Most studies using high-throughput in vitro cell-based bioassays tested chemicals up to a certain fixed concentration. It would be more appropriate to test up to concentrations predicted to elicit baseline toxicity because this is the minimal toxicity of every chemical. Baseline toxicity is also called narcosis and refers to nonspecific intercalation of chemicals in biological membranes, leading to loss of membrane structure and impaired functioning of membrane-related processes such as mitochondrial respiration. In cells, baseline toxicity manifests as cytotoxicity, which was quantified by a robust live-cell imaging method. Inhibitory concentrations for baseline toxicity varied by orders of magnitude between chemicals and were described by a simple quantitative structure activity relationship (QSAR) with the liposome-water partition constant as a sole descriptor. The QSAR equations were remarkably similar for eight reporter gene cell lines of different cellular origin, six of which were used in Tox21. Mass-balance models indicated constant critical membrane concentrations for all cells and all chemicals with a mean of 69 mmol·kglip –1(95% CI: 49–89), which is in the same range as for bacteria and aquatic organisms and consistent with the theory of critical membrane burden of narcosis. The challenge of developing baseline QSARs for cell lines is that many confirmed baseline toxicants are rather volatile. We deduced from cytotoxicity experiments with semi-volatile chemicals that only chemicals with medium-air partition constants >10,000 L/L can be tested in standard robotic setups without appreciable loss of effect. Chemicals just below that cutoff showed crossover effects in neighboring wells, whereas the effects of chemicals with lower medium-air partition constants were plainly lost. Applying the “volatility cut-off” to >8000 chemicals tested in Tox21 indicated that approximately 20% of Tox21 chemicals could have partially been lost during the experiments. We recommend applying the baseline QSARs together with volatility cut-offs for experimental planning of reporter gene assays, that is, to dose only chemicals with medium-air partition constants >10,000 at concentrations up to the baseline toxicity level.

Bioassays show promise as a complementary approach to chemical analysis to assess the efficacy of wastewater treatment processes as they can detect the mixture effects of all bioactive chemicals in a sample. We investigated the treatment efficacy of ten Australian wastewater treatment plants (WWTPs) covering 42% of the national population over seven consecutive days. Solid-phase extracts of influent and effluent were subjected to an in vitro test battery with six bioassays covering nine endpoints that captured the major modes of action detected in receiving surface waters. WWTP influents and effluents were compared on the basis of population- and flow-normalised effect loads, which provided insights into the biological effects exhibited by the mixture of chemicals before and after treatment. Effect removal efficacy varied between effect endpoints and depended on the treatment process. An ozonation treatment step had the best treatment efficacy, while WWTPs with only primary treatment resulted in poor removal of effects. Effect removal was generally better for estrogenic effects and the peroxisome proliferator-activated receptor than for inhibition of photosynthesis, which is consistent with the persistence of herbicides causing this effect. Cytotoxicity and oxidative stress response provided a sum parameter of all bioactive chemicals including transformation products and removal was poorer than for specific endpoints except for photosynthesis inhibition. Although more than 500 chemicals were analysed, the detected chemicals explained typically less than 10% of the measured biological effect, apart from algal toxicity, where the majority of the effect could be explained by one dominant herbicide, diuron. Overall, the current study demonstrated the utility of applying bioassays alongside chemical analysis to evaluate loads of chemical pollution reaching WWTPs and treatment efficacy.

The combined algae test (CAT) is a 96-well plate-based algal toxicity assay with the green algae Raphidocelis subcapitata that combines inhibition of 24h population growth rate with inhibition of photosynthesis detected after 2h and 24h with pulse-amplitude modulated (PAM) fluorometry using a Maxi-Imaging PAM. The CAT has been in use for more than a decade but had limitations due to incompatibilities of the measurements of the two biological endpoints on the same microtiter plates. These limitations could be overcome by increasing growth rates and doubling times on black, clear-bottom 96-well plates by application of dichromatic red/blue LED illumination. Different robotic dosing approaches and additional data evaluation approaches helped to further expand the applicability domain of the assay. The CAT differentiates between non-specifically acting compounds and photosynthesis inhibitors, such as photosystem II (PSII)-herbicides. PSII-herbicides acted immediately on photosynthesis and showed growth rate inhibition at higher concentrations. If growth was a similar or more sensitive endpoint than photosynthesis inhibition, this was an indication that the tested chemical acted non-specifically or a mixture or a water sample was dominated by chemicals other than PSII-herbicides acting on algal growth. We fingerprinted the effects of 45 chemicals on photosynthesis inhibition and growth rate and related the effects of the single compounds to designed mixtures of these chemicals detected in water samples and to Running Head: Combined algae test and mixtures This article is protected by copyright. All rights reserved. Accepted Article the effects directly measured in water samples. Most of the observed effects in the water samples could be explained by known photosystem II inhibitors such as triazines and phenylurea herbicides. The improved setup of the CAT gave consistent results with the previous method but has lower cost, higher throughput and higher precision. This article includes online-only Supplemental Data.

In vitro bioassays indicative of different modes of action demonstrate the high quality of produced drinking water.

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