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.

Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure concentration analysis.

The zebrafish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, which is required by various regulations for environmental risk assessment of chemicals. We investigated the reliability of the embryo test by probing organic industrial chemicals with a wide range of physicochemical properties, toxicities, and modes of toxic action. Moreover, the relevance of using measured versus nominal (intended) exposure concentrations, inclusion of sublethal endpoints, and different exposure durations for the comparability with reported fish acute toxicity was explored. Our results confirm a very strong correlation of zebrafish embryo to fish acute toxicity. When toxicity values were calculated based on measured exposure concentrations, the slope of the type II regression line was 1 and nearly passed through the origin (1 to 1 correlation). Measured concentrations also explained several apparent outliers. Neither prolonged exposure (up to 120 h) nor consideration of sublethal effects led to a reduced number of outliers. Yet, two types of compounds were less lethal to embryos than to adult fish: a neurotoxic compound acting via sodium channels (permethrin) and a compound requiring metabolic activation (allyl alcohol).

Development of a partition-controlled dosing system for cell assays.

Hydrophobic and volatile chemicals have proven to be difficult to dose in cell assays. Cosolvents are often needed to dissolve these chemicals in cell culture medium. Moreover, the free concentration of these chemicals in culture medium may diminish over time due to metabolism, evaporation, and nonspecific binding to well plate surfaces and serum constituents. The aim of this study was to develop a partition-controlled dosing system to maintain constant concentrations of benzo(a)pyrene, 1,2-dichlorobenzene, and 1,2,4-trichlorobenzene in an ethoxyresorufin-O-deethylase (EROD) assay and a cytotoxicity assay with the rainbow trout (Oncorhynchus mykiss) cell lines RTL-W1 and RTgill-W1. Polydimethylsiloxane (PDMS) sheets were loaded with test chemicals in a spiked methanol/water solution and placed in the wells, filled with culture medium, of a 24-well culture plate. Cells were grown on inserts and were subsequently added to the wells with the PDMS sheets. The system reached equilibrium within 24 h, even for the very hydrophobic chemical benzo(a)pyrene. The reservoir of test chemical in PDMS was large enough to compensate for the loss of >95% of the test chemical from the culture medium. The PDMS sheets maintained medium concentrations constant for >72 h. Nominal median effect concentrations (EC(50)) were 1.3-7.0 times lower in the partition-controlled dosing systems than in conventional assays spiked using dimethyl sulfoxide (DMSO) as a carrier solvent, thus indicating that the apparent sensitivity of the bioassay increased when controlled and constant exposure conditions could be assured. The EC(50) values of the test chemicals based on free concentrations were estimated in the partition-controlled dosing systems using measured PDMS-bare culture medium partition coefficients. Results indicated that 61, 70, and 99.8% of 1,2-diclorobenzene, 1,2,4-trichlorobenzene, and benzo(a)pyrene were bound to serum constituents in the culture medium.

Effects of solvents and dosing procedure on chemical toxicity in cell-based in vitro assays.

Due to the implementation of new legislation, such as REACh, a dramatic increase of animal use for toxicity testing is expected and the search for alternatives is timely. Cell-based in vitro assays are promising alternatives. However, the behavior of chemicals in these assays is still poorly understood. We set out to quantify the exposure and associated toxicity of chemicals with different physicochemical properties toward a fish gill cell line when different solvents and procedural steps are used to introduce test chemicals to cells. Three chemicals with a range of hydrophobicity and volatility were selected and delivered in three different solvents using two common dosing procedures. Toxicity tests were coupled with chemical analysis to quantify the chemical concentrations within culture wells. The impact of solvents and dosing procedure was greatest for the most volatile and hydrophobic test chemical. We show that certain combinations of the test chemical, solvent, and procedural steps can lead to inhomogeneous distribution of the test chemical and thus differing degrees of bioavailability, resulting in quantitative differences in apparent toxicity.

Facilitated transport of polychlorinated biphenyls and polybrominated diphenyl ethers by dissolved organic matter.

The exchange rate of hydrophobic organic chemicals between the aqueous phase and a sorbent (e.g., soil, organism, passive sampler) is relevant for distribution processes between environmental compartments, including organisms. Dissolved phases such as humic acids, proteins, and surfactants can affect the transfer of such chemicals between the aqueous and sorbent phases by sorption and desorption processes. In this study, the desorption of polychlorinated biphenyls and polybrominated diphenyl ethers from a polymer phase to an aqueous medium was monitored at different humic acid concentrations. The rate of release of the chemical by the polymer phase demonstrates thatthe chemical sorbed to dissolved humic acid contributed significantly to the total mass transfer when the affinity for the humic acid was sufficiently high. This illustrates that environmentally relevant humic acid concentrations can facilitate transport of hydrophobic organic chemicals. The consequences of these facilitated transport mechanisms for uptake into passive samplers are discussed, in particular in situations where equilibration is very slow or when exposure varies in time or space.

Poly(dimethylsiloxane) as passive sampler material for hydrophobic chemicals: effect of chemical properties and sampler characteristics on partitioning and equilibration times.

Information about sampling rates and equilibration times of passive samplers is essential in their calibration in field monitoring studies as well as sorption studies. The kinetics of a sampler depends on the distribution coefficient between the sampler material and aqueous phase and the exchange rates of chemicals between these phases. In this study, the elimination kinetics of four poly(dimethylsiloxane) (PDMS) passive samplers with different surface-volume ratios are compared. The samplers were loaded with polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) that cover a broad range of hydrophobicities. The surface-volume ratios of the samplers could largely explain the observed kinetics. Furthermore, a simple diffusion-based model illustrates that the exchange of chemicals was limited by diffusion through the aqueous diffusion layer surrounding the sampler. On the basis of this simple diffusion model, equilibration times are predicted for organic chemicals that vary in hydrophobicity and samplers with different dimensions and polymeric phases. This information is of importance in the selection of a passive sampler for a specific purpose.

Measurement of the free concentration of octylphenol in biological samples with negligible depletion-solid phase microextraction (nd-SPME): analysis of matrix effects.

A negligible depletion-solid phase microextraction (nd-SPME) method is presented to measure free concentrations of octylphenol in biological samples. Potential confounding factors, such as matrix effects, are studied as well. Fouling of the fibre appears to occur, but it does not seem to reduce or enhance the measured uptake of octylphenol. In the setup applied here, without any agitation, it has also been found that there is a large effect of protein presence on the kinetics of octylphenol uptake. In addition, an apparent affinity constant of octylphenol for bovine serum albumin was determined.

Sediment dilution method to determine sorption coefficients of hydrophobic organic chemicals.

Sorption coefficients of hydrophobic organic chemicals (HOC) to sediments and soils can easily be underestimated in traditional batch experiments, especially because analysis of the aqueous concentration often includes compounds sorbed to colloidal organic matter. In this work, a "sediment dilution approach" has been combined with measurements of freely dissolved concentrations to determine sorption coefficients of five chlorobenzenes and two chloroanilines in spiked sediment and of two unknown chemicals in field-contaminated sediment. A range of sediment suspensions with different sediment-water ratios was made. Freely dissolved concentrations in these suspensions were measured by negligible depletion solid-phase microextraction (nd-SPME). Sediment-water sorption coefficients (KD) were derived from the decrease of the freely dissolved concentrations as a function of the "dilution factor" (DF = volume water/mass sediment). The determined sorption coefficients were very similar to literature values. The experimental setup provides sorption coefficients without the need for total extractions, and the negligible depletion SPME technique does not require phase separation. The proposed method might be an alternative for batch equilibrium experiments to determine sorption coefficients.

Toward more useful in vitro toxicity data with measured free concentrations.

In vitro assays and computer models are promising alternatives for in vivo animal testing, but the power of these alternative methods to predict in vivo risk is still very limited. One step forward is to make the outcome of in vitro assays (such as median effect concentrations (EC50 values)) independent of assay conditions such as protein content. Here we show that measured free concentrations of chemicals in the in vitro assay medium result in system-independent EC50 values. We introduce a very simple method to measure free concentrations in miniature test systems using negligible depletion solid-phase microextraction. The generated data are much more suitable for extrapolation to in vivo, provide unbiased input for computational methods (for example, quantitative structure-activity relationships), and can shed an entirely different light on the activity of environmental contaminants.

Measured pore-water concentrations make equilibrium partitioning work--a data analysis.

There is an increasing body of evidence that the bioaccumulation of sediment-associated hydrophobic organic compounds (HOCs) is strongly influenced by sequestration. At present, it is not known how equilibrium partitioning theory (EqP), the most commonly employed approach for describing sediment bioaccumulation can be applied to sediments with sequestered contaminants. In this paper, we present freely dissolved pore-water concentrations of HOCs. These data were employed to interpret sediment bioaccumulation and sequestration data in order to arrive at a process based evaluation of EqP. The data analysis suggests that sediment bioaccumulation of compounds up to log K(ow) 7.5 in Tubificidae can be described as bioconcentration from pore-water. In addition, the pore-water concentrations of HOCs (4.5 < log K(ow) < 7.5) are established by equilibrium partitioning between the rapidly desorbing HOCs fraction in the sediment and the pore-water. Taken together, these findings indicate that EqP is a conceptually correct representation of sediment bioaccumulation, provided that sequestration is accounted for. This implies that the risk assessment of sediment-associated HOCs can be significantly simplified: With a method at hand for measuring freely dissolved pore-water concentrations of HOCs, it appears that HOCs' body residues in sediment dwelling organisms can be estimated on the basis of concentrations in pore-water and bioconcentration factors.

Biomimetic solid-phase microextraction to predict body residues and toxicity of chemicals that act by narcosis.

A biomimetic extraction technique using solid-phase microextraction (SPME) fibers has been developed for the risk assessment of contaminants with a narcotic mode of action. Our goal is to apply this technique in the future for the prediction of total baseline toxicity of environmental water and effluent samples. Validation of this method requires establishing the relationship between contaminant accumulation and toxicity in biota and accumulation in the surrogate solid phase (the SPME fiber coating). For this purpose, we determined the median lethal concentration (LC50) values for Chironomus riparius midge larvae exposed to two halogenated aromatic compounds separately and measured body residues in the exposed larvae. Solid-phase microextraction fibers with an 85-microm polyacrylate (PA) coating served as the surrogate hydrophobic phase, mimicking the uptake of the compounds by midge larvae. The toxicant concentrations in SPME fibers measured directly by gas chromatography/mass spectrometry (GC-MS) or calculated from the SPME fiber-water partition coefficient, K(SPME) were related to the toxicant concentrations found in midge larvae. Our results demonstrated that the biomimetic SPME method enables the estimation of body residues in biota and prediction of the degree of baseline toxicity of a water medium.

Bioconcentration of organic chemicals: is a solid-phase microextraction fiber a good surrogate for biota?

When organic chemicals are extracted from a water sample with solid-phase microextraction (SPME) fibers, the resulting concentrations in exposed fibers are proportional to the hydrophobicity of the compounds. This fiber accumulation is analogous to the bioconcentration of chemicals observed in aquatic organisms. The objective of this study was to investigate the prospect of measuring the total concentration in SPME fibers to estimate the total body residue in biota for the purpose of risk assessment. Using larvae of the midge, Chironomus riparius and disposable 15-microm poly(dimethylsiloxane) fibers, we studied the accumulation and accumulation kinetics of a number of narcotic compounds with a range of log K(ow) between 3 and 6. The fibers, which have a larger surface area-to-volume ratio, had consistently higher uptake and elimination rate constants (k1 and k2, respectively) than midge larvae and accumulated the chemicals 5 times faster. Comparison of the relationships of the partition coefficients K(PDMS-water) and K(midge-water) (lipid-normalized) to log K(ow) for all compounds yielded a factor of 28 for translating fiber concentrations to biota concentrations. This factor can be used to estimate internal concentrations in biota for compounds structurally similar to the compounds in this study. The exact chemical domain to which this factor can be applied needs to be defined in future research.