K.A.C. De Schamphelaere

Environmental risk assessment of metals: tools for incorporating bioavailability.

In this paper, some of the main processes and parameters which affect metal bioavailability and toxicity in the aquatic environment and its implications for metal risk assessment procedures will be discussed. It has become clear that, besides chemical processes (speciation, complexation), attention should also be given to physiological aspects for predicting metal toxicity. The development of biotic ligand models (BLMs), which combine speciation models with more biologically oriented models (e.g. GSIM), has offered an answer to this need. The various BLMs which have been developed and/or refined for a number of metals (e.g. Cu, Ag, Zn) and species (algae, crustaceans, fish) are discussed here. Finally, the potential of the BLM approach is illustrated through a theoretical exercise in which chronic zinc toxicity to Daphnia magna is predicted in three regions, taking the physico-chemical characteristics of these areas into account.

Environmental risk assessment of zinc in European freshwaters: A critical appraisal

A risk assessment report (RAR) on zinc and zinc compounds has recently been prepared in the framework of the European Union (EU) Council Regulation 793/93/EEC on Existing Chemicals. The EU Scientific Committee on Human and Environmental Risks (SCHER) has, however, expressed some fundamental, science-based concerns about the approach followed and the conclusions. The main objective of the present study was to assess the potential environmental risks associated with current use patterns of Zn in nine EU river basins in Germany, France and Belgium, thereby using more advanced methodologies which are largely in line with the recommendations made by SCHER. This included (i) avoiding working with measured Zn concentrations from monitoring stations that were potentially influenced by point sources and/or historical contamination, (ii) the full bioavailability normalization of all chronic ecotoxicity data to river basin specific physico-chemistry using biotic ligand models (BLM), prior to deriving predicted no effect concentrations (PNEC) with the species sensitivity distribution (SSD) approach, and (iii) the use of a probabilistic framework for risk characterization. Further, a total risk approach instead of an added risk approach was used, and the PNEC was equated to the HC5-50 without an additional assessment factor. Based on monitoring data we estimated predicted environmental concentrations (PEC) for the different EU river basins between 1.3 and 14.6 microg dissolved Zn/L. PNEC values varied between 22.1 and 46.1 microg dissolved Zn/L. This resulted in deterministic risk characterization ratios (RCR) that were below 1 in all river basins, suggesting that there is no deterministic regional risk associated with current use patterns of Zn in these river basins. With the probabilistic approach we identified rather limited risks, i.e., between <0.4 and 18.3%. When the EU RAR approach was applied to the same monitoring datasets, deterministic risks were found in different river basins. A detailed analysis showed that this different deterministic conclusion of risk is mainly due to the fact that the EU RAR (i) uses an additional assessment factor of 2 to derive the PNEC and (ii) uses a more conservative approach for implementing bioavailability (BioF approach). We argue that the larger conservatism in the EU RAR mainly originates from decisions made to deal in a pragmatic way with (i) uncertainty related to the across-species extrapolation of BLMs and (ii) the relatively high sensitivity of some multi-species toxicity studies.

Biotic ligand model development predicting Zn toxicity to the alga Pseudokirchneriella subcapitata: possibilities and limitations.

Biotic ligand models have been developed for various metals (e.g. Cu, Ag, Zn) and different aquatic species. These models incorporate the effect of physico-chemical water characteristics (major cations, pH, dissolved organic carbon) on the bioavailability and toxicity of the metal. In this study, the individual effects of calcium, magnesium, potassium, sodium and pH on zinc toxicity to the green alga Pseudokirchneriella subcapitata (formerly and better known as Selenastrum capricornutum and Raphidocelis subcapitata) were investigated. Stability constants for binding to algal cells (K(BL)) were derived for those cations affecting zinc toxicity, using the mathematical approach proposed by De Schamphelaere and Janssen [Environ. Sci. Technol. 63, (2002) 48-54]. Potassium proved to be the only cation tested that did not alter zinc toxicity to algae significantly. Log (K(BL)) values for Ca, Mg and Na, derived at pH 7.5, were 3.2, 3.9 and 2.8, respectively. Toxicity tests performed at different pH values (5.5-8.0) indicated that competition between H(+) and Zn(2+) reduces zinc toxicity. However, the observed relationship between (H(+)) and the 72h-EbC(50) [expressed as microM (Zn(2+))] is not linear and suggests that pH affects the physiology of the biotic ligand. Although, in general, our findings seem to suggest that zinc toxicity to algae can be modelled as a function of key water characteristics, the results also demonstrate that the part of the conventional BLM-hypothesis-i.e. that the binding characteristics of the biotic ligand are independent of the test medium characteristics-is not valid for algae. The observed pH-dependent change of stability constants should therefore be further investigated and incorporated in future BL-modelling efforts with algae.

Uncertainties in the Environmental Risk Assessment of Metals

As life has evolved in the presence of metals, the assessment of the potential adverse effects of metals on ecosystems requires a different approach than those presently used for man-made organic substances. This article provides a brief review of applications and limitations of current techniques and presents, based on recent research results, suggestions for improving the scientific relevance and accuracy of environmental risk assessments of metals. The importance of the following factors responsible for major uncertainties in current environmental risk assessments of metals are discussed: factors affecting metal bioavailability and toxicity, the potential importance of deficiency effects (for essential metals), and field extrapolation of laboratory toxicity data. Possible (regulatory) consequences of inaccurately assessing the natural background concentrations of metals and acclimatization/adaptation potential of laboratory organisms and resident communities are illustrated using examples of recent research, hypothesis development, and a probabilistic environmental risk assessment.

Non-simultaneous ecotoxicity testing of single chemicals and their mixture results in erroneous conclusions about the joint action of the mixture

The ecotoxicity of binary chemical mixtures with a common mode of action is often predicted with the concentration addition model. The assumption of concentration addition is commonly tested statistically based on results of toxicity experiments with the two single chemicals and their binary mixture. The present simulation study shows that if not all these experiments are performed simultaneously, one has a 20-80% chance of concluding synergism or antagonism while the mixture is actually additive (false positive rate). Truly synergistic or antagonistic mixtures have a 10-50% chance of being falsely categorized as additive (false negative rate). Additionally, false positive rates decrease with increasing experimental error, while false negative rates increase with increasing experimental error. Based on these results, we put forward a number of recommendations for future mixture ecotoxicity evaluation.

Ecotoxicity and uptake of polymer coated gold nanoparticles

Abstract Bioconjugated gold nanoparticles (Au NPs) are a promising tool for pharmaceutical applications. However, the ecotoxicity of these types of NPs has hardly been studied. We investigated the ecotoxicity and uptake of 4–5 nm Au NPs to which two types of polymer coatings were attached. One coating was an amphiphilic polymer only and the other an amphiphilic coating to which 10 kDa polyethylene glycol chains were attached. In both 72 h algal growth inhibition tests with the alga Pseudokirchneriella subcapitata and in 24 h resazurin cytotoxicity tests with the rainbow trout gill cell line RTGill-W1, the pegylated Au NPs were found less toxic compared to the amphiphilic coated particles. No uptake or direct interaction between particles and algal cells was observed. However, uptake/adsorption in fish gill cells reached up to >106 particles/cell after 1 h and particles were eliminated for ≥96% after 24 h depuration. Both particle types were found within membrane enclosed vesicles in the cytoplasm of RTgill-W1 cells.

The chronic toxicity of molybdate to freshwater organisms, I: generating reliable effects data

The European Union regulation on Registration, Evaluation, Authorization and Restriction of Chemical substances (REACH) (EC, 2006) requires the characterization of the chronic toxicity of many chemicals in the aquatic environment, including molybdate (MoO(4)(2-)). Our literature review on the ecotoxicity of molybdate revealed that a limited amount of reliable chronic no observed effect concentrations (NOECs) for the derivation of a predicted no-effect concentration (PNEC) existed. This paper presents the results of additional ecotoxicity experiments that were conducted in order to fulfill the requirements for the derivation of a PNEC by means of the scientifically most robust species sensitivity distribution (SSD) approach (also called the statistical extrapolation approach). Ten test species were chronically exposed to molybdate (added as sodium molybdate dihydrate, Na(2)MoO(4)·2H(2)O) according to internationally accepted standard testing guidelines or equivalent. The 10% effective concentrations (EC10, expressed as measured dissolved molybdenum) for the most sensitive endpoint per species were 62.8-105.6 (mg Mo)/L for Daphnia magna (21day-reproduction), 78.2 (mg Mo)/L for Ceriodaphnia dubia (7day-reproduction), 61.2-366.2 (mg Mo)/L for the green alga Pseudokirchneriella subcapitata (72h-growth rate), 193.6 (mg Mo)/L for the rotifer Brachionus calyciflorus (48h-population growth rate), 121.4 (mg Mo)/L for the midge Chironomus riparius (14day-growth), 211.3 (mg Mo)/L for the snail Lymnaea stagnalis (28day-growth rate), 115.9 (mg Mo)/L for the frog Xenopus laevis (4day-larval development), 241.5 (mg Mo)/L for the higher plant Lemna minor (7day-growth rate), 39.3 (mg Mo)/L for the fathead minnow Pimephales promelas (34day-dry weight/biomass), and 43.2 (mg Mo)/L for the rainbow trout Oncorhynchus mykiss (78day-biomass). These effect concentrations are in line with the few reliable data currently available in the open literature. The data presented in this study can serve as a basis for the derivation of a PNEC(aquatic) that can be used for national and international regulatory purposes and for setting water quality criteria. Using all reliable data that are currently available, a HC(5,50%) (median hazardous concentration affecting 5% of the species) of 38.2 (mg Mo)/L was derived with the statistical extrapolation approach.

Direct determination of Zn in individual Daphnia magna specimens by means of solid sampling high-resolution continuum source graphite furnace atomic absorption spectrometry

In this work, the capabilities of solid sampling high-resolution continuum source graphite furnace atomic absorption spectrometry (GFAAS) for the direct determination of the Zn body burden in individual specimens of Daphnia magna (small aquatic invertebrates of approx. 2 mm length that are widely used in ecotoxicological research) were investigated. It was concluded that this technique offers interesting features for this type of application, permitting the fast (approx. 2 min for the measurement of a single sample) and practically contamination-free analysis of these invertebrates, as the only preparation required is drying. Moreover, one of the traditional disadvantages of line source-GFAAS, the narrow linear range achieved using a particular set of experimental conditions, can be overcome with this technique by the judicious selection of the detector pixels used for signal quantification, such that the linear range can be extended simply by reprocessing the data, without the need to carry out any additional measurement. This feature is particularly interesting for this application, since every Daphnia magna specimen can be monitored only once, and thus, there is no possibility to alter the conditions and repeat the analysis for samples displaying a Zn content outside the linear range. In this case, the use of central and/or side pixels permits achieving a working range between 5 and 400 ng for the 307.590 nm Zn line, which is fit-for-purpose. Analysis of two biological certified reference materials (SRM 1549 Non-fat milk powder and SRM 1577b Bovine liver) was also investigated, and accuracy could be demonstrated when carrying out the calibration versus aqueous standard solutions.

Waterborne versus dietary zinc accumulation and toxicity in Daphnia magna: A synchrotron radiation based X-ray fluorescence imaging approach

Recent studies have suggested that exposure of the freshwater invertebrate Daphnia magna to dietary Zn may selectively affect reproduction without an associated increase of whole body bioaccumulation of Zn. The aim of the current research was therefore to investigate the hypothesis that dietary Zn toxicity is the result of selective accumulation in tissues that are directly involved in reproduction. Since under field conditions simultaneous exposure to both waterborne and dietary Zn is likely to occur, it was also tested if accumulation and toxicity under combined waterborne and dietary Zn exposure is the result of interactive effects. To this purpose, D. magna was exposed during a 16-day reproduction assay to Zn following a 5 × 2 factorial design, comprising five waterborne concentrations (12, 65, 137, 207, and 281 μg Zn/L) and two dietary Zn levels (49.6 and 495.9 μg Zn/g dry wt.). Tissue-specific Zn distribution was quantified by synchrotron radiation based confocal X-ray fluorescence (XRF). It was observed that the occurrence of reproductive inhibition due to increasing waterborne Zn exposure (from 65 μg/L to 281 μg/L) was accompanied by a relative increase of the Zn burdens which was similar in all tissues considered (i.e., the carapax, eggs, thoracic appendages with gills and the cluster comprising gut epithelium, storage cells and ovaries). In contrast, the impairment of reproduction during dietary Zn exposure was accompanied by a clearly discernible Zn accumulation in the eggs only (at 65 μg/L of waterborne Zn). During simultaneous exposure, bioaccumulation and toxicity were the result of interaction, which implies that the tissue-specific bioaccumulation and toxicity following dietary Zn exposure are dependent on the Zn concentration in the water. Our findings emphasize that (i) effects of dietary Zn exposure should preferably not be investigated in isolation from waterborne Zn exposure, and that (ii) XRF enabled us to provide possible links between tissue-specific bioaccumulation and reproductive effects of Zn.

Multi-linear regression analysis, preliminary biotic ligand modeling, and cross species comparison of the effects of water chemistry on chronic lead toxicity in invertebrates.

The current study examined the chronic toxicity of lead (Pb) to three invertebrate species: the cladoceran Ceriodaphnia dubia, the snail Lymnaea stagnalis and the rotifer Philodina rapida. The test media consisted of natural waters from across North America, varying in pertinent water chemistry parameters including dissolved organic carbon (DOC), calcium, pH and total CO(2). Chronic toxicity was assessed using reproductive endpoints for C. dubia and P. rapida while growth was assessed for L. stagnalis, with chronic toxicity varying markedly according to water chemistry. A multi-linear regression (MLR) approach was used to identify the relative importance of individual water chemistry components in predicting chronic Pb toxicity for each species. DOC was an integral component of MLR models for C. dubia and L. stagnalis, but surprisingly had no predictive impact on chronic Pb toxicity for P. rapida. Furthermore, sodium and total CO(2) were also identified as important factors affecting C. dubia toxicity; no other factors were predictive for L. stagnalis. The Pb toxicity of P. rapida was predicted by calcium and pH. The predictive power of the C. dubia and L. stagnalis MLR models was generally similar to that of the current C. dubia BLM, with R(2) values of 0.55 and 0.82 for the respective MLR models, compared to 0.45 and 0.79 for the respective BLMs. In contrast the BLM poorly predicted P. rapida toxicity (R(2)=0.19), as compared to the MLR (R(2)=0.92). The cross species variability in the effects of water chemistry, especially with respect to rotifers, suggests that cross species modeling of invertebrate chronic Pb toxicity using a C. dubia model may not always be appropriate.