Helena Olsman

Biological and chemical determination of dioxin-like compounds in sediments by means of a sediment triad approach in the catchment area of the river Neckar.

To evaluate the sediment quality of selected sites in the catchment area of the River Neckar, an integrative assessment approach was used to assess the ecological hazard potential of dioxin-like sediment compounds. The approach is based on 7-ethoxyresorufin-O -deethylase (EROD) induction in embryonic chicken liver culture and comprehensive chemical analyses of polycyclic aromatic hydrocarbons (priority PAHs according to the US Environmental Protection Agency). The majority of the sediment extracts exhibited high potencies as EROD-inducers. In one sediment sample, which was influenced by a sewage treatment plant, a very high concentration of 930 ng bioassay 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD) equivalents (bio-TEQs )/g organic carbon could be determined. However, in none of the samples, more than 6% of the EROD-inducing potency could be explained by the PAHs analyzed chemically. Thus, non-analyzed compounds with EROD-inducing potency were present in the extracts. A fractionation of sediment samples according to pH allowed to localize the major part of EROD-inducing compounds in the neutral fractions. However, a significant portion of the EROD induction could also be explained by the acidic fractions. Following the concept of the Sediment Quality Triad according to Chapman, in situ alterations of macrozoobenthos were examined. A comparison of the results predicted by the EROD assay and chemical analyses with alterations in situ , as measured by means of the saprobic index and the ecotoxicological index according to Carmargo, revealed a high ecological relevance of the results of bioassays and chemical analyses for major sites.

Relative differences in aryl hydrocarbon receptor-mediated response for 18 polybrominated and mixed halogenated dibenzo-P-dioxins and -furans in cell lines from four different species†

As a consequence of ubiquitous use of brominated organic chemicals, there is a concern for persistent or increasing environmental levels of polybrominated dibenzo-p-dioxins/furans (PBDD/Fs) and mixed polychlorinated and polybrominated dibenzo-p-dioxins/furans (PXDD/Fs). Hence, there is a need to broaden the toxicological and environmental knowledge about these compounds, as a basis for risk assessment. In the study presented here, the relative potencies (REPs) for 18 PBDD/F and PXDD/ F congeners were determined in four dioxin-specific bioassays from different species: dioxin receptor chemically activated luciferase expression assay (DR-CALUX, rat hepatoma cells), TV101L (human hepatoma cells), and GPC.2D (guinea pig adenoma cells), as well as ethoxyresorufin-O-deethylase induction in the fish cell line RTL-W1 (rainbow trout liver cells). The bioassay specific REP factors presented here enable the assessment of the contribution from PBDD/Fs and PXDD/Fs to total 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TEQs: toxic equivalents), using bioassay analysis. The PBDD/Fs were found to be equally potent as their chlorinated analogues in the three mammalian assays, whereas the PXDD/Fs showed relatively higher potencies. Of special concern were the 2,3,7,8-substituted penta- and tetrahalogenated congeners, for which mean REPs were > or =1. The 2-B-1,3,7,8-CDD (2-bromo-1,3,7,8-tetrachlorodibenzo-p-dioxin) was up to three times more potent than TCDD in individual experiments (on weight basis). The RTL-W1 was less sensitive to the tested compounds with overall 10-fold lower REPs than the mammalian cell lines. Although the REP factors exhibited species-specific differences, overall resembling rank orders of dioxin-like potency were obtained.

Fate of ah receptor agonists during biological treatment of an industrial sludge containing explosives and pharmaceutical residues

Goal, Scope and BackgroundSweden is meeting prohibition for deposition of organic waste from 2005. Since 1 million tonnes of sludge is produced every year in Sweden and the capacity for incineration does not fill the demands, other methods of sludge management have to be introduced to a higher degree. Two biological treatment alternatives are anaerobic digestion and composting. Different oxygen concentrations result in different mi-crobial degradation pathways and, consequently, in a different quality of the digestion or composting residue. It is therefore necessary to study sludge treatment during different oxygen regimes in order to follow both degradation of compounds and change in toxicity. In this study, an industrial sludge containing explosives and pharmaceutical residues was treated with anaerobic digestion or composting, and the change in toxicity was studied. Nitroaromatic compounds, which are the main ingredients of both pharmaceutical and explosives, are well known to cause cytotoxicity and genotoxicity. However, little data are available concerning sludge with nitroaromatics and any associated dioxin-like activity. Therefore, we studied the sludge before and after the treatments in order to detect any changes in levels of Ah receptor (AhR) agonists using two bioassays for dioxin-like compounds.MethodsAn industrial sludge was treated with anaerobic digestion or composting in small reactors in a semi-continuous manner. The same volume as the feeding volume was taken out daily and stored at -20° C. Sample preparation for the bioassays was done by extraction using organic solvents, followed by clean up with silica gel or sulphuric acid, yielding two fractions. The fractions were dissolved in DMSO and tested in the bioassays. The dioxin-like activity was measured using the DR-CALUX assay with transfected H4IIE rat hepatoma pGudluc cells and an EROD induction assay with RTL-W1 rainbow trout liver cells.Results and DiscussionThe bioassays showed that the sludge contained AhR agonists at levels of TCDD equivalents (TEQs) higher than other sludge types in Sweden. In addition, the TEQ values for the acid resistant fractions increased considerably after anaerobic digestion, resulting in an apparent formation of acid resistant TEQs in the anaerobic reactors. Similar results have been reported from studies of fermented household waste. There was a large difference in effects between the two bioassays, with higher TEQ levels in the RTL-W1 EROD assay than in the DR-CALUX assay. This is possibly due to a more rapid metabolism in rat hepatocytes than in trout hepatocytes or to differences in sensitivities for the AhR agonists in the sludge. It was also demonstrated by GC/FID analysis that the sludge contained high concentrations of nitroaromatics. It is suggested that nitroaromatic metabolites, such as aromatic amines and nitroanilines, are possible candidates for the observed bioassay effects. It was also found that the AhR agonists in the sludge samples were volatile.ConclusionsThe sludge contained fairly high concentrations of volatile AhR agonists. The increase of acid resistant AhR agonist after anaerobic digestion warrants further investigations of the chemical and toxic properties of these compounds and of the mechanisms behind this observation.Recommendation and OutlookThis study has pointed out the benefits of using different types of mechanism-specific bioassays when evaluating the change in toxicity by sludge treatment, in which measurement of dioxin-like activity can be a valuable tool. In order to study the recalcitrant properties of the compounds in the sludge using the DR-CALUX assay, the exposure time can be varied between 6 and 24 hours. The properties of the acid-resistant AhR agonists formed in the anaerobic treatment have to be investigated in order to choose the most appropriate method for sludge management.

Fractionation and Determination of Ah Receptor (AhR) Agonists in Organic Waste After Anaerobic Biodegradation and in Batch Experiments with PCB and decaBDE (8 pp)

Goals, Scope and BackgroundAnaerobic digestion of organic household waste can lead to an increase in dioxin-like content, as determined by dioxin-specific bioassays. This may be a result of bioactivation of Ah receptor (AhR) agonists into more potent congeners. Work towards identifying the contributing compound groups is important in order to understand the mechanisms and to assess the relevance behind this increase in dioxin-like toxicity, since the residue can be used as a soil fertilising agent. The aim with the present work was to identify compound groups with AhR agonistic properties that caused the previously reported increase in dioxin-like activity after anaerobic biodegradationMethodsFirstly, chemical fractionation combined with dioxin bioassay testing was used to find bioactive classes of compounds. Secondly, batch digestion experiments with an externally added polychlorinated biphenyl (PCB) mixture (Clophen A50) and with decabrominated diphenyl ether (decaBDE), respectively, were studied as a possible process for transformation of precursors into more potent, dioxin-like compounds. Mesophilic (37ºC) and thermophilic (55ºC) anaerobic digestion were studied. Two different dioxin-specific bioassays were used to analyse AhR agonists in the biodegraded material, the CELCAD and the DR-CALUX. Results and DiscussionAhR agonist activity was detected in both di- and polyaromatic fractions of digestate extracts, which indicated that a diverse mixture of compounds contributed to the bioassay responses. No quantifiable activities were induced by the monoaromatic fractions. Further fractionation based on planarity revealed higher concentrations of AhR agonists than what was detected after the first fractionation, probably due to non-additive biological interactions of compounds in the extract that were removed in the second fractionation. These results showed significant activity in the non-planar diaromatic fractions and in the co-planar fractions of both diaromates and polyaromates. In the batch experiment with externally added PCB, an increase in dioxin-like activity was seen after 21 days of digestion at mesophilic conditions. After completed digestion, the content of AhR agonists was equal to the start concentration. PCB analysis with GC-MS indicated that dehalogenation of PCBs occurred in the digestors. The batch experiment with decaBDE showed no significant changes in TEQ-concentrations over time.ConclusionsThe results show that the previously reported increase of AhR agonists during mesophilic anaerobic digestion is probably due to an accumulation of several different groups of AhR agonists, both diaromatic and polyaromatic, and both co-planar and non-planar. Batch experiments with externally added PCBs and decaBDE, respectively, did not result in any accumulation of AhR agonist activity after completed digestion, even though chemical analysis indicate a dechlorination of PCBs. Complex, unfractionated extracts were difficult to test using the bioassay approach. Removal of AhR antagonists or otherwise interacting compounds during fractionation may yield bio-TEQ values that are much higher than in the original extract.Recommendations and PerspectiveOur results indicate that the environmental risk that AhR agonists may pose concerning large-scale anaerobic digestion of organic household waste probably depends on the efficiency of the digester and the sludge residence time. In order to obtain reliable results with the bioassays, an extensive cleanup and fractionation procedure is necessary. Without clean up and fractionation, there is a risk for false negatives and misleading conclusions. DR-CALUX and CELCAD were both suitable for these kinds of studies, provided that suitable fractionation methods are used.