Effect‐directed analysis of mutagens and ethoxyresorufin‐O‐deethylase inducers in aquatic sediments

Abstract

Sediment extracts from a creek in the Neckar river basin (Germany), which received the discharge of treated hospital wastewater, were found to exhibit strong aromatic hydrocarbon (Ah) receptor-mediated effects in a rainbow trout liver cell line (RTL-WI) as well as high mutagenicity in the Salmonella/microsome assay after fractionation. The crude extract did not exhibit a clear mutagenic response. Apparently, cleanup or fractionation before mutagenicity testing is necessary to minimize the risk of false-negative results. Effect-directed fractionation and analysis were applied to characterize and identify the toxicants that cause these effects. Major ethoxyresorufin-O-deethylase induction potency and mutagenicity were detected in different polyaromatic fractions, indicating different sets of toxicants that induce metabolic activation and mutagenicity. Dioxin-like halogenated aromatic hydrocarbons, including polychlorinated biphenyls, naphthalenes, dibenzo-p-dioxins and furans, and priority polycyclic aromatic hydrocarbons, contributed to Ah receptor-mediated activity only to a minor extent. Benzo[a]pyrene, benzo[a]fluoranthene, and perylene could be confirmed as important contributors to mutagenicity. The nonpriority pollutants 11H-indeno[2,1,7-cde]pyrene, a methylbenzo[e]pyrene, and a methylperylene were tentatively identified as major components, representing 82% of the peak area of a highly mutagenic fraction of the sediment extract. This suggests that hazard and risk assessment of complex environmental mixtures should make increasing attempts to identify and consider hazardous key pollutants rather than focusing on a priori-selected key pollutants alone.

Effect-directed analysis (EDA) in aquatic ecotoxicology: state of the art and future challenges

“…For example, bioassays such as the rat hepatoma cell-line H4IIE test or EROD induction in RTL-W1 cells are widely used to assess the exposure to dioxin-like chemicals that bind to the aryl hydrocarbon receptor (AhR) (Brack et al 2005b(Brack et al , 2008aEngwall et al 1999;Hollert et al 2002;Kammann et al 2005b;Olsman et al 2007b). However, often the chemically detected dioxin-like equivalents (2,3,7,8-TCDD equivalents = TEQs) cannot explain the biological activity measured with the bioassay (bio-TEQ) (e.g., Brack et al 2005bBrack et al , 2008a.…”

“…For example, bioassays such as the rat hepatoma cell-line H4IIE test or EROD induction in RTL-W1 cells are widely used to assess the exposure to dioxin-like chemicals that bind to the aryl hydrocarbon receptor (AhR) (Brack et al 2005b(Brack et al , 2008aEngwall et al 1999;Hollert et al 2002;Kammann et al 2005b;Olsman et al 2007b). However, often the chemically detected dioxin-like equivalents (2,3,7,8-TCDD equivalents = TEQs) cannot explain the biological activity measured with the bioassay (bio-TEQ) (e.g., Brack et al 2005bBrack et al , 2008a. Similarly, attempts to correlate genotoxicity of complex environmental samples measured by tests such as the Ames, the micronucleus, or the comet assay with PAH concentrations often fail (Broman et al 1994;Gustavsson et al 2007;Kosmehl et al 2004;Reifferscheid et al 2008;Vahl et al 1997), suggesting a contribution of other nonregulated mutagens to the observed biological effect.…”

“…In a number of studies assessing sediments by EDA, it could be demonstrated, for example, that in addition to priority pollutants several non-regulated PAHs, including perylene and benzo [a]fluoranthene, 11H-indeno[2,1,7-cde]pyrene, a methylbenzo[e]pyrene, and a methyl perylene were Fig. 1 Principle of effect-directed identification of toxicants (redrawn from Brack 2003) present at significant concentrations in the analyzed samples (Brack et al 2005b). Furthermore, Fernandéz et al (1992) showed that more polar compounds, including several polycyclic quinones and nitroquinones, as well as nitro-PAHs, contributed significantly to the mutagenic effects of marine sediments.…”

“…Finally, as shown in several studies, assessing crude extracts by means of cell-based bioassays may be limited due to general (cyto)toxicity that can mask mechanismspecific effects (Brack et al 2005b;Hollert et al 2002;Kammann et al 2005b). In these situations, the issue of overlying toxicity in the crude extract can be overcome by fractionation approaches resulting in a separation of compounds with different modes of actions.…”

Endocrine disruptor screening: regulatory perspectives and needs

“…A number of studies have been conducted to characterize the endocrine potential of municipal effluents [24,25], surface water [26][27][28] and sediments [26,27,29,30], and there has been increasing awareness of the need for testing approaches targeting EDCs to be included into current environmental assessments and monitoring [31][32][33][34][35][36] …”