Repeatability and Reproducibility of the RTgill-W1 Cell Line Assay for Predicting Fish Acute Toxicity.

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 (EC50) 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.

Determining high-quality critical body residues for multiple species and chemicals by applying improved experimental design and data interpretation concepts.

Ecotoxicological effect data are generally expressed as effective concentrations in the external exposure medium and do thus not account for differences in chemical uptake, bioavailability, and metabolism, which can introduce substantial data variation. The Critical Body Residue (CBR) concept provides clear advantages, because it links effects directly to the internal exposure. Using CBRs instead of external concentrations should therefore reduce variability. For compounds that act via narcosis even a constant CBR has been proposed. Despite the expected uniformity, CBR values for these compounds still show large variability, possibly due to biased and inconsistent experimental testing. In the present study we tested whether variation in CBR data can be substantially reduced when using an improved experimental design and avoiding confounding factors. The aim was to develop and apply a well-defined test protocol for accurately and precisely measuring CBR data, involving improved (passive) dosing, sampling, and processing of organisms. The chemicals 1,2,4-trichlorobenzene, 1,2,3,4-tetrachlorobenzene, 2,3,4-trichloroaniline, 2,3,5,6-tetrachloroaniline, 4-chloro-3-methylphenol, pentylbenzene, pyrene, and bromophos-methyl were tested on Lumbriculus variegatus (California blackworm), Hyalella azteca (scud), and Poecilia reticulata (guppy), which yielded a high-quality database of 348 individual CBR values. Medians of CBR values ranged from 2.1 to 16.1 mmol/kg wet weight (ww) within all combinations of chemicals and species, except for the insecticide bromophos-methyl, for which the median was 1.3 mmol/kg ww. The new database thus covers about one log unit, which is considerably less than in existing databases. Medians differed maximally by a factor of 8.4 between the 7 chemicals but within one species, and by a factor of 2.6 between the three species but for individual chemicals. Accounting for the chemicals' internal distribution to different partitioning domains and relating effects to estimated concentrations in the target compartment (i.e., membrane lipids) was expected to but did not decrease the overall variability, likely because the surrogate partition coefficients for membrane lipid, storage lipid, protein, and carbohydrate that were used as input parameters did not sufficiently represent the actual partitioning processes. The results of this study demonstrate that a well-designed test setup can produce CBR data that are highly uniform beyond chemical and biological diversity.

Adsorption of polar, nonpolar, and substituted aromatics to colloidal graphene oxide nanoparticles.

We conducted batch adsorption experiments to understand the adsorptive properties of colloidal graphene oxide nanoparticles (GONPs) for a range of environmentally relevant aromatics and substituted aromatics, including model nonpolar compounds (pyrene, phenanthrene, naphthalene, and 1,3-dichlorobenzene) and model polar compounds (1-naphthol, 1-naphthylamine, 2,4-dichlorophenol, and 2,4-dinitrotoluene). GONPs exhibited strong adsorption affinities for all the test compounds, with distribution coefficients on the order of 10(3)-10(6) L/kg. Adsorption to GONPs is much more linear than to carbon nanotubes (CNTs) and C60, likely because GO nanoflakes are essentially individually dispersed (rendering adsorption sites of similar adsorption energy) whereas CNT/C60 are prone to bundling/aggregation. For a given compound GONPs and CNTs often exhibit different adsorption affinities, which is attributable to the differences in both the morphology and surface chemistry between the two nanomaterials. Particularly, the high surface O-content of GONPs enables strong H-bonding and Lewis acid-base interactions with hydroxyl- and amino-substituted aromatics.

Predicting fish acute toxicity using a fish gill cell line-based toxicity assay.

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.

Effect-directed assessment of the bioaccumulation potential and chemical nature of Ah receptor agonists in crude and refined oils.

Recent studies have indicated that in addition to narcosis certain chemicals in crude oils and refined petroleum products may induce specific modes of action, such as aryl hydrocarbon receptor (AhR) agonism. The risks these toxic compounds pose to organisms depend on internal exposure levels, as driven by the chemicals' bioaccumulation potential. Information on this potential however is lacking, as the chemicals' identity mostly is unknown. This study showed that AhR agonists bioaccumulate from oil-spiked sediments into aquatic worms and persist in the worms for at least several weeks. Chemical fractionations of eight pure oils into saturates, aromatics, resins, and asphaltenes (SARA), followed by effect-directed analyses using in vitro reporter gene assays revealed that the agonists predominantly are aromatic and resin-like chemicals. Some of the compounds were easily metabolized in vitro, while others were resistant to biotransformation. HPLC-assisted hydrophobicity profiling subsequently indicated that the AhR-active chemicals had a high to extremely high bioaccumulation potential, considering their estimated logK(ow) values of 4 to >10. Most of the AhR agonism, however, was assigned to compounds with logK(ow) of 5-8. These compounds were present mainly in the mid to high boiling point fractions of the oils (C(14)-C(32) alkane range), which are usually not being considered (the most) toxic in current risk assessment. The fractionations further revealed considerable oil and fraction-dependent antagonism in pure oils and SARA fractions. The results of this study clearly demonstrate that crude oils and refined petroleum products contain numerous compounds that can activate the AhR and which because of their likely persistence and extremely high bioaccumulation potential could be potential PBT (persistent, bioaccumulative and toxic) or vPvB (very persistent and very bioaccumulative) substance candidates. Many chemicals were identified by GC-MS, but the responsible individual compounds could not be exactly identified in the complex mixtures of thousands of compounds. Because this obstructs a classical PBT risk assessment, our results advocate an adapted risk assessment approach for complex mixtures in which low concentrations of very potent compounds are responsible for mixture effects.

Ecotoxicological effects of activated carbon addition to sediments.

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.

Analysis of freely dissolved alcohol ethoxylate homologues in various seawater matrixes using solid-phase microextraction.

Solid-phase microextraction fibers (SPME) were tested as tools to determine freely dissolved alcohol ethoxylate (AE) surfactants in seawater matrixes. Partitioning of a wide range of AE homologues into a 35-mum polyacrylate fiber coating was linearly related to aqueous concentrations as low as submicrograms per liter, with high reproducibility. The exposure time needed to reach equilibrium between aqueous phase and the SPME fiber depended on the fiber-water partitioning coefficient (Kfw) of the AE homologue. Specific attention was given to the influence of various matrixes on the analysis via SPME. The presence of sediment increases the uptake kinetics of AE homologues for which diffusion in the aqueous phase is rate limiting. The Kfw in equilibrated systems was not affected by the presence of other homologues, micelles, or varying amounts of sediment phase. SPME is therefore a suitable tool for analysis of AE in sorption studies and sediment toxicity tests. A strong linear relation was observed between Kfw and the hydrophobicity of the AE homologue, using estimated octanol-water partition coefficients. This relation can be used to predict the partitioning coefficient of any AE homologue to the SPME fiber, which facilitates the analysis of complex mixtures.

Solid-phase microextraction to predict bioavailability and accumulation of organic micropollutants in terrestrial organisms after exposure to a field-contaminated soil.

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.

In vitro and in vivo antiestrogenic effects of polycyclic musks in zebrafish.

The polycyclic musks 6-acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN) and 1,2,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyran (HHCB) are used as fragrance ingredients in perfumes, soaps, and household cleaning products. They are known to be ubiquitously present in the aquatic environment, and because of their lipophilic nature, they tend to bioaccumulate in aquatic biota. In surface waters, concentrations between 1 ng/L and 5 microg/L have been found, depending mainly on the proportion of sewage effluents in the water. In fish, under normal environmental conditions, concentrations in the microgram per kilogram fresh weight (fw) range are found. In a previous study we showed that AHTN and HHCB exert mainly antiestrogenic effects on the human estrogen receptor alpha (ERalpha) and ERbeta in an in vitro reporter gene assay. In the current study, we assessed the in vitro antiestrogenic effects of both musks on zebrafish ERs. Antagonism was observed on zfERbeta, and more pronounced on the newly cloned zfERgamma. Using a transgenic zebrafish assay, we studied antiestrogenicity of the musks in vivo. Dose-dependent antagonistic effects were observed at concentrations of 0.1 and 1 microM AHTN and HHCB. GC-MS analysis showed that the musks bioaccumulated in the fish, with internal concentrations (15-150 mg/kg fw) which were roughly 600 times higher than the nominal test doses. To our knowledge, this is the first time that environmental contaminants are shown to be antiestrogenic in an in vivo fish assay that focuses solely on ER-mediated effects. This makes the transgenic zebrafish assay a promising tool for the rapid detection of both estrogenic and antiestrogenic effects of chemicals in fish.

Bioconcentration and acute toxicity of polycyclic musks in two benthic organisms (Chironomus riparius and Lumbriculus variegatus).

In the current study, the bioconcentration behavior and acute toxicity of two polycyclic musks, Tonalide 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN) and Galaxolide 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexa-methylcyclopenta[gamma]-2-benzopyran (HHCB), were studied in two benthic organisms. Polycyclic musks are frequently used fragrances, and they have been detected in different compartments of the environment. The aim of this study was to fill some empirical data gaps for AHTN and HHCB for benthic organisms. Results show that differences exist between both organisms. Chironomus riparius exhibited bioconcentration factors (BCFs) for AHTN and HHCB substantially lower than predicted for nontransformed organics. The BCFs for both chemicals increased after coexposure of the organism to the cytochrome P450 inhibitor piperonyl butoxide. Thus, the low BCF values were the result of rapid biotransformation of AHTN and HHCB in the midge larvae. Bioconcentration kinetics indicated that both chemicals induced their own cytochrome P450-mediated metabolism. Acute toxicity of AHTN to midge larvae was reduced compared to predicted baseline toxicity and was similar for HHCB. Bioconcentration of AHTN and HHCB in the worm (Lumbriculus variegatus) is in agreement with predictions based on the octanol-water partition coefficients of these chemicals. Acute toxicity was found to be similar to predicted values for baseline toxicity. Summarizing, for AHTN and HHCB, acute toxicity and bioconcentration behavior in L. variegatus was in accordance with predicted data for nontransformed organics. In C. riparius, bioconcentration as well as toxicity were reduced.