The Amphibian Short-Term Assay: Evaluation of a New Ecotoxicological Method for Amphibians Using Two Organophosphate Pesticides Commonly Found in Nature-Assessment of Behavioral Traits.

Neurotoxic pesticides are used worldwide to protect crops from insects; they are recognized to impact nontarget organisms that live in areas surrounded by treated crops. Many biochemical and cell-based solutions have been developed for testing insecticide neurotoxicity. Nevertheless, such solutions provide a partial assessment of the impact of neurotoxicity, neglecting important phenotypic components such as behavior. Behavior is the apical endpoint altered by neurotoxicity, and scientists are increasingly recommending including behavioral endpoints in available tests or developing new methods for assessing contaminant-induced behavioral changes. In the present study, we extended an existing protocol (the amphibian short-term assay) with a behavioral test. To this purpose, we developed a homemade device along with an open-source computing solution for tracking trajectories of Xenopus laevis tadpoles exposed to two organophosphates insecticides (OPIs), diazinon (DZN) and chlorpyrifos (CPF). The data resulting from the tracking were then analyzed, and the impact of exposure to DZN and CPF was tested on speed- and direction-related components. Our results demonstrate weak impacts of DZN on the behavioral components, while CPF demonstrated strong effects, notably on speed-related components. Our results also suggest a time-dependent alteration of behavior by CPF, with the highest impacts at day 6 and an absence of impact at day 8. Although only two OPIs were tested, we argue that our solution coupled with biochemical biomarkers is promising for testing the neurotoxicity of this pesticide group on amphibians. Environ Toxicol Chem 2023;42:1595-1606. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The Amphibian Short-Term Assay: Evaluation of a New Ecotoxicological Method for Amphibians Using Two Organophosphate Pesticides Commonly Found in Nature-Assessment of Biochemical, Morphological, and Life-History Traits.

Amphibia is the most threatened class among vertebrates, with >40% of the species threatened with extinction. Pollution is thought to alter amphibian population dynamics. With the growing interest in behavioral ecotoxicology, the neurotoxic organophosphate pesticides are of special concern. Understanding how exposure to neurotoxics leads to behavioral alterations is of crucial importance, and mechanistic endpoints should be included in ecotoxicological methods. In the present study, we tested an 8-day assay to evaluate the toxicity of two organophosphates, diazinon and chlorpyrifos, on Xenopus laevis, that is, on biochemical, morphological, and life-history traits related to locomotion capacities. The method involves measuring biomarkers such as glutathione-S-transferase (GST) and ethoxyresorufin-O-deethylase (EROD; two indicators of the detoxifying system) in the 8-day-old larvae as well as acetylcholinesterase (AChE) activity (involved in the nervous system) in 4-day-old embryos and 8-day-old larvae. Snout-to-vent length and snout-to-tail length of 4-day-old embryos and 8-day larvae were recorded as well as the corresponding growth rate. Fin and tail muscle widths were measured as well for testing changes in tail shape. Both tests showed effects of both organophosphates on AChE activity; however, no changes were observed in GST and EROD. Furthermore, exposure to chlorpyrifos demonstrated impacts on morphological and life-history traits, presaging alteration of locomotor traits. In addition, the results suggest a lower sensitivity to chlorpyrifos of 4-day-old embryos compared to 8-day-old larvae. Tests on other organophosphates are needed to test the validity of this method for the whole organophosphate group. Environ Toxicol Chem 2022;41:2688-2699. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Multigenerational effects of the anticancer drug tamoxifen and its metabolite 4-hydroxy-tamoxifen on Daphnia pulex.

Tamoxifen and its metabolite 4-hydroxy-tamoxifen (4OHTam) are two potent molecules that have anticancer properties on breast cancers. Their medical use is expected to increase with the increasing global cancer rate. After consumption, patients excrete tamoxifen and the 4OHTam metabolite into wastewaters, and tamoxifen has been already detected in wastewaters and natural waters. The concentrations of 4OHTam in waters have never been reported. A single study reported 4OHTam effects on the microcrustacean Daphnia pulex. The effects of tamoxifen and 4OHTam over more than two generations are unknown in aquatic invertebrates. The main goal of this study was to assess the long-term sensitivity of the microcrustacean D. pulex over four generations, based on size, reproduction, viability and the intrinsic rate of natural increase (r). Additional experiments were carried out to observe whether the effects of tamoxifen and 4OHTam were reversible in the next generation after descendants were withdrawn from chemical stress (i.e., recovery experiment), and whether the lowest test concentration of each chemical induced toxic effects when both concentrations were combined (i.e., mixture experiments). Our results showed that tamoxifen and 4OHTam induced the adverse effects at environmentally relevant concentrations. Tamoxifen and 4OHTam impaired size, viability, reproduction and the r in four generations of treated D. pulex, but these effects were not clearly magnified over generations. Tamoxifen was more potent than 4OHTam on D. pulex. When used in a mixture, the combination of tamoxifen and 4OHTam induced effects in offspring, whereas no effects were observed when these chemicals were tested individually. In the recovery experiment, the reproduction and size were reduced in offspring withdrawn from chemical exposures. Our results suggested that tamoxifen and its metabolite may be a relevant pharmaceutical to consider in risk assessment.

The anticancer drug metabolites endoxifen and 4-hydroxy-tamoxifen induce toxic effects on Daphnia pulex in a two-generation study.

Although pharmaceutical metabolites are found in the aquatic environment, their toxicity on living organisms is poorly studied in general. Endoxifen and 4-hydroxy-tamoxifen (4OHTam) are two metabolites of the widely used anticancer drug tamoxifen for the prevention and treatment of breast cancers. Both metabolites have a high pharmacological potency in vertebrates, attributing prodrug characteristics to tamoxifen. Tamoxifen and its metabolites are body-excreted by patients, and the parent compound is found in sewage treatment plan effluents and natural waters. The toxicity of these potent metabolites on non-target aquatic species is unknown, which forces environmental risk assessors to predict their toxicity on aquatic species using knowledge on the parent compounds. Therefore, the aim of this study was to assess the sensitivity of two generations of the freshwater microcrustacean Daphnia pulex towards 4OHTam and endoxifen. Two chronic tests of 4OHTam and endoxifen were run in parallel and several endpoints were assessed. The results show that the metabolites 4OHTam and endoxifen induced reproductive and survival effects. For both metabolites, the sensitivity of D. pulex increased in the second generation. The intrinsic rate of natural increase (r) decreased with increasing 4OHTam and endoxifen concentrations. The No-Observed Effect Concentrations (NOECs) calculated for the reproduction of the second generation exposed to 4OHTam and endoxifen were <1.8 and 4.3 µg/L, respectively, whereas the NOECs that were calculated for the intrinsic rate of natural increase were <1.8 and 0.4 µg/L, respectively. Our study raises questions about prodrug and active metabolites in environmental toxicology assessments of pharmaceuticals. Our findings also emphasize the importance of performing long-term experiments and considering multi-endpoints instead of the standard reproduction outcome.

Shotgun ecotoxicoproteomics of Daphnia pulex: biochemical effects of the anticancer drug tamoxifen.

Among pollutants released into the environment by human activities, residues of pharmaceuticals are an increasing matter of concern because of their potential impact on ecosystems. The aim of this study was to analyze differences of protein expression resulting from acute (2 days) and middle-term (7 days) exposure of aquatic microcrustacean Daphnia pulex to the anticancer drug tamoxifen. Using a liquid chromatography-mass spectrometry shotgun approach, about 4000 proteins could be identified, providing the largest proteomics data set of D. pulex published up to now. Considering both time points and tested concentrations, 189 proteins showed a significant fold change. The identity of regulated proteins suggested a decrease in translation, an increase in protein degradation and changes in carbohydrate and lipid metabolism as the major effects of the drug. Besides these impacted processes, which reflect a general stress response of the organism, some other regulated proteins play a role in Daphnia reproduction. These latter results are in accordance with our previous observations of the impact of tamoxifen on D. pulex reproduction and illustrate the potential of ecotoxicoproteomics to unravel links between xenobiotic effects at the biochemical and organismal levels. Data are available via ProteomeXchange with identifier PXD001257.

Toxicity assessment of oil-contaminated freshwater sediments.

The performance of four microscale toxicity bioassays conducted on whole sediments was evaluated during a bioremediation project undertaken in 1999-2000 on a crude oil-contaminated freshwater shoreline of the St. Lawrence River, Quebec, Canada. The toxicity tests assessed included: (1) the Microtox solid-phase assay (MSPT), (2) the Biotox Flash solid-phase test (Flash), (3) the algal solid-phase assay (ASPA), and 4) the Ostracodtoxkit solid-phase assay. Data generated with these assays were compared with those obtained using the standard endobenthic amphipod (Hyalella azteca) bioassay. Bioanalytical comparisons indicated that all five solid-phase tests were useful in detecting the toxicity of oiled sediments; however, statistical analyses distinguished a difference in response between the invertebrate (amphipod and Ostracodtoxkit) and bacterial luminescence tests (MSPT and Flash). Based on these results, it is recommended that careful selection of biotests be made in the design of the test battery for assessment of residual oil sediment toxicity. Time-series toxicity data generated with ASPA indicated that oiled sediments in the freshwater wetlands of the St. Lawrence River remained toxic to phytoplankton for at least 65 weeks and that remediation treatment was able to accelerate detoxification by 16 weeks.