Cadmium Compartmentalization in the Pulmonate Snail Lymnaea stagnalis: Improving Our Understanding of Exposure.

In ecotoxicology, analytical compartmentalization analysis can be used to better understand metal sequestration and detoxification. Metals are typically found in two main compartments, biologically detoxified metal (BDM) and metal sensitive fractions (MSF). The purpose of this study was to analyze the subcellular distribution of cadmium (Cd) in Lymnaea stagnalis. Adult snails were exposed to three concentrations of Cd for 56 days as part of a global ring test for L. stagnalis. At the end of the 56-day exposure, organisms were separated in two sections (viscera and foot). Each section was subsequently divided by differential centrifugation into five total fractions including (metal rich granules, debris, Organelles, heat denatured proteins, and heat stable proteins) followed by Cd analysis. The concentration in each compartment, BDM, MSF, and bioconcentration factors were estimated as well. There was significantly higher bioconcentration of Cd in the viscera section compared with the foot. Cadmium accumulation in all five fractions also increased with increasing exposure concentrations. Cadmium accumulated the most in the heat denatured protein fraction (enzymes) and accumulated the least in the heat stable protein fraction (metallothionein-like proteins). The MSF compartment (~65%) was in higher proportion than the BDM (~30%), but only in the lowest Cd exposure concentration was there a significant difference between these compartments. The results indicated that, in general, there was more Cd accumulated in the metal sensitive fractions, and that the detoxification mechanisms were not efficient enough to avoid toxicity at the two highest concentrations. This study provides evidence that improves our understanding of Cd tissue distribution in freshwater gastropods.

Optimizing the design of a reproduction toxicity test with the pond snail Lymnaea stagnalis.

This paper presents the results from two ring-tests addressing the feasibility, robustness and reproducibility of a reproduction toxicity test with the freshwater gastropod Lymnaea stagnalis (RENILYS strain). Sixteen laboratories (from inexperienced to expert laboratories in mollusc testing) from nine countries participated in these ring-tests. Survival and reproduction were evaluated in L. stagnalis exposed to cadmium, tributyltin, prochloraz and trenbolone according to an OECD draft Test Guideline. In total, 49 datasets were analysed to assess the practicability of the proposed experimental protocol, and to estimate the between-laboratory reproducibility of toxicity endpoint values. The statistical analysis of count data (number of clutches or eggs per individual-day) leading to ECx estimation was specifically developed and automated through a free web-interface. Based on a complementary statistical analysis, the optimal test duration was established and the most sensitive and cost-effective reproduction toxicity endpoint was identified, to be used as the core endpoint. This validation process and the resulting optimized protocol were used to consolidate the OECD Test Guideline for the evaluation of reproductive effects of chemicals in L. stagnalis.

A cost or a benefit? Counterintuitive effects of diet quality and cadmium in Lymnaea stagnalis.

Diet quality can have a strong impact on organismal fitness although diet quality is infrequently considered as a factor in toxicity tests. The purpose of this study was to assess how diets differing in nutritional content affect sensitivity to Cd as measured by several sublethal responses related directly to bioenergetics. We evaluated feeding rate, growth rate, behavior and macronutrient content in the pond snail Lymnaea stagnalis exposed to Cd and fed two different diets. Hatchlings were fed either lettuce or high-caloric pellets, and exposed to 5, 10, and 20 µg/L Cd for 12 days. Length and weight were measured at the beginning and end of the test. The amount of food eaten and behavior were determined every two days. Total lipids, proteins and carbohydrates were biochemically measured at test end. For the second part of the study, snails were fed either lettuce or pellets for 2 weeks and then exposed to high challenge concentrations of Cd. Growth coefficients based on length and weight were significantly higher for snails fed pellets. In addition, snails exposed to Cd had significantly smaller growth coefficients than those in the control for both diets. Total carbohydrates and lipids were higher for snails fed pellets while the protein content was not significantly affected by Cd or diets. Even though snails fed pellets grew significantly faster, contrary to expectations they were significantly more sensitive to Cd compared to those fed lettuce. This study provides evidence that a bioenergetics-based approach can been used to better understand how diet can affect the ecotoxicity of chemical stressors to freshwater gastropods.

Polycyclic aromatic hydrocarbon concentrations, mutagenicity, and Microtox® acute toxicity testing of Peruvian crude oil and oil-contaminated water and sediment.

The oil industry is a major source of contamination in Peru, and wastewater and sediments containing oil include harmful substances that may have acute and chronic effects. This study determined polycyclic aromatic hydrocarbon (PAH) concentrations by GC/MS, mutagenicity using TA98 and TA100 bacterial strains with and without metabolic activation in the Muta-ChromoPlate™ test, and Microtox® 5-min EC50 values of Peruvian crude oil, and water and sediment pore water from the vicinity of San José de Saramuro on the Marañón River and Villa Trompeteros on the Corrientes River in Loreto, Peru. The highest total PAH concentration in both areas was found in water (Saramuro = 210.15 µg/ml, Trompeteros = 204.66 µg/ml). Total PAH concentrations in water from San José de Saramuro ranged from 9.90 to 210.15 µg/ml (mean = 66.48 µg/ml), while sediment pore water concentrations ranged from 2.19 to 70.41 µg/ml (mean = 24.33 µg/ml). All water samples tested from Saramuro and Trompeteros sites, and one out of four sediment pore water samples from Trompeteros, were found to be mutagenic (P < 0.001). One sediment pore water sample in Saramuro was determined to have a measurable toxicity (Microtox EC50 = 335.1 mg/l), and in Trompeteros, the EC50 in water and sediment pore water ranged from 25.67 to 133.86 mg/l. Peruvian crude oil was mutagenic using the TA98 strain with metabolic activation, and the EC50 was 17.18 mg/l. The two areas sampled had very high PAH concentrations that were most likely associated with oil activities, but did not lead to acute toxic effects. However, since most of the samples were mutagenic, it is thought that there is a greater potential for chronic effects.

Parental diet affects embryogenesis of the great pond snail (Lymnaea stagnalis) exposed to cadmium, pyraclostrobin, and tributyltin.

Diet quality has a strong impact on life-history traits, but it is not usually considered as a factor in toxicity tests. The purpose of the present study was to evaluate how diets differing in nutritional content affect sensitivity to cadmium, pyraclostrobin, and tributyltin in Lymnaea stagnalis offspring. Three groups were fed a different diet each: lettuce, high-caloric pellets, or a combination of both. Snails fed pellets and both diets had similar growth; however, snails fed lettuce showed lower growth until the fourth month. Egg masses were collected from adult snails fed each diet and exposed to 3 concentrations of either cadmium, pyraclostrobin, or tributyltin. We quantified time to hatch, hatching success, and the developmental stages of embryos. We measured fecundity in adults and total lipids, carbohydrates, and proteins in adult snails and egg masses. Adult snails fed different diets produced a similar number of egg masses, but the number of eggs per egg mass in snails fed pellets was lower than for snails fed the other 2 diets. We found that adult snails fed pellets had a higher lipid content compared to snails fed the other 2 diets. However, egg masses from parental snails fed pellets did not hatch, including those from the controls. Interestingly, egg masses exposed to the lowest concentration of tributyltin had low hatching success. These observations on offspring performance suggest that there are important diet effects that can strongly influence responses that could be diet- and chemical-dependent. Environ Toxicol Chem 2018;37:2428-2438. © 2018 SETAC.

Diet quality affects chemical tolerance in the freshwater snail Lymnaea stagnalis.

Organisms generally select high-quality diets to obtain maximal energy while devoting the least amount of time and energy. Diets, however, can vary in natural systems. In ecotoxicological testing, the effect of diet type on organismal responses to toxicants has not been explored despite the potential for dietary effects to influence toxicological endpoints. We first evaluated diet quality using growth rate and sensitivity to the fungicide pyraclostrobin of Lymnaea stagnalis fed lettuce (common laboratory diet), turtle pellets (high nutrient composition), and a combination diet of both food items. We also measured the macronutrient content of snails raised on the multiple diets to determine how diet may have impacted energy allocation patterns. Finally, we evaluated whether snails discernibly preferred a particular diet. Snails fed high-nutrient and combination diets grew larger overall than snails fed a lettuce-only diet. Snails fed the high-nutrient and combination diets, both juvenile and adult, were significantly more tolerant to pyraclostrobin than snails fed lettuce. When measured for macronutrient content, snails raised on high-nutrient and combination diets had significantly higher carbohydrate content than snails fed lettuce. Despite the strong effects of diet type, snails did not exhibit a clear diet choice in preference trials. Dietary composition clearly influences growth rate, sensitivity, and macronutrient content of Lymnaea stagnalis. These results suggest that the nutritional environment has potentially strong impacts on toxicant sensitivity. Environ Toxicol Chem 2018;37:1158-1167. © 2017 SETAC.

Transgenerational endpoints provide increased sensitivity and insight into multigenerational responses of Lymnaea stagnalis exposed to cadmium.

Ecotoxicology provides data to inform environmental management. Many testing protocols do not consider offspring fitness and toxicant sensitivity. Cadmium (Cd) is a well-studied and ubiquitous toxicant but little is known about the effects on offspring of exposed parents (transgenerational effects). This study had three objectives: to identify endpoints related to offspring performance; to determine whether parental effects would manifest as a change in Cd tolerance in offspring and how parental exposure duration influenced the manifestation of parental effects. Adult snails were exposed to Cd 0, 25, 50, 100, 200 and 400 µg Cd/L for eight weeks. There were effects on adult endpoints (e.g., growth, reproduction) but only at the highest concentrations (>100 µg/L). Alternatively, we observed significant transgenerational effects at all Cd concentrations. Surprisingly, we found increased Cd tolerance in hatchlings from all parental Cd exposure concentrations even though eggs and hatchlings were in Cd-free conditions for 6 weeks. Explicit consideration of offspring performance adds value to current toxicity testing protocols. Parental exposure duration has important implications for offspring effects and that contaminant concentrations that are not directly toxic to parents can cause transgenerational changes in resistance that have significant implications for toxicity testing and adaptive responses.

Energetic endpoints provide early indicators of life history effects in a freshwater gastropod exposed to the fungicide, pyraclostrobin.

Organismal energetics provide important insights into the effects of environmental toxicants. We aimed to determine the effects of pyraclostrobin on Lymnaea stagnalis by examining energy allocation patterns and life history traits. Juvenile snails exposed to pyraclostrobin decreased feeding rate and increased apparent avoidance behaviors at environmentally relevant concentrations. In adults, we found that sublethal concentrations of pyraclostrobin did not affect reproductive output, however, there were significant effects on developmental endpoints with longer time to hatch and decreased hatching success in pyraclostrobin-exposed egg masses. Further, there were apparent differences in developmental effects depending on whether mothers were also exposed to pyraclostrobin suggesting this chemical can exert intergenerational effects. Pyraclostrobin also affected protein and carbohydrate content of eggs in mothers that were exposed to pyraclostrobin. Significant effects on macronutrient content of eggs occurred at lower concentrations than effects on gross endpoints such as hatching success and time to hatch suggesting potential value for these endpoints as early indicators of ecologically relevant stress. These results provide important insight into the effects of a common fungicide on important endpoints for organismal energetics and life history.