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.

Development of a new ecotoxicological assay using the testate amoeba Euglypha rotunda (Rhizaria; Euglyphida) and assessment of the impact of the herbicide S-metolachlor.

An ever-increasing diversity of potentially toxic chemical compounds are being developed and released into the environment as a result of human activities (e.g. agriculture, drugs, and cosmetics). Among these, pesticides have been shown to affect non-targeted wildlife since the 1960s. A range of ecotoxicological tests are used to assess the toxicity of pesticides on various model organisms. However most model organisms are metazoans, while the majority of Eukaryotes are unicellular microorganisms known as protists. Protists are ubiquitous organisms of key functional roles in all ecosystems but are so far little studied with respect to pesticide impact. To fill this gap, we developed a new ecotoxicological test based on Euglypha rotunda, a common soil amoeba, grown in culture flask with Escherichia coli as sole food source. We tested this assay with the herbicide S-metolachlor, which is known to affect cell division in seedling shoots and roots of weeds. Reproducible growth conditions were obtained for E. rotunda. The growth of E. coli was not affected by the herbicide. The growth of E. rotunda was affected by the herbicide in a non-linear way, growth being significantly reduced at ca. 15 µg/L, but not at 150 µg/L. Our results show the potential for using soil protists in ecotoxicology and adds to the growing body of evidence for non-linear impacts of pesticides on non-target organisms. With the acquisition of additional data, the protocol should be suitable for standard ecotoxicological tests.

Comparison of specific versus literature species sensitivity distributions for herbicides risk assessment.

Species sensitivity distributions (SSDs) are an important predictive tool in risk assessment. Usually, literature data are used to build SSDs that are mostly based on planktonic species. But, to get adequate protective thresholds for environmental communities, one could argue that SSD should be built on ecotoxicological data obtained from species found in the ecosystem that should be protected. This is particularly true when benthic algae are of concern. Due to the lack of literature data, building SSD on benthic microalgae is difficult. This paper aims in comparing SSDs, and thus protective thresholds (hazardous concentration that affects 5% of the species of a community, HC5), built on ecotoxicological data obtained (1) from literature and (2) with specific bioassays on benthic diatoms from a lake. Thresholds were derived for protection against four herbicides separately and for a mixture of them. Sensitivity data obtained from literature were statistically lower than the specific data for all herbicides: Species tested in the literature were usually more sensitive (mainly chlorophytes), leading to more protective lower HC5. The HC5 thresholds (literature or specific) derived for protection against the mixture were also compared to the observed sensitivity of an assemblage of benthic diatom species exposed to an increasing range of herbicide mixture concentrations. We observed that one species within the assemblage (Fragilaria rumpens) was affected at a concentration below both the literature and the specific HC5 thresholds.

Dynamics and environmental risk assessment of the herbicide glyphosate and its metabolite AMPA in a small vineyard river of the Lake Geneva catchment.

The use of pesticides may lead to environmental problems, such as surface water pollution, with a risk for aquatic organisms. In the present study, a typical vineyard river of western Switzerland was first monitored to measure discharged loads, identify sources, and assess the dynamic of the herbicide glyphosate and its metabolite aminomethylphosphonic acid (AMPA). Second, based on river concentrations, an associated environmental risk was calculated using laboratory tests and ecotoxicity data from the literature. Measured concentrations confirmed the mobility of these molecules with elevated peaks during flood events, up to 4970 ng/L. From April 2011 to September 2011, a total load of 7.1 kg was calculated, with 85% coming from vineyards and minor urban sources and 15% from arable crops. Compared with the existing literature, this load represents an important fraction (6-12%) of the estimated amount applied because of the steep vineyard slopes (∼10%). The associated risk of these compounds toward aquatic species was found to be negligible in the present study, as well as for other rivers in Switzerland. A growth stimulation was nevertheless observed for the algae Scenedesmus vacuolatus with low concentrations of glyphosate, which could indicate a risk of perturbation in aquatic ecosystems, such as eutrophication. The combination of field and ecotoxicity data allowed the performance of a realistic risk assessment for glyphosate and AMPA, which should be applied to other pesticide molecules.

Sediment contamination assessment in urban areas based on total suspended solids.

Sediment represents an important compartment in surface waters. It constitutes a habitat or spawning site for many organisms and is an essential trophic resource for higher level organisms. It can be impacted by anthropogenic activities, particularly through urban wet-weather discharges like stormwater and combined sewer overflows. An approach was presented for assessing the risks caused by urban wet-weather discharges to the sediment compartment based on total suspended solids (TSS). TSS is routinely measured in field surveys and can be considered as a tracer for urban wet-weather contamination. Three assessment endpoints linked with TSS were proposed: a) siltation of the riverbed, b) oxygen demand due to organic matter degradation and c) accumulation of ecotoxic contaminants on the riverbed (heavy metals, PAHs). These criteria were translated in terms of the maximal TSS accumulation load and exposure time (percentage of time exceeding the accumulation criteria) to account for sediment accumulation dynamics and resuspension in streams impacted by urban wet-weather discharges. These assessment endpoints were implemented in a stochastic model that calculates TSS behavior in receiving waters and allows therefore an assessment of potential impacts. The approach was applied to three Swiss case studies. For each, good agreement was found between the risk predictions and the field measurements confirming the reliability of the approach.

Methodology to account for uncertainties and tradeoffs in pharmaceutical environmental hazard assessment.

Many pharmaceutical products find their way into receiving waters, giving rise to concerns regarding their environmental impact. A procedure was proposed that enables ranking of the hazard to aquatic species and human health due to such products. In the procedure, hazard assessment is based on five of the pharmaceutical product's individual physico-chemical properties. These properties are aggregated using the weighted Euclidian distance as the utility function. The weights and physico-chemical properties are considered as random variables. Physico-chemical property uncertainty criteria are obtained from a literature review. Weight uncertainty is based on a hazard ranking from a panel of experts, the histogram of which is converted into a continuous probability density function using statistical Kernel smoothing technique. The hazard-ranking procedure was applied to a list of common pharmaceuticals used in Switzerland. The procedure is target-specific. Two rankings were presented: One giving priority to environmental protection and the other to human health. For most substances, the hazard rank depends on the target. For the Swiss case study, the ranking procedure led to the conclusion that the hormones ethinylestradiol and testosterone, along with the antibiotic erythromycin A, should be in all cases included in risk-assessment methodologies, environmental concentration estimates and regular measurement campaigns. The methodology proposed is flexible and can be extrapolated to other substances and groups of experts.

Occurrence and fate of micropollutants in the Vidy Bay of Lake Geneva, Switzerland. Part I: priority list for environmental risk assessment of pharmaceuticals.

Pharmaceuticals are substances designed to have a biological effect in humans. Their presence in the environment, especially in surface waters, is of increasing concern because of their potential risk to non-target species. A large number of pharmaceuticals are on the market; for example, approximately 2,000 active ingredients are approved in Europe, and many of them have already been detected in surface water. It is therefore crucial to select the substances that may do the most harm to the environment prior to performing measurements and extensive risk assessment. In the present study, a method to determine a list of pharmaceuticals to survey in surface water is proposed. Inclusion of substances on the list was based on a screening procedure, the analytical feasibility, and previous knowledge of pharmaceuticals detected in water. The screening procedure proposed here is an improvement on the standard procedure of the European Medicine Evaluation Agency (EMEA). It is designed to decrease the number of pharmaceuticals to be evaluated in a stepwise manner, thus decreasing the number of data necessary for the evaluation. We applied our approach to determine a list of 37 pharmaceuticals and four hormones to survey in a specific region of Switzerland, the Lake Geneva area, and discussed the advantages and weak points of the method.

Cost-effective experimental design to support modeling of concentration-response functions.

Modeling concentration-response function became extremely popular in ecotoxicology during the last decade. Indeed, modeling allows determining the total response pattern of a given substance. However, reliable modeling is consuming in term of data, which is in contradiction with the current trend in ecotoxicology, which aims to reduce, for cost and ethical reasons, the number of data produced during an experiment. It is therefore crucial to determine experimental design in a cost-effective manner. In this paper, we propose to use the theory of locally D-optimal designs to determine the set of concentrations to be tested so that the parameters of the concentration-response function can be estimated with high precision. We illustrated this approach by determining the locally D-optimal designs to estimate the toxicity of the herbicide dinoseb on daphnids and algae. The results show that the number of concentrations to be tested is often equal to the number of parameters and often related to the their meaning, i.e. they are located close to the parameters. Furthermore, the results show that the locally D-optimal design often has the minimal number of support points and is not much sensitive to small changes in nominal values of the parameters. In order to reduce the experimental cost and the use of test organisms, especially in case of long-term studies, reliable nominal values may therefore be fixed based on prior knowledge and literature research instead of on preliminary experiments.

Risk assessment of herbicide mixtures in a large European lake.

Lake Geneva is one of the largest European lakes with a surface area of 580 km2. Its catchment area covers 7400 km2, of which approximately 20% is arable land. Monitoring campaigns have been carried out in 2004 and 2005 to determine the contamination of the lake by pesticides. The results highlight the widespread presence of herbicides in water, the measured concentrations for most substances remaining constant in 2004 and 2005. However, for some individual herbicides the concentrations increased drastically (e.g., the herbicide foramsulfuron). We assessed the environmental risk of the herbicides detected in the lake using water quality criteria recently determined for the Swiss environmental protection agency. Furthermore, we assessed the risk of herbicide mixtures, grouped based upon their mode of action. Generally, the risk estimated for all single substances is low, except for some sulfonylurea compounds. For these substances, the measured concentrations are higher than the predicted no-effect concentration. Impact on the flora of the lake can therefore not be excluded. When mixtures of pesticides with similar mode of action are taken into account, the risk remains lower than the mixture water quality criteria for all groups, but can reach as high as one third of this quality criteria. A further step would therefore be to assess the risk of the total pesticide mixture, including similar and dissimilar modes of action.

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.