Fundamentals of wildlife dosimetry and lessons learned from a decade of measuring external dose rates in the field.

Methods for determining the radiation dose received by exposed biota require major improvements to reduce uncertainties and increase precision. We share our experiences in attempting to quantify external dose rates to free-ranging wildlife using GPS-coupled dosimetry methods. The manuscript is a primer on fundamental concepts in wildlife dosimetry in which the complexities of quantifying dose rates are highlighted, and lessons learned are presented based on research with wild boar and snakes at Fukushima, wolves at Chornobyl, and reindeer in Norway. GPS-coupled dosimeters produced empirical data to which numerical simulations of external dose using computer software were compared. Our data did not support a standing paradigm in risk analyses: Using averaged soil contaminant levels to model external dose rates conservatively overestimate the dose to individuals within a population. Following this paradigm will likely lead to misguided recommendations for risk management. The GPS-dosimetry data also demonstrated the critical importance of how modeled external dose rates are impacted by the scale at which contaminants are mapped. When contaminant mapping scales are coarse even detailed knowledge about each animal's home range was inadequate to accurately predict external dose rates. Importantly, modeled external dose rates based on a single measurement at a trap site did not correlate to actual dose rates measured on free ranging animals. These findings provide empirical data to support published concerns about inadequate dosimetry in much of the published Chernobyl and Fukushima dose-effects research. Our data indicate that a huge portion of that literature should be challenged, and that improper dosimetry remains a significant source of controversy in radiation dose-effect research.

Mercury Exposure Is Associated with Urbanization but not Salmonella Status in White Ibis (Eudocimus albus).

Mercury (Hg) is a ubiquitous contaminant in wetlands that can cause immunosuppression in birds, which may increase susceptibility to colonization with Salmonella spp. Previously, we found that White Ibis (Eudocimus albus), a recently urbanized wading bird, shed Salmonella spp. at a higher prevalence when captured at urban sites, compared with natural sites. In this study, we sought to determine if Hg burdens in ibis are related to Salmonella status or degree of urbanization or both. We analyzed feathers from 94 ibis in Palm Beach County, Florida, USA, along an urbanization gradient (0-68% urbanization) and from individuals with confirmed Salmonella spp. status (shedding or not shedding). We detected Hg in all ibis feathers (0.22-8.47 mg/kg; mean=1.96 mg/kg; SD=1.94). The Hg concentration was not significantly correlated to Salmonella spp. shedding status (Wilcoxon rank sum test, W=1170; P=0.596) but was negatively associated with capture site urbanization level (R2=0.327; P=0.026). Our findings may suggest that the immunosuppressive effects of Hg do not affect Salmonella shedding in the ibis or that Hg burdens were too low to affect Salmonella shedding status. Further, ibis that were captured in high urbanization sites appeared to have a lower risk of Hg exposure than ibis that were captured within low urbanization sites.

Assessing springtime vertebrate prey of sympatric mesopredators in the southeastern United States using metabarcoding analysis.

Coyotes (Canis latrans) colonized the eastern United States over the last century and formed a 3-species predator guild with bobcats (Lynx rufus) and gray foxes (Urocyon cinereoargenteus) across much of the southeastern United States. Diets among the three species vary along with respective impacts on game species such as white-tailed deer (Odocoileus virginianus) and wild turkeys (Meleagris gallopavo). To determine predation impacts on vertebrate prey and dietary overlap in consumption of prey items, we assessed diets of coyote, bobcat, and gray fox during spring, coinciding with white-tailed deer fawning and wild turkey nesting and brood rearing. We sampled across three sites along the Savannah River in South Carolina from mid-May through mid-June of 2020-2021. We collected 180 scat samples along 295.9 kilometers (71.1-122.4 km/site) of unpaved secondary roads and used DNA metabarcoding to determine vertebrate diet items. We identified predator species of scat using DNA metabarcoding and species-specific mtDNA fragment analysis (153 were coyote, 20 bobcat, and seven gray fox). Overall, we found evidence that two species, coyote and bobcat, consumed deer while all three consumed turkeys. Frequency of deer in the diet varied across sites for coyotes from 62-86% and wild turkey was present with a frequency of occurrence of 9% for coyotes, 5% for bobcats, and 14% for gray fox. Vertebrate diet specialization was evident across predator species with high frequency of deer in coyote diets, rabbits and small mammals in bobcat diets, and herpetofauna in gray fox diets. During deer fawning and wild turkey nesting and brood rearing, dietary overlap appears to be mediated by disparate selection of prey items, which reduced competition among coyotes, bobcats, and gray foxes. Use of DNA metabarcoding may augment our understanding of dietary preferences within this predator guild by providing increased resolution of diet composition among important game species.

Divergence in heritable life history traits suggests potential for local adaptation and trade-offs associated with a coal ash disposal site.

Globally, human activities have resulted in rapid environmental changes that present unique challenges for wildlife. However, investigations of local adaptation in response to simultaneous exposure to multiple anthropogenic selection pressures are rare and often generate conflicting results. We used an in situ reciprocal transplant design within a quantitative genetic framework to examine how adaptive evolution and phenotypic plasticity contribute to the persistence of an amphibian population inhabiting an environment characterized by high levels of multiple toxic trace elements. We found evidence of phenotypic divergence that is largely consistent with local adaptation to an environment contaminated with multiple chemical stressors, tied to potential trade-offs in the absence of contaminants. Specifically, the population derived from the contaminated environment had a reduced risk of mortality and greater larval growth and in the contaminated environment, relative to offspring from the naïve population. Further, while survival in the uncontaminated environment was not compromised in offspring from the contaminant-exposed population, they did show delayed development and reduced growth rates over larval development, relative to the naïve population. We found no evidence of reduced additive genetic variation in the contaminant-exposed population, suggesting long-term selection in a novel environment has not reduced the evolutionary potential of that population. We also saw little evidence that past selection in the ASH environment had reduced trait plasticity in the resident population. Maternal effects were prominent in early development, but we did not detect any trends suggesting these effects were associated with the maternal transfer of toxic trace elements. Our results demonstrate the potential for adaptation to multiple contaminants in a wild amphibian population, which may have facilitated long-term persistence in a heavily impacted environment.

Isolation and characterization of 13 microsatellite loci for the Neotropical otter, Lontra longicaudis, by next generation sequencing.

The Neotropical otter, Lontra longicaudis, is an ecologically important species for freshwater ecosystems that is threatened due to habitat destruction and hunting. However, there is limited information regarding the population sizes, genetic diversity, genetic structure and gene flow of the species, which is crucial for the elaboration of conservation plans. The aim of this study was to isolate and characterize microsatellites for L. longicaudis, using Illumina paired-end-sequencing. Initial amplification tests were performed in 48 loci, out of which, 13 yielded high-quality PCR products and thus were further evaluated. Genetic diversity and discrimination power of the 13 microsatellite loci was assessed using 19 non-invasive samples collected in the Jamapa basin in Veracruz, Mexico and blood samples from six captive individuals. All loci were polymorphic, the number of alleles per locus ranged from 4 to 10, the observed heterozygosity from 0.21 to 0.69, and the expected heterozygosity from 0.55 to 0.82. The combined set of 13 microsatellites showed a high power for discriminating among individuals (probability of identity PID = 1.551 × 10-16) and among siblings (probability of identity of siblings PIDSIB = 3.349 × 10-06). A combination of nine loci are sufficient to discriminate among siblings with high confidence (PIDSIB < 0.0001). The new set of microsatellites for the Neotropical otter reported here will provide a useful genetic tool to assess population genetic patterns and ecological parameters of the species.

Mating dynamics and multiple paternity in a long-lived vertebrate.

Multiple paternity is relatively common across diverse taxa; however, the drivers and implications related to paternal and maternal fitness are not well understood. Several hypotheses have been offered to explain the occurrence and frequency of multiple paternity. One set of hypotheses seeks to explain multiple paternity through direct and indirect benefits including increased genetic diversity or enhanced offspring fitness, whereas another set of hypotheses explains multiple paternity as a by-product of sexual conflict and population-specific parameters such as density. Here, we investigate mating system dynamics in a historically studied population of the American alligator (Alligator mississippiensis) in coastal South Carolina. We examine parentage in 151 nests across 6 years and find that 43% of nests were sired by multiple males and that male reproductive success is strongly influenced by male size. Whereas clutch size and hatchling size did not differ between singly sired and multiply sired nests, fertility rates were observed to be lower in multiply sired clutches. Our findings suggest that multiple paternity may exert cost in regard to female fitness, and raise the possibility that sexual conflict might influence the frequency of multiple paternity in wild alligator populations.

Acute toxicity of copper to the larval stage of three species of ambystomatid salamanders.

Copper (Cu) appears to be consistently more toxic to anuran species relative to other vertebrate taxa. There are limited Cu toxicity data for salamanders; of the few studies conducted on salamanders, most examined Cu effects on the embryonic, but not the larval, stage. We performed acute toxicity experiments, to quantify LC50s, on Harrison stage 46 larvae (free swimming hatchlings with egg yolk completely absorbed) of three ambystomatid salamander species. Each LC50 experiment used exposure concentrations of 10, 20, 30, 40, 50, and 60 µg/L with 10 replicates per concentration each containing one larva. We found very high toxicity for all species compared to previously published research on the embryo stage. Specifically, the 4-d LC50s for Ambystoma tigrinum and A. opacum were 35.3 and 18.73 µg/L, respectively. The same Cu concentrations caused similar toxicity to A. talpoideum (LC50 = 47.88 µg/L), but exposures required up to 48 d to elicit the same level of mortality. A time-to-event analysis indicated that time to mortality was significantly affected by Cu concentration. Additionally, for A. talpoideum, we observed that elevated levels of Cu decreased growth rate. Comparisons with previously reported Cu toxicity for embryos suggest that, as with fish, Cu may be more toxic to larval salamander stages than for embryos. Further, our data suggest that Cu is an important environmental contaminant that deserves increased scrutiny on the potential for population-level effects where contamination has occurred in wetlands and streams inhabited by salamanders.

GPS-coupled contaminant monitors on free-ranging Chernobyl wolves challenge a fundamental assumption in exposure assessments.

Measurements of external contaminant exposures on individual wildlife are rare because of difficulties in using contaminant monitors on free-ranging animals. Most wildlife contaminant exposure data are therefore simulated with computer models. Rarely are empirical exposure data available to verify model simulations, or to test fundamental assumptions inherent in exposure assessments. We used GPS-coupled contaminant monitors to quantify external exposures to individual wolves (Canis lupus) living within the Belarus portion of Chernobyl's 30-km exclusion zone. The study provided data on animal location and contaminant exposure every 35 min for 6 months, resulting in ~6600 individual locations and 137Cs external exposure readings per wolf, representing the most robust external exposure data published to date on free ranging animals. The data provided information on variation in external exposure for each animal over time, as well as variation in external exposure among the eight wolves across the landscape of Chernobyl. The exposure data were then used to test a fundamental assumption in screening-level risk assessments, espoused in guidance documents of the U.S. Environmental Protection Agency and U.S. Department of Energy, - Mean contaminant concentrations conservatively estimate individual external exposures. We tested this assumption by comparing our empirical data to a series of simulations using the ERICA modeling tool. We found that modeled simulations of mean external exposure (10.5 mGy y-1), based on various measures of central tendency, under-predicted mean exposures measured on five of the eight wolves wearing GPS-contaminant monitors (i.e., 12.3, 26.3, 28.0, 28.8 and 35.7 mGy y-1). If under-prediction of exposure occurs for some animals, then arguably the use of averaged contaminant concentrations to predict external exposure is not as conservative as proposed by current risk assessment guidance. Thus, a risk assessor's interpretation of simulated exposures in a screening-level risk analysis might be misguided if contaminant concentrations are based on measures of central tendency. We offer three suggestions for risk assessors to consider in order to reduce the probability of underestimating exposure in a screening-level risk assessment.

Variation in metal tolerance associated with population exposure history in Southern toads (Anaxyrus terrestris).

Human activities have radically shaped the global landscape, affecting the structure and function of ecosystems. Habitat loss is one of the most visible changes to the landscape and a primary driver of species declines; however, anthropogenic environmental contamination also threatens population persistence, but is not as readily observed. Aquatic organisms are especially susceptible to chemical perturbations, which can negatively impact survival and fitness related traits. Some populations have evolved tolerance to chemical stressors, which could mitigate the consequences associated with contamination. Amphibians are experiencing global declines due to multiple stressors and are particularly at risk to aquatic chemical stressors due to their permeable skin and reliance on wetlands for reproduction and larval development. However, amphibians also have substantial plasticity in response to environmental variation. We designed our study to examine whether tolerance to heavy metals is greater in Southern toad (Anaxyrus terrestris) larvae from wetlands with a history of contamination. Considering many of the most common trace elements elicit acute toxicity by disrupting osmotic- and ionic-regulation, we hypothesized that alterations to these aspects of physiology resulting from multigenerational exposure to trace element mixtures would be the most likely routes by which tolerance would evolve. We used copper (Cu) as a proxy for heavy metal exposure because it is a widely distributed aquatic stressor known to cause osmotic stress that can also cause mortality at levels commonly encountered in the environment. We found considerable within and among population variation in Cu tolerance, as measured by time to death. Larvae from populations living in sites contaminated with mixtures of heavy metals associated with coal fly ash were no more tolerant to Cu than those from reference sites. However, larvae from a population inhabiting a constructed wetland complex with high Cu levels were significantly more tolerant; having half the risk of mortality as reference animals. This wetland complex was created < 20 years ago, thus if elevated Cu tolerance in this population is due to selection in the aquatic habitat, such adaptation may occur rapidly (i.e. ∼10 generation). Our results provide evidence that amphibians may be able to evolve tolerance in response to trace element contamination, though such tolerance may be specific to the combination of contaminants present.

Delayed effects and complex life cycles: How the larval aquatic environment influences terrestrial performance and survival.

Species with complex life cycles are susceptible to environmental stressors across life stages, but the carryover and latent effects between stages remain understudied. For species with biphasic life histories, such as pond-breeding amphibians, delayed effects of aquatic conditions can influence terrestrial juveniles and adults directly or indirectly, usually mediated through fitness correlates such as body size. We collected adult southern toads (Anaxyrus terrestris) from 2 source populations-a natural reference wetland and a metal-contaminated industrial wetland-and exposed their offspring to 2 aquatic stressors (a metal contaminant, copper [Cu], and a dragonfly predator cue) in outdoor mesocosms (n = 24). We then reared metamorphs in terraria for 5 mo to examine delayed effects of early life stage environmental conditions on juvenile performance, growth, and survival. Larval exposure to Cu, as well as having parents from a contaminated wetland, resulted in smaller size at metamorphosis-a response later negated by compensatory growth. Although Cu exposure and parental source did not affect larval survival, we observed latent effects of these stressors on juvenile survival, with elevated Cu conditions and metal-contaminated parents reducing postmetamorphic survival. Parental source and larval Cu exposure affected performance at metamorphosis through carryover effects on body size but, 1 mo later, latent effects of parental source and larval predator exposure directly (i.e., not via body size) influenced performance. The carryover and latent effects of parental source population and aquatic Cu level on postmetamorphic survival and juvenile performance highlight the importance of conducting studies across life stages and generations. Environ Toxicol Chem 2018;37:2660-2669. © 2018 SETAC.

SURVEY OF AQUATIC TURTLES ON THE SAVANNAH RIVER SITE, SOUTH CAROLINA, USA, FOR PREVALENCE OF RANAVIRUS.

: Ranaviruses have the ability to infect amphibians, fish, and reptiles, and they have caused multiple amphibian die-off events in the US and Europe. Their prevalence in amphibian populations is much more commonly studied than in chelonian populations. We examined blood samples ( n=286) from eight aquatic turtle species collected during 2008-14 on the Savannah River Site, South Carolina, US, as part of long-term mark-recapture efforts. Previous studies in the southeastern US found high prevalence of Ranavirus in amphibians, but we did not detect Ranavirus in any of the turtles sampled, suggesting the absence of the virus or its presence at a very low prevalence in aquatic turtles across the Savannah River Site during the years tested.

Conservation genetics of the eastern yellow-bellied racer (Coluber constrictor flaviventris) and bullsnake (Pituophis catenifer sayi): River valleys are critical features for snakes at northern range limits.

On the North American Great Plains, several snake species reach their northern range limit where they rely on sparsely distributed hibernacula located in major river valleys. Independent colonization histories for the river valleys and barriers to gene flow caused by the lack of suitable habitat between them may have produced genetically differentiated snake populations. To test this hypothesis, we used 10 microsatellite loci to examine the population structure of two species of conservation concern in Canada: the eastern yellow-bellied racer (Coluber constrictor flaviventris) and bullsnake (Pituophis catenifer sayi) in 3 major river valleys in southern Saskatchewan. Fixation indices (FST) showed that populations in river valleys were significantly differentiated for both species (racers, FST = 0.096, P = 0.001; bullsnakes FST = 0.045-0.157, P = 0.001). Bayesian assignment (STRUCTURE) and ordination (DAPC) strongly supported genetically differentiated groups in the geographically distinct river valleys. Finer-scale subdivision of populations within river valleys was not apparent based on our data, but is a topic that should be investigated further. Our findings highlight the importance of major river valleys for snakes at the northern extent of their ranges, and raise the possibility that populations in each river valley may warrant separate management strategies.

Relationships of mercury concentrations across tissue types, muscle regions and fins for two shark species.

Mercury (Hg) exposure poses a threat to both fish and human health. Sharks are known to bioaccumulate Hg, however, little is known regarding how Hg is distributed between different tissue groups (e.g. muscle regions, organs). Here we evaluated total mercury (THg) concentrations from eight muscle regions, four fins (first dorsal, left and right pectorals, caudal-from both the inner core and trailing margin of each fin), and five internal organs (liver, kidney, spleen, heart, epigonal organ) from two different shark species, bonnethead (Sphyrna tiburo) and silky shark (Carcharhinus falciformis) to determine the relationships of THg concentrations between and within tissue groups. Total Hg concentrations were highest in the eight muscle regions with no significant differences in THg concentrations between the different muscle regions and muscle types (red and white). Results from tissue collected from any muscle region would be representative of all muscle sample locations. Total Hg concentrations were lowest in samples taken from the fin inner core of the first dorsal, pectoral, and caudal (lower lobe) fins. Mercury concentrations for samples taken from the trailing margin of the dorsal, pectoral, and caudal fins (upper and lower lobe) were also not significantly different from each other for both species. Significant relationships were found between THg concentrations in dorsal axial muscle tissue and the fin inner core, liver, kidney, spleen and heart for both species as well as the THg concentrations between the dorsal fin trailing margin and the heart for the silky shark and all other sampled tissue types for the bonnethead shark. Our results suggest that biopsy sampling of dorsal muscle can provide data that can effectively estimate THg concentrations in specific organs without using more invasive, or lethal methods.

Genomic data detect corresponding signatures of population size change on an ecological time scale in two salamander species.

Understanding the demography of species over recent history (e.g. <100 years) is critical in studies of ecology and evolution, but records of population history are rarely available. Surveying genetic variation is a potential alternative to census-based estimates of population size, and can yield insight into the demography of a population. However, to assess the performance of genetic methods, it is important to compare their estimates of population history to known demography. Here, we leveraged the exceptional resources from a wetland with 37 years of amphibian mark-recapture data to study the utility of genetically based demographic inference on salamander species with documented population declines (Ambystoma talpoideum) and expansions (A. opacum), patterns that have been shown to be correlated with changes in wetland hydroperiod. We generated ddRAD data from two temporally sampled populations of A. opacum (1993, 2013) and A. talpoideum (1984, 2011) and used coalescent-based demographic inference to compare alternate evolutionary models. For both species, demographic model inference supported population size changes that corroborated mark-recapture data. Parameter estimation in A. talpoideum was robust to our variations in analytical approach, while estimates for A. opacum were highly inconsistent, tempering our confidence in detecting a demographic trend in this species. Overall, our robust results in A. talpoideum suggest that genome-based demographic inference has utility on an ecological scale, but researchers should also be cognizant that these methods may not work in all systems and evolutionary scenarios. Demographic inference may be an important tool for population monitoring and conservation management planning.

Integrating copper toxicity and climate change to understand extinction risk to two species of pond-breeding anurans.

Chemical contamination is often suggested as an important contributing factor to amphibian population declines, but direct links are rarely reported. Population modeling provides a quantitative method to integrate toxicity data with demographic data to understand the long-term effects of contaminants on population persistence. In this study we use laboratory-derived embryo and larval toxicity data for two anuran species to investigate the potential for toxicity to contribute to population declines. We use the southern toad (Anaxyrus terrestris) and the southern leopard frog (Lithobates sphenocephalus) as model species to investigate copper (Cu) toxicity. We use matrix models to project populations through time and quantify extinction risk (the probability of quasi-extinction in 35 yr). Life-history parameters for toads and frogs were obtained from previously published literature or unpublished data from a long-term (>35 yr) data set. In addition to Cu toxicity, we investigate the role of climate change on amphibian populations by including the probability of early pond drying that results in catastrophic reproductive failure (CRF, i.e., complete mortality of all larval individuals). Our models indicate that CRF is an important parameter for both species as both were unable to persist when CRF probability was >50% for toads or 40% for frogs. Copper toxicity alone did not result in significant effects on extinction risk unless toxicity was very high (>50% reduction in survival parameters). For toads, Cu toxicity and high probability of CRF both resulted in high extinction risk but no synergistic (or greater than additive) effects between the two stressors occurred. For leopard frogs, in the absence of CRF survival was high even under Cu toxicity, but with CRF Cu toxicity increased extinction risk. Our analyses highlight the importance of considering multiple stressors as well as species differences in response to those stressors. Our models were consistently most sensitive to juvenile and adult survival, further suggesting the importance of terrestrial stages to population persistence. Future models will incorporate multiple wetlands with different combinations of stressors to understand if our results for a single wetland result in a population sink within the landscape.

Development of microsatellite markers for buffalograss (Buchloë dactyloides; Poaceae), a drought-tolerant turfgrass alternative.

PREMISE OF THE STUDY: Buchloë dactyloides (Poaceae) is an important component of Great Plains prairies and a popular drought-tolerant turfgrass alternative in North America. This species comprises an autopolyploid series, and microsatellite primers were developed to understand the distribution of genetic variation among cytotypes and across its large geographic range. METHODS AND RESULTS: Fifteen microsatellite loci were designed and successfully amplified in six B. dactyloides populations. Within-population genetic diversity was comparatively high, consistent with B. dactyloides' life history. Allelic variation at 13 loci was consistent with the cytotype established in chromosome-counted samples. CONCLUSIONS: This variable, interpretable set of loci allows for the determination of multilocus genotype in B. dactyloides individuals of varying cytotype. Data such as these from a range-wide sample set can provide important insights for germplasm conservation and crop improvement in this ecologically and economically important species.

Patterns of amphibian infection prevalence across wetlands on the Savannah River Site, South Carolina, USA.

Amphibian diseases, such as chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) and ranaviral disease caused by ranaviruses, are often linked to global amphibian population declines, yet the ecological dynamics of both pathogens are poorly understood. The goal of our study was to determine the baseline prevalence, pathogen loads, and co-infection rate of Bd and ranavirus across the Savannah River Site (SRS) in South Carolina, USA, a region with rich amphibian diversity and a history of amphibian-based research. We tested over 1000 individuals, encompassing 21 amphibian species from 11 wetlands for both Bd and ranavirus. The prevalence of Bd across individuals was 9.7%. Using wetland means, the mean (±SE) Bd prevalence was 7.9 ± 2.9%. Among toad species, Anaxyrus terrestris had 95 and 380% greater odds of being infected with Bd than Scaphiopus holbrookii and Gastrophryne carolinensis, respectively. Odds of Bd infection in adult A. terrestris and Lithobates sphenocephalus were 75 to 77% greater in metal-contaminated sites. The prevalence of ranavirus infections across all individuals was 37.4%. Mean wetland ranavirus prevalence was 29.8 ± 8.8% and was higher in post-metamorphic individuals than in aquatic larvae. Ambystoma tigrinum had 83 to 85% higher odds of ranavirus infection than A. opacum and A. talpoideum. We detected a 4.8% co-infection rate, with individuals positive for ranavirus having a 5% higher occurrence of Bd. In adult Anaxyrus terrestris, odds of Bd infection were 13% higher in ranavirus-positive animals and odds of co-infection were 23% higher in contaminated wetlands. Overall, we found the pathogen prevalence varied by wetland, species, and life stage.

Effects of metal and predator stressors in larval southern toads (Anaxyrus terrestris).

Natural and anthropogenic stressors typically do not occur in isolation; therefore, understanding ecological risk of contaminant exposure should account for potential interactions of multiple stressors. Realistically, common contaminants can also occur chronically in the environment. Because parental exposure to stressors may cause transgenerational effects on offspring, affecting their ability to cope with the same or novel environmental stressors, the exposure histories of generations preceding that being tested should be considered. To examine multiple stressor and parental exposure effects we employed a 2 × 2 × 2 factorial design in outdoor 1000-L mesocosms (n = 24). Larval southern toads (Anaxyrus terrestris), bred from parents collected from reference and metal-contaminated sites, were exposed to two levels of both an anthropogenic (copper-0, 30 µg/L Cu) and natural (predator cue - present/absent) stressor and reared to metamorphosis. Toads from the metal-contaminated parental source population were smaller at metamorphosis and had delayed development; i.e., a prolonged larval period. Similarly, larval Cu exposure also reduced size at metamorphosis and prolonged the larval period. We, additionally, observed a significant interaction between larval Cu and predator-cue exposure on larval period, wherein delayed emergence was only present in the 30-µg/L Cu treatments in the absence of predator cues. The presence of parental effects as well as an interaction between aquatic stressors on commonly measured endpoints highlight the importance of conducting multistressor studies across generations to obtain data that are more relevant to field conditions in order to determine population-level effects of contaminant exposure.

Environmental levels of Zn do not protect embryos from Cu toxicity in three species of amphibians.

Contaminants often occur as mixtures in the environment, but investigations into toxicity usually employ a single chemical. Metal contaminant mixtures from anthropogenic activities such as mining and coal combustion energy are widespread, yet relatively little research has been performed on effects of these mixtures on amphibians. Considering that amphibians tend to be highly sensitive to copper (Cu) and that metal contaminants often occur as mixtures in the environment, it is important to understand the interactive effects that may result from multiple metals. Interactive effects of Cu and zinc (Zn) on amphibians have been reported as antagonistic and, conversely, synergistic. The goal of our study was to investigate the role of Zn in Cu toxicity to amphibians throughout the embryonic developmental period. We also considered maternal effects and population differences by collecting multiple egg masses from contaminated and reference areas for use in four experiments across three species. We performed acute toxicity experiments with Cu concentrations that cause toxicity (10-200 µg/L) in the absence of other contaminants combined with sublethal concentrations of Zn (100 and 1000 µg/L). Our results suggest very few effects of Zn on Cu toxicity at these concentrations of Zn. As has been previously reported, we found that maternal effects and population history had significant influence on Cu toxicity. The explanation for a lack of interaction between Cu and Zn in this experiment is unknown but may be due to the use of sublethal Zn concentrations when previous experiments have used Zn concentrations associated with acute toxicity. Understanding the inconsistency of amphibian Cu/Zn mixture toxicity studies is an important research direction in order to create generalities that can be used to understand risk of contaminant mixtures in the environment.