Sub-lethal toxicity of five disinfection by-products on microalgae determined by flow cytometry - Lines of evidence for adverse outcome pathways.

Standardised tests are often used to determine the ecotoxicity of chemicals and focus mainly on one or a few generic endpoints (e.g. mortality, growth), but information on the sub-cellular processes leading to these effects remain usually partial or missing. Flow cytometry (FCM) can be a practical tool to study the physiological responses of individual cells (such as microalgae) exposed to a stress via the use of fluorochromes and their morphology and natural autofluorescence. This work aimed to assess the effects of five chlorine-based disinfection by-products (DBPs) taken individually on growth and sub-cellular endpoints of the green microalgae Raphidocelis subcapitata. These five DBPs, characteristic of a chlorinated effluent, are the following monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), bromochloroacetic acid (BCAA) and 1,1-dichloropropan-2-one (1,1-DCP). Results showed that 1,1-DCP had the strongest effect on growth inhibition (EC50 = 1.8 mg.L-1), followed by MCAA, TCAA, BCAA and DCAA (EC50 of 10.1, 15.7, 27.3 and 64.5 mg.L-1 respectively). Neutral lipid content, reactive oxygen species (ROS) formation, red autofluorescence, green autofluorescence, size and intracellular complexity were significantly affected by the exposure to the five DBPs. Only mitochondrial membrane potential did not show any variation. Important cellular damages (>10%) were observed for only two of the chemicals (BCAA and 1,1-DCP) and were probably due to ROS formation. The most sensitive and informative sub-lethal parameter studied was metabolic activity (esterase activity), for which three types of response were observed. Combining all this information, an adverse outcome pathways framework was proposed to explain the effect of the targeted chemicals on R. subcapitata. Based on these results, both FCM sub-cellular analysis and conventional endpoint of algal toxicity were found to be complementary approaches.

One and multi-compartments toxico-kinetic modeling to understand metals' organotropism and fate in Gammarus fossarum.

The use of freshwater invertebrates for biomonitoring has been increasing for several decades, but little is known about relations between external exposure concentration of metals and their biodistribution among different tissues. One and multi-compartments toxicokinetic (TK) models are powerful tools to formalize and predict how a contaminant is bioaccumulated. The aim of this study is to develop modeling approaches to improve knowledge on dynamic of accumulation and fate of Cd and Hg in gammarid's organs. Gammarids were exposed to dissolved metals (11.1 ± 1.2 µg.L-1 of Cd or 0.27 ± 0.13 µg.L-1 of Hg) before a depuration phase. At each sampling days, their organs (caeca, cephalon, intestine and remaining tissues) were separated by dissection before analyses. Results allowed us to determine that i) G.fossarum takes up Cd as efficiently as the mussel M.galloprovincialis, but eliminates it more rapidly, ii) organs which accumulate and depurate the most, in terms of concentrations, are caeca and intestine for both metals; iii) the one-compartment TK models is the most relevant for Hg, while the multi-compartments TK model allows a better fit to Cd data, demonstrating dynamic transfer of Cd among organs.