Deciphering structure and aggregation in asphaltenes: hypothesis-driven design and development of synthetic model compounds.

Asphaltenes comprise the heaviest and least understood fraction of crude petroleum. The asphaltenes are a diverse and complex mixture of organic and organometallic molecules in which most of the molecular constituents are tightly aggregated into more complicated suprastructures. The bulk properties of asphaltenes arise from a broad range of polycyclic aromatics, heteroatoms, and polar functional groups. Despite much analytical effort, the precise molecular architectures of the material remain unresolved. To understand asphaltene characteristics and reactivity, the field has turned to synthetic model compounds that mirror asphaltene structure, aggregation behavior, and thermal chemistry, including the nucleation of coke. Historically, molecular asphaltene modeling was limited to commercial compounds, offering little illumination and few opportunities for hypothesis-driven research. More recently, however, rational molecular design and modern organic synthesis have started to impact this area. This review provides an overview of commercially available model compounds but is principally focused on the design and synthesis of structurally advanced and appropriately functionalized compounds to mimic the physical and chemical behavior of asphaltenes. Efforts to model asphaltene aggregation are briefly discussed, and a prognosis for the field is offered. A referenced tabulation of the synthetic compounds reported to date is provided.

Enantioselective Synthesis of Polycyclic γ-Lactams with Multiple Chiral Carbon Centers via Ni(0)-Catalyzed Asymmetric Carbonylative Cycloadditions without Stirring.

γ-Lactam derivatives with multiple contiguous stereogenic carbon centers are ubiquitous in physiologically active compounds. The development of straightforward and reliable synthetic routes to such chiral structural motifs in a stereocontrolled manner should thus be of importance. Herein, we report a strategy to construct polycyclic γ-lactam derivatives that contain more than two contiguous stereogenic centers in an enantioselective as well as atom-economic manner. Moreover, we have achieved the first enantioselective synthesis of strigolactam derivative GR-24, a racemic variant of which is a potential seed germination stimulator and plant-growth regulator. A key of the procedure presented here is a nickel(0)/chiral phosphoramidite-catalyzed asymmetric [2+2+1] carbonylative cycloaddition between readily accessible ene-imines and carbon monoxide, which proceeded enantioselectively to furnish up to 90% ee (>99% ee after recrystallization). The results of mechanistic studies, including the isolation of a chiral heteronickelacycle, support that the enantioselectivity on the two contiguous carbon atoms of the γ-lactams is determined during the oxidative cyclization on nickel(0).

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.

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.

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.

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.

Hepatic and renal trace element concentrations in American alligators (Alligator mississippiensis) following chronic dietary exposure to coal fly ash contaminated prey.

Little is known about the propensity of crocodilians to bioaccumulate trace elements as a result of chronic dietary exposure. We exposed 36 juvenile alligators (Alligator mississippiensis) to one of four dietary treatments that varied in the relative frequency of meals containing prey from coal combustion waste (CCW)-contaminated habitats vs. prey from uncontaminated sites, and evaluated tissue residues and growth rates after 12 mo and 25 mo of exposure. Hepatic and renal concentrations of arsenic (As), cadmium (Cd) and selenium (Se) varied significantly among dietary treatment groups in a dose-dependent manner and were higher in kidneys than in livers. Exposure period did not affect Se or As levels but Cd levels were significantly higher after 25 mo than 12 mo of exposure. Kidney As and Se levels were negatively correlated with body size but neither growth rates nor body condition varied significantly among dietary treatment groups. Our study is among the first to experimentally examine bioaccumulation of trace element contaminants in crocodilians as a result of chronic dietary exposure. A combination of field surveys and laboratory experiments will be required to understand the effects of different exposure scenarios on tissue residues, and ultimately link these concentrations with effects on individual health.

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.

Steroid-Derived Naphthoquinoline Asphaltene Model Compounds: Hydriodic Acid Is the Active Catalyst in I2-Promoted Multicomponent Cyclocondensation Reactions.

A multicomponent cyclocondensation reaction between 2-aminoanthracene, aromatic aldehydes, and 5-α-cholestan-3-one has been used to synthesize model asphaltene compounds. The active catalyst for this reaction has been identified as hydriodic acid, which is formed in situ from the reaction of iodine with water, while iodine is not a catalyst under anhydrous conditions. The products, which contain a tetrahydro[4]helicene moiety, are optically active, and the stereochemical characteristics have been examined by VT-NMR and VT-CD spectroscopies, as well as X-ray crystallography.

Multi-Level Effects of Low Dose Rate Ionizing Radiation on Southern Toad, Anaxyrus [Bufo] terrestris.

Despite their potential vulnerability to contaminants from exposure at multiple life stages, amphibians are one of the least studied groups of vertebrates in ecotoxicology, and research on radiation effects in amphibians is scarce. We used multiple endpoints to assess the radiosensitivity of the southern toad (Anaxyrus [Bufo] terrestris) during its pre-terrestrial stages of development -embryonic, larval, and metamorphic. Toads were exposed, from several hours after oviposition through metamorphosis (up to 77 days later), to four low dose rates of 137Cs at 0.13, 2.4, 21, and 222 mGy d-1, resulting in total doses up to 15.8 Gy. Radiation treatments did not affect hatching success of embryos, larval survival, or the length of the larval period. The individual family variation in hatching success of embryos was larger than the radiation response. In contrast, newly metamorphosed individuals from the higher dose-rate treatments had higher mass and mass/length body indices, a measure which may relate to higher post-metamorphic survival. The increased mass and index at higher dose rates may indicate that the chronic, low dose rate radiation exposures triggered secondary responses. Additionally, the increases in growth were linked to a decrease in DNA damage (as measured by the Comet Assay) in red blood cells at a dose rate of 21 mGy d-1 and a total dose of 1.1 Gy. In conclusion, the complex effects of low dose rates of ionizing radiation may trigger growth and cellular repair mechanisms in amphibian larvae.

Development of an on-animal separation-based sensor for monitoring drug metabolism in freely roaming sheep.

The development of an on-animal separation-based sensor that can be employed for monitoring drug metabolism in a freely roaming sheep is described. The system consists of microdialysis sampling coupled to microchip electrophoresis with electrochemical detection (MD-ME-EC). Separations were accomplished using an all-glass chip with integrated platinum working and reference electrodes. Discrete samples from the microdialysis flow were introduced into the electrophoresis chip using a flow-gated injection approach. Electrochemical detection was accomplished in-channel using a two-electrode isolated potentiostat. Nitrite was separated by microchip electrophoresis using reverse polarity and a run buffer consisting of 50 mM phosphate at pH 7.4. The entire system was under telemetry control. The system was first tested with rats to monitor the production of nitrite following perfusion of nitroglycerin into the subdermal tissue using a linear probe. The data acquired using the on-line MD-ME-EC system were compared to those obtained by off-line analysis using liquid chromatography with electrochemical detection (LC-EC), using a second microdialysis probe implanted parallel to the first probe in the same animal. The MD-ME-EC device was then used on-animal to monitor the subdermal metabolism of nitroglycerin in sheep. The ultimate goal is to use this device to simultaneously monitor drug metabolism and behavior in a freely roaming animal.

Scalable, chromatography-free synthesis of alkyl-tethered pyrene-based materials. Application to first-generation "archipelago model" asphaltene compounds.

In this paper, we report a highly efficient, scalable approach to the total synthesis of conformationally unrestricted, electronically isolated arrays of alkyl-tethered polycyclic aromatic chromophores. This new class of modular molecules consists of polycyclic aromatic "islands" comprising significant structural fragments present in unrefined heavy petroleum, tethered together by short saturated alkyl chains, as represented in the "archipelago model" of asphaltene structure. The most highly branched archipelago compounds reported here share an architecture with first-generation dendrimeric constructs, making the convergent, chromatography-free synthesis described herein particularly attractive for further extensions in scope and applications to materials chemistry. The syntheses are efficient, selective, and readily adaptable to a multigram scale, requiring only inexpensive, "earth-abundant" transition-metal catalysts for cross-coupling reactions and extraction and fractional crystallization for purification. This approach avoids typical limitations in cost, scale, and operational practicality. All of the archipelago compounds and synthetic intermediates have been fully characterized spectroscopically and analytically. The solid-state structure of one archipelago model compound has been determined by X-ray crystallography.

Lethal and sublethal measures of chronic copper toxicity in the eastern narrowmouth toad, Gastrophryne carolinensis.

Many metals are acutely toxic to aquatic organisms at high concentrations and for some metals, such as copper (Cu), even low-level chronic contamination may be cause for conservation concern. Amphibian susceptibility to Cu has been examined in only a few species, and susceptibility is highly variable. The lethal and sublethal effects were examined of chronic aqueous Cu exposure on embryonic and larval eastern narrowmouth toads, Gastrophryne carolinensis. Copper levels as low as 10 µg Cu/L reduced embryonic and larval survival. Embryonic survivorship varied within- and between-source populations, with embryos derived from uncontaminated-wetland parents having greater survival at lower Cu levels than embryos from parents from a metal-contaminated constructed wetland. At 30 µg/L, embryos from the contaminated site had greater survival. Overall survival from oviposition to metamorphosis was 68.9% at 0 µg/L and 5.4% at 10 µg/L. Similarly, embryos exposed to ≥50 µg/L demonstrated developmental delays in transition from embryo to free-swimming larva. These results demonstrate a negative population-specific response to environmentally relevant levels of Cu.

Effects of copper exposure on hatching success and early larval survival in marbled salamanders, Ambystoma opacum.

The creation of wetlands, such as urban and industrial ponds, has increased in recent decades, and these wetlands often become enriched in pollutants over time. One metal contaminant trapped in created wetlands is copper (Cu(2+)). Copper concentrations in sediments and overlying water may affect amphibian species that breed in created wetlands. The authors analyzed the Cu concentration in dried sediments from a contaminated wetland and the levels of aqueous Cu released after flooding the sediments with different volumes of water, mimicking low, medium, and high pond-filling events. Eggs and larvae of Ambystoma opacum Gravenhorst, a salamander that lays eggs on the sediments in dry pond beds that hatch on pond-filling, were exposed to a range of Cu concentrations that bracketed potential aqueous Cu levels in created wetlands. Embryo survival varied among clutches, but increased Cu levels did not affect embryo survival. At Cu concentrations of 500 µg/L or greater, however, embryos hatched earlier, and the aquatic larvae died shortly after hatching. Because Cu concentrations in sediments increase over time in created wetlands, even relatively tolerant species such as A. opacum may be affected by Cu levels in the posthatching environment.

32 species validation of a new Illumina paired-end approach for the development of microsatellites.

Development and optimization of novel species-specific microsatellites, or simple sequence repeats (SSRs) remains an important step for studies in ecology, evolution, and behavior. Numerous approaches exist for identifying new SSRs that vary widely in terms of both time and cost investments. A recent approach of using paired-end Illumina sequence data in conjunction with the bioinformatics pipeline, PAL_FINDER, has the potential to substantially reduce the cost and labor investment while also improving efficiency. However, it does not appear that the approach has been widely adopted, perhaps due to concerns over its broad applicability across taxa. Therefore, to validate the utility of the approach we developed SSRs for 32 species representing 30 families, 25 orders, 11 classes, and six phyla and optimized SSRs for 13 of the species. Overall the IPE method worked extremely well and we identified 1000s of SSRs for all species (mean = 128,485), with 17% of loci being potentially amplifiable loci, and 25% of these met our most stringent criteria designed to that avoid SSRs associated with repetitive elements. Approximately 61% of screened primers yielded strong amplification of a single locus.

Microdialysis sampling coupled to microchip electrophoresis with integrated amperometric detection on an all-glass substrate.

The development of an all-glass separation-based sensor using microdialysis coupled to microchip electrophoresis with amperometric detection is described. The system includes a flow-gated interface to inject discrete sample plugs from the microdialysis perfusate into the microchip electrophoresis system. Electrochemical detection was accomplished with a platinum electrode in an in-channel configuration using a wireless electrically isolated potentiostat. To facilitate bonding around the in-channel electrode, a fabrication process was employed that produced a working and a reference electrode flush with the glass surface. Both normal and reversed polarity separations were performed with this sensor. The system was evaluated in vitro for the continuous monitoring of the production of hydrogen peroxide from the reaction of glucose oxidase with glucose. Microdialysis experiments were performed using a BASi loop probe with an overall lag time of approximately five minutes and a rise time of less than 60 seconds.

Within- and among-population level differences in response to chronic copper exposure in southern toads, Anaxyrus terrestris.

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.

Maternal transfer of contaminants and reduced reproductive success of southern toads ( Bufo [Anaxyrus] terrestris ) exposed to coal combustion waste.

Bioaccumulation of contaminants and subsequent maternal transfer to offspring are important factors that affect the reproductive success of wildlife. However, maternal transfer of contaminants has rarely been investigated in amphibians. We examined maternal transfer of trace elements in southern toads ( Bufo[Anaxyrus] terrestris) residing in two locations: (1) an active coal combustion waste (CCW) disposal basin and adjacent 40-ha floodplain contaminated with CCW over 35 years ago and (2) an uncontaminated reference site. Our study is among the few to document tissue concentration-dependent maternal transfer of contaminants and associated adverse effects in amphibians. We found that females collected from the CCW-contaminated area had elevated concentrations of Ni, Se, and Sr; these females also transferred elevated levels of Cu, Pb, Se, and Sr to their eggs compared to females from the reference site. Overall reproductive success, estimated as a function of clutch size and offspring viability, was reduced by 27% in clutches collected from parents from the contaminated site compared to the reference site. Offspring viability negatively correlated with female and/or egg concentrations of Se and Ni. Reproductive success negatively correlated with Se and Cu concentrations in females, and Se concentrations in eggs. Our study highlights how exposure to CCW can negatively affect amphibian reproduction.