Juvenile food limitation in standardized tests: a warning to ecotoxicologists.

Standard ecotoxicological tests are as simple as possible and food sources are mainly chosen for practical reasons. Since some organisms change their food preferences during the life-cycle, they might be food limited at some stage if we do not account for such a switch. As organisms tend to respond more sensitively to toxicant exposure under food limitation, the interpretation of test results may then be biased. Using a reformulation of the von Bertalanffy model to analyze growth data of the pond snail Lymnaea stagnalis, we detected food limitation in the early juvenile phase. The snails were held under conditions proposed for a standardized test protocol, which prescribes lettuce as food source. Additional experiments showed that juveniles grow considerably faster when fed with fish flakes. The model is based on Dynamic Energy Budget (DEB) theory, which allows for mechanistic interpretation of toxic effects in terms of changes in energy allocation. In a simulation study with the DEB model, we compared the effects of three hypothetical toxicants in different feeding situations. The initial food limitation when fed with lettuce always intensified the effect of the toxicants. When fed with fish flakes, the predicted effect of the toxicants was less pronounced. From this study, we conclude that (i) the proposed test conditions for L. stagnalis are not optimal, and require further investigation, (ii) fish flakes are a better food source for juvenile pond snails than lettuce, (iii) analyzing data with a mechanistic modeling approach such as DEB allows identifying deviations from constant conditions, (iv) being unaware of food limitation in the laboratory can lead to an overestimation of toxicity in ecotoxicological tests.

Biochemical effects of nonylphenol polyethoxylate adjuvant, Diquat herbicide and their mixture on the three-spined stickleback (Gasterosteus aculeatus L.).

This study examined the response of 7-ethoxyresorufine-O-deethylase, glutathione-S-transferase, glutathione peroxidase, glutathione content, level of thiobarbituric acid reactive compounds and circulating vitellogenin, in three-spined sticklebacks after 21 days of exposure to Diquat herbicide, commercial nonylphenol polyethoxylate adjuvant and mixture between Diquat and adjuvant. The results showed that adjuvant exerted more important oxidative effects than Diquat and that mixture effects were unlike to single additivity. This study argues for ecotoxicological risk assessment of adjuvants and mixtures of adjuvants and pesticides.

Invertebrates in testing of environmental chemicals: are they alternatives?

An enlarged interpretation of alternatives in toxicology testing includes the replacement of one animal species with another, preferably a nonmammalian species. This paper reviews the potential of invertebrates in testing environmental chemicals and provides evidence of their usefulness in alternative testing methodologies. The first part of this review addresses the use of invertebrates in laboratory toxicology testing. Problems in extrapolating results obtained in invertebrates to those obtained from vertebrates are noted, suggesting that invertebrates can essentially be used in addition to rather than as replacements for vertebrates in laboratory toxicity tests. However, evaluation of the ecologic impact of environmental chemicals must include defining end points that may frequently differ from those classically used in biomedical research. In this context, alternative approaches using invertebrates may be more pertinent. The second part of the review therefore focuses on the use of invertebrates in situ to assess the environmental impact of pollutants. Advantages of invertebrates in ecotoxicologic investigation are presented for their usefulness for seeking mechanistic links between effects occurring at the individual level and consequences for higher levels of biologic organization (e.g., population and community). In the end, it is considered that replacement of vertebrates by invertebrates in ecotoxicity testing is likely to become a reality when basic knowledge of metabolic, physiologic, and developmental patterns in the latter will be sufficient to assess the effect of a given chemical through end points that could be different between invertebrates and vertebrates.

13th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies for ecotoxicity.

There is growing public pressure to minimize the use of vertebrates in ecotoxicity testing; therefore, effective alternatives to toxicity tests causing suffering are being sought. This report discusses alternatives and differs in some respects from the reports of the other three groups because the primary concern is with harmful effects of chemicals at the level of population and above rather than with harmful effects upon individuals. It is concluded that progress toward the objective of minimizing testing that causes suffering would be served by the following initiatives--a clearer definition of goals and strategies when undertaking testing procedures; development of alternative assays, including in vitro test systems, that are based on new technology; development of nondestructive assays for vertebrates (e.g., biomarkers) that do not cause suffering; selection of most appropriate species, strains, and developmental stages for testing procedures (but no additional species for basic testing); better integrated and more flexible testing procedures incorporating biomarker responses, ecophysiological concepts, and ecological end points (progress in this direction depends upon expert judgment). In general, testing procedures could be made more realistic, taking into account problems with mixtures, and with volatile or insoluble chemicals.

Mesocosms in ecotoxicology (1): Outdoor aquatic systems.

Mesocosms have been used in aquatic ecotoxicology for approximately 20 years and were sometimes claimed to be essential tools, especially for regulatory purposes. The term aquatic mesocosm currently describes indoor and outdoor artificial streams or experimental ponds and enclosures. The use of mesocosms refines the classical methods of ecotoxicological risk assessment because mesocosms provide conditions for a better understanding of environmentally relevant effects of chemicals. They make it possible to assess effects of contaminants by looking at the parts (individuals, populations, communities) and the whole (ecosystems) simultaneously. Ecotoxicological investigations in mesocosms will not entirely replace the use of laboratory animals. However, they allow tests to be performed on species that are not of major societal concern, but which play key roles in the structure and function of ecosystems. In this respect, mesocosms allow nondestructive measurements of integrated endpoints. They also appear as potent tools to predict changes at the highest levels of organization (population, community, and ecosystem) from measurements of individual endpoints. However, after a period of extensive use, regulatory studies using large-scale mesocosms were more or less abandoned at the beginning of the 1990s, mainly because their cost-effectiveness was questionable. This review covers key features of outdoor aquatic mesocosms that can be critical for their use in environmental risk assessment of chemicals and emphasizes the optimization of their use for such purpose. The originality of mesocosms is mainly based on the combination of ecological realism, achieved by introduction of the basic components of natural ecosystems, and facilitated access to a number of physicochemical, biological, and toxicological parameters that can be controlled to some extent. This characteristic determines various features of the systems such as the minimal size required, initial physicochemical and biological composition, or choice of model species for ecotoxicological investigations. Ecological maturity of mesocosms affects the degree of variability of both physicochemical and biological parameters used to investigate the impact of contaminants. Adequate time is required to establish a number of interacting functional groups. The choice of appropriate time scales must be considered in the selection of both study duration and sampling frequency. Whatever the system used, duration of experiments should be sufficient to identify both direct and indirect effects on populations and communities. The choice of the experimental design should be based on the objectives of the study rather than on theoretical considerations. In addition to classical parametric statistical methods, nonparametric approaches and multivariate analysis may significantly improve data processing. Realism, representativity, and replicability of mesocosms are critical for evaluating their usefulness in both risk and impact assessment procedures. Each natural ecosystem is unique because its structure and function mainly depend on local factors. Therefore, there is a conceptual opposition between realism and replicability when applied to mesocosms. Considering the objectives of most mesocosm studies, replicability should be preferred to realism. Replicability may be achieved, in part, by a relative simplification of the systems. Reconstituted systems do not need to exactly simulate natural conditions at all levels, but key features at both structural and functional levels should be preserved as they ensure ecological representativity. Reliability of information on ecotoxicological effects of chemicals tested in aquatic mesocosms closely depends on the representativity of biological processes or structures that are likely to be affected. Extrapolation from small experimental systems to the real world seems generally more problematic than the use of larger systems in which more complex interacti

Esterases as biomarkers in Nereis (Hediste) diversicolor exposed to temephos and Bacillus thuringiensis var. israelensis used for mosquito control in coastal wetlands of Morbihan (Brittany, France).

Since 1998, a biomonitoring programme has been implemented to assess the potential impact of chemical mosquito control on macroinvertebrates of the coastal wetlands of Morbihan (Brittany, France). Acetylcholinesterase and carboxylesterases were used as biomarkers to assess the effects of Abate 500e (a.i. temephos) and Vectobac 12 AS (a.i. endotoxins of Bacillus thuringiensis var. israelensis, Bti) in Nereis (Hediste) diversicolor. Esterase inhibition revealed a marked impact of temephos, suggesting preferential contamination of the worms through the food. In Bti-exposed N. diversicolor, random variations of esterase activities were observed, that could not be attributed to the larvicide. However, esterases only reflected indirect physiological effects of Bti, and further investigations are needed to identify biomarkers more specific of Bti endotoxins.

Influence of environmental factors on the response of a natural population of Daphnia magna (Crustacea: Cladocera) to spinosad and Bacillus thuringiensis israelensis in Mediterranean coastal wetlands.

The present study was undertaken to assess the impact of a candidate mosquito larvicide, spinosad (8, 17 and 33 microg L(-1)) on a field population of Daphnia magna under natural variations of water temperature and salinity, using Bti (0.16 and 0.50 microL L(-1)) as the reference larvicide. Microcosms (125 L) were placed in a shallow temporary marsh where D. magna was naturally present. The peak of salinity observed during the 21-day observation period may have been partly responsible for the decrease of daphnid population density in all the microcosms. It is also probably responsible for the absence of recovery in the microcosms treated with spinosad which caused a sharp decrease of D. magna abundance within the first two days following treatment whereas Bti had no effect. These results suggest that it may be difficult for a field population of daphnids to cope simultaneously with natural (water salinity and temperature) and anthropogenic (larvicides) stressors.

Effects of spinosad and Bacillus thuringiensis israelensis on a natural population of Daphnia pulex in field microcosms.

Spinosad, a candidate biological larvicide for mosquito control, was evaluated for its effects on a field population of Daphnia pulex, using Bacillus thuringiensis serovar israelensis (Bti) as a reference larvicide. Microcosms (125L enclosures) were placed in a shallow temporary oligohaline marsh where D. pulex was present. Three concentrations of spinosad (8, 17 and 33 microg L(-1)) and two concentrations of Bti (0.16 and 0.50 microL L(-1)) were applied (5 replicates per concentration, including the controls). Effects of larvicides on D. pulex were evaluated after 2, 4, 7, 14 and 21d of exposure, through measurements of abundance and individual size. Dissipation of spinosad from the water phase was rapid. Four days after treatment, residue concentration represented 11.8%, 3.9% and 12.7% of the initial exposure level for the nominal concentrations of 8, 17 and 33 microg L(-1), respectively. Spinosyns A and D dissipated at similar rates. Analysis of abundance and size structure of the D. pulex population showed an impact of spinosad. Both survival and size structure were affected. However, at the lowest concentration (8 microg L(-1)), population recovered after the first week. In microcosms treated with Bti, the abundance of D. pulex was not affected but the size structure of the population changed after 21d. As compared to laboratory tests, the use of in situ microcosms improved the environmental risk assessment of larvicides, taking into account the influence of environmental factors (e.g., temperature, light, salinity) and intrinsic capacity of recovery of D. pulex under field conditions.

Effects of parasitism and pesticide exposure on characteristics and functions of hemocyte populations in the freshwater snail Lymnaea palustris (Gastropoda, Pulmonata).

Morphological characteristics and functions of hemocytes were used to compare the immunological effects of biological and chemical stress in the freshwater snail Lymnaea palustris. Animals were either infected by a trematode parasite (Metaleptocephalus sp.), or exposed to environmental contaminants, namely atrazine and hexachlorobenzene (HCB). Three populations of circulating hemocytes, morphologically and cytochemically distinct (round cells, hyalinocytes, granulocytes), were identified in both control and parasitized or pesticide-exposed snails. After 6 h of exposure, HCB and atrazine resulted in 8-fold increases in the mean total number of hemocytes, whereas only a 2.2-fold increase was observed 6 h after cercaria emission in parasitized snails. The impact of HCB was limited to the first 24 h of exposure, whereas long-lasting effects of atrazine were observed. Hyalinocytes and, to a lesser extent, round cells contributed most to the increases in hemocyte density in pesticide-exposed snails. Parasitism and atrazine treatment resulted in significant increases of lectin-stained hemocytes, whereas exposure to HCB did not affect the percentages of stained and unstained cells. Hemocyte phagocytic activity increased in HCB-exposed snails but with no concomitant change of the oxidative burst. Opposite results were obtained in atrazine-treated snail hemocytes, with unchanged phagocytosis and decreased phorbol 12-myristate 13-acetate-stimulated production of reactive oxygen intermediates. No increase in phagocytosis, or in the production of reactive oxygen intermediates, was observed in hemocytes from parasitized snails. Infection with the immunologically compatible trematode parasite Metaleptocephalus sp. and exposure to atrazine generated similar reactions from circulating hemocytes, whereas a different response pattern was observed in HCB-exposed snails.

The role of biomarkers in environmental assessment (5). Invertebrate populations and communities.

Although a number of biomarkers of pollutant exposure have been identified in invertebrate species, direct linkage with changes at population and/or community levels are poorly documented and, despite the ecological importance of invertebrates, there is no conclusive evidence that individual measurements of biochemical parameters may allow the effects of pollutants on populations and communities to be predicted. Among the various biochemical parameters used as biomarkers in invertebrates exposed to pollutants in the field, only those for which changes at population or community level can be suggested are discussed in the present review. At population and community levels, the development of resistance to pesticides and changes in behaviour, reproduction and development are analysed as putative consequences of biochemical and physiological alterations. Limits to the use of biochemical parameters as biomarkers of invertebrate exposure to pollutants are discussed. Future research trends and experimental approaches to the validation of invertebrate biomarkers in environmental pollution assessment are suggested.

Variability of physicochemical and biological parameters between replicated outdoor freshwater lentic mesocosms.

Micro- and mesocosms are frequently required in regulatory procedures of aquatic risk assessment for pesticides. However, many questions are still a matter of debate with regard to the use of these systems for environmental risk assessment, especially considering the inter-system variability of the measured parameters and its consequences on experimental design and data analysis. In this paper, variability of physico-chemical and biological parameters measured during two long-term experiments (8 to 9 months) in uncontaminated outdoor freshwater lentic mesocosms (8 m3) is analysed. Consequences on the design of ecotoxicity tests in mesocosms and on data analysis are also addressed. Water temperature, pH, dissolved oxygen concentration and concentration of suspended solids exhibited a very low variability whereas nutrient concentrations displayed elevated levels of variability. Among biological parameters, those measured at the individual level were less variable than those measured at the community level. Functional descriptors frequently exhibited a lower inter-mesocosm variability than structural descriptors. Aggregation of data proved to significantly reduce inter-mesocosm variability. The results indicate that univariate statistical methods may be used for physico-chemical or species-level (e.g. biometric parameters) data which exhibit a moderate inter-mesocosm variability. The use of multivariate techniques is suggested for other levels of investigation. Nevertheless, variability is not sufficient to identify useful parameters. The sensitivity towards chemicals and ecological relevance of descriptors within the experimental context must also be considered.

Outdoor experimental ponds (mesocosms) designed for long-term ecotoxicological studies in aquatic environment.

Outdoor artificial ponds (mesocosms) of 12 m3 were designed for long-term ecotoxicological studies. Sediment, macrophytes (Typha angustifolia and Elodea canadensis), and free and caged freshwater snails [Lymnaea palustris (Müller)] and wood lice (Asellus aquaticus L.) were collected in nearby natural ecosystems and introduced in the mesocosms. Sixty goldfish (Carassius auratus L.) were caged in each pond. Introduced species developed and reproduced in every mesocosm. Animal species (mainly insects and amphibians) spontaneously colonized the ponds, developed, and reproduced. The resulting communities qualitatively resemble those living in natural lentic systems in the surrounding area. Homogenity in physical and chemical conditions and in abundance of phytoplanktonic, periphytic, and macroinvertebrate communities between the different mesocosms was assessed during the stabilization period (8 months). Except for periphyton biomass, no divergent evolution was observed between the ponds. Mesocosm water was slightly eutrophic, alkaline (mean pH: 8.47 +/- 0.09), and moderately hard and mineralized. The homogenous and realistic environmental conditions and high ecological representativity of the outdoor experimental ponds were suitable for extensive ecotoxicological studies. Considerations on the choice and origin of introduced species and on possible interactive effects of the transfer of organisms from natural environments, maintainance conditions, and pollutant exposure are discussed.