The aim of this review is to examine how the choice of test species and study design employed in the use of in situ approaches in ecological risk assessment can maximize the ecological relevance of data. We provide a framework to define and assess ecological relevance that permits study designs to remain focused on the ecological question being addressed. This framework makes explicit the linkages between effects at lower levels of biological organization and higher-order ecological effects at the population, community, and ecosystem levels. The usefulness of this framework is illustrated by reference to specific examples from aquatic ecotoxicology. The use of models as both interpretive and predictive tools is discussed, with suggestions of appropriate methods for different protection goals.

This study evaluated a short-term sublethal endpoint for in situ toxicity assays for estuarine sediments, based on postexposure feeding of the polychaete Hediste (Nereis) diversicolor Müller. A method for precisely quantifying postexposure feeding rates of H. diversicolor was established under laboratory conditions using Artemia franciscana Kellog nauplii. The sensitivity of the postexposure feeding response to copper was investigated by comparing postexposure feeding rates to growth and lethality. The 48-h and 96-h median lethal concentration (LC50) of copper were 241 and 125 microg/L, respectively, whereas the 48-h median inhibitory concentration (IC50) for postexposure feeding and the 20-d IC50 for growth were 52 and 25 microg/L of copper, respectively. The influence of different exposure conditions (substrate, temperature, salinity, food availability, and light) on H. diversicolor postexposure feeding was assessed; temperature and salinity were found to influence significantly postexposure feeding. The effectiveness of the proposed in situ assay was investigated by deploying it at two reference and six contaminated Portuguese estuaries. A 48-h exposure period was followed by a 1-h postexposure feeding period. High organism recoveries (89-100%) were obtained. Postexposure feeding was depressed significantly (17-90%) at all contaminated sites relatively to reference sites. The proposed in situ assay with H. diversicolor was shown to be a potential useful tool for estuarine sediment toxicity testing.

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EXECUTIVE SUMMARYOne of the most internationally used bioassays for toxicity screening of chemicals and for toxicity monitoring of effluents and contaminated waters is the acute toxicity test with daphnid crustaceans, and in particular that performed with Daphnia magna. Standard methods have been developed for this assay that were gradually endorsed by national and international organisations dealing with toxicity testing procedures, in view of its application within a regulatory framework. As for all toxicity tests, the organisms used for the acute D. magna assay have to be obtained from live stocks which are cultured in the laboratory on live food (micro-algae). Unsurprisingly the various standard protocols of this particular assay differ -at least to a certain extent -with regard to the test organism culturing conditions. In addition, some technical aspects of the toxicity test such as the effect criterion (mortality of immobility), the exposure time, the type of dilution water, etc., also vary from one standard to another. Although this particular assay is currently used in many countries, the technical and biological problems inherent in year-round culturing and availability of the biological material and the culturing/maintenance costs of live stocks restrict its application to a limited number of highly specialised laboratories. This fundamental bottleneck in toxicity testing triggered investigations which brought forward the concept of "microbiotests" or "small-scale" toxicity tests. "Culture/maintenance free" aquatic microbiotests with species of different phylogenetic groups were developed in the early 1990s at the Laboratory for Environmental Toxicology and Aquatic Ecology at the Ghent University in Belgium. These assays which were given the generic name "Toxkits", are unique in that they employ dormant stages ("cryptobiotic eggs") of the test species, which can be stored for long periods of time and "hatched" at the time of performance of the assays. One of these microbiotests is the Daphtoxkit F magna, which is currently used in many laboratories worldwide for research as well as for toxicity monitoring purposes. The microbiotest technology has several advantages in comparison to the "traditional" tests based on laboratory cultures, especially its independence of the stock culturing burden. However, the acceptance (or possible non-acceptance) of performing assays with test organisms obtained from "dormant eggs" should be clearly dictated by the "sensitivity" and "precision" criteria of the former assays in comparison to the latter. The first part of this review therefore thoroughly reviews the scientific literature and of data obtained from various laboratories for assays performed with either D. magna test organisms obtained from lab cultures or hatched from dormant eggs. Attention has focused on data of quality control tests performed on reference chemicals, and in particular on potassium dichromate (K 2 Cr 2 O 7 ) for which an acceptability range of 0.6-2.1 mg·L -1 has been set in ISO standard 6...

This study aimed at evaluating the potential of an in situ algal bioassay for routine toxicity estimates of potentially contaminated estuarine environments using the microalga Phaeodactylum tricornutum immobilized in alginate beads. The influence of the initial cell density in the beads and of salinity on algal growth was first investigated. The potential of the proposed bioassay was evaluated by comparing laboratory with in situ results. A good growth performance of P. tricornutum was observed at all starting densities of beads. Although the growth rate of P. tricornutum was significantly affected by salinity, acceptability criteria currently adopted in algal assays were met, indicating the suitability of P. tricornutum as a test species for bioassays in estuaries. The large differences observed between the laboratory and in situ responses of P. tricornutum were most probably due to the temperature and light conditions less favorable for algal growth in the field and to the lack of representativeness of water samples compared to the field fluctuating conditions. These results showed the need for in situ assessments, especially in estuarine environments influenced by tides. To a lesser extent, the bioassay itself may also have been responsible for the laboratory and field differences. Further improvements in the bioassay chambers and procedures were also discussed.

This study proposes an ecologically relevant and cost-effective phytoplankton growth assay for routine in situ toxicity assessments. Assay procedures were developed applying, to the extent possible, the rationale behind the design of standard algal assays. Chlorella vulgaris was selected as test species because it grows well immobilized in alginate beads and has a wide geographic distribution. The performance of the assay in a freshwater system impacted by acid mine drainage demonstrated the suitability of assay chambers and procedures. The test system, made of inexpensive materials, allowed the rapid and easy deployment of the assay. The deployment of extra chambers at reference sites provided the ability to periodically check whether algal growth had already reached recommended growth criteria (time at which the assay should end). By deploying chambers filled with control medium at all sites, temperature was identified to explain 95% of the variation in growth. By using an artificial nutrient source shown capable of promoting algal growth according to recommended standards, toxicity from the mine effluent was distinguish from in situ nutrient limitation effects. The very good agreement (r2 = 90%) between mean in situ growth rates estimated by microscopy and by spectrophotometry and their similar coefficient of variation showed the latter to be a suitable straightforward methodology for assay endpoint estimation.

Most laboratory tests may underestimate adverse effects in real scenarios of contamination because they imply the forced exposure of organisms to contaminants, thus overlooking the possibility of emigration. Avoidance from contaminants has been observed in several aquatic organisms, and avoidance-based tests have been recommended to be included in risk assessment studies. To reduce uncertainty in the extrapolation of laboratory derived results, the first aim of the present study was to compare both the median avoidance concentration and the lowest-observed-effect gradient (LOEG) values of atrazine for the cladoceran Daphnia magna, between an already developed 1.1-m-long system and a scaled-up system, three times longer. Second, the present study aimed at evaluating the population immediate decline--the proportion of the population that disappears (avoids and, if not, dies)--through the integration of the relationships between lethality and avoidance versus contaminant concentration. Daphnia magna significantly avoided atrazine, during 12-h exposures, with similar results in the original and scaled-up systems. The population immediate decline at the 48-h median lethal concentration would be 94%. Even at a concentration eliciting only 5% mortality, the population immediate decline would be over 50%. Achieving a higher pertinence of avoidance results and a better understanding of the LOEG values and their time dependence, scaling up the system further both spatially and temporally, and modeling explicit spatial dynamics in exposure and organism movement in space and time are needed.

Purpose The present study presents data on the screening phase (tier 1) of a site-specific ecological risk assessment in a former smelter area heavily contaminated with metals (Santo Amaro, Bahia, Brazil). Joining information from three lines of evidence (LoE), chemical, ecotoxicological, and ecological, integrated risk values were calculated to rank sites within the area and identify those that may need further investigation in tier 2. Materials and methods Eleven points were selected up to 1,000 m from the smelter. Three reference points were 3 and 9 km away from the area. Risk values for the chemical LoE were derived from calculating the toxic pressure based on total metal concentrations. Those for the ecotoxicological LoE were based on avoidance (Folsomia candida and Eisenia andrei) and eluate tests (Daphnia magna acute test and Microtox) whereas for the ecological LoE the bait lamina test, soil basal respiration, and vegetation cover were used to derive risk values. Results and discussionThe chemical LoE showed high risk in those points inside the area where metal loadings exceeded in much the existing soil screening values. Ecotoxicological tools showed a variable response, with tests on soil organisms inducing a higher risk (again at sites inside the smelter and with sandy soils) than tests on eluates. The three parameters composing the ecological LoE revealed a concordant response, despite the lower sensitivity of the vegetation cover. A high risk on this LoE was also observed on those sampling points where a high chemical risk was calculated. Conclusions Integrated risk was low outside the smelter area. Inside, a high spatial heterogeneity of risk levels was observed, related to the non homogeneous deposition of smelting residues. Very high risk levels, associated with sandy soils and residue deposits, suggest the need to proceed with remediation actions. However, the uncertainties associated with the contradictory information given by certain LoEs for certain sampling points show the need to confirm potential risks in a tier 2 analysis.