Headspace-Free Setup of in Vitro Bioassays for the Evaluation of Volatile Disinfection By-Products

Abstract

The conventional setup of in vitro bioassays in microplates does not prevent the loss of volatile compounds, which hampers the toxicological characterization of waterborne volatile disinfection by-products (DBPs). To minimize the loss of volatile test chemicals, we adapted four in vitro bioassays to a headspace-free setup using eight volatile organic compounds (four trihalomethanes, 1,1-dichloroethene, bromoethane, and two haloacetonitriles) that cover a wide range of air-water partition coefficients. The nominal effect concentrations of the test chemicals decreased by up to three orders of magnitude when the conventional setup was changed to a headspace-free setup for the bacterial cytotoxicity assay using bioluminescence inhibition of Vibrio fischeri. The increase of apparent sensitivity correlated significantly with the air-water partition coefficient. Purge and trap GC/MS analysis revealed a reduced loss of dosed volatile compounds in the headspace free setup (78-130% of nominal concentration) compared to a substantial loss in the conventional set up (2-13% of the nominal concentration). The experimental effect concentrations converged with the headspace-free setup to the effect concentrations predicted by a QSAR model, confirming the suitability of the headspace-free approach to minimize the loss of volatile test chemicals. The analogue headspace-free design of the bacterial bioassays for genotoxicity (umuC assay) and mutagenicity (Ames fluctuation assay) increased the number of compounds detected as genotoxic or mutagenic from one to four and zero to two, respectively. In a bioassay with a mammalian cell line applied for detecting the induction of the Nrf-2-mediated oxidative stress response (AREc32 assay), the headspace-free setup improved the apparent sensitivity by less than one order of magnitude, presumably due to the retaining effect of the serum components in the medium, which is also reflected in the reduced aqueous concentrations of compounds. This study highlights the importance of adapting bioanalytical test setups when volatile/semivolatile compounds are present in the sample to avoid the loss of chemicals and thus to avoid underestimating the toxicity of mixtures and complex environmental samples.

Bioanalytical and chemical evaluation of disinfection by-products in swimming pool water

“…HAAs (Pals et al 2013). HAAs could only explain ≤1 % of oxidative stress response, which is similar to a recent study that quantified 269 individual chemicals in environmental samples including drinking water (Escher et al 2013).…”

“…Although bioassays have the ability to capture mixture effect, the sample preparation by SPE has its own limitation as it can only retain non-volatile and semivolatile compounds, and not able to capture volatile compounds. Our group is presently working on developing methods to assess volatile chemicals with cell-based bioassays (Stalter et al 2013) and to extract volatiles from water samples without use of excessive amounts of solvents (work in progress).…”

“…The AREc32 cell line was demonstrated to be sensitive to chemicals inducing oxidative stress (Natsch 2010) and has been previously applied for water quality monitoring (Escher et al 2013). All water samples, including the pool water samples, drinking water and surface water, induced the oxidative stress response within the same order of magnitude but effects were higher than in ultrapure water ( Figure 3B).…”

Overview of Disinfection By-products and Associated Health Effects

“…Global indicators of toxicity (cytotoxicity, genotoxicity, etc. ), combined with chemical analysis of disinfection by-products, have recently been used to address the complex nature of DBP mixtures [32,33]. However, observational studies in humans still lack methods to address these complexities, and there have been few attempts that have demonstrated a clear improvement over traditional evaluations [34].…”

Bioanalytical Approaches in Assessing Transformation Products

“…An issue for bioanalytical assays has been the ability to assay volatile TPs. A new method developed by Escher's laboratory that allows the use of closed systems for cell based assays was shown to be a workable solution 56. This method was successfully applied to a cytotoxicity test with Vibrio fisheri, the UmuC assay for genotoxicity and the AREc32 assay for induction of oxidative stress.…”