Passive equilibrium sampling of chemical mixtures from different human post-mortem tissues (liver, brain (cerebrum and cerebellum), adipose tissue) and blood was combined with instrumental analysis using direct sample introduction (DSI) GC-MS/MS and bioanalytical profiling using in vitro bioassays targeting the activation of the aryl hydrocarbon receptor (AhR-CALUX), the adaptive stress response (AREc32) and cytotoxicity. The tissues stemmed from pathology samples collected in two German cities and covered males and females aged 21 to 100 with a mean age of 67 years. Neutral organic chemicals were extracted using polydimethylsiloxane (PDMS) at mass ratios of tissue to PDMS of approximately 6 for blood, 3 for adipose tissue and 10 for liver and brain. Amounts of chemicals in PDMS were converted to lipid-associated concentrations using previously measured partition constants that were chemical-independent despite covering eight orders of magnitude in hydrophobicity. Up to 35 of 99 targeted chemicals were detected in 6 tissues of 16 individuals (88 samples in total), among them legacy persistent organic pollutants (POP) such as DDT and derivatives and polychlorinated biphenyls (PCB) but also modern pesticides and chemicals present in consumer products. POPs were highest in adipose tissue and lipid-associated concentrations increased with age, while concentrations of fragrance materials such as galaxolide were independent of age. In tissues from the same individual, chemical concentrations mostly increased from similar levels in brain and blood to higher levels in liver and highest in adipose tissue. However, easily degradable chemicals such as phenanthrene were mainly detected in blood and brain, and very hydrophilic chemicals were least abundant in adipose tissue. The passive sampling method allows a direct comparison of chemical burden between different tissues and may have forensic applications, for example to study internal distributions or to use one tissue type as a proxy for others. The sum of concentrations of the detected chemicals was positively correlated with the bioassay responses but mixture modeling showed that the detected chemicals explained less than 2% of the activation of the AhR and less than 0.5% of cytotoxicity. This means that more than 10,000 chemicals would need to be included in an analytical method to capture all the effects with many chemicals potentially being below detection limits but still contributing to mixture effects. Therefore, we propose a smart combination of chemical analysis and bioassays to quantify priority chemicals but use bioassay responses as effect-scaled concentrations to capture the entire exposome in future epidemiological studies.

Equilibrium passive sampling employing polydimethylsiloxane (PDMS) as a sampling phase can be used for the extraction of complex mixtures of organic chemicals from lipid-rich biota. We extended the method to lean tissues and more hydrophilic chemicals by implementing a mass-balance model for partitioning between lipids, proteins, and water in tissues and by accelerating uptake kinetics with a custom-built stirrer that effectively decreased time to equilibrium to less than 8 days even for a homogenized liver tissue with an only 4% lipid content. The partition constants log K-lipid/PDMS between tissues and PDMS were derived from measured concentration in PDMS and the mass-balance model and were very similar for 40 neutral chemicals with octanol-water partition constants 1.4 < log K-ow < 8.7, that is, log K-lipid/PDMS of 1.26 (95% CI, 1.13-1.39) for the adipose tissue, 1.16 (1.00-1.33) for the liver, and 0.58 (0.42-0.73) for the brain. This conversion factor can be applied to interpret chemical analysis and in vitro bioassays after additionally accounting for a small fraction of coextracted lipids of <0.7% of the PDMS weight. PDMS is more widely applicable for passive sampling of mammalian tissues than previously thought, both, in terms of diversity of chemicals and the range of lipid contents of tissues and, therefore, an ideal method for human biomonitoring to be combined with in vitro bioassays.

Solvent extracts of mammalian tissues and blood contain a large amount of co-extracted matrix components, in particular lipids, which can adversely affect instrumental analysis. Clean-up typically degrades non-persistent chemicals. Alternatively, passive sampling with the polymer polydimethylsiloxane (PDMS) has been used for a comprehensive extraction from tissue without altering the mixture composition. Despite a smaller fraction of matrix being co-extracted by PDMS than by solvent extraction, direct analysis of PDMS extracts was only possible with direct sample introduction (DSI) GC-MS/MS, which prevented co-extracted matrix components entering the system. Limits of quantitation (LOQ) ranged from 4 to 20 pg μL−1 ethyl acetate (PDMS extract) for pesticides and persistent organic pollutants (POPs). The group of organophosphorus flame retardants showed higher LOQs up to 107 pg μL−1 due to sorption to active sites at the injection system. Intraday precision ranged between 1 and 10%, while the range of interday precision was between 1 and 18% depending on the analyte. The method was developed using pork liver, brain, and fat as well as blood and was then applied to analyze human post-mortem tissues where polychlorinated biphenyls (PCBs) as well as dichlorodiphenyltrichloroethane (DDT) and DDT metabolites were detected.

The Microtox assay, a bioluminescence inhibition assay with the marine bacterium Aliivibrio fischeri, is one of the most popular bioassays for assessing the cytotoxicity of organic chemicals, mixtures and environmental samples. Most environmental chemicals act as baseline toxicants in this short-term screening assay, which is typically run with only 30 min of exposure duration. Numerous Quantitative Structure-Activity Relationships (QSARs) exist for the Microtox assay for nonpolar and polar narcosis. However, typical water pollutants, which have highly diverse structures covering a wide range of hydrophobicity and speciation from neutral to anionic and cationic, are often outside the applicability domain of these QSARs. To include all types of environmentally relevant organic pollutants we developed a general baseline toxicity QSAR using liposome-water distribution ratios as descriptors. Previous limitations in availability of experimental liposome-water partition constants were overcome by reliable prediction models based on polyparameter linear free energy relationships for neutral chemicals and the COSMOmic model for charged chemicals. With this QSAR and targeted mixture experiments we could demonstrate that ionisable chemicals fall in the applicability domain. Most investigated water pollutants acted as baseline toxicants in this bioassay, with the few outliers identified as uncouplers or reactive toxicants. The main limitation of the Microtox assay is that chemicals with a high melting point and/or high hydrophobicity were outside of the applicability domain because of their low water solubility. We quantitatively derived a solubility cut-off but also demonstrated with mixture experiments that chemicals inactive on their own can contribute to mixture toxicity, which is highly relevant for complex environmental mixtures, where these chemicals may be present at concentrations below the solubility cut-off.

In numerous studies on the toxicity of ionisable organic chemicals, it has been shown that the toxicity was typically higher, when larger fractions of the neutral species were present. This observation was explained in some cases by slower uptake of charged species. In other cases it was suggested that the neutral species has intrinsically higher toxicity than the charged species or is alone responsible for the toxicity. However, even permanently charged and organic chemicals with multiple acid and base functional groups and zwitterions are toxic. We set out to reconcile the divergent views and to compare the various existing models for describing the pH-dependence of toxicity with the goal to derive one model that is valid independent of the type and number of charges on the molecule. To achieve this goal we measured the cytotoxicity of 18 acidic, 15 basic and 9 multiprotic/zwitterionic pharmaceuticals at pH 5.5 to pH 9 with the bioluminescence inhibition test using Aliivibrio fischeri (Microtox assay). This assay is useful for an evaluation of various models to describe pH-dependent toxicity because the majority of chemicals act as baseline toxicants in this 30 min cytotoxicity assay. Therefore baseline toxicity with constant membrane concentrations of the sum of all chemical species of approximately 200 mmol kglip-1 served for the validation of the suitability of the various tested models. We confirmed that most tested pharmaceuticals acted as baseline toxicants in this assay at all examined pH values, when toxicity was modeled with a mixture model of concentration addition between the neutral species and all charged species. An ion trapping model, that assumes that the membrane permeability of charged species is kinetically limited, improved model predictions for some pharmaceuticals and pH values. However, neither unhindered uptake nor no uptake of the charged species were ideal models; the reality lies presumably between the two limiting cases with a slower uptake of the charged species than the neutral species. For practical applications a previously developed QSAR model with the ionisation-corrected liposome-water distribution ratio as the sole physicochemical descriptor proved to be generally applicable for all ionisable organic chemicals including those with multiple charges and zwitterions.