Dr Paul C Turner
email: p.c.turner@leeds.ac.uk

Victoria J Burley

Joseph A Rothwell

Kay L M White

Janet E Cade

Christopher P Wild

DEOXYNIVALENOL: RATIONALE FOR DEVELOPMENT AND APPLICATION OF A URINARY BIOMARKER

Keywords: Biscuits, Bread, Cereals and grain biomarker, deoxynivalenol, diet, mycotoxin, urine

with how diverse a diet is available, and the degree of food sufficiency. Deoxynivalenol (DON) is a Fusarium mycotoxin that frequently contaminates wheat, corn and barley in temperate regions of the world. A number of acute poisoning incidences have been linked to DON contaminated foods, and chronic exposure to lower levels of DON has been predicted in many regions. DON is a potent animal toxin, and exposure in humans may cause gastroenteritis, growth faltering and immune toxicity. The ability to conduct accurate exposure assessment at the individual level is required in order to fully understand the potential health consequences of human exposure. To date, such exposure biomarkers have been lacking for many important mycotoxins, including DON. To better assess exposure to DON at the individual level we have developed a robust urinary assay, incorporating immunoaffinity column (IAC) enrichment and LC-MS detection. Further refinement of this urinary assay, by inclusion of 13 C-DON as an internal standard was then undertaken, and tested within the UK. Urinary DON was frequently observed in the urine and was significantly associated with cereal intake. A dietary intervention study demonstrated that avoiding wheat in the diet markedly reduced urinary levels of DON. This biomarker requires further validation, but our initial data suggest it may provide a useful tool in epidemiological investigations of the potential health consequences of this common environmental toxin.

INTRODUCTION

There are around 100,000 fungal species, many of which are beneficial to the environment or useful in medicine and certain food production processes. A number of fungal species can also lead to the production of structurally diverse and highly potent toxins known as mycotoxins [START_REF] Cast | Potential economic costs of mycotoxins in the United States[END_REF]. There are many naturally occurring mycotoxins, with five of major agricultural importance: aflatoxins, deoxynivalenol (DON), fumonisins, ochratoxin A and zearalenone [START_REF] Miller | Fungi and mycotoxins in grain: Implications for stored product research[END_REF]. Mycotoxin contamination of food is often heterogeneous [START_REF] Cirilloy | Natural cooccurrence of deoxynivalenol and fumonisins B1 and B2 in Italian marketed foodstuffs[END_REF] which hinders exposure assessment using classical epidemiological approaches that utilise food diaries and food frequency questionnaires [START_REF] Meky | Deoxynivalenolinduced immunomodulation of human lymphocyte proliferation and cytokine production[END_REF].

With the exception of the extensive developmental and validation work conducted over many years for aflatoxin (reviewed by Wild andTurner 2001, 2002;[START_REF] Groopman | Validation of Exposure and Risk Biomarkers: Aflatoxin as A Case Study. Biomarkers of Environmentally Associated Disease[END_REF][START_REF] Groopman | Aflatoxin and hepatitis B virus biomarkers: a paradigm for complex environmental exposures and cancer risk[END_REF], a lack of biological markers, or biomarkers, still restricts our understanding of exposure at the individual level for the other mycotoxins commonly encountered through diet. As a consequence there is often awareness of frequent exposure to many potent mycotoxins, but a lack of appropriate 'tools' to conduct robust epidemiological studies to assess the potential health consequences.

DON is a Fusarium mycotoxin found primarily on cereal crops that are used for human consumption and in feeds for animals [START_REF] Cast | Potential economic costs of mycotoxins in the United States[END_REF]. Due to the stability of DON to processing and cooking [START_REF] Bullerman | Effect of processing on Fusarium mycotoxins[END_REF][START_REF] Creppy | Update of survey, regulation and toxic effects of mycotoxins in Europe[END_REF][START_REF] Sugita-Konishi | Effect of cooking process on the deoxynivalenol content and its subsequent cytotoxicity in wheat products[END_REF] human exposure is predicted to occur frequently [START_REF] Li | Mycotoxins (trichothecenes, zearalenone and fumonisins) in cereals associated with human red mold intoxications stored since 1989 and 1991 in China[END_REF][START_REF] Canady | Deoxynivalenol[END_REF][START_REF] Scoop | Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states[END_REF]. The toxicity of DON has been clearly established in animal studies, and was recently reviewed with a view to understanding the human health risks from natural dietary exposure [START_REF] Schlatter | Toxicity data relevant for hazard characterization[END_REF][START_REF] Pestka | Deoxynivalenol: toxicology and potential effects on humans[END_REF]. DON and other trichothecenes bind to the 60S ribosomal subunit, which inhibits translation and activates a signaling pathway known as the ribotoxic stress response [START_REF] Rotter | Toxicology of deoxynivalenol (vomitoxin)[END_REF][START_REF] Pestka | Cellular and molecular mechanisms for immune modulation by deoxynivalenol and other trichothecenes: unraveling a paradox[END_REF]. Feed refusal and vomiting are two of the most common toxic symptoms following DON ingestion in several species [START_REF] Who | Selected Mycotoxins: Ochratoxin, Tricothecenes, Ergot[END_REF], and the emetic symptom resulted in the alternative name for DON of 'vomitoxin'. There are species differences in the susceptibility to DON with swine being the most sensitive, chickens and turkeys intermediate, and ruminants having a higher tolerance [START_REF] Pestka | Deoxynivalenol: toxicology and potential effects on humans[END_REF]. In swine, reduced feed consumption and lower weight gain are observed following ingestion of feeds containing DON at < 2 ppm, whilst higher doses can cause vomiting, reduction in body weight and even death [START_REF] Rotter | Toxicology of deoxynivalenol (vomitoxin)[END_REF][START_REF] Prelusky | Effect of intraperitoneal infusion of deoxynivalenol on feed consumption and weight gain in the pig[END_REF]. Consumption of DON contaminated cereals has been associated with several major outbreaks of human intoxication in certain parts of the world [START_REF] Bhat | Outbreak of trichothecenes mycotoxicosis associated with consumption of mould-damaged wheat production in Kashmir Valley, India[END_REF][START_REF] Luo | Food poisoning caused by Fusarium toxins[END_REF][START_REF] Who | Selected Mycotoxins: Ochratoxin, Tricothecenes, Ergot[END_REF]. The symptoms observed include nausea, vomiting, diarrhea, headache, salivation, dizziness and convulsions [START_REF] Who | Selected Mycotoxins: Ochratoxin, Tricothecenes, Ergot[END_REF]. Epidemiological data from food poisoning incidents in China between 1961 and 1991 imply that DON may have a role in acute human toxicity [START_REF] Luo | Food poisoning caused by Fusarium toxins[END_REF]. DON contamination in food was also implicated in a large-scale human toxicosis in the Kashmir Valley of India [START_REF] Bhat | Outbreak of trichothecenes mycotoxicosis associated with consumption of mould-damaged wheat production in Kashmir Valley, India[END_REF] in which ~50,000 people reported vomiting and dizziness amongst other symptoms.

DON also has potent effects on the immune system [START_REF] Bondy | Immunomodulation by fungal toxins[END_REF][START_REF] Meky | Deoxynivalenolinduced immunomodulation of human lymphocyte proliferation and cytokine production[END_REF][START_REF] Gray | Transcriptional regulation of deoxynivalenol-induced IL-8 expression in human monocytes[END_REF], including suppression of the normal immune response to pathogens. In addition DON exposed mice exhibit elevation of serum IgA-immune complexes, mesangial IgA accumulation, and hematuria (reviewed by [START_REF] Pestka | Deoxynivalenol-induced IgA production and IgA nephropathyaberrant mucosal immune response with systemic repercussions[END_REF][START_REF] Pestka | Deoxynivalenol: toxicology and potential effects on humans[END_REF]. These are all hallmarks of IgA nephropathy (Berger's disease), the most common type of glomerulonephritis worldwide, which is of unknown etiology.

Increased levels of serum IgA have also been observed in DON exposed swine (Tiemann et , 2006[START_REF] Goyarts | On the effects of a chronic deoxynivalenol intoxication on performance, haematological and serum parameters of pigs when diets are offered either for ad libitum consumption or fed restrictively[END_REF]. In a recent review of human epidemiological data the possibility of DON-induced gastroenteritis, growth faltering and immunotoxicity, with a possible increase in susceptibility to infectious disease, were highlighted [START_REF] Pestka | Deoxynivalenol: toxicology and potential effects on humans[END_REF]. The variations in species susceptibility to this toxin may in part reflect differences in intestinal detoxification to de-epoxy DON (Yoshizawa et al., 1986). A recent small study suggests that humans lack this putative detoxification route [START_REF] Sundstol-Eriksen | Lack of de-epoxidation of type B trichothecenes in incubates with human faeces[END_REF]. This observation is suggestive that humans could be one of the species that are more sensitive to DON, though further investigations are warranted.

EXPOSURE ASSESSMENT

In order to understand the health effects of DON it is essential to be able to accurately assess intake at the individual level. Exposure assessment for dietary toxins has traditionally been approached by measuring levels of food contamination combined with estimates of food intake, thus providing population based estimates. A recent scientific cooperation [START_REF] Scoop | Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states[END_REF] task surveyed the frequency of foods, predominantly cerealbased, that were contaminated by 16 Fusarium mycotoxins in EU member states. A total of 44,670 food samples were analysed, including eleven countries providing data on DON.

Fusarium mycotoxins were predominantly associated with wheat and maize-based foods.

DON was the most frequently observed Fusarium mycotoxin, with 57% of the 11,022 samples tested for DON being found to be contaminated. Wheat and maize were contaminated with DON most frequently (61% and 89% of samples tested, respectively). Barley, oats and rye were also contaminated albeit slightly less frequently. France had the highest recorded level for DON contamination of wheat (50,000µg/Kg), whilst Sweden had the highest mean level of DON in wheat (1427µg/Kg). For maize the highest level recorded was again in France (8,850µg/Kg), which also had the highest mean level (1056µg/Kg). In the UK more than 600 samples were tested for DON, with around 50% positive, and a contamination range from 10 -2261µg/Kg for positive samples [START_REF] Scoop | Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states[END_REF]. Based on the national consumption of four major wheat based food items (bread, breakfast cereals, biscuits and cakes), both a mean DON intake and a high DON intake (based on consuming a mean amount of each food, and an amount of each food corresponding to the 95 th percentile of intake, respectively) were estimated in µg/day, using the mean level of contamination for each item [START_REF] Scoop | Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states[END_REF]. Data were available for both adult males and females, and are summarized in Table 1. The majority of exposure in adults was predicted to come from bread and breakfast cereals, due to their widespread contamination with DON and the frequent consumption of these items. Additional important sources of exposure are likely to include products containing wheat flour (excluding the items in Table 1) for example pasta, corn flour and possibly cereal-based snacks [START_REF] Scoop | Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states[END_REF]. It should be noted that it is unlikely that any given individual will consume all of the food groups at the high level of consumption, and in the UK, average dietary intakes for DON (assuming an average body weight of 70.1kg) were estimated at 176ng/kg(bw)/day and 142ng/kg(bw)/day, for males and females respectively. Based on a greater food intake per unit body mass, higher mean DON intakes were estimated for infants and young children (range 400-500ng/kg(bw)/day), with bread, breakfast cereals and cereal based snacks predicted to provide the major contributions to exposure. These estimates of intake are below the recommended TDI of 1000ng/kg(bw)/day [START_REF] Scf | Opinion of the Scientific Committee on Food on Fusarium toxins. Part 6: Group evaluation of T-2 toxin, HT-2 toxin, nivalenol and deoxynivalenol: SCF/CS/CNTM/MYC/27 Final[END_REF]. However, these data represent mean levels, and both the quantity of food and the contamination levels of foods were so variable that estimates of exposure may be 10 to 20fold higher in individuals consuming the most contaminated foods. When compared to the In a more global assessment of DON the Joint (FAO/WHO) Expert Committee on Food Additives (JECFA) also estimated dietary intakes using food consumption and contamination data [START_REF] Canady | Deoxynivalenol[END_REF]. Five world regions were created, European (which also includes Australia, Canada, US and New Zealand), Latin America, Middle Eastern, Far Eastern and African. Data for the exposure estimates were often limited, particularly outside of Europe, though crude estimates of international exposure are provided. The JECFA estimates are based on cereal consumers only, whilst it should be noted that the SCOOP data are for the population as a whole. The estimates range from a mean of 780ng/Kg(bw)/day in the African region (albeit based on data from South Africa alone) to 2400ng/Kg(bw)/day in the Middle eastern diet, with the European region having an estimated intake of 1400ng/Kg(bw)day. These alternative estimates suggest that globally the TDI for DON will be frequently exceeded. Whilst the TDI is based on a "No Observed Adverse Effect Level" in animals and incorporates a safety factor, humans may be particularly sensitive to DON compared to animals as discussed above [START_REF] Sundstol-Eriksen | Lack of de-epoxidation of type B trichothecenes in incubates with human faeces[END_REF]. The SCOOP (2003) report additionally highlighted some of the main limitations in its estimate of DON intake, and suggested that the total dietary intake by country should generally be considered an underestimate.

Biomarker approach to exposure assessment: As mentioned above, a major obstacle to studying DON and human health is the heterogeneous nature of cereal contamination, which makes the estimation of exposure at the individual level difficult. Given the uncertainties in the assessment of mycotoxin exposure from dietary data, biomarkers have been suggested to offer a better approach [START_REF] Montesano | Hepatocellular carcinoma: From gene to public health[END_REF][START_REF] Turner | Fumonisin Contamination of food: progress in development of biomarkers to better assess human health[END_REF]Wild and Turner 2001). Mycotoxin exposure biomarkers potentially provide data at the individual level and may establish the prevalence and range of exposure in a population [START_REF] Groopman | Validation of Exposure and Risk Biomarkers: Aflatoxin as A Case Study. Biomarkers of Environmentally Associated Disease[END_REF][START_REF] Turner | Fumonisin Contamination of food: progress in development of biomarkers to better assess human health[END_REF]. In response, the development of a urinary exposure biomarker for DON was initiated based on our understanding of the absorption and clearance of DON in animals. The toxicokinetics of DON in several animal species has previously been reviewed [START_REF] Pestka | Deoxynivalenol: toxicology and potential effects on humans[END_REF]. The absorption of DON following ingestion is species dependant. Swine are particularly sensitive to DON, and following DON ingestion, up to 80% is absorbed [START_REF] Rotter | Toxicology of deoxynivalenol (vomitoxin)[END_REF] and plasma concentrations peak within 15-30 min [START_REF] Prelusky | Pharmacokinetic fate of 14C-labeled deoxynivalenol in swine[END_REF]. In contrast, in ruminants, which are relatively resistant to DON toxicity, a smaller percentage of the parent compound is absorbed; in sheep 6-10% [START_REF] Prelusky | Excretion profiles of the mycotoxin deoxynivalenol, following oral and intravenous administration to sheep[END_REF] and in dairy cows <1% [START_REF] Prelusky | Nontransmission of deoxynivalenol (vomitoxin) to milk following oral administration to dairy cows[END_REF]. This lower level of uptake may in part reflect the greater capacity for bacterial derived deepoxidation of DON to DOM-1 within the ruminant gut [START_REF] Rotter | Toxicology of deoxynivalenol (vomitoxin)[END_REF]. The elimination half-life for DON in most species is relatively rapid, and the majority of DON and DON metabolites are excreted within about 24 hours [START_REF] Pestka | Deoxynivalenol: toxicology and potential effects on humans[END_REF].

The DON-glucuronide metabolite is observed in the serum and urine of swine [START_REF] Goyarts | Bioavailability of the Fusarium toxin deoxynivalenol (DON) from naturally contaminated wheat for the pig[END_REF], and ruminants [START_REF] Prelusky | Nontransmission of deoxynivalenol (vomitoxin) to milk following oral administration to dairy cows[END_REF][START_REF] Cote | Excretion of deoxynivalenol and its metabolite in milk, urine, and feces of lactating dairy cows[END_REF], and the urine of rodents [START_REF] Meky | Development of a urinary biomarker of human exposure to deoxynivalenol[END_REF]. In swine the serum clearance profile for DON only and DON + DON-glucuronide are similar [START_REF] Goyarts | Bioavailability of the Fusarium toxin deoxynivalenol (DON) from naturally contaminated wheat for the pig[END_REF]. DON and DON-glucuronide excretion in the urine represented 37% and 50% of the ingested DON in rat [START_REF] Meky | Development of a urinary biomarker of human exposure to deoxynivalenol[END_REF] and swine [START_REF] Goyarts | Bioavailability of the Fusarium toxin deoxynivalenol (DON) from naturally contaminated wheat for the pig[END_REF], respectively.

The rapid clearance of DON from serum did not make this a good matrix to assess dietary exposure in humans. The high level of transfer of DON to urine, combined with the relative ease of collection however made this a more suitable matrix. In the first stage of development the clearance of DON plus a DON-glucuronide conjugate were examined in a rat model, and utilised immunoaffinity enrichment and LC-MS detection [START_REF] Meky | Development of a urinary biomarker of human exposure to deoxynivalenol[END_REF]. Immuno-affinity enrichment resulted in the loss of the DON-glucuronide and thus to maximize the measure of total DON, the urine samples were pre-incubated with βglucuronidase to release the parent toxin.

In an initial test of a series of 15 female residents in Linxian County, Henan Province or Gejiu, Yunnan Province, China all urines were positive for DON; median 30ng/ml: range 4-94ng [START_REF] Meky | Development of a urinary biomarker of human exposure to deoxynivalenol[END_REF]. To our knowledge, this was the first report of DON in human urine. The urinary DON levels were consistent with those expected based on the range of cereal contamination in this region [START_REF] Luo | Comparative study on the natural occurrence of Fusarium mycotoxins (trichothecenes and zearalenone) in corn and wheat from high-and low-risk areas for human esophageal cancer in China[END_REF]. The hydrolysis conditions used were such that several µg of DON-glucuronide in urine would be readily hydrolysed to release DON [START_REF] Meky | Development of a urinary biomarker of human exposure to deoxynivalenol[END_REF]. Given that the Chinese samples contained ng/ml concentrations, we had considerable confidence that all DON-glucuronide present would be hydrolysed. In addition, increasing the β-glucuronidase concentration >20-fold gave no significant increase in DON recovery (unpublished observation). Further refinement of this urinary assay, by inclusion of 13 C-DON as an internal standard was then undertaken using a UK population dataset. In the UK, wheat is the main cereal crop responsible for human exposure to DON [START_REF] Scoop | Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states[END_REF]. Based on this knowledge we examined firstly, whether urinary DON could be detected in the UK population and, secondly, whether levels of urinary DON would be reduced by avoidance of wheat based products in a short-term intervention study. This intervention study is reviewed below; additional methodological detail, study design and study results have been recently published (Turner et al., 2007a). 2.

The study design reflects the observation that in animal models urinary DON predominantly reflects transfer over the first 24 to 48 hours. The wheat restricted food items in this study were primarily bread, breakfast cereal, cakes/biscuits and pasta, and the suggested alternatives were rice and potatoes.

Urinary Analysis: Urinary DON was measured as described in detail by Turner et al., (2007a). In brief, urine samples were spiked with 13 C-DON as an internal standard, and then digested overnight with 23,000 units β-glucuronidase (Type-IX, Sigma, Poole Dorset), to allow assessment of "total DON" (i.e. the amount of DON plus DON-glucuronide).

Digests were then enriched for DON using immunoaffinity columns (IAC), and analysed by HPLC with mass spectrometric detection using selective ion recording. All DON levels in the urine samples were quantified by reference to the internal standard. The limit of quantification (LOQ) in urine for this analysis was 0.6ng DON /ml urine. Two aliquots of a urine sample with previously measured DON concentration were also spiked with the internal standard and analysed with each batch of test samples as a quality control (QC).

The mean QC value was 10.4ng/ml with a CV of 6.1%. As first morning urine samples were used the final DON concentration in ng DON/ml of urine was adjusted using a measure of creatinine concentration in mg/ml of urine and subsequently data were expressed as ng DON/mg creatinine (ng/mg). 3. The geometric mean level during normal diet (7.2ng DON/mg creatinine; 95% CI 4.9-10.5ng/mg) was 11 fold higher (p<0.001) than that during the intervention (0.6ng/mg: 95% CI 0.4-0.9ng/mg). During the consumption of the 'normal' diet bread, breakfast cereals, biscuits, cakes, and pasta were consumed in amounts consistent with that previously reported for the UK population (Hendersen et al., 2002).

Overall bread was the most frequent source of wheat-based foods (see Table 4). The intervention was implemented between days three and six and the recorded diary information on days five and six indicated good compliance with a reduced frequency of consumption of wheat-based foods. In order to compensate for this reduction there was a marked increase in the consumption of both rice and potatoes (see Table 4). For comparison, data from the Chinese urine samples have been included in Table 3. The samples from China appear to have slightly higher levels of urinary DON, though we should additionally point out that these Chinese data are not adjusted for recoveries, as an internal standard was not available at the time of analysis. Creatinine data were also not available so this adjustment was not made.

More recently we have undertaken a larger survey of urinary DON in the UK (Turner et al., 2007b). Urine samples were available from the 2000-2001 UK adult National Diet and Nutrition Survey (NDNS) [START_REF] Henderson | The National Diet and Nutrition Survey: adults aged 19 to 64 years[END_REF] ). The hypothesis was that cereals would provide the major contribution to DON exposure, thus in this study individuals were selected following stratification into low, medium and high cereal intake groups. Stratification was based on items that provided the major sources of cereal in the UK diet; these included breads (wholemeal, white, soft grain, other), breakfast cereals (high-fibre and other), pasta, pizza, fruit pies, biscuits and buns/cakes. One hundred subjects were identified for each of the following cereal consumption groups: low (mean 107g cereal/day; range 88-125), medium (mean 179g/day; range 162-195) and high (mean 300g/day; range 276-325). DON was detected in 296 of 300 (98.7%) urine samples, and the mean and range of urinary DON levels (arithmetic mean of 9.0ng DON/mg creatinine; range non detectable to 48.2ng/mg) were similar to that reported within the 'normal diet phase' of the intervention study discussed above. Cereal intake was significantly associated with urinary DON (p<0.0005), with geometric mean urinary levels of 7.2 ng/mg (95%CI: 6.2-8.2ng/mg); 9.2ng/mg (95%CI: 8.1-10.5ng/mg) and 10.9ng/mg (95%CI: 9.5-12.4ng/mg) for low, medium and high intake groups respectively, after adjustment for sex and BMI, see Table 3.

Crude estimates of daily DON intake in the UK were reported by Turner et al., (2007aTurner et al., ( , 2007b)). These were based on: (i) the measure of urinary DON, (ii) an assumption that 35-50% the ingested DON was being excreted in the urine [START_REF] Meky | Development of a urinary biomarker of human exposure to deoxynivalenol[END_REF]Goyarts and Danicke 2006), and (iii) an assumption that the urinary DON originated from DON intake in the previous 24 hours. Based on these assumptions the mean intake was calculated to be around half of the recommended TDI for DON ingestion which was set at 1000ng/kg(bw) [START_REF] Scf | Opinion of the Scientific Committee on Food on Fusarium toxins. Part 6: Group evaluation of T-2 toxin, HT-2 toxin, nivalenol and deoxynivalenol: SCF/CS/CNTM/MYC/27 Final[END_REF] with some individuals expected to exceed this intake. However, these values should be interpreted with caution, partly because of the uncertainty in the percentage transfer of DON into human urine and the fact that the kinetics of human urinary excretion of DON and its metabolites has not been established. In addition, it should be noted that specific sub-groups in the population, such as vegetarians and children, may have higher exposures than the general population.

FUTURE WORK

Our biomarker data for exposure to DON in the UK are in good agreement with the estimates from food analysis [START_REF] Scoop | Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states[END_REF], in that exposure is both frequent and predominantly associated with wheat consumption. However, whilst the food analysis data allows estimates at a population level our biomarker approach allows measures of exposure at the individual level. Given that a wide range of exposures are predicted within a population, the assessment of individual exposures within a study population is critical in order to understand the health consequences. The SCOOP (2003) survey predicts that in European countries such as France, Germany and The Netherlands, exposure to DON will be higher than the UK. In a more global consideration, Europe as a region does not have the highest predicted mean level of DON exposure [START_REF] Canady | Deoxynivalenol[END_REF]. Our biomarker data suggest DON exposure has been under-estimated by the SCOOP survey, and this reinforces concerns about the potential health consequences in the UK and elsewhere. This urinary biomarker may also be useful in providing improved understanding of some of the acute poisoning incidents which have been potentially associated with DON contaminated foods [START_REF] Luo | Food poisoning caused by Fusarium toxins[END_REF][START_REF] Bhat | Outbreak of trichothecenes mycotoxicosis associated with consumption of mould-damaged wheat production in Kashmir Valley, India[END_REF]. However, there are still a number of outstanding questions related to fully validating the urinary measure as a reliable exposure biomarker in order to conduct informative epidemiological studies. Jelinek and colleagues reviewed the occurrence of the most important mycotoxins that naturally contaminate foods for human consumption [START_REF] Jelinek | Worldwide occurrence of mycotoxins in foods and feeds--an update[END_REF]. Their report details extensive DON contamination of grains, especially wheat, and further highlights the concern of co-contamination of grains by Fusarium toxins, especially for DON and nivalenol. They recommended that more information on co-occurrence of Fusarium toxins in cereals should be obtained in order to understand the potential additive or synergistic effects of such exposure mixtures. We envisage that our initial development of a urinary marker for DON will add impetus to the development of additional mycotoxin exposure biomarkers. It is possible that as this field continues to expand, additional matrices may be investigated including sera, saliva or faeces. It is also envisaged that biomarkers of the effects of mycotoxin exposures will follow from this development. It is likely that one or more of the 'omics' approaches will provide a wealth of data to further understand and develop such biomarkers and point to new avenues of research involving mechanisms of action. These latter markers of biological effect may be more generic with respect to similar Fusarium mycotoxins such as DON and nivalenol, and thus have the utility of providing important information on the effect of co-exposures.

In cereals, mixtures of different classes of mycotoxin naturally occur, and these may include deoxynivalenol (and other tricothecenes), fumonisins, ochratoxin A, zearalenone and aflatoxin (reviewed by [START_REF] Jelinek | Worldwide occurrence of mycotoxins in foods and feeds--an update[END_REF][START_REF] Speijers | Combined toxic effects of mycotoxins[END_REF]. Understanding the risks associated with exposure to mixtures of mycotoxins is still in its infancy even in animal studies. In humans such studies require validated methods of exposure assessment in order to compare exposure to toxins both individually and in combination with health outcomes. In this context accurate analytical determination of a mycotoxin in a biological sample does not translate to a valid marker of exposure. Rather, the biomarker must be shown to correlate with mycotoxin intake at the individual level as was originally demonstrated for aflatoxins (Wild et al., 1992) and is currently being addressed in our own group for deoxynivalenol (Turner et al., 2007a,b) and fumonisin B1 [START_REF] Gong | Detection of fumonisin B1 in Mexican urine samples using liquid chromatography-mass spectrometry -a possible biomarker of dietary fumonisin exposure[END_REF].

Eventually methodological advances such as applied to the simultaneous measurement of mycotoxin mixtures from cereals (Lattanzio et al., 2007a,b) may be of value for more rapid multi-toxin bio-monitoring.

SUMMARY

To date there is overwhelming evidence that certain dietary staples based on cereal crops worldwide are contaminated with DON. In order to better estimate exposure we have developed a urinary biomarker assay [START_REF] Meky | Development of a urinary biomarker of human exposure to deoxynivalenol[END_REF]Turner et al., 2007a). The methodology is robust, with high specificity and sensitivity, incorporating immunoaffinity isolation of DON and LC separation with mass spectrometric detection. Furthermore, the incorporation of an internal standard allows adjustment for recovery. Preliminary data from the UK (Turner et al., 2007a,b) reveal an almost ubiquitous exposure to DON. The specific activities outlined above indicate the research required to more fully validate this biomarker as a quantitative measure of exposure. This will provide an important foundation to further investigate the possible health effects of DON exposure. These latter investigations need to consider that there are likely to be sub-groups of people who experience particularly high exposures, warranting future targeted surveys of these potentially 'high risk' groups. One of these sub-groups may be children for whom exposure is predicted to be higher than adults, and in whom there may be an increased sensitivity to exposure. Finally, given the reported poisoning episodes in China and India, and the higher average exposure levels in the Middle and Far East, it will be important to target future collaborative research which focuses on potential health concerns within these higher exposure regions.

  dietary contaminants in most regions of the world. The frequency of exposure to the different families of mycotoxins is often dependent upon geographic location, national wealth and related agricultural and regulatory infrastructure, combined

  levels of exposure were predicted for some of the other countries within the EU, notably France, The Netherlands and Germany[START_REF] Scoop | Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states[END_REF].

  The study took place over seven days with 25 healthy adult volunteers, and involved two days (Day 1 and 2) of normal diet and four days (Day 3-6) of a wheat restricted diet. Suggestions for alternative starch based food items and meals were provided for the wheat restriction phase. Food diaries were kept for Days 1 and 2 (normal diet) and Days 5 and 6 (intervention diet). A first morning void urine sample was obtained on Day 3 to represent the 'normal diet', and on Day 7 to represent the 'intervention diet', see Table

  paired samples were available for urine taken corresponding to the normal diet and following the four-day intervention trial. During consumption of the normal diet all individuals had detectable urinary DON, whilst during the intervention fewer samples were positive (9 of 25; 36%) The mean levels of urinary DON in both 'normal' and 'intervention' samples are shown in Table

  , permitting comparison of urinary DON excretion with specific dietary information collected for each individual. Urine samples hour) were collected from 1724 individuals aged 19-64 years of age. In addition individuals completed a detailed seven-day weighed food diary (Office for National Statistics 2005

  is the relationship at the individual level between DON intake and the urinary biomarker? This is probably the most fundamental question to be answered in the further validation steps. To date we have only sought to compare cereal intake with urinary DON levels and this limits the ability to interpret biomarker levels in a quantitative manner in terms of DON consumption. Second, what are the pharmacokinetics of DON and DONglucuronide excretion, and, what are the consequent temporal variations in this biomarker? To date only single time point measures of urinary DON during the consumption of a 'normal diet' have been determined for a given individual. This information is important in being able to interpret urinary DON biomarker data in relation to recent or typical exposure levels. It will be necessary to include measures of the temporal variation in both DON intake and the urinary measure as part of these assessments. Third, does the ratio of DON to DON-glucuronide in urine vary by individual? The current methodology employed for measuring urinary DON involves the digestion of any DON-glucuronide present, to the parent compound, and thus the assay provides a combined measure of free and deconjugated DON, or "total" urinary DON. Genetic variation in this conjugation step may have a bearing on the toxicity of DON, and the DON/DON-glucuronide ratio should be assessed both between individuals and within individuals at different levels of DON exposure.

Table 1 : Predicted intake of DON from four specific food items (adapted from SCOOP 2003)

 1

	Food item	Mean* DON intake	High** DON intake
		µg / day	µg / day
		Male	Female	Male	Female
	Breakfast cereal	3.99	2.89	10.58	7.37
	Bread	7.46	4.69	14.50	8.77
	Biscuits	0.55	0.47	1.54	1.27
	Cakes	0.55	0.43	1.41	1.10

*Mean based on mean intake and mean contamination of specific foods (see SCOOP 2003) **High based on 95 th percentile intake and mean contamination of specific foods (see

[START_REF] Scoop | Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states[END_REF]

.

Table 2 : Design of wheat restriction intervention (Turner et al., 2007a)

 2

	Page 19 of 28	Food Additives and Contaminants
	Day	Diet	Food Diary	First morning urine*
	One	Normal	Yes	/
	Two	Normal	Yes	/
	Three	Intervention	/	Yes
	Four	Intervention	/	/
	Five	Intervention	Yes	/
	Six	Intervention	Yes	/
	Seven	Normal	/	Yes
	Lighter shaded area represents 'normal' dietary period and urine collection.
	Darker shaded area represents 'intervention' dietary period and urine collection.
	* First morning urines are predicted to reflect intake in the previous 24-48hours.

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 3 : Assessment of urinary DON in UK and Chinese samples (modified from Meky et al., 2003 and Turner et al., 2007a,b)

 3

	Detection rate	DON (ng/ml)*	DON (ng/mg)**	Reference
		Mean (range)	GM (95% CI)

Table 4 -Average frequency of consumption of dietary staples in 25 individuals consuming their normal diet (average of two days), and during the wheat reduction intervention diet (average of two days) (modified from Turner et al., 2007a).

 4 Wild C.P., Andersson C., O'Brian N.M., Wilson L., Woods J.A., (2001) A critical evaluation of the application of biomarkers in epidemiological studies on diet and health. Br. J. Nutr. 86 Suppl 1:S37-53. Wild C.P., Hudson G.J., Sabbioni., G., Chapot B., Hall A.J., Wogan G.N., Whittle H., Montesano R., Groopman J.D. (1992) Dietary intake of aflatoxins and the level of albumin-bound aflatoxin in peripheral blood in The Gambia, West Africa. Cancer Epidemiol Biomarkers Prev. 1(3):229-34. Wild C.P. and Turner P.C. (2001). Exposure biomarkers in chemoprevention studies of liver cancer. Eds: Miller, A.B., Bartsch, H., Bofetta, P., Dragsted, L., and Vanio, H. Biomarkers in Cancer Chemoprevention (IARC Scientific Publications No 154) International Agency for Research on Cancer, Lyon. pp 215-.222. Wild C.P. and Turner P.C. (2002) The Toxicology of Aflatoxins as a basis for Public Health Decisions. Mutagenesis 17(6): 471-481. Yoshizawa, T., Cote, L. M., Swanson, S. P., and Buck, W. B. (1986). Confirmation of DOM-1, a de-epoxidation metabolite of deoxynivalenol in biological fluids of lactating cows. Agric. Biol. Chem. 50: 227-229.

		Normal Diet	Intervention Diet
	FOOD GROUP	(%) Average Frequency	(%) Average Frequency	# p -value
	Bread	22 / 25 (88)	1.5 / 25 (6)	<0.001
	Breakfast Cereal*	9 / 25 (36)	0 / 25 (0)	<0.001
	Cakes / Biscuits	18 / 25 (72)	4.5 / 25 (18)	<0.001
	Pasta	6 / 25 (24)	0 / 25 (0)	<0.001
	Potatoes	9 / 25 (32)	15 / 25 (60)	0.016
	Rice**	3 / 25 (12)	17.5 / 25 (70)	<0.001
	# T test for difference in frequency of consumption for Days 1 & 2 combined compared to Days 5 & 6
	combined
	*Excluding Rice Crispies breakfast cereal and porridge
	** Including Rice Crispies breakfast cereal

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants


Noelia ; Fajar
email: fac@tandf.co.uk

OPTIMIZATION OF MICROWAVE-ASSISTED EXTRACTION WITH SAPONIFICATION FOR DETERMINATION OF POLYBROMINATED FLAME RETARDANTS IN AQUACULTURE SAMPLES

Keywords: Chemometrics, Chromatography -GC, Extraction, Reference materials Microwave-Assisted-Extraction with saponification, polybrominated 23 biphenyls, polybrominated diphenyl ethers, Experimental design, Desirability functions, 24 aquaculture samples. 25

The efficiency of microwave-assisted extraction with saponification (MAES) for 6 determination of seven polybrominated flame retardants (polybrominated biphenyls, 7

PBBs, and polybrominated diphenyl ethers, PBDEs) in aquaculture samples is described 8 and compared with microwave-assisted extraction (MAE). Chemometric techniques 9 based on experimental designs and desirability functions were used for simultaneous 10 optimization of the operational parameters used in both MAES and MAE processes. 11 Application of MAES to this group of contaminants in aquaculture samples, which had 12 not been previously applied to this type of analytes, was shown to be superior to MAE 13 in terms of extraction efficiency, extraction time and lipid content extracted from 14 complex matrices (0.7 % as against 18% for MAE extracts). PBBs and PBDEs were 15

determined by gas chromatography with micro-electron capture detection (GC-µECD). 16

The quantification limits for the analytes were 40 -750 pg g -1 (except for which 17 was 1.43 ng g -1 ). Precision for MAES-GC-µECD (%RSD <11%) was significantly 18 better than for MAE-GC-µECD (%RSD <20%). The accuracy of both optimized 19 methods was satisfactorily demonstrated by analysis of appropriate certified reference 20 material (CRM), 22 Introduction 27

Brominated organic compounds such as polybrominated biphenyls (PBBs) and 28 polybrominated diphenyl ethers (PBDEs) have been detected in a wide variety of biota 29 including Arctic wildlife, marine fish and shellfish in the Pacific region, which 30 demonstrates that these compounds are global bioaccumulated contaminants (Zhao et al. 31 2007;[START_REF] Braune | Levels and 312 trends of organochlorines and brominated flame retardants in Ivory Gull eggs from the 313 Canadian Arctic, 1976 to 2004[END_REF]). The increasing consumption of farmed fish makes the 32 determination of polybrominated flame retardants in the fish and the identification of 33 the original sources of contamination essential for evaluating the exposure to these 34 compounds via diet and ultimately for protecting public health (ATSDR, 2004;Sjödin 35 et al. 2004;[START_REF] Stapleton | Instrumental methods and challenges in quantifying 387 polybrominated diphenyl ethers in environmental extracts: a review[END_REF][START_REF] Hites | 350 flame retardants in Northern Fulmar (Fulmarus glacialis) from the Faroe Islands. 352[END_REF]. 36 37 Although there are sensitive methods for determining PBBs and PBDEs (Covaci et al. 38 2007;[START_REF] Eljarrat | Sample handling and analysis of brominated flame 334 retardants in soil and sludge samples[END_REF][START_REF] Covaci | Determination of brominated flame 327 retardants, with emphasis on polybrominated diphenyl ethers (PBDEs) in 328 environmental and human samples-a review[END_REF], the complexity of environmental and 39 biological matrices is a major problem for an accurate quantification of low levels of 40 PBBs and PBDEs [START_REF] Hites | Polybrominated diphenyl ethers in the environment and in people: A 347 meta-analysis of concentrations[END_REF]. The required extraction step still represents an 41 analytical challenge. Classical techniques, such as the Soxhlet extraction, are time-42 consuming (24 h) and need large solvent volumes (250 mL) [START_REF] Covaci | Recent developments in the 331 analysis of brominated flame retardants and brominated natural compounds[END_REF]Vives 43 et al.2007;[START_REF] Braune | Levels and 312 trends of organochlorines and brominated flame retardants in Ivory Gull eggs from the 313 Canadian Arctic, 1976 to 2004[END_REF]. Application of microwave energy during the extraction 44 (microwave-assisted extraction, MAE) can significantly accelerate and improve the 45 yield of the process using lower volumes of solvent [START_REF] Bayen | Determination of polybrominated diphenyl ethers in 309 marine biological tissues using microwave-assisted extraction[END_REF]Belange et al. 46 of PCBs from marine sediments [START_REF] Numata | Investigation of 365 saponification for determination of polychlorinated biphenyls in marine sediments[END_REF], for analysis of trichlorobenzenes 49 in fish [START_REF] Wittmann | Trace 398 analysis of trichlorobenzenes in fish by microwave-assisted extraction and gas 399 chromatography-electron-capture detection[END_REF], and PBDEs from fish and milk samples (Ohta et al. 50 2002). Nevertheless, clean-up of the extract using solid phase extraction (SPE) is 51 required as an additional step. 52

53

The application of microwave energy combined with alkaline decomposition has been 54

previously used in developing techniques for sample preparation, such as microwave-55 assisted saponification (MAS) followed by LLE to extract PCBs or n-alkanes and 56 polycyclic aromatic hydrocarbons (PAHs) from mussels; microwave-assisted 57 decomposition (MAD) combined with MAS or MAE to eliminate organochloride 58 pesticides from sediments and mussels; and simultaneous MAE of PAHs from fish 59 samples and hydrolysis of lipids with KOH [START_REF] Xiong | Microwave-assisted extraction or saponification 402 combined with microwave-assisted decomposition applied in pre-treatment of soil or 403 mussel samples for the determination of polychlorinated biphenyls[END_REF][START_REF] Zhang | Sample pre-treatment with microwave-assisted 414 techniques[END_REF]60 Hernandez-Borges et al. 2006;[START_REF] Pena | Optimization 372 of a microwave-assisted extraction method for the analysis of polycyclic aromatic 373 hydrocarbons from fish samples[END_REF]. A MAE procedure that includes gentle 61 saponification enables shortening the analysis time and avoiding the alkaline 62 decomposition of organochlorine compounds in oyster samples [START_REF] Carro | 323 Microwave-assisted extraction and mild saponification for determination of 324 organochlorine pesticides in oyster samples[END_REF]. 63

64

The aim of the present study was to develop a new method to extract PBBs and PBDEs 65 from feed intended for farmed fish, and from scallops, clams and mussels using 66 microwave energy combined with saponification (MAES) and to compare its efficiency 67 with MAE. The results showed that MAES does not need additional clean-up steps 68 because the lipid content of the extracts is lowered by a factor of twenty six as 69 compared with MAE. Brominated compounds were determined by GC-µECD due to 70 Sulphuric acid (99 %), silica gel (60 Å pore size, 0.040-0.063 mm, 230-400 mesh), 79 HPLC-grade methanol and ethanol (99.9%) were from Merck (Darmstadt, Germany). 80

Anhydrous sodium sulphate (99%) was from BDH (Poole, UK). A mixture of PBDEs 81 (10 µg mL -1 ) in cyclohexane: 2',4,42.5%),[82][83][84][85][86][87][88][89][90][91][92][93][94][95][96][97][98][99]2',4,4',39.3%), 2',4,4', pentabromodiphenyl ether, 10.9%), 2',4,4',5,84 1.9%), 2',4,4',5,2.7%) was supplied by Dr. 85 Ehrenstorfer (Augsburg, Germany). 2',4,97%) were purchased as solids, from Supelco 87 (Bellefonte, PA, USA). A stock PBBs standard solution (400 ng mL -1 ) was used to 88 prepare the working solutions by dilution, except for the PBDEs (1400 ng mL -1 ), which 89 were used at the starting concentration. Optimization experiments were carried out on a pooled (composite) sample of a 50 g 97 homogenate of small trout feed spiked with 100 mL of n-hexane containing 1200 µL of 98 the mixture of PBDEs at 10 µg.mL -1 , 105 µL of BB-15 solution at 22.0 µg.mL -1 , and 99 150 µL of BB-49 solution at 14.2 µg.mL -1 . The n-hexane was removed by evaporation 100 to air-dryness. In preliminary experiments, samples of 0.5, 1 and 1.5 g were taken to 101 evaluate the homogenity. Relative standard deviations lower than 10% for all the 102 studied compounds were obtained when the amount of sample used for analysis was 103 above 1 g, and therefore a minimum sample size of 1 g was used for subsequent 104 experiments. Then samples were stored at room temperature in glass bottles out of light 105 exposure until the analysis. 106 107 A certified reference material (CRM) WMF-01, which contains organic contaminants in 108 fish tissue, was obtained from Wellington Laboratories (Guelph, ON, Canada). 109 110

MAES and MAE conditions. Experimental design 111

Microwave-assisted extraction was carried out in a Millestone Ethos oven (Bergamo,112 Italy). The nature and volume of solvent (hexane 15 mL mixed with 1 mL water) and 113 the power (400 w) were fixed according the results of a previous study about the 114 determination of fifteen representative organochlorine pesticides, polychlorinated 115 [START_REF] Carro | Microwave-318 assisted extraction followed by headspace solid-phase microextraction and gas 319 chromatography with mass spectrometry detection (MAE-HSSPME-GC-MS/MS) for 320 determination of polybrominated compounds in aquaculture samples[END_REF]). The other experimental conditions 116 were established on the basis of the results of the optimization studies. 117 118 The use of chemometric tools such as experimental design improves yields at all stages 119 of the analytical process, and good quality data can be obtained with minimal 120 experimental effort [START_REF] Hernández-Borges | 343 Optimization of the mcrowave-assisted saponification and extraction of organic 344 pollutants from marine biota using experimental design and artificial neural networks[END_REF][START_REF] Pena | Optimization 372 of a microwave-assisted extraction method for the analysis of polycyclic aromatic 373 hydrocarbons from fish samples[END_REF]Serôdio et al. 121 2007). We used a central composite design of a spherical domain with α = 1.682 for 122 varied from zero (undesirable response) to 100 (optimal response). The overall 141 desirability function was estimated as the geometric average of the individual 142 desirability functions. NEMROD © W software was used for the generation and the 143 evaluation of the experimental designs [START_REF] Mathieu | NemrodW Version[END_REF]. This software incorporates 144 an algorithm in the optimization process to find the maximum of the overall desirability 145 function corresponding to the optimal experimental conditions. 146 147

Clean-up of extracts 148

The extracts produced by MAES or MAE were subjected to clean-up to avoid 149 interferences and to protect the chromatographic columns from damage. Among the 150 several clean-up procedures developed for organohalogenated pollutants extracts, those 151 based on Solid-Phase Extraction (SPE) are the most convenient in practice (Karlsson et 152 al. 2006;[START_REF] Gómara | Fractionation of 339 chlorinated and brominated persistent organic pollutants in several food samples by 340 pyrenyl-silica liquid chromatography prior to GC-MS determination[END_REF][START_REF] Carro | Microwave-318 assisted extraction followed by headspace solid-phase microextraction and gas 319 chromatography with mass spectrometry detection (MAE-HSSPME-GC-MS/MS) for 320 determination of polybrominated compounds in aquaculture samples[END_REF]. In this case, 3 g of acid silica gel were 153 used to guarantee lipid removal. Acidic silica gel was prepared adding 4 g of H 2 SO 4 to 6 154 g of silica gel. [START_REF] Rodil | Multicriteria optimisation of a 375 simultaneous supercritical fluid extraction and clean-up procedure for the determination 376 of persistent organohalogenated pollutants in aquaculture samples[END_REF]. With the optimized parameters, the lipid content of 155 the extracts after SPE was less than 0.06% and 0.04% of the original lipid content as 156 measured gravimetrically when MAE (18%) or MAES (0.7%) were respectively 157 applied. The extract was concentrated to 0. The clean extracts were analysed by GC-µECD on an Agilent Technologies 6980N 164 (Avondale, PA, USA) gas chromatograph equipped with a microelectron capture 165 detector. Gas chromatography was carried out on a 30 m x 0.32 mm i.d. HP-5 (5% 166 phenyl-methylpolysiloxane) (Agilent Technologies) fused silica column (0.25 µm film 167 thickness). Helium (purity 99.999%) (Carburos Metálicos, A Coruña, Spain) was used 168 as carrier gas, at a constant pressure of 13 psi. The initial temperature was 60°C, held 169 for 2 min, then ramped at 30°C min -1 up to 170°C, and held for 2 min, followed by a 170 second ramp of 12°C min -1 up to 280°C and held for 10 min. The injector, operated in 171 splitless mode for 1 min, and detector temperatures were fixed at 250ºC and 285°C, 172 respectively. 173 174

Results and discussion 175

Optimization of extraction procedures 176 MAES 177

The results of the central composite design (Table I) indicated that low values of the 178 three factors have a negative effect on extraction yield. The only factor with a direct 179 statistically significant effect for all regression models, except those for BDE-153 and 180 BDE-154, was the volume of KOH solution. Significant interactions between the main 181 factors were also observed. Thus, a multi-criteria methodology that incorporates certain 182 compromise experimental conditions to fulfil the expectations of the analyst was 183 required (Yusá et al. 2006a and2006b;[START_REF] Carro | Microwave-318 assisted extraction followed by headspace solid-phase microextraction and gas 319 chromatography with mass spectrometry detection (MAE-HSSPME-GC-MS/MS) for 320 determination of polybrominated compounds in aquaculture samples[END_REF]. 184 185 Individual desirability functions were defined for each analyte and these individual 186 functions were combined to an overall desirability function that reflects the success in 187 of 1M KOH (methanolic or ethanolic) [START_REF] Wittmann | Trace 398 analysis of trichlorobenzenes in fish by microwave-assisted extraction and gas 399 chromatography-electron-capture detection[END_REF][START_REF] Ohta | Comparison of polybrominated diphenyl ethers in 369 fish,vegetables, and meats and levels in human milk of nursing women in Japan[END_REF]203 Llompart et al. 2001), we studied the effect of KOH concentration in the MAES of the 204 seven analytes of interest. Thus, under the optimal conditions we found for MAES, 205

Performance evaluation of the analytical method 225

To best of our knowledge, there are no reports on the application of MAES combined 226 with GC-µECD to the analysis of PBBs and PBDEs in aquaculture samples. The 227 performance of the method was tested using spiked trout feed samples. The retention 228 times, linear ranges, coefficients of determination (R 2 ) of calibration lines, and limits of 229 detection and quantification are listed in Table II. Good correlations were obtained 230 within the intervals studied, with coefficients of determination equal to or above 0.997 231 (with the exceptions of . The limits of detection (LODs), 232 defined as the minimum amount of analyte that produces a peak with a signal-to-noise 233 ratio of 3, were below 220 pg.g -1 except for BB-15 (430 pg.g -1 ). The limits of 234 quantification (LOQs) were below 750 pg.g -1 except for BB-15 (1.43 ng.g -1 ) (Table II). 235 samples applying other analytical procedures (Karlsson et al. 2006;[START_REF] Bayen | Determination of polybrominated diphenyl ethers in 309 marine biological tissues using microwave-assisted extraction[END_REF]237 Naert et al. 2007). 238

[Insert Table II about here] 239 240 Analytical quality parameters of the proposed MAES were assessed with spiked trout 241 feed. Unspiked trout feed samples were previously analyzed in triplicate for the 242 compounds of interest and none of the investigated analytes were detected. Typical 243 chromatograms obtained for small trout feed with and without spiking, CRM WMF-01 244 and a standard solution (200 ng mL -1 of BB-15 and BB-49, respectively, and 700 ng 245 mL -1 of the total penta-BDE standard) are shown in Fig. 3. 246 [Insert Fig. 3 about here] 247 248

Comparison between MAES and MAE techniques. 249

To evaluate the applicability of the proposed MAES method for determining PBBs and 250 PBDEs in aquaculture samples, the accuracy (% recovery) and the precision (% RSD) 251 of the results were compared with those obtained when MAE was used. The 252 comparative study was made using spiked small trout feed samples. Six independent 253 extractions were performed to evaluate the reproducibility of the experimentally 254 optimized extraction methods. The right panel of Table II shows the average recoveries 255 and precision obtained for each analyte after applying MAES or MAE. The recovery 256 and the repeatability were improved by the use of MAES owing to its higher efficiency 257 to destroy lipids (Table II were quantified by GC-µECD, presenting amounts of 1.48 ± 0.1, 1.23 ± 0.01 and 1.57 ± 285 0.07 ng g -1 , respectively, which were quantified by GC-MS/MS ( 0.5 ng g -1 for LOQ of 286 BB-49 and 0.8 and 2.5 ng g -1 for LOD and LOQ of respectively). 287

[Insert Table III about

  been applied to conventional liquid-liquid extraction (LLE) 48

  of the method, a certified reference material (WMF-01) was 72 analysed applying MAES and MAE and the values of PBDEs concentrations were 73 compared[START_REF] Stapleton | 390 Determination of polybrominated diphenyl ethers in environmental standard reference 391 materials[END_REF]

  and trout and samples of scallop, clam and mussel were obtained from 93 cultivation areas on the Galician coast (Spain). The samples were triturated and 94 homogenised in a grinder before processing. 95 96

  MAES optimization. The design consisted of three factors with five levels: extraction 123 temperature (50, 65, 85, 105 and 120 ºC), extraction time (1, 3, 6, 9 and 11 min) and 124 volume of solution of 1M KOH in methanol (5, 9, 15, 21 and 25 mL). The proposed 125 experimental design (14 experiments + three central points) is shown in Table I. For 126 MAE optimization, five levels for extraction temperature (25, 40, 65, 90 and 105 ºC) 127 and four levels for extraction time (3, 6, 7 and 9 min) were selected using a pentagonal 128 design with three central points. In both cases, the mathematical models were 129 constructed considering the % recovery of each compound studied as the response. The 130 experimental plan and the responses obtained are shown in Table I. 131 132 [Insert Table I about here] 133 134 These experimental designs led to response surfaces that served for a first graphical 135 approach to optimization. Moreover, the use of multi-criteria optimization based on the 136 construction of a desirability function for each individual response enabled the 137 identification of the best operational conditions to simultaneously extraction of PBBs 138and PBDEs using MAES or MAE[START_REF] Lewis | Pharmaceutical Experimental Design[END_REF]. Each individual desirability 139 from a family of linear or exponential continuous functions, and 140

  5 mL in a TurboVap II Station (Zymark, 158 Hopkinton, MA, USA) and finally dried in a nitrogen blow down concentrator. The 159 complete schemes for MAES and MAE are shown in Fig.

  compounds from samples. In this case, non-linear partial 188 desirability functions were chosen for the responses to be maximized. Values below 50 189 % were considered not acceptable (zero desirability), whereas values above 70 % were 190 considered optimal, although the achievement of greater recoveries by using these 191 functions can be expected. The maximum of the overall desirability function 192 corresponded to 65 ºC and 3 min of extraction temperature and time, and 9 mL of 1 M 193 KOH in methanol, and the predicted % recoveries at this point ranged from 72 to 84 %. 194 Under these experimental conditions the constraints imposed on the individual 195 desirability functions are totally fulfilled and an overall desirability of 80 % was 196 reached. The global desirability was very sensitive to small changes in the analysed 197 factors, as can be seen from contour plots of isodesirability curves (Fig.2A,2B, and 198 2C). Grey regions represent null desirability and, thus, processes reported in the literature generally use a solution 202

  experiments (n=4) were carried out with 1M, 2M and a saturated solution of KOH. The 206 best recoveries were obtained with 2M KOH and with the saturated solution. Since the 207 lowest relative standard deviations (RSDs) were observed for 2 M KOH, the pentagonal design (TableI) showed that high values of both factors 213 have a positive effect in the extraction yields, but only the extraction time had a 214 statistically significant effect for BDE-153. According to the information reported by 215 the design (TableI), the experiment run 1 (75 ºC and 9 min) provides the best extraction 216 yields for PBBs and PBDEs. Non-linear partial desirability functions were easily built 217 supporting these results. Fig. 2D shows the two-dimensional plot of this overall 218 desirability function in the experimental domain studied; grey zones corresponding to 219 zero desirability. The maximum in the desirability surface appears at 75 ºC and 9 min of 220 extraction. Since this optimum is at the upper limit of the design, some additional 221 experiments with the extraction time extended to 12 and 15 min were made. No better 222 recoveries were achieved, proving that 9 min is indeed a global maximum. 223 224

  were lower than most of previously reported for biological 236

Figure 1 .Figure 2 .Figure 3 .Figure 4 .

 1234 Figure 1. Scheme of sample preparation for MAES and MAE procedures. 423

uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y Table I. Experimental plans and responses obtained (% recovery) in each experiment. Central composite design for MAES optimization % Recovery

	Page 23 of 26		Food Additives and Contaminants			Page 22 of 26
	50000					BDE-47		BDE-99		A
	30000 40000 160 Run 140 20000 1	KOH 1M Volume (mL) BB-15 9	BB-49 temperature (ºC) Extraction 65	Extraction time (min) 3	BB-15 BB-49 BDE-47 BDE-100 BDE-99 BDE-154 BDE-153 BDE-100 66.30 69.31 74.79 70.22 77.97 78.10 83.54
	0 10000 Area response 10000 20000 30000 40000 50000 40 60 80 100 120 -1 ) 2 3 4 5 6 Concentration (ng g 7 8 20 9 10 0 11 Area response 20000 30000 50000 12 13 14 15 40000 16 17	12 BB-15 21 9 9 9 21 21 21 F 15 o 15 r 15 12 BDE-47 15 5 P BDE-100 25 15 e 105 13 13 65 105 65 105 65 105 50 120 85 85 85 85 85 15 85 e r BDE-99 BB-49 15 85	14 14	BDE-47 BDE-154 3 9 9 3 3 9 9 6 6 1 11 6 6 6 6 6	15 15 57.12 60.90 16 BDE-100 16 MAE BDE-99 MAES WMF-01 58.90 60.26 61.58 61.39 64.32 72.43 70.74 49.11 52.06 50.85 46.93 58.47 55.05 44.73 52.61 52.07 48.38 62.18 61.38 65.64 64.41 65.45 52.81 70.27 69.35 60.50 77.89 53.53 BDE-153 56.24 61.92 60.74 83.27 77.55 76.90 41.91 48.56 48.83 46.05 58.17 58.17 61.61 62.85 63.29 50.91 61.06 60.17	17 54.81 58.02 68.57 48.86 57.72 52.89 61.45 62.88 68.27 75.68 60.66 68.19 48.83 59.62 60.97 58.63	18 BDE-154 18 BDE-154 60.43 63.05 74.43 53.96 61.07 56.78 66.08 68.07 73.59 80.96 66.61 72.83 58.83 63.76 66.90 63.13	19 19 61.05 BDE-153 BDE-153 61.75 73.09 54.66 64.29 59.15 65.76 71.41 72.82 86.97 70.19 69.30 55.50 65.38 68.46 63.62	B C	min min 63.85 69.02 79.36 57.23 67.47 62.46 71.22 72.28 79.92 93.09 75.70 71.65 60.09 69.15 71.82 68.10
	10000
	0	12	13 R		14		15	16		17	18	19	min
	Area response 0 10000 30000 40000 50000 20000		BDE-47 w v e i e	BDE-100	BDE-99		BDE-154	BDE-153	D
		12	13		14		15	16		17	18	19
						O n l			min
								y

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.

Pentagonal design for MAE optimization % Recovery Run Extraction temperature (ºC) Extraction time (min) BB-15 BB-49 BDE-47 BDE-100 BDE-99 BDE-154 BDE-153

	1	65	9	88.63 121.48	77.51	84.56	75.25	71.64	71.83
	2	105	7	73.01 108.60	68.47	77.23	67.10	61.69	60.47
	3	90	3	74.86 108.26	64.26	68.68	59.77	54.49	52.85
	4	40	3	73.59 110.22	65.33	67.12	59.11	55.39	54.92
	5	25	7	76.03 113.27	68.60	69.79	64.00	57.42	55.93
	6	65	6	77.01 106.01	60.73	61.90	55.63	48.82	49.64
	7	65	6	83.16 122.50	70.53	71.51	61.27	54.17	53.21
	8	65	6	74.13 106.07	65.95	69.44	59.70	55.43	52.11

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Karlsson M, Ericsson I, van Bavel B, Jensen, M. Dam J-K. 2006. Levels of brominated