Investigation into the effects of pesticides on amphibians

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Amphibian population decline is a recognised phenomenon spanning at least the last 40 years, and it is likely that a number of factors have contributed, including environmental contamination. Amphibians are vulnerable to agrochemical uptake as they must breed in water, and often spend the aquatic phase of their lifecycle in agricultural water bodies, which may contain a complex mixture of biologically active chemicals. Endocrine disrupting compounds may cause reproductive effects in humans and wildlife, although the link between pesticides and endocrine disruption is largely unknown. Therefore, in this study, the role of pesticides in endocrine disruption, in relation to amphibian metamorphosis and reproductive development, was investigated. To achieve this objective, population data were used to select suitable field sites, water from which was tested for endocrine activity using the yeast estrogen/androgen screen, hepatocyte culture (estrogenic response), and a transgenic Xenopus test (thyroid disruption). Toad (Bufo bufo) specimens from a subsample of these sites were used to compare morphology, thyroidal, and gonadal development of caged and wild-caught tadpoles/metamorphs, to their laboratory-raised counterparts. In addition, environmentally relevant pesticides were tested for endocrine effects in vitro, and a short-term in vivo exposure was used to assess the predictive ability of the in vitro screens in Xenopus. Mortality of Bufo bufo was high in both laboratory-reared and caged individuals, which hindered the interpretation of results due to low n values. However, laboratory-reared individuals from different sites had distinct morphology and gonadal differentiation, possibly suggesting maternal transfer, a latent effect of the pond environment, and/or genetic effects. In addition, caged and wild-caught individuals were smaller, metamorphosed later, and had retarded gonadal differentiation or increased incidence of intersex, compared to their laboratory-reared counterparts. Extracts of water samples from these sites were predominantly anti-estrogenic, and/or anti-androgenic in yeast based assays, and this was also the effect observed in response to environmentally relevant pesticides tested in the same assays. Pesticides also affected ovarian steroidogenesis in vitro, and pentachlorophenol had a reprotoxic effect on adult female Xenopus laevis. Data reported in this study suggest there may be endocrine disrupting effects in native amphibians in the agricultural landscape, although further investigation is needed to confirm these findings

Competitive androgen receptor antagonism as a factor determining the predictability of cumulative antiandrogenic effects of widely used pesticides

Copyright @ 2012 National Institute of Environmental Health Sciences.This article has been made available through the Brunel Open Access Publishing Fund.Background: Many pesticides in current use have recently been revealed as in vitro androgen receptor (AR) antagonists, but information about their combined effects is lacking.
Objective: We investigated the combined effects and the competitive AR antagonism of pesticide mixtures.
Methods: We used the MDA-kb2 assay to test a combination of eight AR antagonists that did not also possess AR agonist properties (“pure” antagonists; 8 mix: fludioxonil, fenhexamid, ortho-­phenylphenol, imazalil, tebuconazole, dimetho­morph, methiocarb, pirimiphos-methyl), a combina­tion of five AR antagonists that also showed agonist activity (5 mix: cyprodinil, pyrimethanil, vinclozolin, chlor­propham, linuron), and all pesticides combined (13 mix). We used concentration addition (CA) and independent action (IA) to formu­late additivity expectations, and Schild plot analyses to investigate competitive AR antagonism.
Results: A good agreement between the effects of the mixture of eight “pure” AR antagonists and the responses predicted by CA was observed. Schild plot analysis revealed that the 8 mix acted by competi­tive AR antagonism. However, the observed responses of the 5 mix and the 13 mix fell within the “prediction window” boundaries defined by the predicted regression curves of CA and IA. Schild plot analysis with these mixtures yielded anomalous responses incompatible with competitive receptor antagonism.
Conclusions: A mixture of widely used pesticides can, in a predictable manner, produce combined AR antagonist effects that exceed the responses elicited by the most potent component alone. Inasmuch as large populations are regularly exposed to mixtures of anti­androgenic pesticides, our results underline the need for considering combination effects for these substances in regulatory practice.
This article is made available through the Brunel Open Access Publishing Fund. This work was funded by the European Commission, FP7 programme (CONTAMED, grant 212502).

Widely Used Pesticides with Previously Unknown Endocrine Activity Revealed as in Vitro Antiandrogens

Background: Evidence suggests that there is widespread decline in male reproductive health and that antiandrogenic pollutants may play a significant role. There is also a clear disparity between pes¬ticide exposure and data on endocrine disruption, with most of the published literature focused on pesticides that are no longer registered for use in developed countries. Objective: We used estimated human exposure data to select pesticides to test for antiandrogenic activity, focusing on highest use pesticides. Methods: We used European databases to select 134 candidate pesticides based on highest expo-sure, followed by a filtering step according to known or predicted receptor-mediated antiandrogenic potency, based on a previously published quantitative structure–activity relationship (QSAR) model. In total, 37 pesticides were tested for in vitro androgen receptor (AR) antagonism. Of these, 14 were previously reported to be AR antagonists (“active”), 4 were predicted AR antagonists using the QSAR, 6 were predicted to not be AR antagonists (“inactive”), and 13 had unknown activity, which were “out of domain” and therefore could not be classified with the QSAR (“unknown”). Results: All 14 pesticides with previous evidence of AR antagonism were confirmed as antiandrogenic in our assay, and 9 previously untested pesticides were identified as antiandrogenic (dimethomorph, fenhexamid, quinoxyfen, cyprodinil, λ-cyhalothrin, pyrimethanil, fludioxonil, azinphos-methyl, pirimiphos-methyl). In addition, we classified 7 compounds as androgenic. Conclusions: Due to estimated antiandrogenic potency, current use, estimated exposure, and lack of previous data, we strongly recommend that dimethomorph, fludioxonil, fenhexamid, imazalil, ortho-phenylphenol, and pirimiphos-methyl be tested for antiandrogenic effects in vivo. The lack of human biomonitoring data for environmentally relevant pesticides presents a barrier to current risk assessment of pesticides on humans.European Commission (grant 21250

Non-destructive methods to assess health of wild tropical frogs (túngara frogs: <i>Engystomops pustulosus</i>) in Trinidad reveal negative impacts of agricultural land

Amphibians are threatened globally with at least 43% of species declining and the most important stressor being habitat loss or degradation. Amphibians inhabiting highly biodiverse tropical regions are disproportionately threatened; however, the effects of landscape alterations on amphibian health are virtually unknown. In this study, we utilised non-destructive techniques to compare size (weight, snout-vent length [SVL]), body condition, male secondary sexual features (forelimb width, nuptial pad length) and breeding success (egg number, fertility [percentage fertilised eggs], hatching success) in túngara frogs (Engystomops pustulosus) collected from reference (n = 5), suburban (n = 6) and agricultural (n = 4) sites in Trinidad; characterised by presence/absence of crops/houses. All measured endpoints were negatively impacted in frogs collected from agricultural sites. The largest effect was observed for hatching success (2.77-fold lower) and egg number (2.5-fold lower). Less pronounced effects were observed on male frogs (weight: 1.77-fold lower; SVL: 1.18-fold lower; forelimb width: 1.33-fold lower; nuptial pad length: 1.15-fold lower). Our findings demonstrate negative impacts of agricultural sites on túngara frog health, with the number of viable offspring reduced by almost one third. The methods outlined here are technically simple and low-cost and thereby have potential for application to other species in order to investigate the potential impacts of habitat degradation on amphibian health. Furthermore, as these methods are non-destructive, they could be used to investigate the potential contribution of frog size and/or reproductive capability as a causative factor contributing to population declines in threatened species, which is particularly pressing in tropical regions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11356-022-20105-4

Nuptial pad (“breeding gland”) morphology is related to non-random mating in wild male common frogs (<i>Rana temporaria</i>)

Androgen levels are closely linked with breeding in male amphibians. Development of the nuptial pad is driven by androgens and is believed to have importance for determining mating success in anurans, but this has not been tested in wild populations. We investigated the association between nuptial pad morphology (length, colour) and mating (amplexus) success in wild male common frogs (Rana temporaria) in the UK (Devon in Southern England and central Scotland). Once active breeding had been confirmed, pond water (25 L) was placed in 1–6 replicate mesocosms (66 cm diameter circular, black plastic tubs) in situ. Eight male and two female frogs were placed into each mesocosm, and success observed by identifying the male frog(s) in amplexus. The length of nuptial pads for male frogs observed in amplexus was greater than those that did not achieve amplexus. There was no difference in the absolute dark colour of nuptial pads (determined by red/green/blue analysis, Adobe photoshop©) for male frogs observed in amplexus versus those that did not achieve amplexus. However, within each mesocosm, the nuptial pad was relatively darker for winning male frogs compared to losing male frogs. Overall, 91% of winning male frogs from Devon, and 89% winning male frogs from Scotland, possessed either a longer and/or a darker nuptial pad, compared with frogs that did not achieve amplexus. These data suggest that features of nuptial pad morphology are associated with amplexus (and thus mating) success in male common frogs. Given that nuptial pads have been identified in all anuran amphibian species analysed to date, nuptial pad morphology may contribute to non-random mating strategies across a broad range of species

Effects of common pesticides on prostaglandin D2 (PGD2) inhibition in SC5 mouse sertoli cells, evidence of binding at the cox-2 active site, and implications for endocrine disruption

Background: There are concerns that diminished prostaglandin action in fetal life could increase the risk of congenital malformations. Many endocrine-disrupting chemicals have been found to suppress prostaglandin synthesis, but to our knowledge, pesticides have never been tested for these effects. Objectives: We assessed the ability of pesticides that are commonly used in the European Union to suppress prostaglandin D2 (PGD2) synthesis. Methods: Changes in PGD2 secretion in juvenile mouse Sertoli cells (SC5 cells) were measured using an ELISA. Coincubation with arachidonic acid (AA) was conducted to determine the site of action in the PGD2 synthetic pathway. Molecular modeling studies were performed to assess whether pesticides identified as PGD2-active could serve as ligands of the cyclooxygenase-2 (COX-2) binding pocket. Results: The pesticides boscalid, chlorpropham, cypermethrin, cyprodinil, fenhexamid, fludioxonil, imazalil (enilconazole), imidacloprid, iprodione, linuron, methiocarb, o-phenylphenol, pirimiphos- methyl, pyrimethanil, and tebuconazole suppressed PGD2 production. Strikingly, some of these substances—o-phenylphenol, cypermethrin, cyprodinil, linuron, and imazalil (enilconazole)— showed potencies (IC50) in the range between 175 and 1,500 nM, similar to those of analgesics intended to block COX enzymes. Supplementation with AA failed to reverse this effect, suggesting that the sites of action of these pesticides are COX enzymes. The molecular modeling studies revealed that the COX-2 binding pocket can accommodate most of the pesticides shown to suppress PGD2 synthesis. Some of these pesticides are also capable of antagonizing the androgen receptor. Conclusions: Chemicals with structural features more varied than previously thought can suppress PGD2 synthesis. Our findings signal a need for in vivo studies to establish the extent of endocrinedisrupting effects that might arise from simultaneous interference with PGD2 signaling and androgen action

Investigation into the effects of pesticides on amphibians

Amphibian population decline is a recognised phenomenon spanning at least the last 40 years, and it is likely that a number of factors have contributed, including environmental contamination. Amphibians are vulnerable to agrochemical uptake as they must breed in water, and often spend the aquatic phase of their lifecycle in agricultural water bodies, which may contain a complex mixture of biologically active chemicals. Endocrine disrupting compounds may cause reproductive effects in humans and wildlife, although the link between pesticides and endocrine disruption is largely unknown. Therefore, in this study, the role of pesticides in endocrine disruption, in relation to amphibian metamorphosis and reproductive development, was investigated. To achieve this objective, population data were used to select suitable field sites, water from which was tested for endocrine activity using the yeast estrogen/androgen screen, hepatocyte culture (estrogenic response), and a transgenic Xenopus test (thyroid disruption). Toad (Bufo bufo) specimens from a subsample of these sites were used to compare morphology, thyroidal, and gonadal development of caged and wild-caught tadpoles/metamorphs, to their laboratory-raised counterparts. In addition, environmentally relevant pesticides were tested for endocrine effects in vitro, and a short-term in vivo exposure was used to assess the predictive ability of the in vitro screens in Xenopus. Mortality of Bufo bufo was high in both laboratory-reared and caged individuals, which hindered the interpretation of results due to low n values. However, laboratory-reared individuals from different sites had distinct morphology and gonadal differentiation, possibly suggesting maternal transfer, a latent effect of the pond environment, and/or genetic effects. In addition, caged and wild-caught individuals were smaller, metamorphosed later, and had retarded gonadal differentiation or increased incidence of intersex, compared to their laboratory-reared counterparts. Extracts of water samples from these sites were predominantly anti-estrogenic, and/or anti-androgenic in yeast based assays, and this was also the effect observed in response to environmentally relevant pesticides tested in the same assays. Pesticides also affected ovarian steroidogenesis in vitro, and pentachlorophenol had a reprotoxic effect on adult female Xenopus laevis. Data reported in this study suggest there may be endocrine disrupting effects in native amphibians in the agricultural landscape, although further investigation is needed to confirm these findings.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

A review of non-destructive biomonitoring techniques to assess the impacts of pollution on reproductive health in frogs and toads

In anuran amphibians (frogs and toads), evidence linking pollution to population declines is limited, in particular through impaired reproduction. Here we review the evidence for pollutant-induced alterations on reproductive endpoints in wild anurans with a particular focus on the application of non-destructive endpoints including on sex ratios, male reproductive phenotypes (data are too scarce for females) and reproductive outputs (reflective of mating success). Data evidencing alterations in sex ratio in wild anurans are scarce, however, both feminisation and masculinisation in response to pollution have been reported (seven studies). Male nuptial pad morphology and calling behaviour display high sensitivity to pollutant-exposure and are important features determining male breeding success, however there is considerable variation in these endpoints and inconsistencies in the responses of them to pollution are reported in wild anurans. Data for clutch size are insufficient to assess sensitivity to pollutants (five studies only). However, hatch success and offspring fitness (tadpole survival/development) are sensitive to pollution, with clear linkages to population stability. In conclusion, there are a wide range of non destructive measures with good potential for application to assess/monitor reproductive health in wild anurans, however, a greater understanding of pollutant effects on these endpoints is needed. There measures deserve wider application as they are relatively simple and inexpensive to implement, and as they can be applied non-destructively are widely applicable to our declining anuran populations