To Örebro University

Risk assessment of chemicals is still primarily focusing on single compound evaluation, even if environmental contamination consists of a mixture of pollutants. The concentration addition (CA) and independent action (IA) models have been developed to predict mixture toxicity. Both models assume no interaction between the components, resulting in an additive mixture effect. In the present study, the embryo toxicity test (OECD TG no. 236) with zebrafish embryos (Danio rerio) was performed to investigate whether the toxicity caused by binary, ternary, and quaternary mixtures of organic (Benzo[a]pyrene, perfluorooctanesulfonate, and 3,3´,4,4´,5-pentachlorobiphenyl 126) and inorganic (arsenate) pollutants can be predicted by CA and IA. The acute toxicity and sub-lethal alterations such as lack of blood circulation were investigated. The models estimated the mixture toxicity well and most of the mixtures were additive. However, the binary mixture of PFOS and PCB126 caused a synergistic effect, with almost a ten-fold difference between the observed and predicted LC₅₀-value. For most of the mixtures, the CA model was better in predicting the mixture toxicity than the IA model, which was not expected due to the chemicals' different modes of action. In addition, some of the mixtures caused sub-lethal effects not observed in the single compound toxicity tests. The mixture of PFOS and BaP caused a division of the yolk and imbalance was caused by the combination of PFOS and As and the ternary mixture of PFOS, As, and BaP. Interestingly, PFOS was part of all three mixtures causing the mixture specific sub-lethal effects. In conclusion, the present study shows that CA and IA are mostly resulting in good estimations of the risks that mixtures with few components are posing. However, for a more reliable assessment and a better understanding of mixture toxicity, further investigations are required to study the underlying mechanisms.

Polycyclic aromatic compounds (PACs) are prevalent in the environment, typically found in complex mixtures and high concentrations. Our understanding of the effects of PACs, excluding the 16 priority polycyclic aromatic hydrocarbons (16 PAHs), remains limited. Zebrafish embryos and in vitro bioassays were utilized to investigate the embryotoxic, behavioral, and molecular effects of a soil sample from a former gasworks site in Sweden. Additionally, targeted chemical analysis was conducted to analyze 87 PACs in the soil, fish, water, and plate material. CALUX® assays were used to assess the activation of aryl hydrocarbon and estrogen receptors, as well as the inhibition of the androgen receptor. Larval behavior was measured by analyzing activity during light and darkness and in response to mechanical stimulation. Furthermore, qPCR analyses were performed on a subset of 36 genes associated with specific adverse outcomes, and the total lipid content in the larvae was measured. Exposure to the sample resulted in embryotoxic effects (LC50 = 0.480 mg dry matter soil/mL water). The mixture also induced hyperactivity in darkness and hypoactivity in light and in response to the mechanical stimulus. qPCR analysis revealed differential regulation of 15 genes, including downregulation of opn1sw1 (eye pigmentation) and upregulation of fpgs (heart failure). The sample caused significant responses in three bioassays (ERα-, DR-, and PAH-CALUX), and the exposed larvae exhibited elevated lipid levels. Chemical analysis identified benzo[a]pyrene as the predominant compound in the soil and approximately half of the total PAC concentration was attributed to the 16 PAHs. This study highlights the value of combining in vitro and in vivo methods with chemical analysis to assess toxic mechanisms at specific targets and to elucidate the possible interactions between various pathways in an organism. It also enhances our understanding of the risks associated with environmental mixtures of PACs and their distribution during toxicity testing.