Investigating the Joint Effects of Pesticides and Ultraviolet B Radiation in Xenopus laevis and Other Amphibians.

Exposure to multiple stressors often results in higher toxicity than one stressor alone. Examining joint effects of multiple stressors could provide more realistic exposure scenarios and a better understanding of the combined effects. In amphibian toxicology, simultaneous exposure to some pesticides and ultraviolet B (UVB) radiation has been suggested to be detrimental and more harmful in amphibian early-life stages than either stressor alone. Therefore, it is important to investigate the joint effects of these two stressors and provide data that could lead to more informed risk assessment. Here we describe how to set up a co-exposure to pesticides and ultraviolet B radiation to examine their joint toxicity in amphibian embryos and larvae, focusing on Xenopus laevis with notes on other amphibian species. With modifications, the methods may be applied to other types of chemicals or other aquatic organisms of interest.

Legacy Contaminants in Aquatic Biota in a Stream Associated with Nuclear Weapons Material Production on the Savannah River Site.

Former nuclear weapons material production at the U.S. Department of Energy's Savannah River Site (SRS) has resulted in contamination of certain terrestrial and aquatic ecosystems on site with legacy wastes such as radiocesium (137Cs), tritium (3H), and metals. We collected fish and invertebrates from five beaver ponds (sites) above, adjacent, and downgradient of three SRS facilities (H-, F-, and C-Areas) to evaluate whether the accumulation of metals and radionuclides in biota were associated with specific facility operations and if the measured levels could pose risks to aquatic organisms. We compared concentrations of various metals, 137Cs, and 3H in fish, as well as in water (3H only), among sites along the stream gradient. Fish collected from sites adjacent to H-Area had significantly higher 137Cs concentrations compared to fish from other sites. Both biota and water samples indicated significantly greater levels of 3H in sites adjacent to and downstream of C-Area. Concentrations of zinc (Zn), copper (Cu), and mercury (Hg) in some samples exceeded effects levels reported for fish and may pose a risk to fish populations. This study reported fish tissue concentrations of 137Cs and 3H, which have not been documented extensively in ecotoxicological studies. Our results suggested that industrial operations such as nuclear material production at SRS could have long-lasting impact on the aquatic ecosystem via the release of radionuclides and metals, and long-term monitoring of physiological effects and population level impact in biota exposed to these contaminants are recommended.

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.

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.

Site-specific water quality criteria for aquatic ecosystems: A case study of pentachlorophenol for Tai Lake, China.

Given the widely varying types of aquatic ecosystems and bioavailability of chemicals, it is important to develop site-specific water quality criteria (WQC) to ensure criteria are neither over- nor under-protective. In the study, using pentachlorophenol (PCP) as an example, several approaches to derive site-specific WQC were investigated, including the conventional species sensitivity distribution (SSD), weighted SSD based on the proportion of each trophic level, and water effect ratio (WER) method. When corrected to a pH of 7.8, the conventional SSD approach resulted in criteria maximum concentration (CMC) and criteria continuous concentration (CCC) of 18.11 and 1.74 µg/L, respectively. If SSD was weighted according to the current species composition in Tai Lake, the CMC and CCC were 32.81 and 4.48 µg/L, respectively. However, available data suggest that many sensitive species inhabiting Tai Lake during 1980s were disappeared. Considering the species composition of the healthier ecosystem in 1980s, the CMC and CCC were 10.99 and 0.38 µg/L, respectively, which provide more protective water quality standards. Water effect ratio (WER) was further used to correct for co-occurrence of other toxicants and factors affecting bioavailability of PCP. A final WER of 4.72 was applied to adjust the criteria derived by using the weighted SSD for the 1980s aquatic community, and the final CMC and CCC obtained were 51.87 and 1.79 µg/L, respectively, at a pH of 7.8. Water quality criteria derived using the 1980s species composition and adjusted with WER were deemed the most appropriate WQC for water management and aquatic life protection. Merits of the various approaches for developing WQC for protection of aquatic species were discussed.

Joint effects of pesticides and ultraviolet-B radiation on amphibian larvae.

A combination of multiple stressors may be linked to global amphibian declines. Of these, pesticides and UVB radiation co-exposures were examined on the African clawed frog (Xenopus laevis) to provide information that may be useful for amphibian conservation. The independent action model and inferential statistics were used to examine interactions between pesticides (malathion, endosulfan, α-cypermethrin, or chlorothalonil) and environmentally relevant UVB exposures. UVB radiation alone caused 35-68% mortality and nearly 100% of malformations. Pesticides and UVB had additive effects on larval mortality; however, several non-additive effects (antagonistic and synergistic interactions) were observed for total body length. Insecticides mainly affected axial development, whereas UVB radiation caused high incidence of edema, gut malformations, and abnormal tail tips. These results suggest that sublethal developmental endpoints were more sensitive for detecting joint effects. This work has implications for amphibian risk assessments for ecosystems where pesticides and high UVB radiation may co-occur.

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.

Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos.

Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50 µg/L endosulfan or 0, 2.5, and 5.0 µg/L α-cypermethrin for 96 h, with environmentally relevant exposures of UVB radiation during the last 7 h of the 96 h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides increased transcript abundance of CSA and MUTL. In addition, mRNA abundance of HSP70 and GADD45α were increased by endosulfan and mRNA abundance of XPG was increased by α-cypermethrin. XPC, HR23B, XPG, and GADD45α exhibited elevated mRNA concentrations whereas there was a reduction in MUTL transcript concentrations in UVB-alone treatments. It appeared that even though expression of XPC and CSA were induced by exposure to UVB or pesticides, XPA was the limiting factor in the NER pathway. Our results suggest that pesticides may increase the accumulation of UVB-induced DNA photo-adducts and one likely mechanism is the alteration of critical NER gene expression. The present study provides important implications for evaluating the combined risks of pesticide usage and potentially increasing UVB radiation in aquatic ecosystems.

Distinct expression profiles of stress defense and DNA repair genes in Daphnia pulex exposed to cadmium, zinc, and quantum dots.

The ever-increasing production and use of nanocrystaline semiconductors (Quantum dots; QDs) will inevitably result in increased appearance of these nanomaterials in the aquatic environment. However, the behavior and potential toxicity of heavy metal constituted nanoparticulates in aquatic invertebrates is largely unknown, especially with regard to molecular responses. The freshwater crustacean Daphnia pulex is a well-suited toxicological and ecological model to study molecular responses to environmental stressors. In this study, D. pulex were exposed for 48 h to sublethal doses of QDs (25% and 50% of LC50) with differing spectral properties (CdTe and CdSe/ZnS QDs) and Cd and Zn salts. Our data suggest that acute exposure to both CdSO4 and Cd-based QDs leads to Cd uptake in vivo, which was biologically supported by the observation of increased expression of metallothionein (MT-1). Furthermore, Cd, Zn, and CdSe/ZnS QDs induced different patterns of gene expression regarding stress defense and DNA repair, which furthers our knowledge regarding which response pathways are affected by nanoparticulate forms of metals versus ionic forms in aquatic crustaceans.

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.

Bioaccumulation of fullerene (C60) and corresponding catalase elevation in Lumbriculus variegatus.

Fullerene (C(60)), with its unique physical properties and nanometer size, has been mass-produced for many applications in recent decades. The increased likelihood of direct release into the environment has raised interest in understanding both the environmental fate and corresponding biological effects of fullerenes to living organisms. Because few studies have emphasized fullerene uptake and resulting biochemical responses by living organisms, a toxicity screening test and a 28-d bioaccumulation test for Lumbriculus variegatus were performed. No mortality was observed in the range of 0.05 mg C(60) /kg dry sediment to 11.33 mg C(60) /kg dry sediment. A biota-sediment accumulation factor of micron-sized fullerene agglomerates (µ-C(60)) was 0.032 ± 0.008 at day 28, which is relatively low compared with pyrene (1.62 ± 0.22). Catalase (CAT) activity, an oxidative stress indicator, was elevated significantly on day 14 for L. variegatus exposed to µ-C(60) (p = 0.034). This peak CAT activity corresponded to the highest body residues observed in the present study, 199 ± 80 µg C(60) /kg dry weight sediment. Additionally, smaller C(60) agglomerate size increased bioaccumulation potential in L. variegatus. The relationship between C(60) body residue and the increased CAT activity followed a linear regression. All results suggest that C(60) has a lower bioaccumulation potential than pyrene but a higher potential to induce oxidative stress in L. variegatus.

Effects of chlorothalonil on development and growth of amphibian embryos and larvae.

Chlorothalonil is a broad spectrum fungicide widely used in agricultural and urban environments, yet little is known regarding its effects on amphibians. We examined effects of chlorothalonil on growth, malformations, and mortality in embryos and larvae of Xenopus laevis and Spea multiplicata, and assessed variation in sensitivity among aquatic organisms using a species sensitivity distribution (SSD). Chlorothalonil induced gut malformations in X. laevis embryos and inhibited growth. Tail degeneration was observed in larvae of both species and reduced tail length to total length ratios occurred at environmentally relevant concentrations (5.9 and 11.0 µg/L). The mechanism of tail degeneration is unclear, but alteration in the expression of genes involved in tail resorption is a hypothesized mechanism. Larval amphibians were more sensitive than invertebrates and fish. Based on our results and the range of reported environmental concentrations, chlorothalonil may pose a risk to larval amphibians in certain habitats and scenarios.

Correlation between heavy metals and turtle abundance in ponds near the Paducah Gaseous Diffusion Plant, Kentucky, USA.

Reptiles are declining globally, and environmental contamination has been suggested as a contributing factor; however, few studies have investigated the relationship between contamination and reptile populations. We performed a mark-recapture study at ponds near the Paducah Gaseous Diffusion Plant (PGDP), Kentucky, to determine if heavy metals had an impact on turtle populations. We measured concentrations of cadmium, chromium, copper, lead, and mercury in red-eared slider turtle (Trachemys scripta elegans) tissues and pond sediment and determined the correlation between metal concentrations and red-eared slider density. Metal concentrations measured in the current study were low, and turtle density was not significantly correlated with metal concentrations in tissues or sediment. However, we observed a trend of decreasing turtle density in ponds that had greater metal concentrations. Sex ratio and proportion of juveniles were significantly different among ponds, but it is unclear if these differences are related to contamination associated with the PGDP.

Lethal and sublethal effects of three insecticides on two developmental stages of Xenopus laevis and comparison with other amphibians.

It has been suggested that Xenopus laevis is less sensitive than other amphibians to some chemicals, and therefore, that the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) may have limited use in risk assessments for other amphibians. However, comparisons are based mostly on results of FETAX, which emphasizes embryos. Larval X. laevis may be more sensitive to chemicals than embryos and may serve as a better life stage in risk assessments. The present study was conducted to determine the lethal and sublethal effects of 3 insecticides (malathion, endosulfan, and α-cypermethrin) on X. laevis embryos and larvae and to compare toxicity of X. laevis with that of other amphibians. All 3 insecticides have different modes of action, and they caused mortality, malformations, and growth inhibition in both developmental stages. Compared with embryos, larvae were more sensitive to endosulfan and α-cypermethrin but not to malathion. Xenopus laevis larvae had low sensitivity to endosulfan, median sensitivity to malathion, and high sensitivity to α-cypermethrin/cypermethrin relative to other larval amphibians. Our results suggest that X. laevis larvae may generate more protective toxicity estimates in risk assessments than embryos. Xenopus laevis may have limited use in evaluating risk of organochlorine insecticides to other amphibians but may provide useful toxicity thresholds for pyrethroid and perhaps organophosphorus insecticides.

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.

Accumulation of polychlorinated biphenyls (PCBs) and evaluation of hematological and immunological effects of PCB exposure on turtles.

Concentrations of total polychlorinated biphenyls (PCBs), Aroclor 1260, and 26 congeners were measured in liver, fat, and eggs of red-eared slider turtles (Trachemys scripta elegans) collected from ponds near or on the Paducah Gaseous Diffusion Plant (PGDP), Kentucky, USA. Concentrations of total PCBs (wet mass) ranged from 0.002 to 0.480 mg/kg, 0.028 to 0.839 mg/kg, and 0.001 to 0.011 mg/kg in liver, fat, and eggs, respectively. Concentrations of Arochlor 1260 did not exceed 0.430, 0.419, and 0.007 mg/kg in liver, fat, and eggs, respectively. Exposure to PCBs in red-eared sliders collected from the PGDP is characterized by low concentrations of moderately chlorinated mono-ortho and di-ortho congeners (PCB 153, 180, and 118). Although PCB concentrations measured in the current study were low, chronic exposure to PCBs may have altered hematology and immunity of the turtles examined. Total white blood cell count and number of heterophils were negatively correlated with concentrations of total PCBs and Arochlor 1260, respectively. However, disease and other contaminants in the study area may influence the results. Because little is known regarding the influence of PCBs on hematology and immune function in turtles, additional study is needed to better evaluate results observed in the current study.

Metal accumulation and evaluation of effects in a freshwater turtle.

A variety of contaminants have been detected in aquatic and terrestrial environments around the Paducah Gaseous Diffusion Plant (PGDP), Kentucky. The presence of these contaminants at the PGDP may pose a risk to biota, yet little is known about the bioaccumulation of contaminants and associated effects in wildlife, especially in aquatic turtles. The current study was initiated to evaluate: (1) the accumulation of heavy metals (Cd, Cr, Cu, Pb, and Hg) in aquatic ecosystems associated with the PGDP using red-eared slider turtle (Trachemys scripta elegans) as biomonitors; (2) maternal transfer of heavy metals; and (3) potential hematological and immunological effects resulting from metal accumulation. A total of 26 turtles were collected from 7 ponds located south, adjacent, and north of the PGDP. Liver Cu concentrations were significantly different among ponds and Cu concentrations in eggs were positively correlated with female Cu concentrations in kidney. The concentrations of heavy metals measured in turtle tissues and eggs were low and, based on previous studies of reptiles and established avian threshold levels of heavy metals, did not appear to have adverse effects on aquatic turtles inhabiting ponds near the PGDP. However, total white blood cell counts, heterophil to lymphocyte ratio, and phytohemagglutinin stimulation index were correlated with metal concentrations. Because other factors may affect the hematological and immunological indices, further investigation is needed to determine if these effects are associated with metal exposure, other contaminants, or disease.