Influence of in Vitro Assay Setup on the Apparent Cytotoxic Potency of Benzalkonium Chlorides.

The nominal concentration is generally used to express concentration-effect relationships in in vitro toxicity assays. However, the nominal concentration does not necessarily represent the exposure concentration responsible for the observed effect. Surfactants accumulate at interphases and likely sorb to in vitro system components such as serum protein and well plate plastic. The extent of sorption and the consequences of this sorption on in vitro readouts is largely unknown for these chemicals. The aim of this study was to demonstrate the effect of sorption to in vitro components on the observed cytotoxic potency of benzalkonium chlorides (BAC) varying in alkyl chain length (6-18 carbon atoms, C6-18) in a basal cytotoxicity assay with the rainbow trout gill cell line (RTgill-W1). Cells were exposed for 48 h in 96-well plates to increasing concentration of BACs in exposure medium containing 0, 60 µM bovine serum albumin (BSA) or 10% fetal bovine serum (FBS). Before and after exposure, BAC concentrations in exposure medium were analytically determined. Based on freely dissolved concentrations at the end of the exposure, median effect concentrations (EC50) decreased with increasing alkyl chain length up to 14 carbons. For BAC with alkyl chains of 12 or more carbons, EC50's based on measured concentrations after exposure in supplement-free medium were up to 25-times lower than EC50's calculated using nominal concentrations. When BSA or FBS was added to the medium, a decrease in cytotoxic potency of up to 22 times was observed for BAC with alkyl chains of eight or more carbons. The results of this study emphasize the importance of expressing the in vitro readouts as a function of a dose metric that is least influenced by assay setup to compare assay sensitivities and chemical potencies.

Wastewater characterisation by combining size fractionation, chemical composition and biodegradability.

The potential for resource recovery from wastewater can be evaluated based on a detailed characterisation of wastewater. In this paper, results from fractionation and characterisation of two distinct wastewaters are reported. Using tangential flow filtration, the wastewater was fractionated into 10 size fractions ranging from 1 kDa to 1 mm, wherein the chemical composition and biodegradability were determined. Carbohydrates were dominant in particulate size fractions larger than 100 µm, indicating a potential of cellulose recovery from these fractions. While the particulate size fractions between 0.65 and 100 µm show a potential as a source for biofuel production due to an abundance of saturated C16 and C18 lipids. Both wastewaters were dominated by particulate (>0.65 µm), and oligo- and monomeric (<1 kDa) COD. Polymeric (1-1000 kDa) and colloidal (1000 kDa-0.65 µm) fractions had a low COD content, expected due to degradation in the sewer system upstream of the wastewater treatment plant. Biodegradation rates of particulate fractions increase with decreasing size. However, this was not seen in polymeric fractions where degradation rate was governed by chemical composition. Analytical validation of molecular weight and particle size distribution showed below filter cut-off retention of particles and polymers close to nominal cut-off, shifting the actual size distribution.