Alwyn R Fernandes
email: a.fernandes@csl.gov.uk

Martin Rose

S White

D N Mortimer

M Gem

C Vaughan

A Greaves

PCBs) in fish oil dietary supplements: Occurrence and human exposure in the UK

Keywords: Survey, Exposure Additives/Contaminants: Dioxins, PCBs Dioxins, PCDD/Fs, PCBs, fish oil, WHO-TEQ, ICES-7 PCBs

HAL is

Introduction

Fish oils derived from the liver or tissue (body oil) of cold-water fish species such as cod, salmon, halibut etc. have enjoyed widespread use as dietary supplements and licensed medicines. These products are commercially available at retail outlets and by mail-order and are commonly sold either as capsules, liquid oils or as oils in combination with more palatable diluents such as fruit syrups. Fish oils are known to be a source of Vitamins A and D, as well as long chain fatty acids, particularly Omega-3 fatty acids.

Over the last two decades, the popularity of these products has increased concurrently with scientific research that has investigated the benefits of these oils in addressing a number of human maladies, including cardiovascular disease, stroke, high blood pressure, renal injury, rheumatoid arthritis and autoimmune disorders [START_REF] Tran | Fish Oil" in Gale Encyclopaedia of Alternative Medicine[END_REF][START_REF] Mori | Effects of varying dietary fat, fish and fish oils on blood lipids in a randomised trial in men at risk of heart disease[END_REF], Donadio et al 1994[START_REF] Schmitz | Omega-3 fatty acids in ESRD: should patients with ESRD eat more fish[END_REF][START_REF] Mason | Fish oils -an update[END_REF]. At least some of the benefits of fish oils are thought to be due to the long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the presence and levels of which are increasingly being included on the labels of fish oil products. The latter compound is essential for the development of the central nervous system, whilst the former is used by the body to produce prostaglandins and is also incorporated into cell lipids, including those in the cell membrane. In recent years, these products have been specifically targeted towards children suffering from dyslexia, dyspraxia, attention-deficit hyperactivity disorder (ADHD) and other similar disorders.

One of the main non-clinical disadvantages of using fish oils as dietary supplements, which has emerged in recent years, is the presence of environmental contaminants in these products. The natural habitat of these fish species -the continental shelf specially around the North Sea and the Atlantic Ocean, has experienced the build-up of persistent organic contaminants such as PCBs, dioxins, polybrominated diphenylethers (PBDEs), polycyclic aromatic hydrocarbons (PAHs) and various pesticides through atmospheric deposition, run-off and pollution from shipping. This has resulted in an enrichment of these pollutants, particularly in coastal sediments and, consequently, in the marine food chain. Many fish species bio-accumulate these contaminants, which are lipophilic and thus associate with lipid-rich tissues such as the liver and oil-rich muscle. As noted earlier, fish oil dietary supplements are produced from such tissues and the initial crude product can contain very high levels of these contaminants. The actual concentrations observed are dependent not only on the species but also on the geographical location [START_REF] Brevik | Levels of PCDDs and PCDFs in crude and processed fish oils in relation to origin and cleaning method[END_REF], with products sourced from the Southern Hemisphere generally showing lower levels of environmental contamination than those from Northern Hemisphere locations such as parts of the North Sea and especially the Baltic Sea, which is very contaminated with dioxins. The age of the fish and type of tissue used may also influence the In an effort to remove these contaminants, the industry employs a number of refining processes. General refining may include processes such as odour removal by steam stripping, exclusion of high molecular weight compounds by cold filtration and removal of other undesirable contaminants by activated charcoal filtration, as well as other chemical methods [START_REF] Brevik | Levels of PCDDs and PCDFs in crude and processed fish oils in relation to origin and cleaning method[END_REF]). However, molecular distillation (using a temperature range of 180 -220 0 C at < 100Pa) is far more effective at removing halogenated contaminants, especially PCBs. A drawback of some of the refining processes is that beneficial components of the fish oils, such as EPA and DHA, are removed at the same time as the contaminants, and the refining process employed must therefore strike a balance between contaminant removal and omega-3 fatty acid retention. Thus, some manufacturers have invested in developing newer (undisclosed) purification methods that are able to achieve this balance and are claiming success.

An alternative means of reducing the dietary exposure to consumers but without further purification is, of course, to reduce the manufacturer recommended dosage, especially for children.

The last survey of fish oil dietary supplements for PCBs and dioxins, carried out by the Ministry of Agriculture, Fisheries and Food in the UK in the mid-1990s, studied levels of these contaminants in retail products purchased in 1994 and 1996.

Combined (dioxin plus dioxin-like PCB) concentrations for cod liver oils ranged from 7.3 ->44 ng WHO-TEQ/kg in 1994 and from 18 -41 ng WHO-TEQ/kg in 1996 (MAFF 1997). The Tolerable Daily Intake (TDI) at the time was 10 pg I-TEQ/kg bodyweight/day, and a typical consumer taking the manufacturer recommended dose would not have exceeded this level except in the case of toddlers (because of low body mass). In combination with the whole diet in 1997, however, all groups would have exceeded the upper end of the 1998 WHO recommended TDI range of 1-4 pg WHO-TEQ/kg bw/day [START_REF] Leeuwen | Assessment of the health risk of dioxins:reevaluation of the TDI[END_REF].

Those findings were followed by discussions between the regulators, the Department of Health, the Medicines Control Agency and representatives of the fish oils industry in the UK with the aim of exploring measures that would help to reduce the dioxin and PCB exposure from fish oil-based dietary supplements. This study represents continued monitoring of dioxin and PCB levels in fish oil dietary supplements. It will demonstrate the effectiveness of improved purification procedures employed by manufacturers as well as establish current levels in these products within the UK.

Sampling and Analysis

A total of 33 dietary supplement products, comprising mostly cod liver oils but also salmon oils, other fish oils and formulations of cod liver oil with orange syrup or lemon flavouring, were purchased from retail outlets and by mail order during 2001-02. It was assumed that sampling would be representative, as these products are distributed nationally across the UK. Products that were sampled in previous surveys in the UK (MAFF 1997) were included where they were still available for purchase, along with a number of additional products. On receipt at the laboratory, each sample was given a unique laboratory reference number and the sample details logged into a database.

Product expiry dates were checked to ensure validity of the shelf-life of the samples during the measurement period. Information on recommended dosage provided on Samples were stored sealed in their original containers at ambient temperature prior to analysis. Where samples were sold as capsules, the casings were excluded from the analytical sample.

The method used for the extraction and analysis of samples have been reported previously [START_REF] Fernandes | Summary of Investigation of Dioxins Furans and PCBs in Farmed Salmon, Wild Salmon, Farmed Trout and Fish Oil Capsules[END_REF]. In brief, samples were fortified with 13 C-labelled analogues of target compounds and exhaustively extracted using mixed organic solvents. Ortho substituted PCBs were separated from non-ortho substituted PCBs and PCDD/Fs by fractionation on activated carbon. The two fractions were further purified using adsorption chromatography on alumina. Analytical measurement was carried out using high resolution gas chromatography/ high resolution mass spectrometry (HRGC-HRMS) for the PCDD/Fs and non-ortho substituted PCBs (IUPAC Nos. 77,81,126 and 169) and high resolution gas chromatography/ low resolution mass spectrometry (HRGC-LRMS) for the ortho substituted PCBs (IUPAC Nos. 18,28,31,47,49,51,52,99,101,105,114,118,123,128,138,153,156,157,167,180,189).

All analyses and results were UKAS accredited to ISO 17025 standards. Sample batches included a blank and the following BCR (Bureau of community reference) reference materials: CRM 350 (mackerel oil) for PCBs and RM 534 (milk) for the PCDD/Fs. Further quality assurance measures included the successful participation, during the course of this study, in international inter-comparison exercises (FAPAS 2002, Dioxins in Food, 2000, Dioxins in Food 2001) on dioxins and dioxin-like PCBs, which included cod liver and cod liver oil among the test matrices.

Results and Discussion

All of the samples that formed part of this study showed detectable levels of PCDD/Fs and PCBs apart from one cod liver oil sample in which the PCDD/Fs were below detection limits. The reporting limit quoted for non-ortho-PCBs and dioxin congeners was the limit of determination that prevailed in that instance. The limit varied for different congeners but, at its lowest value, was 0.02 ng/kg fat. For the ortho-PCBs, a reporting limit of 0.10 µg/kg fat was applied.

Insert Table 1 here

Summary data for all individual samples, presented as Σ WHO-TEQ for each class of PCDD/Fs, non-ortho-PCBs, and ortho-PCBs, as well as total Σ PCDD/F+PCB WHO-TEQ, is presented in Table 1. Details of the origin and brand of the products is given elsewhere (FSA 2002). The Σ ICES 7 PCBs is also included. The WHO-TEQ data were calculated using the appropriate WHO-TEF values (Van den Berg et al. 1998) and summed to give Σ WHO-TEQ (all WHO-TEQ data quoted in this work has been calculated in this way). Data are reported as upper-bound, which means that, in cases where congeners were not detected, they were assumed to be present at a concentration equivalent to the detection limit. As most of the samples in this survey were oils, whole weight basis TEQs would be equivalent to the fat weight basis data and have therefore not been reported separately. One sample that was a mixture (of unspecified proportions) of cod liver oil and orange syrup has been excluded from the statistical analysis which is shown in Table 2.

Insert Table 2 here

These data show that the major contribution to the total Σ WHO-TEQ comprises the PCBs, with approximately 55% from the non-ortho-PCBs and 24% from the ortho- PCBs, the remainder coming from the PCDD/F contribution. This is in agreement with observations on fish oil and fish in general, as reported by other workers (Fernandes et al 2004b, Falandysz et al 1994[START_REF] Jiminez | Levels of PCDDs, PCDFs and non-ortho PCBs in dietary supplement fish oil obtained in Spain[END_REF][START_REF] Jacobs | Investigation of selected Organic Pollutants in Farmed Atlantic Salmon, Salmon Aquaculture feed and fish oil componenets of the feed[END_REF]CFIA 2004, Bordajandi et al 2000, MAFF 1999). In particular, when compared to another contemporaneous survey of fish oils commissioned by the Food Safety Authority of Ireland (FSAI, 2002), the contribution from PCDD/Fs to the ΣWHO-TEQ is the same, at 21% (Table 3). The data are not directly comparable with the UK data. The apparent differences between the countries are largely due to the range and relative quantities of products and brands surveyed. In the UK survey, twelve of the 32 samples are derived from a single manufacturer's product. These samples showed consistently lower TEQ levels than the majority of other fish oil supplements. The Irish survey, on the other hand, only contains one sample of this product.

Consequently, the UK data show a much higher proportion of lower concentrations compared to the Irish data. Therefore, the UK data has a lower mean and median, and is more skewed. The Irish data shows a much more even distribution with good agreement between the mean and median values and a very low skewness.

Nevertheless, the results for the two countries are broadly similar. Both sets of data are positively skewed, and the observed TEQ levels are of the same order of magnitude. The distributions for both sets of PCB WHO-TEQ data are shown in Figure 1, which gives histograms for both sets of data.

Insert Table 3 here

Insert Figure 1 here

The congener profiles for samples in this study are generally similar with the PCDD/Fs dominated by the presence of 2378-TCDF, and with a few samples showing higher levels of OCDD. Among the PCBs, 77 and 126 show the highest levels of occurrence among the non-ortho PCBs and the ICES 7 congeners (particularly 101, Deleted: -11 ng/kg WHO-TEQ for the Irish results). Both sets of data are positively skewed, although the UK data show a much higher proportion of lower concentrations (median 0.9 ng/kg WHO-TEQ) compared to the Irish data, which show similar mean and median values (3.6 and 3.2 ng/kg WHO-TEQ respectively). However, data for the PCB TEQ show slightly different ranges with a lower range of 0.2 -29.8 for the Irish data compared to 1.1 -41.5 ng/kg PCB TEQ. These lower values can be ascribed to the greater proportion of samples of products with lower concentrations in the UK survey. [DN: This last sentence doesn't seem to follow] This view is supported by the lower median value of 4.8 ng/kg and a skewness value of 1.7. The Irish data, on the other hand, show a much more even distribution with good agreement between the mean and median values and a very low skewness. Both sets of data will, however, be influenced by the variation in the range of products and brands surveyed, and the data sets are therefore more notable for their similarity rather than their differences. 118, 138 and 153) dominate the ortho substituted PCBs. In general these observations are similar to those observed in other studies (Jacobs et al, Sandell and Kokkenen, FSAI 2002b) A more detailed assessment within individual analyte groups shows that, in order of contribution, PCB 126 is the largest contributor to total Σ WHO-TEQ followed by PCB 118 and PCB 156. 1,2,3,7,2,3,4,7,2,3,7,3,7,8-TCDF also make significant, though smaller contributions. It must be emphasised that these observations refer to the fish oil data in the group of samples discussed in this paper and may reflect the effects of the purification procedures used by manufacturers, rather than the original contaminant levels in the oil. An important implication that follows is that the reduction in levels observed in this data set compared to previous data may be the result of better purification procedures or more selective sourcing of products based on geography, rather than actual reductions in the levels of contamination in the crude oil products. This view needs to be balanced with the observations on gradually declining dioxin and PCB levels in fish in general (Fernandes et al 2004b).

Maximum limits

In May 2001, the EU Scientific Committee on Food (SCF) adopted an opinion on the Risk Assessment of Dioxins and Dioxin-like PCBs in Food based on available scientific information [START_REF] Scf | Opinion of the Scientific Committee on Food, on the Risk Assessment of Dioxins and Dioxin-like PCBs in Food[END_REF]. The SCF fixed a tolerable weekly intake (TWI) for dioxins and dioxin-like PCBs of 14 pg WHO-TEQ/kg body weight. Exposure estimates indicate that a considerable proportion of the Community population still exceeded the tolerable intake. More than 90% of human exposure is through the diet.

The EU therefore adopted a strategy to reduce the presence of dioxins and dioxin-like PCBs in food. The strategy focussed on two aspects -current and future measures to It is evident from the data for PCDD/Fs presented in Table 1, that 12 products contained PCDD/Fs at concentrations in excess of what was to become the limit. Of these, 11 were cod liver oils and one was a salmon oil. A further cod liver oil product exceeded the future action limit. On the other hand, approximately 64% of the products surveyed contained PCDD/F concentrations below the limit. In view of the intention to bring dioxin-like PCBs into the Regulation, assessment is included of the dioxin-like PCB concentrations of the products surveyed. At the time of writing, the current proposal is for a total WHO-TEQ limit (dioxins plus dioxin-like PCBs) of 10 ng/kg WHO-TEQ in marine oils intended for human consumption, based on a contribution of up to 8 from dioxin-like PCBs. On this basis, 13 of the products tested would be in exceedence. This limit will be reviewed in 2007 with the expectation of a significant reduction, which is likely to require the introduction of new purification techniques by producers of fish oil supplements. Norway already applies a national limit of 3 ng/kg TEQ for dioxin-like PCBs, although it should be noted that consumption of fish oil, as well as fish, is particularly high in Norway. Furthermore, Norwegian oil producers already use molecular distillation techniques to reduce the PCB content. According to the data in Table 1, only 8 products contain PCB concentrations below 3 ng/kg WHO-TEQ.

The WHO-TEQ values for cod liver oil (the majority of products -82% of the samples) in this survey (2001)(2002) ranged from 0.18 -8.4 (mean 2.7, median 0.9) ng/kg for PCDD/Fs. This compares to a range of 1.6 -10 (mean 5.7, median 6.7) ng/kg WHO-TEQ for cod liver oil products surveyed in 1996 (MAFF 1997). The decrease in levels indicates a trend towards lower PCDD/F levels relative to earlier surveys. This is borne out by the fact that the median exceeds the mean in the earlier survey, indicating that a greater number of samples were towards the higher end of the range of measured values. A very similar decrease in mean and median can be observed for PCBs, with the mean concentration in cod liver oil falling from 25 to 10 ng/kg ΣPCB WHO-TEQ and the median even more significantly, from 28 to 5.1 (FSA 2002). This is in line with the general downward trend for dioxins and PCBs in food in the UK (Fernandes et al. 2004b), although levels of PCBs remain higher than the corresponding levels of dioxins. The general fall may reflect either alternative sourcing of crude oils and/or the introduction or increased use of carbon filtration for the processing of fish oils, since carbon is known to remove dioxins and furans more effectively than it does PCBs. In any event, to achieve lower PCB levels in fish oil in the future, it is highly probable that producers will need to make further technological changes. The UK Food standards Agency (FSA) has estimated a range of exposures (FSA 2002) for a wide section of the population ranging from schoolchildren to adults and the elderly, based on the data obtained from this survey, and these are summarized in Table 4. The summary distinguishes exposure from cod liver oil products and from other fish oil products, such as salmon oil. These two headings cover bottled product as well as capsules. The exposures were estimated by taking into account the suggested dosages for each of the age groups for which the products were recommended. The summary also provides estimate ranges for consumption of the products alone as well as in combination with the whole of the diet. Estimates for the dietary contribution were made from figures for mean dietary exposure as well as high level dietary exposure and were based on the 1997 Total Diet Study (FSA 2000), the most recent data available at the time.

Insert Table 4 here

Deleted: Thus where to some extent contaminant exclusion methodologies have had some success in reducing the dioxin component of the WHO-TEQ, the emphasis should now move to excluding PCBs as well, from these products. As crude fish oils are not used it is difficult to identify a trend in these data that might reflect a decline in environmental or marine levels of these contaminants. The ranges summarized in Table 4 show that exposure resulting from cod liver oil products was generally higher than that from other fish oil products, although it is important to bear in mind that there were only 6 products in the latter group. The data also show that the consumption of some products at the manufacturer recommended doses would result in exposures exceeding the UK TDI and would provide a higher intake of dioxins than from the whole diet (FSA 2002). The 1997 TDS provided figures for an average dietary exposure of 1.8 pg WHO-TEQ/kg bodyweight/day for adults and 2.2 pg WHO-TEQ/kg bodyweight/day for schoolchildren. Subsequent estimates by the FSA (FSA 2003) show that intakes had apparently fallen by around 50% between 1997 and 2001. Products with PCDD/F and PCB concentrations leading to exposure at the upper end of the ranges given for fish oil supplements would therefore make a significant contribution to the total exposure, amounting to twice the TDI for two of the products. On the advice of the COT, the UK Food Standards Agency requested manufacturers to withdraw the affected batches of those two products (FSA 2002). Fish oil supplement producers were also advised to consider measures to further reduce dioxin and dioxin-like PCB exposure from their products, for example by differential sourcing of fish oils with lower contaminant contents, more effective purification of the oils or changes to recommended dosages and age ranges of products if they were not already doing so (FSA 2002). The Agency also reiterated the need for dosages and age recommendations to be clearly stated in order to minimize the potential for excessive intakes of dioxins and PCBs. A more recent exposure assessment resulting from fish oil consumption made in Norway (Norwegian Scientific Committee for Food Safety, 2004) reports a dioxin and dioxinlike PCB intake of 0.2 pg WHO-TEQ/kg bodyweight/week for adults and 0.9 pg WHO-TEQ/kg bodyweight/week for children. This value is based on the consumption of a popular brand of fish oil and may not reflect the variation observed in the Deleted: exposure Deleted: the number of other fish oil products that were estimated for exposure were 6 as compared to 18 cod liver oil products Deleted: table Deleted:

Deleted: ody

Conclusions

This survey of fish oil dietary supplements for dioxins/furans and dioxin-like PCBs demonstrates continued surveillance in the UK of a product that can make a significant contribution to the total human exposure to these contaminants. The data obtained for dioxins and dioxin-like PCBs are in good agreement with a contemporaneous survey of fish oils carried out by the Food Safety Authority of Ireland (FSAI).

The results of this survey suggest that, while some products contain lower PCDD/F levels compared with earlier surveys, dioxin and dioxin-like PCB concentrations for a number of products in this survey would exceed a proposed EU limit of 10 ng/kg total WHO-TEQ, whilst the majority of products exceed an existing Norwegian limit of 3 ng/kg WHO-TEQ for dioxin-like PCBs. Since a future review is likely to drive limits down towards the values applied by Norway, fish oil purification processes will probably need to be modified to achieve a significant reduction of PCB levels.

Estimates of human exposure to different population groups resulting from consumption of the fish oil products alone, as well as in combination with an average diet, show that some products would give exposures in excess of the TDI.

Consequently, the UK FSA recommended that the relevant manufacturers withdraw affected batches of the products from sale. The toxicological significance of the exposure data for these products needs to be balanced with the many beneficial effects that they are reported to have on human health. It is desirable to continue to benefit from these products, whilst reducing exposure to contaminants. It is therefore recommended that surveillance of these products for PCBs and dioxins should continue.

Formatted

  Deleted: depositions [DN: I don't know but I imagine that run-off is more significant than aerial deposition for PCBs. I should need to check with DEFRA if this is important.] with data suggesting that oil derived from the liver is often more contaminated than muscle tissue oil.

  was recorded, together with any other relevant sample details.

  The data are Deleted: therefore Deleted: directly comparable with the UK data, showing similar concentration ranges for the dioxins (0.2 Deleted: Deleted: -8.4 ng/kg WHO-TEQ, compared to 0.2 Deleted:

  of these compounds to the environment and the setting of maximum and target levels for them in food and animal feed(Verstraete 2002, Council Regulation 2375/2001, Commission Recommendation 2002). Current measures included the setting of maximum permissible limits of PCDD/Fs in a range of foods including fish oils, whereas future measures concentrated on the target PCDD/F level reductions to be achieved in the future, as well as the inclusion of dioxin-like PCBs in the current PCDD/F maximum limits. Thus, from July 2002 limits were set for PCDD/Fs, with a view to including dioxin-like PCBs by 31 December 2004. Due to the complexity of negotiations, this is unlikely to have been achieved before the second half of 2005. Since July 2002, and subsequent to the collection of samples for this survey, the maximum permissible and target levels applying to PCDD/Fs in fish oil intended for human consumption have been 2 ng/kg and 1.5 ng/kg WHO-TEQ, respectively.

Deleted:

  The strategy focussed on two aspects -current and future measures to Deleted: to establish Deleted: of these contaminants Deleted: One of the Deleted: food products that has been assigned Deleted: limits Deleted: is Deleted: Deleted: . The levels that have been set Deleted: are Deleted: as the maximum limit Deleted: as the target limit Deleted:

  As the maximum limits are based on toxicity considerations, this value is similar to the existing PCDD/F TEQ limit of 2 ng/kg. The data for dioxin-like PCB WHO-TEQ (non-ortho + ortho, the UK Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment (COT) issued a statement (DOH 2001) on human dietary exposure to dioxins and dioxin-like PCBs, whereby the UK tolerable daily intake (UK TDI) for combined dioxin and dioxin-like PCBs was revised from 10 pg/kg bodyweight/day to 2 pg/kg TEQ bodyweight/day. This value is in line with that recommended by the EU SCF for dioxins and dioxin-like PCBs of 14 pg WHO-TEQ/kg bw/week[START_REF] Scf | Opinion of the Scientific Committee on Food, on the Risk Assessment of Dioxins and Dioxin-like PCBs in Food[END_REF] or the WHO/JECFA recommended limit of 70 pg WHO-TEQ/kg bw/month (FAO/WHO 2001).

  suggests Deleted: even on their own Deleted: from Deleted: to Deleted: that occur Deleted: exposure Deleted: only Deleted: it was recommended by Deleted: that Deleted: at the upper end of the exposure range Deleted: It was also recommended that f Deleted: with approaches such as Deleted:. It should be recognized that a number of the products with exposures at the lower end of the ranges may already be using these approaches.Deleted: accepted the COT members advice and PCBs in these products. As observed earlier, the gradually declining dioxin and PCB levels in fish or better purification techniques, (or a combination of the two) may account for these reported exposure values.

  Council Regulation (EC) No 2375/2001 of 29/11/2001 amending Commission Regulation No 466/2001 setting Maximum levels for certain contaminants in foodstuffs, Official Journal of the European Communities, L321 06.12.2001, p1-5. Department of Health (DOH), (2001). Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment. Statement on the Tolerable Daily Intake for dioxins and dioxin-like polychlorinated biphenyls. Dept of Health. Available at:www.food.gov.uk/multimedia/pdfs/cot-diox-full. Dioxins in Food (2000), "Intercalibration on Dioxins in Food -An International Study" National Institute of Public Health, Oslo, Norway, ISBN 82-7364-167-8 Dioxins in Food (2001) "Interlaboratory Comparison on Dioxins in Food -Second round of an International Study" National Institute of Public Health, Oslo, Norway, ISBN 82-7364-178-3 Donadio J, Bergstralh E, Offord K, Spencer D and Holley K (1994) A controlled trial of fish oil in nephropathy. New England Journal of Medicine, 331, 1194-1199. Falandysz J, Tanabe S and Tatsukawa R. (1994) Most toxic and highly bioaccumulative PCB congeners in cod-liver oil of Baltic origin processed in Poland during the 1970s and 1980s, their TEQ-values and possible intake. The Science of the Total Environment, 145, 207 -212. FAPAS  (2002), Environmental Contaminants Report 0612. Series 6, Round 12. FAPAS, Central Science Laboratory, Sand Hutton, York, UK..

  Van den Berg M., Birnbaum L., Bosveld A.T.C., Brunström B., Cook P., Feeley M., Giesy J.P., Hanberg A., Hasegawa R., Kennedy S.W., Kubiak T., Larsen J.C., van Leeuwen F.X.R., Liem A.K.D., Nolt C., Peterson R.E., Poellinger L., Safe S., Schrenk D. Tillitt D., Tysklind M., Younes M., Waern F. and Zacharewski T. (1998) Toxic Equivalency Factors (TEFs) for PCBs PCDDs and PCDFs for humans and wildlife. Environmental Health Perspectives, 106, 775-792 Verstraete F (2002), Development and implementation of an EC strategy on dioxins, furans and dioxin-like PCBs in food and feed. Organohalogen Compounds, 55, 1-4.

  Figure 1. Data distribution for UK and Irish studies

Table 1 :

 1 Concentrations of dioxins and PCBs (upper bound) in individual fish oil products. (n=33)

	Sample No	Σ Σ Σ Σ PCDD/F WHO-TEQ	Σ Σ Σ Σ non-ortho PCB WHO-TEQ	Σ Σ Σ Σ o-PCB WHO-TEQ	Σ Σ Σ Σ Total WHO-TEQ	Σ Σ Σ Σ o-PCBs & non-ortho TEQ PCB WHO-	Σ Σ Σ Σ ICES 7 PCBs
		ng/kg fat	ng/kg fat	ng/kg fat	ng/kg fat	ng/kg fat	µg/kg fat
	5897	4.84	7.73	2.06	14.6	9.79	73.1
	5898	4.58	10.3	3.11	18.0	13.4	115
	5899	1.52	9.17	2.18	12.9	11.4	53.2
	5900	2.54	7.58	2.07	12.2	9.65	53.1
	5901	4.55	31.3	10.1	46.0	41.5	267
	5902	0.38	1.17	1.69	3.24	2.86	81.2
	5903	0.85	0.56	2.93	4.34	3.49	115
	5908	0.23	2.48	0.53	3.24	3.01	22.6
	5909	0.86	0.82	0.3	1.98	1.12	8.3
	5910	5.9	20.3	7.4	33.6	27.7	224
	5911	5.29	17.1	4.91	27.3	22.0	146
	6465	6.47	10.0	3.24	19.7	13.3	124
	6466	0.87	0.98	3.81	5.66	4.79	177
	6467	0.49	1.92	0.33	2.74	2.25	12.0
	6468	0.77	1.72	0.41	2.9	2.13	14.6
	6469	5.11	8.33	2.8	16.2	11.1	101
	6470	3.92	9.82	3.1	16.8	12.9	93.9
	6471	0.61	1.25	1.86	3.72	3.11	65.5
	6472	0.93	2.68	2.39	6.0	5.07	81.9
	6473	0.5	1.24	1.89	3.63	3.13	65.6
	6474	0.73	2.24	2.93	5.9	5.17	107
	6475	0.53	1.65	2.81	4.99	4.46	103
	6476	0.57	2.15	2.95	5.67	5.1	107
	6477	0.7	0.62	1.8	3.12	2.42	87.7
	6478	0.73	1.06	1.86	3.65	2.92	89.3
	6479	0.18	0.67	2.37	3.22	3.04	98.4
	6480	6.34	14.2	4.62	25.1	18.8	153
	6481	8.4	18.5	6.04	33.0	24.6	196
	6710	7.87	19.1	5.83	32.8	24.9	196
	6711*	0.32	0.17	0.28	0.77	0.45	7.4
	6712	0.22	0.78	1.03	2.03	1.81	43.8
	6731	1.33	3.17	0.83	5.33	4.0	30.8
	6995	0.31	0.58	0.96	1.85	1.54	39.5

Table 2 :

 2 Summary of concentration data with descriptive statistics One sample not included due to unspecified fish oil content. Skewness -extent of asymmetry of a distribution relative to the mean

	Parameter N=32* Mean Median Skewness Minimum 25th Percentile 50th Percentile 75th percentile Maximum SD Mean+2sd % contribution to Σ Σ Σ ΣWHO-TEQ * Page 22 of 25 Σ Σ Σ Σ PCDD/F Σ Σ Σ Σ non-ortho PCBs Σ Σ Σ Σ o-PCBs Σ Σ Σ Σ WHO-TEQ Σ Σ Σ Σ ICES 7 PCBs ng/kg fat weight 2.5 6.6 2.8 11.9 98.3 0.9 2.4 2.4 5.7 91.6 1.0 1.5 1.6 1.3 0.8 0.2 0.6 0.3 1.9 8.3 0.6 1.1 1.8 3.2 53.2 0.9 2.4 2.4 5.7 91.6 4.6 9.9 3.1 17.1 117.5 8.4 31.3 10.1 46.0 266.6 2.6 7.7 2.2 11.8 63.0 7.6 21.9 7.2 35.6 224.4 21 55 24 Food Additives and Contaminants

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Table 3 :

 3 Comparison with recent literature data(FSAI, 2002).

	Page 23 of 25		Food Additives and Contaminants
	Parameter	Σ Σ Σ Σ PCDD/F TEQ	Σ Σ Σ Σ o-PCB & non-ortho PCB TEQ	Σ Σ Σ Σ WHO-TEQ
	UK, n=32
	Ireland, n=15	UK	Ireland	UK	Ireland	UK	Ireland
	Mean	2.5	3.6	9.4	13.4	11.9	17.0
	Median	0.9	3.1	4.8	14.0	5.7	17.9
	Skewness	1.0	1.0	1.7	0.2	1.3	0.2
	Minimum	0.2	0.2	1.1	0.2	1.9	0.6
	Maximum	8.4	11.0	41.5	29.8	46.0	37.6
	SD	2.6	3.1	9.6	8.3	11.8	10.9
	Mean+2sd	7.6	9.7	28.7	30.0	35.6	38.7
	% contribution to
	Σ Σ Σ Σ WHO-TEQ	21	21	79	79

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Table 4 :

 4 Human exposure arising from fish oil supplement consumption.

	Exposure in pg WHO-TEQ/kg bodyweight/day
	Population group	Cod liver oil products	Other fish oil products
	Fish oil supplements only
	Children, 4-18 yrs	0.02 to 10	0.04 to 4.9
	Adults	0.02 to 7.1	0.04 to 1.7
	Elderly	0.01 to 6.9	0.03 to 1.6
	Fish oil supplements + average diet in 1997
	Children, 4-18 yrs	1.4 to 14	1.4 to 8.1
	Adults	1.8 to 8.9	1.8 to 3.5
	Elderly	1.3 to 8.6	1.3 to 3.3
	Fish oil supplements + high level diet in 1997
	Children, 4-18 yrs	2.5 to 16	2.5 to 11
	Adults	3.1 to 10	3.1 to 4.8
	Elderly	2.3 to 9.8	2.3 to 4.5

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Acknowledgements

The authors are grateful to the UK Food Standards Agency for funding this work


Toine Frank

Henk Bovee

Gerrit Bor

Henri H Heskamp

Ron Laurentius Hoogenboom

Michel W F Nielen

Toine F H Bovee
email: toine.bovee@wur.nl

Ron L A P Hoogenboom

Validation and application of a robust yeast estrogen bioassay for the screening of estrogenic activity in animal feed

Keywords: Screening -biosensor, Receptors Additives/Contaminants: Veterinary drug residues -anabolic steroids, Oestrogens, Veterinary drug residues -zeranol, Drug residues -hormones animal feed, estrogens, pharmaceutical waste, validation, yeast bioassay

Estrogens can be present in animal feed by inclusion of ingredients containing phytoestrogens but also by hormone abuse or even by incidents in which pharmaceutical waste is mixed into feed. The 2002 MPA-incident in the European Union showed that animal feed was contaminated with medroxyprogesterone 17-acetate (MPA), due to syrup that was contaminated with pharmaceutical waste [START_REF] Van Leengoed | The weakest link: medroxyprogesterone acetate in pig feed[END_REF]. It should be emphasised that waste from pharmaceutical steroid production might contain estrogens as well, since the female birth control pill and tablets for hormone replacement therapy (HRT) both contain large amounts of estrogens. Estrogens are known be used illegally to increase meat production, as it stimulates the growth of calves [START_REF] Meyer | Immunoaffinity chromatography and a biotinstreptavidin amplified enzymeimmunoassay for sensitive and specific estimation of estradiol-17-beta[END_REF][START_REF] Guarda | Pathologico-anatomic findings in thymus gland and female sex organs of slaughtered calves after treatment with anabolically effective substances[END_REF][START_REF] Smith | Implant sequence effects in intact male Holstein veal calves: live and slaughter traits[END_REF]. The use of growth promoters for fattening purposes in cattle has been banned in the European Union since 1988 (EC Council Directive 96/22). Countries outside the EU have a list of substances that are not prohibited or have no legislation at all. The EU ban prohibits all substances having hormonal action and does not provide a black-list of substances. In practice residue analysis, while aiming at consumer protection, fair trade and enforcement of the ban, is still carried out on specific target compounds (EC Council Directive 96/23) and can thus fulfil the control only to a limited extent. Histopathological effects in tissues can be examined and used to establish to use of growth promoting agents [START_REF] Groot | Histopathological effects of boldenone in cattle[END_REF]. However, these tests are only useful at the end of the food production, as they can not be used to test preparations, urine or animal feed samples. Thus they are not suited to be used as an early warning system. Alternatively, receptor based assays can be used to detect all compounds having affinity for a given receptor (Garcia-Reyero et al., 2000;[START_REF] Mueller | Overview of in vitro tools to assess the estrogenic and antiestrogenic activity of phytoestrogens[END_REF]Michelini et al., 2004). In contrast to receptor binding assays, reporter gene bioassays also mimic the transactivation step and can distinguish between receptor agonists and receptor antagonists [START_REF] Mueller | Overview of in vitro tools to assess the estrogenic and antiestrogenic activity of phytoestrogens[END_REF]. This feature is very helpful in detecting known and unknown compounds, as receptor stimulation plays a key role in the mechanism of action of growth promoters. Several assays have been developed for this purpose, using both mammalian and yeast cells. In general transcription activation assays based on mammalian or more particular human cell lines have been shown to be more sensitive than yeast based assays, and may be able to identify compounds that require human metabolism for activation into their active state [START_REF] Legner | Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line[END_REF][START_REF] Hoogenboom | Estrogenic activity of estradiol and its metabolites in the ER-CALUX assay with human T47 breast cells[END_REF][START_REF] Sonneveld | Comparison of in vitro and in vivo screening models for androgenic and estrogenic activities[END_REF]).

However, yeast based assays have several other advantages. These include low costs and easier handling, lack of known endogenous receptors that may compete with the activity under investigation, use of media that are devoid of steroids [START_REF] Breithofer | Regulation of human estrogen receptor by phytoestrogens in yeast and human cells[END_REF][START_REF] Graumann | Monitoring of estrogen mimics by a recombinant yeast assay: synergy between natural and synthetic compounds?[END_REF][START_REF] Witters | Detection of estrogenic activity in Flemish surface waters using an in vitro recombinant assay with yeast cells[END_REF], and last but not least, yeast cells assays are extremely robust and survive extracts from dirty sample matrices such as urine [START_REF] Bovee | Validation of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein, for the screening of estrogenic activity in calf urine[END_REF]Nielen et al., submitted). Recently we developed a novel yeast estrogen bioassay (REA), stably expressing human estrogen receptor α (hERα) and expressing yeast enhanced green fluorescent protein (yEGFP) in response to estrogens.

The only handling required to perform this assay is the addition of the yeast suspension to a sample extract, followed by fluorescence measurement after 24 h. This assay is completely performed in a 96 well plate and fluorescence is measured directly in intact yeast cells in a plate reader. This assay is relatively simple and sensitive, as shown by an EC50 value for E2β of 0.7 nM (Bovee et al., 2004a). Furthermore, to investigate the specificity of the assay, a large number of chemically different compounds with known estrogenic, but also other hormonal activities were tested. All these estrogenic compounds caused a dose-related increase in the production of green fluorescent protein, whereas the gestagens progesterone and medroxyprogesterone 17-acetate showed no response and the androgen 17β-testosterone only showed a very weak response (Bovee et al., 2004b). This bioassay has been fully validated for calf urine [START_REF] Bovee | Validation of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein, for the screening of estrogenic activity in calf urine[END_REF] and recently acquired an ISO17025 accreditation status in The Netherlands for this matrix.

The present study describes the validation of the assay for animal feed. Regulation (EC) No 882/2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules, that shall apply from 1 January 2006, prescribes that laboratories involved in the analysis of official samples should work in accordance with internationally approved procedures or criteria-based performance standards and use methods of analysis that have, as far as possible, been validated. Therefore, the yeast estrogen bioassay for the screening of estrogenic activity in animal feed was validated as the Commission Decision of 12 August 2002 of the EC prescribes in EC Decision 2002/657. As this decision prescribes that validation of a qualitative screening method must include verification of the detection capability (CCβ), specificity and stability, these performance characteristics are determined in the present study. 17β-Estradiol (E2β), 17α-ethynylestradiol (EE2), diethylstilbestrol (DES), zearalenone and equol were chosen as model compounds, because E2β, EE2 and DES are priority compounds for both hormone abuse and pharmaceutical waste and because of the differences in their polarity, with equol being the most polar and DES being the most apolar compound. Furthermore, zearalenone and equol are natural compounds that may occur in animal feed. Zearalenone is an estrogenic mycotoxin and equol is an estrogenic metabolite of the less estrogenic phytoestrogen daidzein. As representatives for feed samples, 7 wet pulp feed samples (pigs), 7 dry grinded feed samples (ruminants, poultry, pigs) and 6 regular milk replacer samples (calves) were used. In this study we also present long-term performance data for the screening of estrogenic activity in animal feed. The applicability of this early warning bioassay system was further demonstrated by screening and identifying the estrogenic activity found in wet pulp feed samples of the MPA-incident and in the routine screening of fish feed samples.

Experimental

Chemicals

Water was purified using a Milli-Q Gradient A10 system (Millipore, Bedford, MA, USA).

Acetonitrile and methanol were from Biosolve (Valkenswaard, The Netherlands). Ammonium sulphate, dimethyl sulfoxide, sodium acetate and sodium carbonate were obtained from Merck (Darmstadt, Germany). The compounds 17β-estradiol (E2β), 17αethynylestradiol (EE2), zearalenone, 17β-testosterone, progesterone and L-leucine were purchased from Sigma,. equol from Apin Chemicals (Abingdon, U.K.), diethylstilbestrol ammonium sulphate (1.7 g per l), dextrose (20 g per l) and ammonium sulphate (5 g per l) and was supplemented with L-leucine (60 mg per l).

Samples and sample treatment

As representatives for 20 animal feed samples, 7 wet pulp feed samples which are normally used to feed pigs, 7 dry grinded (1 mm) feed samples which are normally used to feed ruminants, poultry and pigs and 6 regular milk replacers which are normally used to feed calves were used. The extraction procedure described below is partly comparable to the procedure that is commonly applied to the isolation of steroids from urine samples [START_REF] Blankvoort | Detection of hormonal anabolic compounds in calf urine and unverified growth-promoting preparations: Application of the AR-LUX bioassay for screening and determination of androgenic activity[END_REF][START_REF] Nielen | Bioassay-directed identification of estrogen residues in urine by liquid chromatography electrospray quadrupole time-of-flight mass spectrometry[END_REF]. Stock solutions of E2β, EE2, DES, zearalenone, equol, testosterone and progesterone were prepared in DMSO (respectively 0.5; 0.5; 0.5; 100, 10000, 50 and 50 µg ml -1

). Feed samples were spiked with E2β, EE2 and DES at 5 and 10 ng per g, zearalenone at 1250 ng per g and equol at 200 µg per g.

For extraction, 1 g of blank feed and spiked feed samples were mixed with 4 ml methanol and 4 ml sodium acetate pH 4.8. Samples were incubated for 10 min in an ultrasonic bath and subsequently mixed for 15 min head over head. Samples were centrifuged at 3500 g and 4 ml of the upper liquid phase was brought in a glass tube. Next, the pH was adjusted to 4.8 using 4 N acetic acid and the extract was subjected to solid phase extraction (SPE) on a C18 column. This column was previously conditioned with 2.5 ml methanol and after that with 2.5 ml sodium acetate pH 4.8. Subsequently, this column was washed with 1.5 ml 10% (w/v) sodium carbonate solution, 3.0 ml water, 1.5 ml sodium acetate pH 4.8, 3.0 ml water and finally with 2 ml methanol/water (50/50 v/v). The column was air-dried and eluted with 4 ml acetonitrile. The eluate was applied to an NH 2column that was previously conditioned with 3.0 ml acetonitrile. The acetonitrile eluate thus obtained was evaporated to 3 ml under a stream of nitrogen gas. A 100 µl or 200 µl aliquot of this extract, equivalent to respectively 0.017 or 0.033 g animal feed, was transferred to a 96 well plate in triplicate and 50 µl of a 4% DMSO solution was added to each well. To remove the acetonitrile, the plate was dried overnight in a fume cupboard and was then ready to be screened on estrogenic activities with the yeast estrogen bioassay. In the same way and in each series a reagent blank was prepared.

Yeast estrogen bioassay

The yeast cytosensor expressing the human estrogen receptor α (hERα) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to estrogens was developed in-house and has been described previously (Bovee et al., 2004a(Bovee et al., ., 2004b)). In short, an agar plate containing the selective MM/L medium was inoculated with the yeast ERα cytosensor from a frozen -80°C stock (20% glycerol v/v). The plate was incubated at 30°C for 24-48 h and then stored at 4°C. The day before running the assay, a single colony of the yeast cytosensor was used to inoculate 10 ml of selective MM/L medium. This culture was grown overnight at 30°C with vigorous orbital shaking at 225 rpm. At the late log phase, the yeast ERα cytosensor was diluted in MM/L, giving an OD at 604 nm in the range of 0.07-0.13. For exposure in 96 well plates, aliquots of 200 µl of this diluted yeast culture were pipetted into each well, already containing the extracts of the feed samples (see section 2.2). A 17β-estradiol dose-response curve was included in each exposure experiment. Aliquots of 200 µl of the diluted yeast culture were pipetted into each well of a 96 well plate and exposure to different doses of 17β-estradiol was performed through the addition of 2 µl of 17β-estradiol stock solutions in DMSO. Each feed sample extract and each 17β-estradiol stock was assayed in triplicate. Exposure was performed for 0 h and 24 h. Fluorescence at these time intervals was measured directly in a CytoFluor Multi-Well Plate Reader (Series 4000, PerSeptive Biosystems) using excitation at 485 nm and measuring emission at 530 nm. The densities of the yeast culture at these time intervals were also determined by measuring the OD at 630 nm.

This was done to check whether a feed sample was toxic for yeast. ) were analysed in the bioassay in order to determine the decision limit CCα and the detection capability CCβ of the yeast estrogen bioassay. In each series an extract of a reagent blank was made as well and was also analysed in the bioassay. .

Assay validation and data analysis

Stability

For the determination of the stability of the feed samples, aliquots of 1 g of a blank and spiked wet feed sample were stored at -20°C and aliquots of 1 g of a blank and spiked dry feed sample and of a blank and spiked milk replacer were stored at room temperature in the dark. At certain times, 1 g aliquots of these three blank and spiked feed samples:

E2β or EE2 at 5 ng g -1 , DES at 10 ng g -1 , zearalenone at 1.25 µg g -1 and equol at 200 µg g -1

, were taken and extracts were made and 200 µl aliquots were analysed in the bioassay.

Results and discussion

The performance characteristics detection capability CCβ, specificity and stability of the yeast estrogen bioassay for the screening of estrogenic activity in animal feed were determined in order to validate the bioassay and to test its possible use to screen low levels of estrogens in animal feed. There is no permitted limit for estrogens, but for the routine screening an action level of 5 ng of 17β-estradiol per gram feed was selected for validation. This is very low, as with illegal use or accidents with pharmaceuticals, much higher contents are expected. Theoretically, assuming no recovery loss during the sample treatment, the 200 µl extract, equivalent to 0.033 g of feed, of the 5 ng g -1 17β-estradiol spike that is added to a well in a final well volume of about 250 µl, results in a final concentration of 2.45 nM 17β-estradiol in the well. We have shown previously (Bovee et al., 2004b) that in the yeast bioassay the concentration where half-maximal activation is reached (EC50), is about 0.7 nM for 17β-estradiol and so theoretically the 5 ng g ) and zearalenone and equol were spiked at higher levels, respectively 1.25 and

200 µg g -1
.

With the used clean-up procedure it is expected that the more apolar compounds will have slightly lower recoveries. DES, the most apolar compound tested, was therefore also spiked at the two times higher level of 10 ng g -1 .

In the routine screening of animal feed samples, the 200 µl extracts were occasionally (<1% of the samples) cytotoxic. In order to be able to screen these extracts on estrogenic activity, 100 µl instead of 200 µl aliquots of these extracts were analysed in the bioassay.

The 100 µl aliquots never showed cytotoxicity. In a second validation experiment, feed samples were spiked with E2β, EE2 and DES at 10 ng g -1

, zearalenone at 1.25 µg g -1 and equol at 200 µg g -1 and 100 µl aliquots of the extracts, equivalent to 0.017 g feed, were

analysed in the bioassay.

Detection capability (CCβ) with 200 µl aliquots of the feed sample extracts

In this experiment feed samples were spiked with E2β and EE2 at 5 ng g -1

, DES at 5 and 10 ng g -1

, zearalenone at 1.25 µg g -1 and equol at 200 µg g -1

.

Table I shows the results of the yeast estrogen bioassay of the 20 blank and the 20 spiked feed samples using 200 µl of the extracts. The signals are the responses obtained after 24 h of exposure that are corrected for the responses obtained at 0 h (t24-t0) and for the response (t24-t0) obtained with a reagent blank. All these responses are the mean of a triplicate and in general the %CV of these triplicates is less than 5% (data not shown). After 24 h of exposure there were no differences in the OD at 630 nm, meaning that no toxic effects on the yeast could be observed (data not shown). Most blank feed samples have corrected responses that have a negative value (see Table I). These negative values are due to the relatively low responses of these blank feed samples when compared to the corresponding reagent blank, for which they are corrected. Also the 17β-estradiol standard curve corrected for the response obtained with a reagent blank starts with low negative values (see Figure 1).

Furthermore, these negative values are very low when compared to the signals that were measured. For example, the t0 and t24 measurements of sample #1 were respectively 101 ± 3 (standard deviation) and 307 ± 2, resulting in a t24-t0 signal that was 206 ± 2.

The corresponding reagent blank gave a signal of 222 ± 4 and as a result the corrected signal for blank feed #1 is -16. However, the signals of the blank dry feed samples are the lowest and although there is no toxic effect, these extracts probably contain substances that give a little inhibitory effect in the bioassay. The mean response value of the 20 blank feed samples X B is -28 with a corresponding standard deviation S B of 35. The calculated decision limit CCα, being the mean plus 3 times the standard deviation, for the corrected fluorescence response is therefore 77. The use of the assay can be seen as a qualitative ON/OFF method. Samples giving a signal lower than the CCα are compliant: OFF.

Samples giving a response higher than the determined CCα are suspect: ON. The results in Table I demonstrate that the blank feed samples fulfil the CCα criterion, meaning that all blank feed samples give a response that is lower than the CCα. All blank samples are thus compliant (method result: OFF). For DES spiked at 5 ng per gram feed, 10 of the 20 spiked samples gave a response that is lower than the determined CCα of 77. This compound does not fulfil the CCβ criterion at the 5 ng g -1 level. However, DES spiked at 10 ng g -1 fulfils the CCβ criterion, as all 20 spiked samples gave a response that is higher than the CCα and are thus suspect (method result: ON). All but two of the E2β, EE2, zearalenone and equol spikes gave a response that is higher than the determined CCα and are thus classified as suspect samples. Only sample #4 for E2β and sample #4 for EE2 showed a response that is lower than the CCα and are thus classified as compliant.

These are false compliant results. However, overall the results fulfil the CCβ criterion, meaning that at least 19 out of the 20 spiked samples gave a signal above the CCα and are thus classified as suspect (method result: ON) (β=5%).

[INSERT In general the spikes in milk replacer gave about the same mean signals (X) as all 20 feed samples, spikes in dry feed gave lower responses and spikes in wet feed showed higher responses. Probably the recovery of the spiked compounds is best from wet feed and recoveries from milk replacer and dry feed are lower. Recovery losses with the applied extraction procedure are mainly determined by the relative polarity of a compound and the interaction with matrix particles. So, the lower recoveries from milk replacer and dry feed could be expected as these matrices contain rather apolar particles, just as the compounds tested, and so interactions from the compounds with matrix particles are probably stronger for milk replacer and dry feed and are the strongest for DES, being the most apolar compound of the compounds tested. These recovery losses and/or the possible inhibitory effect in the bioassay of the dry feed extracts are probably the reason for the lower responses found with especially the DES spikes in dry feed.

However, the bioassay method is not affected to such an extent that it does disturb the qualitative compliant/suspect (OFF/ON) screening principle of the method.

[INSERT FIGURE 1 ABOUT HERE]

Detection capability (CCβ) with 100 µl aliquots of the feed sample extracts

In this experiment feed samples were spiked with 10 ng g -1 of E2β, EE2 and DES, 1.25 µg g -1 of zearalenone or 200 µg g -1 of equol. Table II shows the results of the yeast estrogen bioassay of the 20 blank and the 20 spiked feed samples using 100 µl of the extracts. All these responses are the mean of a triplicate and in general the %CV of these triplicates was less than 5% (data not shown). Again, and as expected, there were no differences in the OD at 630 nm after 24 h of exposure, meaning that no toxic effects on the yeast were observed (data not shown). Just as for the 200 µl aliquots, 100 µl aliquots of the extracts of the blank feed samples have corrected responses with a negative value (see Table II). The mean response value of the 20 blank feed samples X B was -11 with a corresponding standard deviation (S B ) of 16. As a result the calculated decision limit CCα for the corrected fluorescence response was calculated to be 37. The results in [

INSERT TABLE II ABOUT HERE]

Although the validation was performed as a qualitative screening method for the determination of estrogenic activity in animal feed and is used as an ON/OFF assay, the responses of the feed extracts can be converted to concentrations using a 17β-estradiol standard dose-response curve that is also corrected for a reagent blank (see Figure 1).

When the CCα of 37 is calculated from this standard curve this quantitative approach results in a concentration of 0. gram was calculated and thus the recovery of the method for this compound was 45%.

The recovery for equol could not be calculated as this compound was spiked with a higher amount, resulting in a mean response value that was equal to the maximum response of the 17β-estradiol standard dose-response curve. Although this quantitative approach is beyond the scope of this paper and is not needed for the validation of a qualitative screening method, it showes that the recovery of the sample treatment is about 45% for 17β-estradiol, 17α-ethynylestradiol and zearalenone. For DES, the most apolar compound tested, the recovery is about 25%.

Specificity and interference

The specificity of the yeast estrogen bioassay was determined with three blank animal feed samples, one wet, one dry and one milk replacer that were spiked with a high dose of 17β-testosterone or progesterone (1000 ng g -1

). Interference was checked with these three blank animal feed samples by spiking them with a low dose of E2β, EE2, DES at ng g -1

, zearalenone at 1.25 µg g -1 and equol at 200 µg g The stability of the estrogenic compounds in animal feed samples was determined with three animal feed samples, one wet, one dry and one milk replacer that were spiked.

Aliquots of 1 g of a blank and spiked wet feed sample were stored at -20°C and aliquots of 1 g of a blank and spiked dry feed sample and of a blank and spiked milk replacer were stored at room temperature in the dark. At certain times, 1 g aliquots of these three IV. Only the equol spike in the wet feed that was stored at room temperature in the dark for 55 days gave a corrected response of -15 that was below that of the CCα value of 77. This is a false compliant. The corresponding blank wet feed gave a response of 379 and is a false suspect. However, it is much more likely that there was an interchange of these two samples. The 200 µl extracts of the dry feed at day 0 were toxic to the yeast cells. This could be seen at the OD at 630 nm, while the OD was about 1.0 for the controls, the wet feed and the milk replacer, the OD at 630 nm with 200 µl extracts of this dry feed was below 0.6 (data not shown). Extracts are considered to be toxic if the OD measured at 630 nm is less than 70% of the OD of the control samples. Extracts of day 0 were kept at 4°C and from day 2 on, exposures with the extracts of this dry feed were performed with 100 µl instead of 200 µl. As a consequence, a CCα value of 37 instead of 77 was applied. The 100 µl aliquots of this dry feed sample were analysed and were never cytotoxic. The 200 µl aliquots of this dry feed sample were not always cytotoxic and when not cytotoxic they fulfilled the criteria (data not shown). The data in Table IV demonstrate that the feed samples can be stored at their specific conditions for up to 98 This method is in routine use at RIKILT for more than one year now. In that period, the method was applied more than 40 times to series of about 20 feed samples and showed good robustness: feed extracts were almost never cytotoxic (less than 1% of the samples) and when cytotoxic, the 100 µl method could be used. Reagent blanks were always found compliant and the spiked reagent blanks were always screened suspect.

Figure 2 shows a control chart, showing the decision limit CCα and the corrected responses for the reagent blank, blank feed control, spiked reagent blank and the spiked feed control. Only once a blank feed control was found suspect and once a spiked feed control was found compliant. However, the extract of this spiked feed control was coloured. As this control feed was used several times, showing no colour and when spiked was always found suspect, this indicates that only once the clean-up procedure did not work optimally, resulting in a coloured extract that gave a signal just below the determined CCα. The one time that a blank feed control was found suspect, the signal was just above the determined CCα. Just as for the stability study, the data in Figure 2 demonstrate that the method is robust and that the outcome of the assay, compliant/suspect, is reproducible.

[INSERT FIGURE 2 ABOUT HERE]

The method is validated as a qualitative screening method for estrogenic activity in feed and thus suspect screened samples need to be confirmed. Therefore, we have set up a liquid chromatochraphy (LC) system that fractionates feed extracts using a dual 96 well fraction collector system via effluent splitting. The LC-fractions in the first 96 well plate are used for the detection of estrogenic activity using the yeast estrogen bioassay. The suspect well numbers are then correlated with the LC retention time and results from the analysis of the corresponding "suspect" wells in the second 96 well plate with QTOFMS/MS with exact mass measurement. Figure 3 shows this generic setup for the fractionation and identification of (un)known bioactive substances using LC/bioassay/QTOFMS(/MS) [START_REF] Nielen | Bioassay-directed identification of estrogen residues in urine by liquid chromatography electrospray quadrupole time-of-flight mass spectrometry[END_REF]Nielen et al., in press).

[

INSERT FIGURE 3 ABOUT HERE]

During the MPA-incident in the European Union in 2002 there were problems with pregnant pigs in breeding farms [START_REF] Van Leengoed | The weakest link: medroxyprogesterone acetate in pig feed[END_REF]. To determine the cause of the problems, wet feed samples were send to RIKILT for testing. Part of this testing was to investigate these samples with our estrogen bioassay. The samples were screened suspect in the estrogen bioassay. Knowing that the estrogen bioassay is specific for estrogens, based on the fact that other hormones like 17β-testosterone, progesterone and medroxyprogesterone 17-acetate (MPA) do not give a response in this test (Bovee et al., 2004b), the responsible substances had to be estrogenic. It was decided to fractionate an extract. Figure 4 shows the estrogenicity biogram of a fractionated wet feed and shows that fraction number 24 and 25 are estrogenic. In this system the corresponding retention time of these fractions was equal to that of 17β-estradiol. This was the first indication that hormones could be responsible for the problems in the pregnant pigs and that 17β-estradiol might be involved. This was confirmed by GC/MS analysis. The identified 17β-estradiol turned out to be part of the problem, since early thereafter it became clear that MPA was the main issue in the feed contamination. As hormones used for hormone replacement therapy (HRT) and the female contraceptive are two major pharmaceuticals, both containing large amounts of estrogens, this demonstrates the applicability of the bioassay method as an early warning system for pharmaceutical waste in animal feed. This is even true in cases where the estrogens are part of the contamination, because the test is not influenced by the presence of other hormones, i.e. there is no interference. It also demonstrates that the bioassay can be used to identify a fraction that contains the estrogenic activity.

[INSERT FIGURE 4 ABOUT HERE]

Figure 5 shows the estrogenicity biogram of a fractionated fish feed that was found suspect in our routine estrogen bioassay method. Figure 5 shows that fraction number 22 and 23 are estrogenic. In this system (see Figure 3) the corresponding retention time from 7.00 to 7.66 minutes was equal to that of 17β-estradiol. Using the LC/bioassay/MS setup the 17β-estradiol was indeed confirmed by exact mass measurement in both fractions. This demonstrates the applicability of the bioassay method as an early warning system for hormone abuse. It also demonstrates that the bioassay can be used as an inexpensive LC detector to identify a fraction that contains the estrogenic activity. The natural steroids are orally not very active, but the 17-alkylated steroids, like the oral contraceptive 17α-ethynylestradiol, are usually orally active in mammalians and besides as waste, might thus be abused in animal feed. Less well known is the illegal use of hormones in aquaculture. In eel the natural estrogen 17β-estradiol is orally effective.

Besides the growth effect, 17β-estradiol affects the sex determination of eels. After 1 year, 70% of eels fed with 60 mg 17β-estradiol/kg in their diet were identified as female, compared to 26% of the control group [START_REF] Degani | Effects of 17β-estradiol and grouping on sex determination of European eels[END_REF]. In another study these percentages were 88% in the group treated with 17β-estradiol and 6% in the control group. This latter study also showed that after 600 days, males reached a weight of 114,1 ± 4.3 and the females 171 ± 11.7 (g ± SE) [START_REF] Degani | Growth differences and growth hormone expression in male and female European eels[END_REF]. Thus the abuse of 17β-estradiol for the growth of eel can be very lucrative.

Conclusions

The data presented and the determined performance characteristics prove that the yeast estrogen bioassay can detect low levels of 17β-estradiol (5 ng g -1

), 17α-ethynylestradiol

(5 ng g -1

), diethylstilbestrol (10 ng g High levels of the androgen 17β-testosterone or the gestagen progesterone did not give a response in the bioassay, nor did they interfere with the screening result of spiked estrogens at low levels. The data demonstrate that the bioassay is specific for estrogenic substances. The observation that feed samples could be stored at their specific conditions for up to 98 days without influencing the screening result showed that estrogens in feed are stable. The stability study also demonstrates that the outcome of the assay, compliant/suspect, is reproducible and that the procedure is robust. As all the performance characteristics met the criteria that were put forward in EC Decision 2002/657 for validation of a qualitative screening method, the above described cleanup/yeast estrogen bioassay procedure is proven to be valid for the determination of estrogenic activity in animal feed. The clean-up procedure for feed samples is relatively simple and the yeast estrogen bioassay, using yEGFP as a reporter protein, is sensitive, rapid, convenient and reproducible. Combined this resulted in a low cost bioassay that is suited to be used as a high throughput system for the screening of estrogenic activity in complex animal feed samples. Due to the good sensitivity of the bioassay, only 1 gram of feed is enough to be processed. This results in a 3 ml acetonitrile extract of which 0.6 ml Mean (X) fluorescence response in the yeast estrogen bioassay of 20 blank and 20 spiked feed samples and the determination of the decision limit CCα using 200 µl aliquots of the extracts for exposure. Mean (X) fluorescence response in the yeast estrogen bioassay of 20 blank and 20 spiked feed samples and the determination of the decision limit CCα using 100 µl aliquots of the extracts for exposure. a The specificity of the yeast estrogen bioassay was determined with three blank feed samples that were spiked with a high dose of 17β-testosterone (A) or progesterone (B), both at a level of 1000 ng per gram. Interference was checked with the same three feed samples by spiking them with a low dose of E2β, EE2, DES, zearalenone or equol in combination with the high dose of either 17β-testosterone or progesterone. Exposures were performed with 200 µl aliquots of the extracts. In bold and grey the samples that not fulfil the criterion. a Exposures were performed with 100 µl aliquots of the extracts, because 200 µl of these extracts were toxic for the yeast cells. The corresponding CCα value is 37 (see Table 1).

b Exposures were performed with 200 µl aliquots of the extracts. The corresponding CCα value is 77 (see Table 2). In bold and grey the samples that not fulfil the criterion.

F o r P e e r R e v i e w O n l y

F o r P e e r R e v i e w O n l y

#DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0!

  from ICN. Isolute NH 2 extraction columns (100 mg) from IST (Hengoed, U.K.) and Bond Elut C18 solid phase extraction columns (500 mg) from Varian (Harbor City, CA, USA). Dextrose and yeast nitrogen base without amino acids and without ammonium sulphate were obtained from Difco (Detroit, MI, USA). The minimal medium with L-leucine medium (MM/L) consisted of yeast nitrogen base without amino acids and without

  blank and spiked feed samples: E2β or EE2 at 5 and extracts were made and 200 µl aliquots were analysed in the bioassay. Fluorescence signals at 24 h were corrected for the signals obtained at 0 h and the response of a reagent blank. Results are shown in Table

  disrupting the screening result, assuming that the blank and equol spike of the wet feed were interchanged at day 55.[INSERT TABLE IV ABOUT HERE]3.5 Application of the yeast estrogen bioassay for the screening of estrogenic activity in animal feed

[

  by using 200 µl of the extracts. All 20 blank feed samples gave a response lower than the determined CCα of 77 and thus there were no false suspect results. Signals of all 20 DES, zearalenone and equol spikes were higher than the determined CCα of 77 and thus fulfilled the CCβ criterion. Signals of 19 out of 20 17β-estradiol and 17α-ethynylestradiol spikes were higher than the determined CCα of 77 and thus also fulfilled the CCβ criterion. When samples are cytotoxic, 100 µl of the extracts instead of 200 µl can be used. As a consequence, the applied CCα must be 37 instead of 77 and the action levels of 17β-estradiol and 17α-ethynylestradiol change from 5 to 10 ng per gram of feed.

Figure 1 .

 1 Figure 5.

Figure 2 .

 2 Figure 2. Longterm response (n=48) in the yeast estrogen bioassay of the reagent blank, the reagent blank spiked with 5 ng 17β-estradiol per gram, the blank feed sample and a blank feed sample spiked with 5 ng 17β-estradiol per gram.

Figure 3 .

 3 Figure 3. Generic setup for the fractionation and identification of unknown bioactive substances using LC/bioassay/QTOFMS(/MS).

Figure 4 .

 4 Figure 4. Estrogenicity biogram of a wet feed sample from the MPA-incident that was found suspect in the yeast estrogen bioassay. Extracts were separated on a C18-column and fractions of 0.33 minutes were collected and tested in the bioassay.

Figure 5 .

 5 Figure 5. Estrogenicity biogram of a fish feed sample that was suspect in the routine screening. Extracts were separated on a C18-column and fractions of 0.33 minutes were collected and tested in the bioassay.

a

  Mean, SD and CCα (CCα = Mean of the blank + 3.0*SD of the blank) are determined from 20 feed samples. In bold and grey the samples that not fulfil the criterion.(b) The extracts were prepared on two different days and were analysed in the yeast estrogen bioassay in one exposure. These sample preparations and the exposure were performed within a time period of three weeks.

Figure 1 .

 1 Figure 1. Response of the yeast estrogen cytosensor after a 24 h exposure to 17β-estradiol. Exposure was started by adding 2 µl of a 17β-estradiol stock solution in DMSO to 200 µl of a yeast culture. Fluorescence was determined after 0 and 24 hours as described in Experimental (see section 2.3 Yeast estrogen bioassay). Fluorescence signals are the mean of a triplicate with sd and are corrected for the signals obtained at 0 h and a reagent blank.

F

  a response that was lower than the CCα. As the rate of false suspect results is 1% (α=1%), it is not expected to find a false suspect result. As can be seen in TableII, all but two of the E2β, EE2, DES, zearalenone and equol spikes gave a response that was higher than the determined CCα and were thus classified as suspect samples. Only sample #2 for DES and sample #19 for equol gave a response that was lower than the CCα and were thus classified as compliant. These are false compliant results. Sample #2 for DES is a real false compliant, but in sample #19 the equol was possibly not spiked. However, all spikes fulfil the CCβ criterion, meaning that at least 19 out of the 20 spiked samples gave a signal above the CCα and are thus classified as suspect (β=5%).

	feed samples gave
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	13

Table II demonstrate that the blank feed samples fulfil the CCα criterion, meaning that all blank

  Whenlooking at the data in TableIII, they show that neither the androgen 17β-testosterone nor the gestagen progesterone gave a response in the bioassay, as the blank samples gave about the same response as the corresponding 17β-testosterone and progesterone spiked samples. Results in Table III also show that both of these compounds do not interfere with the screening result of the estrogenic compounds E2β, DES, EE2, zearalenone or equol, as all signals of the estrogen spiked samples are well above the CCα value of 77. Moreover, responses of the estrogen spiked samples are about the same as spiking them in combination with the high dose of either 17β-testosterone or

	progesterone. Despite the one false suspect result, the data demonstrate the specificity of
	the bioassay.
	[INSERT TABLE III ABOUT HERE]
	3.4 Stability of estrogens in feed
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

-1 in combination with the high dose of either 17β-testosterone or progesterone. Extracts were made and 200 µl aliquots were analysed in the bioassay. Fluorescence signals at 24 h were corrected for the signals obtained at 0 h and the response of a reagent blank. Results are shown in Table

III

.

Strictly seen, the milk replacer spiked with 1000 ng progesterone per gram gave a false suspect result. However, this milk replacer spiked with 1000 ng progesterone per gram gave a signal of 80 that was just above the determined decision limit CCα of 77.

  the bioassay screening in triplicate while the remaining 2.4 ml can be used for identification and conformation using the LC/bioassay/QTOFMS setup. This method is in routine use at RIKILT for more than one year now. It showed good robustness: feed extracts were almost never cytotoxic (less than 1% of the samples).

	Flu o re sce n ce	F o r P e Sample pretreatment LC-fractions MPA-feed is needed for Reagent blank and blank feed controls were always found compliant and the spiked reagent blank and spiked feed controls were always screened suspect. The examples of the MPA-incident with wet pig feed and the fish feed demonstrate the applicability of the bioassay method as an early warning system for pharmaceutical waste and hormone abuse respectively. F o r P e F o r P e F o r P e F o r P e 150 350 550 750 Fluorescence F o r P e and clean-up 600 LC-fractions fish feed Feed control chart 200 400 600 800 1000 Fluorescence decision limit CCα=77 spiked reagent blank spiked blank feed reagent blank blank feed F o r P e Gradient Liquid Chromatography Optional on-line QTOF MS/MS Flo w F o r P e 0 100 200 300 400 500 -50 Retention time (min) e Fraction nummer 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 P 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 200 250 300 F l u o r F 100 e 150 s c e o n c r 50 e
		e r R e v i e w e r R e v i e w e r R e v i e w e r R e v i e w e r R e v i e w 0.01 0.1 1 Concentration E2 [nM] 10 e r R e v i e w 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 100 1000 e r Bioactivity screening e r e r Week number R e v i e Bioassay plate R R Collection plate 0.001 -50 -200 Figure 1. 0 1 3 5 7 9 Identification off-line e e -(LC)QTOFMS/MS -GC(HR)MS(/MS) v i e v i e Figure 2. w w w
		Figure 3.	O n l O n l O n l O n l O n l O n l O n l O n l O n l
		Figure 4.	y y y y y y y y y
		20 24 27 28

  Mean, SD and CCα are determined from 20 feed samples. The extracts were prepared on six different days and were analysed in the yeast estrogen bioassay in five separate exposures. These sample preparations and exposures were performed within a time period of 3 months. In bold and grey the samples that not fulfil the criterion.Specificity and interference of the yeast estrogen bioassay, checked with feed samples spiked with a high dose of 17β-testosterone (A) or progesterone (B).

		Food Additives and Contaminants	Page 32 of 51
	Table III
	Feed sample # Blank feed E2β 10 ng g -1 EE2 10 ng g -1 DES 10 ng g -1 zearalenone 1.25 µg g -1 equol 200 µg g -1 Feed sample Addition a Blank feed E2β 10 ng g -1 EE2 10 ng g -1 DES 10 ng g -1 zearalenone 1.25 µg g -1 equol 200 µg g -1	day 1 2 milk dry wet milk dry milk wet wet wet dry milk dry wet wet dry day 2 day 3 day 4 1 3 4 5 6 7 8 9 10 11 12 13 14 15 -11 12 -4 9 -32 -10 -4 -6 -34 -26 4 -31 -5 -6 -36 274 223 547 493 268 270 609 521 640 672 891 696 1046 899 249 529 596 849 590 644 day 5 day 6 16 17 18 19 20 dry dry wet milk milk 6 -37 1 -10 5 332 205 632 635 245 355 635 565 700 726 910 668 1034 878 277 537 566 933 546 621 68 36 208 129 80 71 341 292 335 303 418 148 558 421 96 193 118 587 116 263 116 92 216 152 125 139 379 292 379 533 734 389 544 358 147 221 351 601 247 226 313 353 632 736 339 466 734 520 742 887 1101 920 1039 938 383 860 842 1011 -15 366 O n l y wet feed milk replacer B none A B none A B -3 -61 -58 -61 58 68 80 335 373 370 349 1273 1271 1127 340 377 348 357 692 891 1148 182 283 329 255 739 683 638 200 241 201 193 400 363 334 F o r P e e r R e v i e w Mean (X) a SD (S) a CCα a -11 16 37 575 236 600 233 239 164 312 180 658 303 dry feed none A -14 -4 328 331 321 329 191 199 181 175 338 382 341 342 378 357 1081 1209 1307
		32

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	Feed sample # Blank feed E2β 5 ng g -1(b) Feed sample # Feed sample Days at room temp. in the dark Mean (X) a SD (S) a -28 35 342 116 Mean (X) a SD (S) a Addition a none Extract volume for exposure (µl) Blank feed -11 16 Blank feed -14 Dry feed blank E2β 10 ng g -1 575 236 E2β 10 ng g -1 328 E2β 5 ng g -1	CCα a 77 dry feed 0 CCα a A 200 37 -4 tox 331 tox	day 1 5 milk dry milk dry milk dry milk dry 1 2 3 4 6 7 8 -16 9 -9 -101 -28 -43 9 -41 -89 -90 -87 -13 -14 9 10 11 12 13 dry dry dry milk milk wet wet wet wet wet wet wet day 2 14 15 16 17 18 19 20 -7 -15 -12 -4 -6 -2 -9 300 221 360 64 320 372 452 247 128 203 363 411 468 480 447 438 424 406 361 365 day 1 day 2 day 3 day 4 day 5 day 6 wet feed milkreplacer 0 2 7 14 28 55 98 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 milk dry wet milk dry milk wet wet wet dry milk dry wet wet dry dry B none A B none A B 100 a 100 a 100 a 100 a 100 a 100 a 100 a dry wet milk milk -11 12 -4 9 -32 -10 -4 -6 -34 -26 4 -31 -5 -6 -36 6 -37 1 -10 -3 -61 -58 -61 58 68 80 -76 -82 -70 -73 -79 -54 -74 5 274 223 547 493 268 270 609 521 640 672 891 696 1046 899 249 529 596 849 590 644 335 373 370 349 1273 1271 1127 77 77 213 201 213 138 171
	EE2 5 ng g -1(b) DES 5 ng g -1(b) DES 10 ng g -1© zearalenone 1.25 µg g -1© equol 200 µg g -1© EE2 10 ng g -1 DES 10 ng g -1 zearalenone 1.25 µg g -1 equol 200 µg g -1 EE2 10 ng g -1 DES 10 ng g -1 zearalenone 1.25 µg g -1 equol 200 µg g -1 EE2 5 ng g -1 DES 10 ng g -1 zearalenone 1.25 µg g -1 equol 200 µg g -1 Days at room temp. in the dark 314 129 315 285 342 F o r P e e r R e v i e w O n l y 117 299 155 312 51 297 307 466 167 97 237 363 382 357 446 417 398 427 411 328 356 123 87 20 35 -72 36 118 259 52 -16 70 36 57 129 290 255 291 278 310 59 292 181 264 169 853 133 194 270 507 154 180 179 648 139 285 370 301 355 323 401 252 324 101 300 214 304 118 268 253 556 190 160 206 490 247 259 332 343 338 297 297 239 290 77 300 414 470 211 303 299 398 283 233 238 420 269 475 387 387 392 323 395 338 302 600 233 332 205 632 635 245 355 635 565 700 726 910 668 1034 878 277 537 566 933 546 621 239 164 68 36 208 129 80 71 341 292 335 303 418 148 558 421 96 193 118 587 116 263 312 180 116 92 216 152 125 139 379 292 379 533 734 389 544 358 147 221 351 601 247 226 658 303 313 353 632 736 339 466 734 520 742 887 1101 920 1039 938 383 860 842 1011 -15 366 321 329 340 377 348 357 692 891 1148 191 199 182 283 329 255 739 683 638 181 175 200 241 201 193 400 363 334 338 382 341 342 378 357 1081 1209 1307 tox 51 80 193 198 232 141 173 tox 79 81 148 165 213 109 111 tox 46 58 221 229 276 93 411 tox 145 151 478 466 550 232 329 0 2 7 14 28 55 98 Extract volume for exposure (µl) 200 b 200 b 200 b 200 b 200 b 200 b 200 b Milkreplacer blank -16 5 -8 -14 -18 -15 -13 E2β 5 ng g -1 258 335 257 626 629 333 574 EE2 5 ng g -1 242 286 547 627 635 360 590 DES 10 ng g -1 246 360 232 576 594 356 544 zearalenone 1.25 µg g -1 174 242 194 439 582 240 300 equol 200 µg g -1 199 317 320 607 755 331 444 Days at -20 °C 0 2 7 14 28 55 98 Extract volume for exposure (µl) 200 b 200 b 200 b 200 b 200 b 200 b 200 b Wet Feed blank -1 -1 28 -1 -8 379 -16 E2β 5 ng g -1 313 487 363 682 719 403 588 EE2 5 ng g -1 309 377 417 679 772 477 546 DES 10 ng g -1 309 455 433 658 748 477 586 zearalenone 1.25 µg g -1 261 304 329 528 744 434 381 F o r P e e r R e v i e w O n l y equol 200 µg g -1 264 342 285 638 812 -15 430

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	6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 F o r P Uitslag controlemonsters Datum blanco chem blanco voer afkeurgrens + 5 ng/g + 5 ng/g 4/mei/04 77 345 256 13/jul/04 77 347 196 13/jul/04 77 384 226 21/sep/04 77 225 142 18/okt/04 77 684 662 25/okt/04 77 343 279 11/nov/04 77 257 205 11/nov/04 77 308 214 23/nov/04 77 259 154 29/nov/04 77 350 263 6/dec/04 77 419 225 13/dec/04 77 527 392 17/jan/05 77 479 202 31/jan/05 77 760 690 31/jan/05 77 754 789 7/feb/05 77 214 248 22/feb/05 77 466 588 7/mrt/05 77 460 463 21/mrt/05 77 424 252 21/mrt/05 77 474 268 4/apr/05 77 257 100 19/apr/05 77 384 359 25/apr/05 77 349 250 25/apr/05 77 375 222 2/mei/05 77 323 254 23/mei/05 77 340 108 14/jun/05 77 251 84 4/jul/05 77 404 168 18/jul/05 77 322 121 19/sep/05 77 121 110 22/sep/05 77 489 303 22/sep/05 77 413 247 23/sep/05 77 399 331 26/sep/05 77 245 73 18/okt/05 77 234 135 18/okt/05 77 309 176 18/okt/05 77 335 169 19/okt/05 77 317 171 19/okt/05 77 320 151 19/okt/05 77 392 482 27/okt/05 77 412 286 27/okt/05 77 262 247 27/okt/05 77 196 188 27/okt/05 77 380 226 27/okt/05 77 478 273 27/okt/05 77 557 345 27/okt/05 77 326 103 27/okt/05 77 343 116 77 77 77 77 77	t.o.v. blanco DMSO max ijklijn 653 607 607 389 895 570 799 799 807 683 449 596 485 1039 1039 938 1026 407 765 765 760 1076 504 504 1022 748 898 1112 762 1293 949 949 484 700 759 759 759 759 759 759 839 839 839 839 839 839 839 839	t.o.v. blanco gist blanco chem -28 -25 -43 -18 7 9 2 -15 -46 -10 -81 -23 15 44 31 14 -9 27 -3 -1 -45 -20 12 -12 4 -39 19 -11 -31 3 -105 38 -24 -7 -24 -20 -56 -44 -91 -16 -33 -116 -132 -5 -106 -29 -6 -8	blanco voer -16 -61 -37 -38 9 -76 24 -12 -19 93 -11 18 -12 -44 -34 19 -18 20 57 54 0 3 -18 -18 11 -8 -19 -19 -29 -6 26 36 3 -86 -7 -7 -7 -9 -15 -3 -36 -50 -12 -11 -20 4 -20 -21	rest t.o.v. blanco chem opm. 10 ppb en 100ul van 2 ml 5 ppb en 100 µl van 2 ml (10 ppb en 100ul van 2 ml) 5 ppb en 200 µl van 3 ml wissel-ijklijn wissel-ijklijn wissel-ijklijn wissel-ijklijn,gekleurd extract, voer hoog signaal op t=0

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	77
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	0.39
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	Page 51 of 51														Page 52 of 51
																WAARNEMINGENFORMULIER RSVA0943 WAARNEMINGENFORMULIER RSVA0943
	1
	2															0.00
	3	WAARNEMINGENFORMULIER GIST BIO-ASSAY ESTROGENEN FRACTIEVERZAMELAAR WAARNEMINGENFORMULIER GIST BIO-ASSAY ESTROGENEN WAARNEMINGENFORMULIER GIST BIO-ASSAY ESTROGENEN			PAG. 1 PAG. 2 PAG. 3		DATUM PLAATnr. DATUM PLAATnr. DATUM PLAATnr.	10/29/2004 LABJOURNAAL REA44-5 LOGBOEK 10/29/2004 LABJOURNAAL REA44-5 LOGBOEK MS 10/29/2004 LABJOURNAAL REA44-5 LOGBOEK		XXXX/1427 XXXX/1427 XXXX/1427	RUWE DATA FILE T0 RUWE DATA FILE T24 RUWE DATA FILE T0 0 RUWE DATA FILE T24 RUWE DATA FILE T0 0 RUWE DATA FILE T24	rea445t0 rea44524 rea445t0 rea44524 rea445t0 rea44524	0.33 0.66
	4	VERSIE: UITVANG HPLC-MSMS SYSTEEM VERSIE: UITVANG HPLC-MSMS SYSTEEM VERSIE: UITVANG HPLC-MSMS SYSTEEM									RUWE DATA MAP RUWE DATA MAP RUWE DATA MAP	REA08 REA08 REA08	MS-FILE MS-FILE MS-FILE	0 0	1.00
	5	PIPETTEERSCHEMA RESULTAAT		MONSTER RIK131926										1.33 1.66
	6	FRACTIE NR.	1 UITVANGVENSTER 2	3 RESULTAAT	4 CORRECTIE	5	UITSLAG	6	7	8	9	10	11	12	2.00 2.33
	7	FRACTIE 1	a FRACTIE 1 b FRACTIE 2 VAN 0.00	FRACTIE 16 FRACTIE 15 TOT 0.33	FRACTIE 17 FRACTIE 18 296	FRACTIE 32 FRACTIE 31 BLANCO 34	FRACTIE 33 FRACTIE 34	FRACTIE 48 FRACTIE 47	FRACTIE 49 FRACTIE 50	FRACTIE 64 FRACTIE 63	FRACTIE 65 FRACTIE 66	Blanco Gist Blanco Gist	Blanco Gist 600 pM E2B	Blanco Gist Blanco Gist	2.66 3.00
	8	FRACTIE 2 FRACTIE 3	c FRACTIE 3 d FRACTIE 4 0.33 0.66	FRACTIE 14 FRACTIE 13 0.66 1.00	FRACTIE 19 FRACTIE 20 294 302	FRACTIE 30 FRACTIE 29 32 40	FRACTIE 35 FRACTIE 36	FRACTIE 46 FRACTIE 45	FRACTIE 51 FRACTIE 52	FRACTIE 62 FRACTIE 61	FRACTIE 67 FRACTIE 68	Blanco Gist Blanco Gist	600 pM E2B 600 pM E2B	Blanco Gist Blanco Gist	3.33 3.66
	9	FRACTIE 4 FRACTIE 5	e FRACTIE 5 f FRACTIE 6 1.00 1.33	FRACTIE 12 FRACTIE 11 1.33 1.66	FRACTIE 21 FRACTIE 22 298 298	FRACTIE 28 FRACTIE 27 36 36	FRACTIE 37 FRACTIE 38	FRACTIE 44 FRACTIE 43	FRACTIE 53 FRACTIE 54	FRACTIE 60 FRACTIE 59	FRACTIE 69 FRACTIE 70	Blanco Gist Blanco Gist	Blanco Gist Blanco Gist	Blanco Gist Blanco Gist	4.00 4.33
	10	FRACTIE 6	g FRACTIE 7 1.66	FRACTIE 10 2.00	FRACTIE 23 321	FRACTIE 26 59	FRACTIE 39 RANDEFFECT??, HOGE OD FRACTIE 42	FRACTIE 55	FRACTIE 58	Blanco Gist	Blanco Gist	Blanco Gist	Blanco Gist	4.66
	11	FRACTIE 7 FRACTIE 8	h FRACTIE 8 2.00 2.33	FRACTIE 9 2.33 2.66	FRACTIE 24 310 333	FRACTIE 25 48 71	FRACTIE 40 RANDEFFECT??, HOGE OD FRACTIE 41	FRACTIE 56	FRACTIE 57	Blanco Gist	Blanco Gist	Blanco Gist	Blanco Gist	5.00 5.33
	12	FRACTIE 9 FRACTIE 10	2.66 3.00	3.00 3.33	335 294	73 32										5.66 6.00
	13	RUWE DATA FRACTIE 11 FRACTIE 12	3.33 3.66	3.66 4.00	286 289	24 27										6.33 6.66
	T0 T24 BEREKENING T24 -T0 SIGNAAL a 99 1 b 99 c 97 d 96 e 96 f 97 g 98 h 120 1 a 395 b 393 c 399 d 394 e 394 f 418 g 408 h 453 1 a 296 b 294 c 302 d 298 e 298 f 321 g 310 h 333 CONTROLES CONTROLE GEMIDDELD Blanco Gist 262 600 pM E2 326 ACCEPTATIECRITERIA UITSLAG STDEV 2 100 107 97 98 100 100 102 122 2 387 370 356 389 389 386 396 457 2 287 263 259 291 289 286 294 335 6 19 FRACTIE 13 4.00 4.33 FRACTIE 14 4.33 4.66 FRACTIE 15 4.66 5.00 FRACTIE 16 5.00 5.33 FRACTIE 17 5.33 5.66 FRACTIE 18 5.66 6.00 FRACTIE 19 6.00 6.33 FRACTIE 20 6.33 6.66 FRACTIE 21 6.66 7.00 FRACTIE 22 7.00 7.33 FRACTIE 23 7.33 7.66 FRACTIE 24 7.66 8.00 FRACTIE 25 8.00 8.33 FRACTIE 26 8.33 8.66 FRACTIE 27 8.66 9.00 FRACTIE 28 9.00 9.33 FRACTIE 29 9.33 9.66 FRACTIE 30 9.66 10.00 FRACTIE 31 10.00 10.33 FRACTIE 32 10.33 10.66 FRACTIE 33 10.66 11.00 FRACTIE 34 11.00 11.33 FRACTIE 35 11.33 11.66 FRACTIE 36 11.66 12.00 FRACTIE 37 12.00 12.33 FRACTIE 38 12.33 12.66 FRACTIE 39 12.66 13.00 FRACTIE 40 13.00 13.33 FRACTIE 41 13.33 13.66 FRACTIE 42 13.66 14.00 FRACTIE 43 14.00 14.33 FRACTIE 44 14.33 14.66 FRACTIE 45 14.66 15.00 FRACTIE 46 15.00 15.33 FRACTIE 47 15.33 15.66 FRACTIE 48 15.66 16.00 FRACTIE 49 16.00 16.33 FRACTIE 50 16.33 16.66 FRACTIE 51 16.66 17.00 FRACTIE 52 17.00 17.33 FRACTIE 53 17.33 17.66 FRACTIE 54 17.66 18.00 FRACTIE 55 18.00 18.33 FRACTIE 56 18.33 18.66 FRACTIE 57 18.66 19.00 FRACTIE 58 19.00 19.33 FRACTIE 59 19.33 19.66 FRACTIE 60 19.66 20.00 FRACTIE 61 20.00 20.33 FRACTIE 62 20.33 20.66 FRACTIE 63 20.66 21.00 FRACTIE 64 21.00 21.33 FRACTIE 65 21.33 21.66 FRACTIE 66 21.66 22.00 FRACTIE 67 22.00 22.33 FRACTIE 68 22.33 22.66 FRACTIE 69 22.66 23.00 FRACTIE 70 23.00 23.43 1e VALIDATIE F 3 4 99 99 99 99 100 104 98 98 95 97 100 96 98 95 96 95 3 4 383 374 389 377 394 378 395 382 391 380 638 370 626 373 413 380 3 4 284 275 290 278 294 274 297 284 296 283 538 274 528 278 317 285 BLANCO CORRECTIE 63 CRITERIIUM o 5 105 104 102 103 100 99 97 98 5 396 390 383 395 370 375 380 384 5 291 286 281 292 270 276 283 286 261.25 VOLDOET r 103 6 101 100 101 102 100 98 100 6 383 387 389 385 374 372 373 382 6 280 286 289 284 272 272 275 282 P e 7 102 100 105 101 100 99 95 101 7 377 373 364 366 367 356 358 365 7 275 273 259 265 267 257 263 264 e 8 105 101 102 101 100 97 98 98 8 383 367 364 361 365 354 359 368 8 278 266 262 260 265 257 261 270 r 9 99 99 97 99 96 95 94 99 9 370 363 358 359 362 353 346 357 9 271 264 261 260 266 258 252 258 1e VALIDATIE R 10 102 97 101 97 98 96 97 105 10 373 363 364 364 359 356 357 373 10 271 266 263 267 261 260 260 268 e 11 97 98 95 99 94 94 96 99 11 367 439 427 403 356 351 364 367 11 270 341 332 304 262 257 268 268 v 12 101 100 100 98 96 97 99 102 12 372 369 369 369 363 357 361 379 12 271 269 269 271 267 260 262 277 2e VALIDATIE i e w O n l y 291 29 259 -3 263 1 287 25 284 22 290 28 294 32 297 35 296 34 538 276 VERDACHT , BEVAT MOGELIJK 17-B ESTRADIOL 528 266 VERDACHT , BEVAT MOGELIJK 17-B ESTRADIOL 317 55 285 23 278 16 274 12 283 21 284 22 274 12 278 16 275 13 291 29 286 24 281 19 292 30 270 8 276 14 283 21 286 24 282 20 275 13 272 10 272 10 284 22 289 27 286 24 280 18 275 13 273 11 259 -3 265 3 267 5 257 -5 263 1 264 2 270 8 261 -1 257 -5 265 3 260 -2 262 0 266 4 278 16 271 9 264 2 261 -1 260 -2 266 4 258 -4 2e VALIDATIE 7.00 LC-fractions fish feed o r R e v i e w O n l y 7.33 7.66 8.00 8.33 8.66 9.00 9.33 9.66 10.00 10.33 15.66 16.00 16.33 16.66 17.00 17.33 17.66 18.00 18.33 18.66 19.00 19.33 19.66 20.00 20.33 20.66 21.00 21.33 21.66 22.00 22.33 22.66 23.00 150 200 250 300 F l u o r e s c 59 F 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 14.66 15.00 15.33 r 13.66 14.00 14.33 e 12.66 13.00 13.33 -50 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Retention time (min) e 10.66 11.00 11.33 12.00 0 12.33 e 100 n c P 11.66 50 e

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Food Additives and Contaminants

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© The extracts were prepared on six different days (see Table2) and the 200 µl aliquots were analysed in the yeast estrogen bioassay in one exposure. These sample preparations and the exposure were performed within a time period of 3 months.

Acknowledgements

This project was financially supported by the Dutch Ministry of Agriculture, Nature and Food Quality.