Microplastics (MPs) have been reported to threaten a wide variety of terrestrial, marine, and freshwater organisms. However, knowledge about the effects of MPs on anuran amphibians, one of the most threatened taxa worldwide, is still limited. To assess the effects of MPs on the growth and survival of the Italian agile frog (Rana latastei) and green toad (Bufotes balearicus), we exposed tadpoles to three different concentrations (1, 7, and 50 mg L⁻¹) of an environmental relevant mixture of microplastics (HPDE, PVC, PS and PES), recording data on their activity level, weight and mortality rates. While the effects of MPs on green toad tadpoles were negligible, Italian agile frog tadpoles were severely affected both in terms of growth and activity level, with high mortality rates even at the lowest MP density (1 mg L⁻¹). Our results suggest that MP contamination of freshwater habitats may contribute to the ongoing decline of anuran amphibians.

Despite intensive ecotoxicological research, we still know relatively little about the ecological impacts of many environmental contaminants. Filling these knowledge gaps is particularly important regarding amphibians, because they play significant roles in freshwater and terrestrial ecosystems, and their populations are declining worldwide. In this study, we investigated two pollutants that have been poorly studied in ecotoxicology despite their widespread occurrence in surface waters: the herbicide terbuthylazine and the pharmaceutical drug carbamazepine. We exposed two anuran species throughout their larval development to each of two environmentally relevant concentrations of each pollutant, and recorded mortality and 17 sub-lethal endpoints up to several months after exposure. Mortality was low and unrelated to treatment. In agile frogs (Rana dalmatina), we found that treatment with 0.3 μg/L terbuthylazine decreased tadpole activity and reduced fat bodies in juveniles, whereas treatment with 50 μg/L carbamazepine decreased spleen size and increased spleen pigmentation. In common toads (Bufo bufo), treatment with 0.003 μg/L terbuthylazine increased body mass at metamorphosis, treatment with 0.3 μg/L terbuthylazine increased the size of optic tecta, and treatment with 0.5 μg/L carbamazepine decreased hypothalamus size. Treatment with 50 μg/L carbamazepine reduced the feeding activity of toad tadpoles, decreased their production of anti-predatory bufadienolide toxins, and increased their body mass at metamorphosis; juvenile toads in this treatment group had reduced spleen pigmentation. Neither treatments affected the time to metamorphosis, post-metamorphic body mass, or sex ratios significantly. These results show that environmental levels of both terbuthylazine and carbamazepine can have several sub-lethal effects on anurans, which may be detrimental to individual fitness and population persistence in natural conditions. Our findings further highlight that toxic effects cannot be generalized between chemicals of similar structure, because the terbuthylazine effects we found do not conform with previously reported effects of atrazine, a related and extensively studied herbicide.

The application of road deicing salts has led to the salinization of freshwater ecosystems in northern regions worldwide. Increased chloride concentrations in lakes, streams, ponds, and wetlands may negatively affect freshwater biota, potentially threatening ecosystem services. In an effort to reduce the effects of road salt, operators have increased the use of salt alternatives, yet we lack an understanding of how these deicers affect aquatic communities. We examined the direct and indirect effects of the most commonly used road salt (NaCl) and a proprietary salt mixture (NaCl, KCl, MgCl2), at three environmentally relevant concentrations (150, 470, and 780 mg Cl−/L) on freshwater wetland communities in combination with one of three biotic stressors (control, predator cues, and competitors). The communities contained periphyton, phytoplankton, zooplankton, and two tadpole species (American toads, Anaxyrus americanus; wood frogs, Lithobates sylvaticus). Overall, we found the two road salts did not interact with the natural stressors. Both salts decreased pH and reduced zooplankton abundance. The strong decrease in zooplankton abundance in the highest NaCl concentration caused a trophic cascade that resulted in increased phytoplankton abundance. The highest NaCl concentration also reduced toad activity. For the biotic stressors, predatory stress decreased whereas competitive stress increased the activity of both tadpole species. Wood frog survival, time to metamorphosis, and mass at metamorphosis all decreased under competitive stress whereas toad time to metamorphosis increased and mass at metamorphosis decreased. Road salts and biotic stressors can both affect freshwater communities, but their effects are not interactive.

We conducted a mesocosm study to assess the individual and interactive effects of previous maternal exposure and larval exposure to trace element-laden sediments on southern toads (Bufo terrestris). Previous maternal exposure to coal combustion wastes (CCW) reduced larval survival to metamorphosis up to 57% compared to larvae of unexposed females. Larvae reared on CCW accumulated significant concentrations of trace elements resulting in extended larval periods, reduced growth rates, and reduced mass at metamorphosis. However, the effects were dependent on age of sediments, suggesting the effects of contaminants from CCW may be partially ameliorated over time through the reduced bioavailability of trace elements in aged CCW. Most importantly, maternal exposure to contaminants coupled with larval exposure to fresh CCW interacted to reduce survival to metamorphosis by 85% compared to reference conditions. Our study yields further evidence that disposal of CCW in aquatic basins potentially creates ecological traps for some amphibian populations.

Dichlorodiphenyltrichloroethanes (DDTs), Polychlorinated biphenyls (PCBs), and halogenated flame retardants (HFRs) were measured in aquatic, amphibious, and terrestrial wildlife collected from an e-waste contaminated pond and its surrounding region. The species-specific bioaccumulation and maternal transfer of chemicals in the watersnake were investigated. Total concentrations of target chemicals ranged from 1.3 × 103 to 4.8 × 105 ng g−1 lipid weight. PCBs were the predominant (72–95%) contaminants, followed by polybrominated biphenyl ethers (PBDEs, 4–27%). The concentrations of PCBs and HFRs except decabromodiphenyl ethane (DBDPE) were higher in aquatic organisms and terrestrial birds than in amphibians and lizards. Relatively high DDT levels were observed in the terrestrial birds and toads, but high DBDPE was found in the aquatic species except for waterbird eggs. Species-specific congeners profiles for PCB and PBDE and isomeric composition for dechlorane plus were observed. These results indicated a habitat-dependent accumulation among different species. Maternal transfer examined by the ratio of egg to carcass for watersnakes indicated multi-linear correlations between maternal transfer potential and octanol-water partition coefficient (log KOW) of chemicals. The same maternal transfer efficiencies were found for chemicals with log KOW between 6 and 8, then the maternal transfer potential rapidly decreased with increasing of log KOW.

Environmental contaminants are implicated in the global decline of amphibian populations. Copper (Cu) is a widespread contaminant that can be toxic at concentrations just above the normal physiological range. In the present study we examined the effects of chronic Cu aqueous exposure on embryos and larvae of southern toads, Anaxyrus (Bufo) terrestris. Measurable levels of Cu were found in larvae, with tissue concentrations up to 27.5 μg Cu/g dry mass. Aqueous concentrations of Cu as low as 10 μg/L significantly reduced survival to the free-swimming stage and no larvae reached metamorphosis at concentrations above 15 μg/L. Clutches from populations with prior Cu exposure had the lowest survivorship. Among several populations there was significant variation in survivorship at different levels of Cu. More data are needed to understand the underlying causes of within- and among-population resilience to anthropogenic stressors.

Diazinon is an extensively applied organophosphate pesticide, and nonylphenol is one of the major degradation products of nonylphenol polyethoxylates which are commonly used as surfactant in pesticide formulations. Both pollutants are widely distributed and often coexist in agroecosystems, where they might cause toxic effects to wild biota. This study assessed single and joint toxicity of binary mixtures of these organic compounds on the early development of Rhinella arenarum by means of a standardized test. Joint toxicity of diazinon/nonylphenol mixtures were assessed in embryos and larvae exposed to three different proportions at different exposure times. Embryo and larval toxicity was time-dependent, and larvae were significantly more sensitive than embryos to both compounds. For both embryos and larvae, nonylphenol was between 11 and 18 times more toxic than diazinon. Joint toxicity of the chemicals showed a tendency to be significantly higher than the predicted by additivity effects highlighting the threat that diazinon/nonylphenol mixtures represent for Rhinella arenarum populations.

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.

Agricultural fungicide application in Argentina has increased twice since 2008, with Maxim® XL (2.5% fludioxonil +1% metalaxyl-M) as one of the most used fungicide formulation. The toxicity of this pesticide on Rhinella arenarum was assessed by means of continuous (from embryo and larval development) and 24-h pulse exposure standardized bioassays. Lethality was concentration- and exposure time-dependent. Maxim® XL caused a progressive lethal effect along the bioassays with higher toxicity on embryos than larvae, obtaining 50% lethal concentrations at 96, 336, and 504 h of 10.85, 2.89, and 1.71 mg/L for embryos, and 43.94, 11.79, and 5.76 mg/L for larvae respectively. Lethal 504-h no observed effect concentration values for embryos and larvae were 1 and 2.5 mg/L respectively. A stage-dependent toxicity of Maxim® XL was also demonstrated within the embryo development, with early stages more sensitive than the later ones, and blastula as the most sensitive developmental stage. The risk quotients obtained for chronic risk assessment determined a potential threat for the survival and continuity of R. arenarum populations under these conditions. The results indicate that the levels of the fungicide reaching amphibian habitats could be risky for the early development of this amphibian species. This study also emphasizes the necessity to evaluate the chronic effects of fungicides in pesticide risk assessment.

Copper is a common aqueous pollutant that is known to cause oxidative stress and disrupt the thyroid axis in amphibians. In the present study, tadpoles of the Chinese toad (Bufo gargarizans) were exposed to 1, 6.4, 32, and 64 μg L⁻¹ of copper from Gosner stages 26 to 42. We aimed to examine the influence of copper on thyroid hormone-responsive and stress-associated gene expression in the hind-limb, tail, and liver of B. gargarizans tadpoles. Exposure to 64 μg L⁻¹ copper decreased percent metamorphosis and increased length of both hind-limb and tail of B. gargarizans tadpoles at Gs 42. In addition, according to real-time PCR results, exposure to 64 μg L⁻¹ copper induced downregulation of Dio2, Dio3, TRα, and TRβ mRNA levels in all tissues examined. We inferred that copper might induce a considerable reduction of TH levels through downregulation of Dio2 and Dio3 mRNA levels in peripheral tissues. Decreased TH levels may then decrease the expressions of TRα and TRβ. Also, HSP, SOD, and PHGPx transcript levels were measured to assess cellular stress which might affect TH signaling and metamorphosis. We found that copper significantly downregulated the level of HSP, SOD, and PHGPx transcripts in the hind-limb and tail. This demonstrates that high concentrations of copper could disrupt the antioxidant system of B. gargarizans tadpoles and increase oxidative damage. Therefore, we conclude that copper could disrupt the antioxidant system and cause thyroid hormone disruption in B. gargarizans tadpoles.