Understanding the influence of multiple pollutant stressors on the decline of freshwater mussels in a biodiversity hotspot.

The Clinch River watershed of the upper Tennessee River Basin of Virginia and Tennessee, USA supports one of North America's greatest concentrations of freshwater biodiversity, including 46 extant species of native freshwater mussels (Order Unionida), 20 of which are protected as federally endangered. Despite the global biological significance of the Clinch River, mussel populations are declining in some reaches, both in species richness and abundance. The aim of this study was to evaluate the exposure of adult resident mussels to a suite of inorganic and organic contaminant stressors in distinct sections of the Clinch River that encompassed a range of mussel abundance and health. To provide insight into the potential role of pollutants in the decline of mussels, including within a previously documented "zone of mussel decline", the mainstem Clinch River (8 sites) and its tributaries (4 sites) were examined over two consecutive years. We quantified and related metals and organic contaminant concentrations in mussels to their associated habitat compartments (bed sediment, suspended particulate sediment, pore water, and surface water). We found that concentrations of organic contaminants in resident mussels, particularly the suite of 42 polycyclic aromatic hydrocarbons (PAHs) analyzed, were related to PAH concentrations in all four habitat (media) compartments. Further, PAH concentrations in mussel tissue (range 37.8-978.1 ng/g dry weight in 2012 and 194.3-1073.7 ng/g dry weight in 2013) were negatively related to the spatial pattern in mussel densities (rs = -0.64, p ≤ 0.05 in 2012 and rs = -0.83, p ≤ 0.05 in 2013) within the river, and were highest in the "zone of mussel decline". In contrast, the suite of 22 metals analyzed in resident mussels were largely unrelated to the spatial pattern of variation of metals in the four habitat compartments except for Manganese (Mn; range 3630.5-23,749.2 µg/g dry weight in 2012 and 1540.4-12,605.8 µg/g dry weight in 2013) in surface water (rs = 0.58, p < 0.1) and pore water (rs = 0.76, p ≤ 0.05). This study revealed that PAHs and Mn are important pollutant stressors to mussels in the Clinch River and that they are largely being delivered through the Guest River tributary watershed. Accordingly, future conservation and management efforts would benefit by identifying, and ideally mitigating, the sources of PAHs, Mn, and other current or legacy mining-associated pollutants to the mainstem river and its tributaries.

Validation of Bioavailability-Based Toxicity Models for Metals.

Regulatory jurisdictions worldwide are increasingly incorporating bioavailability-based toxicity models into development of protective values (PVALs) for freshwater and saltwater aquatic life (e.g., water quality criteria, standards, and/or guidelines) for metals. Use of such models for regulatory purposes should be contingent on their ability to meet performance criteria as specified through a model-validation process. Model validation generally involves an assessment of a model's appropriateness, relevance, and accuracy. We review existing guidance for validation of bioavailability-based toxicity models, recommend questions that should be addressed in model-validation studies, discuss model study type and design considerations, present several new ways to evaluate model performance in validation studies, and suggest a framework for use of model validation in PVAL development. We conclude that model validation should be rigorous but flexible enough to fit the user's purpose. Although a model can never be fully validated to a level of zero uncertainty, it can be sufficiently validated to fit a specific purpose. Therefore, support (or lack of support) for a model should be presented in such a way that users can choose their own level of acceptability. We recommend that models be validated using experimental designs and endpoints consistent with the data sets that were used to parameterize and calibrate the model and validated across a broad range of geographically and ecologically relevant water types. Environ Toxicol Chem 2019;39:101-117. © 2019 SETAC.

Assessing toxicity of contaminants in riverine suspended sediments to freshwater mussels.

The Clinch River in Virginia and Tennessee, USA, is well known for its diverse native freshwater mussel assemblages; however, notable declines in mussel populations in recent decades have prompted much concern and subsequent research. The authors examined the toxicity of recently deposited sediments on juveniles of the freshwater mussel Epioblasma brevidens by collecting time-integrated sediment samples from the water column with sediment traps from 11 sites in the Clinch River basin, including 6 sites within an 88-km reach deemed a "mussel zone of decline." Mussels were exposed to the riverine sediments and to 3 control sediments for 28 d; survival, shell length, and biomass were then assessed. Sediment treatment (i.e., river location) had a significant effect on mussel survival (p < 0.01) and biomass (p = 0.02) but did not affect length (p = 0.37), and sediments from 2 of the tributaries were the most toxic. Inorganic and organic analyses of sediments indicated the presence of metals and polycyclic aromatic hydrocarbons at all sites. Manganese was negatively correlated with mussel survival and biomass, as was ammonia with survival and total organic carbon with biomass. Current land uses in the watershed indicate that fossil fuel mining and agriculture may be associated with elevated manganese and ammonia, respectively. The authors found that sediments collected with sediment traps over relatively short deployment durations can help elucidate recent contaminant influx and its potential for inducing toxicity in benthic organisms. Environ Toxicol Chem 2017;36:395-407. © 2016 SETAC.

Use of toe clips as a nonlethal index of mercury accumulation and maternal transfer in amphibians.

Nonlethal indices of contaminant exposure can facilitate research on the accumulation and effects of contaminants in wildlife. Here, we tested the efficacy of using amputated toes ("toe clips"), a common byproduct when marking amphibians in population and genetic studies, to determine mercury (Hg) concentrations in amphibians. We examined total mercury (THg) concentrations in American toads (Bufo americanus) collected along a contamination gradient at a Hg-contaminated field site. We found significant positive correlations between toe THg and blood THg concentrations in adult males and females collected in two different years. We also found that blood and toe clips could be used to predict maternal transfer of Hg, an important mechanism of reproductive toxicity in wildlife. Maternal toe THg concentrations were more highly correlated with egg THg concentrations than were maternal blood THg concentrations. Our results indicate that amputated toes are effective for identifying Hg concentrations in amphibians.

Do effects of mercury in larval amphibians persist after metamorphosis?

Despite widespread concern about the role of environmental contaminants in global amphibian declines, and evidence that post-metamorphic life stages contribute disproportionately to amphibian population dynamics, most studies in amphibian ecotoxicology focus on larval life stages. Studies that focus solely on early life stages may miss important effects of contaminant exposure, such as latent effects that manifest some time after previous exposure. Moreover, it is often assumed that effects observed in amphibian larvae will persist to affect survival or reproduction later in life. We used terrestrial enclosures to determine whether exposure to mercury (Hg) through maternal transfer and/or larval diet had any adverse effects in post-metamorphic American toads (Bufo americanus). We found a 5% difference in size at metamorphosis that was attributed to maternal Hg exposure persisted for 1 year in the terrestrial environment, resulting in a 7% difference at the conclusion of the study. Although patterns of survival differed among treatments through time, we found no overall difference in survival after 1 year. We also found no evidence of emergent latent effects in the terrestrial toads that could be attributed to earlier exposure. Our results indicate that adverse effects of maternal Hg exposure that were observed in larval amphibians may persist to affect later terrestrial life stages but that no novel adverse effects developed when animals were raised in a semi-natural environment. Moreover, we found no evidence of persistent effects of dietary Hg exposure in larvae, highlighting a need for greater focus on maternal effects in amphibian ecotoxicology. Finally, we suggest an increase in the use of longitudinal studies to better understand contaminant impacts to amphibian populations via effects in both aquatic and terrestrial life stages.

Counterbalancing effects of maternal mercury exposure during different stages of early ontogeny in American toads.

Maternal transfer of environmental contaminants is a disadvantageous parental effect which can have long-lasting implications for offspring fitness. We investigated the effects of mercury (Hg) on the reproductive success of female amphibians and the subsequent effects of maternal transfer on the development of their offspring. American toads (Bufo americanus) maternally transferred Hg to their eggs, and there was a negative relationship between Hg concentrations and the percentage of viable hatchlings produced in clutches. However, when we continued to monitor larvae that successfully hatched, we found 21% greater metamorphic success in larvae from Hg-exposed mothers compared to reference larvae. The negative effect in the embryonic stage and positive effect in the larval stage counterbalanced one another, ultimately resulting in no difference in predicted terrestrial recruitment, regardless of maternal Hg exposure. Our findings demonstrate that maternal effects on survival manifesting at different stages in ontogeny have the potential to produce complicated outcomes.

Dietary mercury has no observable effects on thyroid-mediated processes and fitness-related traits in wood frogs.

Mercury (Hg) is a neurotoxicant known to cause developmental and behavioral abnormalities in vertebrates. Increasing evidence suggests that Hg can also disrupt endocrine functions and endocrine-dependent processes. For example, dietary Hg has been shown to delay tail resorption during metamorphic climax in amphibians, a process mediated by thyroid hormones. However, a direct link between Hg, hormone disruption, and developmental delays in amphibians has not been explored. Therefore, we examined the effects of dietary Hg (0.01, 2.5, and 10 µg/g total Hg, dry wt) on thyroid hormone concentrations, development, growth, performance, and survival of wood frogs (Rana sylvatica). Tadpoles accumulated Hg in a concentration-dependent manner; total Hg concentrations in tadpoles at the beginning of metamorphic climax (Gosner stage 42) were 0.03, 1.06, 3.54 µg/g, dry wt, for control, low, and high Hg diets, respectively. During metamorphic climax, tadpoles eliminated 35% of the inorganic Hg from their tissues but retained most of their accumulated methylmercury. Contrary to our predictions, we found no effect of Hg on the duration of tadpole development, size at metamorphosis, tail resorption time, or hopping performance. Consistent with the lack of effects on development, we also detected no differences in whole-body thyroid hormone concentrations among our dietary treatments. Our results, when compared with the effects of Hg on other amphibians, suggest that amphibian species may differ substantially in their sensitivity to dietary Hg, emphasizing the need for data on multiple species when establishing toxicity benchmarks.

Aquatic and terrestrial stressors in amphibians: a test of the double jeopardy hypothesis based on maternally and trophically derived contaminants.

Amphibians with biphasic life histories occupy aquatic and terrestrial habitats at different times in their lives, leading to a double jeopardy of contaminant risk in both habitats. The present study examines individual and interactive effects of mercury exposure to terrestrial adults and aquatic larvae on fitness-related traits of American toads, Bufo americanus. Eggs from reference mothers or contaminated mothers were allowed to hatch and larvae were fed diets of either no added Hg or 2.5 or 10 µg/g total Hg (dry wt). Both dietary and maternal Hg had adverse effects on developing larvae, but there was no interaction between these factors. Dietary Hg had a marginal effect of decreased survival with increasing Hg in the diet. Animals from Hg-exposed mothers weighed 14% less than those from reference mothers, and size at metamorphosis was directly correlated with hopping performance. Animals from Hg-exposed mothers also took longer to complete metamorphosis and had 2.5 times the prevalence of spinal malformations compared with those from reference mothers. Results of the present study demonstrate that amphibians do indeed face a double jeopardy of contaminant exposure stemming from terrestrial and aquatic environments, because both exposure pathways adversely affected developing offspring. The present study also demonstrates that all possible routes of exposure over an organism's life history must be examined to provide a comprehensive picture of the ecological consequences of habitat contamination.

Does maternal exposure to an environmental stressor affect offspring response to predators?

There is growing recognition of the ways in which maternal effects can influence offspring size, physiological performance, and survival. Additionally, environmental contaminants increasingly act as stressors in maternal environments, possibly leading to maternal effects on subsequent offspring. Thus, it is important to determine whether contaminants and other stressors can contribute to maternal effects, particularly under varied ecological conditions that encompass the range under which offspring develop. We used aquatic mesocosms to determine whether maternal effects of mercury (Hg) exposure shape offspring phenotype in the American toad (Bufo americanus) in the presence or absence of larval predators (dragonfly naiads). We found significant maternal effects of Hg exposure and significant effects of predators on several offspring traits, but there was little evidence that maternal effects altered offspring interactions with predators. Offspring from Hg-exposed mothers were 18% smaller than those of reference mothers. Offspring reared with predators were 23% smaller at metamorphosis than those reared without predators. There was also evidence of reduced larval survival when larvae were reared with predators, but this was independent of maternal effects. Additionally, 5 times more larvae had spinal malformations when reared without predators, suggesting selective predation of malformed larvae by predators. Lastly, we found a significant negative correlation between offspring survival and algal density in mesocosms, indicating a role for top-down effects of predators on periphyton communities. Our results demonstrate that maternal exposure to an environmental stressor can induce phenotypic responses in offspring in a direction similar to that produced by direct exposure of offspring to predators.

Interactive effects of maternal and dietary mercury exposure have latent and lethal consequences for amphibian larvae.

Organisms born into the same contaminated environment as their parents can be exposed both maternally and environmentally to contaminants, potentially placing them at greater risk of adverse effects than when exposed via either of the two pathways independently. We examined whether embryonic exposure to maternally derived mercury (Hg) interacts with dietary exposure to negatively influence larval development in American toads ( Bufo americanus ). We collected eggs from breeding pairs at reference and Hg-contaminated sites and monitored performance, development, and survival of larvae fed three experimental Hg diets (total Hg, 0.01, 2.5, and 10 µg/g). The negative sublethal effects of maternal and/or dietary Hg manifested differently, but maternal Hg exposure had a greater overall influence on offspring health than dietary exposure. However, the combination of sublethal effects of the two exposure routes interacted with lethal consequences; larvae exposed to maternal Hg and high dietary Hg experienced 50% greater mortality compared to larvae from reference mothers fed the control diet. This study is the first to demonstrate that the latent effects of maternally transferred contaminants may be exacerbated by further exposure later in ontogeny, findings that may have important implications for both wildlife and human health.

Mercury accumulation along a contamination gradient and nondestructive indices of bioaccumulation in amphibians.

Mercury (Hg) is an important environmental contaminant due to its global distribution, tendency to bioaccumulate, and toxicity in wildlife. However, Hg has received little attention in amphibians compared to other vertebrates. Amphibians vary widely in life history strategies and feeding ecologies, which could influence Hg exposure and accumulation. To determine whether species and life stage affects Hg bioaccumulation, adults from three species (Plethodon cinereus, Eurycea bislineata, and Bufo americanus) and larvae from the latter two species were collected along a contamination gradient on the South River (VA, USA). Total Hg (THg) concentrations in the contaminated site were 3.5 to 22 times higher than in the reference site. Differences were found in THg concentrations in amphibians that were consistent with their habitat requirements and feeding preferences. In general, adults (3,453 +/- 196 ng/g, dry mass) and larvae (2,479 +/- 171 ng/g) of the most river-associated species, E. bislineata, had the highest THg concentrations, followed by B. americanus tadpoles (2,132 +/- 602 ng/g), whereas adults of the more terrestrial B. americanus (598 +/- 117 ng/g) and P. cinereus (583 +/- 178 ng/g) had the lowest concentrations. In addition, nondestructive sampling techniques were developed. For the salamander species, THg concentrations in tail tissue were strongly correlated (r >or= 0.97) with the remaining carcass. A strong positive correlation (r = 0.92) also existed between blood and whole-body THg concentrations in B. americanus. These results suggest that amphibians and their terrestrial predators may be at risk of Hg exposure in this system and that nondestructive methods may be a viable sampling alternative that reduces impacts to local populations.

Bioaccumulation and maternal transfer of mercury and selenium in amphibians.

Amphibian population declines have been documented worldwide and environmental contaminants are believed to contribute to some declines. Maternal transfer of bioaccumulated contaminants to offspring may be an important and overlooked mechanism of impaired reproductive success that affects amphibian populations. Mercury (Hg) is of particular concern due to its ubiquity in the environment, known toxicity to other wildlife, and complex relationships with other elements, such as selenium (Se). The objectives of the present study were to describe the relationships between total Hg (THg), methlymercury (MMHg), and Se in three amphibian species (Plethodon cinereus, Eurycea bislineata cirrigera, and Bufo americanus) along a Hg-polluted river and floodplain, and to determine if B. americanus maternally transfers Hg and Se to its eggs in a tissue residue-dependent manner. Total Hg and MMHg concentrations in all species spanned two orders of magnitude between the reference and contaminated areas, while Se concentrations were generally low in all species at both sites. Strong positive relationships between THg and MMHg in tissues of all species were observed throughout. Both Hg and Se were maternally transferred from females to eggs in B. americanus, but the percentage of the females' Hg body burden transferred to eggs was low compared with Se. In addition, Hg concentrations appeared to positively influence the amount of Se transferred from female to eggs. The present study is the first to confirm a correlation between Hg concentrations in female carcass and eggs in amphibians and among the first to describe co-transference of Se and Hg in an anamniotic vertebrate. The results suggest future work is needed to determine whether maternal transfer of Hg has transgenerational implications for amphibian progeny.

Effects of mercury on behavior and performance of northern two-lined salamanders (Eurycea bislineata).

Mercury (Hg) causes a range of deleterious effects in wildlife, but little is known about its effects on amphibians. Our objective was to determine whether Hg affects performance and behavior in two-lined salamanders (Eurycea bislineata). We collected salamanders from Hg-contaminated and reference sites and assessed speed, responsiveness, and prey capture ability. Mercury concentrations were >17× higher in salamanders from the contaminated sites and were among the highest documented in amphibians. In the first, but not in the second, locomotion trial, we found a significant effect of Hg on speed and responsiveness. In the prey capture experiment, reference salamanders ate approximately twice as many prey items as the contaminated salamanders. Together, our results suggest that sublethal Hg concentrations may negatively affect salamanders by reducing their ability to successfully execute tasks critical to survival. Future work is warranted to determine whether Hg has other sublethal effects on salamanders and whether other amphibians are similarly affected.

Influence of feeding ecology on blood mercury concentrations in four species of turtles.

Mercury is a relatively well-studied pollutant because of its global distribution, toxicity, and ability to bioaccumulate and biomagnify in food webs: however, little is known about bioaccumulation and toxicity of Hg in turtles. Total Hg (THg) concentrations in blood were determined for 552 turtles representing four different species (Chelydra serpentina, Sternotherus odoratus, Chrysemys picta, and Pseudemys rubriventris) from a Hg-contaminated site on the South River (VA, USA) and upstream reference sites. Methylmercury and Se concentrations also were determined in a subset of samples. Because the feeding ecology of these species differs drastically, stable isotopes of carbon (delta13C) and nitrogen (delta15N) were employed to infer the relationship between relative trophic position and Hg concentrations. Significant differences were found among sites and species, suggesting that blood can be used as a bioindicator of Hg exposure in turtles. We found differences in THg concentrations in turtles from the contaminated site that were consistent with their known feeding ecology: C. serpentina > or = S. odoratus > C. picta > P. rubriventris. This trend was generally supported by the isotope data, which suggested that individual turtles were feeding at more than one trophic level. Methylmercury followed similar spatial patterns as THg and was the predominant Hg species in blood for all turtles. Blood Se concentrations were low in the system, but a marginally positive relationship was found between THg and Se when species were pooled. The blood THg concentrations for the turtles in the present study are some of the highest reported in reptiles, necessitating further studies to investigate potential adverse effects of these high concentrations.