Genotoxic effects of combined multiple stressors on Gammarus locusta haemocytes: Interactions between temperature, pCO2 and the synthetic progestin levonorgestrel.

Climate change and pharmaceutical contamination are two priority research topics due to their impacts in the aquatic ecosystems and in the food chain structure. In the bottom of many food chains are the invertebrates, like the amphipods, which are important environmental and ecotoxicological models. In this study, we combined the increase of temperature [ambient and warming temperature], pCO2 [normocapnia and hypercapnia] and the synthetic progestin levonorgestrel (LNG) [environmentally relevant concentration (10 ng L-1) and 100-fold higher (1000 ng L-1)] to evaluate the genotoxic effects on the amphipod Gammarus locusta haemocytes, using the comet assay technique. Additionally, the study examined protective/potentiating effects of the three tested factors against hydrogen peroxide (H2O2)-induced DNA damage in haemocytes after ex vivo exposure. Our data revealed no significant effects of any of the three stressors on DNA damage of G. locusta haemocytes or protection against H2O2-induced DNA damage after twenty-one days exposure. Only a significant effect of the solvent was visible, since it was able to induce higher DNA damage (i.e. strand breaks) on exposed individuals. On the other hand, LNG exposure seemed to induce a slight increase of DNA damage after H2O2 exposure. Our findings suggest that more short-term studies to conclude about the genotoxicity and/or protective effects of the stress factors in G. locusta should be made, attending to the fast turnover rate of repairing cells that could have masked impacts seen only after the end of the experiment.

Interactive effects of increased temperature, pCO2 and the synthetic progestin levonorgestrel on the fitness and breeding of the amphipod Gammarus locusta.

Given the lack of knowledge regarding climate change-chemical exposure interactions, it is vital to evaluate how these two drivers jointly impact aquatic species. Thus, for the first time, we aimed at investigating the combined effects of increased temperature, pCO2 and the synthetic progestin levonorgestrel on survival, growth, consumption rate and reproduction of the amphipod Gammarus locusta. For that, a full factorial design manipulating temperature [ambient temperature and warming (+4 °C)], pCO2 [normocapnia and hypercapnia (Δ pH 0.5 units)] and the progestin levonorgestrel (LNG: L1 - 10 ngLL-1 and L2 - 1000 ngLL-1, control - no progestin and solvent control - vehicle ethanol (0.01%)) was implemented for 21 days. G. locusta was strongly negatively affected by warming, experiencing higher mortality rates (50-80%) than in any other treatments. Instead, growth rates were significantly affected by interactions of LNG with temperature and pCO2. It was observed, in the short-term (7d) that under ambient temperature (18 °C) and hypercapnic conditions (pH 7.6), the LNG presence promoted the amphipod's growth, while in the medium-term (21d) this response was not observed. Relative consumption rates (RCRs), during the first week were higher than in the third week. Furthermore, in the first week, RCRs were negatively affected by higher temperature while in the third week, RCRs were negatively affected by acidification. Furthermore, it was observed a negative effect of higher temperature and acidification on G. locusta fecundity, contrarily to LNG. Concluding, the impact of increased temperature and pCO2 was clearly more adverse for the species than exposure to the synthetic progestin, however, some interactions between the progestin and the climate factors were observed. Thus, in a future scenario of global change, the presence of LNG (and other progestins alike) may modulate to a certain level the effects of climate drivers (and vice-versa) on the gammarids fitness and reproduction.