Jeanne Garric

Ecotoxicological impact of pharmaceuticals found in treated wastewaters: study of carbamazepine, clofibric acid, and diclofenac

In four countries (France, Greece, Italy, and Sweden) occurrence in sewage treatment plant (STP) effluents and ecotoxicity of the pharmaceuticals carbamazepine, clofibric acid, and diclofenac were investigated. Bioassays were performed on bacteria, algae, microcrustaceans, and fishes in order to calculate their predicted no-effect concentrations (PNEC) and to perform a first approach of risk characterization. For this aim, risk has been estimated by the predicted environmental concentration/PNEC ratio and the measured environmental concentration/PNEC ratio. First, regarding the PNEC, carbamazepine appears to be the more hazardous compound. Second, even though it is demonstrated that carbamazepine, clofibric acid, and diclofenac have been detected in effluents, only carbamazepine have been detected in all sewage treatment plants with the greatest concentrations. Third, risk quotients greater than unity were calculated only for carbamazepine, suggesting that risk for the water compartment is expected.

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.

Anticancer drugs in surface waters: what can we say about the occurrence and environmental significance of cytotoxic, cytostatic and endocrine therapy drugs?

This study considers the implications and research needs arising from anticancer (also referred to as antineoplastic) drugs being released into the aquatic environment, for the entire therapeutic classes used: cytotoxic, cytostatic and endocrine therapy drugs. A categorization approach, based on French consumption amounts, allowed to highlight parent molecules and several metabolites on which further occurrence and ecotoxicological studies should be conducted. Investigations of consumption trends at a national and a local scale show an increase in the use of anticancer drugs between 2004 and 2008, thus leading to increased levels released in the environment. It therefore appears necessary to continue surveying their presence in surface waters and in wastewater treatment plant (WWTP) effluents. Furthermore, due to the rise of anticancer home treatments, most of the prescribed molecules are now available in town pharmacies. Consequently, hospital effluents are no longer the main expected entry route of anticancer drugs into the aquatic environment. Concerning ecotoxicological risks, current knowledge remains insufficient to support a definitive conclusion. Risk posed by cytotoxic molecules is still not well documented and it is not possible to conclude on their long-term effects on non-target organisms. To date, ecotoxicological effects have been assessed using standardized or in vitro assays. Such tests however may not be suitable for anticancer drugs, and further work should focus on full-life cycle or even multigenerational tests. Environmental significance (i.e. occurrence and effects) of cytostatics (protein kinases inhibitors and monoclonal antibodies), if any, is not documented. Protein kinases inhibitors, in particular, deserve further investigation due to their universal mode of action. Finally, concerning endocrine therapy drugs, molecules such as antiestrogen Tamoxifen and its active metabolites, could be of concern. Overall, to accurately assess the ecotoxicological risk of anticancer drugs, we discuss the need to break away from tests on isolated molecules and to test effects of mixtures at the low ng.l(-1) range.

Physiological and behavioural responses of Gammarus pulex (Crustacea: Amphipoda) exposed to cadmium.

The aim of this study was to investigate the effects of cadmium on physiological and behavioural responses in Gammarus pulex. In a first experiment, cadmium LC50s for different times were evaluated in 264 h experiment under continuous mode of exposure (LC50(96 h)=82.1 microgL(-1), LC50(120 h)=37.1 microgL(-1), LC50(168 h)=21.6 microgL(-1), LC50(264 h)=10.5 microgL(-1)). In a second experiment, the physiological and behavioural responses of the amphipod exposed to cadmium (0, 7.5 and 15 microgL(-1)) were investigated under laboratory conditions. The mortality and the whole body cadmium concentration of organisms exposed to cadmium were significantly higher than in controls. Concerning physiological responses, cadmium exposure exerted a significant decrease on osmolality and haemolymph Ca(2+) concentration, but not on haemolymph Na(+) and Cl(-) concentrations, whereas the Na(+)/K(+)-ATPase activity was significantly increased. Behavioural responses, such as feeding rate, locomotor and ventilatory activities, were significantly reduced in Cd exposed organisms. Mechanism of cadmium action and consequent energetic reallocation in favour of maintenance functions (i.e., osmoregulation) are discussed. The results of this study indicate that osmolality and locomotor activity in G. pulex could be effective ecophysiological/behavioural markers to monitor freshwater ecosystem and to assess the health of organisms.

Acetylcholinesterase activity in Gammarus fossarum (Crustacea Amphipoda): linking AChE inhibition and behavioural alteration.

Relations between whole-body acetylcholinesterase (AChE) inhibition and changes in feeding and locomotor behaviours were investigated in adult male Gammarus fossarum during short-term exposure (96h) to the organophosphorous pesticide chlorpyrifos (CPE) and the carbamate pesticide methomyl (MT). AChE activity was measured after 24, 48 and 96h of exposure. The feeding rate was assessed after 48 and 96h of exposure and locomotor activity was measured at the end of the experiment. A concentration-dependent decrease of AChE activity and behavioural parameters was observed for both CPE and MT. However, these two compounds presented dissimilar modes of action since MT-induced effects appeared rapidly during the first 48h of the experiment and remained constant until the end of experiment, contrary to CPE-induced effects, which occurred gradually during the last 48h. For CPE, significant mortality was observed from 50% AChE inhibition, contrary to MT for which no mortality was observed despite 66% inhibition. These results suggest that, for CPE, the observed mortality was not directly related to AChE inhibition but that an additional toxic mode of action occurred. On the contrary, the feeding rate and locomotion impairment were directly correlated to levels of AChE inhibition for both chemicals, except for the lowest concentrations of MT for which an induction of the behavioural parameters was observed. Although CPE and MT have different modes of action (acting as indirect and direct inhibitors), the relations between AChE activity and inhibition of behavioural parameters were relatively close for these two compounds. This study provides a basis to interpret the biomarker AChE at the upper biological organisation level, on both the feeding rate and locomotor behaviour, which are known to be relevant ecological responses.

Progestagens for human use, exposure and hazard assessment for the aquatic environment

Little information is available on the environmental occurrence and ecotoxicological effects of pharmaceutical gestagens released in the aquatic environment. Since eighteen different gestagens were found to be used in France, preliminary exposure and hazard assessment were done. Predicted environmental concentrations (PECs) suggest that if parent gestagens are expected to be found in the ng l(-1) range, some active metabolites could be present at higher concentrations, although limited data on metabolism and environmental fate limit the relevance of PECs. The biological effects are not expected to be restricted to progestagenic activity. Both anti-androgenic activity (mainly for cyproterone acetate, chlormadinone acetate and their metabolites) and estrogenic activity (mainly for reduced metabolites of levonorgestrel and norethisterone) should also occur. All these molecules are likely to have a cumulative effect among themselves or with other xenoestrogens. Studies on occurrence, toxicity and degradation time are therefore needed for several of these compounds.

Fluoxetine effects assessment on the life cycle of aquatic invertebrates

Fluoxetine is a serotonin re-uptake inhibitor, generally used as an antidepressant. It is suspected to provoke substantial effects in the aquatic environment. This study reports the effects of fluoxetine on the life cycle of four invertebrate species, Daphnia magna, Hyalella azteca and the snail Potamopyrgus antipodarum exposed to fluoxetine spiked-water and the midge Chironomus riparius exposed to fluoxetine-spiked sediments. For D. magna, a multi-generational study was performed with exposition of newborns from exposed organisms. Effects of fluoxetine could be found at low measured concentrations (around 10microgl(-1)), especially for parthenogenetic reproduction of D. magna and P. antipodarum. For daphnids, newborns length was impacted by fluoxetine and the second generation of exposed individuals showed much more pronounced effects than the first one, with a NOEC of 8.9microgl(-1). For P. antipodarum, significant decrease of reproduction was found for concentrations around 10microgl(-1). In contrast, we found no effect on the reproduction of H. azteca but a significant effect on growth, which resulted in a NOEC of 33microgl(-1), expressed in nominal concentration. No effect on C. riparius could be found for measured concentrations up to 59.5mgkg(-1). General mechanistic energy-based models showed poor relevance for data analysis, which suggests that fluoxetine targets specific mechanisms of reproduction.

Induction of Fish Vitellogenin and Alterations in Testicular Structure: Preliminary Results of Estrogenic Effects in Chub (Leuciscus cephalus)

Laboratory and field studies were carried out in order to assess, in a common cyprinid freshwater fish, the chub (Leuciscus cephalus), the feasibility of biological methods to detect and quantify estrogenic effects. In the laboratory the effects of 17beta estradiol were investigated on Vitellogenin (Vtg) induction and on testis organisation. Vtg was quantified by immunodetection using an established ELISA for carp (Cyprinus carpio) Vtg. Estradiol exposure resulted in a significant and rapid increase in plasma Vtg in both male and female chub, indicating that vitellogenic response in the chub is sensitive to estrogen(s). Histological examination of the testis in males exposed to estradiol also showed effects on the testis. Preliminary field studies on wild chub showed that fish living in a polluted river had elevated concentrations of plasma Vtg and had some alterations in the testis compared with wild fish from a reference site. Together these preliminary laboratory and field results show that chub could be an interesting fish species for further studies of biological effects of environmental estrogens in the aquatic environment.

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.

Environmental risk assessment for the serotonin re-uptake inhibitor fluoxetine: Case study using the European Risk Assessment Framework

The serotonin re-uptake inhibitor fluoxetine was selected for an environmental risk assessment, using the most recent European guideline (EMEA 2006) within the European Union (EU)-funded Environmental Risk Assessment of Pharmaceuticals (ERAPharm) project due to its environmental persistence, acute toxicity to nontarget organisms, and unique pharmacokinetics associated with a readily ionizable compound. As a widely prescribed psychotropic drug, fluoxetine is frequently detected in surface waters adjacent to urban areas because municipal wastewater effluents are the primary route of entry to aquatic environments. In Phase I of the assessment, the initial predicted environmental concentration of fluoxetine in surface water (initial PEC(SW)) reached or exceeded the action limit of 10 ng/L, when using both a default market penetration factor and prescription data for Sweden, Germany, and the United Kingdom. Consequently, a Phase II risk assessment was conducted in which green algae were identified as the most sensitive species with a NOEC of <0.6 microg/L. From this value, a predicted no effect concentration for surface waters (PNEC(SW)) of 0.012 microg/L was derived. The PEC/PNEC ratio was above the trigger value of 1 in worst-case exposure scenarios indicating a potential risk to the aquatic compartment. Similarly, risks of fluoxetine for sediment-dwelling organisms could not be excluded. No risk assessment was conducted for the terrestrial compartment due to a lack of data on effects of fluoxetine on soil organisms. The need for a separate risk assessment for the main metabolite of fluoxetine, norfluoxetine, was not conducted because of a lack of fate and effect studies. Based on published data, fluoxetine and norfluoxetine appeared to have a low to moderate bioaccumulation potential, which should be confirmed in formal studies according to OECD guidelines. Exposure assessments for fluoxetine according to the current framework rely heavily on K(OC) and K(OW) values. This approach is problematic, because fluoxetine is predominantly a cationic substance at environmental pH values. Consequently, the fate of fluoxetine (and other ionic substances) cannot be predicted using partition coefficients established for nonionic compounds. Further, published estimates for partition coefficients of fluoxetine vary, resulting in considerable uncertainties in both the exposure and environmental risk assessments of fluoxetine.