Peter A. Behnisch

Brominated dioxin-like compounds: in vitro assessment in comparison to classical dioxin-like compounds and other polyaromatic compounds.

Recently, several countries agreed to adopt the Stockholm convention on persistent organic pollutants (POPs). One future obligation will be to add other POPs as new evidence becomes available. In vitro cell-based bioassays offer a rapid, sensitive, and relatively inexpensive solution to screen possible POP candidates. In the present study, we investigated the aryl hydrocarbon (Ah)-receptor activity of several dioxin-like POPs by using the Micro-EROD (Ethoxy-Resorufin-O-Deethylase) and DR-CALUX (Dioxin-Responsive-Chemical Activated Luciferase gene eXpression) bioassays, which are two state-of-the-art methods. The Micro-EROD system used in our study utilizes a wild-type rat liver cell line (rat liver H4IIEC3/T cells), while the DR-CALUX bioassay consists of a genetically modified rat hepatoma H4IIE cell line that incorporates the firefly luciferase gene coupled to dioxin-responsive elements (DREs) as a reporter gene. In the case of the DR-CALUX bioassay, we used an exposure time of 24 h, whereas we used a 72-h exposure time in the Micro-EROD bioassay. The aim of this study was to compare conventional dioxin-like POPs (such as polychlorinated dibenzodioxins and -furans, PCDD/Fs and coplanar polychlorinated biphenyls, PCBs) with several other classes of possible candidates to be added to the current toxicity equivalent factor (TEF) model in the future. Therefore, this study compares in vitro CYP1A1 (Micro-EROD bioassay) and firefly luciferase induction (DR-CALUX bioassay) in several mixed polyhalogenated dibenzodioxins and -furans (PXDD/Fs; X=Br, Cl, or F), alkyl-substituted polyhalogenated dibenzodioxins and -furans (PMCDD/Fs; M=methyl), polyhalogenated biphenyls (PXBs, X=Br, Cl ), polybrominated diphenyl ethers (PBDEs), pentabromophenols (PBPs), and tetrabromobisphenol-A (TBBP-A). We also evaluate congener-specific relative potencies (REPs) and efficacies (% of TCDD(max)) and discuss the dose-response curves of these compounds, as well as the dioxin-like potency of several other Ah-receptor agonists, such as those of the polyaromatic hydrocarbons (PAHs) and polychlorinated naphthalenes (PCNs). The highest REP values were found for several PXDD/F congeners, followed by some coplanar PXBs, trichlorinated PCDD/Fs, PAHs, PBDE-126, 1-6-HxCN, and some brominated flame retardants (TBBP-A). These in vitro investigations indicate that further research is necessary to evaluate more Ah-receptor agonists for dioxin-like potency.

Estrogenic and dioxin-like potency in each step of a controlled landfill leachate treatment plant in Japan.

The estrogenic activity (by E-screen bioassay), the concentrations of PCBs, PCDDs/PCDFs (and their resulting toxicity equivalents, TEQ) and several endocrine disrupting chemicals (EDCs: e.g., bisphenol A, nonylphenol, Butyl benzylpthalate (BBP), di-n-butylphthalate (DBP), 17alpha-ethynyl-estradiol or 4-octylphenol) have been analyzed from leachates of each step (before treatment, after biodegradation/sedimentation and after charcoal treatment) of a controlled landfill leachate treatment plant. The comparison of the effluent of the examined landfill leachate treatment plant with water from a nearby river in this study indicated no additional dioxin-like (e.g., TEQ: 0.027 compared to 1.01 pg TEQ/l; PCBs: 1.2 compared to 3.9 ng/l) or estrogenic impact (2.8 compared to 3.5 ng estradiol equivalents EE/l; analyzed by E-screen bioassay) from the leachate treatment plant into the surrounding water environment. The impact of dioxin-like compounds from uncleaned leachates into the final cleaned effluents could be sufficiently reduced by the leachate treatment plant for PCDDs (75%), PCDFs (62%), dioxin-like PCBs (97%), and the sum of TEQ (78%). The leachate treatment plant also achieved a reduction of the estrogenic activity as determined by E-screen (from 4.8 to 2.8 ng EE/l = 42%), by GC/MS for bisphenol A (>96% and nonylphenol (>98%) or by ELISA for estradiol (>80%). Additionally, for the validation of the E-screen, five known endocrine disrupting chemicals (EDCs: bisphenol A, BBP, DBP, 17 alpha-ethynyl-estradiol, 4-octylphenol) were analyzed. The EC50 values and estradiol equivalents factors (EEFs) for the five EDCs determined in this study were comparable to previously published data. The combined biological and chemical trace analysis data have provided valuable information on the relative contribution of natural, synthetic, and non-steroidal anthropogenic chemicals to the estrogenic and dioxin-like activity in leachates from a wastewater treatment plant, and water from a nearby river.

Melting and incineration plants of municipal waste - Chemical and biochemical diagnosis of thermal processing samples (emission, residues)

Control of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) in emissions and thermal residues from incinerators has been a cause of public concern for more than one decade. Recently, several studies showed that other persistent organic pollutants (POPs) such as coplanar polychlorinated biphenyls (co-PCBs) also have dioxin-like activity and are released from incinerators. Therefore, the present study was aimed at making a risk assessment about dioxin-like activity in extracts of thermal waste residues (e.g. combustion gas; fly ash, slag) from incineration and melting processes in Germany and Japan. For this purpose, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs), coplanar polychlorinated biphenyls (co-PCBs), polychlorinated naphthalenes (PCNs) and polyaromatic hydrocarbons (PAHs) were analyzed by chemical analysis. Additionally, 2,3,7,8-TCDD equivalents (EROD-TEQs) were determined byin vitro Micro-EROD bioassay using rat H4IIE hepatoma cells. EROD-TEQs could be correlated to I-TEQ values (from PCDD/Fs/co-PCBs) analyzed by chemical analysis resulting in a maximal sixfold higher estimate. Our study indicates minor influences of co-PCBs, PAHs and PCNs to the sum of dioxin-like toxicity in the extracts of thermal waste residues as determined here. Furthermore, we showed that the levels of dioxins and co-PCBs contained in slag from melting processes and bottom ashes from incineration processes were lower by 1–2 orders of magnitude than that in fly ash.

In Vitro Bioassays for Dioxin and Dixin-like Compounds.

ハロゲン化芳香族炭化水素化合物 (PHAHs: Polyhalogenated aromatic hydrocarbons) による様々な毒性および生物学的反応の多くはアリル炭化水素受容体 (AhR: aryl hydrocarbon recepter) を介することが知られている。EROD (ethoxyresorufin-O-deethylase) およびCALUX (chemical-activated luciferase gene expression) バイオアッセイはPHAHsによるAhRを介した遺伝子発現反応を利用した感度の高い生物学的測定法であり, 未知物質も含め, 環境・廃棄物試料のような混合物中に含まれるダイオキシン様活性物質を包括的に検出することができる。ダイオキシン様物質を検出するためのAh結合バイオアッセイについて, 培養細胞によるEROD法およびCALUX法を中心に, 背景, 歴史, イムノアッセイとの比較, ハロゲン化芳香族炭化水素化合物, および環境・廃棄物試料への分析応用例等について概説した。ERODまたはCALUXバイオアッセイをGC/MSによる個別定量と併用すること, あるいは化学分析前のプレスクリーニングに用いることにより, 環境・廃棄物試料などに含有されるダイオキシン様物質を包括的に理解するうえで有用と考えられる。