DNA damage in Gammarus fossarum sperm as a biomarker of genotoxic pressure: intrinsic variability and reference level.

In the perspective of a biomonitoring application for assessing genotoxicity of freshwater ecosystems, the Comet assay has recently been developed on spermatozoa in the amphipod Gammarus fossarum, in order to propose a sensitive and reliable genotoxicity biomarker in an ecologically relevant freshwater species. The appropriate use of a genotoxicity biomarker requires good knowledge of its basal level and its natural variability related to intrinsic biotic and environmental abiotic factors. We propose a procedure for which the lowest biomarker variability related to methodological and intrinsic biotic factors is obtained and a reference value of biomarker basal response taking into account its spatio-temporal changes has been defined. A strong impact of spermatogenesis status and exposure time on the response to genotoxicant pressure was observed. These reports led us to select a standard organism, i.e., the mature male gammarid in precopula. No effect of temperature and conductivity on baseline DNA damage was observed in the laboratory for the tested range (6-24 °C and 300/600 µS cm⁻¹). Similarly, no spatio-temporal change relative to season or the physico-chemical characteristics of the water was recorded during the field survey. On the basis of these results, a reference level with maximal threshold values has been proposed for the standard gammarid.

DNA damage in caged Gammarus fossarum amphipods: a tool for freshwater genotoxicity assessment.

The aim of this study was to propose a tool for freshwater environmental genotoxicity assessment using Gammarus fossarum, a high ecologically relevant species. In a first part, gammarids were caged upstream and downstream wastewater treatment plant effluent output. The sensitivity of genotoxic responses of haemocytes, oocytes and spermatozoa was compared using the Comet assay. Spermatozoa appeared to be the most sensitive, suitable and relevant cell type for genotoxicity risk assessment. In a second part, a watershed-scale study was conducted over 2 years to evaluate the applicability of our caging procedure. The genotoxic impact of a contamination was followed, taking into account seasonal variability. DNA damage in spermatozoa exhibited low basal level and low variability in control upstream sites, providing a reliable discrimination of polluted sites. Finally, DNA damage in caged G. fossarum has been proved to be a sensitive and reproducible tool for freshwater genotoxicity assessment.

In situ biomonitoring of freshwater quality using the New Zealand mudsnail Potamopyrgus antipodarum (Gray) exposed to waste water treatment plant (WWTP) effluent discharges.

Mollusk species have been shown to be sensitive to various endocrine disrupting compounds (EDC) at environmentally relevant concentrations. Waste water treatment plant (WWTP) effluents are a major source of potential or known EDC in the aquatic environment. The aim of this study was to develop an in situ exposure method using the New Zealand mudsnail Potamopyrgus antipodarum (Molluska, Hydrobiidea) to assess the impact of water quality on the life traits of this species, by focusing on its reproduction. The impact of three WWTP discharges on three different receiving rivers was studied. The effects of WWTP effluent on adult survival, weight, reproduction and vertebrate-like sex-steroid levels in snails were monitored for three to four weeks. Although the physicochemical and hydrological parameters varied greatly between the rivers, the caging experiments allowed us to detect significant impairment of the life traits of snails when exposed downstream of the WWTPs discharge. While adult survival was not affected by exposure, reproduction was significantly impacted downstream from the WWTP effluent discharges (60-70% decrease of embryos without shells after three to four weeks exposure) independently of the river. Modulations of steroid levels proved to be an informative parameter with an increase of testosterone downstream of the discharges, and increases and decreases of 17beta-estradiol levels according to site. The endpoints used proved to be an adapted method for field exposures and allowed the discrimination between upstream and downstream sites.

Acetylcholinesterase activity in Gammarus fossarum (Crustacea Amphipoda) Intrinsic variability, reference levels, and a reliable tool for field surveys.

The appropriate use of an enzyme activity as a biomarker requires good knowledge of its basal level and its natural variability related to intrinsic biotic and environmental abiotic factors. In view of using whole-body acetylcholinesterase (AChE) activity in Gammarus fossarum as a reliable biomarker of exposure to anti-cholinesterase agents in aquatic ecosystems, (i) the effects of the main biotic (sex, reproductive status, and weight) and abiotic (water temperature) factors on the basal activity level of this enzyme were measured in the laboratory and (ii) the spatio-temporal variability of basal enzyme activity was followed in wild populations over a 1-year period. The results show no direct effect of sex. However, significant differences in AChE activity were observed between females depending on gonadal and embryonic development. A strong negative correlation between the AChE activity levels and organism body weight was observed. Indeed, AChE activity decreases drastically during the early life stages and tends to stabilise in larger individuals. These reports led us to select a standard organism (male; weight range, 15-20mg) to minimise inter-individual variability. No effect of temperature on basal AChE activity was observed in the laboratory for the tested range (6-24 degrees C). Similarly, no spatio-temporal change relative to season or the physico-chemical characteristics of the water (such as conductivity and temperature) was recorded during the field survey. On the basis of field-collected data, we defined the standard organism having a reference activity level with minimal and maximal threshold values. Finally, the value of AChE activity normalisation by protein contents is discussed.

Assessing the risk of metal mixtures in contaminated sediments on Chironomus riparius based on cytosolic accumulation.

Sediments usually contain mixtures of trace metals introduced via natural geochemical processes and anthropogenic activities. Kinetics and effects of these metals are strongly dependent both on the composition of the mixture and on the physico-chemical characteristics of the sediment. Relating effects to metal concentration may consequently be advised. However, total accumulation may be a poor predictor of metal toxicity for Chironomus riparius exposed to contaminated field sediments. As an alternative, we proposed to relate effects on Chironomus growth with cytosolic metal accumulation, measured in larvae after a short exposure period. Dose-response relationships were derived for zinc, copper, and cadmium through single-metal exposure data analysed with toxicokinetics and toxicodynamics models. They permitted, on the basis of cytosolic accumulation measures, to predict successfully the effects of mixtures of cadmium, zinc, and copper on the growth of larvae exposed to spiked sediments, as well as to field sediments in which zinc and copper were assumed to be predominant.

Dynamic energy budget as a basis to model population-level effects of zinc-spiked sediments in the gastropod Valvata piscinalis.

This paper presents original toxicity test designs and mathematical models that may be used to assess the deleterious effects of toxicants on Valvata piscinalis (Mollusca, Gastropoda). Results obtained for zinc, used as a reference toxicant, are presented. The feeding behavior, juvenile survival, growth, age at puberty, onset of reproduction, number of breedings during the life cycle, and fecundity were significantly altered when the snails were exposed to zinc-spiked sediments. Dynamic energy budget models (DEBtox) adequately predicted the effects of zinc on the V. piscinalis life cycle. They also provided estimates for lifecycle parameters that were used to parameterize a demographic model, based on a Z-transformed life-cycle graph. The effect threshold for the population growth rate (lambda) was estimated at 259 mg/kg dry sediment of zinc, showing that significant changes in abundance may occur at environmental concentrations. Significant effects occurring just above this threshold value were mainly caused by the severe impairment of reproductive endpoints. Sensitivity analysis showed that the value of lambda depended mainly on the juvenile survival rate. The impairment of this latter parameter may result in extinction of V. piscinalis. Finally, the present study highlights advantages of the proposed modeling approach in V. piscinalis and possible transfer to other test species and contaminants.

Rearing and estimation of life-cycle parameters of the tubicifid worm Branchiura sowerbyi: application to ecotoxicity testing.

This paper provides original collection, acclimatizing, rearing and toxicity test methods for the freshwater worm Branchiura sowerbyi, an alternative species to Tubifex tubifex for ecotoxicity evaluation of sediments. Influence of the substrate, type of food, and feeding level on individual performances was assessed in short-term tests, in order to set up optimal culture and test conditions. Low-size particles and high organic matter content favoured the growth and reproduction of B. sowerbyi. The relative contribution of sediments and fish food to the individual food intake was assessed using a foraging efficiency model based upon the dynamic energy budget theory. Individual performances were optimal when the substrate plus fish food provided the energy equivalent to 5 mg Tetramin per worm per day, which is the ad libitum food level for adults at 21 degrees C. The life-cycle of the worm was fully characterized using a life-cycle test conducted under the previously defined optimal conditions. Hatching rates were low (32%), whereas newborn and juveniles exhibited high survival (>80%) and growth (2.4 mg/day in juveniles) rates. Age at puberty was low (60 days) when compared to the maximal life span (1100 days) as predicted using a Weibull model. Adults reproduced every other month with a constant fecundity (0.16 cocoon/worm/day). The mean values of the life-cycle parameters and their variability and reproducibility among laboratory studies were discussed in order to identify relevant endpoints to be used in ecotoxicity tests. Survival, juvenile growth, and fecundity may constitute suitable test endpoints, whereas hatching rate and adult growth should not be used as endpoints in B. sowerbyi.

Development of a zebrafish 4-day embryo-larval bioassay to assess toxicity of chemicals.

A 4-day embryo-larval zebrafish test, from blastula stage to hatching included, was developed. The observations of embryo developmental were made at different development stages, for which morphological, physiological, and behavioral endpoints were selected and quantified for unexposed and exposed embryos. The sensitivity and the ability of these endpoints to inform about mode of action (MoA) were established in testing three model toxicants with well-known toxic effects (propranolol, malathion, cadmium). Lethal, sublethal (heart rate/edema, spontaneous movements, and hatching rate/time disturbance), and teratogenic effects were detected for all the studied compounds. This bioassay allows characterization of impairments at different biological levels: neuromuscular, physiological, morphological, and behavioral, and brings useful information about the toxic MoA of the chemicals on nontarget organisms. In this sense to answers the chemical industries and international organization (EMEA) requirements for the environmental risk assessment of new chemicals and pharmaceuticals.

Deriving effects on Chironomus population carrying capacity from standard toxicity tests.

Translating effects from individual to population level is a crucial issue in ecological risk assessment of bed sediments. We first propose a modeling package to tackle this question for the species Chironomus riparius with biology- and ecology-based models to deduce effects on chironomid production per generation or per year. However, this approach requires the performance of many toxicity tests. Currently, the information available to perform this scaling is limited to a few data from standardized tests, mainly growth and survival after a certain period of time and sometimes emergence after 28 d. We propose here to simplify our model to make it able to deal with only data from standardized tests. The daily death rate is assumed to be constant throughout each instar, the effects on growth to be independent of time, and the effects on reproduction to be negligible. The reliability of this approach is first tested by comparing the population effects on organisms exposed to copper-spiked sediments calculated with the simple model and the more exhaustive one from which it was derived. The simple model just slightly underestimated the effects at the population level. Second, data from tests performed either with unpolluted sediment or with field sediment with different degrees of contamination are used to propose an effect threshold for populations. It appears that control sediments lead to less than 20% effects at population level, which may help define a natural variability range. For polluted sediments, we could find effects up to 42%. Implications of these findings are discussed in a sediment risk assessment perspective.

Development of rearing and testing protocols for a new freshwater sediment test species: the gastropod Valvata piscinalis.

This paper aimed at proposing rearing and testing protocols for Valvata piscinalis, a new potential species for sediment toxicity testing. Such tests were developed since this species reliably represents the bio/ecological characteristics of other gastropods. It may thus be representative of their sensitivity to chemicals. V. piscinalis was successfully cultured in our laboratory for six generations. Cultures provided a high productivity for a low working time and low costs. The tests conditions we proposed seemed to be relevant for the development of reliable tests with this species. Indeed, hatching probability of egg-capsules, as well as embryo, newborn and juvenile survival rates, were close to 100%. Moreover, growth rates and fecundity were significantly higher than in field and in other laboratory studies. Partial life-cycle tests on clean sediments were achieved for various feeding levels to determine survival, growth and reproduction patterns, ad libitum feeding level and life cycle parameters values. Ad libitum feeding levels for newborn, juveniles and adults were 0.1, 0.4 and 0.8 mg Tetramin/individual/working day. Growth tests with zinc-spiked sediments provided a no-effect concentration and a lowest effect concentration of respectively 200 and 624 mg zinc/kg dry sediment. Other growth tests on spiked sediments we ran at our laboratory with second, third and fourth instars larvae of Chironomus riparius pointed out that V. piscinalis was more sensible to zinc than the chironomid, which is a routine test species in ecotoxicology. According to these results, V. piscinalis is a promising candidate species for sediment toxicity testing.

Body residues: a key variable to analyze toxicity tests with Chironomus riparius exposed to copper-spiked sediments.

Fourth instar Chironomus riparius larvae were exposed to four sediments spiked with copper. Length and copper concentration in the tissue were monitored daily. Kinetics data analysis was performed by fitting a one-compartment model. The analysis of growth data was performed using an energy-based approach which estimates a no-effect concentration (NEC). The elimination rate of copper did not depend on the sediment characteristics, whereas the bioconcentration factor differed by a factor of 10 among the tested sediments, accounting for differences in copper bioavailability. Consequently, the NEC expressed in term of exposure concentrations differed substantially between sediments. In contrast, the NEC expressed in terms of body residues did not depend on the sediment characteristics. This work links, for the first time, a mechanistic effects model with the critical body residue approach, and could contribute to develop relevant tools for sediment risk assessment.

Modelling of the life cycle of Chironomus species using an energy-based model.

Little is known about the differences between the species of the genus Chironomus relatively to their life cycle strategies. This knowledge is however crucial to fully understand the response of the Chironomus community to field perturbations. Here, we proposed to study four Chironomus species by using an energy-based model to describe growth, emergence and reproduction. We used data from the literature for two species (Chironomus plumosus and Chironomus tentans) and data from our experiments for two other species (Chironomus prasinus and Chironomus riparius). We showed that our model is able to accurately describe the life-history attributes for all the species tested, which suggests that Chironomus species have the same fundamental characteristics (low maintenance energetic costs, isomorphism), which makes possible the building of a common modelling framework to assess effects of toxicants at individual and population level. The species showed a few differences relatively to the parameters of the models with possible consequences when assessing effects of chemicals on Chironomus community. For instance, due to differences in growth parameters, C. riparius population dynamics should be more sensitive to effects on individual growth than C. prasinus or C. plumosus ones.

Environmental risk assessment of six human pharmaceuticals: are the current environmental risk assessment procedures sufficient for the protection of the aquatic environment?

In this study, exposure and ecotoxicity data of six human pharmaceuticals (carbamazepine, clofibric acid, diclofenac, ofloxacin, propranolol, and sulfamethoxazole) were collected, including our own experimental data and literature data. From this data collection, the two-tiered European draft guideline on the environmental risk assessment of human pharmaceuticals was tested. Measured environmental concentrations in effluents from France and in effluents and surface waters from Germany were compared to the predicted environmental concentrations (PECs) in both countries. In a similar manner, predicted no-effect concentrations (PNECs) derived from acute data and PNECs derived from chronic data were estimated for each pharmaceutical and corresponding PEC/PNEC ratios then were compared in both countries. Globally, results demonstrated that all environmental concentrations (predicted or measured) for each considered pharmaceutical exceeded the 10-ng/L cutoff value, which requires the implementation of the second-tier assessment based on ecotoxicity data. Moreover, the six pharmaceuticals showed a relatively limited acute toxicity, and carbamazepine and propranolol were inaccurately identified as having negligible risks under the current European draft procedure. Such results lead to discussion of the actual procedure on pharmaceuticals, especially on the need of appropriate ecotoxicity tests.

Energy-based modeling as a basis for the analysis of reproductive data with the midge (Chironomus riparius).

We propose a biologically based approach to analyze reproductive data for the midge (Chironomus riparius). We showed in a previous study that its larval development can be divided in two distinct phases regarding the use of energy: The somatic growth period and the gametic growth period. We hypothesize here the gametic period to be a crucial period for the energetic investment for reproduction. To test this, we performed several assays with different feeding programs. Our results confirmed that energetic investment for reproduction mainly occurs during the gametic growth period. We could then propose energy-based models to analyze reproductive data. We assumed the effects to result from a perturbation in the use of energy. Two models corresponding to different physiological modes of actions--decreased efficiency of feeding (i.e., feeding decrease model) or increased cost of egg production (i.e., egg-cost increase model)--were built and used for the analysis of data obtained after an exposure to an artificially copper-spiked sediment. During this experiment, different exposure patterns were performed to investigate whether effects on reproduction resulted mainly from an exposure that occurs during the gametic growth period or from an exposure that occurs during the somatic growth period. These exposure patterns led to similar effects on reproduction, which suggests that the toxicant-induced stress persists during the whole life cycle, even in case of exposure occurring only during part of it. Both the feeding decrease model and egg-cost increase model were able to describe the data. We showed that addition of copper in the sediment, even in low quantity (<6.5 mg/kg), might affect reproduction at the individual level. If no-effect concentrations were derived from these results, this may lead to an overprotective value. A relevant no-effect concentration could be derived from further investigations at the population level.

A model to understand the confounding effects of natural sediments in toxicity tests with Chironomus riparius.

Recently, we built a model to link feeding input with growth, emergence, and reproduction of the midge Chironomus riparius exposed to an artificial sandy sediment. This model is based on assumptions about both feeding behavior and use of energy. Here, we show how it can be used for toxicity tests with natural sediments to understand and model the influence of sediment characteristics. We measured growth, emergence, and reproduction of chironomids exposed in beakers to four unpolluted natural sediments and three feeding conditions (no feeding, 0.2 mg Tetramin/larva/d, and 1.4 mg Tetramin/larva/d) and compared the results with data obtained on our artificial sandy sediment. Sediment characteristics had lower influence on growth than feeding level, but their influence could not be neglected. First, we could distinguish between sandy sediments and other sediments. This difference resulted in a significant delay of about 18 h in the growth curves. Second, in case of food limitation, chironomids could use the organic materials in the sediment, provided that the C:N ratio of the sediment was less than 14. Our model proved to be able to incorporate those two phenomena. As for reproduction, we observed a better reproduction (measured in number of eggs per mass) for natural sediments than for artificial sediments. We showed that this difference could be due to the lipid content of the natural sediments.

Modelling toxicity and mode of action of chemicals to analyse growth and emergence tests with the midge Chironomus riparius.

We present a new growth test data analysis for toxicity tests with the midge Chironomus riparius. The analysis is based on mathematical models which proved to be able to predict growth and emergence of non-exposed organisms for diverse feeding levels or densities. Here, we adapt these models to account for toxicity. We distinguish between two modes of action of the compounds: decrease of feeding or increase of growth energy costs. The models are used to analyse growth data with organisms exposed to copper spiked artificial sediments. Both models provide a good fitting of the data in the case of feeding ad libitum, but only the growth costs model can account for effects of copper in the case of food limitation. We also show that the threshold of effects do not depend on the age (the no effect concentrations (NOEC) are 6, 7 and 9 mg/kg, respectively, for second, third and fourth instars larvae), but that, as soon as this threshold is exceeded, fourth instar larvae are less affected by copper than earlier larvae. Our models constitute a step towards a more biologically relevant analysis of standardized tests, which should facilitate both the understanding of the mechanisms of toxicity and the change of scale from the individual to the population.

A modeling approach to link food availability, growth, emergence, and reproduction for the midge Chironomus riparius.

We present models to link feeding with growth, emergence, and reproduction of the midge Chironomus riparius. These models are based on assumptions about the biology of this species and distinguish between males and females. The assumptions are the isomorphism of the chironomidae, the fact that much more energy is used for growth than for maintenance, and the existence of a maximum length for male and female larvae that does not depend on food availability. We supported our assumptions by experimental data and estimated the parameters of the model. We then successfully predicted the length pattern of 2-d-old larvae exposed in an artificial sediment to different feeding levels with different starting densities and also linked emergence time and growth pattern. We found our model to be consistent with data from another study and another species (Chironomus plumosus). As for reproduction, the mean number of eggs per mass was described as a linear function of feeding quantity. Our models could be used in sediment risk assessment to choose feeding level, to build effects models, or to predict the effects of toxicants at the population level.