascorbic acid. Activated protein kinase C, phosphatidylinositol 3-kinase, and the mitogen-activated protein kinases such as ERK1/2 and p38MAPK play a role in eliciting Ca(2+) influx and cell death. Roundup decreased the levels of reduced glutathione (GSH) and increased the amounts of thiobarbituric acid-reactive species (TBARS) and protein carbonyls. Also, exposure to glyphosate-Roundup stimulated the activity of glutathione peroxidase, glutathione reductase, glutathione S-transferase, γ-glutamyltransferase, catalase, superoxide dismutase, and glucose-6-phosphate dehydrogenase, supporting downregulated GSH levels. Glyphosate has been described as an endocrine disruptor affecting the male reproductive system; however, the molecular basis of its toxicity remains to be clarified. We propose that Roundup toxicity, implicated in Ca(2+) overload, cell signaling misregulation, stress response of the endoplasmic reticulum, and/or depleted antioxidant defenses, could contribute to Sertoli cell disruption in spermatogenesis that could have an impact on male fertility.
30. Marc, Julie,Mulner-Lorillon, Odile, and Belle, Robert “Glyphosate-Based Pesticides Affect Cell Cycle Regulation,” Biology of the Cell 96, no. 3 (April 2004): 245-49. Abstract: Cell-cycle dysregulation is a hallmark of tumor cells and human cancers. Failure in the cell-cycle checkpoints leads to genomic instability and subsequent development of cancers from the initial affected cell. A worldwide used product Roundup 3plus, based on glyphosate as the active herbicide, was suggested to be of human health concern since it induced cell cycle dysfunction as judged from analysis of the first cell division of sea urchin embryos, a recognized model for cell cycle studies. Several glyphosate-based pesticides from different manufacturers were assayed in comparison with Roundup 3plus for their ability to interfere with the cell cycle regulation. All the tested products, Amega, Cargly, Cosmic, and Roundup Biovert induced cell cycle dysfunction. The threshold concentration for induction of cell cycle dysfunction was evaluated for each product and suggests high risk by inhalation for people in the vicinity of the pesticide handling sprayed at 500 to 4000 times higher dose than the cell-cycle adverse concentration.
31. Hokanson, R., Fudge, R. et al. 2007. “Alteration of estrogen-regulated gene expression in human cells induced by the agricultural and horticultural herbicide glyphosate”. Hum ExpToxicol 26(9): 747–752. Abstract: Gene expression is altered in mammalian cells (MCF-7 cells), by exposure to a variety of chemicals that mimic steroid hormones or interact with endocrine receptors or their co-factors. Among those populations chronically exposed to these endocrine disruptive chemicals are persons, and their families, who are employed in agriculture or horticulture, or who use agricultural/horticultural chemicals. Among the chemicals most commonly used, both commercially and in the home, is the herbicide glyphosate. Although glyphosate is commonly considered to be relatively non-toxic, we utilized in vitro DNA microarray analysis of this chemical to evaluate its capacity to alter the expression of a variety of genes in human cells. We selected a group of genes, determined by DNA microarray analysis to be dysregulated, and used quantitative real-time PCR to corroborate their altered states of expression. We discussed the reported function of those genes, with emphasis on altered physiological states that are capable of initiating adverse health effects that might be anticipated if gene expression were significantly altered in either adults or embryos exposed in utero.
32. Thongprakaisang, Siriporn, et al., “Glyphosate Induces Human Breast Cancer Cells Growth via Estrogen Receptors,” Food and Chemical Toxicology 59 (September 2013): 129-36. Abstract: Glyphosate is an active ingredient of the most widely used herbicide and it is believed to be less toxic than other pesticides. However, several recent studies showed its potential adverse health effects to humans as it may be an endocrine disruptor. This study focuses on the effects of pure glyphosate on estrogen receptors (ERs) mediated transcriptional activity and their expressions. Glyphosate exerted proliferative effects only in human hormone-dependent breast cancer, T47D cells, but not in hormone-independent breast cancer, MDA-MB231 cells, at 10⁻¹² to 10⁻⁶M in estrogen withdrawal condition. The proliferative concentrations of glyphosate that induced the activation of estrogen response element (ERE) transcription activity were 5-13 fold of control in T47D-KBluc cells and this activation was inhibited by an estrogen antagonist, ICI 182780, indicating that the estrogenic activity of glyphosate was mediated via ERs. Furthermore, glyphosate also altered both ERα and β expression. These results indicated that low and environmentally relevant concentrations of glyphosate possessed estrogenic activity. Glyphosate-based herbicides are widely used for soybean cultivation, and our results also found that there was an additive estrogenic effect between glyphosate and genistein, a phytoestrogen in soybeans. However, these additive effects of glyphosate contamination in soybeans need further animal study.
33. Hong, C.-C.; Tang, B.-K.; Hammond, G.L.; Tritchler, D.; Yaffe, M.; Boyd, N.F. “Cytochrome P450 1A2 (CYP1A2) activity and risk factors for breast cancer: A cross-sectional study”. Breast Cancer Res. 2004, 6, R352-R365. Abstract: In both menopausal groups CYP1A2 activity was positively related to smoking and levels of sex hormone binding globulin. In premenopausal women, CYP1A2 activity was also positively related to insulin levels, caffeine intake, age, and plasma triglyceride levels, and negatively related with total cholesterol levels and body mass index. In postmenopausal women CYP1A2 activity was positively associated with insulin-like growth factor-1, and negatively associated with plasma triglyceride, high-density lipoprotein cholesterol, and age at menarche.These results suggest that CYP1A2 activity is correlated with hormones, blood lipids, and lifestyle factors associated with breast cancer risk, although some of the observed associations were contrary to hypothesized directions and suggest that increased CYP1A2 function may be associated with increased risk for breast cancer.
34. Samsel A, Seneff S.“Glyphosate, pathways to modern diseases IV: Cancer and related”. Journal of Biological Physics and Chemistry 15(2015) 121–159 Abstract: Glyphosate is the active ingredient in the pervasive herbicide, Roundup, and its usage, particularly in the United States, has increased dramatically in the last two decades, in step with the widespread adoption of Roundup®-Ready core crops. The World Health Organization recently labeled glyphosate as “probably carcinogenic.” In this paper, we review the research literature, with the goal of evaluating the carcinogenic potential of glyphosate. Glyphosate has alarge number of tumorigenic effects on biological systems, including direct damage to DNA insensitive cells, disruption of glycine homeostasis, succinate dehydrogenase inhibition, chelation of manganese, modification to more carcinogenic molecules such as N-nitrosoglyphosate and glyoxylate, disruption of fructose metabolism, etc. Epidemiological evidence supports strong temporal correlations between glyphosate usage on crops and a multitude of cancers that are reaching epidemic proportions, including breast cancer, pancreatic cancer, kidney cancer, thyroid cancer, liver cancer, bladder cancer and myeloid leukaemia. Here, we support these correlations through an examination of Monsanto’s early studies on glyphosate, and explain how the biological effects of glyphosate could induce each of these cancers. We believe that the available evidence warrants a reconsideration of the risk/benefit trade-off with respect to glyphosate usage to control weeds, and we advocate much stricter regulation of glyphosate.
Non-Hodgkin’s Lymphoma (NHL)
35. Eriksson, M., Hardell, L., Carlberg, M., Akerman, M. 2008. “Pesticide exposure as risk factor for non-Hodgkin lymphoma including histopathological subgroup analysis” Int J Cancer. Oct 1 2008;123(7): 1657–1663. Abstract: We report a population based case-control study of exposure to pesticides as risk factor for non-Hodgkin lymphoma (NHL). Male and female subjects aged 18-74 years living in Sweden were included during December 1, 1999, to April 30, 2002. Controls were selected from the national population registry. Exposure to different agents was assessed by questionnaire. In total 910 (91 %) cases and 1016 (92%) con 51. Regarding phenoxyacetic acids highest risk was calculated for MCPA; OR 2.81, 95% CI 1.27-6.22, all these cases had a latency period >10 years. Exposure to glyphosate gave OR 2.02, 95% CI 1.10-3.71 and with >10 years latency period OR 2.26, 95% CI 1.16-4.40. Insecticides overall gave OR 1.28, 95% CI 0.96-1.72 and impregnating agents OR 1.57, 95% CI 1.07-2.30. Results are also presented for different entities of NHL. In conclusion our study confirmed an association between exposure to phenoxyacetic acids and NHL and the association with glyphosate was considerably strengthened.
Tetragenic (Birth Defects)
36. Lajmanovich, Raafael C., Sandoval, M.T., Peltzer, Paola M. , “Induction of Mortality and Malformation in Scinaxnasicus Tadpoles Exposed to Glyphosate Formulations”. Bulletin of Environmental Contamination Toxicology 70 no.3 (March 2003): 612-18. Abstract: Larval maldevelopment (craniofacial and mouth deformities, eye abnormalities and bent curved tails) occurred in all tests and increased with time and GLY-F concentration. ... Malformation were minimal at 3.07 mg/L exposed for one day, whereas greater that 90% were malformed at a GLY-F level of 7.5 mg/L. The current test confirmed the malformation effects of GLY-F on tadpoles.
37. Paganelli, Alejandra et al., “Glyphosate-Based Herbicides Produce Teratogenic Effects on Vertebrates by Impairing Retinoic Acid Signaling,” Chemical Research in Toxicology 23, no. 10 (August 2010): 1586-95. Abstract: The broad spectrum herbicide glyphosate is widely used in agriculture worldwide. There has been ongoing controversy regarding the possible adverse effects of glyphosate on the environment and on human health. Reports of neural defects and craniofacial malformations from regions where glyphosate-based herbicides (GBH) are used led us to undertake an embryological approach to explore the effects of low doses of glyphosate in development. Xenopuslaevis embryos were incubated with 1/5000 dilutions of a commercial GBH. The treated embryos were highly abnormal with marked alterations in cephalic and neural crest development and shortening of the anterior−posterior (A-P) axis. Alterations on neural crest markers were later correlated with deformities in the cranial cartilages at tadpole stages. Embryos injected with pure glyphosate showed very similar phenotypes. Moreover, GBH produced similar effects in chicken embryos, showing a gradual loss of rhombomere domains, reduction of the optic vesicles, and microcephaly. This suggests that glyphosate itself was responsible for the phenotypes observed, rather than a surfactant or other component of the commercial formulation. A reporter gene assay revealed that GBH treatment increased endogenous retinoic acid (RA) activity in Xenopus embryos and cotreatment with a RA antagonist rescued the teratogenic effects of the GBH. Therefore, we conclude that the phenotypes produced by GBH are mainly a consequence of the increase of endogenous retinoid activity. This is consistent with the decrease of Sonic hedgehog (Shh) signaling from the embryonic dorsal midline, with the inhibition of otx2 expression and with the disruption of cephalic neural crest development. The direct effect of glyphosate on early mechanisms of morphogenesis in vertebrate embryos opens concerns about the clinical findings from human offspring in populations exposed to GBH in agricultural fields.
38. Marc, Julie.; Mulner-Lorillon, O.; Boulben, S.; Hureau, D.; Durand, G.; Bellé, R. “Pesticide roundup provokes cell division dysfunction at the level of CDK1/cyclin B activation”. Chem. Res. Toxicol. 2002, 15, 326–331. Abstract: To assess human health risk from environmental chemicals, we have studied the effect on cell cycle regulation of the widely used glyphosate-containing pesticide Roundup. As a model system we have used sea urchin embryonic first divisions following fertilization, which are appropriate for the study of universal cell cycle regulation without interference with transcription. We show that 0.8% Roundup (containing 8 mM glyphosate) induces a delay in the kinetic of the first cell cleavage of sea urchin embryos. The delay is dependent on the concentration of Roundup. The delay in the cell cycle could be induced using increasing glyphosate concentrations (1-10 mM) in the presence of a subthreshold concentration of Roundup 0.2%, while glyphosate alone was ineffective, thus indicating synergy between glyphosate and Roundup formulation products. The effect of Roundup was not lethal and involved a delay in entry into M-phase of the cell cycle, as judged cytologically. Since CDK1/cyclin B regulates universally the M-phase of the cell cycle, we analyzed CDK1/cyclin B activation during the first division of early development. Roundup delayed the activation of CDK1/cyclin B in vivo. Roundup inhibited also the global protein synthetic rate without preventing the accumulation of cyclin B. In summary, Roundup affects cell cycle regulation by delaying activation of the CDK1/cyclin B complex, by synergic effect of glyphosate and formulation products. Considering the universality among species of the CDK1/cyclin B regulator, our results question the safety of glyphosate and Roundup on human health.
39. Marc, Julie,Bellé, R.; Morales, J.; Cormier, P.; Mulner-Lorillon, O. “Formulated glyphosate activates the DNA-response checkpoint of the cell cycle leading to the prevention of G2/M transition”. Toxicoogicall. Sciences. 2004, 82, 436–442. Abstract: A glyphosate containing pesticide impedes at 10 mM glyphosate the G2/M transition as judged from analysis of the first cell cycle of sea urchin development. We show that formulated glyphosate prevented dephosphorylation of Tyr 15 of the cell cycle regulator CDK1/cyclin B in vivo, the end point target of the G2/M cell cycle checkpoint. Formulated glyphosate had no direct effect on the dual specific cdc25 phosphatase activity responsible for Tyr 15 dephosphorylation. At a concentration that efficiently impeded the cell cycle, formulated glyphosate inhibited the synthesis of DNA occurring in S phase of the cell cycle. The extent of the inhibition of DNA synthesis by formulated glyphosate was correlated with the effect on the cell cycle. We conclude that formulated glyphosate's effect on the cell cycle is exerted at the level of the DNA-response checkpoint of S phase. The resulting inhibition of CDK1/cyclin B Tyr 15 dephosphorylation leads to prevention of the G2/M transition and cell cycle progression.
40. Antoniou, Michael et al., “Teratogenic Effects of Glyphosate-Based Herbicides: Divergence of Regulatory Decisions from Scientific Evidence.” Journal of Environmental and Analytical Toxicology (2012):S4:006. Abstract: The publication of a study in 2010 showing that a glyphosate herbicide formulation and glyphosate alone caused malformations in the embryos of Xenopuslaevis and chickens caused a scientific and political controversy. Debate centered on the effects of the production and consumption of genetically modified Roundup® Ready® soy, which is engineered to tolerate applications of glyphosate herbicide. This study, along with others indicating teratogenic and reproductive effects from glyphosate herbicide exposure, was rebutted by the German Federal Office for Consumer Protection and Food Safety, BVL, as well as in industry-sponsored papers. These rebuttals relied partly on unpublished industry-sponsored studies commissioned for regulatory purposes, which, it was claimed, showed that glyphosate is not teratogenic or a reproductive toxin. However, examination of the German authorities’ draft assessment report (DAR) on the industry studies, which underlies glyphosate’s EU authorisation, revealed further evidence of glyphosate’s teratogenicity. Nevertheless, the German and EU authorities minimized these findings in their assessment and set a potentially unsafe acceptable daily intake (ADI) level for glyphosate. This paper reviews the evidence on the teratogenicity and reproductive toxicity of glyphosate herbicides and concludes that a new and transparent risk assessment needs to be conducted by scientists who are independent of industry and of the regulatory bodies that were involved in the existing authorisation of glyphosate.
41. Arbuckle, T.E.; Lin, Z.; Mery, L.S. “An exploratory analysis of the effect of pesticide exposure on the risk of spontaneous abortion in an Ontario farm population”. Environ. Health Persp.2001, 109, 851–857. Abstract:The toxicity of pesticides on human reproduction is largely unknown--particularly how mixtures of pesticide products might affect fetal toxicity. The Ontario Farm Family Health Study collected data by questionnaire on the identity and timing of pesticide use on the farm, lifestyle factors, and a complete reproductive history from the farm operator and eligible couples living on the farm. A total of 2,110 women provided information on 3,936 pregnancies, including 395 spontaneous abortions. To explore critical windows of exposure and target sites for toxicity, we examined exposures separately for preconception (3 months before and up to month of conception) and post conception (first trimester) windows and for early (< 12 weeks) and late (12-19 weeks) spontaneous abortions. We observed moderate increases in risk of early abortions for preconception exposures to phenoxy acetic acid herbicides [odds ratio (OR) = 1.5; 95% confidence interval (CI), 1.1-2.1], triazines (OR = 1.4; 95% CI, 1.0-2.0), and any herbicide (OR = 1.4; 95% CI, 1.1-1.9). For late abortions, preconception exposure to glyphosate (OR = 1.7; 95% CI, 1.0-2.9), thiocarbamates (OR = 1.8; 95% CI, 1.1-3.0), and the miscellaneous class of pesticides (OR = 1.5; 95% CI, 1.0-2.4) was associated with elevated risks. Post conception exposures were generally associated with late spontaneous abortions. Older maternal age (> 34 years of age) was the strongest risk factor for spontaneous abortions, and we observed several interactions between pesticides in the older age group using Classification and Regression Tree analysis. This study shows that timing of exposure and restricting analyses to more homogeneous endpoints are important in characterizing the reproductive toxicity of pesticides.
42. Rauh, Virginia et al., “Rain Anomalies in Children Exposed Prenatally to a Common Organophosphate Pesticide,” Proceedings of the National Academy of Sciences of the United State of America 109, no. 20 (May 2012). Abstract: Prenatal exposure to chlorpyrifos (CPF), an organophosphate insecticide, is associated with neurobehavioral deficits in humans and animal models. We investigated associations between CPF exposure and brain morphology using magnetic resonance imaging in 40 children, 5.9–11.2 y, selected from a nonclinical, representative community-based cohort. Twenty high-exposure children (upper tertile of CPF concentrations in umbilical cord blood) were compared with 20 low-exposure children on cortical surface features; all participants had minimal prenatal exposure to environmental tobacco smoke and polycyclic aromatic hydrocarbons. High CPF exposure was associated with enlargement of superior temporal, posterior middle temporal, and inferior postcentralgyri bilaterally, and enlarged superior frontal gyrus, gyrus rectus, cuneus, and precuneus along the mesial wall of the right hemisphere. Group differences were derived from exposure effects on underlying white matter. A significant exposure × IQ interaction was derived from CPF disruption of normal IQ associations with surface measures in low-exposure children. In preliminary analyses, high-exposure children did not show expected sex differences in the right inferior parietal lobule and superior marginal gyrus, and displayed reversal of sex differences in the right mesial superior frontal gyrus, consistent with disruption by CPF of normal behavioral sexual dimorphisms reported in animal models. High-exposure children also showed frontal and parietal cortical thinning, and an inverse dose–response relationship between CPF and cortical thickness. This study reports significant associations of prenatal exposure to a widely used environmental neurotoxicant, at standard use levels, with structural changes in the developing human brain.
43. Benachour, N., Séralini, G. E. 2009. “Glyphosate formulations induce apoptosis and necrosis in human umbilical, embryonic, and placental cells”. Chem Res Toxicol 22: 97–105. Abstract: We have evaluated the toxicity of four glyphosate (G)-based herbicides in Roundup formulations, from 10(5) times dilutions, on three different human cell types. This dilution level is far below agricultural recommendations and corresponds to low levels of residues in food or feed. The formulations have been compared to G alone and with its main metabolite AMPA or with one known adjuvant of R formulations, POEA. HUVEC primary neonate umbilical cord vein cells have been tested with 293 embryonic kidney and JEG3 placental cell lines. All R formulations cause total cell death within 24 h, through an inhibition of the mitochondrial succinate dehydrogenase activity, and necrosis, by release of cytosolic adenylate kinase measuring membrane damage. They also induce apoptosis via activation of enzymatic caspases 3/7 activity. This is confirmed by characteristic DNA fragmentation, nuclear shrinkage (pyknosis), and nuclear fragmentation (karyorrhexis), which is demonstrated by DAPI in apoptotic round cells. G provokes only apoptosis, and HUVEC are 100 times more sensitive overall at this level. The deleterious effects are not proportional to G concentrations but rather depend on the nature of the adjuvants. AMPA and POEA separately and synergistically damage cell membranes like R but at different concentrations. Their mixtures are generally even more harmful with G. In conclusion, the R adjuvants like POEA change human cell permeability and amplify toxicity induced already by G, through apoptosis and necrosis. The real threshold of G toxicity must take into account the presence of adjuvants but also G metabolism and time-amplified effects or bioaccumulation. This should be discussed when analyzing the in vivo toxic actions of R. This work clearly confirms that the adjuvants in Roundup formulations are not inert. Moreover, the proprietary mixtures available on the market could cause cell damage and even death around residual levels to be expected, especially in food and feed derived from R formulation-treated crops.
Chronic Kidney Disease
44. Jayasumana, Channa, Gunatilake, Sarath, and Senanyake, Priyantha, “Glyphosate, Hard Water and Nephrotixic Metals: Are they the Culprits Behind the Epidemic of Chronic Kidney Disease of Unknown Etiology in Sri Lanka?” International Journal of Environmental Research and Public Health 11, no. 2 (February 2014): 2125-47. Abstract: The current chronic kidney disease epidemic, the major health issue in the rice paddy farming areas in Sri Lanka has been the subject of many scientific and political debates over the last decade. Although there is no agreement among scientists about the etiology of the disease, a majority of them has concluded that this is a toxic nephropathy. None of the hypotheses put forward so far could explain coherently the totality of clinical, biochemical, histopathological findings, and the unique geographical distribution of the disease and its appearance in the mid-1990s. A strong association between the consumption of hard water and the occurrence of this special kidney disease has been observed, but the relationship has not been explained consistently. Here, we have hypothesized the association of using glyphosate, the most widely used herbicide in the disease endemic area and its unique metal chelating properties. The possible role played by glyphosate-metal complexes in this epidemic has not been given any serious consideration by investigators for the last two decades. Furthermore, it may explain similar kidney disease epidemics observed in Andra Pradesh (India) and Central America. Although glyphosate alone does not cause an epidemic of chronic kidney disease, it seems to have acquired the ability to destroy the renal tissues of thousands of farmers when it forms complexes with a localized geo environmental factor (hardness) and nephrotoxic metals.
Skin and Oral Effects
45. Mariager, T.P.; Madsen, P.V.; EbbehŅj, N.E.; Schmidt, B.; Juhl, A. “Severe adverse effects related to dermal exposure to a glyphosate-surfactant herbicide”. Clin.Toxicol.(Phila.).2013, 51, 111–113. Abstract: This is a case of severe chemical burns following prolonged accidental exposure to a glyphosate-surfactant herbicide. The patient developed local swelling, bullae and exuding wounds. Neurological impairment followed affecting finger flexion and sensation with reduced nerve conduction. Imaging revealed oedema of the soft tissue and juxta-articular osteopenia, and a causal relationship to exposure is suggested.
46. Deo, S.P., Shetty, P., “Accidental Burns of Oral Mucosa by Herbicide,” JNMA J. Nepal Med. Assoc. 2012, 52, 40-42. Abstract: Glyphosate (GlySH) is a broad spectrum, nonselective herbicide, widely used in agriculture. This case report describes a 25-year-old man presenting with extensive chemical burns and ulceration of the oral cavity as a result of accidental exposure to GlySH. This paper aims to illustrate the typical appearance of GlySH related chemical mucosal burn and to demonstrate the severity of the corrosive effect of GlySH which need team approach to prevent unfavorable sequelae such as microstomia.
47. Kruger, Monika, et al 2014, “Detection of glyphosate residues in animals and humans”, Environmental and Analytical Toxicology, 4(2). Abstract: In the present study glyphosate residues were tested in urine and different organs of dairy cows as well as in urine of hares, rabbits and humans using ELISA and Gas Chromatography-Mass Spectroscopy (GC-MS). The correlation coefficients between ELISA and GC-MS were 0.96, 0.87, 0.97and 0.96 for cattle, human, and rabbit urine and organs, respectively. The recovery rate of glyphosate in spiked meat using ELISA was 91%. Glyphosate excretion in German dairy cows was significantly lower than Danish cows. Cows kept in genetically modified free area had significantly lower glyphosate concentrations in urine than conventional husbandry cows. Also glyphosate was detected in different organs of slaughtered cows as intestine, liver, muscles, spleen and kidney. Fattening rabbits showed significantly higher glyphosate residues in urine than hares. Moreover, glyphosate was significantly higher in urine of humans with conventional feeding. Furthermore, chronically ill humans showed significantly higher glyphosate residues in urine than healthy population. The presence of glyphosate residues in both humans and animals could haul the entire population towards numerous health hazards, studying the impact of glyphosate residues on health is warranted and the global regulations for the use of glyphosate may have to be re-evaluated.
48. Defarge N, Takács E, Lozano VL, Mesnage R, Spiroux de Vendômois J, Séralini GE, Székács A., “Herbicides Disrupt Aromatase Activity in Human Cells below Toxic Level Co-Formulants in Glyphosate-Based ls.” Int J Environ Res Public Health. 2016 Feb 26;13(3). Abstract: Pesticide formulations contain declared active ingredients and co-formulants presented as inert and confidential compounds. We tested the endocrine disruption of co-formulants in six glyphosate-based herbicides (GBH), the most used pesticides worldwide. All co-formulants and formulations were comparably cytotoxic well below the agricultural dilution of 1% (18-2000 times for co-formulants, 8-141 times for formulations), and not the declared active ingredient glyphosate (G) alone. The endocrine-disrupting effects of all these compounds were measured on aromatase activity, a key enzyme in the balance of sex hormones, below the toxicity threshold. Aromatase activity was decreased both by the co-formulants alone (polyethoxylated tallow amine-POEA and alkyl polyglucoside-APG) and by the formulations, from concentrations 800 times lower than the agricultural dilutions; while G exerted an effect only at 1/3 of the agricultural dilution. It was demonstrated for the first time that endocrine disruption by GBH could not only be due to the declared active ingredient but also to co-formulants. These results could explain numerous in vivo results with GBHs not seen with G alone; moreover, they challenge the relevance of the acceptable daily intake (ADI) value for GBHs exposures, currently calculated from toxicity tests of the declared active ingredient alone.
3. Impact on the Environment
49. Nicolas, V., N. Oestreicher, and C. Vélot(2016). “Multiple effects of a commercial Roundup® formulation on the soil filamentous fungus Aspergillusnidulansat low doses: evidence of an unexpected impact on energetic metabolism.” Abstract: Soil microorganisms are highly exposed to glyphosate-based herbicides (GBH), especially to Roundup® which is widely used worldwide. However, studies on the effects of GBH formulations on specific non-rhizosphere soil microbial species are scarce. We evaluated the toxicity of a commercial formulation of Roundup® (R450), containing 450 g/L of glyphosate (GLY), on the soil filamentous fungus Aspergillus nidulans, an experimental model microorganism. The median lethal dose (LD50) on solid media was between 90 and 112 mg/L GLY (among adjuvants, which are also included in the Roundup® formulation), which corresponds to a dilution percentage about 100 times lower than that used in agriculture. The LOAEL and NOAEL (lowest- and no-observed-adverse-effect levels) associated to morphology and growth were 33.75 and 31.5 mg/L GLY among adjuvants, respectively. The formulation R450 proved to be much more active than technical GLY. At the LD50 and lower concentrations, R450 impaired growth, cellular polarity, endocytosis, and mitochondria (average number, total volume and metabolism). In contrast with the depletion of mitochondrial activities reported in animal studies, R450 caused a stimulation of mitochondrial enzyme activities, thus revealing a different mode of action of Roundup® on energetic metabolism. These mitochondrial disruptions were also evident at a low dose corresponding to the NOAEL for macroscopic parameters, indicating that these mitochondrial biomarkers are more sensitive than those for growth and morphological ones. Altogether, our data indicate that GBH toxic effects on soil filamentous fungi, and thus potential impairment of soil ecosystems, may occur at doses far below recommended agricultural application rate. Environmental Science and Pollution Research, pp.1-12 DOI 10.1007/s11356-016-6596-2.
50. Bergström L, Börjesson E &StenströmJ (2011) Laboratory and Lysimeter,“Studies of Glyphosate and Aminomethylphosphonic Acid in a Sand and a Clay Soil”Journal of Environmental QualityVol 40 pp 98– 108. Abstract: Due to the increasing concern about the appearance of glyphosate [N-(phosphonomethyl)glycine] and its major metabolite aminomethylphosphonic acid (AMPA) in natural waters, batch laboratory and lysimeter transport studies were performed to assess the potential for leaching of the compounds in two agricultural soils. Unlabeled and 14C-labeled glyphosate were added at a rate corresponding to 1.54 kg a.i. ha(-1) on undisturbed sand and clay columns. Leachate was sampled weekly during a period of 748 d for analyses of glyphosate, AMPA, total 14C, and particle-bound residues. Topsoil and subsoil samples were used for determination of glyphosate adsorption, glyphosate degradation, and formation of AMPA and its degradation. The influence of adsorption on glyphosate degradation was confirmed, giving very slow degradation rate in the clay soil (half-life 110-151 d). The kinetics of AMPA residues suggest that although AMPA is always more persistent than glyphosate when formed from glyphosate, its degradation rate can be faster than that of glyphosate. The kinetics also suggest that apart from glyphosate being transformed to AMPA, the sarcosine pathway can be just as significant. The long persistence of glyphosate was also confirmed in the lysimeter study, where glyphosate+AMPA residues constituted 59% of the initial amount of glyphosate added to the clay soil 748 d after application. Despite large amounts of precipitation in the autumn and winter after application, however, these residues were mainly located in the topsoil, and only 0.009 and 0.019% of the initial amount of glyphosate added leached during the whole study period in the sand and clay, respectively. No leaching ofAMPA occurred in the sand, whereas 0.03 g ha(-1) leached in the clay soil.
51. Simonsen L, Fomsgard IS, Svensmark B &SplidNH. 2008. “Fate and availability of glyphosate and AMPA in agricultural soil”. Journal of Environmental Science and Health Part B43: 365-375. Abstract: The fate of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) was studied in soil. Labeled glyphosate was used to be able to distinguish the measured quantities of glyphosate and AMPA from the background values since the soil was sampled in a field where glyphosate had been used formerly. After addition of labeled glyphosate, the disappearance of glyphosate and the formation and disappearance of AMPA were monitored. The resulting curves were fitted according to a new EU guideline. The best fit of the glyphosate degradation data was obtained using a first-order multi compartment (FOMC) model. DT(50) values of 9 days (glyphosate) and 32 days (AMPA) indicated relatively rapid degradation. After an aging period of 6 months, the leaching risk of each residue was determined by treating the soil with pure water or a phosphate solution (pH 6), to simulate rain over a non-fertilized or fertilized field, respectively. Significantly larger (p < 0.05) amounts of aged glyphosate and AMPA were extracted from the soil when phosphate solution was used as an extraction agent, compared with pure water. This indicates that the risk of leaching of aged glyphosate and AMPA residues from soil is greater in fertilized soil. The blank soil, to which 252 g glyphosate/ha was applied 21 months before this study, contained 0.81 ng glyphosate/g dry soil and 10.46 ng AMPA/g dry soil at the start of the study. Blank soil samples were used as controls without glyphosate addition. After incubation of the blank soil samples for 6 months, a significantly larger amount of AMPA was extracted from the soil treated with phosphate solution than from that treated with pure water. To determine the degree of uptake of aged glyphosate residues by crops growing in the soil, (14)C-labeled glyphosate was applied to soil 6.5 months prior to sowing rape and barley seeds. After 41 days, 0.006 +/- 0.002% and 0.005 +/- 0.001% of the applied radioactivity was measured in rape and barley, respectively.
52. Eker, S., Ozturk, L., Yazici, A., Erenoglu, B., Römheld, V., Cakmak, I., 2006. “Foliarapplied glyphosate substantially reduced uptake and transport of iron and manganese in sunflower (Helianthus annuus L.)”J. Agric. Food Chem., 2006, 54 (26), pp 10019–10025. Abstract: Evidence clearly shows that cationic micronutrients in spray solutions reduce the herbicidal effectiveness of glyphosate for weed control due to the formation of metal−glyphosate complexes. The formation of these glyphosate−metal complexes in plant tissue may also impair micronutrient nutrition of nontarget plants when exposed to glyphosate drift or glyphosate residues in soil. In the present study, the effects of simulated glyphosate drift on plant growth and uptake, translocation, and accumulation (tissue concentration) of iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu) were investigated in sunflower (Helianthus annuus L.) plants grown in nutrient solution under controlled environmental conditions. Glyphosate was sprayed on plant shoots at different rates between 1.25 and 6.0% of the recommended dosage (i.e., 0.39 and 1.89 mM glyphosate isopropylamine salt). Glyphosate applications significantly decreased root and shoot dry matter production and chlorophyll concentrations of young leaves and shoot tips. The basal parts of the youngest leaves and shoot tips were severely chlorotic. These effects became apparent within 48 h after the glyphosate spray. Glyphosate also caused substantial decreases in leaf concentration of Fe and Mn while the concentration of Zn and Cu was less affected. In short-term uptake experiments with radiolabeled Fe (59Fe), Mn (54Mn), and Zn (65Zn), root uptake of 59Fe and 54Mn was significantly reduced in 12 and 24 h after application of 6% of the recommended dosage of glyphosate, respectively. Glyphosate resulted in almost complete inhibition of root-to-shoot translocation of 59Fe within 12 h and 54Mn within 24 h after application. These results suggest that glyphosate residues or drift may result in severe impairments in Fe and Mn nutrition of nontarget plants, possibly due to the formation of poorly soluble glyphosate−metal complexes in plant tissues and/or rhizosphere interactions.
53. Springett,.AJ.& Gray, R.A.J. 1992. “Effect of repeated low doses of biocides on the earthworm Aporrecto deacaliginosa in laboratory culture”. Soil Biology and Biochemistry24: 1739-1744. Abstract: Herbicides containing glyphosate are widely used in agriculture and private gardens, however, surprisingly little is known on potential side effects on non-target soil organisms. In a greenhouse experiment with white clover we investigated, to what extent a globally-used glyphosate herbicide affects interactions between essential soil organisms such as earthworms and arbuscularmycorrhizal fungi (AMF). We found that herbicides significantly decreased root mycorrhization, soil AMF spore biomass, vesicles and propagules. Herbicide application and earthworms increased soil hyphal biomass and tended to reduce soil water infiltration after a simulated heavy rainfall. Herbicide application in interaction with AMF led to slightly heavier but less active earthworms. Leaching of glyphosate after a simulated rainfall was substantial and altered by earthworms and AMF. These sizeable changes provide impetus for more general attention to side-effects of glyphosate-based herbicides on key soil organisms and their associated ecosystem services.
54. Kremer RJ & Means NE. 2009. “Glyphosate and glyphosate -resistant crop interactions with rhizosphere microorganisms”. European Journal of Agronomy 31: 153-161, Abstract: Current crop production relies heavily on transgenic, glyphosate-resistant (GR) cultivars. Widespread cultivation of transgenic crops has received considerable attention. Impacts of glyphosate on rhizosphere microorganisms and activities are reviewed based on published and new data from long-term field projects documenting effects of glyphosate applied to GR soybean and maize. Field studies conducted in Missouri, U.S.A. during 1997–2007 assessed effects of glyphosate applied to GR soybean and maize on root colonization and soil populations of Fusarium and selected rhizosphere bacteria. Frequency of root-colonizing Fusarium increased significantly after glyphosate application during growing seasons in each year at all sites. Roots of GR soybean and maize treated with glyphosate were heavily colonized by Fusarium compared to non-GR or GR cultivars not treated with glyphosate. Microbial groups and functions affected by glyphosate included Mn transformation and plant availability; phytopathogen–antagonistic bacterial interactions; and reduction in nodulation. Root-exuded glyphosate may serve as a nutrient source for fungi and stimulate propagule germination. The specific microbial indicator groups and processes were sensitive to impacts of GR crops and are part of an evolving framework in developing polyphasic microbial analyses for complete assessment of GR technology that is more reliable than single techniques or general microbial assays.
55. Kanissery, R.G., A. Welsh, and G.K. Sims (2015). “Effect of Soil Aeration and Phosphate Addition on the Microbial Bioavailability of Carbon-14-Glyphosate” Abstract: The adsorption, desorption, degradation, and mineralization of C-glyphosate [-(phosphonomethyl)glycine] were examined in Catlin (a fine-silty, mixed, superactive, mesic Oxyaquic Argiudoll), Flanagan (a fine, smectitic, mesic Aquic Argiudoll), and Drummer (a fine-silty, mixed, superactive, mesic Typic Endoaquoll) soils under oxic and anoxic soil conditions. With the exception of the Drummer soil, soil aeration did not significantly alter the adsorption pattern of C-glyphosate to soils. Herbicide desorption was generally enhanced with anaerobiosis in all the soil types. Anoxic soils demonstrated slower microbial degradation and mineralization kinetics of C-glyphosate than oxic soils in all the soil types studied. Phosphate additions significantly reduced the adsorption of C-glyphosate to soils irrespective of soil aeration and confirmed the well-established competitive adsorption theory. The addition of soil phosphate stimulated degradation only in anoxic soils. The results from this research highlight the importance of soil redox conditions as an important factor affecting the bioavailability and mobility of glyphosate in soils. Journal of Environmental Quality, January 8, 2015, Technical Reports, Biomediation and Biodegradation.
56. Newman, M.M., N. Hoilett, N. Lorenz, R.P. Dick, M.R. Liles, C. Ramsier, and J.W. Kloepper(2016). “Glyphosate effects on soil rhizosphere-associated bacterial communities.” Abstract: Glyphosate is one of the most widely used herbicides in agriculture with predictions that 1.35 million metric tons will be used annually by 2017. With the advent of glyphosate tolerant (GT) cropping more than 10 years ago, there is now concern for non-target effects on soil microbial communities that has potential to negatively affect soil functions, plant health, and crop productivity. Although extensive research has been done on short-term response to glyphosate, relatively little information is available on long-term effects. Therefore, the overall objective was to investigate shifts in the rhizosphere bacterial community following long-term glyphosate application on GT corn and soybean in the greenhouse. In this study, rhizosphere soil was sampled from rhizoboxes following 4 growth periods, and bacterial community composition was compared between glyphosate treated and untreated rhizospheres using next-generation barcoded sequencing. In the presence or absence of glyphosate, corn and soybean rhizospheres were dominated by members of the phyla Proteobacteria, Acidobacteria, and Actinobacteria. Proteobacteria (particularly gammaproteobacteria) increased in relative abundance for both crops following glyphosate exposure, and the relative abundance of Acidobacteria decreased in response to glyphosate exposure. Given that some members of the Acidobacteria are involved in biogeochemical processes, a decrease in their abundance could lead to significant changes in nutrient status of the rhizosphere. Our results also highlight the need for applying culture-independent approaches in studying the effects of pesticides on the soil and rhizosphere microbial community. Science of the Total Environment, 543:155–160.
57. Casabeę, N., L. Piola, J. Fuchs, M.L. Oneto, L. Pamparato, S. Basack, R. Gimenez, R. Massaro, J.C. Papa, and E. Kesten(2007). “Ecotoxicological assessment of the effects of glyphosate and chlorpyrifos in an Argentine soya field.” Abstract: The objective of this work was to evaluate the effects of chlorpyrifos on earthworms and on soil functional parameters. An integrated laboratory-field study was performed in a wheat field in Argentina, sprayed with chlorpyrifos at two recommended application rates (240 or 960 g ha-1 a.i.). Laboratory tests included neutral red retention time, comet assay (single cell gel electrophoresis), and avoidance behavior, each using the earthworm Eisenia andrei exposed in soil collected 1 or 14 days after pesticide application, and the bait-lamina test. Field tests assessed organic matter breakdown using the litterbag and bait-lamina assays. Earthworm populations in the field were assessed using formalin application and hand-sorting. The neutral red retention time and comet assays were sensitive biomarkers to the effects of chlorpyrifos on the earthworm E. andrei; however, the earthworm avoidance test was not sufficiently robust to assess these effects. Feeding activity of soil biota, assessed by the bait lamina test, was significantly inhibited by chlorpyrifos after 97 days, but recovered by the 118th day of the test. Litterbag test showed no significant differences in comparison to controls. Earthworm abundance in the field was too low to adequately test the sensitivity of this assessment endpoint. Journal of Soils and Sediment, 8, pp.1–8.
58. Contardo-Jara V., E. Klingelmann, and C. Wiegand(2009).”Bioaccumulation of glyphosate and its formulation Roundup ultra in Lumbriculus variegatus and its effects on biotransformation and antioxidant enzymes.” Abstract: The bioaccumulation potential of glyphosate and the formulation Roundup Ultra, as well as possible effects on biotransformation and antioxidant enzymes in Lumbriculus variegatus were compared by four days exposure to concentrations between 0.05 and 5 mg L(-1) pure glyphosate and its formulation. Bioaccumulation was determined using (14)C labeled glyphosate. The bioaccumulation factor (BCF) varied between 1.4 and 5.9 for the different concentrations, and was higher than estimated from logP(ow). Glyphosate and its surfactant POEA caused elevation of biotransformation enzyme soluble glutathione S-transferase at non-toxic concentrations. Membrane bound glutathione S-transferase activity was significantly elevated in Roundup Ultra exposed worms, compared to treatment with equal glyphosate concentrations, but did not significantly differ from the control. Antioxidant enzyme superoxide dismutase was significantly increased by glyphosate but in particular by Roundup Ultra exposure indicating oxidative stress. The results show that the formulation Roundup Ultra is of more ecotoxicological relevance than the glyphosate itself. Environmental Pollution, 157(1):57–63.
59. Correia, F. V., and J.C. Moreira (2010). “Effects of glyphosate and 2,4-D on earthworms (Eiseniafoetida) in laboratory tests.” Abstract: Laboratory tests were conducted to compare the effects of various concentrations of glyphosate and 2,4-D on earthworms (Eisenia foetida) cultured in Argissol during 56 days of incubation. The effects on earthworm growth, survival, and reproduction rates were verified for different exposure times. Earthworms kept in glyphosate-treated soil were classified as alive in all evaluations, but showed gradual and significant reduction in mean weight (50%) at all test concentrations. For 2,4-D, 100% mortality was observed in soil treated with 500 and 1,000 mg/kg. At 14 days, 30%–40% mortality levels were observed in all other concentrations. No cocoons or juveniles were found in soil treated with either herbicide. Glyphosate and 2,4-D demonstrated severe effects on the development and reproduction of Eisenia foetida in laboratory tests in the range of test concentrations. Bulletin of Environmental Contamination and Toxicology, 85, pp.264–268.
60. Druart, C., M. Millet, R. Scheifler, O. Delhomme, and A. de Vaufleury(2011). “Glyphosate and glufosinate-based herbicides: Fate in soil, transfer to, and effects on land snails.” Abstract: The aim of this work was to assess the transfer and effects of two widely used herbicides on the land snail Helix aspersa during long-term exposure under laboratory conditions. Newly hatched snails were exposed for 168 days to soil and/or food contaminated with a formulation of glyphosate (Bypass®) or glufosinate (Basta®) at the recommended field doses and also at 10-fold this dose. Herbicide degradation patterns showed that snails were mainly exposed during the first 28 days. The DT50 of glyphosate and glufosinate was established at 10.6 and 3.7 days, respectively. No significant effects on survival and growth were determined. Concerning genital tract maturation of the snails, exposure to herbicides tended to decrease the development of the albumen gland (inhibition of 43.5 Ī 32.8%). The presence of glyphosate (6 mg kg−1 dry weight) was demonstrated in snails exposed continuously to this active ingredient at the highest concentration in their food. These results showed a low effect of herbicides at relevant concentrations in soil but the detection of residues in tissues indicated a potential risk of transfer to the food chain. This chronic toxicity bioassay could complete the available tests to assess toxicity of contaminants, and more particularly pesticides, in soil. Journal of Soils and Sediments, 11(8):1373–1384.
61. García-Pérez, J.A., E. Alarcón, Y. Hernández, and C. Hernández (2016). “Impact of litter contaminated with glyphosate-based herbicide on the performance of Pontoscolexcorethrurus, soil phosphatase activities and soil pH” Abstract: The bioavailability of glyphosate from plant residues may be high in the soil matrix. In order to determine the impact of litter contaminated with glyphosate (GLY) on the growth and cocoon production of the earthworm Pontoscolex corethrurus as well as on acid and basic phosphatase activities and soil pH, a commercial glyphosate-based herbicide was applied to three types of litter mixed with soil. Earthworms from a GLY-free coffee plantation were grown for 132 days in microcosms in a two-factorial design. The first factor was glyphosate (GLY; 21.6 g of glyphosate per kg of litter, with two levels (−GLY, +GLY), and the second factor was type of litter (Coffea arabica, Musa cavendishii, Inga vera). All studied factors (except days*litter*glyphosate interaction) had significant effects on earthworm biomass (P < 0.05). Treatments with Coffea and Musa litter -GLY had significantly higher biomass (P < 0.05), whereas treatments with Inga litter -GLY and +GLY had the lowest biomass. A decline in earthworm biomass was detected in all treatments with glyphosate (+GLY) after 72 days. No significant effect on cocoon production was detected for the main factors or their interaction. Litter type showed a marginal effect (P = 0.06); the average (Ī SE) number of cocoons decreased from Musa (5.8 Ī 2.9) to Coffea (1.3 Ī 0.56) and Inga (0.17 Ī 0.17). Soil pH significantly (P = 0.006) decreased at the end of the experiment from 4.4 Ī 0.17 in -GLY to 3.9 Ī 0.06 in +GLY. Acid phosphatase activity increased in treatments with Musa and Inga +GLY (P < 0.05), while alkaline phosphatase activity in the same substrates was higher in treatments with -GLY (P < 0.05). We conclude that repeated application of litter contaminated with glyphosate negatively affects earthworm vitality and increases soil acidity and acid phosphatase activity. Applied Soil Ecology, available online 15 March 2016.
62. Gaupp-Berghausen, M., M. Hofer, B. Rewald, and J.G. Zaller(2015). “Glyphosate-based herbicides reduce the activity and reproduction of earthworms and lead to increased soil nutrient concentrations.”, Abstract: Herbicide use is increasing worldwide both in agriculture and private gardens. However, our knowledge of potential side-effects on non-target soil organisms, even on such eminent ones as earthworms, is still very scarce. In a greenhouse experiment, we assessed the impact of the most widely used glyphosate-based herbicide Roundup on two earthworm species with different feeding strategies. We demonstrate, that the surface casting activity of vertically burrowing earthworms (Lumbricus terrestris) almost ceased three weeks after herbicide application, while the activity of soil dwelling earthworms (Aporrectodea caliginosa) was not affected. Reproduction of the soil dwellers was reduced by 56% within three months after herbicide application. Herbicide application led to increased soil concentrations of nitrate by 1592% and phosphate by 127%, pointing to potential risks for nutrient leaching into streams, lakes, or groundwater aquifers. These sizeable herbicide-induced impacts on agroecosystems are particularly worrisome because these herbicides have been globally used for decades. Nature: Scientific Reports, 5:12886, DOI:10.1038/srep12886.
63. Gimsing, A.L., Borggaard, O.K., Jacobsen, O.S., Aamand, J and Sorensen (2004). “Chemical and microbiological soil characteristics controlling glyphosate mineralization in Danish surface soils.” Abstract: The results showed that glyphosate is initially adsorbed mostly in the upper 2 cm. It is than transported and adsorbed after few days in deeper soil horizons with concomitant increasing content of its metabolite aminomethylphosphonic acid. Moreover, Fe-oxides seem to be a key parameter for glyphosate and aminomethylphosphonic adsorption in soils. This study confirmed previous studies: the analysis showed lower contents of dithionite-soluble and Fe-oxides for the Chernozem, with consequently lower adsorption of glyphosate and aminomethylphosphonic as compared with the Cambisol and the Stagnosol. Applied Soil Ecology, 2, pp. 233–242. DOI:10.1016/j.apsoil.2004.05.007
Aquatic Life and Terrestrial Amphibians
64. Fabio Leonardo Meza-Joya et al., “Toxic, cytotoxic, and genotoxic effects of a glyphosate formulation (Roundup®SL–Cosmoflux®411F) in the direct-developing frog Environmental and Molecular Mutagenesis,,The aerial spraying of glyphosate formulations in Colombia to eradicate illegal crops has generated great concern about its possible impact on nontarget organisms, particularly amphibians. This study evaluated the toxic, cytotoxic, and genotoxic effects of a glyphosate formulation (Roundup®SL–Cosmoflux®411F) in the direct-developing frog Eleutherodactylusjohnstonei by estimating the median lethal application rate (LC50), median hemolytic application rate (HD50), and extent of DNA damage using the in vitro and in vivo Comet assays. Toxicity results indicated that the application rate [37.4 Ķg acid equivalent (a.e.)/cm2] equivalent to that used in aerial spraying (3.74 kg a.e./ha) is not lethal in male and female adult frogs, whereas neonates are highly sensitive. Glyphosate formulation at application rates above 5.4 Ķg a.e./cm2 (in vivo) and concentrations above 95 Ķg a.e./mL (in vitro) showed clear evidence of cytotoxicity. In vivo and in vitro exposure of E. johnstonei erythrocytes to the glyphosate formulation induced DNA breaks in a dose-dependent manner with statistically significant values (P < 0.05) at all doses tested. DNA damage initially increased with the duration of exposure and then decreased, suggesting that DNA repair events were occurring during in vivo and in vitro exposures. These results are discussed from the perspective of possible ecotoxicological risks to anuran species from exposure to glyphosate formulation.
65. Relyea, R.A. 2005c. “The lethal impact of roundup on aquatic and terrestrial amphibians”. Ecological Applications15: 1118–1124. Abstract: The global decline in amphibian diversity has become an international environmental problem with a multitude of possible causes. There is evidence that pesticides may play a role, yet few pesticides have been tested on amphibians. For example, Roundup is a globally common herbicide that is conventionally thought to be nonlethal to amphibians. However, Roundup has been tested on few amphibian species, with existing tests conducted mostly under laboratory conditions and on larval amphibians. Recent laboratory studies have indicated that Roundup may be highly lethal to North American tadpoles, but we need to determine whether this effect occurs under more natural conditions and in post-meta-morphic amphibians. I assembled communities of three species of North American tadpoles in outdoor pond mesocosms that contained different types of soil (which can absorb the pesticide) and applied Roundup as a direct overspray. After three weeks, Roundup killed 96–100% of larval amphibians (regardless of soil presence). I then exposed three species of juvenile (post-metamorphic) anurans to a direct overspray of Roundup in laboratory containers. After one day, Roundup killed 68–86% of juvenile amphibians. These results suggest that Roundup, a compound designed to kill plants, can cause extremely high rates of mortality to amphibians that could lead to population declines.
66. Relyea, RA. 2005b. “The impact of insecticides and herbicides on the biodiversity and productivity of aquatic communities”. Ecological Applications, 15: 618. Abstract: Pesticides constitute a major anthropogenic addition to natural communities. In aquatic communities, a great majority of pesticide impacts are determined from single-species experiments conducted under laboratory conditions. Although this is an essential protocol to rapidly identify the direct impacts of pesticides on organisms, it prevents an assessment of direct and indirect pesticide effects on organisms embedded in their natural ecological contexts. In this study, I examined the impact of four globally common pesticides (two insecticides, carbaryl [Sevin] and malathion; two herbicides, glyphosate [Roundup] and 2,4-D) on the biodiversity of aquatic communities containing algae and 25 species of animals.Species richness was reduced by 15% with Sevin, 30% with malathion, and 22% with Roundup, whereas 2,4-D had no effect. Both insecticides reduced zooplankton diversity by eliminating cladocerans but not copepods (the latter increased in abundance). The insecticides also reduced the diversity and biomass of predatory insects and had an apparent indirect positive effect on several species of tadpoles, but had no effect on snails. The two herbicides had no effects on zooplankton, insect predators, or snails. Moreover, the herbicide 2,4-D had no effect on tadpoles. However, Roundup completely eliminated two species of tadpoles and nearly exterminated a third species, resulting in a 70% decline in the species richness of tadpoles. This study represents one of the most extensive experimental investigations of pesticide effects on aquatic communities and offers a comprehensive perspective on the impacts of pesticides when nontarget organisms are examined under ecologically relevant conditions.
67. Kelly, D.W., Poulin, R., Tompkins, D.M.& Townsend, C.R. (2010). “Synergistic effects of glyphosate formulation and parasite infection on fish malformations and survival”. Journal of Applied Ecology47: 498–504. Abstract: This is the first study to show that parasites and glyphosate can act synergistically on aquatic vertebrates at environmentally relevant concentrations, and that glyphosate might increase the risk of disease in fish. Our results have important implications when identifying risks to aquatic communities and suggest that threshold levels of glyphosate currently set by regulatory authorities do not adequately protect freshwater systems. Anthropogenic stressors such as pollutants can modulate levels of parasitic infections in aquatic animals by suppressing host immunity or through some other mechanisms. One such mechanism could involve increases in either the quantity or quality of infective stages produced by parasites. We investigated the effect of exposure of infected snails, Potamopyrgusantipodarum, to different concentrations of the widely-used herbicide glyphosate, on (i) the production of infective cercariae by three trematode species, Coitocaecumparvum, Apatemon sp. and an undescribed renicolid, and (ii) the survival of cercariae of the latter species. For all three trematode species, infected snails exposed over a month to low (0.36 mg a.i. L−1) or medium (3.6 mg a.i. L−1) formulated glyphosate concentrations released between 1.5 and 3 times more cercariae per day than snails under control conditions. The similar pattern seen in all trematodes suggests a general weakening of the host benefiting any of its parasites rather than some parasite species-specific mechanism. In addition, the survival of renicolidcercariae improved with increasing glyphosate concentrations, with cercariae living about 50% longer in the medium concentration (3.6 mg a.i. L−1) than in control conditions. Our results demonstrate a clear interaction between glyphosate pollution and parasitism by trematodes in freshwater systems, occurring at glyphosate concentrations recorded in aquatic habitats, and within the environmental exposure limit allowed in New Zealand freshwaters. Future risk assessments and toxicity tests need to consider indirect impacts resulting from infections to invertebrate and vertebrate species penetrated by cercariae and serving as second intermediate hosts of trematodes.
68. Mineau, Pierre, “Direct Losses of Birds to Pesticides – Beginnings of a Quantification”, USDA Forest Service Gen. Tech. Rep. PSW-GTR-191. 2005 Abstract: Recent analyses and modeling of avian pesticide field studies have led to the conclusion that bird kills are regular and frequent in insecticide-treated fields. Unfortunately, data are seldom adequate to quantify this mortality. Also, mortality is expected to be highly variable in response to varying bird presence in and around treated fields. Studies reporting kills of birds in cornfields treated with granular formulations of carbofuran provide a good example of the types of calculations that are needed to estimate direct bird losses in farm fields. Several studies provide the means to correct carcass counts for search efficiency and scavenging. Based on typical Midwest cornfields, use of granular carbofuran resulted in an estimated annual mortality rate of 3.0 to 16 songbirds per ha of treated field, the higher number corresponding to better field edge habitat. Much larger kills also occurred where fields bordered non-crop habitat more suitable for birds. At the peak of its popularity in the United States, this single product was conservatively giving rise to an estimated annual loss of 17-91 million birds in cornfields alone. Granular carbofuran formulations continue to be sold and used in most Latin American countries on a wide variety of crops.
69. Glusczak, L, Dos Santos,Miron D, Moraes, B.S., Simões, R.R., Schetinger, M.R.C., Morsch, V.M. &Loro, V.L., 2007. “Acute effects of glyphosate herbicide on metabolic and enzymatic parameters of silver catfish (Rhamdiaquelen)”Comparative Biochemistry and Physiology Part C 146: 519-524. Abstract: Silver catfish (Rhamdiaquelen; Teleostei) were exposed to commercial formulation Roundup, a glyphosate herbicide: 0 (control), 0.2 or 0.4 mg/L for 96 h. Fish exposed to glyphosate showed an increase in hepatic glycogen, but a reduction in muscle glycogen at both concentrations tested. Glucose decreased in liver and increased in muscle of fish at both herbicide concentrations. Glyphosate exposure increased lactate levels in liver and white muscle at both concentrations. Protein levels increased in liver and decreased in white muscle while levels of ammonia in both tissues increased in fish at both glyphosate concentrations. Specific AChE activity was reduced in brain after treatments, no changes were observed in muscle tissue. Catalase activity in liver did not change during of exposure. Fish exposed to glyphosate demonstrated increased TBARS production in muscle tissue at both concentrations tested. For both glyphosate concentrations tested brain showed a reduction of TBARS after 96 h of exposure. The present results showed that in 96 h, glyphosate changed AChE activity, metabolic parameters and TBARS production. The parameters measured can be used as herbicide toxicity indicators considering environmentally relevant concentration.
70. Moore, L.J., L. Fuentes, J. H. Rodgers, Jr., W.W. Bowerman, G. K. Yarrow, W.Y. Chao, and W.C. Bridges, Jr. (2012). “Relative toxicity of the components of the original formulation of Roundup® to five north american anurans”. Abstract: The responses of five North American frog species that were exposed in an aqueous system to the original formulation of Roundup were compared. Carefully designed and un-confounded laboratory toxicity tests are crucial for accurate assessment of potential risks from the original formulation of Roundup to North American amphibians in aquatic environments. The formulated mixture of this herbicide as well as its components, isopropylamine (IPA) salt of glyphosate and the surfactant MON 0818 (containing polyethoxylated tallowamine (POEA)) were separately tested in 96 h acute toxicity tests with Gosner stage 25 larval anurans. Rana pipiens, R. clamitans, R. catesbeiana, Bufo fowleri, and Hyla chrysoscelis were reared from egg masses and exposed to a series of 11 concentrations of the original formulation of Roundup herbicide, nine concentrations of MON 0818 and three concentrations of IPA salt of glyphosate in static (non-renewal) aqueous laboratory tests. LC50 values are expressed as glyphosate acid equivalents (ae) or as mg/L for MON 0818 concentrations for comparison between the formulation and components. R. pipiens was the most sensitive of five species with 96 h-LC50 values for formulation tests, for the five species, ranging from 1.80 to 4.22 mg ae/L, and MON 0818 exposures with 96 h-LC50 values ranging from 0.68 to 1.32 mg/L. No significant mortality was observed during exposures of 96 h for any of the five species exposed to glyphosate IPA salt at concentrations up to 100 times the predicted environmental concentration (PEC). These results agree with previous studies which have noted that the surfactant MON 0818 containing POEA contributes the majority of the toxicity to the herbicide formulations for fish, aquatic invertebrates, and amphibians. These study results suggest that anurans are among the most sensitive species, and emphasize the importance of testing the herbicide formulation in addition to its separate components to accurately characterize the toxicity and potential risk of the formulation. Ecotoxicology and Environmental Safety, 78:128–133.
71. Salbego, J., A. Pretto, C.R. Gioda, C.C. de Menezes, R. Lazzari, J.R. Neto, B. Baldisserotto, and V.L. Loro(2010). “Herbicide formulation with glyphosate affects growth, acetylcholinesterase activity, and metabolic and hematological parameters in piava (Leporinus obtusidens)”. Abstract: The teleost fish Leporinus obtusidens (piava) was exposed to different concentrations of Roundup, a commercial herbicide formulation containing glyphosate (0, 1, or 5 mg L(-1)), for 90 days. Acetylcholinesterase (AChE) activity was verified in brain and muscle. Hepatic and muscular metabolic parameters as well as some hematological parameters were determined. The results showed that brain AChE activity was significantly decreased in fish exposed to 5 mg L(-1) Roundup, whereas muscular AChE activity was not altered. Both Roundup concentrations significantly decreased liver glycogen without altering the muscle glycogen content. Hepatic glucose levels were reduced only in fish exposed to 5 mg L(-1) Roundup. Lactate levels in the liver and muscle significantly increased in fish exposed to both Roundup concentrations. Hepatic protein content remained constant at 1 mg L(-1) but increased at 5 mg L(-1) Roundup. In the muscle however, protein content decreased with increasing exposure concentration. The herbicide exposure produced a decrease in hematological parameters at both concentrations tested. The majority of observed effects occur at environmental relevant concentrations, and in summary, the results show that Roundup affects brain AChE activity as well as metabolic and hematologic parameters of piavas. Thus, we can suggest that long-term exposure to Roundup causes metabolic disruption in Leporinus obtusidens. Archives of Environmental Contamination and Toxicology 58(3): 740–745.
72. Sandrini, J.Z., R.C. Rola, F.M. Lopes, H.F. Buffon, M.M. Freitas, C.M.G. Martins, and C.E. Rosa (2013). “Effects of glyphosate on cholinesterase activity of the mussel Perna perna and the fish Danio rerio and Jenynsia multidentata: In vitro studies”. Abstract: Although the herbicide glyphosate [N-(phosphonomethyl)glycine] is not classified as an acethylcholinesterase inhibitor, some studies have reported reduction in the acethylcolinesterase activity after in vivo exposure to both its pure form and its commercial formulations. Considering this controversy, the objective of the present study was to investigate, in vitro, the effects of glyphosate exposure on cholinesterase activity of the brown mussel Perna perna and of two fish species: zebrafish Danio rerio and onesided livebearer Jenynsia multidentata. For this purpose, samples of different tissues (brain and muscle for fish; gills and muscle for mussel) were homogenized and pre-incubated with different glyphosate concentrations before cholinesterase activity determination. Results demonstrated that cholinesterase from different fractions of all species tested was inhibited by glyphosate. The concentrations of glyphosate that inhibits 50% of cholinesterase activity (IC50) ranged from 0.62 mM for P. perna muscle to 8.43 mM for J. multidentata brain. According to this, cholinesterase from mussel seems to be more sensitive to glyphosate exposure than those from the fish D. rerio and J. multidentata. Aquatic Toxicology, 130-131:171–173.
73. Cattaneo, R., B. Clasen, V.L. Loro, C.C. de Menezes, A. Pretto, B. Baldisserotto, A. Santi, and L.A. de Avila (2011). “Toxicological responses of Cyprinus carpio exposed to a commercial formulation containing glyphosate.” Abstract: The effects of commercial glyphosate herbicide formulation on the activity of acetylcholinesterase (AChE) enzyme and oxidative stress were studied in the fish Cyprinus carpio exposed for 96 h to 0.0, 0.5, 2.5, 5.0 and 10.0 mg/L and then allowed to equal recovery period in water without herbicide. The activity of AChE was inhibited in the brain and in the muscle after exposure. However, after recovery period brain and muscle AChE activity increased. Brain thiobarbituric acid reactive species (TBARS) were measured as an indicator of oxidative stress. Increased TBARS levels were observed with all concentrations tested of the glyphosate formulation, and remained increased after the recovery period. The results recorded clearly indicate lipid peroxidation and anti-AChE action induced by Roundup® exposure. Bulletin of Environmental Contamination and Toxicology, 87(6): 597–602.
74. Costa L.D.F., K.C. Miranda, M.P. Severo, and L.A. Sampaio(2008). “Tolerance of juvenile Pompano trachinotus marginatus to acute ammonia and nitrite exposure at different salinity levels.” Abstract: The present study was conducted to estimate the acute toxicity of unionized ammonia–nitrogen (NH3–N) and nitrite–nitrogen (NO2–N) to juvenile pompano Trachinotus marginatus (0.86 Ī 0.21 g) at different salinity levels: 5, 10 (equivalent to its isosmotic point), and 30š. Fish were acclimated to the different salinities for 10 days and fed ad libitum daily. Groups of five fishes were exposed to five concentrations of NH3–N and NO2–N for 96 h plus control groups for each salinity, where no toxicant was added. Test concentrations ranged from 0.28 to 3.53 mg NH3–N/L and 24.8 to 191.1 mg NO2–N/L with three replicates per treatment. Tests were run using a standard semi-static system with 100% daily renewal of water and toxicants. The results were based on mortality data registered in different concentrations tested, using the software Trimmed Spearman Karber method. The median lethal concentrations (LC50) after 96 h of exposure to NH3–N were 0.66 (0.53–0.81), 1.87 (1.65–2.12) and 1.06 (0.94–1.20) mg NH3–N/L for 5, 10, and 30š. The 96 h LC50 to NO2–N were 39.94 (36.39–43.84), 116.68 (112.52–121.00) and 37.55 (20.91–67.44) mg NO2–N/L for 5, 10, and 30š. Acute toxicity of NH3–N and NO2–N to pompano was affected by salinity. Results of the present study show that pompano reared at an isosmotic environment are less sensitive to NH3–N and NO2–N. Tolerance to NH3–N is compromised at reduced salinities, while toxicity of NO2–N is similar at 5 and 30š. Aquaculture, 285(1-4): 270–272.
75. Cuhra, M., T. Traavik, and T. Bohn (2013). “Clone- and age-dependent toxicity of a glyphosate commercial formulation and its active ingredient in Daphnia magna.” 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 EC(50) (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. EC(50) 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 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. Ecotoxicology, 22:251–262, DOI 10.1007/s10646-012-1021-1.
76. Folmar, L.C., H.O. Sanders, and A.M. Julin(1979). “Toxicity of the herbicide glyphosate and several of its formulations to fish and aquatic invertebrates.” Abstract: Studies were Initiated to determine the acute toxicity of technical grade glyphosate (MONC?573), the isopropylamine salt of glyphosate (MONOI39), the formulated herbicide Roundup (MON2139),and the Roundup surfactant (MON0818) to four aquatic invertebrates and four fishes: daphnids (Daphnia magna), scuds (G.aI1'l;TJ1arus pseudolimnaeus) 1 midge larvae (Crlironomu.s p,lumosus), mayfly nymphs (Ephemerella ~eri), rainbow trout (Salmo gairdneri), fathead minnows (Pimephaes promelas), channel catfish (Ictalurus punctatus), and bluegills (Lepomis macrochirus). Acute toxicities for Roundup ranged from 2.3 mg/l (96-h LC50, fathead minnow) to 43 mg/l (L.8-h EC50, mature scuds). Toxicities of the surfactant were similar to those of the Roundup formulation. Technical glyphosate was considerably less toxic than Roundup or the surfactant; for midge larvae the 48-h EC50 was 55 mg/l and for rainbow trout the 96-h LC50 was 140 mg/l. Roundup was more toxic to rainbow trout and bluegil1s at the higher test temperatures, and at pH 7.5 than at pH 6.5. Toxicity did not increase at pH 8.5 or 9.5. Eyed eggs were the least sensitive life stage, but toxicity increased markedly as the fish entered the sac fry and early swim-up stages. No changes in fecundity or gonadosomatic index were observed in adult rainbow trout. Archives of Environmental Contamination and Toxicology, 8(3):269–278.
77. Frontera, J.L., I. Vatnick, A. Chaulet, and E. M. Rodriguez (2011). “Effects of glyphosate and polyoxyethylenamine on growth and energetic reserves in the freshwater crayfish Cheraxquadricarinatus (Decapoda, Parastacidae).” Abstract: Freshwater crayfish Cherax quadricarinatus have a high commercial value and are cultured in farms where they are potentially exposed to pesticides. Therefore, we examined the sublethal effects of a 50-day exposure to glyphosate acid and polyoxyethylenamine (POEA), both alone and in a 3:1 mixture, on the growth and energetic reserves in muscle, hepatopancreas and hemolymph of growing juvenile crayfish. Exposure to two different glyphosate and POEA mixtures caused lower somatic growth and decreased muscle protein levels. These effects, caused by both compounds interacting in the mixture, could also be synergistic because they were expressed even at the lowest concentration. The decrease in protein levels could be related to the greater use of other energy reserves. This hypothesis is supported by the decrease in muscle glycogen stores due to glyphosate exposure and the decrease in lipid reserves associated with exposure to POEA.Archives of Environmental Contamination and Toxicology, 61(4):590–598.
78. Fuentes, L., L.J. Moore, J.H. Rodgers, W.W. Bowerman, G. K. Yarrow, and W. Y. Chao (2011). “Comparative toxicity of two glyphosate formulations (original formulation of Roundup ® and Roundup Weathermax ®) to six North American larval anurans.” Abstract: The toxicity of two glyphosate formulations (the original formulation of Roundup® and Roundup WeatherMAX®) to six species of North American larval anurans was evaluated by using 96-h static, nonrenewal aqueous exposures. The 96-h median lethal concentration values (LC50) ranged from 1.80 to 4.22 mg acid equivalent (ae)/L and 1.96 to 3.26 mg ae/L for the original formulation of Roundup and Roundup WeatherMAX, respectively. Judged by LC50 values, four species were more sensitive to Roundup WeatherMAX exposures, and two species were more sensitive to the original formulation. Two of six species, Bufo fowleri (p < 0.05, F = 14.89, degrees of freedom [df] = 1) and Rana clamitans (p < 0.05, F = 18.46, df = 1), had significantly different responses to the two formulations tested. Increased sensitivity to Roundup WeatherMAX likely was due to differences in the surfactants or relative amounts of the surfactants in the two formulations. Potency slopes for exposures of the original formulation ranged from 24.3 to 92.5% mortality/mg ae/L. Thresholds ranged from 1.31 to 3.68 mg ae/L, showing an approximately three times difference in the initiation of response among species tested. For exposures of Roundup WeatherMAX, slopes ranged from 49.3 to 84.2% mortality/mg ae/L. Thresholds ranged from 0.83 to 2.68 mg ae/L. Margins of safety derived from a simulated direct overspray were above 1, except for one species in exposures of Roundup WeatherMAX. Laboratory data based on aqueous exposures are conservative because of the lack of environmental ligands; however, these tests provide information regarding the relative toxicity between these two Roundup formulations. Environmental Toxicology and Chemistry, 30(12):2756–2761.
79. Glusczak, L., V.L. Loro, A. Pretto, B.S. Moraes, A. Raabe, M.F. Duarte, M.B. da Fonseca, C.C. de Menezes, D.M.D. Valladao(2011). “Acute exposure to glyphosate herbicide affects oxidative parameters in Piava (Leporinusobtusidens).” Abstract: In recent years, commercial glyphosate herbicide formulations have been widely used in agriculture to control aquatic weeds. These pesticides may result in disruption of ecological balance, causing damage to nontarget organisms including fish. Teleostean fish (Leporinus obtusidens) were exposed to commercial glyphosate herbicide formulation at 0 (control), 3, 6, 10 or 20 mg L−1 for 96 h. The effects of herbicide on plasmatic metabolic parameters, thiobarbituric acid reactive substances (TBARS), catalase activity, protein carbonyl, and mucus layer parameters were studied. Plasmatic glucose and lactate levels increased but protein levels showed reduction after herbicide exposure. TBARS levels in brain showed a reduction at all tested concentrations. However, liver demonstrated increased TBARS levels at all tested concentrations, whereas in white muscle TBARS production did not change after exposure to herbicide. Fish exposed to all concentrations of glyphosate showed increase in liver catalase activity and protein carbonyl. Herbicide exposure increased protein and carbohydrate levels of the mucus layer at all tested concentrations. The present results showed that, in 96 h, glyphosate changed toxicological parameters analyzed in piava. Parameters measured in this study may be useful in environmental biomonitoring.Archives of Environmental Contamination and Toxicology, 61: 624–630.
80. Glusczak, L., dos Santos, D. Miron, B.S. Moraes, R.R. Simoes, M.R.C. Schetinger, V.M. Morsch, and V.L. Loro(2007). “Acute effects of glyphosate herbicide on metabolic and enzymatic parameters of silver catfish (Rhamdiaquelen).” Abstract: We investigated the effects of four commercial formulations of herbicides (glyphosate [GLY], metsulfuron-methyl [MET], bispyribac-sodium [BIS], and picloram [PIC]) individually, and in three 50:50 mixtures (GLY–MET, GLY–BIS, GLY–PIC) on the common toad Rhinella arenarum (Anura: Bufonidae) tadpoles. Enzymatic parameters such as, glutathione S-transferase (GST), butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) activities, as well as erythrocyte nuclear abnormalities (ENA) were studied. Interactions between herbicides in mixtures were evaluated and classified as additive, synergistic, or antagonistic. Toxicity results (48-h LC50) showed that PIC was the most toxic herbicide, followed by BIS, GLY, and MET, while GLY–PIC was the most toxic mixture, followed by GLY–BIS, and GLY–MET. All commercial herbicide formulations and their mixtures significantly inhibited BChE activity in exposed tadpoles. The AChE activity was also inhibited by all herbicides and their mixtures, except by GLY–BIS. The inhibition of GST activity was only significant for GLY, MET, PIC, and GLY–MET. A significant increase in the frequency of ENA was found for tadpoles exposed either to commercial herbicide formulations or to mixtures, except for GLY. All the mixtures showed synergism for BChE activity while for AChE only the GLY–MET and GLY–PIC mixtures acted synergistically. GLY–MET showed synergism for GST, whereas for ENA, the mixture GLY–BIS was antagonistic. This study with R. arenarum tadpoles demonstrates that the interactions between three of the most intensively used herbicides in soybean crops results in synergistic effects on mortality and neurotoxicity and synergistic or additive effects in genotoxicity. Comparative Biochemistry and Physiology- Pharmacology, Toxicology and Endocrinology, 146(4):519–524.
81. Guilherme, S., I. Gaivao, M.A. Santos, and M. Pacheco (2012). “DNA damage in fish (Anguilla anguilla) exposed to a glyphosate-based herbicide -elucidation of organ-specificity and the role of oxidative stress.” Mutation Research, Abstract: Organophosphate herbicides are among the most dangerous agrochemicals for the aquatic environment. In this context, Roundup(®), a glyphosate-based herbicide, has been widely detected in natural water bodies, representing a potential threat to non-target organisms, namely fish. Thus, the main goal of the present study was to evaluate the genotoxic potential of Roundup(®) in the teleost fish Anguilla anguilla, addressing the possible causative involvement of oxidative stress. Fish were exposed to environmentally realistic concentrations of this herbicide (58 and 116 μgL(-1)) during one or three days. The standard procedure of the comet assay was applied to gill and liver cells in order to determine organ-specific genetic damage. Since liver is a central organ in xenobiotic metabolism, nucleoids of hepatic cells were also incubated with a lesion-specific repair enzyme (formamidopyrimidine DNA glycosylase - FPG), in order to recognise oxidised purines. Antioxidants were determined in both organs as indicators of pro-oxidant state. In general, both organs displayed an increase in DNA damage for the two Roundup(®) concentrations and exposure times, although liver showed to be less susceptible to the lower concentration. The enzyme-modified comet assay showed the occurrence of FPG-sensitive sites in liver only after a 3-day exposure to the higher Roundup(®) concentration. The antioxidant defences were in general unresponsive, despite a single increment of catalase activity in gills (116 μgL(-1), 3-day) and a decrease of superoxide dismutase activity in liver (58 μgL(-1), 3-day). Overall, the mechanisms involved in Roundup(®)-induced DNA strand-breaks showed to be similar in both organs. Nevertheless, it was demonstrated that the type of DNA damage varies with the concentration and exposure duration. Hence, after 1-day exposure, an increase on pro-oxidant state is not a necessary condition for the induction of DNA-damaging effects of Roundup(®). By increasing the duration of exposure to three days, ROS-dependent processes gained preponderance as a mechanism of DNA-damage induction in the higher concentration. Genetic Toxicology and Environmental Mutagenesis, 743: 1–9.
82. Guilherme, S., I. Gaivao, M.A. Santos, and M. Pacheco (2010). “European eel (Anguilla anguilla) genotoxic and pro-oxidant responses following short-term exposure to Roundup® - a glyphosate-based herbicide.” Abstract: The glyphosate-based herbicide, Roundup, is among the most used pesticides worldwide. Due to its extensive use, it has been widely detected in aquatic ecosystems representing a potential threat to non-target organisms, including fish. Despite the negative impact of this commercial formulation in fish, as described in literature, the scarcity of studies assessing its genotoxicity and underlying mechanisms is evident. Therefore, as a novel approach, this study evaluated the genotoxic potential of Roundup to blood cells of the European eel (Anguilla anguilla) following short-term (1 and 3 days) exposure to environmentally realistic concentrations (58 and 116 microg/l), addressing also the possible association with oxidative stress. Thus, comet and erythrocytic nuclear abnormalities (ENAs) assays were adopted, as genotoxic end points, reflecting different types of genetic damage. The pro-oxidant state was assessed through enzymatic (catalase, glutathione-S-transferase, glutathione peroxidase and glutathione reductase) and non-enzymatic (total glutathione content) antioxidants, as well as by lipid peroxidation (LPO) measurements. The Roundup potential to induce DNA strand breaks for both concentrations was demonstrated by the comet assay. The induction of chromosome breakage and/or segregational abnormalities was also demonstrated through the ENA assay, though only after 3-day exposure to both tested concentrations. In addition, the two genotoxic indicators were positively correlated. Antioxidant defenses were unresponsive to Roundup. LPO levels increased only for the high concentration after the first day of exposure, indicating that oxidative stress caused by this agrochemical in blood was not severe. Overall results suggested that both DNA damaging effects induced by Roundup are not directly related with an increased pro-oxidant state. Moreover, it was demonstrated that environmentally relevant concentrations of Roundup can pose a health risk for fish populations. Mutagenesis, 25: 523–530.
83. Hagner, M., S. Hallman, L. Jauhiainen, R. Kemppainen, S. Ram, K. Tiikkala, and H. Setal(2015). “Birch (Betula spp.) wood biochar is a potential soil amendment to reduce glyphosate leaching in agricultural soils” Abstract: Glyphosate (N-(phosphonomethyl) glycine), a commonly used herbicide in agriculture can leach to deeper soil layers and settle in surface- and ground waters. To mitigate the leaching of pesticides and nutrients, biochar has been suggested as a potential soil amendment due to its ability to sorb both organic and inorganic substances. However, the efficiency of biochar in retaining agro-chemicals in the soil is likely to vary with feedstock material and pyrolysis conditions. A greenhouse pot experiment, mimicking a crop rotation cycle of three plant genera, was established to study the effects of pyrolysis temperature on the ability of birch (Betula sp.) wood originated biochar to reduce the leaching of (i) glyphosate, (ii) its primary degradation product AMPA and (iii) phosphorus from the soil. The biochar types used were produced at three different temperatures: 300 °C (BC300), 375 °C (BC375) and 475 °C (BC475). Compared to the control treatment without biochar, the leaching of glyphosate was reduced by 81%, 74% and 58% in BC300, BC375 and BC475 treated soils, respectively. The respective values for AMPA were 46%, 39% and 23%. Biochar had no significant effect on the retention of water-soluble phosphorus in the soil. Our results corroborate earlier findings on pesticides, suggesting that biochar amendment to the soil is a promising way to reduce also the leaching of glyphosate. Importantly, the ability of biochar to adsorb agro-chemicals depends on the temperature at which feedstock is pyrolysed. Journal of Environmental Management, 164:46–52.
Rain and Atmosphere
84. Chang, Feng-chih, Simcik, Matt F., Capel, Paul D., “Occurrence and fate of the herbicide glyphosate and its degradate aminomethylphosphonic acid in the atmosphere”. Environ Toxicol Chem. 2011 Mar;30(3):548-55. Abstract: This is the first report on the ambient levels of glyphosate, the most widely used herbicide in the United States, and its major degradation product, aminomethylphosphonic acid (AMPA), in air and rain. Concurrent, weekly integrated air particle and rain samples were collected during two growing seasons in agricultural areas in Mississippi and Iowa. Rain was also collected in Indiana in a preliminary phase of the study. The frequency of glyphosate detection ranged from 60 to 100% in both air and rain. The concentrations of glyphosate ranged from<0.01 to 9.1 ng/m(3) and from<0.1 to 2.5Ķg/L in air and rain samples, respectively. The frequency of detection and median and maximum concentrations of glyphosate in air were similar or greater to those of the other high-use herbicides observed in the Mississippi River basin, whereas its concentration in rain was greater than the other herbicides. It is not known what percentage of the applied glyphosate is introduced into the air, but it was estimated that up to 0.7% of application is removed from the air in rainfall. Glyphosate is efficiently removed from the air; it is estimated that an average of 97% of the glyphosate in the air is removed by a weekly rainfall ≥ 30 mm.
85. Chang FC, Simcik MF and Capel P., 2011. “Occurrence and fate of the herbicide glyphosate and its degradate Aminomethylphosphonic acid in the atmosphere”,Environmental Toxicology and Chemistry30 : 548–555 Abstract: This is the first report on the ambient levels of glyphosate, the most widely used herbicide in the United States, and its major degradation product, aminomethylphosphonic acid (AMPA), in air and rain. Concurrent, weekly integrated air particle and rain samples were collected during two growing seasons in agricultural areas in Mississippi and Iowa. Rain was also collected in Indiana in a preliminary phase of the study. The frequency of glyphosate detection ranged from 60 to 100% in both air and rain. The concentrations of glyphosate ranged from <0.01 to 9.1 ng/m(3) and from <0.1 to 2.5 Ķg/L in air and rain samples, respectively. The frequency of detection and median and maximum concentrations of glyphosate in air were similar or greater to those of the other high-use herbicides observed in the Mississippi River basin, whereas its concentration in rain was greater than the other herbicides. It is not known what percentage of the applied glyphosate is introduced into the air, but it was estimated that up to 0.7% of application is removed from the air in rainfall. Glyphosate is efficiently removed from the air; it is estimated that an average of 97% of the glyphosate in the air is removed by a weekly rainfall ≥ 30 mm.
Water and Watersheds
86. Kaiser, Kristine, “Preliminary Study of Pesticide Drift into the Maya Mountain Protected Areas of Belize” Bull Environ ContamToxicol. 2011 Jan; 86(1): 56–59. Abstract: In Belize, Central America, many farms surrounding the Protected Areas of the Maya Mountains rely heavily on the application of agrochemicals. The purpose of this study was to test whether orographic drift of glyphosate and organophosphates into the nearby Maya Mountain Protected Areas occurred by collecting phytotelmic water from seven sites over 3 years. Regardless of location within the Maya Mountain Protected Areas, glyphosate was present; organophosphates were more common at ridge sites. Although glyphosate concentrations were low, due to the number of threatened species and the human use of stream water outside the Maya Mountain Protected Areas, better understanding of these effects is warranted.
87. Mercurio, Philip, Flores, Florita, Mueller, Jochen F., Carter, Steve, Negri, Andrew P.“Glyphosate persistence in seawater”,Marine Pollution Bulletin, Vol. 85, issue 2, 30 August 2014, Pages 385–390. Abstract: Glyphosate is one of the most widely applied herbicides globally but its persistence in seawater has not been reported. Here we quantify the biodegradation of glyphosate using standard “simulation” flask tests with native bacterial populations and coastal seawater from the Great Barrier Reef. The half-life for glyphosate at 25 °C in low-light was 47 days, extending to 267 days in the dark at 25 °C and 315 days in the dark at 31 °C, which is the longest persistence reported for this herbicide. AMPA, the microbial transformation product of glyphosate, was detected under all conditions, confirming that degradation was mediated by the native microbial community. This study demonstrates glyphosate is moderately persistent in the marine water under low light conditions and is highly persistent in the dark. Little degradation would be expected during flood plumes in the tropics, which could potentially deliver dissolved and sediment-bound glyphosate far from shore.
88. Love, Bradley J, Einheuser, Matthew D., Nejadhashemi, A. Pouyan, “Effects on aquatic and human health due to large scale bioenergy crop expansion”.Sci Total Environ. 2011 Aug 1 ;409(17):3215-29. Abstract: In this study, the environmental impacts of large scale bioenergy crops were evaluated using the Soil and Water Assessment Tool (SWAT). Daily pesticide concentration data for a study area consisting of four large watersheds located in Michigan (totaling 53,358km(2)) was estimated over a six year period (2000-2005). Model outputs for atrazine, bromoxynil, glyphosate, metolachlor, pendimethalin, sethoxydim, triflualin, and 2,4-D model output were used to predict the possible long-term implications that large-scale bioenergy crop expansion may have on the bluegill (Lepomismacrochirus) and humans. Threshold toxicity levels were obtained for the bluegill and for human consumption for all pesticides being evaluated through an extensive literature review. Model output was compared to each toxicity level for the suggested exposure time (96-hour for bluegill and 24-hour for humans). The results suggest that traditional intensive row crops such as canola, corn and sorghum may negatively impact aquatic life, and in most cases affect the safe drinking water availability. The continuous corn rotation, the most representative rotation for current agricultural practices for a starch-based ethanol economy, delivers the highest concentrations of glyphosate to the stream. In addition, continuous canola contributed to a concentration of 1.11ppm of trifluralin, a highly toxic herbicide, which is 8.7 times the 96-hour ecotoxicity of bluegills and 21 times the safe drinking water level. Also during the period of study, continuous corn resulted in the impairment of 541,152km of stream. However, there is promise with second-generation lignocellulosic bioenergy crops such as switchgrass, which resulted in a 171,667km reduction in total stream length that exceeds the human threshold criteria, as compared to the base scenario. Results of this study may be useful in determining the suitability of bioenergy crop rotations and aid in decision making regarding the adaption of large-scale bioenergy cropping systems.
89. Battaglin WA, Meyer MT, Kuivila KM, and Dietze JE. “Glyphosate and Its Degradation Product AMPA Occur Frequently and Widely in U.S. Soils, Surface Water, Groundwater, and Precipitation”. Journal of the American Water Resources Association (JAWRA) 2014, 50, 275-290. Abstract: Looking at a wide range of geographical locations, researchers from the US Geological Survey (USGS) analyzed 3732 water and sediment samples and 1081 quality assurance samples collected between 2001 and 2010 from 38 states in the US and the district of Columbia. They found glyphosate in 39.4 % of samples (1470 out of 3732) and its metabolite AMPA (α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) in 55% of samples. Water samples included streams, groundwater, ditches and drains, large rivers, soil water, lakes, ponds and wetlands, precipitation, soil and sediment, and waste water treatment plants.
90. Battaglin, W.A.; Kolpin, D.W.; Scribner, E.A.; Kuivila, K.M.; Sandstrom, M.W. “Glyphosate, other herbicides, and transformation products in midwestern streams”, 2002. J. Am. Water Resour. Assoc2005, 41, 323–332. Abstract: The use of glyphosate has increased rapidly, and there is limited understanding of its environmental fate. The objective of this study was to document the occurrence of glyphosate and the transformation product minomethylphosphonic acid (AMPA) in Midwestern streams and to compare their occurrence with that of more commonly measured herbicides such as acetochlor, atrazine, and metolachlor. Water samples were collected at sites on 51 streams in nine Midwestern states in 2002 during three runoff events: after the application of pre-emergence herbicides, after the application of post-emergence herbicides, and during harvest season. All samples were analyzed for glyphosate and 20 other herbicides using gas chromatography/mass spectrometry or high performance liquid chromatography/mass spectrometry. The frequency of glyphosate and AMPA detection, range of concentrations in runoff samples, and ratios of AMPA to glyphosate concentrations did not vary throughout the growing season as substantially as for other herbicides like atrazine, probably because of different seasonal use patterns. Glyphosate was detected at or above 0.1 μg/l in 35 percent of pre-emergence, 40 percent of post-emergence, and 31 percent of harvest season samples, with a maximum concentration of 8.7 μg/l. AMPA was detected at or above 0.1 μg/l in 53 percent of pre-emergence, 83 percent of post-emergence, and 73 percent of harvest season samples, with a maximum concentration of 3.6 μg/l. Glyphosate was not detected at a concentration at or above the U.S. Environmental Protection Agency’s maximum contamination level (MCL) of 700 μg/l in any sample. Atrazine was detected at or above 0.1 μg/l in 94 percent of pre-emergence, 96 percent of post-emergence, and 57 percent of harvest season samples, with a maximum concentration of 55 μg/l. Atrazine was detected at or above its MCL (3 μg/l) in 57 percent of pre-emergence and 33 percent of post-emergence samples.
91. Sasal, M.C. et al, “Glyphosate loss by runoff and its relationship with phosphorus fertilization”. Journal of Agriculture and Food Chemistry, 2015 May 13;63(18):4444-8. Abstract: The aim of this study was to evaluate the relationship between glyphosate and phosphate fertilizer application and their contribution to surface water runoff contamination. The study was performed in AquicArgiudoll soil (Tezanos Pinto series). Four treatments were assessed on three dates of rainfall simulation after fertilizer and herbicide application. The soluble phosphorus in runoff water was determined by a colorimetric method. For the determination of glyphosate and aminomethylphosphonic acid (AMPA), a method based on fluorenylmethyloxycarbonyl (FMOC) group derivatization, solid phase extraction (SPE) purification, and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was employed. The application of phosphorus fertilizer resulted in an increased loss of glyphosate by runoff after 1 day of application. These results suggest the need for further study to understand the interactions and to determine appropriate application timing with the goal of reducing the pollution risk by runoff.
92. Jain , Sunil, Sharma, Dr. Gunwant, Mathur, Dr. Y.P. “Effects of Pesticides on hormone and enzyme systems of aqua population: a view over Anasagar lake, Ajmer”, IOSR Journal Of Environmental Science, Toxicology And Food Technology (IOSR) Volume 1, Issue 5 (Nov. -Dec. 2012), PP 24-28. Abstract: Water sources are particularly at risk to contamination by pesticides because of the accumulation and distribution of contaminating substances in sediments of rivers, lakes, and ponds. Potential sources of this impact in water bodies include municipal sewage and agricultural runoff (pesticides and herbicides). The enzyme and hormone disrupting capabilities of pesticides and related chemicals are suspected to be some of the factors contributing to the decline of fish, amphibian and reptile populations of water bodies. In most cases the cause are assumed to result from man-made pollutants in the aquatic environment. The lake Anasagar is a perennial, shallow fresh wetland situated in the heart of Ajmercity. It is degraded because of anthropogenic activities including input of detergents, pesticides and other chemicals due to agricultural activities, sewage disposal and human settlement around the lake. A large area of the catchment of Lake Anasagar is being used for agriculture particularly Trapabispinosa as the primary cash crop and vegetable crops such as Cauliflower, Tomato, Cabbage, Brinjal, Okra etc .leading to input of organo chlorine pesticides in the Lake. It has been indicated that many pesticides in the aquatic environment are capable of disrupting the endocrine systems of aqua life. Some pesticides and related chemicals are persistent in the environment and are accumulated in the fatty tissue of organisms and increase in concentration as they move up through the food web. These chemicals are substances that can cause adverse effects by interfering in some way with the body’s hormones or chemical messengers and even disrupting the sexual development of aqua population even at extremely low doses ,reducing the cholinesterase activity of amphibians and reptiles, causing disease and reproductive failure in fish populations. Concern over the decline of amphibians globally has highlighted the importance of using this group as a bioindicator of environmental contamination and climate change.
93. Laetz, CathyA. et al., “The Synergistic Toxicity of Pesticide Mixtures: Implications for Risk Assessment and the Conservation of Endangered, Pacific Salmon” Environmental Health Perspectives 117, No.3 (March 2009) 348-53. Abstract: Mixtures of organophosphate and carbamate pesticides are commonly detected in freshwater habitats that support threatened and endangered species of Pacific salmon (Oncorhynchus sp.). These pesticides inhibit the activity of acetylcholinesterase (AChE) and thus have potential to interfere with behaviors that may be essential for salmon survival. Although the effects of individual anticholin-esterase insecticides on aquatic species have been studied for decades, the neurotoxicity of mixtures is still poorly understood. We assessed whether chemicals in a mixture act in isolation (resulting in additive AChE inhibition) or whether components interact to produce either antagonistic or synergistic toxicity. We measured brain AChE inhibition in juvenile coho salmon (Oncorhynchuskisutch) exposed to sublethal concentrations of the organophosphates diazinon, malathion, and chlorpyrifos, as well as the carbamatescarbaryl and carbofuran. Concentrations of individual chemicals were normalized to their respective median effective concentrations (EC50) and collectively fit to a nonlinear regression. We used this curve to determine whether toxicologic responses to binary mixtures were additive, antagonistic, or synergistic. We observed addition and synergism, with a greater degree of synergism at higher exposure concentrations. Several combinations of organophosphates were lethal at concentrations that were sublethal in single-chemical trials.Single-chemical risk assessments are likely to underestimate the impacts of these insecticides on salmon in river systems where mixtures occur. Moreover, mixtures of pesticides that have been commonly reported in salmon habitats may pose a more important challenge for species recovery than previously anticipated.
94. Birch H., Mikkelson P.S., Jenson J.K and LützhŅft, 2011. “Micropollutants in stormwater runoff and combined sewer overflow in the Copenhagen area, Denmark”. Water Science and Technology64:485-493. Abstract: Stormwater runoff contains a broad range of micropollutants. In Europe a number of these substances are regulated through the Water Framework Directive, which establishes Environmental Quality Standards (EQSs) for surface waters. Knowledge about discharge of these substances through stormwater runoff and combined sewer overflows (CSOs) is essential to ensure compliance with the EQSs. Results from a screening campaign including more than 50 substances at four storm water discharge locations and one CSO in Copenhagen are reported here. Heavy metal concentrations were detected at levels similar to earlier findings, e.g., with copper found at concentrations up to 13 times greater than the Danish standard for surface waters. The concentration of polyaromatic hydrocarbons (PAHs) exceeded the EQSs by factors up to 500 times for storm water and 2,000 times for the CSO. Glyphosate was found in all samples whilst diuron, isoproturon, terbutylazine and MCPA were found only in some of the samples. Diethylhexylphthalate (DEHP) was also found at all five locations in concentrations exceeding the EQS. The results give a valuable background for designing further monitoring programmes focusing on the chemical status of surface waters in urban areas
95. Villeneuve A., Larroudé S &HumbertJF., 2011. “Herbicide contamination of freshwater ecosystems: impact on microbial communities”. In: Pesticides – Formulations, Effects, Fate.Stoytcheva M. (Ed.) pp. 285-312, In Tech Abstract: In France, the “InstitutFranćais de l’Environnement” (French Institute for Environment; now known as the Service de l’Observationet des Statistiques, SOeS) has published data on the contamination of water by pesticides for every year since 1998. Their annual report is based on monitoring 453 pesticides at 2023 sampling points (groundwater and rivers). In 2007, pesticides were detected at almost 91% of the sampling points, but usually at mean annual concentrations of <0.5 μg/L. The highest concentrations were found in regions with intensive agriculture (South-West, Center-North and North of France) and the lowest in regions (South-East and South of Massif Central) characterized by less intensive agriculture or by the presence of large areas of natural environments. The pesticide most often detected in French streams was AMPA (aminomethylphosphonic acid), which is the primary degradation product of glyphosate. There is a lot of data is available about the contamination of freshwater ecosystems by herbicides and also on the direct or indirect impact of these compounds on microbial communities living in these ecosystems. These compounds appear to affect the structure and composition of these communities, and also the metabolism of the microorganisms involved. It is very difficult to evaluate the consequences of such changes on the whole functioning of freshwater ecosystems, but there can be no doubt that it is significantly affected by herbicide contamination, because microbial communities play a key role in these ecosystems. This impact is probably reinforced by the fact that freshwater ecosystems are simultaneously subjected to other selective pressures. For example, herbicide pollution is generally concomitant with pollution by mineral nutrients (phosphorus and nitrogen), which also influence the structure and the functioning of microbial communities.
96. Borggaard, O.K.; and A.L. Gimsing(2008). “Fate of glyphosate in soil and the possibility of leaching to ground and surface waters: A review.” Abstract: The very wide use of glyphosate to control weeds in agricultural, silvicultural and urban areas throughout the world requires that special attention be paid to its possible transport from terrestrial to aquatic environments. The aim of this review is to present and discuss the state of knowledge on sorption, degradation and leachability of glyphosate in soils. Difficulties of drawing clear and unambiguous conclusions because of strong soil dependency and limited conclusive investigations are pointed out. Nevertheless, the risk of ground and surface water pollution by glyphosate seems limited because of sorption onto variable-charge soil minerals, e.g. aluminum and iron oxides, and because of microbial degradation. Although sorption and degradation are affected by many factors that might be expected to affect glyphosate mobility in soils, glyphosate leaching seems mainly determined by soil structure and rainfall. Limited leaching has been observed in non-structured sandy soils, while subsurface leaching to drainage systems was observed in a structured soil with preferential flow in macropores, but only when high rainfall followed glyphosate application. Glyphosate in drainage water runs into surface waters but not necessarily to groundwater because it may be sorbed and degraded in deeper soil layers before reaching the groundwater. Although the transport of glyphosate from land to water environments seems very limited, knowledge about subsurface leaching and surface runoff of glyphosate as well as the importance of this transport as related to ground and surface water quality is scarce. Pest Management Science, 64:441–456, DOI:10.1002/ps.1512.
97. Bergman,Ake,Heinde, Jerrold J., Jobling , Susan, Kidd, Karen A., Zoeller, R. Thomas, “State of the Science of Endocrine Disrupting Chemicals”, Inter-OrganisationProgramme for the Sound Management of Chemicals (IOMC), with The Participating Organisations FAO, ILO, UNDP, UNEP, UNIDO, UNITAR, WHO, World Bank and OECD, 2012, Excerpt: There is a worldwide loss of species or reduced population numbers of amphibians, mammals, birds, reptiles, freshwater and marine fishes and invertebrates. Endocrine Disruptor Chemicals have been shown to negatively affect body systems that are critical for the health and survival of wildlife. The current body burdens of POPs such as PCBs, organochlorine pesticides and methylmercury in some fish-eating birds and marine mammal populations are at levels known to cause effects on breeding and on the immune system some of these populations are threatened or endangered. Legal, technical and ethical constraints to working with wildlife, notably those listed under endangered species legislation, prevent research to investigate chemical causes of population declines in these animals. An increasing number of chemicals to which wildlife are exposed have been shown to interfere with the hormonal and immune systems of wildlife species. Most of these chemicals are not monitored in ecosystems. Exposed wildlife populations are often not monitored either. Experimental animal studies have shown that many chemicals can interfere with the development and function of endocrine systems, leading to effects on behaviour, fecundity, growth, survival and disease resistance. This increases the probability that exposure to EDCs could lead to population-level effects in wildlife.
98. Pleasants J.N. and Oberhauser K.S., 2012.“Milkweed loss in agricultural fields because of herbicide use: effect on the monarch butterfly population”. Insect Conservation and Diversity Volume 6, Issue 2, 135–144, March 2013, Abstract: The size of the Mexican overwintering population of monarch butterflies has decreased over the last decade. Approximately half of these butterflies come from the U.S. Midwest where larvae feed on common milkweed. There has been a large decline in milkweed in agricultural fields in the Midwest over the last decade. This loss is coincident with the increased use of glyphosate herbicide in conjunction with increased planting of genetically modified (GM) glyphosate-tolerant corn (maize) and soybeans (soya). We investigate whether the decline in the size of the overwintering population can be attributed to a decline in monarch production owing to a loss of milkweeds in agricultural fields in the Midwest. We estimate Midwest annual monarch production using data on the number of monarch eggs per milkweed plant for milkweeds in different habitats, the density of milkweeds in different habitats, and the area occupied by those habitats on the landscape. We estimate that there has been a 58% decline in milkweeds on the Midwest landscape and an 81% decline in monarch production in the Midwest from 1999 to 2010. Monarch production in the Midwest each year was positively correlated with the size of the subsequent overwintering population in Mexico. Taken together, these results strongly suggest that a loss of agricultural milkweeds is a major contributor to the decline in the monarch population. The smaller monarch population size that has become the norm will make the species more vulnerable to other conservation threats.
99. Mao, W.; Schuler, M.A.; Berenbaum, M.R. “CYP9Q-mediated detoxification of acaricides in the honey bee (Apismellifera)”. Proc. Natl. Am. Soi. 2011, 108, 12657–12662. Abstract: Although Apismellifera, the western honey bee, has long encountered pesticides when foraging in agricultural fields, for two decades it has encountered pesticides in-hive in the form of acaricides to control Varroa destructor, a devastating parasitic mite. The pyrethroid tau-fluvalinate and the organophosphate coumaphos have been used for Varroa control, with little knowledge of honey bee detoxification mechanisms. Cytochrome P450-mediated detoxification contributes to pyrethroid tolerance in many insects, but specific P450s responsible for pesticide detoxification in honey bees (indeed, in any hymenopteran pollinator) have not been defined. We expressed and assayed CYP3 clan midgut P450s and demonstrated that CYP9Q1, CYP9Q2, and CYP9Q3 metabolize tau-fluvalinate to a form suitable for further cleavage by the carboxylesterases that also contribute to tau-fluvalinate tolerance. These in vitro assays indicated that all of the three CYP9Q enzymes also detoxify coumaphos. Molecular models demonstrate that coumaphos and tau-fluvalinate fit into the same catalytic pocket, providing a possible explanation for the synergism observed between these two compounds. Induction of CYP9Q2 and CYP9Q3 transcripts by honey extracts suggested that diet-derived phytochemicals may be natural substrates and heterologous expression of CYP9Q3 confirmed activity against quercetin, a flavonoid ubiquitous in honey. Up-regulation by honey constituents suggests that diet may influence the ability of honey bees to detoxify pesticides. Quantitative RT-PCR assays demonstrated that tau-fluvalinate enhances CYP9Q3 transcripts, whereas the pyrethroidbifenthrin enhances CYP9Q1 and CYP9Q2 transcripts and represses CYP9Q3 transcripts. The independent regulation of these P450s can be useful for monitoring and differentiating between pesticide exposures in-hive and in agricultural fields.
100. Foulk, K.E.; Reeves, C. Identifying the role of glyphosate-containing herbicides on honeybee mortality rates and colony collapse disorder. In Proceedings of Junior Science, Engineering, and Humanities Symposium, Camdenton, MO, USA, 2009; 2–23. Abstract: Samples of bees and honey of hives from colonies that have and have not been affected by Colony Collapse Disorder were collected from beekeepers located throughout the State of Missouri. These samples were analyzed using a liquid-chromatography mass spectrometer to detect traces of glyphosate and its metabolite aminomethylphosphonic acid. Nine hives, built according to a design described by Dr. Marion Ellis of the University of Nebraska-Lincoln, were filled with bees, and the nine hives were divided into three groups of three hives each. Each group was fed one of the following: sugar water, sugar water with glyphosate, or sugar water with Roundup QuikPro®. The amount of glyphosate added was based on a study performed by Blackburn and Boutin (2003). All hives were kept in a dark, humidified room located in the science department in Camdenton High School, Camdenton, MO. Data on mortality were collected and subjected to a two-way Analysis of Variance (ANOVA). Samples of bees and comb from each experimental hive were analyzed using a liquid-chromatography mass spectrometer (USGS Water Research Facility, Lawrence, KS). The results of this study indicate that the mortality rate of bees that were fed glyphosate-supplemented diets, both pure glyphosate and Roundup QuikPro®, were significantly greater than that of hives without glyphosate supplemented diets (p < 0.001).
101.Balbuena, M. S., L. Tison, M-L.Hahn, U. Greggers, R. Menzel, and W. M. Farina (2015).”Effects of sublethal doses of glyphosate on honeybee navigation.” Abstract: Glyphosate (GLY) is a herbicide that is widely used in agriculture for weed control. Although reports about the impact of GLY in snails, crustaceans and amphibians exist, few studies have investigated its sublethal effects in non-target organisms such as the honeybee Apis mellifera, the main pollen vector in commercial crops. Here, we tested whether exposure to three sublethal concentrations of GLY (2.5, 5 and 10 mg l(-1): corresponding to 0.125, 0.250 and 0.500 μg per animal) affects the homeward flight path of honeybees in an open field. We performed an experiment in which forager honeybees were trained to an artificial feeder, and then captured, fed with sugar solution containing traces of GLY and released from a novel site either once or twice. Their homeward trajectories were tracked using harmonic radar technology. We found that honeybees that had been fed with solution containing 10 mg l(-1) GLY spent more time performing homeward flights than control bees or bees treated with lower concentrations. They also performed more indirect homing flights. Moreover, the proportion of direct homeward flights performed after a second release from the same site increased in control bees but not in treated bees. These results suggest that, in honeybees, exposure to levels of GLY commonly found in agricultural settings impairs the cognitive capacities needed to retrieve and integrate spatial information for a successful return to the hive. Therefore, honeybee navigation is affected by ingesting traces of the most widely used herbicide worldwide, with potential long-term negative consequences for colony foraging success. Journal of Experimental Biology, 218:2799-2805.
102.Herbert, L.T., D.E. Vázquez, A. Arenas and W.M. Farina (2014). “Effects of field-realistic doses of glyphosate on honeybee appetitive behavior.” Abstract: Glyphosate (GLY) is a broad-spectrum herbicide used for weed control. The sub-lethal impact of GLY on non-target organisms such as insect pollinators has not yet been evaluated. Apis mellifera is the main pollinator in agricultural environments and is a well-known model for behavioural research. Honeybees are also accurate biosensors of environmental pollutants and their appetitive behavioural response is a suitable tool with which to test sub-lethal effects of agrochemicals. We studied the effects of field-realistic doses of GLY on honeybees exposed chronically or acutely to the herbicide. We focused on sucrose sensitivity, elemental and non-elemental associative olfactory conditioning of the proboscis extension response (PER), and foraging-related behaviour. We found a reduced sensitivity to sucrose and learning performance for the groups chronically exposed to GLY concentrations within the range of recommended doses. When olfactory PER conditioning was performed with sucrose reward with the same GLY concentrations (acute exposure), elemental learning and short-term memory retention decreased significantly compared with controls. Non-elemental associative learning was also impaired by an acute exposure to GLY traces. Altogether, these results imply that GLY at concentrations found in agro-ecosystems as a result of standard spraying can reduce sensitivity to nectar reward and impair associative learning in honeybees. However, no effect on foraging-related behaviour was found. Therefore, we speculate that successful forager bees could become a source of constant inflow of nectar with GLY traces that could then be distributed among nestmates, stored in the hive and have long-term negative consequences on colony performance. Journal of Experimental Biology, 217:3457-3464.
103.Annetta, R., H. R. Habibib and A. Hontela(2014). “Impact of glyphosate and glyphosate-based herbicides on the freshwater environment.” Journal of Applied Toxicology 34:458-479. Abstract: Glyphosate [N-(phosphonomethyl) glycine] is a broad spectrum, post emergent herbicide and is among the most widely used agricultural chemicals globally. Initially developed to control the growth of weed species in agriculture, this herbicide also plays an important role in both modern silviculture and domestic weed control. The creation of glyphosate tolerant crop species has significantly increased the demand and use of this herbicide and has also increased the risk of exposure to non-target species. Commercially available glyphosate-based herbicides are comprised of multiple, often proprietary, constituents, each with a unique level of toxicity. Surfactants used to increase herbicide efficacy have been identified in some studies as the chemicals responsible for toxicity of glyphosate-based herbicides to non-target species, yet they are often difficult to chemically identify. Most glyphosate-based herbicides are not approved for use in the aquatic environment; however, measurable quantities of the active ingredient and surfactants are detected in surface waters, giving them the potential to alter the physiology of aquatic organisms. Acute toxicity is highly species dependent across all taxa, with toxicity depending on the timing, magnitude, and route of exposure. The toxicity of glyphosate to amphibians has been a major focus of recent research, which has suggested increased sensitivity compared with other vertebrates due to their life history traits and reliance on both the aquatic and terrestrial environments. This review is designed to update previous reviews of glyphosate-based herbicide toxicity, with a focus on recent studies of the aquatic toxicity of this class of chemicals.
104.Arunakumara, K. I. U., B. C. Walpola and M. Yoon (2013). “Metabolism and degradation of glyphosate in aquatic cyanobacteria: A review.” African Journal of Microbiology Research, 7(32):4084-4090. Abstract: Use of glyphosate (N-phosphonomethylglycine), a broad-spectrum, non-selective, post-emergence herbicide has been increased steadily with the introduction of genetically modified glyphosate-resistant crops. Increased reliance on herbicides for suppressing weeds and aggressive marketing have also contributed substantially to rising demand for glyphosate. Degradation of glyphosate was basically done by soil microorganisms; however, once the herbicide reached to the aquatic systems, cyanobacterial strains were reported to be involved in the process of biodegradation. Upon glyphosate exposure, a remarkable tolerance was reported in many strains, where cell proliferation was found to be completely unaffected by the herbicide at the concentration of micromolar to millimolar range. However, the mechanism through which cyanobacteria exhibit the tolerance seemed to be widely varied and species-dependent. Carrier-independent uptake of glyphosate has been suggested as the resistance mechanism at micromolar level concentrations. Presence of resistant form of the target enzyme EPSP (5-enolpyruvylshikimate-3-phosphate) and the ability of some strains to metabolize glyphosate have also been reported to be responsible for the tolerance. A remarkable ability to degrade glyphosate has been identified from some cyanobacterial strains such as Spirulina spp. where degradative pathway was however reported to be different from those exhibited in other bacteria. Exploitation of cyanobacteria in biological treatments of waste water contaminated with glyphosate has not yet been reported, mainly due to lack of research evidence on as to how cyanobacteria deal with biodegradation of glyphosate under field conditions.
105.Vera M.S. et al (2012). “Direct and indirect effects of the glyphosate formulation GlifosatoAtanor® on freshwater microbial communities”.EcotoxicologyVol 21 pp 1805-16. Abstract: Glyphosate-based formulations are among the most widely used herbicides in the world. The effect of the formulation GlifosatoAtanor(®) on freshwater microbial communities (phytoplankton, bacterioplankton, periphyton and zooplankton) was assessed through a manipulative experiment using six small outdoor microcosms of small volume. Three of the microcosms were added with 3.5 mg l(-1) of glyphosate whereas the other three were left as controls without the herbicide. The treated microcosms showed a significant increase in total phosphorus, not fully explained by the glyphosate present in the GlifosatoAtanor(®). Therefore, part of the phosphorus should have come from the surfactants of the formulation. The results showed significant direct and indirect effects of GlifosatoAtanor(®) on the microbial communities. A single application of the herbicide caused a fast increase both in the abundance of bacterioplankton and planktonic picocyanobacteria and in chlorophyll a concentration in the water column. Although metabolic alterations related to oxidative stress were induced in the periphyton community, the herbicide favored its development, with a large contribution of filamentous algae typical of nutrient-rich systems, with shallow and calm waters. An indirect effect of the herbicide on the zooplankton was observed due to the increase in the abundance of the rotifer Lecane spp. as a consequence of the improved food availability given by picocyanobacteria and bacteria. The formulation affected directly a fraction of copepods as a target. It was concluded that the GlifosatoAtanor(®) accelerates the deterioration of the water quality, especially when considering small-volume water systems.
106.Mensah, P.K. W.J. Muller, C.G. Palmer (2011). “Acute toxicity of Roundup herbicide to three life stages of the freshwater shrimp Caridinanilotica (Decapoda: Atyidae)”. Abstract: Glyphosate based herbicides, including Roundup®, are frequently used in the chemical control of weeds and invading alien plant species in South Africa. These herbicides ultimately get into water courses directly or indirectly through processes such as drifting, leaching, surface runoff and foliar spray of aquatic nuisance plants. Despite their widespread use, no water quality guideline exists to protect indigenous South African freshwater organisms from the toxic effects of these herbicides. The toxicity of the herbicide Roundup® was assessed using three different life stages of the freshwater shrimp Caridina nilotica, a prevalent species in South African freshwater ecosystems. Neonate (<7 days post hatching (dph)), juvenile (>7 dph and <20 dph) and adult (>40 dph) shrimps were exposed to varying concentrations (1.5–50 mg/L acid equivalence (a.e.)) of the herbicide in 48 and 96 h acute toxicity tests in order to determine the most sensitive life-stage. The results showed neonates to be more sensitive to Roundup® than both juveniles and adults with mean 96 h LC50 values of 2.5, 7.0 and 25.3 mg/L a.e. respectively. The estimated 96 h LC50 of neonates is much lower than the application rate (20–30 mg/L a.e.), although the application’s impact will depend on the dilution rate of the applied concentration in the environment. All three life-stages of unexposed animals exhibited active and coordinated movement but exposed shrimps were erratic and slow in their movements, with neonates showing most of these behavioral irregularities. This study shows that low levels of the herbicide Roundup® may adversely affect C. nilotica health and survival. Thus, the herbicide should be carefully managed to minimize any negative impact on non-target freshwater organisms. Physics and chemistry of the Earth, 36:905-909.
107. Perez, G.L., A. Torremorell, H. Mugni, P. Rodriguez, M.S. Vera, M. Nascimento, L. Allende, J. Bustingorry, R. Escaray, M. Ferraro (2007). “Effects of the herbicide Roundup on freshwater microbial communities: A mesocosm study” . Abstract: The impact of the widely used herbicide glyphosate has been mainly studied in terrestrial weed control, laboratory bioassays, and field studies focusing on invertebrates, amphibians, and fishes. Despite the importance of phytoplankton and periphyton communities at the base of the aquatic food webs, fewer studies have investigated the effects of glyphosate on freshwater microbial assemblages. We assessed the effect of the commercial formulation Roundup using artificial earthen mesocosms. The herbicide was added at three doses: a control (without Roundup) and two treatments of 6 and 12 mg/L of the active ingredient (glyphosate). Estimates of the dissipation rate (k) were similar in the two treatments (half-lives of 5.77 and 7.37 d, respectively). The only two physicochemical parameters showing statistically significant differences between treatments and controls were the downward vertical spectral attenuation coefficient kd(λ), where λ is wavelength, and total phosphorus concentration (TP). At the end of the experiment, the treated mesocosms showed a significant increase in the ratio kd(490 nm)/kd(550 nm) and an eightfold increase in TP. Roundup affected the structure of phytoplankton and periphyton assemblages. Total micro- and nanophytoplankton decreased in abundance in treated mesocosms. In contrast, the abundance of picocyanobacteria increased by a factor of about 40. Primary production also increased in treated mesocosms (roughly by a factor of two). Similar patterns were observed in the periphytic assemblages, which showed an increased proportion of dead : live individuals and increased abundances of cyanobacteria (about 4.5-fold). Interestingly, the observed changes in the microbial assemblages were captured by the analysis of the pigment composition of the phytoplankton, the phytoplankton absorption spectra, and the analysis of the optical properties of the water. The observed changes in the structure of the microbial assemblages are more consistent with a direct toxicological effect of glyphosate rather than an indirect effect mediated by phosphorus enrichment.. Ecological Applications, 17(8): 2310–2322.
108.Romero, D.M., M.C.R. de Molina, and A.B. Juarez (2011). “Oxidative stress induced by a commercial glyphosate formulation in a tolerant strain of Chlorella kessleri (algae)”. Abstract: We studied the toxicity of a glyphosate formulation and provide evidence of metabolic alterations due to oxidative stress caused in a Chlorella kessleri tolerant strain by exposure to the herbicide. After 96 h of exposure to increasing concentrations of the herbicide (0-70 mg L(-1)) with alkylaryl polyglycol ether surfactant, growth was inhibited (EC50-96 h 55.62 mg L(-1)). Glyphosate increased protein and malondialdehyde content which was significantly higher than in the control at 50-70 mg L(-1). Superoxide dismutase and catalase activities and reduced glutathione levels increased in a concentration-dependant manner. Morphological studies showed increases in vacuolisation and in cell and sporangia sizes. The glyphosate formulation studied has a cytotoxic effect on C. kessleri through a mechanism that would involve the induction of oxidative stress. Upon glyphosate exposure, oxidative stress parameters such as SOD and CAT activities and MDA level could be more sensitive biomarkers than usually tested growth parameters in C. kessleri Ecotoxicology and Environmental Safety, 74(4):741–747.
4. The Need for New Regulatory Action
109.Colburn, Theo, “A case for revisiting the safety of pesticides: a closer look at neurodevelopment” Environ Health Persp. 2006 Jan;114(1):10-17 Abstract: The quality and quantity of the data about the risk posed to humans by individual pesticides vary considerably. Unlike obvious birth defects, most developmental effects cannot be seen at birth or even later in life. Instead, brain and nervous system disturbances are expressed in terms of how an individual behaves and functions, which can vary considerably from birth through adulthood. In this article I challenge the protective value of current pesticide risk assessment strategies in light of the vast numbers of pesticides on the market and the vast number of possible target tissues and end points that often differ depending upon timing of exposure. Using the insecticide chlorpyrifos as a model, I reinforce the need for a new approach to determine the safety of all pesticide classes. Because of the uncertainty that will continue to exist about the safety of pesticides, it is apparent that a new regulatory approach to protect human health is needed.