Aryl hydrocarbon receptor (AhR) activity in water: Looking beyond dioxin-like toxicity

In vitro bioassays indicative of activation of the aryl hydrocarbon receptor (AhR) are commonly applied to water, sediment and biota. Many equate AhR activity in water to dioxin-like toxicity, but hydrophobic dioxin-like chemicals will not be present in water extracts. Aryl hydrocarbon receptor activity has been observed in wastewater, surface water, drinking water and recycled water, with a diverse range of environmental contaminants, including pesticides, pharmaceuticals and industrial chemicals, contributing to the observed AhR activity. Therefore, this critical perspective advocates for a change in mindset from dioxin-like toxicity to sensitive indicator of chemicals that activate xenobiotic metabolism when AhR assays are applied to water samples. Firstly, we proposed a new environmentally relevant reference compound to express AhR activity in water to replace widely used hydrophobic reference compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a)pyrene. The fluorescent dye 7-diethylamino-4-methylcoumarin (DEAMC) was identified as a suitable water-soluble reference compound after considering occurrence in wastewater, common mixture effect drivers and specificity. Iceberg modeling, where the predicted mixture effect is compared to the measured effect, demonstrated that very different chemicals activate AhR in water and sediment, supporting the need for a water-soluble reference compound. Effect-based trigger values (EBT) differentiate between acceptable and poor chemical water quality based on the bioassay response, with EBTs for AhR assays primarily derived for surface water to protect ecological health. However, the detection of AhR activity in drinking water and recycled water supports the need for robust EBTs for drinking water to protect human health. We derived a new human EBT (humanEBT) using established read-across methods and applying a mixture factor based on the fraction of AhR activity explained in water. Overall, this perspective clearly demonstrates why we need to look beyond dioxin-like toxicity when applying activation of AhR assays to water samples.