Probabilistic approaches to accounting for data variability in the practical application of bioavailability in predicting aquatic risks from metals.

The biotic ligand model (BLM) theoretically enables the derivation of environmental quality standards that are based on true bioavailable fractions of metals. Several physicochemical variables (especially pH, major cations, dissolved organic carbon, and dissolved metal concentrations) must, however, be assigned to run the BLM, but they are highly variable in time and space in natural systems. This article describes probabilistic approaches for integrating such variability during the derivation of risk indexes. To describe each variable using a probability density function (PDF), several methods were combined to 1) treat censored data (i.e., data below the limit of detection), 2) incorporate the uncertainty of the solid-to-liquid partitioning of metals, and 3) detect outliers. From a probabilistic perspective, 2 alternative approaches that are based on log-normal and Γ distributions were tested to estimate the probability of the predicted environmental concentration (PEC) exceeding the predicted non-effect concentration (PNEC), i.e., p(PEC/PNEC>1). The probabilistic approach was tested on 4 real-case studies based on Cu-related data collected from stations on the Loire and Moselle rivers. The approach described in this article is based on BLM tools that are freely available for end-users (i.e., the Bio-Met software) and on accessible statistical data treatments. This approach could be used by stakeholders who are involved in risk assessments of metals for improving site-specific studies.

Speciation and bioavailability of dissolved copper in different freshwaters: comparison of modelling, biological and chemical responses in aquatic mosses and gammarids.

Biological and chemical measurements were performed in mesocosms to investigate the bioavailability of copper, with a greater emphasis on the effects of competing ions and copper speciation. Measurements were achieved in three different natural waters for two aquatic species (Gammarus pulex and Fontinalis antipyretica) along a copper gradient concentration: natural concentration, spiked at 5 and 15 µg L(-1). Aquatic mosses exhibited high enrichment rates that were above the background levels compared to gammarids. The accumulation of copper in F. antipyretica is better correlated to the weakly complexed copper concentrations measured using differential pulse anodic stripping voltammetry (DPASV) and diffusive gradient in thin film (DGT) than to the free copper concentration measured using an ion selective electrode (ISE). In unspiked natural waters, the presence of dissolved organic ligands strongly controls the metal speciation and consequently largely minimised the impact of competing cations on the accumulation of Cu in mosses. Furthermore, the BioMet Biotic Ligand Model (BLM) successfully describes the site-specific copper bioaccumulation for the freshwater mosses studied. However, the comparison of the results with a previous study appears to indicate that the adsorption/desorption of Cu in mosses is impacted by seasons. This highlights a limit of the BioMet model in which the physiological state of aquatic organisms is not considered. No toxic effect of Cu exposure on lipid peroxidation was observed in the mosses and gammarids regardless of the site and the concentration considered. However, the oxidative stress measured in the mosses via their guaiacol peroxidase (GPX) activity increased in the case where internalised Cu reached maximal values, which suggests a threshold effect on the GPX activity.

Assessment of the LuminoTox leachate phase assay as a complement to the LuminoTox solid phase assay: effect of fine particles in natural sediments.

The LuminoTox solid phase assay (Lum-SPA), developed to assess the potential ecotoxicity of natural polluted sediments, employs stabilized thylakoids isolated from spinach plant extracts. When thylakoids are exposed to polluted samples, the action of pollutants can interfere with transmission of chlorophyll fluorescence linked to Photosystems I and II, causing a decrease in fluorescence emission. To differentiate between bulk and leachate effects, we developed a complementary assay of the Lum-SPA named the LuminoTox leachate phase assay (Lum-LPA). Twelve natural sediments were selected on the basis of their potential ecotoxicity and different quantities of fines. Results showed that in the Lum-LPA, the IC50s were always lower than those observed in the Lum-SPA. Significant inverse correlations were found between the IC50s obtained with the Lum-SPA and the percentage of fines (silt+clay fraction). In order to identify the cause of the higher ecotoxicity found in the Lum-LPA, we tested the supernatant of the liquid phase recovered after centrifugation (3000 g), and it appeared far less toxic than the Lum-LPA filtrates. To confirm the hypothesis that sensitivity in the Lum-LPA could be due to the presence of very fine particles, Photosynthetic Enzyme Complexes (PECs) were exposed to two kinds of clay materials with and without copper (0.6 mg L(-1)). Similar results were obtained in the Lum-LPA for both cases, revealing the sensitivity of this test to very fine particles.

New methodological improvements in the Microtox® solid phase assay.

The classic Microtox® solid phase assay (MSPA) based on the inhibition of light production of the marine bacteria recently renamed Aliivibrio fischeri suffers from various bias and interferences, mainly due to physico-chemical characteristics of the tested solid phase. To precisely assess ecotoxicity of sediments, we have developed an alternative method, named Microtox® leachate phase assay (MLPA), in order to measure the action of dissolved pollutants in the aqueous phase. Two hypotheses were formulated to explain the observed difference between MSPA and MLPA results: a real ecotoxicity of the solid phase or the fixation of bacteria to fine particles and/or organic matter. To estimate the latter, flow cytometry analyses were performed with two fluorochromes (known for their ability to stain bacterial DNA), allowing correction of MSPA measurements and generation of new (corrected) IC50. Comparison of results of MLPA with the new IC50 MSPA allows differentiating real ecotoxic and fixation effect in classic MSPA especially for samples with high amount of fines and/or organic matter.