Thierry Caquet

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.

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.

Hypoxia, hyperoxia and exposure to potassium dichromate or fenitrothion alter the energy metabolism in Chironomus riparius Mg. (Diptera: Chironomidae) larvae.

Short-term (24 h) effects of four stressors (hypoxia, hyperoxia, potassium dichromate, fenitrothion) on the activity of the electron transport system (ETS) and total lipid, glycogen and protein contents were assessed in 4th instar larvae of Chironomus riparius. Hypoxia and hyperoxia caused an increase in ETS activity and protein content. Glycogen content decreased when larvae were placed under hypoxic conditions. ETS activity increased following exposure to 2 microg x l(-1) of fenitrothion. It decreased in larvae exposed to 20 microg x l(-1) of this insecticide. A decrease in lipid and glycogen contents was observed in larvae exposed to potassium dichromate or fenitrothion. Changes in ETS activity and lipid and glycogen contents may be related to the activation of the respiratory chain due to an increase in energy cost associated with homeostatic phenomena, such as detoxification processes. These results suggest that some parameters related to energy metabolism, such as ETS activity and lipid and glycogen contents, may be used as biomarkers of environmental disturbance in Chironomus riparius larvae.

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.

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.

Heterosis and inbreeding depression in bottlenecked populations: a test in the hermaphroditic freshwater snail Lymnaea stagnalis

Small population size is expected to induce heterosis, due to the random fixation and accumulation of mildly deleterious mutations, whereas within‐population inbreeding depression should decrease due to increased homozygosity. Population bottlenecks, although less effective, may have similar consequences. We tested this hypothesis in the self‐fertile freshwater snail Lymnaea stagnalis, by subjecting experimental populations to a single bottleneck of varied magnitude. Although patterns were not strong, heterosis was significant in the most severely bottlenecked populations, under stressful conditions. This was mainly due to hatching rate, suggesting that early acting and highly deleterious alleles were involved. Although L. stagnalis is a preferential outcrosser, inbreeding depression was very low and showed no clear relationship with bottleneck size. In the less reduced populations, inbreeding depression for hatching success increased under high inbreeding. This may be consistent with the occurence of synergistic epistasis between fitness loci, which may contribute to favour outcrossing in L. stagnalis.

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.

Chitobiase activity as an indicator of altered survival, growth and reproduction in Daphnia pulex and Daphnia magna (Crustacea: Cladocera) exposed to spinosad and diflubenzuron

Chitobiase is involved in exoskeleton degradation and recycling during the moulting process in arthropods. In aquatic species, the moulting fluid is released into the aqueous environment, and chitobiase activity present therein can be used to follow the dynamics of arthropod populations. Here, chitobiase activity was used for monitoring the impact of mosquito candidate larvicides on Daphnia pulex and Daphnia magna under laboratory conditions. Both species were exposed to spinosad (2, 4, 8 μg L(-1)) and diflubenzuron (0.2, 0.4, 0.8 μg L(-1)) for 14 days. Bacillus thuringiensis var. israelensis (Bti; 0.25, 0.5, 1 μL L(-1)) was used as the reference larvicide. Chitobiase activity, adult survival, individual growth and fecundity, expressed as the number of neonates produced, were measured every 2 days. Average Exposure Concentrations of spinosad were ten-fold lower than the nominal concentrations, whereas only a slight deviation was observed for diflubenzuron. In contrast to Bti, spinosad and diflubenzuron significantly affected both species in terms of adult survival, and production of neonates. As compared to D. pulex, D. magna was more severely affected by diflubenzuron, at low and medium concentrations, with reduced adult growth and much lower chitobiase activity. Chitobiase activity was positively correlated with the individual body length, number of neonates produced between two consecutive observation dates, and number of females and neonates. In addition, the significant positive correlations between chitobiase activity measured on the last sampling date before the first emission of neonates and the cumulative number of neonates produced during the whole observation period strongly support the potential of the activity of this chitinolytic enzyme as a proxy for assessing the dynamics of arthropod populations exposed to larvicides used for mosquito control.

Characterization of superoxide dismutase activity in Chironomus riparius Mg. (Diptera, Chironomidae) larvae--a potential biomarker.

The activities of superoxide dismutase (SOD) isoenzymes were measured in fourth instar larvae of Chironomus riparius Mg. Three types of superoxide dismutase were identified: Cu,Zn-SOD in hemolymph and postmitochondrial fraction; Mn-SOD in mitochondrial fraction and presumably Fe-SOD in postmitochondrial fraction. The latter could have an endosymbiotic or a parasitic origin. Extracellular and cytosolic SOD activities, especially Cu,Zn-SOD, tended to increase in the last phase of larval development, independently of protein or hemoglobin contents. This supposes that SOD activity in Ch. riparius larvae is probably activated at the end of fourth instar stage. Cu,Zn-SOD and Mn-SOD activities showed a significant increase under severe hypoxia and slight hyperoxia. Oxygen radical scavengers such as SOD may play a role in the increased tolerance of Ch. riparius to oxidative stress. These results suggest that the specific induction of some SOD isoenzymes could be used as a biomarker of environmental disturbance such as oxidative stress initiated by xenobiotics.

An integrated environmental approach to investigate biomarker fluctuations in the blue mussel Mytilus edulis L. in the Vilaine estuary, France

Estuarine areas represent complex and highly changing environments at the interface between freshwater and marine aquatic ecosystems. Therefore, the aquatic organisms living in estuaries have to face highly variable environmental conditions. The aim of this work was to study the influence of environmental changes from either natural or anthropogenic origins on the physiological responses of Mytilus edulis. Mussels were collected in the Vilaine estuary during early summer because this season represents a critical period of active reproduction in mussels and of increased anthropogenic inputs from agricultural and boating activities into the estuary. The physiological status of the mussel M. edulis was evaluated through measurements of a suite of biomarkers related to: oxidative stress (catalase, malondialdehyde), detoxication (benzopyrene hydroxylase, carboxylesterase), neurotoxicity (acetylcholinesterase), reproductive cycle (vitelline, condition index, maturation stages), immunotoxicity (hemocyte concentration, granulocyte percentage, phagocytosis, reactive oxygen species production, oxidative burst), and general physiological stress (lysosomal stability). A selection of relevant organic contaminant (pesticides, (polycyclic aromatic hydrocarbons, polychlorobiphenyls) was measured as well as environmental parameters (water temperature, salinity, total suspended solids, turbidity, chlorophyll a, pheopigments) and mussel phycotoxin contamination. Two locations differently exposed to the plume of the Vilaine River were compared. Both temporal and inter-site variations of these biomarkers were studied. Our results show that reproduction cycle and environmental parameters such as temperature, organic ontaminants, and algal blooms could strongly influence the biomarker responses. These observations highlight the necessity to conduct integrated environmental approaches in order to better understand the causes of biomarker variations.