Keith Scudamore

Clare M Hazel

Sue Patel

Fiona Scriven

Keith ; Scudamore

Mycotoxins Kas

Hazel

K A Scudamore

C M Hazel #

Deoxynivalenol and other Fusarium mycotoxins in bread, cake and biscuits produced from UK-grown wheat under commercial and pilot scale conditions

Keywords: Mycotoxins -fusarium, Mycotoxins -trichothecenes, Mycotoxinszearalenone Food Types: Bakery products, Biscuits, Bread, Cereals Cereals, Fusarium, mycotoxin, bread, cake, biscuits, processing

Deoxynivalenol and other Fusarium mycotoxins in bread, cake and biscuits produced from UK-grown wheat under commercial and pilot scale conditions.

are largely stable and survive processing, when concentrations were determined on an 'as is' basis as stipulated in legislation, levels in finished products were usually lower than in the starting flour due to the dilution effect of other ingredients such as fat, sugar and water. Thus mean concentrations of DON in bread were reduced by about 35% and 39% in white and wholemeal bread respectively which are in close agreement with the reduction required by the regulations although the changes that occur during milling white and wholemeal flour from whole wheat also need to be taken into account. The reduction of DON during cake manufacture is greater than for bread because flour makes up only about 25% of the starting ingredients. However, in biscuit production, particularly for crackers for which flour constitutes about 90% of the recipe ingredients, the reduction indicated by the regulations is not achieved. It is concluded that for some commercial processes, the whole-wheat or flour ingredients used will need to contain DON levels lower than those set by legislation to ensure that the final products will still meet statutory limits. Limited results with consignments containing low concentrations of ZON and NIV suggest that their stability and survival are similar to those for DON.

Introduction

Cereals including wheat are often infected by Fusarium fungi in the field that can cause diseases such as Fusarium head blight [START_REF] Edwards | Influence of agricultural practices on fusarium infection of cereals and subsequent contamination of grain by trichothecene mycotoxins[END_REF]) and the formation of mycotoxins that include deoxynivalenol, zearalenone, nivalenol, HT2-toxin (HT2) and T2-toxin (T2). Many mycotoxins are inherently chemically stable so may survive processing to remain in the products purchased by the consumer. It is thus important to know the extent to which this occurs to assist in assessing the risk that Fusarium mycotoxins might pose to the consumer. To achieve this it is necessary to study both the occurrence of the mycotoxins in primary agricultural crops such as wheat and the effect of processing and food manufacturing on their concentrations in the retail products (Hazel and Patel 2003).

The consumer can then be protected by the implementation and observation of appropriate legal limits such as have already been introduced in the EC for DON and ZON in wheat and in other cereals (EC 2006). The need of, and values for, further legislation to address HT2 and T2 are currently being considered. Different limits are set for mycotoxins at selected stages during their processing so that for wheat there are values at intake, in milled ingredients, in retail products and for baby and infant foods (Table 1). Legal limits refer to the product "as is" and the acceptance of a product must also take into consideration the measurement uncertainty.

INSERT TABLE 1

While considerable data are available (e.g. [START_REF] Gareis | Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states, European Commission[END_REF] to show the frequency and levels of Fusarium mycotoxins, particularly DON, occurring in wheat there is much less information on their transfer to wheat-based products such as bread, cakes, pastries and biscuits at the retail point. [START_REF] Samar | Effect of fermentation on naturally occurring deoxynivalenol (DON) in Argentinean bread processing technology[END_REF] found that fermentation reduced naturally occurring deoxynivalenol in Argentinean bread processing technology within a pilot scale plant. French bread and Vienna bread were prepared from wheat flour naturally contaminated with deoxynivalenol at 150 µg kg -1 in which dough was fermented at 30-50º C according to standard procedures used in Argentina. The maximum reduction obtained in dough at 50º C was 46% for the Vienna bread and 41% for French bread. This agreed with a study by [START_REF] Neira | The effects of bakery processing on natural deoxynivalenol contamination[END_REF] in which 8 types of product were prepared in a low technology bakery that showed a significant reduction of deoxynivalenol during the bread making process.

However, [START_REF] Gilbert | The analysis and occurrence of Fusarium mycotoxins in the United Kingdom and their fate during food processing[END_REF] showed that 80% of deoxynivalenol survived in both spiked and naturally contaminated wheat although Abbas et al. 1985 obtained variable losses from 19 to 69%. Studies have further suggested that DON may sometimes be present in cereals in a bound form and that an acid solvolysis procedure may be required to release this bound mycotoxin [START_REF] Liu | Solvolysis procedures for the determination of bound residues of the mycotoxin deoxynivalenol in fusarium species infected grain of two winter wheat cultivars pre-infected with barley yellow dwarf virus[END_REF]. [START_REF] Boyacioglu | Additives affect deoxynivalenol (vomitoxin) flour during baking[END_REF] examined the effect of additives such as potassium bromate, L-ascorbic acid, sodium bisulphite, L-cysteine, ammonium phosphate at varying levels on deoxynivalenol during baking. The reduction was about 7% in flour without additives. Potassium bromate and L-ascorbic acid had no effect, but sodium bisulphite, L-cysteine and ammonium phosphate resulted in about 40% reduction.

ZON is also usually regarded as stable or only partly decomposed by heat.

However, Matsuura and Yoshizawa 1981 found ZON was reduced in bread by approximately 40% while about 50% was lost during the production of noodles although it is unclear whether their results for bread were corrected for water content. However, baking at 170ºC did not degrade ZON or NIV (Tanake et al. 1986). During extrusion of maize, Scudamore et al. 2008a found that ZON was reduced particularly under low moisture conditions (13.5%) but remained virtually unchanged when forming pellets under high moisture extrusion conditions (28%).

In related studies (Scudamore and Patel 2008b) the occurrence and fate of DON during commercial preparation of whole-wheat breakfast cereals has shown that exhaustive cleaning regimes can remove up to about 50% of DON from wheat received directly from UK farms and that this could be further reduced in whole-wheat breakfast cereals. However, on investigation this was shown to be principally due to solution of DON in aqueous liquid run off from the cookers, and not to chemical breakdown.

Materials and Methods

Bread manufacture and sampling

The manufacture of bread from naturally contaminated flour was carried out in a pilot scale commercial bakery with 4 kg mixes containing 2.25 kg flour using the Chorleywood Process [START_REF] Chamberlain | The Chorleywood Bread Process[END_REF][START_REF] Axford | The Chorleywood Process[END_REF]. In summary this process involves mixing the ingredients of the dough typically for a few minutes to a set work input in order to achieve a dough temperature ex-mixer of 30°C, the dough was divided, moulded and proved for a maximum of 60 minutes at 40-45 There is a very large range of cake types and two different recipes were examined here; a plain Genoa (cake 1) in which the ingredients were baked for 15 minutes at 170°C and a stressed high ratio cake (cake 2) baked for longer (30 minutes) but at a lower temperature of 150°C. The ingredients in both cake recipes consisted of flour, sugar, egg, water, margarine and other ingredients High ratio refers to the sugar to flour ratio while stress is a property of the dough. The dry ingredients were mixed first and then the liquids added and mixed. The mixtures were deposited immediately, stood for a set period of time then baked in the oven. The cakes were allowed to cool, ground together in a food homogeniser and stored at -20C until required for analysis.

°C

Biscuit manufacture

Semi-sweet biscuits were produced by using a recipe containing about 70% flour, the remaining ingredients being mainly fats and sugar. The analytical methods were UKAS (ISO 17025) accredited. Analyses were conducted with a spiked sample, i.e. a known amount of toxin was added to each sample matrix each day prior to extraction, clean up and determination for each batch of 1-5 samples. The spike level was 200 µg kg -1 for DON and 50 µg kg -1 for ZON. These results were used to assess recovery and all reported results

were corrected using the values obtained. Recoveries in the range 70-110% were considered acceptable. The limit of detection and limit of determination respectively for each mycotoxin were 5 µg kg -1 and 10 µg kg -1 for DON, NIV and other trichothecenes and 1.5 µg kg -1 and 3 µg kg -1 respectively for ZON. The limit of detection is defined as 3 times the electronic baseline noise and the limit of determination as 6 times baseline noise. Calibration curves for each mycotoxin were plotted with the lowest calibration points respectively being equivalent to 10 and 3 for DON and ZON. After analysis samples were retained and stored at -20°C.

In house reference material (wheat and maize) contaminated at 220 and 550 µg/kg of DON was spiked with 200 and 500 µg/kg respectively. Typical mean recovery was 89% with a coefficient of variability of 8.1% for 10 replicates. For fumonisins, maize in house reference material contaminated at 498 µg kg -1 was then spiked at 400 µg kg -1 . Typical mean recovery was 86% with a coefficient of variability of 6.5% for 10 replicates. For ZON, ground maize in house reference material contaminated at 50 µg kg -1 of ZON was spiked at 53 µg kg -1 . Typical mean recovery was 88% with a coefficient of variability of 7.2 % for 10 replicates.

Results and Discussion

Tables 2 and3 show the levels of DON in white bread and wholemeal bread manufactured from commercial consignments of white and wholemeal flour respectively. Legislation applies to ingredients and products on an 'as is' basis, that is, the concentration determined without any correction for moisture content or the presence of other components. The analytical values obtained for flour and for the bread produced are converted to a dry weight values. Changes in concentrations are then presented 'as is' (relevant for legislation) and to show the true change in concentration (using a standard moisture content of 12.5% for flour and 40% for bread). The mean of 13 and 7 baking runs respectively for white bread and wholemeal bread show that the 'as is' levels detected in bread were 35% and 39% lower than the concentration in the flour. The EC limits for DON in flour and bread are 750 µg kg -1 and 500 µg kg -1 respectively, Table 1, indicating that a 33% fall in concentration should be expected. The results reported here are thus consistent with the current legislative limits which in themselves are required to take into account measurement uncertainty..

INSERT TABLE 2

The maximum concentration of DON found in commercial consignments of flour over the period of these studies was 284 µg kg -1 , which is approximately 40% of the maximum allowed limit. When the values for DON of each flour and the corresponding bread sample are plotted, Figure 1, a linear relationship is obtained with an R 2 value of 0.91. This implies that results can be extrapolated safely up to the EC statutory limit.

INSERT FIGURE 2

The results for bread in Tables 2 and3 have also been corrected for moisture content. When this is done the mean concentrations show an actual loss of only 5% for DON in white bread and 11% for the wholemeal bread although the changes in concentration obtained between runs were quite variable. Any further correction to take into account the presence of other minor ingredients would only increase the calculated values in bread by a small amount so that it is clear that DON is virtually unaffected by the baking procedure. However, there is a large variability in the milling fractionation pattern between different consignments of wheat. On 6 occasions samples were taken at intermediate stages in the bread making process including the dough after mixing and after dividing, from the dough after its first poof and after its final proof. After correcting for the moisture content at each stage the results confirm that there is no significant change in DON during these intermediate stages. These results are not shown. Elsewhere Edwards et al. (in press) have shown that during milling flour from whole wheat a mean concentration of DON is reduced by about 30 % for white flour but this is virtually unchanged with wholemeal flour because the latter consists of all the components of the whole grain. This emphasises that millers and bakers cannot use wholemeal wheat containing DON at the legislative limit if they are to produce wholemeal bread within the legal limits and would need to lower the acceptable concentration in the unprocessed wheat.

INSERT TABLE 3

INSERT TABLE 4

Table 4 gives the results for cake manufacture. On an 'as is basis DON in both types of cake was reduced on average by at least 55% so that the reduction expected from the statutory limits is easily met. The results are calculated on the basis that flour only constitutes 255 of the cake produces, the other ingredients being mostly fat, sugar and water. While the total number of bakes was small, the relative concentration of DON in the 2 Genoa type cakes compared with the flour was lower than those in the high stress type of cake. When the other ingredients are considered there is close to 100% survival of DON in the Genoa cake but an apparent increase in DON levels in the high stress cake bakes. However, the starting concentrations of DON in flour are low and the increases seen are only equivalent to about 20-30 µg kg -1 of the mycotoxin. The reason for this is unclear but could be due to the release of 'bound' DON or the presence of DON in one or more of the other ingredients although these were checked for the presence of DON in selected samples. The apparent small increase in DON during manufacture of cereal products, including breakfast cereals, has been noted in other processes (Scudamore et al. 2008b)

INSERT TABLE 5

The changes in DON and ZON concentrations during the manufacture of biscuits are given in Table 5. Because concentrations of both mycotoxins were low in commercial biscuit flour, portions milled from wheat grown on experimental plots was used to carry out bakes to study DON at levels up to and ZON are not affected during baking. NIV occurred in low concentrations in some of the flour from the experimentally grown wheat and this allowed some limited studies. The results are shown in Table 6. This suggests that there was little loss in sweet biscuits with the reduction in concentration seen being due to the presence of other ingredients. The mean reduction in NIV concentration in crackers was >28% although the results obtained were quite variable ranging from 0 to 42%.

INSERT TABLE 6

The results obtained in this study have been used to calculate the levels expected in the products manufactured when starting with whole wheat containing DON extrapolated to the regulatory limit and are shown in Table 7.

This shows that limits for DON in consumer products are only met in cakes because of the large dilution factor due to other ingredients. White bread and a cracker variety of biscuit would have levels of DON in excess of the statutory limits, while those in wholemeal bread will be higher still because the flour will contain all DON present in the whole wheat. The studies also show that there is quite a large variability in results from batch to batch so allowance for this also needs to be taken into account.

One outcome is that buyers will decide to purchase whole wheat or flour containing levels of DON below the maximum allowed to have confidence that legislation will be met in the susceptible finished products. In some circumstances this will increase costs and/or necessitate wheat being used for other purposes.

  products manufactured from flour involves many different production methods and uses a large range of ingredients based on a complex and specialised food technology. Flour is a combination of protein, starch, sugar, fat and mineral salts but the quality and quantity of the gluten -forming proteins is the most significant factor in determining its suitability for specific products. It is recognised that the behaviour of mycotoxins in laboratory or pilot scale studies may not accurately reflect what occurs in large commercial operations such as in mills, bakeries and other processing plants using naturally contaminated cereals.The work reported here forms part of a Project studying the occurrence and fate of Fusarium mycotoxins in UK cereals, and the changes in mycotoxin concentrations during the commercial and pilot plant manufacture of specimen samples of bread, cake and biscuit. The results are discussed in relation to the existing EC regulations.

  followed by baking for approximately 21 minutes at 210°C. Dough ingredients were flour, yeast, salt, fat, ascorbic acid fungal alpha-amylase and water. The bread was then cooled to 27°C. Samples (500 g) made up of 5 incremental samples were taken post mixing and post proving. A composite final bread sample of four loaves constituted the final product sample. These were mixed together in a food processor and stored frozen at -20ºC until required for analysis.

  Cracker biscuits were manufactured by baking a lean cracker recipe containing about 90% flour and with significantly less fat and sugars. Both commercial flour and flour from experimental trials were used and blended to provide a range of concentrations in batches of between 5 and 10 kg of ingredients. The biscuits and crackers were baked in a 3 zone travelling oven for 5 minutes at a maximum temperature of 245ºC for semi-sweet biscuits and 3 minutes at a maximum temperature of 280ºC for crackers.Analytical methodsDON (and other related trichothecene mycotoxins) was determined following the method of[START_REF] Patel | Survey of ethnic foods for mycotoxins[END_REF] as used by[START_REF] Scudamore | The occurrence and fate of Fusarium mycotoxins during the commercial processing of oats in the UK[END_REF] Ground or chopped samples (20 g) were extracted with 100 ml acetonitrile/water (84:16). An aliquot of the extract was cleaned using a charcoal/alumina column. After taking to dryness and re-dissolving in acetonitrile DON was derivatised to form the trichothecene -trimethyl silyl (TMS) derivatives and determined by GC/MS operating in selected ion mode, using 4 ions for confirmation. ZON was determined by HPLC with fluorescence detection as used by[START_REF] Scudamore | The occurrence and fate of Fusarium mycotoxins during the commercial processing of oats in the UK[END_REF] Ground samples (25 g) were extracted with 125 ml of acetonitrile/water (75/25). The extract was cleaned-up using an immunoaffinity column and determined by HPLC with fluorescence detection..

  limits and to also carry out a very limited study with NIV and ZON. Intermediate concentrations were obtained by blending commercial consignments of flour with experimental consignments of flour. The relative concentrations of DON and ZON in the flour and sweet biscuits were 25% and 30% lower respectively on an 'as is' basis. Because the sweet biscuits studied were produced from ingredients which contained about 70% flour this indicates that there was no loss of either DON or ZON during manufacture. Crackers are produced from a recipe containing about 90% flour so that the mean reduction in concentration of DON on an 'as is' basis indicates again that DON(and probably)

Figure 1 :

 1 Figure 1: Correlation of the percentage of DON in flour with DON in white and wholemeal bread -all bread results included, results on an 'as is' basis

Table 1 .

 1 EC, Maximum permissible limits for DON and ZON in wheat.

	(EC 2006)
		Mycotoxin, µg kg -1
	Wheat component	DON	ZON
	Unprocessed wheat (other than durum wheat)	1250	100
	Wheat intended for direct human consumption,	750	75
	wheat flour, bran and germ as end product
	marketed for direct human consumption
	Bread, pastries, biscuits, cereal snacks and	500	50
	breakfast cereals
	Processed cereal-based and baby foods for infants and young children	200	20

Table 2 .

 2 Change in DON concentration from flour to white bread on an 'as is'

		basis and on a true basis after correction
			µg kg -1		change, %
			flour		white bread
	bake no.	"as is" corrected * "as is" corrected *	"as is"	true
	1	87	99	54	90	-38	-10
	2	89	101	38	63	-57	-38
	3	63	72	42	70	-33	-3
	4	284	323	185	307	-35	-5
	5	284	323	186	309	-34	-4
	6	67	76	56	93	-16	+22
	7	158	180	80	133	-49	-26
	8	61	69	31	51	-49	-26
	9	106	121	52	86	-51	-28
	10	64	73	39	65	-39	-11
	11	169	192	124	206	-27	+7
	12	124	141	119	198	-4	+40
	13	95	108	75	125	-21	+16
	mean					-35	5
	range					-4 to -57 +40 to -38
	* = dry weight basis
	Table 3. Change in DON concentrations from wholemeal flour to wholemeal
		bread on an 'as is' basis and on a true basis after correction
			µg kg -1			change, %
			flour		bread
	Bake no. "as is"	corrected * "as is"	corrected * "as is"	true
	1	81	92	56	93	-31	+1
	2	74	84	51	85	-31	+1
	3	51	58	28	46	-45	-20
	4	110	125	71	118	-35	-6
	5	99	113	56	93	-43	-18
	6	178	203	108	179	-39	-12
	7	232	264	125	208	-46	-21
	mean					-39	-11
	range					-31 to -46 +1 to -21

Table 4 .

 4 Change in DON concentrations from flour to cake on an 'as is' basis and on a true basis after correction. = value used was half the LoQ # =corrected on dry weight basis ## =correction based on a combined factor of moisture dry weight and on flour being 24% -25% of the ingredients

				µg kg -1				change, %
		cake	flour				cake
	type run	"as is" corrected # "as is" corrected ##	"as is"	true
	1	a	115	131	27	136	-76	+4
	1	b	24	27	5 *	25	≥-79	-7
	2	c	75	85	28	141	-63	+168
	2	d	51	58	23	116	-55	+203
	2	e	17	19	10	50	-41	+279
	2	f	19	22	5 *	25	≥-74	+118
	2	g	19	22	5 *	25	≥-74	+118
	1	mean					≥ -77	-1.5
		range					-76 to ≥ -79 +4 to -7
	2	mean					≥ -61	+ 177
		range					-41 to ≥ -74 118 to 279

*

Table 6 .

 6 Change in NIV concentrations occurring during the manufacture of biscuits, on an 'as is' basis

	NIV, µg kg -1

Table 7 .

 7 Concentrations of DON in wheat based bread, cakes and biscuits extrapolated to the statutory limit in whole wheat, µg kg -1

		legislation	study results
		white flour and wholemeal	ex white	ex wholemeal
	whole wheat	1250		1250
	flour	750	875*	1250
	bread	500	569	762
	cake 1 cake 2	500	236 394
	semi sweet biscuits crackers	500	656 779

* = value obtained in related study

(Edwards et al in press)

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Table 5 :

 5 Change in DON and ZON concentrations occurring during the manufacture of biscuits, on an 'as is' basis, µg kg -1

	Mycotoxin, µg/kg
	DON	ZON

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Acknowledgements

These studies were funded by the UK DEFRA, Food Quality and Safety LINK Programme and the UK Food Standards Agency and would not have been possible without the enthusiastic cooperation and assistance from the staff of the cereal companies involved.

Conclusions

DON appears to be very stable in all processes examined. Where results obtained for DON were lower in the products, this was due to a dilution effect from other ingredients including water. This dilution enabled the regulatory limits to be met in the consumer product because legislation applies to results on an 'as is' basis.

Within the limits of analytical variation the level of DON at each intermediate stage in the baking process was the constant when corrected for moisture content.

The baking of white bread from naturally contaminated flour using the Chorleywood Baking Process resulted in little change in DON levels.

Concentrations of DON in both white and wholemeal bread were shown to be reduced by 35-40% during bread making compared to the flour ingredient when concentrations were determined on an 'as is' basis. If moisture content and the presence of other ingredients were taken into account the loss of DON was less than 5 or 11% respectively confirming that it is stable during the processes.

In the absence of ZON in flour, the stability of this mycotoxin could not be determined.

In the same way, the concentration of DON in cakes and biscuits when compared to that in the flour was only reduced by an amount due to dilution with the other ingredients added. Because biscuits are manufactured from ingredients containing a high percentage of flour, the reduction demanded by the legislation would appear not always to be met. However, because allowance must also be made for the uncertainty of the measurement (typically about 44%), the need to reject these products in practice would be extremely unlikely.


The effect of cooking on the concentration and burden of domoic acid in two bivalve molluscs was studied. The Manila clam (Ruditapes philippinarum) and cockle (Cerastoderma edule) were subjected to steaming and boiling, respectively. In both cases, factorial plans were used to evaluate the effects of common cooking methods and the variations likely to take place during the cooking procedure (cooking time and salt concentration in both species, in addition to ethanol percentage in Manila clam). The domoic acid concentration and toxin content were affected by cooking in very different ways in the two species studied. The cockle lost a significant part of its domoic acid content, while the clam did not. Since the weight of the soft tissues in cooked bivalves was lower than in the raw samples in both species, the toxin concentration decreased less than the toxin burden in the cockle, while it increased in the clam, where the toxin burden did not change significantly. Among the cooking variables tested, only cooking time had a noticeable effect on the domoic acid content in the clam and cockle, with the bivalves that were cooked for a longer time having smaller amounts of toxin. It is clear that cooking affects the toxin concentration in bivalves in a way that is species-specific. This characteristic must be taken into account when evaluating epidemiological information, establishing allowable toxin levels and in cases where pre-processing treatments such as cooking or similar methods are used in monitoring systems.

The effect of some habitual cooking processes on the domoic acid concentration in the cockle (Cerastoderma edule) and Manila clam (Ruditapes philippinarum)

Introduction

Filter feeding shellfish such as mussels, clams, oysters and cockles can accumulate domoic acid which is the main toxin responsible for amnesic shellfish poisoning (ASP) in high concentrations. These organisms ingest phytoplankton during algal blooms which are, in some cases, composed of species that produce toxins. Some

Pseudo-nitzschia species produce domoic acid and consequently, when they bloom in the plankton, these species become the primary sources of domoic acid in bivalve shellfish food. In such cases the toxin is accumulated by the bivalves, which when consumed by humans, may produce a type of poisoning known as amnesic shellfish poisoning (ASP), since one of the symptoms of this syndrome is recent memory loss. In 1987, the first episode of this kind of toxicity was recorded in Canada, causing three fatalities. The symptoms included vomiting and diarrhea followed by confusion, memory loss and, in critical cases, coma [START_REF] Wright | Identification of domoic acid, a neuroexcitatory amino acid, in toxic mussels from easten P.E.I[END_REF]).

In September 1994, during a phytoplankton bloom, consisting mainly of Pseudonitzschia australis, ASP toxins were detected in cultured mussels from Galician Rías [START_REF] Míguez | First detection of domoic acid in Galicia (NW Spain)[END_REF]. Since then, the Galician monitoring system has been recording outbreaks of this type of toxicity in spring and autumn practically every year [START_REF] Correa | Establishing special health checks for the harvesting and processing of certain bivalve molluscs with a level of amnesic shellfish poison (ASP) exceeding the limit[END_REF]). The frequent closure of the main shellfish production areas due to these recurrent episodes is having an important economical impact on Galicia and many other areas in Europe.

In order to minimize economic losses while maintaining consumer safety, the maximum allowable level of these toxins needs to be fine-tuned. This is a difficult task, mainly because intoxications of this kind are difficult to detect, but also because, when detection does occur, there is little information on the effect of cooking on the toxin concentration which is generally required to transform the toxicity found in ingested food to raw shellfish toxicity (the latter being the one needed for harvesting regulation).

The effect of cooking (or canning) on the concentration of toxins, other than ASP toxins, in shellfish has been evaluated in several studies. [START_REF] Lawrence | Effect of cooking on the concentration of toxins associated with paralytic shellfish poison in lobster hepatopancreas. Toxicon. 32: 57-64. content of domoic acid in blue mussels, Mytilus edulis[END_REF] reported reductions of Paralytic Shellfish Poisoning toxins (PST) in cooked (steamed or boiled) lobsters, and [START_REF] Berenguer | The effect of commercial processing on the PSP content of naturallycontaminated Acanthocardia tuberculatum[END_REF] and [START_REF] Vieites | Canning process that diminishes paralytic shellfish poison in naturally contaminated mussels (Mytilus galloprovincialis)[END_REF] examined the concentration of the same group of toxins during canning, in Acantocardia tuberculatum and Mytilus galloprovincialis, respectively. More recently, [START_REF] Hess | Tissue distribution, effects of cooking and parameters affecting the extraction of azaspiracids from mussels, Mytilus edulis, prior to analysis by liquid chromatography coupled to mass spectrometry[END_REF] studied the effects of cooking on azaspiracids (AZP toxins), okadaic and dinophysistoxin-2-all of which are lipophilic toxins-in the mussel Mytilus edulis. In relation to ASP toxins, only two recent studies focused on the effect of some cooking processes on the concentration of domoic acid (the main ASP toxin) in shellfish. et al. (2006) found that the toxin concentration in the mussel Mytilus edulis was unaffected or actually underwent a slight increase (20%) by steaming, depending on the location in which the mussels were harvested. In the other shellfish species studied, the Dungeness crab (Cancer magister), [START_REF] Hatfield | The fate of domoic acid in Dungeness Crab (Cancer magister) as a function of processing[END_REF] found a 67-71% reduction of domoic acid concentration in the viscera after the cooking process (boiling in fresh water or 3% salt water). To our knowledge, no studies have been done on other species, including the cockle Cerastoderma edule and the Manila clam Ruditapes philippinarum, which are important species in the shellfish market (Anonymous 2004, Anonymous 2008).

McCarron

Cooking processes have some degree of variability. The amount of water, salt concentration, temperature, time and other factors may sometimes vary, depending on the preferences of the cook. No information has been reported on the effect of these variations on the final toxicity of the cooked food.

Additionally, some monitoring programs have included pre-treatments (i.e. mild steaming) of the samples prior to analysis in order to facilitate shell removal and stabilize the matrix [START_REF] Hess | Tissue distribution, effects of cooking and parameters affecting the extraction of azaspiracids from mussels, Mytilus edulis, prior to analysis by liquid chromatography coupled to mass spectrometry[END_REF]. This is the case, for example, of the monitoring systems in Denmark and Germany which cook the mussels by light steaming before analysis [START_REF] Mccarron | Effects of cooking and heat treatment on concentration and tissue distribution of okadaic acid and dinophysistoxin-2 in mussels (Mytilus edulis)[END_REF]. The current European regulation stipulates that the total content of amnesic toxin in the edible part of molluscs should not exceed 20 mg of domoic acid kg -1 of meat (Community Decision of March 15, 2002), but it does not address the question of pre-treatments. If treatments are found to have a significant effect on the toxicity of the samples and their use is not strictly regulated, they might cause major changes in the current levels of closure used by the different monitoring systems, thus compromising the efforts made to achieve the harmonization of the regulations worldwide.

In this paper we have studied, the effect of: a) the usual cooking procedures, and b) some deviations from the standard procedure likely to take place during the cooking process on domoic acid concentrations in two commercially important species (cockle

Cerastoderma edule and Manila clam Ruditapes philippinarum).

Material and methods

Biological material

Cockles (Cerastoderma edule) and Manila clams (Ruditapes philippinarum) naturally contaminated with ASP toxins were obtained from the Ría de Camariñas (NW Spain).

A 5-kg sample of cockle and a 6-kg sample of Manila clam were taken and transported to the laboratory within a few hours of their collection. There, the individuals were carefully mixed and a number of 15-individual subsamples (depending on the experiment) were obtained completely at random. All individuals were weighed prior to being subjected to the experimental treatments.

Experimental design

The experimental designs used consisted of two parts that focused directly on the two objectives of this work. In order to quantify the effect of variations during the cooking process, 2-level factorial plans with central points were used. The effect of the standard way of cooking was quantified by means of a simple comparison between raw samples and the central points of the factorial designs were planned.

Two different factorial plans were used for each species to account for differences in common cooking methods. Cockles are usually steamed for a short time in a small amount of water (without being submerged), in some cases containing salt. Clams (clams marinière) are usually boiled, with the water containing some salt. Wine is frequently added, but in this experiment it was substituted by 12% (v:v) ethanol.

Consequently the factorial design corresponding to cockles included two factors (steaming time and salt content, each having two levels (plus the central points). As regards opening time, the lowest level was the time it took the cockles to open and the highest level was that time plus 5 min. The salt levels were 0 and 35g L -1 (Figure 1A).

The design for clams included three factors: boiling time, salt content and percentage of ethanol. Boiling and salt levels were the same as in the cockle, and the ethanol percentages were 0% and 6% (Figure 1B). Cockle treatments were performed in duplicate, while clam treatments were carried out without replication, with the exception of 4 central points that were included in both experiments.

All computations were made with Minitab 15 Statistical package.

Chemicals, toxin extraction and analysis

Domoic acid calibration solutions were prepared from certified reference materials Ultrapure water from a Milli-Q system (Millipore) was used in all preparations and manipulations requiring aqueous solutions.

Domoic acid was extracted from shellfish meat following the method of [START_REF] Quilliam | Rapid Extraction and Cleanup for Liquid Chromatographic Determination of Domoic Acid in Unsalted Seafood[END_REF]. The tissues in each sample were homogenized and 4 g of this homogenate were disrupted with Ultraturrax (IKA), for 3 min, with 16 mL of methanol-water (50:50, v:v). The resulting slurry was then centrifuged at 4000 rpm for 10 min and the supernatant filtered through a Millipore filter (Millex-HN, Nylon 0.45µm). The extracts were directly injected in the HPLC. Omitting the SPE-SAX purification step in the method because no interferent was detected by the DAD purity test, and because its use increases the variation coefficient of the determinations.

HPLC analyses were carried out by the method of [START_REF] Quilliam | Rapid Extraction and Cleanup for Liquid Chromatographic Determination of Domoic Acid in Unsalted Seafood[END_REF] with some minor modifications in order to adapt the method to existing equipments: 1) a Waters HPLC system including a photo diode array detector (PDA) was used for analysis; 2) a Lichrospher RP18 125 x 4mm, 5µm, reverse phase C18 column was used; 3) the isocratic elution was carried out with a mobile phase of water, acetonitrile and acetic acid, at a ratio of 89:10:1 v:v:v; and 4) the flow rate was 0.6 mL•min -1 .

Domoic acid was identified by comparing the retention time with that of a reference solution obtained from NRC, Halifax, Canada, and by examining the UV spectrum (maximum at 242nm). It was quantified by means of a comparison of the response of the samples at 242 nm, with the above-mentioned reference solution, and the purity of the peaks was checked by means of the wavelength (242.0 ±2.4nm) and by visual examination of the spectrum.

Results

Experiment A: Steamed Cockles

Effect of standard cooking.

Meat weight decreased (P=0.001) by 38% with the standard cooking process. This decrease was mainly due to dehydration, as no significant amount of particulate matter was found in the cooking water after the process (Figure 2A). The domoic acid burden of the cockle was also substantially reduced (P= 0.001). This reduction was estimated to be 52 % of the initial toxin amount (Figure 2B). The domoic acid concentration also decreased but to a lesser extent than the toxin burden (23%, P=0.032), as would be expected given the fact that toxin burden and meat weight have opposite effects on concentration, as it is the ratio between the two variables).

Effect of variations in cooking conditions

Steaming time was the only cooking variable with a relevant effect on tissue weight and toxin burden, producing a significant decrease in both variables (P = 0.002, in both cases). The other factor tested (salt concentration) and the interaction between the two factors (salt and time) (Figure 3A, B) did not have any statistically significant effects (Table 1).

Although the domoic acid concentrations observed after cooking for the longest time period and with the highest salt level were ca. 10% smaller than those corresponding to the lowest levels of these factors, (Figure 3C), the differences were not statistically significant. Moreover these two variables do not seem to interact with one another, since no statistical significance for the interaction term was found.

Experiment B: Clams marinière

Effect of standard cooking

The response of the meat weight of Manila clams to the cooking process was similar to that of cockles, but the responses of toxin burden and concentration were substantially different. Cooking produced a noticeable reduction (29 %) in the weight of the soft tissues (P = 0.006). As in cockles, this transformation was not due to a loss of particulate matter, since its presence in the cooking liquids was found to be negligible.

The domoic acid burden of the soft tissues remained unchanged after cooking, indicating that it was not degraded or released into the cooking liquid (Figure 4B). As the content of domoic was constant, the toxin concentration was only affected by the decrease in meat weight which, having diminished with cooking, produced a significant increase in concentration (P = 0.045) which was 26.5% higher in cooked molluscs than in raw ones (Figure 4C).

Effect of variations in cooking conditions

Cooking time was found to have a negative effect on the toxin burden and toxin concentration but not on meat weight. In the latter variable a slight decrease (ca. 5% of the average) was observed but it was not statistically significant (Figure 5, Table 2). The above-mentioned effects of time were only found to be significant when the factorial experiment was simplified by removing salt concentration and ethanol concentration as factors.

Discussion

Cooking can affect the domoic acid concentration and burden of different species in different ways. Toxin concentration decreased in the cockle while it increased in the Manila clam. This difference is due to two facts: 1) during cooking, the cockle loses a significant proportion of its toxin, but the Manila clam does not; and 2) in both species the weight of the soft tissues decreased, thus causing the toxin to concentrate in less biomass. In both cases weight undergoes a decrease. However, while in the cockle it partially counteracts the effect of the decrease of toxin burden in the concentration, in the case of Manila clams, where no reduction in toxin burden was detected, it produces an increase in concentration. The two species examined in this study, would seem to be in terms of response to cooking, like the mussel Mytilus edulis (McCarron et al. 2006), since the toxin concentration is maintained or increases only slightly, depending on the balance between the increase produced by weight reduction and the decrease derived from toxin loss.

Although the cooking processes studied here were not exactly the same, they share the same basic characteristics, i.e., they are thermal treatments in an aqueous environment, which would suggest that the differences found are more likely related to the species rather than to the differences in treatment. Only small differences in domoic acid concentration were able to be attributed to the likely deviations from the standard cooking procedure. In fact, only the cooking time seems to have had a significant effect, i.e., a reduction in toxin burden and concentration. Salt concentration during cooking had no effect either, as was also reported by [START_REF] Hatfield | The fate of domoic acid in Dungeness Crab (Cancer magister) as a function of processing[END_REF]

in the Dungeness crab

Cancer magister, where the effect of using salt water (3%NaCl) versus fresh water on the resulting domoic acid content was investigated. In view of the information presented above, it seems clear that the actual banning levels based on raw bivalves might over-or under-protect the consumers when shellfish are cooked. Moreover, epidemiological data based on toxin or toxicity quantification of raw shellfish, should be carefully evaluated if the intoxications are produced by cooked specimens. The use of cooked samples for monitoring purposes or the inclusion of species-specific factors to transform raw toxicity to cooked toxicity would be useful for the optimization of shellfish aquaculture or fisheries while safeguarding consumer health, provided that similar cooking methods are generally used for all species.

It is clear that monitoring the resources using heating-related pre-treatment of samples might in fact be more or less permissive than using raw samples, depending on the species and the toxin group. In the cockle, for example, control carried out using cooked samples would be more permissive than with raw samples and the opposite would be expected to be the case in Manila clams. Mussels Mytilus edulis would probably not be affected at all in view of the results of McCarron et al. (2006). While the approach might be correct, in the sense that it could approach more closely the actual levels that the consumers ingest, the direct introduction of these pre-processing steps without adapting the banning levels, (that are in general defined for raw shellfish) could produce differences in the actual banning levels used in the different monitoring systems or when dealing with different species. This fact should be taken into account in order to harmonize the regulation in the different countries and to prevent food alerts originating from the importation of products subjected to different (not theoretical but practical), banning levels.

Figure 1 .Figure 2 .

 12 Figure 1. Diagram of the two experiments. (A) Effect of cooking on cockle; and (B) on Manila clam. In both cases raw samples are compared to the central point of the factorial part of the experiment. Numbers inside the small squares indicate the number of replicates of each design point. 104x146mm (600 x 600 DPI)

Figure 3 .

 3 Figure 3. Main effects of steaming time and salt concentration on weight (A), domoic acid concentration (B) and burden (C) of the soft tissues (edible part) of the cockle Cerastoderma edule. 553x984mm (96 x 96 DPI)

Figure 4 .Figure 5 .

 45 Figure 4. Weight (A), burden (B) and domoic acid concentration (C) of the soft tissues (edible part) of the Manila clam Ruditapes philippinarum after (cooked) and before (raw) the standard cooking of this species (cooking). 526x980mm (96 x 96 DPI)

Table 1 .

 1 -Results of the analyses of the factorial experiment for cockle, for the two variables studied. The upper part of the table shows the magnitude of the effects, their corresponding coefficients and Ts and their probabilities. The lower part shows the ANOVA of the design indicating the degrees of freedom (DF), the mean square (MS), F and its probability.

				Food Additives and Contaminants					Page 16 of 17
	Analysis of variance		Weight			Burden			DA Concentration
	Term	Effect Coef	T	P	Effect Coef	T	P	Effect Coef	T	P
	Time	-11.5	-5.7	-5.0 0.002	-249.2 -124.6 20.5 0.002	-1.9	-0.95 -1.4 0.211
	Salt	-0.39	-0.2	-0.2 0.868	-58.3 -29.1 -4.6 0.317	-1.3	-0.67 -1.0 0.361
	Time*salt	2.29	1.1	1.0 0.352	63.4	31.7	-1.2 0.279	0.5	0.25	0.4	0.728
	Source	DF	MS	F	P	DF	MS	F	P	DF	MS	F	P
	Main effects	2	132.06 12.51 0.005	2	65501 11.22 0.007	2	5.4325 1.42 0.303
	2-Way Interac.	1	10.49 0.99 0.352	1	8043 1.38 0.279	1	0.5000 0.13 0.728
	Error	7	10.56			7	5840			7	3.8139

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Table 2 .

 2 -Results of the analyses of the factorial experiment for clams (simplified to one factor). The upper part of the table shows the magnitude of the effects, their corresponding coefficients and Ts and their probabilities. The lower part shows the ANOVA of the design indicating the degrees of freedom (DF), the mean square (MS), F and its probability.

	Page 17 of 17
	Analysis of variance		Weight				Burden				DA Concentration
	Term	Effect	Coef	T	P	Effect	Coef	T	P	Effect Coef	T	P
	Time	-2.282 -1.141 -0.92 0.378	-134.04 -67.02 -2.28 0.046	-2.75 -1.37 -2.72 0.022
	Source	DF	MS	F	P	DF	MS	F	P	DF	MS	F	P
	Main effects	1	10.420 0.85 0.378	1	35934 5.19 0.046	1	15.12 7.37 0.022
	Error	9	13.082			9	7297			9	2.163

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Acknowledgements

This research was funded by the Consellería de Innovación e Industria, Xunta de Galicia through the cooperation agreement EPITOX. We have to acknowledge to Mónica Goicoechea, Fabiola Arévalo and Susana Muñiz for their technical assistance. We also gratefully acknowledge the Cofradía de Camariñas for their support.