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.

In situ effects of metal contamination from former uranium mining sites on the health of the three-spined stickleback (Gasterosteus aculeatus, L.).

Human activities have led to increased levels of various pollutants including metals in aquatic ecosystems. Increase of metallic concentrations in aquatic environments represents a potential risk to exposed organisms, including fish. The aim of this study was to characterize the environmental risk to fish health linked to a polymetallic contamination from former uranium mines in France. This contamination is characterized by metals naturally present in the areas (manganese and iron), uranium, and metals (aluminum and barium) added to precipitate uranium and its decay products. Effects from mine releases in two contaminated ponds (Pontabrier for Haute-Vienne Department and Saint-Pierre for Cantal Department) were compared to those assessed at four other ponds outside the influence of mine tailings (two reference ponds/department). In this way, 360 adult three-spined sticklebacks (Gasterosteus aculeatus) were caged for 28 days in these six ponds before biomarker analyses (immune system, antioxidant system, biometry, histology, DNA integrity, etc.). Ponds receiving uranium mine tailings presented higher concentrations of uranium, manganese and aluminum, especially for the Haute-Vienne Department. This uranium contamination could explain the higher bioaccumulation of this metal in fish caged in Pontabrier and Saint-Pierre Ponds. In the same way, many fish biomarkers (antioxidant and immune systems, acetylcholinesterase activity and biometric parameters) were impacted by this environmental exposure to mine tailings. This study shows the interest of caging and the use of a multi-biomarker approach in the study of a complex metallic contamination.

Former uranium mine-induced effects in caged roach: a multiparametric approach for the evaluation of in situ metal toxicity.

To characterize environmental risks linked to former uranium mines in the Limousin region of France, a study was conducted on fish health effects from uranium releases. Two private ponds were compared in this study, one with uranium contamination and one background site, upstream of the mining zone. Roach, Rutilus rutilus, were caged for 28 days in both ponds. Physico-chemical parameters of water and sediments and bioaccumulation of metals in several organs were determined. After 14 and 28 days of caging, immune, oxidative stress, biotransformation, neurotoxicity and physiological parameters were measured. Iron and aluminium were quantified in the water of both sites; however, barium and manganese were only present in the water of the uranium contaminated site. Uranium was present in both sites but at very different concentrations. The sediments from the uranium contaminated site contained high levels of radioactive elements coming from the disintegration chain of uranium. Results of biological parameters indicated stimulation of immune parameters and of oxidative stress and a decrease of AChE in fish caged in the uranium contaminated pond compared to the uranium-free pond. Overall, the results determined roach health status in the context of pollution from poly-metallic mining. The data strengthen our knowledge of the environmental risk assessment associated with radioactive substances in the environment.

Depleted uranium disturbs immune parameters in zebrafish, Danio rerio: an ex vivo/in vivo experiment.

In this study, we investigated the effects of depleted uranium (DU), the byproduct of nuclear enrichment of uranium, on several parameters related to defence system in the zebrafish, Danio rerio, using flow cytometry. Several immune cellular parameters were followed on kidney leucocytes: cell proportion, cell mortality, phagocytosis activity and associated oxidative burst and lysosomal membrane integrity (LMI). Effects of DU were tested ex vivo after 17 h of contact between DU and freshly isolated leucocytes from 0 to 500 µg DU/L. Moreover, adult zebrafish were exposed in vivo during 3 days at 20 and 250 µg DU/L. Oxidative burst results showed that DU increased reactive oxygen species (ROS) basal level and therefore reduced ROS stimulation index in both ex vivo and in vivo experiments. ROS PMA-stimulated level was also increased at 250 µg DU/L in vivo only. Furthermore, a decrease of LMI was detected after in vivo experiments. Cell mortality was also decreased at 20 µg DU/L in ex vivo experiment. However, phagocytosis activity was not modified in both ex vivo and in vivo experiments. A reduction of immune-related parameters was demonstrated in zebrafish exposed to DU. DU could therefore decrease the ability of fish to stimulate its own immune system which could, in turn, enhance the susceptibility of fish to infection. These results encourage the development and the use of innate immune analysis by flow cytometry in order to understand the effects of DU and more generally radionuclides on fish immune system and response to infectious diseases.

In vivo effects of LCO soluble fraction on immune-related functions and gene transcription in the Pacific oyster, Crassostrea gigas (Thunberg).

The effects of a soluble fraction of light cycle oil (LCO) on haemocyte parameters, phenoloxidase (PO) activity and mRNA expression of immune-related genes, in the Pacific oyster, Crassostrea gigas, were tested after seven days of exposure and two weeks of recovery period. Five polycyclic aromatic hydrocarbons (PAHs) out of ten detected in tank water had bioaccumulated at the end of the contamination period. The concentration of PAHs in oyster tissues decreased during the recovery period and 14 days after the exposure, 69% of bioaccumulated PAHs were detected in contaminated oysters. The exposure induced severe oyster mortality (21%), external and internal green colouration of the shell and a significant decrease of PO activity. The mRNA expression of several genes was altered. As a conclusion, a modulation of immune-related parameters was demonstrated using three different approaches, namely cellular (flow cytometry), biochemical (spectrophotometry) and genomics (gene transcription) in oysters after contact with soluble fraction of LCO.

Detection of phenoloxidase activity in early stages of the Pacific oyster Crassostrea gigas (Thunberg).

The presence of phenoloxidase (PO) activity was detected in different developmental stages of the Pacific oyster, Crassostrea gigas. A significant reduction in PO activity was observed from the 6h embryo stage to the day 11 larvae by spectrophotometry. A progressive increase was also observed from the day 13 larvae right through to the juvenile stage. The microscopy studies with '6h embryo' and adult samples confirmed the presence of PO activity. Various modulators of PO activity were used to study the triggering of pro-phenoloxidase (proPO) activating system of C. gigas but also to confirm the exact nature of the monitored activity. The enzyme activation mechanisms appear to differ with the developmental stage: bacterial lipopolysaccharides constitute an early elicitor of the proPO-PO system, whereas a purified trypsin triggers proPO-PO system in C. gigas spat. Phenoloxidase activity was totally suppressed by PO-specific inhibitors such as beta-2-mercaptoethanol, sodium diethyldithiocarbonate and tropolone. This study demonstrated the selective response of PO-like activity by different elicitors and suggested that proPO-PO activating system, which is supposed to play an important function in non-self recognition and host immune reactions in oyster, is expressed early in the Pacific oyster, C. gigas.