The OECD test guideline 203 for determination of fish acute toxicity requires substantial numbers of fish and uses death as an apical end point. One potential alternative are fish cell lines; however, several studies indicated that these appear up to several orders of magnitude less sensitive than fish. We developed a fish gill cell line-based (RTgill-W1) assay, using several measures to improve sensitivity. The optimized assay was applied to determine the toxicity of 35 organic chemicals, having a wide range of toxicity to fish, mode of action and physicochemical properties. We found a very good agreement between in vivo and in vitro effective concentrations. For up to 73% of the tested compounds, the difference between the two approaches was less than 5-fold, covering baseline toxicants but as well compounds with presumed specific modes of action, including reactivity, inhibition of acetylcholine esterase or uncoupling of oxidative phosphorylation. Accounting for measured chemical concentrations eliminated two outliers, the hydrophobic 4-decylaniline and the volatile 2,3-dimethyl-1,3-butadiene, with an outlier being operationally defined as a substance showing a more than 10-fold difference between in vivo/in vitro effect concentrations. Few outliers remained. The most striking were allyl alcohol (2700-fold), which likely needs to be metabolically activated, and permethrin (190-fold) and lindane (63-fold), compounds acting, respectively, on sodium and chloride channels in the brain of fish. We discuss further developments of this assay and suggest its use beyond predicting acute toxicity to fish, for example, as part of adverse outcome pathways to replace, reduce, or refine chronic fish tests.

Activated carbon (AC) addition is a recently developed technique for the remediation of sediments and soils contaminated with hydrophobic organic chemicals. Laboratory and field experiments have demonstrated that the addition of 3-4% of AC can reduce aqueous concentrations and the bioaccumulation potential of contaminants. However, one aspect of the technique that has hardly received any attention is the possible occurrence of secondary, eco(toxico)logical effects, i.e., effects of AC addition on the health, behavior, and habitat quality of local organisms. In the present study, several ecotoxicological effects were investigated in AC-water and AC-enriched (0-25%) sediment systems. It was demonstrated that (i) powdered activated carbons can be toxic to aquatic invertebrates (Lumbriculus variegatus, Daphnia magna, and Corophium volutator) based on different mechanisms and preferably should be washed prior to application; (ii) Asellus aquaticus and Corophium volutator may physically avoid AC-enriched sediments; (iii) exposure of Lumbriculus variegatus to AC-enriched sediments lead to a time and dose-dependent reduction in the worms' lipid content, which was most probably caused by the observation that (iv) worm egestion rates decreased drastically upon AC addition, indicating that the presence of AC disturbed feeding behavior; and (v) there were no obvious effects on the microbiological community structure. All in all, these results suggest potential ecotoxicological effects of powdered AC addition and stress the need for a detailed further investigation of secondary effects of the technique, prior to any large-scale field application.

The risk posed by soil contaminants strongly depends on their bioavailability. In this study, a partition-based sampling method was applied as a tool to estimate bioavailability in soil. The accumulation of organic micropollutants was measured in two earthworm species (Eisenia andrei and Aporrectodea caliginosa) and in 30-microm poly(dimethylsiloxane) (PDMS)-coated solid-phase micro extraction (SPME) fibers after exposure to two field-contaminated soils. Within 10 days, steady state in earthworms was reached, and within 20 days in the SPME fibers. Steady-state concentrations in both earthworm species were linearly related to concentrations in fibers over a 10,000-fold range of concentrations. Measured concentrations in earthworms were compared to levels calculated via equilibrium partitioning theory and total concentrations of contaminants in soil. In addition, freely dissolved concentrations of contaminants in pore water, derived from SPME measurements, were used to calculate concentrations in earthworms. Measured concentrations in earthworms were close to estimated concentrations from the SPME fiber measurements. Freely dissolved concentrations of contaminants in pore water, derived from SPME measurements, were used to calculate bioconcentration factors (BCF) in earthworms. A plot of log BCFs against the octanol-water partition coefficient (log Kow) was linear up to a log Kow of 8. These results show that measuring concentrations of hydrophobic chemicals in a PDMS-coated fiber represents a simple tool to estimate internal concentrations of chemicals in biota exposed to soil.

Ecological investigations revealed differences in breeding success of cormorants (Phalacrocorax carbo) between two colonies in The Netherlands In this study the possible role of organohalogen pollutants was investigated. Thirty‐nine cormorant eggs were collected from two colonies with marked differences in contamination Seventeen cormorant eggs were hatched in an incubator. The respiration rate was monitored regularly during the incubation. Hatchlings were euthanized at day 1 Several morphological parameters were measured PCBs and polychlorinated dibenzo‐p‐dioxins (PCDDs) and dibenzofurans (PCDFs) were analyzed in the yolk sac Blood and liver were collected for analysis of cytochrome P450, ethoxyresorufin‐O‐deethylation (EROD) and pentoxyresorufin‐O‐depentylation (PROD) activities, vitamin A, and thyroid hormone levels. Residue levels differed two‐ to five‐fold for PCBs and 25% for PCDDs and PCDFs between both colonies Birds from the most contaminated colony showed an increased in ovo respiration rate, increased cytochrome P450 and EROD activity, and reduced plasma thyroid hormone and hepatic retinyl palmitate levels. Large interindividual differences were observed for all parameters The data were compared on an individual basis (n = 17) to detect any concentration‐effect relationships. Significant (p < 0 05) concentration‐effect relationships were observed for EROD induction, plasma free thyroxine reduction, yolk sac weight, relative liver weight, and head size. It is concluded these compounds may, at least in part, have played a role in the observed low breeding success of cormorants

Predicting fish acute toxicity of chemicals in vitro is an attractive alternative method to the conventional approach using juvenile and adult fish. The rainbow trout ( Oncorhynchus mykiss ) cell line assay with RTgill-W1 cells has been designed for this purpose. It quantifies cell viability using fluorescent measurements for metabolic activity, cell- and lysosomal-membrane integrity on the same set of cells. Results from over 70 organic chemicals attest to the high predictive capacity of this test. We here report on the repeatability (intralaboratory variability) and reproducibility (interlaboratory variability) of the RTgill-W1 cell line assay in a round-robin study focusing on 6 test chemicals involving 6 laboratories from the industrial and academic sector. All participating laboratories were able to establish the assay according to preset quality criteria even though, apart from the lead laboratory, none had previously worked with the RTgill-W1 cell line. Concentration-response modeling, based on either nominal or geometric mean-derived measured concentrations, yielded effect concentrations (EC 50 ) that spanned approximately 4 orders of magnitude over the chemical range, covering all fish acute toxicity categories. Coefficients of variation for intralaboratory and interlaboratory variability for the average of the 3 fluorescent cell viability measurements were 15.5% and 30.8%, respectively, which is comparable to other fish-derived, small-scale bioassays. This study therefore underlines the robustness of the RTgill-W1 cell line assay and its accurate performance when carried out by operators in different laboratory settings.