Laurent Lagadic

In situ-based effects measures: Determining the ecological relevance of measured responses

Abstract The aim of this review is to examine how the choice of test species and study design employed in the use of in situ approaches in ecological risk assessment can maximize the ecological relevance of data. We provide a framework to define and assess ecological relevance that permits study designs to remain focused on the ecological question being addressed. This framework makes explicit the linkages between effects at lower levels of biological organization and higher-order ecological effects at the population, community, and ecosystem levels. The usefulness of this framework is illustrated by reference to specific examples from aquatic ecotoxicology. The use of models as both interpretive and predictive tools is discussed, with suggestions of appropriate methods for different protection goals.

INFLUENCE OF ISOLATION ON THE RECOVERY OF POND MESOCOSMS FROM THE APPLICATION OF AN INSECTICIDE. II. BENTHIC MACROINVERTEBRATE RESPONSES

The immediate response and recovery of the macrobenthic communities of nonisolated and isolated freshwater outdoor 9 ml mesocosms following an acute stress caused by the addition of deltamethrin were studied over a 14-month period. To discriminate between internal and external recovery mechanisms, half of the treated ponds were covered by 1-mm mesh lids that restricted aerial recolonization. Both structural (abundance of the different taxonomic groups) and functional (litter breakdown) parameters were monitored. Insects were broadly reduced in numbers by deltamethrin addition. In general, noninsect groups were not affected or increased in abundance in deltamethrin-treated ponds, probably because of relative insensitivity to deltamethrin, reduced predation, and lower competition for food. No major change in litter breakdown rates were seen, probably because of functional redundancy among the macrobenthic community. Chironominae larvae recovered in open, treated mesocosms 62 d after deltamethrin addition and most insect groups recovered 84 d after the treatment date. However, the presence of lids significantly reduced insect recovery rate, suggesting that it largely depends on the immigration of winged forms (i.e., external recovery) from surrounding non- or less affected systems. These results indicate that the recovery time of macrobenthic communities in an affected natural pond would depend on spatial characteristics of the landscape and also the season that exposure occurs. Isolated ecosystems would display posttreatment insect recovery dynamics very different from highly connected ones, evolving toward alternate pseudoequilibrium states, possibly with lower biodiversity but with preserved functionality. Consequences for higher tier risk assessment of pesticides are discussed.

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

Effects of environmental concentrations of atrazine on hemocyte density and phagocytic activity in the pond snail Lymnaea stagnalis (Gastropoda, Pulmonata)

Immunotoxicological effects of environmentally relevant concentrations (10, 23, 50, 100 microg/l) of atrazine were studied in Lymnaea stagnalis. Individual hemolymph sampling was performed at 0, 24, 48, 72, 96, 168, 336, 504 and 672 h during exposure. Every atrazine concentration induced a significant increase in the mean number of circulating hemocytes, without any concentration-response relation. A peak (1.6-fold increase) of hemocyte density was observed after 96 h of exposure. After 504 h, the number of hemocytes remained higher only in the snails exposed to the two highest concentrations. Granulocytes contributed most to the increase in hemocyte density in herbicide-exposed snails. Both short- (24 and 96 h) and long-term (504 h) exposures resulted in significant inhibition of hemocyte phagocytic activity upon E. coli. Over the long-term, phagocytosis recovered for the two lowest concentrations. After 504 h of exposure, every herbicide level resulted in a significant reduction of reactive oxygen species production in E. coli-stimulated hemocytes, which was not observed for short-term exposures.

Purification and partial characterization of glutathione S-transferases from insecticide-resistant and lindane-induced susceptible Spodoptera littoralis (Boisd.) larvae

Abstract Cytosolic GST activity was followed after a single topical application of a non-lethal dose of lindane to fourth instar larvae of a susceptible strain of Spodoptera littoralis (Boisd.). The induction reached its peak 8 h after treatment for 1-chloro-2,4-dinitrobenzene (CDNB) conjugating GSTs (1.5-fold), and 12 h after the treatment for 1,2-dichloro-4-nitrobenzene (DCNB) comjugating enzymes (1.3-fold). CDNB conjugating GST activity in an insecticide-resistant strain of S. littoralis was twice that measured in the non-induced suceptible larvae. No significant difference was observed with DCNB. GSTs of both larvae were purified by affinity chromatography using a glutathione-agarose column, which resulted in a 30-fold prufication and a yield of 50–70%. Purified GSTs were characterized using CDNB as the substrate. Apparent K m and V max values calculated for CDNB and reduced glutathione (GSH) were significantly different between the resistant strain and the susceptible one, whether induced by lindane or not. The lindane treatment modified only K m values for CDNB and GSH in susceptible larvae. Monodimensional SDS-PAGE of GSTs purified from both strains revealed a large band of 27 kDa apparent molecular weight.

Influence of isolation on the recovery of pond mesocosms from the application of an insecticide. I. Study design and planktonic community responses

The influence of relative isolation on the ecological recovery of freshwater outdoor mesocosm communities after an acute toxic stress was assessed in a 14-month-long study. A single concentration of deltamethrin was applied to 8 out of 16 outdoor 9-m3 mesocosms to create a rapid decrease of the abundance of arthropods. To discriminate between external and internal recovery mechanisms, four treated and four untreated (control) mesocosms were covered with 1-mm mesh screen lids. The dynamics of planktonic communities were monitored in the four types of ponds. The abundance of many phytoplankton taxa increased after deltamethrin addition, but the magnitude of most increases was relatively small, probably due to low nutrient availability and the survival of rotifers. The greatest impact on zooplankton was seen in Daphniidae and, to a lesser extent, calanoid copepods. Recovery (defined as when statistical analysis failed to detect a difference in the abundance between the deltamethrin-treated ponds and corresponding control ponds for two consecutive sampling dates) of Daphniidae was observed in the water column 105 and 77 d after deltamethrin addition in open and covered mesocosms, respectively, and <42 d for both open and covered ponds at the surface of the sediments. Rotifers did not proliferate, probably because of the survival of predators (e.g., cyclopoid copepods). These results confirm that the recovery of planktonic communities after exposure to a strong temporary chemical stress mostly depends upon internal mechanisms (except for larvae of the insect Chaoborus sp.) and that recovery dynamics are controlled by biotic factors, such as the presence of dormant forms and selective survival of predators.

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.

Bti sprays do not adversely affect non‐target aquatic invertebrates in French Atlantic coastal wetlands

Both the increase in human mobility and climate change contribute to the globalization of vector-borne diseases. Some mosquito species are efficient disease vectors in Europe, thus increasing the risk of epidemic (re)emergence.Bacillus thuringiensis var. israelensis (Bti) is considered as the most efficient larvicide to control mosquito populations with negligible environmental impacts. However, repeated field applications of Bti over many years raise the question of possible long-term effects on non-target invertebrates with putative subsequent alterations of food webs. Environmental effects of Bti have mainly been studied in continental freshwater wetlands. Much less is known for brackish water coastal wetlands. We investigated whether repeated treatments with Bti, applied as VectoBac((R)) WG over seven consecutive years, may affect non-target invertebrate communities in wetlands of the French Atlantic coast. Particular attention was devoted to invertebrates potentially used as food sources by shorebirds and wading birds. Invertebrates were sampled in the water and sediment of control and VectoBac((R))-treated saltmarsh pools between 2006 and 2012. Taxa abundance data were used to calculate community descriptors and to analyse the potential structural changes due to VectoBac((R)) using the principal response curve method and similarity analysis. Physicochemical parameters were measured in the same pools so that homogeneity of the environmental conditions between the control and treated areas could be tested. We demonstrated that long-term use of VectoBac((R)) WG in French Atlantic coastal wetlands had no influence on the temporal evolution of the taxonomic structure and taxa abundance of non-target aquatic invertebrate communities, which is highly driven by abiotic factors. In addition, over the long term, the amount of invertebrates that could be used as food resources by birds is maintained in VectoBac((R))-treated areas.Synthesis and applications. Reduced application rate and targeted spraying of VectoBac((R)) WG in mosquito breeding sites minimize potential environmental impacts of Bacillus thuringiensis var. israelensis (Bti). Even so, surveillance of its possible primary side effects is needed, which requires comparable control and treated areas. Indeed, systematic temporal trends and subtle differences in the range of variation of abiotic factors result in discrepancies between control and treated area in terms of invertebrate abundance, which could be wrongly attributed to VectoBac((R)). Management decisions and mitigation measures may therefore benefit from (i) extending surveillance to a time frame that allows for coverage of the immense temporal variation in taxa abundance and diversity and (ii) the inclusion of environmental variables in the monitoring of non-target animal communities potentially exposed to Bti. Reduced application rate and targeted spraying of VectoBac((R)) WG in mosquito breeding sites minimize potential environmental impacts of Bacillus thuringiensis var. israelensis (Bti). Even so, surveillance of its possible primary side effects is needed, which requires comparable control and treated areas. Indeed, systematic temporal trends and subtle differences in the range of variation of abiotic factors result in discrepancies between control and treated area in terms of invertebrate abundance, which could be wrongly attributed to VectoBac((R)). Management decisions and mitigation measures may therefore benefit from (i) extending surveillance to a time frame that allows for coverage of the immense temporal variation in taxa abundance and diversity and (ii) the inclusion of environmental variables in the monitoring of non-target animal communities potentially exposed to Bti.

Development and validation of an OECD reproductive toxicity test guideline with the pond snail Lymnaea stagnalis (Mollusca, Gastropoda)

The OECD test guideline development program has been extended in 2011 to establish a partial life-cycle protocol for assessing the reproductive toxicity of chemicals to several mollusk species, including the great pond snail Lymnaea stagnalis. In this paper, we summarize the standard draft protocol for a reproduction test with this species, and present inter-comparison results obtained in a 56-day prevalidation ring-test using this protocol. Seven European laboratories performed semi-static tests with cultured snails of the strain Renilys® exposed to nominal concentrations of cadmium chloride (from 53 to 608μgCdL(-1)). Cd concentrations in test solutions were analytically determined to confirm accuracy in the metal exposure concentrations in all laboratories. Physico-chemical and biological validity criteria (namely dissolved oxygen content >60% ASV, water temperature 20±1°C, control snail survival >80% and control snail fecundity >8 egg-masses per snail over the test period) were met in all laboratories which consistently demonstrated the reproductive toxicity of Cd in snails using the proposed draft protocol. Effect concentrations for fecundity after 56days were reproducible between laboratories (68<EC50-56d<124μgL(-1)) and were consistent with literature data. EC50-56d and EC10-56d values were comprised within a factor of 1.8 and 3.6, respectively, which is in the range of acceptable variation defined for reference chemicals in OECD test guidelines for invertebrates. The inter-laboratory reproducibility coefficient of variation (CV) for the Cd LC50-56d values was 8.19%. The inter-laboratory comparison of fecundity within the controls gave a CV of 29.12%, while exposure to Cd gave a CV of 25.49% based on the EC50-56d values. The OECD has acknowledged the success of this prevalidation exercise and a validation ring-test involving 14 laboratories in Europe, North- and South-America is currently being implemented using four chemicals (Cd, prochloraz, trenbolone and tributyltin).

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.