Wastewater treatment efficacy evaluated with in vitro bioassays.

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 for the Evaluation of Mixture Toxicity in Environmental Samples.

The combined algae test is a 96-well plate-based algal toxicity assay with the green algae Raphidocelis subcapitata that combines inhibition of 24-h population growth rate with inhibition of photosynthesis detected after 2 and 24 h with pulse-amplitude modulated (PAM) fluorometry using a Maxi-Imaging PAM. The combined algae test has been in use for more than a decade but has had limitations due to incompatibilities of the measurements of the 2 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 light-emitting diode illumination. Different robotic dosing approaches and additional data evaluation methods helped to further expand the applicability domain of the assay. The combined algae test differentiates between nonspecifically acting compounds and photosynthesis inhibitors, such as photosystem II (PSII) herbicides. The 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 nonspecifically or that 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 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 combined algae test gave results consistent with those of the previous method but has lower costs, higher throughput, and higher precision. Environ Toxicol Chem 2020;39:2496-2508. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Optimization of a pre-metabolization procedure using rat liver S9 and cell-extracted S9 in the Ames fluctuation test.

Many environmental pollutants pose a toxicological hazard only after metabolic activation. In vitro bioassays using cell lines or bacteria have often no or reduced metabolic activity, which impedes their use in the risk assessment. To improve the predictive capability of in vitro assays, external metabolization systems like the liver S9 fraction are frequently combined with in vitro toxicity assays. While it is typical for S9 fractions that samples and testing systems are combined in the same exposure system, we propose to separate the metabolism step and toxicity measurement. This allows for a modular combination of metabolic activation by enzymes isolated from rat liver (S9) or a biotechnological alternative (ewoS9R) with in vitro bioassays that lack metabolic capacity. Benzo(a)pyrene and 2-aminoanthracene were used as model compounds to optimize the conditions for the S9 metabolic degradation/activation step. The Ames assay with Salmonella typhimurium strains TA98 and TA100 was applied to validate the set-up of decoupling the S9 activation/metabolism from the bioassay system. S9 protein concentration of 0.25 mgprotein/mL, a supplement of 0.13 mM NADPH and a pre-incubation time of 100 min are recommended for activation of samples prior to dosing them to in vitro bioassays using the regular dosing protocols of the respective bioassay. EwoS9R performed equally well as Moltox S9, which is a step forward in developing true animal-free in vitro bioassays. After pre-incubation with S9 fraction, chemicals induced bacteria revertants in both the TA98 and the TA100 assay as efficiently as the standard Ames assay. The pre-incubation of chemicals with S9 fraction could serve for a wide range of cellular in vitro assays to efficiently combine activation and toxicity measurement, which may greatly facilitate the application of these assays for chemical hazard assessment and monitoring of environmental samples.

Baseline Toxicity and Volatility Cutoff in Reporter Gene Assays Used for High-Throughput Screening.

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.