Variation in copper sensitivity between laboratory and wild strains of Caenorhabditis elegans.

Ecological risk assessments of chemicals are frequently based on laboratory toxicity data from a small number of model species that may be reared in labs for years or decades. These populations can undergo many processes in the lab including artificial selection, founder effect, and genetic drift, and may not adequately represent their wild counterparts, potentially undermining the goal of protecting natural populations. Here we measure variation in lethality to copper chloride among strains of an emerging model species in toxicology, Caenorhabditis elegans. We tested four wild strains from Chile, Germany, Kenya, and Madeira (Portugal) against several versions of the standard laboratory N2 strain from Bristol, UK used in molecular biology. The four wild strains were more sensitive than any of the N2 strains tested with copper. We also found that the standard N2 strain cultured in the laboratory for >1 year was less sensitive than a recently cultured N2 strain as well as a cataloged ancestral version of the N2 strain. These results suggest that toxicologists should be cognizant of performing toxicity testing with long-held animal cultures, and should perhaps use multiple strains as well as renew cultures periodically in the laboratory. This study also shows that multi-strain toxicity testing with nematodes is highly achievable and useful for understanding variation in intra- and interspecific chemical sensitivity.

Investigating potential toxic effects of pollutants on population growth rates and probability of extinction for a representative squamate.

Chemical contamination has been suggested as an important contributing factor to reptile population declines, but direct links are rarely reported. Population modeling provides a quantitative method to understand the long-term effects of contaminants on population persistence. We created a matrix model for Sceloporus lizards and investigated hypothetical toxic effects by reducing survival and reproductive parameters by 0 to 100% in 10% increments. We report effects on population growth rate (λ) and elasticity values for each stage due to these reductions. We then incorporated stochasticity to the model to simulate the variation seen in demographic data and quantified extinction risk. The deterministic model yielded a λ of 1.07 suggesting stability in some wild Sceloporus populations. A yearly reduction of 20 to 30% in demographic parameters was needed to push λ to decline in both our deterministic and stochastic simulations. Surprisingly, our baseline stochastic simulations had a 30% extinction probability despite a stable deterministic model. We tested three adjustments to the stochastic model, (1) increased survival/fecundity parameters, (2) higher starting densities, and (3) a density-dependent juvenile survival function. The model with density-dependent juvenile growth had the lowest extinction risk. Ultimately, 20 or 30% mortality every year is likely unrealistic, but our results provide insight in linking toxicity to population effects. Ultimately, very little reduction in demographics is needed to cause declines in these populations. Our generalized models provide important tools for screening-level risk assessment of chemical contamination, especially for taxonomic groups that tend to receive less research interest.

Interspecific Variation in Nematode Responses to Metals.

Performing toxicity testing on multiple species with differing degrees of evolutionary relatedness can provide important information on how chemical sensitivity varies among species and can help pinpoint the biological drivers of species sensitivity. Such knowledge could ultimately be used to design better multispecies predictive ecological risk assessment models and identify particularly sensitive species. However, laboratory toxicity tests involving multiple species can also be resource intensive, especially when each species has unique husbandry conditions. We performed lethality tests with 2 metals, copper chloride and zinc chloride, on 5 different nematode species, which are nested in their degree of evolutionary relatedness: Caenorhabditis briggsae, Caenorhabditis elegans, Oscheius myriophila, Oscheius tipulae, and Pristionchus pacificus. All species were successfully cultured and tested concurrently with limited resources, demonstrating that inexpensive, multispecies nematode toxicity testing systems are achievable. The results indicate that P. pacificus is the most sensitive to both metals. Conversely, C. elegans is the least sensitive species to copper, but the second most sensitive to zinc, indicating that species relationships do not necessarily predict species sensitivity. Toxicity testing with additional nematode species and types of chemicals is feasible and will help form more generalizable conclusions about relative species sensitivity. Environ Toxicol Chem 2020;39:1006-1016. © 2020 SETAC.

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.

Toward Sustainable Environmental Quality: Priority Research Questions for North America.

Anticipating, identifying, and prioritizing strategic needs represent essential activities by research organizations. Decided benefits emerge when these pursuits engage globally important environment and health goals, including the United Nations Sustainable Development Goals. To this end, horizon scanning efforts can facilitate identification of specific research needs to address grand challenges. We report and discuss 40 priority research questions following engagement of scientists and engineers in North America. These timely questions identify the importance of stimulating innovation and developing new methods, tools, and concepts in environmental chemistry and toxicology to improve assessment and management of chemical contaminants and other diverse environmental stressors. Grand challenges to achieving sustainable management of the environment are becoming increasingly complex and structured by global megatrends, which collectively challenge existing sustainable environmental quality efforts. Transdisciplinary, systems-based approaches will be required to define and avoid adverse biological effects across temporal and spatial gradients. Similarly, coordinated research activities among organizations within and among countries are necessary to address the priority research needs reported here. Acquiring answers to these 40 research questions will not be trivial, but doing so promises to advance sustainable environmental quality in the 21st century. Environ Toxicol Chem 2019;38:1606-1624. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Investigating spatial patterns of mercury and rodenticide residues in raptors collected near the Charlotte, NC, USA, metropolitan area.

Raptor population growth is dynamic and trends vary across species and by location in the United States. For those species that are declining, it is important to identify potential causes including chemical contaminants. Sampling wild raptors is problematic due to their small population sizes and role as a top predator. Therefore, we obtained liver samples (n = 56) from carcasses of several raptor species, including common species like red-tailed hawks, red-shouldered hawks, barred owls, great horned owls, and osprey that arrived dead or were euthanized from a non-profit rehabilitation center in Charlotte, North Carolina. Raptors were found or collected in South Carolina, North Carolina, and Virginia, but most samples were located near the metropolitan region of Charlotte, NC. We analyzed livers for total mercury residue (mg/kg, dry weight) and five anti-coagulant rodenticides (µg/kg wet weight). Mercury was analyzed using a direct mercury analyzer approach and rodenticides were quantified by LC-MS. Mercury residues were high in piscivorous birds (15.09 mg/kg for osprey and 6.93 mg/kg for great blue herons, dry weight) and relatively high in red-shouldered hawks and one eastern screech owl tested. Six of our samples exceeded a health threshold of 1 mg/kg (wet weight) including three osprey and one each of great blue heron, red-shouldered hawk, and eastern screech owl. Brodifacoum was the only rodenticide consistently detected in our samples. Brodifacoum detections exceeded 75% in barred owls, great horned owls, and red-shouldered hawks. Sixty-nine percent of owl samples were within (or exceeded) a threshold of brodifacoum residue associated with a 10-20% risk of acute toxicity. Correlations between residues and human population density were not significant for either mercury or brodifacoum. Our data suggest that mercury residues for most raptors were not of significant concern with the exception of osprey and possibly red-shouldered hawks. Rodenticide exposures associated with a risk of acute toxicity appear to be common and warrant further investigation.

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.

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.

Insights into reptile dermal contaminant exposure: Reptile skin permeability to pesticides.

There is growing interest in improving ecological risk assessment exposure estimation, specifically by incorporating dermal exposure. At the same time, there is a growing interest in amphibians and reptiles as receptors in ecological risk assessment, despite generally receiving less research than more traditional receptors. Previous research has suggested that dermal exposure may be more important than previously considered for reptiles. We measured reptile skin permeability to four pesticides (thiamethoxam, malathion, tebuthiuron, trifluralin) using ventral skin samples. All four pesticides penetrated the skin but generally had low permeability. There was no apparent relationship between physicochemical properties and permeability coefficients. Malathion had a significantly greater permeability rate at all time points compared to the other pesticides. Tebuthiuron had a greater permeability than thiamethoxam. Reptiles and mammals appear to have similar skin permeability suggesting that dermal exposure estimates for mammals may be representative of reptiles.

Direct and indirect effects of petroleum production activities on the western fence lizard (Sceloporus occidentalis) as a surrogate for the dunes sagebrush lizard (Sceloporus arenicolus).

The dunes sagebrush lizard (Sceloporus arenicolus) is a habitat specialist of conservation concern limited to shin oak sand dune systems of New Mexico and Texas (USA). Because much of the dunes sagebrush lizard's habitat occurs in areas of high oil and gas production, there may be direct and indirect effects of these activities. The congeneric Western fence lizard (Sceloporus occidentalis) was used as a surrogate species to determine direct effects of 2 contaminants associated with oil and gas drilling activities in the Permian Basin (NM and TX, USA): herbicide formulations (Krovar and Quest) and hydrogen sulfide gas (H2S). Lizards were exposed to 2 concentrations of H2 S (30 ppm or 90 ppm) and herbicide formulations (1× or 2× label application rate) representing high-end exposure scenarios. Sublethal behavioral endpoints were evaluated, including sprint speed and time to prey detection and capture. Neither H2S nor herbicide formulations caused significant behavioral effects compared to controls. To understand potential indirect effects of oil and gas drilling on the prey base, terrestrial invertebrate biomass and order diversity were quantified at impacted sites to compare with nonimpacted sites. A significant decrease in biomass was found at impacted sites, but no significant effects on diversity. The results suggest little risk from direct toxic effects, but the potential for indirect effects should be further explored.

Improving reptile ecological risk assessment: oral and dermal toxicity of pesticides to a common lizard species (Sceloporus occidentalis).

Reptiles have been understudied in ecotoxicology, which limits consideration in ecological risk assessments. The goals of the present study were 3-fold: to improve oral and dermal dosing methodologies for reptiles, to generate reptile toxicity data for pesticides, and to correlate reptile and avian toxicity. The authors first assessed the toxicity of different dosing vehicles: 100 µL of water, propylene glycol, and acetone were not toxic. The authors then assessed the oral and dermal toxicity of 4 pesticides following the up-and-down procedure. Neither brodifacoum nor chlorothalonil caused mortality at doses ≤ 1750 µg/g. Under the "neat pesticide" oral exposure, endosulfan (median lethal dose [LD50] = 9.8 µg/g) was more toxic than λ-cyhalothrin (LD50 = 916.5 µg/g). Neither chemical was toxic via dermal exposure. An acetone dosing vehicle increased λ-cyhalothrin toxicity (oral LD50 = 9.8 µg/g; dermal LD50 = 17.5 µg/g), but not endosulfan. Finally, changes in dosing method and husbandry significantly increased dermal λ-cyhalothrin LD50s, which highlights the importance of standardized methods. The authors combined data from the present study with other reptile LD50s to correlate with available avian data. When only definitive LD50s were used in the analysis, a strong correlation was found between avian and reptile toxicity. The results suggest it is possible to build predictive relationships between avian and reptile LD50s. More research is needed, however, to understand trends associated with chemical classes and modes of action.

Unraveling the relative importance of oral and dermal contaminant exposure in reptiles: insights from studies using the western fence lizard (Sceloporus occidentalis).

Despite widespread recognition of significant data deficiencies, reptiles remain a relatively understudied taxon in ecotoxicology. To conduct ecological risk assessments on reptiles frequently requires using surrogate taxa such as birds, but recent research suggests that reptiles have significantly different exposure profiles and toxicant sensitivity. We exposed western fence lizards, Sceloporus occidentalis, to the same quantities of three model chemicals via oral (gavage) and dermal (ventral skin application) exposure for either 24 or 48 hours. Three phthalate esters (di-methyl phthalate [DMP], di-iso-butyl phthalate [DIBP], and di-n-octyl phthalate [DNOP]) were chosen as model chemicals because they represent a gradient of lipophilicity but are otherwise structurally similar. Overall, the more lipophilic phthalates (DIBP and DNOP) were found to have higher concentrations in tissues than the less lipophilic DMP. Significant differences in tissue concentrations between DIBP and DNOP were tissue-dependent, suggesting that delivery to a site of action following exposure is not only a simple function of lipophilicity. In dermal treatments, DMP usually had fewer detections (except in ventral skin samples), suggesting that lipophilicity (log Kow>2) is a requirement for uptake across the skin. In general, tissue residues were greater in oral treatments than dermal treatments (significant in adipose and liver tissue), but differences were driven strongly by differences in DMP which did not appear to be absorbed well across skin. When differences in tissue residue concentrations between oral and dermal exposure did occur, the difference was not drastic. Taken together these results suggest that dermal exposure should be considered in risk assessments for reptilian receptors. Dermal exposure may be an especially important route for reptiles as their ectothermic physiology translates to lower energetic demands and dietary exposure compared to birds and mammals.

The effects of pesticide exposure on ultraviolet-B radiation avoidance behavior in tadpoles.

Effects of contaminants on behavior may have important consequences on wildlife populations because behaviors such as predation, predator avoidance, reproduction, and social interaction can affect population dynamics. As a common environmental stressor, ultraviolet-B (UVB) radiation causes various deleterious effects and some aquatic organisms actively avoid UVB radiation in water. However, the extent to which environmental contaminants can impair UVB avoidance has not been evaluated, which may cause greater UVB exposure and toxicity. In the present study, we used Xenopus laevis tadpoles to determine if acute exposure to sublethal concentrations of agricultural chemicals can alter tadpole response to UVB radiation. We exposed tadpoles to four pesticides (malathion, endosulfan, α-cypermethrin, and chlorothalonil) for 96 h. At the end of the exposure, tadpoles were transferred to tanks divided into UVB and no-UVB areas. We observed tadpoles for 30 min and recorded time spent in the UVB area. We compared the proportion of time tadpoles spent in the UVB area among different concentrations for each pesticide. There was no significant difference between FETAX control and solvent control tadpoles. When combined, control tadpoles spent less than half of the time in the UVB area indicating that X. laevis tadpoles exhibit UVB avoidance behavior. Tadpoles exposed to 5 µg/L endosulfan spent significantly more time under UVB than control tadpoles. Other pesticides had no effect on tadpole UVB avoidance behavior. Our results suggest that some neurotoxic pesticides can affect UVB avoidance in larval amphibians, which may increase their exposure and subsequently the risk of UVB-induced damage. The present study highlights the importance of examining the interaction between two stressors that co-occur across broad spatial scales and to consider behavioral alteration when evaluating the risk of pesticides to amphibians.

Phthalate ester leachates in aquatic mesocosms: implications for ecotoxicity studies of endocrine disrupting compounds.

Aquatic mesocosm experiments have become common throughout the fields of ecology and ecotoxicology. Mesocosm containers are often composed of plastic materials as these are lighter and cheaper than steel cattle tanks. Plastics may contain phthalate esters which may leach from containers, potentially confounding experiments via toxicity or endocrine disruption. The objective of this experiment was to determine the extent to which plastic containers (="tanks") used for mesocosms leach phthalates, and if there are significant differences between tank types and phthalate profiles. We investigated four tank types: fiberglass, polyethylene, poly-vinyl chloride, and polyethylene tanks containing an established aquatic community. We measured six common phthalate esters in water samples collected from each tank every 2 weeks for 8 weeks. There was a significant effect of tank type, time, and time x type interactions on phthalate ester concentrations. Di-(2-ethylhexyl) phthalate was the predominant congener measured in all samples. Fiberglass tanks had greater concentrations of dimethyl phthalate compared to other tank types (more than 600x larger concentration), but no other differences in phthalate profiles among tank types were evident. In addition, tanks with established communities had much higher concentrations of most phthalates at the 6 and 8 week timepoints. Our study confirms that mesocosm tanks of different materials leach phthalates starting immediately after they are filled and continuing for at least 8 weeks, but do so at different rates. Future ecotoxicity experiments should consider the potentially confounding effects of phthalates that may leach from tanks used in experimental mesocosms.

Organochlorine pesticides in squamate reptiles from southern Arizona, USA.

Despite recognition of the lack of reptile ecotoxicology data, the taxon remains poorly studied. Contaminant body burdens are useful in demonstrating exposures to contaminants do occur and may provide insight regarding risks. The purpose of this study was to determine organochlorine pesticide burdens in various tissues of terrestrial reptiles opportunistically collected in Arizona. Heptachlor, DDE, and endrin were the most common analytes detected in fat samples. Liver samples contained methoxychlor and heptachlor at greater frequency than other organochlorines. Investigations into chronic low-level exposures are rare for reptiles and research is needed to determine critical body residues associated with adverse impacts.

Acute toxicity of herbicide formulations and chronic toxicity of technical-grade trifluralin to larval green frogs (Lithobates clamitans).

Fewer toxicity studies have been performed on herbicides than on insecticides despite heavier use of herbicides and evidence of herbicide formulation toxicity to amphibians. We conducted acute and chronic toxicity tests with the herbicide trifluralin (2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)aniline) on tadpoles. Herbicide formulations had lower median lethal concentrations than an insecticide formulation and technical-grade trifluralin. Chronic trifluralin exposure resulted in significantly smaller tadpoles at low concentrations (20 µg/L) compared with controls and 200-µg/L treatments.

Ecological risk of anthropogenic pollutants to reptiles: Evaluating assumptions of sensitivity and exposure.

A large data gap for reptile ecotoxicology still persists; therefore, ecological risk assessments of reptiles usually incorporate the use of surrogate species. This necessitates that (1) the surrogate is at least as sensitive as the target taxon and/or (2) exposures to the surrogate are greater than that of the target taxon. We evaluated these assumptions for the use of birds as surrogates for reptiles. Based on a survey of the literature, birds were more sensitive than reptiles in less than 1/4 of the chemicals investigated. Dietary and dermal exposure modeling indicated that exposure to reptiles was relatively high, particularly when the dermal route was considered. We conclude that caution is warranted in the use of avian receptors as surrogates for reptiles in ecological risk assessment and emphasize the need to better understand the magnitude and mechanism of contaminant exposure in reptiles to improve exposure and risk estimation.

Mercury concentrations in wetlands associated with coal-fired power plants.

There have been contradictory reports of the relative proportion of mercury from coal-fired power plants that deposits locally. Our objective was to determine any local effect of coal-fired power plants on total mercury concentrations in wetland sediment and tadpole samples. Four power plants and 45 wetlands were selected for study. Total mercury concentrations were determined in 75 sediment samples (range: 8-82 ng/g dry weight) and 100 bullfrog (Lithobates catesbeiana) and green frog (Lithobates clamitans) tadpoles (range: 5-318 ng/g wet weight). Tadpole and sediment total mercury did not significantly vary by power plant or distance from the plant. Only one power plant had a significantly greater concentration of total mercury in sediment downwind compared to upwind wetlands. A similar (but non-significant) trend was found for tadpole total mercury surrounding the same plant. Tadpole total mercury was negatively correlated with both tadpole weight and total length. Tadpole and sediment total mercury concentrations were not significantly correlated with one another. The results of the current study suggest that coal-fired power plants are not significantly affecting mercury concentrations in surrounding wetlands.