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.

A seasonal comparison of trace metal concentrations in the tissues of Arctic charr (Salvelinus alpinus) in Northern Québec, Canada.

Ecotoxicological research detailing trace metal contamination and seasonal variation in the tissues of northern fishes such as Arctic charr (Salvelinus alpinus) has been poorly represented in the literature beyond examination of mercury. In an effort to address this, anadromous Arctic charr were collected from the Deception River watershed in the late summer and post-winter season, before quantifying seasonal and organotropic variations in dorsal muscle and liver concentrations of arsenic, cadmium, chromium, copper, nickel, lead, and zinc. Potential linkages with biological variables (fork length, age, and somatic condition) and indicators of feeding behavior (δ13C and δ15N) were also assessed. Trace metal organotropism favouring elevation in liver tissue concentrations was exhibited by cadmium, copper, nickel and zinc, while arsenic, chromium and lead exhibited no significant organotropic variation. Seasonal differences in concentrations were metal and tissue dependent, but generally increased in tissues collected from post-winter sampled Arctic charr. Significant correlations with biological and trophic descriptors were also determined to be element and tissue dependent. These parameters, in addition to season, were incorporated into multi-predictor variable models, where variations in trace metal concentration data were often best explained when season, somatic condition, and trophic descriptors were included. These variables were also of greatest relative importance across all considered trace metals and tissue types. These findings suggest that seasonally linked processes have the greatest influence on trace metal concentrations in anadromous Arctic charr. Future metal-related research on Arctic charr and other northern fish species should further consider these variables when evaluating elemental accumulation.

Subcellular partitioning of metals and metalloids (As, Cd, Cu, Se and Zn) in liver and gonads of wild white suckers (Catostomus commersonii) collected downstream from a mining operation.

In the present study, we examined the subcellular distribution of metals and metalloids (As, Cd, Cu, Se and Zn) in the liver and gonads of wild white suckers (Catostomus commersonii) collected downstream from a metal mining operation (exposure area) and in a reference area. Metal partitioning among potentially metal-sensitive fractions (heat-denatured proteins (HDP), mitochondria and microsomes) and potentially biologically detoxified fractions (heat-stable proteins (HSP) and metal-rich granules) within cells was determined after differential centrifugation, NaOH digestion and heat-denaturation steps. Metal-handling strategies between liver and gonads, and between sexes, were examined. Hepatic metal concentrations were significantly higher in exposed compared to reference fish, especially for Se (14x), Cd (5x) and Cu (3x), and did not vary between sexes. In contrast, gonadal Cd, Cu, Se and Zn concentrations were consistently lower in testes than in ovaries; marked differences in Cd and Se concentrations between exposed and reference fish were observed for both sexes. Overall, metal-handling strategies were similar in both liver (male and female pooled) and female gonads, but differed from those in male gonads, likely due to the different functions assigned to ovaries and testes. Subcellular partitioning of As, Cd and Cu showed that the HSP fraction was most responsive to increased metal exposure, presumably reflecting Cu regulation, and possibly Cd and As detoxification. Zinc concentrations were tightly controlled and mainly found in the HDP fraction. Interestingly, changes in Cd-handling strategy in female gonads were particularly evident, with Cd shifting dramatically from the metal-sensitive HDP fraction in reference fish to the metal-detoxified HSP fraction in exposed fish. It seems that Cd detoxification in female gonads was not fully induced in the less contaminated fish, but became more effective above a threshold Cd concentration of 0.05 nmol/g dry weight. Partitioning of Se was different, with the largest contributor to the total liver and gonad Se burdens being the putative metal-sensitive HDP fraction, suggesting that excess Se in this fraction in exposed fish may lead to Se-related stress. The present subcellular partitioning results demonstrate that metal handling strategies vary among metals, between organs and (in some cases) as a function of metal exposure. They also show promise in identifying metals of potential concern in a risk assessment context.

Application of a multidisciplinary and integrative weight-of-evidence approach to a 1-year monitoring survey of the Seine River.

Quality assessment of environments under high anthropogenic pressures such as the Seine Basin, subjected to complex and chronic inputs, can only be based on combined chemical and biological analyses. The present study integrates and summarizes a multidisciplinary dataset acquired throughout a 1-year monitoring survey conducted at three workshop sites along the Seine River (PIREN-Seine program), upstream and downstream of the Paris conurbation, during four seasonal campaigns using a weight-of-evidence approach. Sediment and water column chemical analyses, bioaccumulation levels and biomarker responses in caged gammarids, and laboratory (eco)toxicity bioassays were integrated into four lines of evidence (LOEs). Results from each LOE clearly reflected an anthropogenic gradient, with contamination levels and biological effects increasing from upstream to downstream of Paris, in good agreement with the variations in the structure and composition of bacterial communities from the water column. Based on annual average data, the global hazard was summarized as "moderate" at the upstream station and as "major" at the two downstream ones. Seasonal variability was also highlighted; the winter campaign was least impacted. The model was notably improved using previously established reference and threshold values from national-scale studies. It undoubtedly represents a powerful practical tool to facilitate the decision-making processes of environment managers within the framework of an environmental risk assessment strategy.

Contribution of aqueous and dietary uptakes to lead (Pb) bioaccumulation in Gammarus pulex: From multipathway modeling to in situ validation.

Although dynamic approaches are nowadays used increasingly to describe metal bioaccumulation in aquatic organisms, the validation of such laboratory-derived modeling is rarely assessed under environmental conditions. Furthermore, information on bioaccumulation kinetics of Pb and the significance of its uptake by dietary route is scarce in freshwater species. This study aims at modeling aqueous and dietary uptakes of Pb in the litter-degrader Gammarus pulex and assessing the predictive quality of multipathway modeling from in situ bioaccumulation data. In microcosms, G. pulex were exposed to environmentally realistic concentrations of Pb (from 0.1 to 10µg/L) in the presence of Pb-contaminated poplar leaves, which were enclosed or not in a net to distinguish aqueous and dietary uptakes. Results show that water and food both constitute contamination sources for gammarids. Establishing biodynamic parameters involved in Pb aqueous and dietary uptake and elimination rates enabled to construct a multipathway model to describe Pb bioaccumulation in gammarids. This laboratory-derived model successfully predicted bioaccumulation measured in native populations of G. pulex collected in situ when local litter was used as dietary exposure source. This study demonstrates not only the suitable applicability of biodynamic parameters for predicting Pb bioaccumulation but also the necessity of taking dietary uptake into account for a better interpretation of the gammarids' contamination in natural conditions.

Seasonal variability and inter-species comparison of metal bioaccumulation in caged gammarids under urban diffuse contamination gradient: implications for biomonitoring investigations.

Although caging of Gammarus species offers promising lines of inquiry to monitor metal bioavailability in freshwaters, the interspecies responsiveness to metal exposures is still unclear. In addition, abiotic factors inherent to transplantation can hamper the interpretation of field bioaccumulation data. To assess the relevance of using gammarids as biomonitors, we investigated the seasonal influence on metal bioaccumulation in two common species, Gammarus pulex and Gammarus fossarum. During four seasons, caged gammarids were deployed on three sites along the Seine River exhibiting a diffuse gradient of multi-metal contamination: a site upstream and two sites downstream from the Paris megacity. For each seasonal deployment, metal concentrations in animals were determined after 7d-exposure in situ (Ag, Cd, Co, Cu, Mn, Ni, Pb and Zn). Results show that the seasonal patterns of metal contaminations are similar between both Gammarus species, and closely related to the river axis' contamination gradient. Statistical analyses indicate that bioaccumulation of essential metals in both species is influenced by season, especially by water temperature. This highlights the necessity to consider this climatic factor inherent to the deployment period for a reliable interpretation of bioaccumulation data in the field. The comparison of accumulation factors suggests that these two species coming from different geochemical origins display similar abilities to internalize metals. This generic responsiveness of caged gammarids supports their use as sentinel organisms to quantify low spatiotemporal variations in metal bioavailabilities.