Pesticides applied to crops and amyotrophic lateral sclerosis risk in the U.S.

BACKGROUND: Environmental exposures are implicated in the etiology of amyotrophic lateral sclerosis (ALS). Application of insecticides, herbicides, and fungicides with neurotoxic properties to crops is permitted in the U.S., however reporting of the quantities is government mandated. OBJECTIVE: To identify pesticides that may be associated with ALS etiology for future study. METHODS: We geospatially estimated exposure to crop-applied pesticides as risk factors for ALS in a large de-identified medical claims database, the SYMPHONY Integrated Dataverse®. We extracted residence at diagnosis of ∼26,000 nationally distributed ALS patients, and matched non-ALS controls. We mapped county-level U.S. Geological Survey data on applications of 423 pesticides to estimate local residential exposure. We randomly broke the SYMPHONY dataset into two groups to form independent discovery and validation cohorts, then confirmed top hits using residential history information from a study of NH, VT, and OH. RESULTS: Pesticides with the largest positive statistically significant associations in both the discovery and the validation studies and evidence of neurotoxicity in the literature were the herbicides 2,4-D (OR 1.25 95 % CI 1.17-1.34) and glyphosate (OR 1.29 95 %CI 1.19-1.39), and the insecticides carbaryl (OR 1.32 95 %CI 1.23-1.42) and chlorpyrifos (OR 1.25 95 %CI 1.17-1.33). SIGNIFICANCE: Our geospatial analysis results support potential neurotoxic pesticide exposures as risk factors for sporadic ALS. Focused studies to assess these identified potential relationships are warranted.

Epigenetic effects of insecticides on early differentiation of mouse embryonic stem cells.

Increasing evidence indicates that many insecticides produce significant epigenetic changes during embryogenesis, leading to developmental toxicities. However, the effects of insecticides on DNA methylation status during early development have not been well studied. We developed a novel nuclear phenotypic approach using mouse embryonic stem cells harboring enhanced green fluorescent protein fused with methyl CpG-binding protein to evaluate global DNA methylation changes via high-content imaging analysis. Exposure to imidacloprid, carbaryl, and o,p'-DDT increased the fluorescent intensity of granules in the nuclei, indicating global DNA methylating effects. However, DNA methylation profiling in cell-cycle-related genes, such as Cdkn2a, Dapk1, Cdh1, Mlh1, Timp3, and Rarb, decreased in imidacloprid treatments, suggesting the potential influence of DNA methylation patterns on cell differentiation. We developed a rapid method for evaluating global DNA methylation and used this approach to show that insecticides pose risks of developmental toxicity through DNA methylation.

Glutathione reductase and catalase as potential biomarkers for synergistic intoxication of pesticides in fish.

Synergy occurs when chemicals give pronounced effect on combination in contrast to their individual effect. The objective of this study was to investigate the synergistic effect of pesticides carbaryl (C) and methyl parathion (MP) on oxidative stress biomarkers viz catalase (CAT), glutathione reductase (GSSG-R) including different enzymes like lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and acetyl cholinesterase (AChE) in different tissues of carps Catla catla. Fishes were exposed to 6.25 mg/L of MP and 2.3 mg/L of C in mixture (one-third of LC50 value). CAT and GSSG-R were studied in gills, brain, liver and muscle of carp were found to be elevated significantly (p < 0.005). LDH activity increased significantly (p < 0.005) in synergistic group, there was a seven-fold (748%) increase in LDH activity in muscle compared to individual studies with same pesticides. Contrary to LDH, sudden decrease in SDH activity was accounted. Significant (p < 0.005) decrease in AChE activity after initial 24 h was remarkable addressing to the shift in neurotransmission pathway in organism. Significant increase was observed in activity of CAT and GSSG-R in all tissues compared to control fishes in individual as well as synergistic (MP + C) group suggesting that CAT and GSSG-R can be a potential biomarker of oxidative stress when studied in combination.

The histopathological, cytopathological and ultrastructural effects of carbaryl on gills of Oreochromis niloticus (Linnaeus, 1758).

Ultrastructural and histopathological reponses in the organs of living organisms are important and useful tools to determine the health condition and the effects of pollutants, such as pesticides, on the organisms. The aim of this study is to determine possible histopathological, cytopathological and ultrastructural alterations in gills of Oreochromis niloticus individuals exposed to 850 µg/L carbaryl standart at 7th, 14th and 21st days with light and electron microscopes. The fish were exposed to carbaryl for 21 days and the histopatological, ultrastructural and cytopathological alterations occuring in the gill tissues of organisms were determined by light, Scanning and Transmission Electron Microscopes (SEM and TEM). At the end of the study, it was observed that carbaryl caused both histopathological and cytopathological changes in the gills of O. niloticus. It has been determined that the most of the pathological changes in the exposed organisms are the metabolic defence reactions.

An amphibian with a contracting range is not more vulnerable to pesticides in outdoor experimental communities than common species.

In areas with heavy pesticide use, it is easy to attribute population declines to environmental contamination. The Blanchard's cricket frogs (Acris blanchardi) is an amphibian experiencing declines and range contractions across its distribution in the Midwest Corn Belt (USA). Experimental studies suggest that cricket frogs are sensitive to pesticides, but there are few studies examining this species' susceptibility to contaminants in realistic environments or comparing relative impacts with other anuran species. I reared 3 summer breeding anurans in outdoor mesocosms posthatching through metamorphosis to examine the effects of 2 insecticides (imidacloprid and carbaryl) and 1 herbicide (glyphosate with polyoxyethylene tallow amine) on larval development and metamorphosis. Cricket frogs were positively affected by insecticide exposure, likely a result of changes in the food web that increased food abundance. However, metamorphosis of green frogs (Lithobates clamitans) and gray tree frogs (Hyla chrysoscelis) appeared unaffected by pesticide exposure. The results of the present study suggest that the impacts of pesticides alone are unlikely to have population-level impacts for the anurans examined. Environ Toxicol Chem 2018;37:2699-2704. © 2018 SETAC.

Histopathological analysis of carbaryl-induced toxicity in the spleen of Levantine frog, Pelophylax bedriagae (Anura: Ranidae).

This study describes the histopathological effects of carbaryl in the spleen of Levantine frog, Pelophylax bedriage. Due to its primary role in immune system, it is important to research the toxic effects of pesticides, which play an important role in environmental pollution, on spleen. To that end, adult frogs were exposed to carbaryl for 96 h. Experimental groups contained low dose, (0.05 mg/g), medium dose (0.1 mg/g) and high dose (0.2 mg/g). After following exposure to carbaryl, the frogs were euthanised and dissected. In low-dose group, there were no important changes in spleen tissue. In medium-dose group, prominence in haemorrhage just below the capsule and an increase in the melanomacrophage number were determined. In high-dose group, in addition to increases in the melanomacrophages, separations in capsule, haemorrhage below capsule and within splenic tissue, sinusoidal enlargement, congestion in dilated sinusoid, hypertrophic plasma cells and fibrosis were determined as important histological lesions in exposed frogs. This study clearly showed that carbaryl caused important histopathological damages in splenic tissue of Pelophylax bedriagae. In view of these findings, it can be said that this insecticide has the capacity to disrupt spleen' functions.

Long term exposure to low dose neurotoxic pesticides affects hatching, viability and cholinesterase activity of Artemia sp.

The brine shrimp Artemia was used as a model organism to test toxicity of several neuroactive pesticides (chlorpyrifos (CLP), chlorpyrifos oxon (CLP ox), diazinon (DZN), carbaryl (CBR)) following exposure to far below than lethal doses. Cysts were exposed to the pesticides in order to test a scenario similar to actual coastal environment contamination, by analyzing different responses. Cysts were rehydrated in water containing the pesticides at concentrations ranging from 10-11 to 10-5 M, for 72, 96 and 192 h, respectively. For these exposure times, morpho-functional and biochemical parameters, such as hatching speed and viability were investigated in the larvae together with cholinesterase (ChE) activity quantification and histochemical localization. Finally, ChE inhibition was also compared with conventional selective ChE inhibitors. Results showed that CLP ox and CBR caused a significant dose-dependent decrease in hatching speed, followed by high percentages of larval death, while CLP and DZN were responsible for irregular hatching patterns. In addition, the pesticides mostly caused larval death some days post-hatching, whereas this effect was negligible for the specific ChE inhibitors, suggesting that part of pesticide toxicity may be due to molecules other than the primary target. ChE activity was observed in the protocerebrum lobes, linked to the development of pair eyes. Such activity was inhibited in larvae exposed to all pesticides. When compared to conventional selective inhibitors of ChE activities, this inhibition demonstrated that the selected pesticides mainly affect acetylcholinesterase and, to a lesser extent, pseudocholinesterases. In conclusion, the brine shrimp is a good model to test the environmental toxicity of long term exposure to cholinergic pesticides, since changes in hatching speed, viability and ChE activity were observed.

Effect of pesticides on microbial communities in container aquatic habitats.

Container aquatic habitats support a specialized community of macroinvertebrates (e.g. mosquitoes) that feed on microbial communities associated with decaying organic matter. These aquatic habitats are often embedded within and around agricultural lands and are frequently exposed to pesticides. We used a microcosm approach to examine the single and combined effects of two herbicides (atrazine, glyphosate), and three insecticides (malathion, carbaryl, permethrin) on microbial communities of container aquatic habitats. MiSeq sequencing of the V4 region of both bacterial and archaeal 16S rRNA gene was used to characterize the microbial communities of indoor microcosms that were either exposed to each pesticide alone, a mix of herbicides, a mix of insecticides, or a mix of all five insecticides. Individual insecticides but not herbicides reduced the microbial diversity and richness and two insecticides, carbaryl and permethrin, also altered the microbial community structure. A mixture of herbicides had no effect on microbial diversity or structure but a mixture of insecticides or all five pesticides reduced microbial diversity and altered the community structure. These findings suggest that exposure of aquatic ecosystems to individual pesticides or their mixtures can disrupt aquatic microbial communities and there is need to decipher how these changes affect resident macroinvertebrate communities.

Aged garlic extract ameliorates immunotoxicity, hematotoxicity and impaired burn-healing in malathion- and carbaryl-treated male albino rats.

Malathion and carbaryl are the most widely used organophosphate and carbamate insecticides, respectively, especially in developing countries; they pose a potential health hazard for both humans and animals. Here, we evaluated the protective effects of an odorless (free from allicin) Kyolic aged garlic extract (AGE, containing 0.1% S-allylcysteine; 200 mg/kg body weight) on the toxicity induced by 0.1 LD50 of malathion (89.5 mg/kg body weight) and/or carbaryl (33.9 mg/kg body weight) in male Wistar rats. Doses were orally administered to animals for four consecutive weeks. The present study showed that AGE completely modulated most adverse effects induced by malathion and/or carbaryl in rats including the normocytic normochromic anemia, immunosuppression, and the delay in the skin-burning healing process through normalizing the count of blood cells (erythrocytes, leucocytes and platelets), hemoglobin content, hematocrit value, blood glucose-6-phosphodehydrogenase activity, weights and cellularity of lymphoid organs, serum γ-globulin concentration, and the delayed type of hypersensitivity response to the control values, and accelerating the inflammatory and proliferative phases of burn-healing. In addition, AGE completely modulated the decrease in serum reduced glutathione (GSH) concentration and the increase in clotting time in malathion alone and carbaryl alone treated rats. Moreover, AGE induced a significant increase (P < 0.001) in serum GSH concentration (above the normal value) and accelerating burn-healing process in healthy rats. In conclusion, AGE was effective in modulating most adverse effects induced in rats by malathion and carbaryl, and hence may be useful as a dietary adjunct for alleviating the toxicity in highly vulnerable people to insecticides intoxication. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 789-798, 2017.

Reciprocal effects of pesticides and pathogens on amphibian hosts: The importance of exposure order and timing.

Ecological communities are increasingly exposed to natural and anthropogenic stressors. While the effects of individual stressors have been broadly investigated, there is growing evidence that multiple stressors are frequently encountered underscoring the need to examine interactive effects. Pesticides and infectious diseases are two common stressors that regularly occur together in nature. Given the documented lethal and sublethal effects of each stressor on individuals, there is the potential for interactive effects that alter disease outcomes and pesticide toxicity. Using larval wood frogs (Lithobates sylvaticus), we examined the reciprocal interaction between insecticides (carbaryl and thiamethoxam) and the viral pathogen ranavirus by testing whether: (1) prior ranavirus infection influences pesticide toxicity and (2) sublethal pesticide exposure increases susceptibility to and transmission of ranavirus. We found that prior infection with ranavirus increased pesticide toxicity; median lethal concentration (LC50) estimates were reduced by 72 and 55% for carbaryl and thiamethoxam, respectively. Importantly, LC50 estimates were reduced to concentrations found in natural systems. This is the first demonstration that an infection can alter pesticide toxicity. We also found that prior pesticide exposure exacerbated disease-induced mortality by increasing mortality rates, but effects on infection prevalence and transmission of the pathogen were minimal. Collectively, our results underscore the importance of incorporating complexity (i.e. order and timing of exposures) into research examining the interactions between natural and anthropogenic stressors. Given the environmental heterogeneity present in nature, such research will provide a more comprehensive understanding of how stressors affect wildlife.

Molecular effects of 1-naphthyl-methylcarbamate and solar radiation exposures on human melanocytes.

Carbaryl (1-naphthyl-methylcarbamate), a broad-spectrum insecticide, has recently been associated with the development of cutaneous melanoma in an epidemiological cohort study with U.S. farm workers also exposed to ultraviolet radiation, the main etiologic factor for skin carcinogenesis. We hypothesized that carbaryl exposure may increase deleterious effects of UV solar radiation on skin melanocytes. This study aimed to characterize human melanocytes after individual or combined exposure to carbaryl (100µM) and solar radiation (375mJ/cm2). In a microarray analysis, carbaryl, but not solar radiation, induced an oxidative stress response, evidenced by the upregulation of antioxidant genes, such as Hemeoxygenase-1 (HMOX1), and downregulation of Microphtalmia-associated Transcription Factor (MITF), the main regulator of melanocytic activity; results were confirmed by qRT-PCR. Carbaryl and solar radiation induced a gene response suggestive of DNA damage and cell cycle alteration. The expression of CDKN1A, BRCA1/2 and MDM2 genes was notably more intense in the combined treatment group, in a synergistic manner. Flow cytometry assays demonstrated S-phase cell cycle arrest, reduced apoptosis levels and faster induction of cyclobutane pyrimidine dimers (CPD) lesions in carbaryl treated groups. Our data suggests that carbaryl is genotoxic to human melanocytes, especially when associated with solar radiation.

Long term effects of carbaryl exposure on antiviral immune responses in Xenopus laevis.

Water pollutants associated with agriculture may contribute to the increased prevalence of infectious diseases caused by ranaviruses. We have established the amphibian Xenopus laevis and the ranavirus Frog Virus 3 (FV3) as a reliable experimental platform for evaluating the effects of common waterborne pollutants, such as the insecticide carbaryl. Following 3 weeks of exposure to 10 ppb carbaryl, X. laevis tadpoles exhibited a marked increase in mortality and accelerated development. Exposure at lower concentrations (0.1 and 1.0 ppb) was not toxic, but it impaired tadpole innate antiviral immune responses, as evidenced by significantly decreased TNF-α, IL-1ß, IFN-I, and IFN-III gene expression. The defect in IFN-I and IL-1ß gene expression levels persisted after metamorphosis in froglets, whereas only IFN-I gene expression in response to FV3 was attenuated when carbaryl exposure was performed at the adult stage. These findings suggest that the agriculture-associated carbaryl exposure at low but ecologically-relevant concentrations has the potential to induce long term alterations in host-pathogen interactions and antiviral immunity.

Developmental neurotoxic effects of two pesticides: Behavior and biomolecular studies on chlorpyrifos and carbaryl.

In recent times, an increased occurrence of neurodevelopmental disorders, such as neurodevelopmental delays and cognitive abnormalities has been recognized. Exposure to pesticides has been suspected to be a possible cause of these disorders, as these compounds target the nervous system of pests. Due to the similarities of brain development and composition, these pesticides may also be neurotoxic to humans. We studied two different pesticides, chlorpyrifos and carbaryl, which specifically inhibit acetylcholinesterase (AChE) in the nervous system. The aim of the study was to investigate if the pesticides can induce neurotoxic effects, when exposure occurs during a period of rapid brain growth and maturation. The results from the present study show that both compounds can affect protein levels in the developing brain and induce persistent adult behavior and cognitive impairments, in mice neonatally exposed to a single oral dose of chlorpyrifos (0.1, 1.0 or 5mg/kg body weight) or carbaryl (0.5, 5.0 or 20.0mg/kg body weight) on postnatal day 10. The results also indicate that the developmental neurotoxic effects induced are not related to the classical mechanism of acute cholinergic hyperstimulation, as the AChE inhibition level (8-12%) remained below the threshold for causing systemic toxicity. The neurotoxic effects are more likely caused by a disturbed neurodevelopment, as similar behavioral neurotoxic effects have been reported in studies with pesticides such as organochlorines, organophosphates, pyrethroids and POPs, when exposed during a critical window of neonatal brain development.

Responses of wheat (Triticum aestivum) and turnip (Brassica rapa) to the combined exposure of carbaryl and ultraviolet radiation.

The increase of ultraviolet (UV) radiation reaching the Earth's surface as a result of increased ozone layer depletion has affected crop production systems and, in combination with pesticides used in agricultural activities, can lead to greater risks to the environment. The impact of UV radiation and carbaryl singly and in combination on Triticum aestivum (wheat) and Brassica rapa (turnip) was studied. The combined exposure was analyzed using the MixTox tool and was based on the conceptual model of independent action, where possible deviations to synergism or antagonism and dose-ratio or dose-level response pattern were also considered. Compared with the control, carbaryl and UV radiation individually led to reductions in growth, fresh and dry weight, and water content for both species. Combined treatment of UV and carbaryl was more deleterious compared with single exposure. For T. aestivum length, no interaction between the 2 stressors was found (independent action), and a dose-level deviation was the best description for the weight parameters. For B. rapa, dose-ratio deviations from the conceptual model were found when length and dry weight were analyzed, and a higher than expected effect on the fresh weight (synergism) occurred with combined exposure.

Highly selective biomarkers for pesticides developed in Eisenia fetida using SELDI-TOF MS.

The repeated use of pesticides, and their subsequent residues, has contributed to severe adverse effects on the environment, including risks to human health. Therefore, it is important to assess the quality of the environment to ensure it remains free from pesticide residues. The six pesticides tested in this study showed high mortality on Eisenia fetida with LC50 values ranging from 7.7 to 37.9 g L(-1). The strongest lethal effect resulted from the organochlorine insecticide endosulfan (LC50=7.7 g L(-1)). Following exposure to the carbamate pesticides, acetylcholinesterase activity in E. fetida decreased dramatically in comparison to the control. Carboxylesterase activity was only lowered in E. fetida exposed to propoxur, when compared to the control. The remaining five pesticides had no significant effect on carboxylesterase activity in E. fetida. In order to discover pesticide-specific biomarkers with differentially expressed proteins after exposure to pesticides, protein patterns of pesticide-treated E. fetida were analyzed using SELDI-TOF MS with Q10 ProteinChips. Protein patterns were compared with their intensities at the same mass-to-charge ratios (m/z). All 42 peaks had intensities with associated p-values less than 0.089, and 40 of these peaks had associated p-values of 0.05. Using SELDI-TOF MS technology, selective biomarkers for the six pesticides tested were found in E. fetida; four proteins with 5425, 5697, 9523, and 9868 m/z were consistently observed in the earthworms following exposure to the carbamates.

Histopathologic changes in liver and kidney tissues induced by carbaryl in Bufotes variabilis (Anura: Bufonidae).

The purpose of this work was to investigate for the first time histopathologic effects of carbaryl in liver and kidney tissues of Bufotes variabilis. After 96h following exposure to carbaryl (low dose: 0.05, medium dose: 0.1 and high dose: 0.2mg/g), the toads were euthanized and dissected. In liver tissue, vacuolization in hepatocytes, necrosis, mononuclear cell infiltration, an increase in melanomacrophage number, enlargement of sinusoids, hemorrhage and congestion were determined in exposed toads. In kidney tissue, mononuclear cell infiltration, hypertrophied Bowman's capsule cells, deformation, vacuolization, karyolysis and necrosis of renal tubule epithelium, brush border destruction, glomerular shrinkage, hemorrhage and fibrosis were observed in carbaryl-treated groups. According to this investigation, carbaryl caused histopathologic damages in liver and kidney tissues of B. variabilis.

Assessment of interactive effects of elevated salinity and three pesticides on life history and behavior of southern toad (Anaxyrus terrestris) tadpoles.

Because habitats are increasingly exposed to multiple stressors simultaneously, assessing the interactive effects of stressors is crucial for understanding how populations respond to human-altered habitats. Salinization of freshwater habitats is increasing and has the potential to interact with other stressors. Chemical pollutants also contribute to habitat degradation in freshwater environments, and both salinity and various pesticides can harm amphibians. The present study used a factorial experiment to investigate the effect of elevated salinity alone and in combination with each of 3 pesticides-atrazine, carbaryl, and glyphosate-on life history and behavior of southern toad larvae (Anaxyrus terrestris). Tadpoles were negatively affected by elevated salinity and by exposure to the insecticide carbaryl, with the most deleterious outcomes associated with both stressors combined. Carbaryl exposure led to reduced survival as well as sublethal effects on growth, activity and feeding behavior, escape response swimming, and time to metamorphosis. Tadpoles reared at elevated salinity were also smaller and less active, and ultimately metamorphosed later and at smaller size. Together, carbaryl and elevated salinity had a synergistic effect, resulting in particularly poor growth, depressed activity and feeding, and sluggish escape swimming among tadpoles exposed to both stressors simultaneously. These results suggest that both elevated salinity and carbaryl represent threats for amphibian populations and that pesticide exposure in salinized habitats may pose a particularly high risk.

Immunotoxicity effects of carbaryl in vivo and in vitro.

Carbaryl is a pesticide for controlling pests in agricultural industry. To determine of immunotoxicity effects of carbaryl, rats were exposure with carbaryl for 28 days. The lymphoid organ weight, lymphocyte proliferation, IL-2, IFN-γ, IL-4, IL-10, IL-1ß and TNF-α cytokines level were measured, respectively. Exposure with carbaryl significantly reduced both thymus and spleen weight and also suppressed lymphocyte proliferation. In addition, carbaryl significantly decreased IL-2, IFN-γ, IL-1ß and TNF-α and also increased IL-4, IL-10 cytokines. These findings suggest that exposure to carbaryl can induce immunotoxicity effects on lymphoid organ weight, suppresses the functions of lymphocyte and macrophage, Th2 polarization in Th1/Th2 balance by reducing of IFN-γ and increasing of IL-4 and IL-10 cytokines. Therefore, carbaryl can contribute to the development of allergic, autoimmune, cancer or infection diseases through immunotoxicity effects and unbalancing of Th1/Th2 immune response however, further study is necessary.

The interactive effects of chytrid fungus, pesticides, and exposure timing on gray treefrog (Hyla versicolor) larvae.

Aquatic organisms are often exposed to a wide variety of perturbations in nature, including pathogens and chemical contaminants. Despite the co-occurrence of these 2 stressors, few studies have examined the effects of chemical contaminants on host-pathogen dynamics. The authors tested the individual and combined effects on gray treefrog (Hyla versicolor) tadpoles of 2 commonly used pesticides (Roundup® and Sevin®) and the pathogenic fungus Batrachochytrium dendrobatidis (Bd). A fully factorial design was used, and tadpoles were exposed to Bd, Roundup, or Sevin alone, or a combination of Bd and either pesticide at 3 points during larval development (early, mid, late). It was predicted that pesticides would mediate the effect of Bd on tadpoles and reduce the likelihood of negative consequences of infection and that timing of exposure would influence these effects. Tadpoles exposed to Bd at the mid point experienced higher survival through metamorphosis than those exposed to Bd at the early or late points, while tadpoles exposed to Sevin at the early point experienced reduced survival compared with those exposed to Roundup or no-pesticide control at the same exposure point. Roundup ameliorated the effects of Bd on survival compared with tadpoles exposed to Bd alone, while there was no interactive effect of Sevin on survival. In addition, Sevin reduced mass of new metamorphs compared with Roundup and reduced snout-vent length compared with all other treatments. The present study supports the hypothesis that pesticides can mitigate the effects of Bd on amphibian hosts and that such effects may depend on the timing of exposure.

In vitro induction/inhibition of germinal vesicle breakdown (GVBD) in frog (Euphlyctis cyanophlyctis) oocytes by endocrine active compounds.

Oocyte maturation is transformation of oocytes into a fertilizable egg. This study examined the effects of four classes of chemicals: 1) acephate (organophosphate); 2) atrazine (herbicide); 3) cypermethrin and fenvalerate (synthetic pyrethroids); and 4) carbaryl (carbamate) on in vitro germinal vesicle breakdown (GVBD) of Euphlyctis cyanophlyctis oocytes. Follicles were isolated and defolliculated from surgically removed ovaries of E. cyanophlyctis and exposed to either progesterone (1 µM/mL) or graded concentrations (1, 5, 10, 15, and 20 µg/mL) of test chemicals. GVBD was evident by the presence of a white spot in the animal pole as well as the absence of germinal vesicles in sectioned heat-fixed oocytes. Percent GVBD was scored every 4 hours until 24 hours. Progesterone induced 77-84% GVBD, compared to 29-33% in controls, at 24 hours. Acephate induced 46-67% GVBD, whereas atrazine elicited 58-77% of GVBD. In cypermethrin or carbaryl- or fenvalerate-exposed oocytes, GVBD was limited to 22-28, 17-29 and 18-24%, respectively. The study infers that some chemical contaminants in the aquatic system may interfere with GVBD in amphibians. Because oocyte maturation is a prerequisite for the production of fertilizable eggs, any alteration in this process potentially impairs the fecundity of females.