Tools for photomotor response assay standardization in ecotoxicological studies: Example of exposure to gentamicin in the freshwater planaria Schmidtea mediterranea.

Photomotor response assay (PMR) is very useful in an ecotoxicological context because it allows evaluation of behavioral response to potential toxic compounds. However, a lack of procedure standardization makes results comparison difficult between labs and organisms. Here, we aimed to propose five different tools to standardize the PMR procedure so that it may be applied to all model species, regarding: (1) the minimum total sample size, (2) the acclimation period, (3) the number and duration of light and dark phases alternation, (4) the measured behavior, and (5) the statistical analysis. As an example of procedure application, we analyzed the effect of an exposure to the antibiotic gentamicin on the locomotion behavior during PMR in an invertebrate species: the asexual freshwater planaria Schmidtea mediterranea. We encourage future studies using PMR to follow these five tools to improve data analysis and results comparability.

Optimization and calibration of behavioural tests on different species of planaria for ecotoxicological studies.

Freshwater planarian are emerging as a valuable in vivo model for (eco) toxicological studies, but the lack of harmonization of procedures between laboratories remains a challenge. This study aimed to optimize, automate and select the best behavioural tests and analyse the potential of different planarian species as models for toxicological assessment. We implemented four tests: exploration, photomotor response, Tapping and Planarian Light Dark Test, on different planaria species using the DanioVision system. We conclude that the exploration assay performed in 24 well-plate at 10,000 lux is the one that is robust and reliable for toxicological studies with planaria. Dugesia japonica and Schmidtea mediterranea have proved to be sensitive models for toxicological screening of organophosphorus pesticides through behavioural analysis. Under necessary experimental conditions, the motility baseline in controls, for both species allowed the detection of behavioural changes, making both good models for behavioural testing in (eco) toxicological context.

Multigenerational responses in the Lymnaea stagnalis freshwater gastropod exposed to diclofenac at environmental concentrations.

Over the last decade, there has been increased concern about the occurrence of diclofenac (DCF) in aquatic ecosystems. Living organisms could be exposed to this "pseudo-persistent" pharmaceutical for more than one generation. In this multigenerational study, we assessed the DCF impact at environmentally relevant concentrations on the life history and behavioral parameters of two offspring generations (F1 and F2) of the Lymnaea stagnalis freshwater gastropod. Snail growth was affected by DCF in the F1 generation, with increased shell sizes of juveniles exposed to 0.1 µg L - 1 concentration and a decreased shell size at 2 and 10 µg L - 1. DCF also lowered food intake, enhanced locomotion activity and reduced the number of eggs/egg mass in the F1 generation. For the F2 generation, shorter time to hatch, faster growth, increased food intake and production of more egg masses/snail were induced by DCF exposure at 10 µg L - 1. Over time, DCF exposure led to maximization of L. stagnalis reproductive function. These results show that multigenerational studies are crucial to reveal adaptive responses to chronic contaminant exposure, which are not observable after short-term exposure.

Long-term exposure to environmental diclofenac concentrations impairs growth and induces molecular changes in Lymnaea stagnalis freshwater snails.

As pharmaceutical substances are highly used in human and veterinary medicine and subsequently released in the environment, they represent emerging contaminants in the aquatic compartment. Diclofenac (DCF) is one of the most commonly detected pharmaceuticals in water and little research has been focused on its long-term effects on freshwater invertebrates. In this study, we assessed the chronic impacts of DCF on the freshwater gastropod Lymnaea stagnalis using life history, behavioral and molecular approaches. These organisms were exposed from the embryo to the adult stage to three environmentally relevant DCF concentrations (0.1, 2 and 10 µg/L). The results indicated that DCF impaired shell growth and feeding behavior at the juvenile stage, yet no impacts on hatching, locomotion and response to light stress were noted. The molecular findings (metabolomics and transcriptomic) suggested that DCF may disturb the immune system, energy metabolism, osmoregulation and redox balance. In addition, prostaglandin synthesis could potentially be inhibited by DCF exposure. The molecular findings revealed signs of reproduction impairment but this trend was not confirmed by the physiological tests. Combined omics tools provided complementary information and enabled us to gain further insight into DCF effects in freshwater organisms.

Effects of the anxiolytic benzodiazepine oxazepam on freshwater gastropod reproduction: a prospective study.

Psychoactive drugs have emerged as contaminants over the last few decades. These drugs are frequently prescribed and poorly eliminated by wastewater treatment plants, and many are present at non-negligible concentrations in surface waters. Several studies have investigated the non-target organism toxicity of one such drug, oxazepam, a benzodiazepine anxiolytic frequently detected in rivers. However, very little is known about the impact of this drug on reproduction. We investigated the effects of environmentally relevant concentrations of oxazepam on Radix balthica, a freshwater gastropod widespread in Europe. We identified the reproductive organs of Radix balthica. We then exposed this gastropod to oxazepam for two months and assessed several reproductive parameters, from reproductive organ status to behavioral parameters. We found that adults exposed to 10 µg/L oxazepam display an increase in the density of spermatozoa, and that adults exposed to 0.8 µg/L oxazepam displayed a decrease in the number of eggs per egg mass over time. By contrast, oxazepam had no effect on shell length, the size of male reproductive organs or social interactions. Finally, a locomotor activity analysis showed the distance covered over time decreased in all conditions of exposure to oxazepam, potentially reflecting a disturbance of exploratory activity. These results shed light on the effects of oxazepam on the reproduction of a non-target freshwater mollusk.

Low concentrations of oxazepam induce feeding and molecular changes in Radix balthica juveniles.

Psychotropics, especially benzodiazepines, are commonly prescribed worldwide. Poorly eliminated at wastewater treatment plants, they belong to a group of emerging contaminants. Due to their interaction with the GABAA receptor, they may affect the function of the nervous system of non-target organisms, such as aquatic organisms. The toxicity of oxazepam, a very frequently detected benzodiazepine in continental freshwater, has been largely studied in aquatic vertebrates over the last decade. However, its effects on freshwater non-vertebrates have received much less attention. We aimed to evaluate the long-term effects of oxazepam on the juvenile stage of a freshwater gastropod widespread in Europe, Radix balthica. Juveniles were exposed for a month to environmentally-relevant concentrations of oxazepam found in rivers (0.8 µg/L) and effluents (10 µg/L). Three main physiological functions were studied: feeding, growth, and locomotion. Additionally, gene expression analysis was performed to provide insights into toxicity mechanisms. There was a strong short-term activation of the feeding rate at low concentration, whereas the high dose resulted in long-term inhibition of food intake. A significant decrease in mortality rate was observed in juveniles exposed to the lowest dose. Shell growth and locomotor activity did not appear to be affected by oxazepam. Transcriptomic analysis revealed global over-expression of genes involved in the nervous regulation of the feeding, digestive, and locomotion systems after oxazepam exposure. The molecular analysis also revealed a possible interference of animal manipulation with the molecular effects induced by oxazepam exposure. Overall, these results improve our understanding of the effects of the psychoactive drug oxazepam on an aquatic mollusc gastropod.

Behavioral responses of three freshwater planaria species to light, visual and olfactory stimuli: Setting the stage for further ecotoxicological studies.

Planarians are freshwater flatworms commonly used as environmental bioindicator due to their sensitivity of response and their ease of culturing in lab. Nevertheless, to date, very few studies describing their behavior have been led. This work aims to fill the literature gap by providing preliminary results through six behavioral challenges (locomotion, exploration, light stress, planarian light/dark test, shoaling and foraging) conducted with three different species Dugesia tigrina, Schmidtea mediterranea and Schmidtea polychroa. The behavioral responses of every species in each of these six assays were recorded and differences between species were highlighted, depending on the assays and conditions. Schmidtea polychroa is less active than the two others and had the highest light aversion. Reactions observed in response to diverse and realistic stimuli helped us to select the most suitable tests and choose the species that seem the most appropriate for future ecotoxicological and neurophysiological tests. Four tests - out of the six tested- seem reliable in order to standardize planarian behavioral tests.