Application of Experimental Polystyrene Partition Constants and Diffusion Coefficients to Predict the Sorption of Neutral Organic Chemicals to Multiwell Plates in in Vivo and in Vitro Bioassays

Abstract

Sorption to the polystyrene (PS) of multiwell plates can affect the exposure to organic chemicals over time in in vitro and in vivo bioassays. Experimentally determined diffusion coefficients in PS (D PS) were in a narrow range of 1.25 to 8.0 · 10–16 m2 s–1 and PS-water partition constants (K PS/w) ranged from 0.04 to 5.10 log-units for 22 neutral organic chemicals. A kinetic model, which explicitly accounts for diffusion in the plastic, was applied to predict the depletion of neutral organic chemicals from different bioassay media by sorption to various multiwell plate formats. For chemicals with log K ow > 3, the medium concentrations decreased rapidly and considerably in the fish embryo toxicity assay but medium concentrations remained relatively constant in the cell-based bioassays with medium containing 10% fetal bovine serum (FBS), emphasizing the ability of the protein- and lipid-rich medium to compensate for losses by multiwell plate sorption. The PS sorption data may serve not only for exposure assessment in bioassays but also to model the contaminant uptake by and release from plastic packaging material and the chemical transport by PS particles in the environment.

Time-Resolved Freely Dissolved Concentrations of Semivolatile and Hydrophobic Test Chemicals in In Vitro Assays—Measuring High Losses and Crossover by Headspace Solid-Phase Microextraction

“…It has been shown that the reduction in losses caused by medium binding can be used as a buffer to stabilize the concentrations in the tests. 6,37 Fischer et al termed this approach 'serum mediated passive dosing'. 6 Schirmer et al 38 At serum concentrations of 10%, the serum acted as a buffer-compartment and retained chemicals with a log K ow of 5 and above in the test medium.…”

“…6,37 Fischer et al termed this approach 'serum mediated passive dosing'. 6 Schirmer et al 38 At serum concentrations of 10%, the serum acted as a buffer-compartment and retained chemicals with a log K ow of 5 and above in the test medium. Even for these highly hydrophobic chemicals, it was still important that the volatility was below a certain level, which increased with increasing hydrophobicity (see Figure 4).…”

“…Losses were for some of the more hydrophobic chemicals slightly larger Chemicals would normally not be expected to be able to crossover from one well to the next when the wells are closed. Recently, measured diffusion coefficients for >20 chemicals (D PS in the range 10 -16 -10 -15 m 2 s -1 ) showed very slow diffusion in polystyrene at 21°C, with diffusion distances of 5-20 µm in 96 hours 6 -well below the thickness of the well walls. In our study, nnitrosodi-n-butylamine was however found in the closed non-spiked wells at multiple timepoints in all three media.…”

“…However, several studies have found that volatile and/or hydrophobic chemicals evaporate from wells, [1][2][3][4] cross over to adjacent wells, 5 sorb to microtiter plates, 1,2,6,7 and sorb to medium constituents. 1,2,6,8 Therefore, the actual exposure of the cells to the test substances is often not known and decreases with time, which can lead to reduced toxicity and assay sensitivity. 1,2,9,10 Changes in assay setup affect the losses and thereby alter the toxicity parameter based on nominal concentrations.…”

Hypo- or hyperactivity of zebrafish embryos provoked by neuroactive substances: a review on how experimental parameters impact the predictability of behavior changes

“…This could be the result of higher well absorption area with respect to volume in 384-well plates leading to higher adsorption. Hence, as would be appropriate for any other test and endpoint, the sorption of chemicals to exposure vessels should be considered by: (1) determining or predicting the real exposure concentration; and (2) estimating a possible loss of the chemical instead of using nominal concentrations only [35,43,44].…”