Shaun White

Brominated and chlorinated dioxins, PCBs and brominated flame retardants in Scottish shellfish: Methodology, occurrence and human dietary exposure

The most commonly consumed shellfish species produced in Scotland - mussels, oysters and scallops - were investigated for the occurrence of a range of brominated and chlorinated contaminants in order to establish current levels and estimate human dietary exposure. Flesh from individual sub-samples was representatively pooled and 35 composites were analysed for brominated and chlorinated dioxins (PBDD/Fs, PCDD/Fs), brominated and chlorinated biphenyls (PBBs, PCBs), polybrominated diphenyl ethers (PBDEs), hexabromocyclododecanes (HBCDs) and tetrabromobisphenol A (TBBPA). The analytical methodology used (13)C(12) labelled surrogates of the target compounds, with GC coupled to (usually) high resolution MS, and LC-MS/MS for HBCD and TBBPA analysis. Positive identifications were made in the majority of samples for most analytes with the exception of TBBPA and most PBDD congeners measured. None of the levels detected for PCDD/F and PCB were above the maximum permitted levels specified in European Union regulations. The levels of brominated furans predominated over brominated dioxins, reflecting the environmental distribution and source emission profiles of these contaminants, and relatively high levels of the tri-brominated congeners were observed. Levels of the flame retardant chemicals reflected current and legacy use, with appreciable concentrations of PBDEs and HBCDs (predominantly alpha-HBCD) but far lower levels of PBBs. TBBPA was not detected in any of the species. In general, mussels and oysters displayed relatively higher levels of contamination than scallops, although the gonad tissue of the latter showed significant levels of brominated dioxins. The estimated adult dietary intakes of PCDD/Fs and PCBs arising from the consumption of a typical portion of these foods in combination with an otherwise average UK diet were in the range 0.5-0.6 pg World Health Organisation (WHO)-toxic equivalent (TEQ)(2005)/kg bodyweight per day. These estimated dietary intakes are well within the Tolerable Daily Intake for dioxins and dioxin-like PCBs of 2 pg WHO-TEQ(2005)/kg bodyweight/day endorsed by the independent expert Committee on Toxicology of Chemicals in Food, Consumer Products and the Environment. The corresponding intakes for sumPBDEs and sumHBCDs were 5.6-6.1 and 5.9-7.9 ng/kg bodyweight/day respectively.

Simultaneous determination of PCDDs, PCDFs, PCBs and PBDEs in food.

Established and comprehensively validated methodology for the analysis of PCDDs, PCDFs and polychlorinated biphenyls (PCBs) in food, animal feed and other matrices is presented. The method achieves the analytical standards of EU protocols (2002/69/EC and 2002/70/EC) that are used to determine the compliance of food and animal feed to maximum permissible levels of chlorinated dioxins in these commodities. The methodology provides WHO-TEQ data for dioxins and PCBs as well as individual concentrations for toxic PCDD/F congeners and >50 commonly occurring PCBs. In addition, the methodology allows the simultaneous determination of individual polybrominated diphenylether (PBDE) congeners. A wide range of (13)Carbon -labelled surrogates allow accurate internal standardisation, and measurements are carried out using high resolution GC coupled to high resolution mass spectrometry except for mono-, tetra, ortho-substituted PCBs where unit resolution mass spectrometry can be used instead. Evidence of internal as well as external validation through the frequent use of reference materials, and successful participation in international inter-comparison exercises over many years is presented. A large number of different food types have been analysed for dioxins and PCBs using this methodology over several years and typical congener profiles for various food matrices are discussed.

Investigation into the formation of PAHs in foods prepared in the home to determine the effects of frying, grilling, barbecuing, toasting and roasting

The effects of frying, grilling, barbecuing, toasting and roasting on the formation of 27 different PAHs in foods were investigated. A total of 256 samples from in-house cooking experiments were produced. There was little evidence of PAH formation during the grilling, frying, roasting and toasting experiments. Comparison with the raw materials used in the experiments showed little or no increase in PAH concentrations for all of the sample types, regardless of distances from the heat source, cooking mediums and intensity of cooking conditions. Barbecuing with charcoal plus wood chips however resulted in the formation of benzo[a]pyrene in most foods; for beef burgers only, barbecuing over charcoal (without the use of wood chips) gave the highest levels. In general PAH levels increased when the food was barbequed closer to the heat source. For sausages cooked over briquettes, and for beef burgers, beef and salmon cooked over charcoal, the concentration of PAHs was lower when the food was closer to the heat source. Cooking time may result in a moderate increase of PAHs in some foods, although concentrations in beef burgers appeared to fall when cooking time was extended by 50-100%.

Dioxins and polychlorinated biphenyls (PCBs) in fish oil dietary supplements: Occurrence and human exposure in the UK

Commercially available fish oil supplements sourced from retail outlets in the UK, as well as by mail order, were surveyed in 2000–02 for dioxin (PCDD/Fs) and polychlorinated biphenyl (PCB) content. Sampled products were representative of market share. The WHO-TEQ values for these products ranged from 0.18 to 8.4 ng kg−1 for ΣPCDD/F and from 1.1 to 41 ng kg−1 for Σ dioxin-like PCBs. The results suggest a downward trend in the levels of dioxins in fish oil supplements over the last decade, since levels for similar products ranged from 0.3 to 10 ng kg−1 for ΣPCDD/F WHO-TEQ in 1996. Levels of ICES (International Council for the Exploration of the Seas) 7 PCBs in the current study ranged from 8.3 to 267 µg kg−1. Subsequent to this survey, European Union legislation has been introduced that includes a maximum limit of 2 ng kg−1 WHO-TEQ for dioxins in fish oil products for human consumption. Twelve of the 33 products reported here would have exceeded this limit. Negotiations are in progress to incorporate dioxin-like PCBs into the European Union regulations. When manufacturer-recommended doses were applied to the observed levels, the estimated upper bound human exposure to dioxins and dioxin-like PCBs from dietary intake of these products ranged from 0.02 to 7.1 pg WHO-TEQ kg−1 body weight day−1 for adults and from 0.02 to 10 pg WHO-TEQ kg−1 body weight day−1 for schoolchildren. This level rises to 1.8–8.9 pg WHO-TEQ kg−1 body weight day−1 for adults and 1.4–14 pg WHO-TEQ kg−1 body weight day−1 for schoolchildren when combined with the average exposure from the whole diet in 1997. Again, subsequent to this survey, the Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment (COT) revised the UK tolerable daily intake (TDI) for mixtures of dioxins and dioxin-like PCBs from 10 to 2 pg WHO-TEQ kg−1 body weight day−1. This is in line with the tolerable weekly intake (TWI) of 14 pg WHO-TEQ kg−1 body weight set by the Scientific Committee on Food (SCF).

Brominated dioxins (PBDD/Fs) and PBDEs in marine shellfish in the UK.

The occurrence of brominated dioxins (PBDD/Fs) and polybrominated diphenyl ethers (PBDEs) was investigated in commonly consumed species of marine shellfish in the UK. Individual samples of Pacific oysters (Crassostrea gigas), native oysters (Ostrea edulis), mussels (Mytilus edulis), scallops (Pecten maximus), and cockles (Cerastoderma edule) were collected from different coastal regions between 2006 and 2007. Samples of a particular species from each site were composited and 60 samples were analysed. Polybrominated dibenzofurans (PBDFs) occurred more frequently and generally at a higher level than polybrominated dibenzodioxins (PBDDs), except for 237-TriBDD, which was the predominant PBDD/F congener in some species, notably oysters. This profile may reflect the environmental distribution of these compounds and the effects of removal mechanisms, such as degradation, selective uptake and metabolism. PBDEs were detected in all samples. The dominant congeners were BDEs 47, 49, 99 and 100 and, to a lesser extent, BDEs 66 and 154. The occurrence of BDE-209 was observed in most samples and appears to be species selective, with the highest values occurring almost exclusively in mussels and cockles. Among the species studied, oysters and mussels displayed relatively higher levels of both sets of contaminants; native oysters, in particular, showed elevated levels of 237-TriBDD (up to 14.5 ng/kg). In general, contaminant levels appeared to be consistent with the extent of local industrialisation with lower levels observed in more remote areas such as the north of Scotland. Polybrominated biphenyls (PBBs) were also measured, and PBBs 49, 52 and 77 were the most frequently detected, although levels were very low. Dietary intakes, estimated for PBDD/Fs, showed that 237-TriBDD from single portions of oysters constituted a high proportion of the total dietary intake of the congener but, otherwise, dietary intakes of PBDD/Fs from shellfish were relatively low.

Butter as an indicator of regional persistent organic pollutant contamination: further development of the approach using polychlorinated dioxins and furans (PCDD/Fs), and dioxin-like polychlorinated biphenyls (PCBs)

The potential for use of butter as a widely available, relatively uniform lipid-rich matrix for the determination of spatial distributions of persistent organic pollutants has already been demonstrated. The present study determines the contributions to toxicity equivalence (TEQ) from polychlorinated dioxins and furans (PCDD/Fs) and polychlorinated biphenyls (PCBs) using butter samples from 24 countries world wide. Concentrations of PCDD/Fs and dioxin-like PCBs ranged from 0.07 to 5.69 pg ΣWHO-TEQ g-1 lipid. For most samples, PCDD/F TEQ fell within ranges reported for European dairy products over the last decade (0.3–2 pg g-1 lipid I-TEQ), though a single sample from Spain was a notable exception. Other than this sample, the highest values were recorded for samples from the Netherlands and Italy, with those from India, China and Tunisia also being relatively high. The contribution from non- ortho -PCBs was particularly significant in samples from Germany, Austria, Italy, the Czech Republic, Tunisia, India and Argentina. Although overall TEQs were generally highest in European and Mediterranean butters, elevated levels were also apparent in industrializing regions of Asia (India, China) and Latin America (Argentina). More detailed regional studies would be necessary to identify likely dioxin and PCB sources in each case. Nevertheless, this study supports the utility of butter as a monitoring matrix that may be especially applicable in regions for which monitoring programmes are currently lacking.

Single-laboratory validation of a GC/MS method for the determination of 27 polycyclic aromatic hydrocarbons (PAHs) in oils and fats

A protocol for the measurement of 27 polycyclic aromatic hydrocarbons (PAHs) in vegetable oils by GC/MS has undergone single-laboratory validation. PAHs were measured in three oils (olive pomace, sunflower and coconut oil). Five samples of each oil (one unfortified, and four fortified at concentrations between 2 and 50 µg kg−1) were analysed in replicate (four times in separate runs). Two samples (one unfortified and one fortified at 2 µg kg−1) of five oils (virgin olive oil, grapeseed oil, toasted sesame oil, olive margarine and palm oil) were also analysed. The validation included an assessment of measurement bias from the results of 120 measurements of a certified reference material (coconut oil BCR CRM458 certified for six PAHs). The method is capable of reliably detecting 26 out of 27 PAHs, at concentration <2 µg kg−1 which is the European Union maximum limit for benzo[a]pyrene, in vegetable oils, olive pomace oil, sunflower oil and coconut oil. Quantitative results were obtained that are fit for purpose for concentrations from <2 to 50 µg kg−1 for 24 out of 27 PAHs in olive pomace oil, sunflower oil and coconut oil. The reliable detection of 2 µg kg−1 of PAHs in five additional oils (virgin olive oil, grapeseed oil, toasted sesame oil, olive margarine and palm oil) has been demonstrated. The method failed to produce fit-for-purpose results for the measurement of dibenzo[a,h]pyrene, anthanthrene and cyclopenta[c,d]pyrene. The reason for the failure was the large variation in results. The likely cause was the lack of availability of 13C isotope internal standards for these PAHs at the time of the study. The protocol has been shown to be fit-for-purpose and is suitable for formal validation by inter-laboratory collaborative study.

Interpretation of studies on the developmental reproductive toxicology of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male offspring.

There have been several studies on the maternal administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and effects in the reproductive tract of male offspring, subsequent to risk assessments undertaken in 2001. This review compares the methodology and results to examine key methodological features, and consistency in reported outcomes. Maternal dosing at >0.8 microg TCDD/kg causes lethality and weight loss, and it is difficult to distinguish between direct and indirect effects of TCDD at these dose levels. Statistically significant effects of maternal doses of <1 microg TCDD/kg (i.e. the dose levels relevant for risk assessment) on prostate weight or epididymal sperm counts in offspring were reported in the minority of studies. The pharmacokinetics of TCDD differs considerably between acute and chronic dosing, and with dose level of TCDD. On the basis of body burden, TCDD had different potency at inducing adverse effects in the only comparison study between acute and chronic dosing. Understanding of the pharmacokinetics of TCDD and relationship to adverse effects in offspring is required. These analyses identify key features of TCDD developmental toxicity in male offspring, and identify data needs for future risk assessment.

Occurrence of dioxins (PCDDs, PCDFs) and polychlorinated biphenyls (PCBs) in wild, farmed and processed fish, and shellfish

Forty-eight composite samples of the most commonly consumed fish and shellfish species were prepared from up to 60 individual subsamples of each species and analysed for chlorinated dioxins (PCDD/Fs) and polychlorinated biphenyls (PCBs). These included 24 species of fresh wild fish, seven of farmed fish, seven of fresh shellfish, and ten processed fish and shellfish products. The ISO 17025-accredited analytical methodology used is consistent with the requirements given in European Commission Directive 2002/69/EC. Concentrations ranged from 0.03 ng kg–1 PCDD/F plus PCB World Health Organization-toxic equivalent quantity (WHO-TEQ) for a sample of surimi, to approximately 6 ng kg–1 for wild pilchards/sardines. The corresponding range for the ΣICES-6 PCBs was 0.04 μg kg–1 to approximately 47 μg kg–1. None of the samples showed concentrations above the European Union maximum permitted limits. Averaged PCDD/F and PCB concentrations for the two groups of farmed and wild fish show that there is little difference between the two categories, although individual species may show variations depending on factors such as the sampling location.

Recombinant expression of aryl hydrocarbon receptor for quantitative ligand-binding analysis

Recombinant expression of the aryl hydrocarbon receptor (AhR) yields small amounts of ligand-binding-competent AhR. Therefore, Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high-level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding-competent AhR was sensitive to the protein concentration of Sf9 extract, and coexpression of the chaperone p23 failed to affect the yield of functional ligand-binding AhR. The expression system yielded high levels of functional protein, with the ligand-binding capacity (Bmax) typically 20-fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity of human and rat AhR were obtained; the Kd for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding-competent AhR, and sufficient AhR for quantitative analysis of ligand binding.