Demonstrating the reliability of in vivo metabolomics based chemical grouping: towards best practice.

While grouping/read-across is widely used to fill data gaps, chemical registration dossiers are often rejected due to weak category justifications based on structural similarity only. Metabolomics provides a route to robust chemical categories via evidence of shared molecular effects across source and target substances. To gain international acceptance, this approach must demonstrate high reliability, and best-practice guidance is required. The MetAbolomics ring Trial for CHemical groupING (MATCHING), comprising six industrial, government and academic ring-trial partners, evaluated inter-laboratory reproducibility and worked towards best-practice. An independent team selected eight substances (WY-14643, 4-chloro-3-nitroaniline, 17α-methyl-testosterone, trenbolone, aniline, dichlorprop-p, 2-chloroaniline, fenofibrate); ring-trial partners were blinded to their identities and modes-of-action. Plasma samples were derived from 28-day rat tests (two doses per substance), aliquoted, and distributed to partners. Each partner applied their preferred liquid chromatography-mass spectrometry (LC-MS) metabolomics workflows to acquire, process, quality assess, statistically analyze and report their grouping results to the European Chemicals Agency, to ensure the blinding conditions of the ring trial. Five of six partners, whose metabolomics datasets passed quality control, correctly identified the grouping of eight test substances into three categories, for both male and female rats. Strikingly, this was achieved even though a range of metabolomics approaches were used. Through assessing intrastudy quality-control samples, the sixth partner observed high technical variation and was unable to group the substances. By comparing workflows, we conclude that some heterogeneity in metabolomics methods is not detrimental to consistent grouping, and that assessing data quality prior to grouping is essential. We recommend development of international guidance for quality-control acceptance criteria. This study demonstrates the reliability of metabolomics for chemical grouping and works towards best-practice.

Assessment of oil refinery wastewater and effluent integrating bioassays, mechanistic modelling and bioavailability evaluation.

Water is used in petroleum oil refineries in significant volumes for cooling, steam generation and processing of raw materials. Effective water management is required at refineries to ensure their efficient and responsible operation with respect to the water environment. However, ascertaining the potential environmental risks associated with discharge of refinery effluents to receiving waters is challenging because of their compositional complexity. Recent European research and regulatory initiatives propose a more holistic approach including biological effect methods to assess complex effluents and surface water quality. The study presented here investigated potential effects of effluent composition, particularly hydrocarbons, on aquatic toxicity and was a component of a larger study assessing contaminant removal during refinery wastewater treatment (Hjort et al 2021). The evaluation of effects utilised a novel combination of mechanistic toxicity modelling based on the exposure composition, measured bioavailable hydrocarbons using biomimetic solid phase microextraction (BE-SPME), and bioassays. The results indicate that in the refinery effluent assessments measured bioavailable hydrocarbons using BE-SPME was correlated with the responses in standard bioassays. It confirms that bioassays are providing relevant data and that BE-SPME measurement, combined with knowledge of other known non-hydrocarbon toxic constituents, provide key tools for toxicity identification. Overall, the results indicate that oil refinery effluents treated in accordance to the EU Industrial Emissions Directive requirements have low to negligible toxicity to aquatic organisms and their receiving environments. Low-cost, animal-free BE-SPME represents a compelling tool for rapid effluent characterization.

Conventional and high resolution chemical characterization to assess refinery effluent treatment performance.

Refinery effluents represent an emission source of hydrocarbons (HCs) and other constituents to the environment. Thus, characterisation of effluent quality in terms of concentrations of key parameters relative to permitted standards is important and for total petroleum hydrocarbons (TPH), the specific composition of the HC mixture can affect its toxicity to aquatic organisms. Therefore, this study was designed to analyse TPH, benzene, toluene, ethyl benzene, xylenes (BTEX), polycyclic aromatic hydrocarbons (PAHs), (bio) chemical oxygen demand, total nitrogen, total suspended solids and selected metals before, and after, treatment steps to demonstrate removal efficiencies across 13 refineries with variable wastewater treatment systems. Final discharge concentrations of the measured parameters were by 97% within the so called Best Available Technique Associated Emission Levels (BAT-AELs). Further, TPH composition was characterised using high-resolution two-dimensional gas chromatography (GCxGC) analysis to understand the mass distribution by carbon number and specific chemical class. Measurements were compared to SimpleTreat model predictions for validation. SimpleTreat successfully predicted the shape of the effluent composition since it is essentially a removal constant applied to the influent composition. The predictions were of similar magnitude as, or were greater than, the effluent concentrations since SimpleTreat is based on typical performance and is intended to be conservative. This was especially true for aromatic constituents. Reduction in potential HC exposures also coincided with a decrease in predicted toxicity using a mechanistic oil toxicity model, PETROTOX. Overall, the results indicate that EU petroleum refineries are likely to achieve a high performance level regarding effluent treatment.

The underlying challenges that arise when analysing short-chain chlorinated paraffins in environmental matrices.

As short-chain chlorinated paraffins (SCCPs) are listed on several monitoring programs, validated methods are essential. However, their complexity and the lack of commercially available certified reference materials (RMs) hinder a proper validation of methods. Instead, one method is usually 'validated' by evaluating performances and results of spiked materials with that of one other method, which could easily lead to unreliable results. This study evaluated four analytical methods with different principles (i.e. comprehensive two dimensional GC coupled to a micro electron capture detector, developed for this study, chloride enhanced atmospheric pressure chemical ionization triple quadrupole time of flight MS (APCI-QToF-HRMS), GC coupled to an electron capture negative ion low resolution MS (GC-ECNI-LRMS) and carbon skeleton GC-MS), investigated the comparability in SCCP determination in spiked and naturally contaminated samples and determined SCCP amounts in candidate RMs for possible certification. The results cast doubt on the use of the most commonly applied method (i.e. GC-ECNI-LRMS), as well as using spiked materials for method validation. The APCI-QToF-HRMS method was found most promising as it achieves the required MS resolution (>21,000), is relatively fast and can detect also other CPs. The suitable identified SCCP levels in the candidate RMs and the agreement in results between the methods bring the first certification of a RM for SCCPs within reach.

Propelling plastics into the circular economy - weeding out the toxics first.

The Stockholm Convention bans toxic chemicals on its persistent organic pollutants (POPs) list in order to promote cleaner production and prevent POPs accumulation in the global environment. The original 'dirty dozen' set of POPs has been expanded to include some of the brominated diphenyl ether flame retardants (POP-BDEs). In addition to cleaner production, there is an urgent need for increased resource efficiency to address the finite amount of raw materials on Earth. Recycling plastic enhances resource efficiency and is part of the circular economy approach, but how clean are the materials we are recycling? With the help of a new screening method and detailed analyses, we set out to investigate where these largely obsolete BDEs were showing up in Dutch automotive and electronics waste streams, calculate mass flows and determine to what extent they are entering the new product chains. Our study revealed that banned BDEs and other toxic flame retardants are found at high concentrations in certain plastic materials destined for recycling markets. They were also found in a variety of new consumer products, including children's toys. A mass flow analysis showed that 22% of all the POP-BDE in waste electrical and electronic equipment (WEEE) is expected to end up in recycled plastics because these toxic, bioaccumulative and persistent substances are currently not effectively separated out of plastic waste streams. In the automotive sector, this is 14%, while an additional 19% is expected to end up in second-hand parts (reuse). These results raise the issue of delicate trade-offs between consumer safety/cleaner production and resource efficiency. As petroleum intensive materials, plastic products ought to be repaired, reused, remanufactured and recycled, making good use of the 'inner circles' of the circular economy. Keeping hazardous substances - whether they are well known POPs or emerging contaminants - out of products and plastic waste streams could make these cycles work better for businesses, people and nature.

Predicting the bioaccumulation of polyaromatic hydrocarbons and polychlorinated biphenyls in benthic animals in sediments.

There were two main objectives in this study. The first was to compare the accuracy of different prediction methods for the chemical concentrations of polyaromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in the organism, based on the measured chemical concentrations existing in sediment dry matter or pore water. The predicted tissue concentrations were compared to the measured ones after 28-day laboratory test using oligochaeta worms (Lumbriculus variegatus). The second objective was to compare the bioaccumulation of PAHs and PCBs in the laboratory test with the in situ bioaccumulation of these compounds. Using the traditional organic carbon-water partitioning model, tissue concentrations were greatly overestimated, based on the concentrations in the sediment dry matter. Use of an additional correction factor for black carbon with a two-carbon model, significantly improved the bioaccumulation predictions, thus confirming that black carbon was important in binding the chemicals and reducing their accumulation. The predicted PAH tissue concentrations were, however, high compared to the observed values. The chemical concentrations were most accurately predicted from their freely dissolved pore water concentrations, determined using equilibrium passive sampling. The patterns of PCB and PAH accumulation in sediments for laboratory-exposed L. variegatus were similar to those in field-collected Lumbriculidae worms. Field-collected benthic invertebrates and L. variegatus accumulated less PAHs than PCBs with similar lipophilicity. The biota to sediment accumulation factors of PAHs tended to decrease with increasing sediment organic carbon normalized concentrations. The presented data yields bioconcentration factors (BCF) describing the chemical water-lipid partition, which were found to be higher than the octanol-water partition coefficients, but on a similar level with BCFs drawn from relevant literature. In conclusion, using the two-carbon model method, or the measured freely dissolved pore water concentrations method is recommended for predicting the bioaccumulation of PAHs and PCBs.

Properties, performance and associated hazards of state-of-the-art durable water repellent (DWR) chemistry for textile finishing.

Following the phase-out of long-chain per- and polyfluoroalkyl substances (PFASs), the textile industry had to find alternatives for side-chain fluorinated polymer based durable water repellent (DWR) chemistries that incorporated long perfluoroalkyl side chains. This phase-out and subsequent substitution with alternatives has resulted in a market where both fluorinated and non-fluorinated DWRs are available. These DWR alternatives can be divided into four broad groups that reflect their basic chemistry: side-chain fluorinated polymers, silicones, hydrocarbons and other chemistries (includes dendrimer and inorganic nanoparticle chemistries). In this critical review, the alternative DWRs are assessed with regards to their structural properties and connected performance, loss and degradation processes resulting in diffuse environmental emissions, and hazard profiles for selected emitted substances. Our review shows that there are large differences in performance between the alternative DWRs, most importantly the lack of oil repellence of non-fluorinated alternatives. It also shows that for all alternatives, impurities and/or degradation products of the DWR chemistries are diffusively emitted to the environment. Our hazard ranking suggests that hydrocarbon based DWR is the most environmentally benign, followed by silicone and side-chain fluorinated polymer-based DWR chemistries. Industrial commitments to reduce the levels of impurities in silicone based and side-chain fluorinated polymer based DWR formulations will lower the actual risks. There is a lack of information on the hazards associated with DWRs, in particular for the dendrimer and inorganic nanoparticle chemistries, and these data gaps must be filled. Until environmentally safe alternatives, which provide the required performance, are available our recommendation is to choose DWR chemistry on a case-by-case basis, always weighing the benefits connected to increased performance against the risks to the environment and human health.

Flame retardants: Dust - And not food - Might be the risk.

Flame retardants (FRs) are used to delay ignition of materials such as furniture and electric and electronic instruments. Many FRs are persistent and end up in the environment. Environmental studies on flame retardants (FRs) took off in the late 1990s. Polybrominated diphenylethers (PBDEs) appeared to be bioaccumulative and were found in many organisms all over the world. When PBDEs were banned or their production voluntarily terminated, alternatives appeared on the market that often had similar properties or were of more concern due to their toxicity such as halogenated phosphorus-based FRs. Here we show that in spite of the ban on PBDEs more brominated FRs are being produced, an increasing number of other FRs is being applied and FR levels in our homes are much higher than in the outdoor environment. While nowadays we live in better isolated houses and sit in front of the computer or television, on flame retarded upholstery, we are at risk due to the toxic effects of a suite of FRs. The high exposure to these substances indoors calls for better risk assessments that include mixture effects.

Exposure to organophosphate and polybrominated diphenyl ether flame retardants via indoor dust and childhood asthma.

Although the ubiquitous detection of polybrominated diphenyl ether (PBDE) and organophosphate flame retardants (PFRs) in indoor dust has raised health concerns, only very few epidemiological studies have assessed their impact on human health. Inhalation of dust is one of the exposure routes of FRs, especially in children and can be hazardous for the respiratory health. Moreover, PFRs are structurally similar to organophosphate pesticides, which have been associated with allergic asthma. Thus, we investigated whether the concentrations of PFRs and PBDEs in indoor dust are associated with the development of childhood asthma. We selected 110 children who developed asthma at 4 or at 8 years old and 110 matched controls from a large prospective birth cohort (BAMSE - Barn, Allergy, Milieu Stockholm Epidemiology). We analyzed the concentrations of 7 PFRs and 21 PBDEs in dust collected around 2 months after birth from the mother's mattress. The abundance rank in dust was as follows: TBOEP⪢TPHP>mmp-TMPP>EHDPHP~TDCIPP>TCEP~TCIPP~BDE-209⪢BDE-99>BDE-47>BDE-153>BDE-183>BDE-100. There was no positive association between the FRs in mattress dust and the development of childhood asthma. In contrast, dust collected from mattresses of the mothers of children who would develop asthma contained significant lower levels of TPHP and mmp-TMPP. This study provides data on a wide range of PFRs and PBDEs in dust samples and development of asthma in children.

Analysis of two alternative organophosphorus flame retardants in electronic and plastic consumer products: resorcinol bis-(diphenylphosphate) (PBDPP) and bisphenol A bis (diphenylphosphate) (BPA-BDPP).

Following the phase-out of polybrominated diphenyl ethers (PBDEs), organophosphorus flame retardants (PFRs) are increasingly used as alternative flame retardants in many products. Data on the presence of two alternative PFRs in consumer products, resorcinol bis (diphenylphosphate) (PBDPP or RDP) and bisphenol A bis (diphenylphosphate) (BPA-BDPP or BDP) is still scarce or non-existing. In this study we propose a simple extraction method and analysis by liquid chromatography-atmospheric pressure chemical ionization (APCI) coupled to a high resolution time-of-flight mass spectrometry (TOF) for plastic consumer products. Detection limits were low enough for trace quantitation in plastic or electronic samples (0.001% and 0.002% w/w for PBDPP and BPA-BDPP, respectively). The APCI source provided better sensitivity and matrix effects than the commonly used ESI source for the analysis of these PFRs. Both PBDPP and BPA-BDPP were detected in 7 of the 12 products purchased in 2012 (at 0.002-0.3% w/w for PBDPP and 0.02-0.18% w/w for BPA-BDPP) while only PBDPP was found in 4 of the 13 products purchased before 2006 (0.005-7.8% w/w). In newly purchased products, PBDPP, BPA-BDPP and triphenyl phosphate (TPHP) were the most frequently detected PFRs. These results support the recent findings of our research group about high concentration levels of PBDPP and BPA-BDPP up to 0.5-1 mg g(-1) in house dust collected on electronic equipment and highlights the need for further research on these two novel PFRs.

Direct probe atmospheric pressure photoionization/atmospheric pressure chemical ionization high-resolution mass spectrometry for fast screening of flame retardants and plasticizers in products and waste.

In this study, we develop fast screening methods for flame retardants and plasticizers in products and waste based on direct probe (DP) atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI) coupled to a high-resolution (HR) time-of-flight mass spectrometer. DP-APPI is reported for the first time in this study, and DP-APCI that has been scarcely exploited is optimized for comparison. DP-APPI was more selective than DP-APCI and also more sensitive for the most hydrophobic compounds. No sample treatment was necessary, and only a minimal amount of sample (few milligrams) was used for analysis that was performed within a few minutes. Both methods were applied to the analysis of plastic products, electronic waste, and car interiors. Polybrominated diphenylethers, new brominated flame retardants, and organophosphorus flame retardants were present in most of the samples. The combination of DP with HR mass spectra and data processing based on mass accuracy and isotopic patterns allowed the unambiguous identification of chemicals at low levels of about 0.025 % (w/w). Under untargeted screening, resorcinol bis(biphenylphosphate) and bisphenol A bis(bisphenylphosphate) were identified in many of the consumer products of which literature data are still very limited.

Optimization and development of analytical methods for the determination of new brominated flame retardants and polybrominated diphenyl ethers in sediments and suspended particulate matter.

With more stringent legislation on brominated flame retardants, it is expected that increasing amounts of substitutes would replace polybrominated diphenylethers (PBDEs). Therefore, the development and optimization of analytical methodologies that allow their identification and quantification are of paramount relevance. This work describes the optimization of an analytical procedure to determine pentabromochlorocyclohexane, tetrabromo-o-chlorotoluene, 2,3,5,6-tetrabromo-p-xylene, tetrabromophthalic anhydride, 2,3,4,5,6-pentabromotoluene, tris(2,3-dibromopropyl)phosphate, decabromodiphenylethane and 1,2-bis(2,4,6-tribromophenoxy)ethane together with PBDEs in sediments and in suspended particulate matter. This method comprises a pressurized liquid extraction followed by three cleanup steps (gel permeation chromatography and solid phase extraction on Oasis™ HLB and on silica cartridges). Gas chromatography-mass spectrometry, using electron capture negative chemical ionization, is used for the final analysis. The proposed method provides recoveries >85%. The method was applied to sediment and suspended particulate matter samples from different locations in the Western Scheldt estuary (the Netherlands). To the best of our knowledge, this is the first time that the occurrence of the additive flame retardants 2,3,5,6-tetrabromo-p-xylene, 3,4,5,6-tetrabromo-o-chlorotoluene and 2,3,4,5,6-pentabromochlorocyclohexane is reported in the literature. The concentrations of these new flame retardants ranged from 0.05 to 0.30 µg/kg dry weight.

Methods for the determination of phenolic brominated flame retardants, and by-products, formulation intermediates and decomposition products of brominated flame retardants in water.

Brominated flame retardants (BFRs) are the chemicals of high importance within the REAch framework. In addition to polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCD) and tetrabromobisphenol A (TBBPA), other BFRs such as bromophenols, intermediates in FR formulation like bromoanilines, and their brominated and non-brominated by-products such as bromoanisoles, bromotoluenes, bromoalkanes and 1,5,9-cyclododecatriene, respectively should be monitored and controlled because of their toxicity and their very low odour and taste thresholds, below sub-nanogram-per liter levels. In the present study several analytical methods for the simultaneous determination, i.e., combining one single sample treatment and one analysis step, of these compounds in water have been developed, optimized and evaluated. The methods involve a (pre-concentration)-extraction technique, such as liquid-liquid (LLE), solid-phase (SPE), headspace (HS) extraction or solid-phase microextraction (SPME), followed by gas chromatography (GC)-mass spectrometry (MS) analysis with either electron capture negative ionization (ECNI) or electron impact (EI) as ionization techniques. ECNI is more sensitive than EI for analytes with more than one bromine atom. HS and SPME were previously optimized by means of a multifactorial experimental design. Extraction temperature and the liquid/headspace volume ratio were the most significant factors in HS extraction. In SPME, the variables studied were the nature of the fiber, the mode of extraction and the extraction temperature. Polydimethylsiloxane (PDMS) fibers appeared to be more suitable than carboxen-polydimethylsiloxane (CAR-PDMS) for the analysis of the target compounds with more than one bromine atom. The extraction of 2,4-dibromoaniline was only achieved in a direct immersion mode, in which the optimal extraction temperature was 60 degrees C. The methods LLE-GC-(ECNI)MS, LLE-GC-(EI)MS, SPE-GC-(ECNI)MS, SPE-GC-(EI)MS, HS-GC-(EI)MS and SPME-GC-(EI)MS were evaluated in terms of linearity, precision, detection limits and trueness. All methods, with the exception of HS-GC-(EI)MS, were linear in a range of at least two orders of magnitude, giving recoveries above 75% and detection limits at the low ng/L level for most of the target analytes. SPE-GC-(ECNI)MS is the most sensitive and reliable method for the determination of most of the bromine compounds, whereas SPE-GC-(EI)MS is the most suitable to quantify the three isomers of 1,5,9-cyclododecatriene. Both methods together with SPME-GC-(EI)MS (for qualitative confirmation) were applied to water samples from the Western Scheldt (The Netherlands), where 2,6-dibromophenol and 2,4,6-tribromoanisole could be detected at levels higher than their respective odour thresholds.

Polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls in fish from the Netherlands: concentrations, profiles and comparison with DR CALUX bioassay results.

Fish from Dutch markets were analysed for concentrations of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) and compared with the new European maximum residue levels (MRLs), set in 2006. In a first study on 11 different fish and shellfish from various locations, concentrations of PCDD/Fs were nearly all below the MRL for PCDD/Fs [4 pg toxic equivalents (TEQ) per gram wet weight (ww)] and nearly all below 8 pg total TEQ/g ww, the new MRL for the sum of PCDD/Fs and DL-PCBs. Some samples exceeded the total TEQ MRL, such as anchovy, tuna and sea bass. Furthermore, 20 (out of 39) wild eel samples exceeded the specific MRL for eel (12 pg total TEQ/g ww), as the study revealed PCDD/F TEQ levels of 0.2-7.9 pg TEQ/g ww and total TEQ values of 0.9 to 52 pg/g ww. TEQ levels in farmed and imported eel were lower and complied with the MRLs. Smoking eel, a popular tradition in the Netherlands, only had marginal effects on PCDD/F and DL-PCB concentrations. Owing to volatilization, concentrations of lower-chlorinated PCBs were reduced to below the limit of quantification after smoking. DL-PCBs contributed 61-97% to the total TEQ in all eel samples. This also holds for other fish and shellfish (except shrimps): DL-PCB contributed (on average) from 53 (herring) to 83% (tuna) to the total TEQ. Principal-component analysis revealed distinctive congener profiles for PCDD/Fs and non-ortho PCBs for mussels, pikeperch, herring and various Mediterranean fish. The application of new TCDD toxic equivalency factors (TEFs) set by the World Health Organization in 2006 (to replace the 1997 TEFs) resulted in lower TEQ values, mainly owing to a decreased mono-ortho PCB contribution. This decrease is most pronounced for eel, owing to the relative high mono-ortho PCB concentrations in eel. Consequently, a larger number of samples would comply with the MRLs when the new TEFs are applied. The DR CALUX(R) assay may be used for screening total TEQ levels in eel, in combination with gas chromatography-high resolution mass spectrometry confirmation of suspected samples. An almost 1:1 correlation was found when the 1997 TEFs were applied, but, surprisingly, a 1.4-fold overestimation occurred with application of the 2006 TEFs.

Long-term exposure of European flounder (Platichthys flesus) to the flame-retardants tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD).

Tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD) are widely used flame retardants that have increasingly been found as contaminants in the aquatic environment. In the present study, European flounder (Platichthys flesus) were chronically exposed to TBBPA; (105 days) and HBCD (78 days), in a wide range including environmentally relevant concentrations. TBBPA was administered via the water, whereas HBCD was administered in food and sediment, or in sediment alone. Chemical analysis of muscle showed an average increase in internal concentrations of approximately two orders of magnitude for both compounds tested. Animals exposed to HBCD via sediment alone (8000 microg/g total organic carbon, TOC) showed a proportional increase of alpha-HBCD in muscle compared to animals exposed via food and sediment. In both studies, exposure to the test compounds did not affect general health and toxicity parameters (behavior, survival, growth rate, relative liver and gonad weight). Hepatic microsomal enzyme activities (TBBPA: EROD; HBCD: EROD, PROD, and BROD) were not induced by any of the tested chemicals. Aromatase activity in male gonads showed a mild increase with rising TBBPA levels. There were no morphological and immunohistochemical indications for increased production of the yolk precursor protein vitellogenin (VTG) in animals exposed to TBBPA and HBCD; immunochemical analysis of plasma VTG levels showed no dose response in animals exposed to TBBPA. In animals exposed to TBBPA, levels of the thyroid hormone thyroxin (T(4)) increased with internal concentrations of the test compound, possibly indicating competition of TBBPA for plasma protein binding. Triiodothyronin (T(3)) levels were not affected and histology showed no signs of altered thyroid gland activity. Other organs investigated (liver, gills, kidney, skin, and gonads) revealed no histological changes related to TBBPA or HBCD exposure. Overall, the present results indicate limited endocrine effects of these widely used flame retardants in a test species representative of European estuaries at environmentally relevant exposure levels and at internal levels up to 4300 ng TBBPA/g wet weight, and 446 microg HBCD/g lipid weight in flounder muscle.

Toxicity of tetrabromobisphenol A (TBBPA) in zebrafish (Danio rerio) in a partial life-cycle test.

Toxicological effects of the widely used flame retardant, tetrabromobisphenol A (TBBPA) were assessed in a partial life-cycle test with zebrafish (Danio rerio). Exposure of adult fish during 30 days to water-borne TBBPA in nominal concentrations ranging from 0 (control) to 1.5 microM was followed by exposure of the offspring in early life stages up to 47 days posthatching (dph) to the same concentrations. Adults exposed to 3 and 6 microM showed severe disorientation and lethargy shortly after beginning of exposure and were euthanized. Because semistatic exposure resulted in fluctuating water concentrations, pooled fish samples were chemically analyzed for internal dose assessment. Egg production was decreased in fish exposed to TBBPA concentrations of 0.047 microM and higher, and a critical effect level of 7.2 microg/g lipid with a lower 5% confidence limit of 3.9 microg/g lipid for 50% decreased egg production was calculated. Histology of adult ovaries indicated a relative increase of premature oocytes in two surviving females exposed to 1.5 microM. Hatching of TBBPA-exposed larvae was decreased except in animals exposed to 0.375 microM. In the highest exposure concentration, early posthatching mortality was high (81%) in larvae and the surviving juveniles showed a significant predominance of the female phenotype. Exposure of eggs from control parents up to 6 microM TBBPA resulted in increasing malformation and pericardial fluid accumulation from 1.5 microM; at higher concentrations, all embryos failed to hatch. The presented results indicate decreased reproductive success in zebrafish at environmentally relevant TBBPA concentrations.

In vivo and in vitro Ah-receptor activation by commercial and fractionated pentabromodiphenylether using zebrafish (Danio rerio) and the DR-CALUX assay.

The present study addresses the toxicity of a commercial pentabrominated diphenylether (PeBDE) flame retardant mixture, DE-71, in a model aquatic vertebrate. Four weeks' exposure of juvenile zebrafish (Danio rerio) to water-borne DE-71 resulted in dose-dependent induction of CYP1A immunoreactivity, predominantly in the endocardium and the endothelium of larger blood vessels, such as ventral aorta and branchial arteries, as well as the larger hepatic and pancreatic blood vessels. To investigate the impact of possible contaminating PBDD/Fs in the DE-71 product, the study was repeated after DE-71 had been fractionated into a non-planar (cleaned PBDEs) and a planar fraction (PBDD/Fs). Zebrafish were exposed under similar conditions to the planar and cleaned DE-71 fractions, and to uncleaned DE-71. In addition, the above fractions were chemically analyzed and tested in a reporter gene assay (DR-CALUX) for their aromatic hydrocarbon-receptor (AhR) stimulating potencies. A relatively strong CALUX response was detected from the planar DE-71 fraction (19.7ng TCDD equivalent (TEQ)/g DE-71), coinciding with a strong induction of CYP1A immunoreactivity in zebrafish. CYP1A immunoreactivity in zebrafish exposed to uncleaned DE-71 was intense, although the CALUX response was 10-fold less compared to the planar fraction. Only weak CYP1A immunoreactivity was found in fish exposed to cleaned DE-71, and none in control animals; no CALUX response was detected in cleaned DE-71. The present findings indicate that chemical impurities of the commercial PeBDE product account for AhR-mediated effects. Analytical isolation of a planar fraction from the commercial product increased the in vitro (DR-CALUX) signal 10 times. Immunohistochemistry showed a strong tissue specific reaction to DE-71 in vivo at these relatively low TEQ levels regardless of chemical pretreatment of the mix, reflecting the sensitivity of CYP1A induction in juvenile zebrafish to AhR agonists.

Determination of in vitro relative potency (REP) values for mono-ortho polychlorinated biphenyls after purification with active charcoal.

The TEF system for dioxin-like compounds has included assignment of TEF values for mono-ortho polychlorinated biphenyls (MO-PCBs). Small traces of aryl hydrocarbon receptor (AhR)-active impurities could result in artifactually higher relative potency (REP) values. MO-PCBs -105, -118, -156, and -167 were purified on an active charcoal column to remove AhR agonists that could be present as impurities. Activation or inhibition of AhR-dependent gene expression by purified MO-PCBs was studied in stably transfected cell lines (H1G1.1c3 mouse, H4G1.1c2 rat hepatoma), containing an AhR-responsive (AhR-EGFP) reporter gene. In addition, EROD activity was used as marker for CYP1A1 activity in these cell lines. MO-PCBs -105, -118, -156 induced AhR-EGFP expression in both rodent cell lines, with PCB-156 (10microM) being most effectively; inducing gene expression to approximately 27% of TCDD (mouse cells) and 62.5+/-3.4% (rat cells) of TCDD. This concurred with increased EROD activity in both cell lines to maxima of 20.5+/-1.5% and 68+/-3.2% of TCDD, respectively. No induction was observed for PCB-167. In the H1G1.1c3 mouse cells, PCB-105, -118 and -156 (10microM) significantly reduced TCDD-induced AhR-EGFP expression to 50.9+/-2.9%, 58.3+/-2.2% and 70.8+/-1.3% of TCDD. Reduced EROD activity was also observed, of 39.3+/-2.8%, 67+/-5% and 48.3+/-4% compared to TCDD. PCB-167 did not result in significant reduction. In rat cells, only PCB-156 resulted in significant decrease in TCDD-induced AhR-EGFP expression of 35%, suggesting species differences play a role. Our results suggest that purification of MO-PCBs is an essential step in determining accurate REP values, and could very likely lead to lower TEF values than those presently assigned by the WHO.

Comprehensive two-dimensional gas chromatography of polybrominated diphenyl ethers.

Comprehensive two-dimensional gas chromatography with micro electron-capture detection (GCxGC-muECD) was evaluated for the separation of 125 polybrominated diphenyl ethers (PBDEs). From among the six column combinations that were evaluated, DB-1x007-65HT was found to be the most suitable because of: (i) the highest number of BDE congeners separated; (ii) the least decomposition of higher brominated congeners; and (iii) the most suitable maximum operating temperature. The separation of the 125 BDE congeners from five hydroxy- and two methoxy-BDEs and nine other brominated flame retardants (polybrominated biphenyls, tetrabromobisphenol-A, methyl-tetrabromobisphenol-A and hexabromocyclododecane) was also studied. Fluorinated BDEs were found to be valuable internal standards for the determination of BDEs because of their very similar physico-chemical properties and excellent separation from the parent BDEs, mainly in the second dimension. GCxGC-time-of-flight MS and GCxGC-muECD were shown to be useful tools to identify decomposition products of nona- and deca-substituted BDEs, which are formed during the GC run. Three nona-BDEs were shown to be the major decomposition products of BDE 209.