Dioxin-like Activity Research Articles

Detection of high PBDD/Fs levels and dioxin-like activity in toys using a combination of GC-HRMS, rat-based and human-based DR CALUX® reporter gene assays

Brominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) are increasingly reported at significant levels in various matrices, including consumer goods that are manufactured from plastics containing certain brominated flame retardants. PBDD/Fs are known ligands for the aryl hydrocarbon receptor (AhR) but are not yet considered in the hazard assessment of dioxin mixtures. The aim of the present study was to determine if PBDD/Fs levels present in plastic constituents of toys could pose a threat to children’s health. PBDD/Fs, unlike their chlorinated counterparts (PCDD/Fs), have not been officially assigned toxic equivalence factors (TEFs) by the WHO therefore, we determined their relative potency towards AhR activation in both human and rodent cell-based DR CALUX® bioassays. This allowed us to compare GC-HRMS PBDD/F congener levels, converted to total Toxic Equivalents (TEQ) by using the PCDD/F TEFs, to CALUX Bioanalytical Equivalents (BEQ) levels present in contaminated plastic constituents from children’s toys. Finally, an estimate was made of the daily ingestion of TEQs from PBDD/Fs-contaminated plastic toys by child mouthing habits. It is observed that the daily ingestion of PBDD/Fs from contaminated plastic toys may significantly contribute to the total dioxin daily intake of young children.

Validity of using a relative potency factor approach for the risk management of dioxin-like polychlorinated naphthalenes.

Here, we characterized the dioxin-like activities of 42 polychlorinated naphthalenes (PCNs) and 6 technical Halowax formulations by using the DR-CALUX (dioxin-responsive chemically activated luciferase expression) assay with rat hepatoma luciferase-expressing H4IIE cells. Of the 42 PCNs examined, 31 showed dioxin-like activities, for which the mass-based REP-EC5TCDD (potency relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin based on the 5% effective concentration determined from the dose-response curve for 2,3,7,8-TCDD) ranged from 0.00000012 to 0.0051, indicating that some of the PCNs (e.g., 1,2,3,6,7,8-HxCN and 1,2,3,4,6,7-HxCN) had dioxin-like activities that were equal to or higher than the WHO-TEFs and the mass-based REP-EC5TCDD reported for dioxins such as octachlorodibenzo-p-dioxin, octachlorodibenzofuran, 3,3',4,4'-tetrachlorobiphenyl (PCB-77), 3,4,4',5-tetrachlorobiphenyl (PCB-81), and 3,3',4,4',5,5'-hexachlorobiphenyl (PCB-169). For PeCNs to OCN with high dioxin-like activities, REPs determined in previous studies were comparable to the REP values obtained in the present study. The TCDD-EQs (2,3,7,8-TCDD equivalents) obtained experimentally for the Halowax formulations decreased in the order HW1051 (37mg/kg)>HW1014 (30mg/kg)>HW1013 (5.6mg/kg)>HW1099 (2.9mg/kg)>HW1001 (0.60mg/kg)>HW1031 (<0.10mg/kg) and were comparable to the theoretical TCDD-EQs calculated by multiplying the concentration and REP of each PCN. In addition, the theoretical TCDD-EQs for PCNs in emission gases produced by thermal processes were below the Japanese emission standard of 0.1-10ng WHO-toxicity equivalent (TEQ)/m3N, and 3 to 4 orders of magnitude lower than the corresponding WHO-TEQ. Based on a comparison of theoretical and experimental TCDD-EQs, we found that our REP-based approach was suitable for the risk management of industrially produced and unintentionally generated dioxin-like PCNs. This approach will be particularly useful for the risk management of unintentionally generated PCNs in emission gases because the contribution of dioxin-like PCNs to the whole dioxin-like toxicity of emission gases can be elucidated.

Method Development for Effect-Directed Analysis of Endocrine Disrupting Compounds in Human Amniotic Fluid.

The developing fetus represents a highly sensitive period of exposure to endocrine disrupting compounds (EDCs). However, risk assessment of EDCs is hampered by the lack of data on direct in utero exposure. In this study, we developed a robust analytical methodology for the identification of a wide range of known and unknown EDCs in full-term amniotic fluid (AF). First, a method for extraction and fractionation of a broad range of polar and nonpolar EDCs was developed and validated. Maximal recoveries of reference compounds and minimal interference from the matrix were achieved with a combination of solid phase extraction and dispersive liquid/liquid extraction. Bioassay analysis using cell-based reporter gene assays revealed estrogenic, androgenic, and dioxin-like activity in AF extract corresponding to 1.4 nmol EEQ/L, 76.6 pmol DHT-EQ/L, and 10.1 pmol TEQ/L, respectively. Targeted analysis revealed 13 xenobiotics, phytoestrogens, and endogenous hormones in the AF extract that partly contributed to the bioassay activity. Separation of the complex mixture of chemicals in the AF extract with reversed-phase chromatographic fractionation and subsequent bioassay analysis revealed activity in fractions over a wide range of polarity, indicating diverse (unidentified) substances with potential ED activity. The method developed here represents the first methodological step in an effect-directed analysis approach to identify unknown biologically active compounds in the fetal environment.

Xenopus laevis as a Bioindicator of Endocrine Disruptors in the Region of Central Chile.

One of the direct causes of biodiversity loss is environmental pollution resulting from the use of chemicals. Different kinds of chemicals, such as persistent organic pollutants and some heavy metals, can be endocrine disruptors, which act at low doses over a long period of time and have a negative effect on the reproductive and thyroid system in vertebrates worldwide. Research on the effects of endocrine disruptors and the use of bioindicators in neotropical ecosystems where pressure on biodiversity is high is scarce. In Chile, although endocrine disruptors have been detected at different concentrations in the environments of some ecosystems, few studies have been performed on their biological effects in the field. In this work, Xenopus laevis (African clawed frog), an introduced species, is used as a bioindicator for the presence of endocrine disruptors in aquatic systems with different degrees of contamination in a Mediterranean zone in central Chile. For the first time for Chile, alterations are described that can be linked to exposure to endocrine disruptors, such as vitellogenin induction, decreased testosterone in male frogs, and histological changes in gonads. Dioxin-like and oestrogenic activity was detected in sediments at locations where it seem to be related to alterations found in the frogs. In addition, an analysis of land use/cover use revealed that urban soil was the best model to explain the variations in frog health indicators. This study points to the usefulness of an invasive species as a bioindicator for the presence of endocrine-disruptive chemicals.

Benzo[a]pyrene and 2,3-benzofuran induce divergent temporal patterns of AhR-regulated responses in zebrafish embryos (Danio rerio)

Biotests like the fish embryo toxicity test have become increasingly popular in risk assessment and evaluation of chemicals found in the environment. The large range of possible endpoints is a big advantage when researching on the mode of action of a certain substance. Here, we utilized the frequently used model organism zebrafish (Danio rerio) to examine regulative mechanisms in the pathway of the aryl-hydrocarbon receptor (AHR) in early development. We exposed embryos to representatives of two chemical classes known to elicit dioxin-like activity: benzo[a]pyrene for polycyclic aromatic hydrocarbons (PAHs) and 2,3-benzofuran for polar O-substituted heterocycles as a member of heterocyclic compounds in general (N-, S-, O-heterocycles; NSO-hets). We measured gene transcription of the induced P450 cytochromes (cyp1), their formation of protein and biotransformation activity throughout the whole embryonic development until 5 days after fertilization. The results show a very specific time course of transcription depending on the chemical properties (e.g. halogenation, planarity, Kow), the physical decay and the biodegradability of the tested compound. However, although this temporal pattern was not precisely transferable onto the protein level, significant regulation in enzymatic activity over time could be detected. We conclude, that a careful choice of time and end point as well as consideration of the chemical properties of a substance are fairly important when planning, conducting and especially evaluating biotests.

What you extract is what you see: Optimising the preparation of water and wastewater samples for in vitro bioassays

The assessment of water quality is crucial for safeguarding drinking water resources and ecosystem integrity. To this end, sample preparation and extraction is critically important, especially when investigating emerging contaminants and the toxicity of water samples. As extraction methods are rarely optimised for bioassays but rather adopted from chemical analysis, this may result in a misrepresentation of the actual toxicity.In this study, surface water, groundwater, hospital and municipal wastewater were used to characterise the impacts of common sample preparation techniques (acidification, filtration and solid phase extraction (SPE)) on the outcomes of eleven in vitro bioassays. The latter covered endocrine activity (reporter gene assays for estrogen, androgen, aryl-hydrocarbon, retinoic acid, retinoid X, vitamin D, thyroid receptor), mutagenicity (Ames fluctuation test), genotoxicity (umu test) and cytotoxicity. Water samples extracted using different SPE sorbents (Oasis HLB, Supelco ENVI-Carb+, Telos C18/ENV) at acidic and neutral pH were compared for their performance in recovering biological effects.Acidification, commonly used for stabilisation, significantly altered the endocrine activity and toxicity of most (waste)water samples. Sample filtration did not affect the majority of endpoints but in certain cases affected the (anti-)estrogenic and dioxin-like activities. SPE extracts (10.4 × final concentration), including WWTP effluents, induced significant endocrine effects that were not detected in aqueous samples (0.63 × final concentration), such as estrogenic, (anti-)androgenic and dioxin-like activities. When ranking the SPE methods using multivariate Pareto optimisation an extraction with Telos C18/ENV at pH 7 was most effective in recovering toxicity. At the same time, these extracts were highly cytotoxic masking the endpoint under investigation. Compared to that, extraction at pH 2.5 enriched less cytotoxicity.In summary, our study demonstrates that sample preparation and extraction critically affect the outcome of bioassays when assessing the toxicity of water samples. Depending on the water matrix and the bioassay, these methods need to be optimised to accurately assess water quality.

Prenatal exposure to persistent organic pollutants in Northern Tanzania and their distribution between breast milk, maternal blood, placenta and cord blood

Human exposure to persistent organic pollutants (POPs) begins during pregnancy and may cause adverse health effects in the fetus or later in life. The present study aimed to assess prenatal POPs exposure to Tanzanian infants and evaluate the distribution of POPs between breast milk, maternal blood, placenta and cord blood. For assessment of prenatal exposure, 48 maternal blood samples from Mount Meru Regional Referral Hospital (MMRRH), Arusha Tanzania, were analyzed for organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), brominated flame retardants (BFRs), dioxin-like (DL) activity and perfluorinated alkyl substances (PFASs). For evaluation of POPs distribution between maternal/infant compartments, breast milk, placenta and cord blood corresponding to the maternal blood were analyzed for OCPs, PCBs and BFRs. In maternal blood, p,p´- DDE was detected in 100% of the samples ranging between 29 and 1890 ng/g lipid weight (lw). PCB-153 was the only PCB detected in maternal blood, with detection rate of 29% and concentrations up to 116 ng/g lw. BDE-47 was detected in 65% of the maternal blood samples, ranging between <LOD and 83.2 ng/g lw. DL activity was measured using Dioxin Responsive CALUX® bioassay. The DL activity was above LOQ in 92% of the samples, ranging from <LOQ to 114 pg CALUX TEQ/g lw. PFASs was dominated by PFOS and PFOA, however, the concentrations were low (range ∑PFASs 0.18–3.14 ng/mL). p,p´-DDE was detected in 100% of the breast milk, placenta and cord blood samples and the concentrations were strongly correlated (r = 0.89–0.98) between all compartments. Maternal blood (MB) had significantly lower p,p´-DDE concentrations (ng/g lw) than cord blood (CB) and breast milk (BM). The median CB/MB ratio was 1.3 and median MB/BM ratio was 0.8. p,p´-DDE concentrations in breast milk and cord blood did not show significant difference and median CB/BM ratio was 1. In addition, the relative p,p`-DDE transfer from maternal blood to breast milk and to cord blood increased when p,p`-DDE concentrations in maternal blood increased. This study shows that Tanzanian infants are exposed to a wide range of POPs during fetal life, which raise concerns for potential health effects. In addition, this study found that maternal blood concentrations may lead to underestimation of prenatal exposure, while breast milk collected close to delivery may be a more suitable indicator of prenatal exposure.

The Role of Endocrine and Dioxin-Like Activity of Extracts of Petroleum Substances in Developmental Toxicity as Detected in a Panel of CALUX Reporter Gene Assays.

Recent evidence suggests that the interaction of polycyclic aromatic hydrocarbons (PAHs), present in some petroleum substances (PS), with particular nuclear-hormone-receptors and/or the dioxin (aryl hydrocarbon receptor [AhR]) receptor, may play a role in the prenatal developmental toxicity (PDT) induced by these substances. To address this hypothesis, we evaluated the possible endocrine and dioxin-like activity of the dimethylsulfoxide (DMSO)-extracts of 9 PS, varying in PAH content, and 2 gas-to-liquid (GTL) products, containing no PAHs but having similar other properties as PS, using a series of Chemical Activated LUciferase gene eXpression (CALUX) assays. The results show that the extracts of PS tested in this study possess various endocrine and dioxin-like activities and these in vitro potencies are associated with the quantity and type of PAHs they contain. All tested DMSO-extracts of PS show a strong AhR agonist activity and rather weak antiprogesterone, antiandrogen, and estrogenic activities. In the assays that evaluate thyroid-related and antiestrogen activity, only minor effects of specific extracts, particularly those with a substantial amount of 4–5 ring PAHs, ie, sample No. 34, 98, and 99, were observed. None of the GTL extracts interacted with the selected receptors. Of all assays, the AhR agonist activity correlates best (R2 = 0.80) with the in vitro PDT of the substances as quantified previously in the embryonic stem cell test, suggesting an important role of the AhR in mediating this effect. Hierarchic clustering of the combined CALUX data clustered the compounds in line with their chemical characteristics, suggesting a PS class-specific effects signature in the various CALUX assays, depending on the PAH profile. To conclude, our findings indicate a high potential for endocrine and dioxin-like activity of some PS extracts which correlates with their in vitro PDT and is driven by the PAHs present in these substances.

Bioaccumulation and Spatiotemporal Trends of Polyhalogenated Carbazoles in Great Lakes Fish from 2004 to 2016.

Polyhalogenated carbazoles (PHCZs) were recently discovered in Great Lakes sediment and other aquatic systems. However, knowledge about their bioaccumulation and potential risks to fish and wildlife remains very limited. The present study investigated PHCZs in Great Lakes lake trout ( Salvelinus namaycush) and walleye ( Sander vitreus; Lake Erie only) composites collected between 2004 and 2016. Median concentrations of ∑PHCZs by lake ranged from 54.7 to 154 ng/g lipid weight or lw (6.8-28.0 ng/g wet weight). Dominant congeners included 3,6-dichlorocarbazole, 1,3,6-tribromocarbazole, and 1,3,6,8-tetrachlorocarbazole. The highest ∑PHCZs concentrations were found in Lakes Michigan and Ontario fish, followed by Lake Huron, whereas Lakes Erie and Superior fish contained the lowest concentrations. Congener profiles of PHCZs also exhibited spatial variations. After age normalization to minimize fish age influence on bioaccumulation rates, fish ∑PHCZs' concentrations declined significantly over time in all lakes except Lake Erie, with slopes ranging from -10.24% to -3.85% per year. The median toxic equivalent (TEQ) of PHCZs due to their dioxin-like activity was determined to range from 8.7 to 25.7 pg/g lw in Great Lakes fish. This study provides the first insight into the bioaccumulation and spatiotemporal trends of PHCZs in Great Lakes and suggests the need for further research on this group of chemicals.

2,3′,4,4′,5-Pentachlorobiphenyl impairs insulin-induced NO production partly through excessive ROS production in endothelial cells

Polychlorinated biphenyls (PCBs) have been reported to be associated with increased risk to hypertension, atherosclerosis, cardiovascular disease, etc. 2,3′,4,4′,5-Pentachlorobiphenyl, known as PCB-118, is a member of coplanar PCBs which renders their structure similar to polychlorinated dibenzo-p-dioxins (PCDDs) and has dioxin-like activity. In our current study, we investigated the effect of PCB-118 exposure on nitric oxide (NO) production and the underlying mechanisms in vitro. Exposure of PCB-118 impaired insulin-induced NO production and endothelial nitric oxide synthase (eNOS) activity in human umbilical vein endothelial cells (HUVECs) with no significant effect on cell viability. Furthermore, PCB-118 treatment induced oxidative stress. In addition, scavenging of reactive oxygen species (ROS) by 10 μM N-acetyl-l-cysteine (NAC) partly rescued impaired insulin-induced eNOS activities and NO productions induced by PCB-118 in HUVECs. Taken together, these results indicate that PCB-118 mediates lower eNOS activity and impairs insulin-induced NO production partly through excessive ROS production in endothelial cells.

Dioxin-like activity of brominated dioxins as individual compounds or mixtures in in vitro reporter gene assays with rat and mouse hepatoma cell lines

In vitro reporter gene assays detecting dioxin-like compounds have been developed and validated since the middle 1990's, and applied to the determination of dioxin-like activities in various samples for their risk management. Data on characterizing the potency of individual brominated dioxins and their activity in mixture with chlorinated dioxins are still limited on the cell-based assay. This study characterized the dioxin-like activities of the 32 brominated dioxins, such as polybrominated dibenzo-p-dioxins, polybrominated dibenzofurans (PBDFs), coplanar polybrominated biphenyls, mixed halogenated dibenzo-p-dioxins and dibenzofurans (PXDFs), as a sole component or in a mixture by DR-CALUX (dioxin-responsive chemically activated luciferase expression) using the rat hepatoma H4IIE cell line and XDS-CALUX (xenobiotic detection systems–chemically activated luciferase expression) assays using the mouse hepatoma H1L6.1 cell line. The 2,3,7,8-TCDD-relative potencies (REPs) of most of the brominated dioxins were within a factor of 10 of the WHO toxicity equivalency factor (WHO-TEF) for the chlorinated analogues. The REPs of a few PXDFs were an order of magnitude higher than the corresponding WHO-TEFs, indicating their toxicological importance. Results with reconstituted mixtures suggest that the activity of brominated and chlorinated dioxins in both CALUX assays was dose-additive. Thus, obtained results indicated the applicability of the CALUX assays as screening tools of brominated dioxins together with their chlorinated analogues.

Toxic potential of organic constituents of submicron particulate matter (PM1) in an urban road site (Barcelona).

Atmospheric particulate matter (PM) is a recognized risk factor contributing to a number of diseases in human populations and wildlife globally. Organic matter is a major component of PM, but its contribution to overall toxicity of PM has not been thoroughly evaluated yet. In the present work, the biological activity of organic extracts from PM1 (particles with less than 1μm of aerodynamic diameter) collected from an urban road site in the centre of Barcelona (NE Spain) was evaluated using a yeast-based assay (AhR-RYA) and different gene expression markers in zebrafish embryos. Dioxin-like activity of the extracts correlated to primary emissions from local traffic exhausts, reflecting weekday/weekend alternance. Expression levels of cyp1a and of gene markers for key cellular processes and development (ier2, fos) also correlated to vehicle emissions, whereas expression of gene markers related to antioxidant defence and endocrine effects (gstal, hao1, ttr) was strongly reduced in samples with strong contribution from regional air masses with aged secondary organic species or with strong influence of biomass burning emissions. Our data suggest that the toxic potential of PM1 organic chemical constituents strongly depends on the emission sources and on the process of ageing from primary to secondary organic aerosols.

Effects of unintentional PCBs in pigments and chemical products on transcriptional activity via aryl hydrocarbon and nuclear hormone receptors

In recent years, some pigments and chemical products have been reported to contain polychlorinated biphenyl (PCB) congeners as unintentional byproducts, and these have also been detected in residential environments from indoor air and house dust. In this study, using in vitro reporter gene assays, we characterized the agonistic and antagonistic activities of a total of 25 PCB congeners contained in pigments (PCB-1 to -16, -20, -35, -40, -52, -56, -77, -101, -126, and -153) against five nuclear hormone receptors, (estrogen receptor (ER) α/β, glucocorticoid receptor (GR), androgen receptor (AR), thyroid hormone receptor (TR) α1) and aryl hydrocarbon receptor (AhR). In the ERα/β assays, 19 and 13 of the 25 PCBs tested showed ERα/β agonistic and/or antagonistic activities, respectively. Relatively potent agonistic activities against ERα/β were found in PCB congeners possessing chlorides at positions 2 and 3. In the GR and AR assays, five and all of the 25 PCB congeners showed antagonistic activity, respectively. Among the anti-androgenic PCB congeners, the activities were more potent in PCB congeners possessing more than three chlorides including consecutive ortho- and meta- or meta- and para-chlorides. In the AhR assay using a sensitive DR-EcoScreen cell line, five of the 25 PCB congeners showed agonistic activity. We newly found that PCB-1, -35 and -56 can act as AhR agonists. Despite these activities among the PCBs, the effects of PCB-11, mainly detected in pigments and chemical products, against these receptors were found to be weaker than those of other tested PCBs. These results suggest that unintentional PCBs in pigments and chemical products might act as agonists and/or antagonists against ERα/β, AR, GR, and AhR, and some of the PCBs might disrupt endocrine functions via multiple receptors and/or simultaneously induce dioxin-like activity via AhR.

In vivo EROD assays with the zebrafish (Danio rerio) as rapid screening tools for the detection of dioxin-like activity

The present study compares two alternative in vivo approaches for the measurement of ethoxyresorufin-O-deethylase (EROD) activity in zebrafish (Danio rerio) following exposure to acetonic model sediment extracts: (1) the live-imaging EROD assay for the direct detection of EROD induction in individual livers via epifluorescence, and (2) the fish embryo EROD assay in subcellular fractions derived from entire zebrafish embryos after in vivo exposure.For toxicity assessment, each sediment extract was tested with the standard fish embryo test (FET). Upon completion of a functioning liver after 72h, the embryos gave a distinct fluorescent signal in the liver, and a corresponding EROD activity could be detected in the fish embryo EROD assay. The exposure time in the live-imaging EROD assay was reduced to 3h, which resulted in a stronger, less variable and more sensitive EROD response. Overall, the live-imaging and the fish embryo EROD assays showed the same tendencies and gave comparable results, e.g. a concentration-dependent increase in EROD activity at concentrations one order of magnitude below concentrations producing macroscopically visible abnormalities. At higher concentrations, however, a decrease of EROD activity was observed in either test. Both tests ranked the three model sediment extracts in the same order. Results indicate that both test systems complement each other and together provide a rapid and reliable in vivo tool to investigate the presence of dioxin-like substances in environmental samples.

Integrated environmental risk assessment of chemical pollution in a Mediterranean floodplain by combining chemical and biological methods.

The Tablas de Daimiel National Park (TDNP) is a unique floodplain ecosystem in central Spain, serving as permanent resting and breeding areas for many waterbird species. In the last decades, this biodiversity hotspot has been severely endangered by poorly treated wastewater discharges from upstream urban communities arriving through its two major contributors, the Cigüela and Guadiana rivers. In this work, we analysed the potential risk of this constant input of micropollutants (estrogens, dioxin-like compounds and other endocrine disruptors) for the resident wildlife. We sampled 12 locations in TDNP and in the nearby Navaseca Pond during 2013, and performed a series of in-vivo and in-vitro bioassays, including Daphnia magna post-exposure feeding inhibition and recombinant yeast-based assays for dioxin-like and estrogenic activities. These results were then compared with the chemical composition of the samples, analysed by GC-MS/MS and LC-MS/MS, and evaluated according to their toxic potential as toxic equivalents or TEQ. The Navaseca Pond, heavily impacted by wastewater from the town of Daimiel, showed the highest levels of toxic compounds, estrogenic activity, and Daphnia toxicity. Conversely, the less impacted TDNP sites showed low residue levels of contaminants, low estrogenicity and dioxin-like activity and negligible toxicity. The results indicates that the current good chemical status of TDNP is menaced by both the inflow of wastewater treatment plants effluents from Guadiana and Cigüela rivers into TDNP tributaries and, as it occurs in the Navaseca Pond, by direct sewage discharges.

Dioxin-like activities, halogenated flame retardants, organophosphate esters and chlorinated paraffins in dust from Australia, the United Kingdom, Canada, Sweden and China

The concentrations of organic flame retardants (FRs) and dioxin-like activities in dust collected from five countries were investigated. The correlations between the concentrations of the different groups of FRs and dioxin-like activities were examined. Chlorinated paraffins (CPs, C9 to C31) were found in the highest concentration (median ∑CP 700 μg/g, range 280–4750 μg/g), followed by organophosphate esters (median ∑13OPEs 56 μg/g, range 21–110 μg/g), halogenated flame retardants (median ∑17HFRs 3.3 μg/g, range 0.87–14 μg/g) and polybrominated diphenyl ethers (median ∑17PBDEs 2.8 μg/g, range 0.46–11 μg/g). There were no significant differences in concentrations of the FRs among the countries but differences in PBDE and CP congener profiles were found. BDE209 predominated in dust from Australia, the UK, Sweden and China, ranging from 50 to 70% of total PBDEs. The lowest percentage of BDE209 was found in the dust from Canada, representing only 20% of total PBDEs. For CPs in dust from Sweden, the long-chain CPs (especially C18 congeners) predominated, while for other countries, medium-chain CPs (especially C14 congeners) predominated. The dioxin-activities of the dusts ranged from 58 to 590 pg CALUX-TEQ/g, and had a median of 200 pg CALUX-TEQ/g. There were significant positive correlations between concentrations of PBDEs and CPs with dioxin-like activities. The dioxin-like activity may be due to the presence of polychlorinated or polybrominated dioxin/furans (PBDD/DFs) or polychlorinated naphthalenes (PCNs) in the dust. The PBDD/DFs are known impurities and degradation product of the penta-BDE mixture, and PCNs are known impurities of CPs which exhibit dioxin-like activities.

Detection of estrogenic and dioxin-like activities in sewage sludge by means of yeast-based assays

Detection of estrogenic and dioxin-like activities in sewage sludge by means of yeast-based assays

Microscale In Vitro Assays for the Investigation of Neutral Red Retention and Ethoxyresorufin-O-Deethylase of Biofuels and Fossil Fuels.

Only few information on the potential toxic effectiveness of biofuels are available. Due to increasing worldwide demand for energy and fuels during the past decades, biofuels are considered as a promising alternative for fossil fuels in the transport sector. Hence, more information on their hazard potentials are required to understand the toxicological impact of biofuels on the environment. In the German Cluster of Excellence “Tailor-made Fuels from Biomass” design processes for economical, sustainable and environmentally friendly biofuels are investigated. In an unique and interdisciplinary approach, ecotoxicological methods are applied to gain information on potential adverse environmental effects of biofuels at an early phase of their development. In the present study, three potential biofuels, ethyl levulinate, 2-methyltetrahydrofuran and 2-methylfuran were tested. Furthermore, we investigated a fossil gasoline fuel, a fossil diesel fuel and an established biodiesel. Two in vitro bioassays, one for assessing cytotoxicity and one for aryl hydrocarbon receptor agonism, so called dioxin-like activity, as measured by Ethoxyresorufin-O-Deethylase, were applied using the permanent fish liver cell line RTL-W1 (Oncorhynchus mykiss). The special properties of these fuel samples required modifications of the test design. Points that had to be addressed were high substance volatility, material compatibility and low solubility. For testing of gasoline, diesel and biodiesel, water accommodated fractions and a passive dosing approach were tested to address the high hydrophobicity and low solubility of these complex mixtures. Further work has to focus on an improvement of the chemical analyses of the fuel samples to allow a better comparison of any effects of fossil fuels and biofuels.

Detoxification of sewage sludge by natural attenuation and implications for its use as a fertilizer on agricultural soils

Sewage Sludges (SS) from wastewater treatment systems constitute a potential alternative to agricultural fertilizers. However, their use is limited by the presence of toxic substances that may represent significant hazards for the environment and for human health. To test the potential of natural processes to attenuate their putative toxic activities, actual SS samples from domestic sewage were buried in holes in a pollution-free environment for different periods of time, up to one year. Aqueous and organic extracts were obtained after each period of natural attenuation, and their respective toxicity was tested for estrogenic and dioxin-like activity by yeast-based bioassays (ER-RYA and AhR-RYA, respectively) and for general toxicity and teratogenicity in zebrafish embryos. Dioxin-like activity was also tested in zebrafish embryos by monitoring the induction of the marker gene cyp1a. Whereas the results showed essentially no estrogenic activity, both dioxin-like activity and embryotoxicity were observed in the initial samples, decreasing significantly after six months of attenuation. Chemical analysis of toxic SS samples showed the presence of low levels of dioxins and furans, and relatively high levels of m- and p-cresol, at concentrations that only partially justify the observed biological effects. Our data indicates the presence of largely uncharacterized hydrophilic compounds with high biological activity in SS, constituting a potential risk of groundwater pollution upon their disposal into the environment. It also shows that this potential impact may be significantly mitigated by attenuation protocols, as the one presented here.

Relative effect potency estimates of dioxin-like activity for dioxins, furans, and dioxin-like PCBs in adults based on cytochrome P450 1A1 and 1B1 gene expression in blood

BackgroundIn the risk assessment of PCDDs, PCDFs, and dioxin-like (DL) PCBs, regulatory authorities support the use of the toxic equivalency factor (TEF)-scheme derived from a heterogeneous data set of the relative effect potency (REPs) estimates. ObjectivesWe sought to determine REPs for dioxin-like compounds (DLCs) using expression of cytochrome P450 (CYP) 1A1 and 1B1 mRNA in human peripheral blood mononuclear cells representing two different pathways. MethodsWe used a sex and age adjusted regression-based approach comparing the strength of association between each DLC and the cytochrome P450 (CYP) 1A1 and 1B1 mRNA expression in 320 adults residing in an organochlorine-polluted area of eastern Slovakia. ResultsWe calculated REPs based on CYP1A1 expression for 4 PCDDs, 8 PCDFs, and 1 PCB congener, and based on CYP1B1 expression for 5 PCDFs and 11 PCB congeners. REPs from CYP1A1 correlated with REPs previously derived from thyroid volume (ρ=0.85; p<0.001) and serum FT4 (ρ=0.77; p=0.009). The 13 log REPs from CYP1A1 correlated with log WHO-TEFs (r=0.63; p=0.015) and 11 log PCB REPs with PCB consensus toxicity factors (CTFs) for compounds with WHO-TEFs (r=0.80; p=0.003). The complete set of derived 56 log REPs correlated with the log CTFs (r=0.77; p=0.001) and log WHO-TEFs (r=0.81; p<0.001). ConclusionsREPs calculated from thyroid and cytochrome P450 endpoints realistically reflect human exposure scenarios because they are based on human chronic and low-dose exposures. While the CYP 1A1 seems more suitable for toxicity evaluation of PCDD/Fs, the CYP 1B1 is more apt for PCDFs and PCBs and reflects different pathways.