Nieves Carro
email: ncarro@intecmar.org

Isabel García

María Ignacio

Ana Mouteira

Levels and distribution pattern of PCB congeners in razor clams Ensis siliqua (Linnaeus, 1758) from Galicia (N.W., Spain) in relation to biometric parameters

Keywords: INTECMAR, Chemical Contamination, INTECMAR, Chemical Contamination Mouteira, Ana, INTECMAR, Chemical Contamination Saavedra, Yolanda, INTECMAR, Chemical contamination Ignacio, Maria, INTECMAR, Chemical Contamination Methods/Techniques: Chemometrics, Chromatography -GC, Chromatography -GC/MS Additives/Contaminants: Environmental contaminants, PCBs PCBs, Biometric parameters, Gas chromatography, Ensis siliqua, Galician coast, PCA (Principal component Analysis)

To establish polychlorinated biphenyl (PCB) levels and distribution as well as their relationship to biometric parameters in the razor clam Ensis siliqua, 15 individual samples collected from two points sited in the Islas Cies, Ría de Vigo (Galicia, Spain), collected monthly in the period from February 2003 to April 2004, were analysed. PCBs (IUPAC nº 31, 28, 52, 101, 118, 153, 105, 138, 156 and 180) were isolated by Soxhlet extraction. The identification and quantification were performed by GC/MS and GC (electron capture detector, ECD), respectively. The isomer concentrations in the razor clam E. siliqua were in the order hexachlorobiphenyls > pentachlorobiphenyls > heptachlorobiphenyls > trichlorobiphenyls > tetrachlorbiphenyls. No correlation was observed between the E. siliqua shell length and PCBs levels. The PCBs 31, 101, 105 and 153 levels were statistically related with some biometric parameters (p<0.05). Multivariate techniques such as Principal Component Analysis (PCA) show clear seasonal differences between samples.

Introduction

Galicia in north-west Spain is an important shellfish producing region, characterized by a peculiar geography with a succession of estuarine bay systems called Rías which give good oceanographic conditions for aquaculture, mainly for farming bivalve molluscs.

The razor clam Ensis siliqua (Linnaeus, 1758) is a benthic bivalve organism that lives in the bottom of sandy sediments in the infralittoral region. It is found in the middle and external zones in the Ría de Vigo [START_REF] Rolán | Moluscos de la Ría de Vigo[END_REF]. The Islas Cies belong to the National Park of Atlantic Islands, they are three islands sited in the mouth of the Ría de Vigo.

Polychlorinated biphenyls (PCBs) are biologically, chemically and physically stable in the environment. They are hydrophobic and persistent contaminants, which bioaccumulate along the food chain [START_REF] Erickson | Physical, chemical, commercial, environmental and biological propierties[END_REF]. The fat content and condition index of mollusk are strongly connected to the reproductive cycle because a part of their lipid content is lost during the spawning period. Moreover, the gonad cycle is closely related to environmental conditions and seasons [START_REF] Hawkins | The mussel mytilus: ecology, physiology, genetics and culture[END_REF][START_REF] Brousseau | Gametogenesis and spawning in intertidal oysters (Crassostrea virginica) from Western Long Island Sound[END_REF]. So, PCBs are generally distributed in marine organisms as a consequence of condition index and lipid content variations, mainly during reproduction periods.

There is a lack of information relating to hydrophobic baseline micropollutant levels in E. siliqua from the Galician coast. Few regional studies about reproductive cycle and biometric parameters of this species in Galicia coast have been reported [START_REF] Darriba | Gametogenic cycle of Ensis siliqua (Linnaeus, 1758) in the Ría de Corcubión, Northwestern Spain[END_REF]. The objective of this work was to study the levels and distribution of 10 PCBs, recommended by the International Council for Exploration of the Seas (ICES) (Duinker et al. 1988) with IUPAC number 31, 28, 52, 101, 118, 153, 105, 138, 156 and

Materials and Methods

Sampling. Biometric parameters

Samples of 15 adults specimens of sizes between 120 and 160 mm coming from two points of The Islas Cíes (Ría de Vigo), Playa de Aras das Rodas and Playa de San Martin, were monthly collected during the period from February 2003 to April 2004 (See Figure 1). Each specimen was measured and the flesh was dissected from the shells. Condition indices were calculated taking account shell and body weights of each sample. CI= (body weight/body + shell weights).

[Insert Figure 1

Standard solutions and calibration

Solutions of Standard stock (crystalline solid) were prepared by weighing a appropriate amount of each standard and diluting to 5 mL with isooctane. Working solutions were made by dilution of the solution of stock. For quantitative gas chromatographic determinations, calibration was performed at five concentration levels for each congener spanning the range of 1-400 ng g -1 and using CB 155 (500 ng g -1 ) as internal standard.

Sample preparation

Samples were homogenized. Homogenates of the flesh were frozen (-30 ºC), freezedried and Soxhlet extracted (5 g of sample; 150 mL of dichloromethane-pentane, 1-1; 8 hours) by duplicate. The sample extract was concentrated under vacuum evaporation to dryness and then redissolved in 1 mL of isooctane. An aliquot of the extract was used to determine gravimetrically the lipid content. Interfering lipids were removed from a suitable portion of the concentrate by column chromatography on alumina (6% deactivated). The sample extract was eluted with n-pentane. The fraction of PCBs was isolated using column chromatography on Silica (1% deactivated) and eluted with isooctane. CB 155 was added as an internal standard prior to analysis by gas chromatography [START_REF] González-Quijano | Determinación de congéneres individuales de bifenilos policlorados en organismos marinos[END_REF].

Chromatographic analysis

Sample extracts (1 µL) were analyzed individually by separate runs by high resolution GC using a Perkin-Elmer Autosystem GC with an electron capture detector (ECD). A TRB-5 (Teknokroma, Spain) 5 % diphenyldimethyl siloxane capillary column (60 m x 0,20 mm i.d. x 0,4 µm phase thickness) was used. The chromatographic conditions were: the column temperature program was 90º C (3 min.) to 215 ºC (40 min.) at 30 ºC/min. and 275ºC (30 min.) at 5ºC/min.; the injector temperature (splitless mode, 1,8 min.) was 270 ºC; the ECD temperature was 365 ºC; carrier gas was hydrogen purchased from Air Liquid (Spain). The confirmation of polychlorinated biphenyl compounds was performed by injecting a 1 µL of sample extract in a Varian Saturn 2000 Gas Chromatograph-Ion Trap Detector Mass Spectrometer (ITD). The experimental conditions and capillary column were identical to those described before; carrier gas was helium (Air Liquid, Spain). The mass conditions were: the trap temperature 170 ºC; the scan range 100-450 m/z; the multiplier delay 6 min.; the emission current 10 µA; the multiplier voltage 1600 V; the maximum ionization time 25000 µs. Data numerical analysis was performed by means of the Minitab 14 statistical package.

Quality control

The QA/QC (Quality Control/ Quality Assurance) procedures included analysis of blanks, duplicates and Standard Reference Materials (SRMs). Curves of calibration have been carried out at five concentration levels using suitably diluted standards (working solutions). Each concentration level has been injected in duplicate and peak heights have been fitted by linear regression. The coefficient of correlation was 0,9999 31, 28, 52, 101, 118, 153, 105, 138, 156 and 180). The sample was manually mixed for 30 min and allowed to air-dry for 7 days and stored in the dark for a month before analysis. The expected final concentrations were calculated to range from 15 to 20 ng.g -1 . The mean recoveries of target compounds were from 80.0 to 101.2 %.

A study of method accuracy was performed on a certified reference material, mussel tissues homogenate, NIST 2977. The PCBs content agreed with the certified value. The certified content for the organochlorine compounds, PCBs 31, 28, 52, 101, 118, 153, 105, 138, 156 and 180 are 3.92 + 0.24, 5.37 +0.44, 8.37 + 0.54, 11.2 + 1.2, 10.5 + 1.0, 14.1 + 1.0, 3.76 + 0.49, 16.6 + 1.6, 0.96 + 0.09 and 6.79 + 0.67ng g -1 ,respectively. Detection limits were calculated as three times the standard deviation of the peak height for 30 determinations of the blank and were for PCBs 31, 28, 52, 101, 118, 153, 105, 138, 156 and 180, 0.04, 0.1, 0.08, 0.08, 0.1, 0.06, 0.07, 0.04, 0.04 and 0.01 ng L -1 , respectively. The limits of quantification were calculated as 10 times the standard deviation of the peak height for 30 determinations of the blank and were for PCBs 31, , 52, 101, 118, 153, 105, 138, 156 and 180, 0.13, 0.32, 0.28, 0.26, 0.33, 0.21, 0.22, 0.13, 0.14 and 0.04 ng L -1 , respectively.

Results and discussion

The average of two analytical replicates of ten individual PCB congener concentrations (IUPAC number 31,28,52,101,118,153,105,138,156 and 180) Galicia 2003). This situation recurs constantly in Galician coast and in other littorals and ecosystems [START_REF] Carro | Possible influence of lipid content on levels of organochlorine compounds in mussels from Galicia coast (Northwestern, Spain). Spatial and temporal distribution patterns[END_REF][START_REF] Olafsdottir | Seasonal fluctuations of organochlorine levels in the common eider (Somateria mollissima) in Iceland[END_REF] The isomer concentrations of E. siliqua body were in the order hexachlorobiphenyls>pentachlorobiphenyls>heptachlorobiphenyls>trichlorobiphenyls> tetrachlorobiphenyls. Hexachlorobiphenyls 153 and 138 were the most prevalent congeners accounting for 47.6% and 19.2% of the total PCBs levels, respectively. This indicates that the biota is exposed predominantly to higher chlorinated PCB formulations. PCBs 153 and 138 are the main constituents in Arochlor 1254 and 1260 because they were the most largely used in commercial mixtures in European countries [START_REF] Ivanov | Characterization of polychlorinated biphenyl isomers in sovol and thrichlorodiphenyl formulations by high resolution gas chromatography techniques[END_REF]. On the other hand, both PCBs belong to the group of difficult to metabolize congeners. These percentages are consistent with the studies of PCBs in other marine organisms [START_REF] Carro | Possible influence of lipid content on levels of organochlorine compounds in mussels from Galicia coast (Northwestern, Spain). Spatial and temporal distribution patterns[END_REF][START_REF] Storelli | Levels and congener pattern of polychlorinated biphenyls in the bubbler of the Mediterranean bottlenose dolphins Tursiops truncatus[END_REF].

In Figure 2 it can be seen that E. siliqua coming from playa de San Martin had higher levels of hexachlorobiphenyl congeners, less mobile and less water soluble compounds, than those coming from playa de A. Rodas. This means that playa de San Martin point is nearer a source of contamination. Low chlorinated biphenyls, the most water soluble compounds, were predominant in biota coming from playa de A. Rodas, point away from a source. This hypothesis is supported by the fact that the mean of ΣPCBs in E. siliqua coming from playa de San Martin (13.24 ng g -1 ) was slightly higher than the mean of ΣPCBs in samples coming from playa de A. Rodas (9.72 ng g -1 ). However, this conclusion cannot be conclusively confirmed because the differences between zones were not statistically significant (see statistical analysis).

[Insert Figure 2 The percentage lipid content was found at the lowest levels during February-April, when the only period of spawning occurred (see the Table I). The average lipid content was similar for samples coming from the two studied points, 5.50% for playa de San Martin and 5.64% for playa de A. Rodas.

Biota coming from playa de San Martin possessed slightly worse biometric characteristic, shell length, 133 mm and condition index, 0.56, compared to E. siliqua from playa de A. Rodas, 136 mm. and 0.59, respectively, it is logical and consistent with PCBs levels. As previously mentioned, the point in playa de San Martin underwent slight conditions of environmental stress. The two studied zones are geographically very close, so biometric parameters were similar.

Statistical analysis.

The statistical analysis considered concentrations of 10 PCBs and 3 biometric parameters as quantitative variables. Levels reported as "Not detected" were assigned of half the detection limit. All data were tested for homogeneity of variance (Kolmogorov Smirnov Test) and analyzed using Analysis of Variance (One Way ANOVA. Fisher's method) in order to check the influence of biometric parameters on chemical variables. This analysis indicated a significant effect (p<0.05) of quantitative biometric parameters, fat content on PCB 101 levels and condition index on PCBs 31 and 153 levels. The relationship of PCB congeners levels to lipid content is very complex, fat nature, extraction methods and difference between individual organisms have an important role which is difficult to know [START_REF] Ewald | Differences between Bligh and Dyer and Soxhlet extractions of PCBs and lipids from fat and lean fish muscle: Implications for data evaluation[END_REF]. Moreover, the levels of PCBs are very low and the analysis methods are not able to pick up the small differences among individuals collected monthly, induced by the variation in lipid. Spatial and temporal trends were also studied using one-way ANOVA. If the variable month was grouped in seasons: spring, summer, autumn and winter, only fat content and PCB 105 levels showed a significant season term using the ANOVA (p=0.00 and p=0.031, respectively). PCBs 101 and 180 levels were statistically influenced by the sampling months (p=0.00). No statistical significance was found for sampling zones, which is logical because both points are in the same estuarine bay, Ría de Vigo, and they have been subjected to similar environmental conditions, exhibiting a similar biological cycle and therefore a similar trend in biometric parameters and not very different PCBs levels. The significant differences between seasons and levels of PCBs and biometric parameters were supported by PCA that provided an easier visualization of the relationship among variables in a large data set. PCA is a multivariate technique that tries to reduce the number of original variables explaining most of variance in the data, it transforms a set of correlated measured variables into a set of a few not correlated components. If PCA was applied to original data, the retained first three principal components explained the 79.6 % of total data set. Table II shows the loading of the variables in the first three components of the PCA, that is to say the contribution of the original variables to each principal component. The first component had high negative (PCBs 101,118,138,156,180 and ΣPCBs), the second component had high negative loading for trichlorobiphenyl compounds (PCBs 31 and 28) and PCB 105 and positive loading for fat content. The third component had high scores for shell length, condition index and PCB 52 (negative ones) and PCB 153 (positive one). In the analysis of the Pearson correlation coefficients, the higher chlorinated biphenyls had a strong correlation between them, the same behavior presented the lower chlorinated biphenyls, it could be due to either the probable similar profile of input and bioaccumulation or the same origin of both kinds of compounds.

[Insert Table II about here].

In Figure 3a it can be seen the PCA scores plot, distribution profiles for the studied samples. In the plane of the first two components, three groups corresponding to the samples collected in spring, summer and autumn appear.

The group of the samples collected in winter (January, February and March) had a great dispersion. The most polluted sample (the highest value of ΣPCBs), collected in February 2003 in playa de San Martin, was defined by negative scores on PC1 and PC2, they had a high concentration of the lower and higher chlorinated biphenyls at the same time. The sample collected in March 2003 in playa de A. Rodas also showed an high negative score for PC2, this sample had the highest levels for the trichlorobiphenyls, PCBs 31 and 28, and for the PCB 105 (see Table II). Samples collected in January 2004 showed high negative scores on PC1, having high concentrations of the higher chlorinated compounds (PCBs 101,118,138,156,180 and ΣPCBs). The first group, formed by samples collected in spring (April, May and June), showed different degree of negative scores on PC2, these samples were contaminated by the lower chlorinated biphenyls and PCB105 in more or less degree depending on the month (May or April), samples from May were situated nearer from the origin of coordinates, then were less contaminated. Samples collected in April 2004 were associated with the negative part of PC1 and to the positive part of PC2, showing a greater contamination with the higher chlorinated compounds and a higher fat content.

The second and first group, samples collected in summer and autumn had positive scores for PC1 and PC2. Summer samples were distributed along PC1, those collected in September were defined by the highest positive scores on PC1 and PC2. These samples were the least contaminated and contained the highest fat amounts, period before spawning.

All samples collected in 2004 were related to the negative part of the PC1 and the positive part of PC2, having high levels of PCBs 101, 118, 138, 156 and 180 and ΣPCBs.

In Figure 3b it can be seen that the samples of the two zones which were studied (playa de San Martin and A. Rodas) were strongly associated with the sampling month, and no spatial and temporal trends were observed, it shows that the contamination by PCBs

  from two points of The Islas Cíes, Ría de Vigo (Playa de Aras das Rodas and Playa de San Martin) during the period from February 2003 to April 2004. Univariate and multivariate techniques were applied in order to check the influence of biometric parameters on PCBs levels and to establish any the spatial and temporal trends.

  , dichloromethane and isooctane for organic trace analysis and aluminium oxide (alumina), silica gel and anhydrous sodium sulfate were purchased from Merck (Darmstadt, Germany). Analytical reagent grade PCB individual congener standards were supplied by Dr. Ehrenstorfer (Augsburg, Germany). A certified reference material, mussel tissues homogenate, purchased from National Institute of Standards and Technology (NIST 2977) was used for quality control.

  target compounds. The linearity range was from 3 to 300 ng g -1 for all studied compounds. Method reproducibility studies were performed for single compounds on five replicate samples (mixtures of PCBs), analyzed by two people on different days. The mean relative standard deviation ranged from 8 to 10 % for the PCBs which were studied. The percentage recovery of PCBs were investigated by an exhaustive Soxhlet extraction of E. Siliqua spiked by slowly pouring it over acetone containing the target compounds (PCBs

  expressed in ng g -1 dry weight (dw) of E. siliqua coming from two studied points and monthly collected in the period from February 2003 to April 2004 is shown in the Table I. The ΣPCBs is the sum of all individual PCB congeners. The levels of ΣPCBs varied in the range of 2.75 to 67.70 ng g -1 . E. siliqua collected in the playa de San Martin in February 2003 and in the playa de A. Rodas in January 2004, the winter months, gave the highest concentarions of ΣPCBs. The lowest value appeared in July 2003 in playa de San Martin. This behaviour could be due to unfavourable meteorological conditions in winter which coincide with extremely high rain fall (162 mm. of total precipitation in January and February 2003 and 106 mm. of total precipitation in January 2004, Anuario climatolóxico de Galicia 2003 y 2004) and rates of re-suspension attributed to storm events that can cause inputs of high PCBs levels. In summer, however, there was hardly any inflow contributions and for this reason the PCBs were found in samples at very low levels (40 mm. of total precipitation in July 2003, Anuario climatolóxico de

  about here]

  was observed between shell length and concentration of PCBs. So, the accumulation of pollutants by E. siliqua organism could have reached a steady state and their elimination rate, mainly caused by their reproductive cycle, would be sufficiently fast to compensate for the dietary intake.

  the studied sampling months and shows no upward or downward trend.[Insert Figure3babout here]ConclusionsDue to the low statistical significance between biometric parameters and levels of these hydrophobic pollutants, it is not necessary to take into account the lipid content or condition index when data are expressed. It is very useful in monitoring programs because biometric parameters are influenced by other complex factors such as reproductive cycle and physiological state (internal factors) and/or meteorological and environmental conditions (external factors) and/or even, analytically speaking, their determination or measurement. This benthic organism can be used as bioindicator in zones where mussels are not cultured because they are able to bioaccumulate contaminants to much higher concentrations than those found in water and to the extent of showing clear seasonal differences in level of PCBs. Another important reason for considering the razor clam as a good bioindicator is that the distribution patterns of PCB congeners are similar to those of other marine organisms such as mussel. This work is a good tool for establishing the background levels of PCBs in E.siliqua from the Galicia coast.

  plot. Distribution of Ensis siliqua samples collected in the different seasons in the plane of the first and second components. 1. Spring (April, May, June), 2. Summer (July, August, September), 3. Autumn (October, November, December), 4. Winter (January, February, March). b. Scores plot. Distribution of Ensis siliqua samples collected in the different months in the plane of the first and second components. 1. January, 2. February, 3. March, 4. April, 5. May, 6. June, 7. July, 8. August, 9. September, 10. October, 11. November, 12. December.

  . TableIshows that samples collected in April 2003 were less contaminated than samples collected in April 2004.

	The two sampling points had very similar concentrations in this month.

[Insert Table I about here]

Table I .

 I Average concentration of PCBs (ng g -1 ) in E. siliqua from two points in The Islas Cies (R. Vigo). Biometric parameters: Shell length (mm), fat (%), condition index.

	San Martin
	Month	Length FAT	CI	ΣPCB CB31 CB28 CB52 CB101 CB118 CB153 CB105 CB138 CB156
	Feb.03	133.93 2.96	0.62	67.7 0.77 0.27 N.D. 0.81	0.88 61.59	0.82	2.34	N.D.
	March 03 132.33 3.17 0.5888 3.27 N.D. 0.14 N.D 0.27	N.D.	2.1	N.D.	0.76	N.D.
	April 03 134.73 2.93 0.6233 6.17 1.92 0.93 0.14 0.32	0.2	1.23	0.09	1.02	0.21
	May-03 128.13 5.84 0.5113 5.73 0.66 0.36 N.D 0.67	0.43	2.06	0.15	1.3	0.1
	July 03 134.33 6.59 0.6255 2.75 0.07 0.04 0.13 0.51	0.48	N.D.	0.22	1.25	N.D.
	Aug.03 133.53 7.14 0.6315 5.34 0.11 N.D 0.14 0.59	0.57	2.04	0.31	1.51	N.D.
	Sept.03 138.53 8.05 0.1892	4.7 0.16 0.14 0.04 0.65	0.47	1.76	N.D.	1.43	N.D.
	Oct.03	120.27 3.54 0.6455 8.85 0.05 0.14 0.23 0.79	0.18	2.29	N.D.	2.76	0.22
	Nov.03 136.87 7.03 0.5849	5.3 0.33 0.11 0.11 0.59	0.39	1.71	N.D.	1.56	N.D.
	Dec.03 Jan.04 April 04 Month Aras das Rodas 137.26 8.3 141 4.43 0.5485 26.48 0.21 0.19 0.46 3.37 0.6189 4.31 0.24 0.1 0.09 0.64 129 6.09 0.6299 18.34 0.07 0.08 0.18 1.9 Length FAT CI F o ΣPCB CB31 CB28 CB52 CB101 CB118 CB153 CB105 CB138 CB156 0.48 N.D. N.D. 2.05 N.D. 1.06 12.51 0.33 0.18 0.66 0.68 7.79 0.2 6.18 0.54 Feb.03 133.73 2.84 0.6591 5.54 0.71 0.34 0.78 0.32 N.D. 2.35 N.D. 1.04 N.D. r
	March 03 143.4 3.95 0.5965 9.04 1.64 1.4 0.06 0.58 April 03 137.87 2.74 0.5782 6.53 0.84 0.35 N.D. 0.52 May-03 128.67 4.42 0.615 5.93 0.68 0.74 N.D. 0.6 P	0.37 0.44 0.46	2.12 2.12 2.03	0.87 0.61 0.08	1.64 1.55 1.34	0.32 0.1 N.D.
	July 03 136.73 6.7 Aug.03 133.27 6.87 0.6394 0.6351 3.86 0.09 N.D 0.11 0.43 4.4 0.86 0.27 0.16 0.4 Sept.03 128.6 7.56 0.2213 3.82 0.1 0.09 0.08 0.35 Oct.03 132.23 6.77 0.6088 5.73 0.12 0.03 0.22 0.57 Nov.03 144.21 7.9 0.6384 4.7 0.07 0.09 0.22 0.55 Dec. 03 136.13 6.52 0.638 11.29 0.52 0.16 0.08 0.74 e e r	0.46 0.35 0.43 0.66 0.39 0.45	1.56 1.35 1.42 1.45 1.17 2.93	0.01 0.05 0.12 N.D. N.D. N.D.	1.14 0.94 1.15 1.9 1.45 3.21	0.01 N.D. 0.02 N.D. N.D. 0.25
	Jan.04 April 04 N.D. Not detected	138.67 5.5 140.1 6	0.5941 36.6 0.27 0.15 0.54 4.55 0.6218 19.18 0.08 0.06 0.2 1.97 R e v i e w 1.23 16.27 0.7 8.03	0.44 0.16	2.84 6.43	1.01 0.43
					O n
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Table I .

 I Average concentration of PCBs (ng g -1 ) in E. siliqua from two points in The Islas Cies (R. Vigo). Biometric parameters: Shell length (mm), fat (%), condition index.

	CB180
	0.22
	N.D.
	0.11
	N.D.
	0.05
	0.07
	0.05
	2.19
	0.5
	0.71 7.51 0.68 CB180 N.D.	F o r
	0.04 N.D. N.D.	P
	0.05 0.02 0.06 0.78 0.76 2.95	e e r
	9.3 1.12 N.D. Not detected	R e v i e w
		O n
		l y

Table II .

 II Loading of the variables in the first three components of the PCA

			Food Additives and Contaminants	Page 18 of 21
	Variable PC1		PC2	PC3
	ΣCB	-0,365		-0,159	0,356
	Length -0,135		-0,054	-0,162
	FAT	0,086		0,423	0,126
	IC	-0,071		-0,134	-0,176
	CB31	0,032		-0,508	-0,205
	CB28	0,032		-0,491	-0,253
	CB52	-0,199		0,119	-0,448
	CB101 -0,422 CB118 -0,396 CB153 -0,273 CB105 -0,238 CB138 -0,173 F o	0,144 0,113 -0,204 -0,381 0,131	-0,171 0,166 0,477 0,188 0,169
	CB156 -0,393 CB180 -0,379	r	0,045 0,147	-0,271 -0,268
			P
			e e r
			R v w e i Map with sampling points. e 177x125mm (300 x 300 DPI)
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	100%
	90%
	80%
	70%
	60%		hepta-CBs
			hexa-CBs
	50% 30% 40%	F r o	penta-CBs tetra-CBs tri-CBs
	10% 20%	e P
	0%	e r Playa S. Martiño	Playa A. Rodas
		R
		e
		v i e
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Acknowledgments

The authors wish to thank the Pathology Department for supplying samples.


Barbieri Giampiero
email: giampiero.barbieri@ssica.it

Frigeri Giorgia

Identification of hidden allergens -detection of pistachio traces in mortadella

Keywords: allergens, pistachio, mortadella, PCR method

An analytical method based on the detection of specific DNA was developed, and was applied to mortadella samples with and without pistachio (Pistacia Vera). The method is proposed for detection of traces of pistachio deriving from previous processes or from accidental contamination, since in predisposed individuals pistachios can cause allergic reactions leading to anaphylactic shock. Three pairs of primers were identified and tested by PCR on mortadella samples prepared with pistachio. Accidental contamination was also simulated. The optimised PCR was able to detect the presence of pistachio even at low concentrations. The primers pair PSTC 1-2 is suggested for unambiguous identification of pistachio in mortadella. The limit of detection for this primers pair was 100 mg/kg. No interference was observed from other spices or ingredients utilised in the formulation of the mortadella. The method enabled the identification of possible traces of pistachio remaining in the production plant after less than thorough washing.

Introduction

Analysis of traces of food allergens is desirable to minimize the risk to consumers.

Sensitive test systems are coming into use for some allergens but some analytical problems remain. The main problems are related to extraction, cross-reactivity, matrix effects and food processing. Enzyme-linked immunosorbent assay (ELISA) is the most frequently used analytical method to detect allergens. DNA-based methods are often controversial since they do not detect allergenic protein, but they have many advantages: good extraction of DNA from some problematic food matrix and its unchanging nature set against variability of protein composition (Poms et al. 2004).

An ingredient sometimes present in certain formulations of mortadella is pistachio (Pistacia Vera), a component that may cause serious allergic reactions in sensitive individuals.

Pistachio belongs to the anacardiaceae family, together with cashew and mango, whereas the common sub-class of the Rosidae groups it with other allergens such as soybean, peanut, English walnut and hazelnut. Cross reactivity with cashew has often been observed in the past (Jansen et al. 1992;[START_REF] Fernandez | Allergy to pistachio: cross-reactivity between pistachio nut and other anacardiaceae[END_REF] and has been confirmed in recent studies (Goetz et al. 2005;Roux et al. 2002); it is probable that the seed storage proteins belonging to 11S globulin and 2S albumins play a key role in clinical cross reactivity (Tawde, 2004).

Since processing of mortadella does not involve other types of nut other than pistachio, it constitutes the only real risk for consumers who are allergic to nuts. For this reason, it is important to be able to rule out its presence even in traces or, at any rate, to indicate its possible presence on the label, in accordance with the law (European Directive 2003/89/EC) Naturally, when it is a stated ingredient, its presence is clearly visible, since the whole nut is utilised in the mixture and can be seen in the slice. There may be pistachio dust even on products where its presence is not stated, owing to dust contamination in the environment in which the ingredients are kept, or to contamination from preceding processes if sufficient steps have not been taken to avoid it. This type of contamination may be difficult to identify and to quantify. A certificate testifying to the absence of pistachio, even in traces, is often required for the exporting of mortadella outside Italy. At present, no analytical methods are available for the specific detection of pistachio and the only immunoenzymatic kits for nuts identification concern hazelnut and peanut (Kiening et al. 2005). The aim of the work reported here was to develop a method of identifying traces of pistachio in mortadella utilising PCR methodology. Analysis with the PCR method for the detection of allergenic species, like all DNA tests is not as specific as analysing for the presence of protein responsible. Nevertheless, with regard of pistachio in mortadella, it constitutes a reasonable guarantee of absence of the relative allergen protein.

Materials and methods.

Preparation of the samples

Pistachio were purchased, either fresh or toasted, from local markets, in order to obtain pistachio DNA reference material. The mortadella, without pistachio (MP0) or with pistachio (MP1), was produced in an industrial plant. The mortadella samples containing pistachio were prepared by mixing in 5 g of whole pistachio nuts for every kg of mixture, as is the custom for the traditional product.

Contamination deriving from less than thorough cleaning of the plants is presumably very slight and thus cannot be quantified. The same goes for contamination due to dust stirred up by draughts of air inside a storage room. The sensitivity to these types of contamination was simulated with two experimental strategies: a) A meat mixture was prepared with the intentional addition of a known and very low quantity of pistachio dust M100 (100 mg/kg) and M40 (40 mg/kg). b) A batch of mortadella (MCONT) without pistachio was prepared on production lines not cleaned after a preceding processing with pistachio, simulating an involuntary contamination of the first mortadella samples produced with the new mixture.

A control test of the cleaning of the production line was carried out as follows: sterile paper swabs lightly soaked in sterile water were rubbed onto the critical points of a mortadella production plant (kneader screw, bagging machine bottom and outlet). DNA was subsequently extracted from these. A further test of the effectiveness of the method was conducted, eliminating all the pieces of pistachio from a slice of mortadella, including the product adjacent to the nut, and mincing the rest, from which DNA was extracted and examined for traces of pistachio.

Mortadella samples of around 12 kg in weight were produced.

From mortadella samples of each batch of production, four slices were obtained in differing positions: two slices from the ends, one from the middle and one three-quarters of the way through the product. Each slice was minced, 100 g was homogenised with sterile water to obtain a slurry, and a sample of around 200 mg was taken. The fresh and toasted pistachios (PSTC) were milled in their turn, and 100 mg of these were utilised for the analysis. The same protocol was used for the DNA extraction of ingredients, flavourings and some types of nuts listed in Table 1 to check their interference with the pistachio test.

Extraction of the DNA and PCR

The samples were incubated in 2 mL test tubes at 37°C overnight, after the addition of 500 µL of extraction buffer ( Tris-HCl 0.01 M pH 8.0, NaCl 0.15M, EDTA 2 mM ), 50 µL of 10% Sodium dodecylsulphate and 5 µL of proteinase K ( 20 mg/mL). After centrifugation for 15 min at 14 000 g, the supernatant was purified with 1 vol of CHCl 3 / isoamylic alcohol 24:1, and then with 1/3 vol of 10% ammonium acetate. From the supernatant obtained by centrifugation (15 min at 14 000 g) the DNA was precipitated with 1 vol of 80% isopropanol. The pellet was washed in 70% ethanol, air-dried, suspended in sterile water solution, measured spectrophotometrically by absorption at 260 nm and diluted to a final concentration of 100 ng/ µL.

In this study, we utilised the sequences of two genes codifying for a protein belonging to the dehydrin family (Golan-Goldhirsh, 2001), and for a ribulose 1,5-bisphosphate carboxylase/oxygenase (Savolainen et al. 2000). For the PCR on the DNA extracted, on the basis of the sequences of two genes codifying for pistachio proteins obtained from the literature, 3 pairs of primers were designed using the Primer3 software (Rozen and Skaletsky, 2000). Two of these are planned on COR gene for dehydrin PSTC1 (5'-GAATACGGGGAGCACAAAGA-3'), and PSTC2 (5'-AACCTCTCGCTAACCCCATT-3') , Thermocycler conditions were as follows. PCR of the primers PSTC 1-2 and 3-4: 35 cycles consisting of DNA denaturation at 94°C for 10 s, primers annealing at 58°C for 20 s, primer extension at 72°C for 15 s; with the primers PSTC 5-6, the annealing T° was lowered to 52°C for 20 s and the extension time at 72°C was prolonged to 30 s. Both protocols have a final extension step at 72°C for 5 min. PCR was carried out in PTC-100 termocycler (MJ Research). The amplification products were visualised with 0.5 µg/mL ethidium bromide after an electrophoretic run on 2% agarose gel. The running condition were constant voltage at 120 V for 1 h in Tris Borate EDTA 0.5X buffer.

Results and discussion

Figure 1 shows the electrophoretic pattern of the three PCRs with which we tested the efficacy of the pairs of primers. These primers were designed to amplify sequences characteristic of the pistachio species and any interference by various commercial flavours the results of controls made on various types of nut with the three pairs of primers: it can be observed that PSTC 1-2 and PSTC 3-4 are specific for pistachio, whereas there is an interference with cashew and Brazil nut for the PCR conducted with PSTC 5-6. This fact, however, is of only relative significance in the case of mortadella analysis, since these are not ingredients utilised in the technology of this cold meat product.

The PCR conducted with all pairs of primers on DNA extracted from the mortadella samples always allowed for the unequivocal identification of the products with pistachio, even those contaminated intentionally (MCONT and M100), and a positive result was also yielded by a sample in which the whole pistachio nuts were manually removed from the slice before the extraction of the DNA. The mere presence of the nut in contact with the meat was sufficient to give a positive signal for the PCR analysis with primer PSTC 1-2.

However, the M40 sample yielded a positive signal only with PSTC 1-2, but was not always detectable with the other pairs of primers, and was detected in only 3 samples out of 12 (data not shown).

The DNA extracted from the swabs taken at various places in the plant was sensitive to the presence of pistachio. This yielded the amplicon of the pistachio with PSTC 1-2 and PSTC 3-4 also after simple washing with water, disappearing only when rinses were done first with soda and then with normal water.

By carrying out a combined amplification of PSTC 1-2, PSTC 3-4 and PSTC 5-6, pistachio can be detected in mortadella samples. This includes accidental contamination in the case of neglect in cleaning the equipment (simulated by MCONT sample), or of crosscontamination by dust from ingredients (simulated by M100 and M40 samples). A contamination of 100 mg/kg of pistachio in mortadella was always detected. The limit of detection is thus close to this value. The presence of traces of pistachio due to accidental contamination can be unmistakably detected using PCR using three pairs of primers (PSTC 1-2, PSTC 3-4, PSTC 5-6). The first pair of primers is extremely sensitive although they yield co-amplified fragments. The low detection limit (100 mg/kg of pistachio in mortadella) that these primers achieve enables the detection in the ingredients of pistachio, even in traces, due to accidental contamination. In addition, the two pairs of primers planned on the COR gene are very specific, not reacting with other types of nut.

Conclusions

The last pair of primers, however, are advantageous because of the presence of only one amplicon in PCR, although the fact that they also give positive signals for cashew and Brazil nut render them less useful beyond the analysis of mortadella. Common ingredients used in the formulation of mortadella did not interfere with pistachio identification, as evidenced by the negative tests conducted both on mortadella samples without pistachio and on the potential ingredients analysed individually. Since no difference was observed in the distribution of pistachio traces during cooking in the bulk of the mortadella, the sample can be taken from any part of the meat. A further use for the analytical method described could be to check on the cleanliness of mortadella production lines after the processing of the product with pistachio and before passing to a subsequent processing without pistachio. Samples taken at the critical points with swabs ensured that no traces of the preceding mixtures remain.

This method can be applied to various food products containing pistacchio (e.g confectionary) or, albeit only in the case of the PSTC 5-6 primers, it could be directed towards the detection of anacardiaceae in general. For the scope of this work, however, the primers pair PSTC 1-2 is suggested to unambiguously identify pistachio in mortadella.

F

  TCTTTGTGCTCCCCGTATTC-3') (GenBank accession number Y07600.2). The last was constructed on rbcL gene of ribulose 1,5-bisphosphate carboxylase/oxygenase PSTC 5 -GACAACTGTGTGGACCGATG-3') and PSTC6(5'-CAGGGCTTTGAACCCAAATA-3') (GenBank accession number AJ235786.1).The Master mix consisted of reaction Buffer 1X (Sigma), MgCl 2 1.4 mM, dNTPs (Eppendorf) 0.2 mM, Primer Forward and Reverse (Invitrogen) 0.25 µM (for PSTC 1-2 and PSTC 3-4) or 0.5 µM (for PSTC 5-6), Taq polymerase (Sigma) 1U and 200 ng of DNA in a final volume of 50 µL.

(

  composition not declared by producer) used in the production of mortadella. Only the pistachio samples gave a positive signal. No difference was observed for samples taken from the differing position of the product (from the middle or the ends).The PSTC 1-2 pair yielded three amplicons, one of which was not justified by the PCR product sequence, whereas the other bands correspond to the predicted amplicons (280 bp and 391 bp), as reported in figure2A. The pair of primers 3-4 provided a 111 bp long amplicon, although sometimes in the presence of high quantities of target DNA it also yielded two extra amplicons (222 bp, 333 bp). In the tests, we utilised all three pairs of primers, although the PCR carried out with PSTC 5-6 (figure2B) resulted as being more suitable on account of the univocal nature of the signal (193 bp), even though in some cases the primers PSTC 1-2 demonstrated greater sensitivity.

  Specificity tests were carried out, as well as on three previously prepared commercial flavours, with some natural ingredients normally utilised in mortadella flavourings. The results are shown in table 1. None of them yielded a positive signal. The same tableshows

Figure 1 .Figure 2A .Figure 2B .

 12A2B Figure 1. PCR carried out with three pairs of primers. a) -d): PSTC 1-2; f) -i): PSTC 3-4; k) -n): PSTC 5-6. b), g), n) are the pistachio samples; a), f), k) are commercial flavour A; c), h), l) are commercial flavour B; d), i), m) are commercial flavour C; e) and j) are marker (50 bp ladder). 96x54mm (200 x 200 DPI)

Table 1 .

 1 Commercial and natural flavours, and nuts tested by PCR PSTC 1-2 , 3-4, 5-6 to test interference with pistachio response.

	Goetz, D.W., Whisman, B. A. and Goetz, A. D. (2005) Cross-reactivity among edible nuts:
	double immunodiffusion, crossed immunoelectrophoresis, and human specific IgE
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	to pistachio nuts. Allergy Proc. 13: 255-258
	o r Kiening, M., Niessner, R., Drs, E., Baumgartner, S., Krska, R., Bremer, M., Tomkies, V.,
	Reece, P., Danks, C., Immer, U., and Weller, M.G. (2005), Sandwich Immunoassays for P the Determination of Peanut and Hazelnut Traces in Foods, Journal of Agricultural and e Food Chemistry, 53: 3321-3327
	e r Poms R.E., Klein C.L., Anklam E., (2004), Methods for allergen analysis in food-A review,
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	e Roux KH., Teuber S.S., Sathe S.K., (2002) Tree nut allergens, Int. Arch. Allergy Immunol. v e i 131:234-244
	Rozen, S and Skaletsky, H.J., (2000) Primer3 on the WWW for general users and for w biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and
	Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386 O l n Source code available at http://fokker.wi.mit.edu/primer3/.
	Savolainen,V., Chase,M.W., Hoot,S.B., Morton,C.M., Soltis,D.E.,Bayer,C., Fay,M.F., de y Bruijn,A.Y., Sullivan,S. and Qiu,Y.L., (2000) Syst. Biol. 49: 306-362
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	for the degree of Master of Science, Dep. Biological Sci. Florida State University

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


Maria-Jose Jose Lopez-Espinosa

Alicia Granada

Patricia Araque

Jose Manuel Molina-Molina

Maria-Carmen Carmen Puertollano

Ana Maria Rivas

Mariana Fernandez

Isabel Cerrillo

Maria Fatima Olea-Serrano

Concepcion Lopez

Jose-Manuel Molina- Molina

Mariana Fernández

María-Fatima Olea-Serrano

Concepción López

Nicolas Olea
email: nolea@ugr.es

Estrogenicity of paper and cardboard used as food containers

Keywords: Bioassay, Chromatography Additives/Contaminants: Bisphenol A, Phthalates Paper and cardboard, phthalates, bisphenol-A, estrogenicity

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Food and drink consumed outside the home represents a large and important part of the Western diet (World Cancer Research Fund and American Institute for Cancer Research, 1997). Take-away food is defined as food packaged immediately prior to sale and consumed shortly thereafter. Boxes, wrappings, cups, and trays used for take-away food are made of paper, cardboard, plastic (polystyrene), and aluminum, among other materials. The paper and cardboard can be new or recycled, grease-proof, printed, varnished, or glued. All of these materials are of particular interest because, although the contact period is usually short, the vast majority of these foods are often served hot and many are oily, favoring the migration of any contaminants in the packaging materials to the food.

Endocrine disrupting activity has been described for some food chemical contaminants such as bisphenol A and phthalates [START_REF] Aurela | Phthalates in paper and board packaging and their migration into Tenax and sugar[END_REF]. The presence of these hormonally active chemical residues in foods has been associated with food packaging materials and food processing [START_REF] Soto | The E-Screen assay as a tool to identify estrogens: An update on estrogenic environmental pollutants[END_REF][START_REF] Castle | Migration studies from paper and board food packaging materials. Part 1. Compositional analysis[END_REF][START_REF] Song | Analytical procedure for quantifying five compounds suspected as possible contaminants in recycled paper/paper board for food packaging[END_REF][START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF][START_REF] Summerfield | Investigation of migration from paper and board into food-development of methods for rapid testing[END_REF]Triantafyllou et al., 2002). Low levels of these chemicals have been found in baby food [START_REF] Petersen | Plasticizers in total diet samples, baby food and infant formulae[END_REF], baby bottles [START_REF] Brede | Increased migration levels of bisphenol A from polycarbonate baby bottles after dishwashing, boiling and brushing[END_REF], and recycled paper (Triantafyllou et al., 2002).

Nevertheless, little attention has been paid to this contamination on the assumption that it carries negligible risk. More recently, there has been a demand for a more rigorous risk assessment, adopting the precautionary principle as a guide for preventive action [START_REF] Kriebel | The precautionary principle in environmental science[END_REF]. In this regard, Vinggaard et al. [START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF] demonstrated the presence of multiple residues in recycled kitchen rolls, which showed a marked estrogenic response in an in vitro yeast estrogen screen. The possibility of similar compounds migrating from paper products used for food packaging has increased concerns about the health risks that may be involved.

Phthalates used in the manufacture, stabilization, modification, and performance of plastic polymers have been found in food [START_REF] Petersen | Plasticizers in total diet samples, baby food and infant formulae[END_REF]Saito et al., 2002). Food may be contaminated by ortho-phthalate esters such us dibuthyl (DBP), butylbenzyl phthalate (BBP), and di-2-ethylhexyl phthalate (DEHP), which are used in polyvinylchloride as plasticizers and in adhesives [START_REF] Song | Analytical procedure for quantifying five compounds suspected as possible contaminants in recycled paper/paper board for food packaging[END_REF], printing inks [START_REF] Castle | Migration of plasticizers from printing inks into foods[END_REF][START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF][START_REF] Summerfield | Investigation of migration from paper and board into food-development of methods for rapid testing[END_REF], and colored laminated films [START_REF] Mcneal | Determination of suspected endocrine disruptors in foods and food packaging[END_REF]. Phthalates have also been detected in virgin and recycled paper used for food packaging [START_REF] Castle | Migration studies from paper and board food packaging materials. Part 1. Compositional analysis[END_REF][START_REF] Sipilainen-Malm | Purity of recycled fibre-based materials[END_REF][START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF][START_REF] Summerfield | Investigation of migration from paper and board into food-development of methods for rapid testing[END_REF], most frequently DBP and DEHP, whereas BBP was not found in paper and cardboard extracts [START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF]. The presence of phthalates in some food has also been associated with the use of PVC tubes in the production of baby food [START_REF] Tsumura | Eleven phthalate esters and di(2-ethylhexyl) adipate in one-week duplicate diet samples obtained from hospitals and their estimated daily intake[END_REF], with cap-sealing resins in bottled foods [START_REF] Hirayama | Analysis of plasticizers in cap-sealing resins for bottled foods[END_REF], and with gloves worn by food handlers [START_REF] Tsumura | Eleven phthalate esters and di(2-ethylhexyl) adipate in one-week duplicate diet samples obtained from hospitals and their estimated daily intake[END_REF]. Finally, baby pacifiers [START_REF] Sugita | Estimation of daily oral exposure to phthalates derived from soft polyvinyl chloride baby toys[END_REF] can also be a source of inadvertent exposure.

Bisphenol-A (BPA), a starting material for the synthesis of polycarbonate and epoxy resins, was detected in autoclavable flasks [START_REF] Krishnan | Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving[END_REF], baby bottles [START_REF] Brede | Increased migration levels of bisphenol A from polycarbonate baby bottles after dishwashing, boiling and brushing[END_REF], reusable water carboys [START_REF] Mcneal | Determination of suspected endocrine disruptors in foods and food packaging[END_REF], and coffee and black tea cans [START_REF] Kang | Bisphenol A migration from cans containing coffee and caffeine[END_REF], among other food materials and containers. The leaching of BPA was related to the temperature, heating time, and type of food contained [START_REF] Kang | Factors influencing the migration of bisphenol A from cans[END_REF]. The estrogenicity of food preserved in cans was related to BPA and oligomers leaching from the inner surface of lacquer-coated cans [START_REF] Brotons | Xenoestrogens released from lacquer coating in food cans[END_REF].

The estrogenicity of kitchen roll extracts was attributed to BPA among other compounds [START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF].

Hence, bisphenols and phthalates may migrate to food packaged in paper products that contain these contaminants [START_REF] Sipilainen-Malm | Purity of recycled fibre-based materials[END_REF][START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF][START_REF] Summerfield | Investigation of migration from paper and board into food-development of methods for rapid testing[END_REF]. Whereas recycled paper used for kitchen rolls contained BPA, attributed to the office waste used, virgin paper did not appear to be an important source of bisphenol-A leaching [START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF]. In contrast, phthalates were found in both virgin and recycled paper, related to offset printing inks, adhesives, and lacquers [START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF].

The E-Screen assay, developed to measure estrogenic activity using cell proliferation as the end point (Soto et el., 1992;[START_REF] Aurela | Phthalates in paper and board packaging and their migration into Tenax and sugar[END_REF], is a common bioassay to identify estrogenic chemicals among environmental pollutants and is among the most sensitive and reproducible assays for estrogenic activity [START_REF] Villalobos | The E-Screen assay: Comparison among different MCF7 cell stocks[END_REF][START_REF] Andersen | Comparison of short-term estrogenicity tests for identification of hormone-disrupting chemicals[END_REF][START_REF] Fang | Quantitative comparisons of in vitro assays for estrogenic activities[END_REF]. The present study aimed to investigate the estrogenicity of paper and cardboard used in take-away food packaging materials by testing their estrogenicity in the E-Screen bioassay. In addition, chemical analyses of the extracts were performed to identify some of the chemicals responsible for observed hormonal effects.

Material and Methods

F

Samples

Forty containers made of paper and cardboard were obtained from take-away food retail establishments in four European countries (Belgium, Italy, Portugal, and Spain). The containers were supplied by CONSEUR: VU-testACHATS-Belgium, DECO-Prostete-Portugal, ALTROconsumo-Italia, and OCU-Compramaestra-Spain. Eighteen containers were pizza boxes and cardboard triangles for individual pizza rations, sixteen were chipped-potato and chicken-finger containers, four were paper bags for sandwiches and fritters, one was for strawberries, and one for floury paste. Thirty-two of the containers were made of cardboard and eight of paper. The containers were wrapped by collecting agents after removal of the food and shipped to our Laboratory of Medical Investigation (Spain) for analysis. Only the side of the container in contact with the food was processed for analysis.

Chemicals and reagents

Chemicals used as standards for the analysis were bisphenol A (BPA), CAS nº: 3253-39-2 (Sigma-Aldrich Quimica S.A.); dibuthylphthalate (DBP), CAS nº: 84-74-2; and diethylhexylphthalate (DEHP), CAS nº: 117-81-7 (Dr Ehrenstorfer Lab., Ausburg, Germany). All solvents used were of high purity grade: acetonitrile, doubly distilled water, chloroform, ethanol, and hexane.

Apparatus

Sample processing

In order to exclude the influence of the inks used on printed surfaces, chemical residues were extracted from the side of the food container in contact with food. Internal parts that were in contact with food were dissected using a scalpel and separated from the internal structure of cardboards. A section weighing between 5 and 10 g was processed.

In brief, the sample was placed in a glass beaker, and 150 ml of distilled water were added and heated for 2 h at 100ºC, following one of the protocols recently proposed for testing migration using food simulates, including water [START_REF] Summerfield | Investigation of migration from paper and board into food-development of methods for rapid testing[END_REF]Triantafyllou et al., 2002). Next, the paper mass was mechanically pressed to remove the absorbed water, which was then filtered through 110 nm Whatman ® filter papers. The extract obtained was concentrated at a reduced pressure to a volume of 2 ml. Then, 10 ml of water and 15 ml of chloroform were added through a decanting funnel. The chloroform phase was filtered through a Na 2 SO 4 anhydro-treated 110 nm Whatman ® filter. The eluted obtained was concentrated at reduced pressure to a volume of 1 ml and dried under a stream of nitrogen. Two ml of ethanol were added to the dry extract and the suspension was divided between two aliquots, 1ml for use in the E-Screen bioassay and 1ml for chemical analysis. The aliquot for chemical analysis was Bioassay for Measuring Estrogenicity MCF-7 cell line: Cloned MCF-7 cancer cells were grown for routine maintenance in Dulbecco's modification of Eagle's medium (DME) supplemented with 10% fetal bovine serum (FBS) (BioWittaker, Walkersville, MD) in an atmosphere of 5% C0 2 /95% air under saturating humidity at 37ºC. The cells were subcultured at weekly intervals using a mixture of 0.05% trypsin and 0.01% EDTA.

Charcoal-dextran treatment of serum. Sex steroids were removed from serum by charcoal-dextran stripping. Briefly, a suspension of 5% charcoal (Norit A, Sigma Chemical Co, St. Louis, MO) with 0.5% dextran T-70 (Pharmacia-LKB, Uppsala, Sweden) was prepared. Aliquots of the charcoal-dextran suspension of similar volume to the serum aliquot to be processed were centrifuged at 1000 g for 10 min.

Supernatants were aspirated, and serum aliquots were mixed with the charcoal pellets. This charcoal-serum mixture was maintained in suspension by rolling at 6 cycles/min at 37º C for 1 h. The suspension was centrifuged at 1000 g for 20 min, and the supernatant was then filtered through a 0.22 µm filter (Millipore). Charcoal dextran-treated bovine and human serum (CDFBS and CDHuS, respectively) were stored at -20º C until needed.

Cell proliferation experiments. MCF7 cells were used in the estrogenicity test following a slight modification [START_REF] Villalobos | The E-Screen assay: Comparison among different MCF7 cell stocks[END_REF] of the original technique [START_REF] Soto | An "in culture" bioassay to assess the estrogenicity of xenobiotics[END_REF]. Briefly, cells were trypsinized and plated in 24-well plates (Limbro, McLean, VA) at initial concentrations of 20 000 cells per well in 5% FBS in DME. Cells were allowed to attach for 24 h and the seeding medium was then replaced with 10% CDFBS or CDHuS-supplemented phenol red-free DME. Different concentrations of extracts were added, and the assay was stopped after 144 h by removing medium from wells, fixing the cells, and staining them with sulforhodamine-B (SRB) as described elsewhere [START_REF] Olea | Estrogenicity of resin-based composites and sealants used in dentistry[END_REF][START_REF] Perez | The estrogenicity of bisphenol A-related diphenylalkanes with various substituents at the central carbon and the hydroxy groups[END_REF]. The linearity of the SRB assay with cell number was verified prior to the cell growth experiments. The 100% proliferative effect (PE) was calculated as the ratio between the highest cell yield obtained with 50 pM of estradiol and the proliferation of hormone-free control cells.

Quantitative evaluation of estrogenicity of paper extracts. One mL of ethanol suspension of paper and cardboard extracts was dried, resuspended in 100 uL of ethanol and then in supplemented phenol red-free medium, vigorously shaken, and left at rest for 30 min; finally, it was passed through a 0.22 µm filter and tested in the estrogenicity assay at dilutions ranging from 1:1 to 1:10.000. Each sample was run in triplicate with a negative (vehicle) and positive (estradiol 50 pM) control in each plate. The proliferative effect (PE) of the extracts was referred to the maximal proliferative effect obtained with estradiol, transformed into estradiol equivalent units (Eeq), and expressed as pM concentration by reading from a dose-response curve prepared using estradiol (concentration range 0.1 pM to 10 nM). Mean cell numbers did not significantly differ from those in steroid-free controls at concentrations below 1 pM estradiol, equivalent to 1 fmol in 1 ml of culture medium. Therefore, 1 fmol estradiol/well was considered the lowest detectable amount of estrogen in this assay. Recovery rates were determined before investigating the BPA, DEHP and DBP content of extracts. Liquid from the paper mass was spiked with 10 -70 µg/mL of BPA, DEHP or DBP and then extracted according to the protocol described above under Sample processing. Recovery rates ranged from 82.4 ± 6.1% to 86.2 ± 4.3%. Quantification was performed by HPLC after determination of the limits of detection (LOD) for each chemical (BPA=22.8 ng/mL; DBP=50µg/mL; DEHP=70µg/mL) and standard curves.

Chemical analysis

Finally, blank tests were performed for each chemical and no detectable amounts were found.

Statistical analysis

A univariate analysis was performed to explore the distribution of the outcome variables, and the potential correlation among the three residues was analyzed. In order to reduce the skewness and improve the adjustment in the linear regression between the estrogenicity and BPA, DBP, and DEHP levels in the extracts, all variables were transformed into their natural logarithms. Central tendency measurements (geometric means, median, and standard deviation) were calculated for the proliferation test and chemical analysis results, and the arithmetic mean was also reported for the purposes of comparison with previous articles. A bivariate analysis was performed to estimate the association between estrogenicity and BPA, DBP, and DEHP levels, and multivariate models were developed to describe associations between each chemical and estrogenicity in the extracts. Values below the LOD were replaced by the corresponding random numbers from a table of randomly assorted digits [START_REF] Snedecor | Statistical methods[END_REF]. All statistical analyses were performed using SPSS-11 (2006) and the STATA Version 9 (2005) statistical software.

Results

Figure 1 shows the proliferative effect of estradiol, BPA, DBP and DEHP on MCF-7 breast cancer cells. The estrogenicity of these chemicals was assessed by determining their relative proliferative potency (RPP) and their relative proliferative effect (RPE) [START_REF] Soto | The E-Screen assay as a tool to identify estrogens: An update on estrogenic environmental pollutants[END_REF]. RPP was 0.03% for BPA and <0.0001% for both phthalates; RPE was 100% for BPA and 30% for both phthalates.

[Insert Figure 1 about here] Table I shows the arithmetic and geometric means, median, standard deviation, range, and frequency of the estrogenicity values of all samples (total) and of cardboard and paper samples separately. Values are expressed in pM estradiol equivalent (Eeq) per weight (g) of material. The frequency of estrogenic response was similar in paper and cardboard samples (87.50% and 90.00%, respectively). The geometric mean estrogenicity was higher in cardboard (13.33 pM estradiol equivalents/g of cardboard) than in paper samples (7.78 pM estradiol equivalents/g of paper). Absence of toxicity in the extracts was demonstrated when any partial agonist effect was shown in the presence of 100 pM of estradiol at maximal dilution of the extract (data not shown).

[Insert Table I about here] Table II shows the arithmetic and geometric means, median, standard deviation, range, and frequency of the content of BPA, DBP, and DEHP in total, cardboard, and paper samples. Values are expressed as ng of residue per g of material; 37.50-100% of analyzed samples showed detectable levels of the studied chemicals. The most frequently detected chemical was DBP, present in 100% of paper samples, followed by DEHP, present in 75.00% and 78.13% of paper and cardboard samples, respectively. BPA was present in half of cardboard samples but was less frequent and less abundant in paper extracts. DBP values were correlated with DEHP levels in the samples (p=0.040; ρ=0.326). The presence of BPA did not appear to be related to any of the phthalates studied.

[Insert Table II about here] Table III shows the bivariate analysis between BPA, DBP, DEHP levels (ng/g of cardboard or paper) and estrogenicity values (pM estradiol equivalents/g cardboard or paper) for total, cardboard, and paper samples separately. No statistically significant association was found between the levels of each chemical and the estrogenicity of the samples for the grouped data. When data for paper and cardboard were considered separately, the relationship of estrogenicity with DEHP concentrations in cardboard and DBP in paper was close to statistical significance (β=0.21; p=0.17 and β=1.25; p=0.06, respectively. III about here] Table IV shows the multivariate analysis for the association between BPA, DBP and DEHP levels (ng/g of cardboard or paper) and estrogenicity values (pM estradiol equivalents/g cardboard or paper) for total, cardboard, and paper samples. In the paper samples (n=8), the multivariate model showed a statistically significant association with DBP (β=1.61; p=0.03) and a close to significant association with DEHP (β=-0.43; p=0.07). When a multivariate model with the two phtalates was constructed, both showed a statistically significant association with estrogenicity (DBP: β=1.68, p=0.01; DEHP: β=0.44, p=0.04) (Data not shown).

[Insert Table IV about here]

Discussion

Nine out ten aqueous extracts of paper and cardboard containers used in take-away food showed a statistically significant proliferative effect on MCF7 human breast cancer cells. The geometric mean estrogenicity of one gram of these materials was similar to that of 11.97 pM of estradiol on MCF7 cells. In terms of surface area, extracts of each square cm resulted in an estrogenicity of 0.60 pM of estradiol.

In an attempt to identify the chemicals responsible for this hormonal effect, the presence of bisphenol A and of phthalates DBP and DEHP was investigated. DBP (geometric mean of 37.39 ng/g material) and DEHP (geometric mean of 341.74 ng/g material) was detected in 67% and 77.5% of total samples, respectively. Several phthalates were previously identified as components of virgin and recycled fibers [START_REF] Sipilainen-Malm | Purity of recycled fibre-based materials[END_REF][START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF]. [START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF] reported DBP in all extracts of kitchen rolls made from virgin (range, 0.1-28 µg/g) or recycled (range 2-22 µg/g) paper at similar levels to those found in the present study (0.0001-10.77 µg/g). It has been suggested that phthalates can derive from printing inks [START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF][START_REF] Summerfield | Investigation of migration from paper and board into food-development of methods for rapid testing[END_REF] or adhesives [START_REF] Song | Analytical procedure for quantifying five compounds suspected as possible contaminants in recycled paper/paper board for food packaging[END_REF] used in food containers, among other sources. In the present study, when walls of food containers could be mechanically separated into layers, our protocol for sample processing only used the internal part in direct contact with food, therefore the sample was not contaminated by inks on printed surfaces. The levels of the two phthalates were correlated (p=0.040; ρ=0.326), suggesting that they might have derived from the same source. Both phthalates would contribute to the estrogenicity of the samples, as their estrogenicity in the E-Screen assay is known [START_REF] Aurela | Phthalates in paper and board packaging and their migration into Tenax and sugar[END_REF]Okubo et al., 2003).

Although the small sample size prevented statistically significant associations from being reached between the estrogenicity and each chemical in the bivariate analysis, when the total sample was divided between cardboard and paper, a clear tendency to significance was found for these associations (See Table III). Given the small sample of paper extracts (n=8), it is remarkable that this association was close to statistical significance (β=1.25; 95% IC from -0.07 to 2.56), with the finding in the multivariate analysis of a positive statistically significant association between estrogenicity and DBP (β=1.61; p=0.03) and a negative association, close to significance, between estrogenicity and DEHP (β=-0.43; p=0.07). Finally, when a multivariate model was constructed with only the two phtalates, a statistical significant association was found between chemical content and E-Screen estrogenicity in both cases (DBP: β=1.68, p=0.01; DEHP: β=0.44, p=0.04). Chemical residues in paper and cardboard vary as a function of the primary processing material and the use of recycled material. Thus, it was proposed that BPA was useful to distinguish between virgin and recycled fibers [START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF]. The BPA levels found in these take-away food packaging materials (0.00005-1.81 µg/g material) were lower than those reported by Vinggaard and coworkers (2000) in recycled kitchen rolls (0.55-24.1 µg/g) but higher than those found in virgin rolls (0.03-0.1 µg/g). BPA levels detected by [START_REF] Ozaki | Chemical analysis and genotoxical safety assesment of paper and paperboard used for food packaging[END_REF] in recycled materials (0.19-26 µg/g) were ten-foldhigher than those found in virgin products [START_REF] Ozaki | Chemical analysis and genotoxical safety assesment of paper and paperboard used for food packaging[END_REF]. Disparities in levels and frequency of BPA residues might be due to variability in the percentage of recycled material and in the composition of the paper. We found the highest BPA concentration in a pizza box made of recycled material. However, no association was found between BPA levels and virgin or recycled types of paper or cardboard. It is worth noting that only 6 out of 40 samples were described as containing recycled fiber according to information printed on the containers. However, investigation by the container suppliers revealed the presence of recycled fibers in all but one sample and a proportion of virgin wood pulp that ranged from 36-98%. Interestingly, the sample with no recycled fibers was negative for the presence of BPA. However, neither the classification of container composition based on manufacturer's information nor our re-classification based on the percentage of virgin fiber served to establish an association between recycled composition and BPA levels. BPA is used in the production of epoxy resins and polycarbonate, and epoxy resins are used in the formulation of printer inks (Ullmans Encyklopadie of Technical Chemistry, 1984). Therefore, printed paper in waste from offices is a possible source of BPA in recycled paper. The presence of BPA in half of the studied samples suggests that this compound is a common contaminant of paper, as reported by other authors [START_REF] Vinggaard | Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[END_REF]. Moreover, the estrogenic activity of [START_REF] Brotons | Xenoestrogens released from lacquer coating in food cans[END_REF][START_REF] Soto | The E-Screen assay as a tool to identify estrogens: An update on estrogenic environmental pollutants[END_REF][START_REF] Olea | Estrogenicity of resin-based composites and sealants used in dentistry[END_REF][START_REF] Perez | The estrogenicity of bisphenol A-related diphenylalkanes with various substituents at the central carbon and the hydroxy groups[END_REF][START_REF] Pulgar | Determination of bisphenol A and related aromatic compounds released from bis-GMA-based composites and sealants by high performance liquid chromatography[END_REF][START_REF] Rivas | Human exposure to endocrine disrupters: standardisation of a marker of estrogenic exposure in adipose tissue. APMIS 109: 185-197. foods[END_REF] might suggest that this compound is a main cause of the activity observed in the E-Screen assay. However, we found no significant correlation between the presence or concentration of BPA in the samples and their estrogenicity. We cannot rule out the presence of other chemicals that may contribute to the estrogenicity of the extract. In addition, the estrogenicity of the extracts may be increased by additive or synergistic effects of the chemicals detected (BPA, DBP, DEHP) or by their interaction with any other hormonally active chemical that might be present in the extracts [START_REF] Rajapakse | Combining xenoestrogens at levels below individual no-observed-effect concentration dramatically enhances steroid hormone action[END_REF].

In Europe, the safety and quality of paper and cardboard used for food packaging are regulated by the general directives for articles intended to come in contact with foodstuffs (Directive 89/109/EEC), but there is no specific EC Directive for paper and cardboard. To our best knowledge, this is the first report of the estrogenicity of paper and cardboard that is in direct contact with take-away food. In-depth investigation is needed into the presence of hormonally active chemicals in these materials alongside a risk assessment evaluation. Our data highlight the usefulness of the E-Screen to estimate the total estrogen hormonal toxicity of paper/cardboard in direct contact with food and to perform an initial screening of suspected samples. In conclusion, the use of food containers made of paper or cardboard warrants closer scrutiny to determine whether chemicals that migrate to food from this type of packaging contribute to the inadvertent exposure of consumers to endocrine-disrupting chemicals.

  HPLC was performed with a Waters Model 501 Millipore apparatus (Marlborough, MA) equipped with two pumps and U6K injector of 500 µl load capacity. The ultraviolet/visible detector was a Waters Model 490 Millipore device with Millennium Chromatography Manager software. A Lichrocart Merck S5 ODS Column (20x 0.4 cm; Merck, Darmstadt, Germany) was used. Gas chromatography-Mass spectrometry was done using a Saturn 2000 ion-trap mass spectrometer from Varian Instrument (Walnut Creek, CA) with automatic injector 8200, SPI/1078; a DB5-MS capillary column (30 m x 0.25 mm) was used. The ion-trap mass spectrometer was operated in SIS impact mode.

  and confirmed by GC/MS after dilution in ethanol and hexane, respectively.

  High performance liquid chromatography. The gradient was: at time= 0 min, 100% phase A (acetonitrile-bidistilled water, 1:1) and 0% phase B (acetonitrile): at time = 15 min, 100% phase B and 0% phase A; and at time = 17 min, 100% phase A and 0% phase B. The elution profile was monitored at 280 nm and 254 nm.Gas chromatography and mass spectrometry. The following working conditions were used: initial column temperature, 50ºC (3min), increased at 30ºC/min to 200ºC (5min), increased at 30ºC/min to 270ºC (8min), and finally increased at 30ºC/min to 310ºC (2min); injector temperature: 250ºC split of 0.01; carrier gas, He (99.999%); injector flow: 1ml/min; and injection volume 2 µl. BPA retention time: 8.16 min; m/z: 213, 228. DBP retention time: 7.10 min; m/z: 149, 223. DHEP retention time: 10.12; m/z: 149, 279.

  BPA in MCF-7 cells

Figure 1 .

 1 Figure 1. Cell proliferation in MCF7 cells. Cells growing in 10% charcoal dextrantreated human serum-supplemented medium were exposed for 144 h to different amounts of estradiol-17ß, BPA, DBP and DEHP. Points represent quadruplicate cultures; bars indicate standard deviations.

  Estimated values of estrogenicity (pM estradiol equivalents Eeq/g of cardboard or paper) and frequency of positive samples in the E-Screen assay of paper and cardboard extracts.

Table II .

 II Levels and frequency of BPA, DBP and DEHP (ng/g of cardboard or paper) in samples.

	Residues		Total samples	Cardboard	Paper
				n=40	n=32	n= 8
	BPA
	Arithmetic mean		97.34	115.32	25.43
	Geometric mean		2.38	2.74	1.35
	Median SD	F o r	60.52 11.97 0.52 16.33	64.24 13.33 0.52 17.68	45.61 7.78 0.49 12.72
	Range Frequency (%)	18.69 12.55 0.05-1817.00 P e 45.00	21.51 13.02 0.05-1817.00 46.88	16.80 12.09 0.08-188.00 37.50
	DBP Arithmetic mean		0.01-355.50 90.00 e r 713.17	0.01-355.50 90.63 706.32	0.08-280.90 87.50 740.49
	Geometric mean		R 37.39	20.97	377.63
	Median SD Range Frequency (%)	e v i 75.62 41.65 e 0.10-10774.00 121.84 35.61 0.10-10774.00 w 67.50 59.38	548.55 3.95 29.10-3049.00 100
	DEHP Arithmetic mean Geometric mean		3901.56 341.74	O 706.32 n l 740.49 y 302.11 559.54
	Median			893.48	814.44	2751.84
	SD			23.27	22.53	31.36
	Range		0.52-61013.00	0.52-61013.00	1.53-10198.00
	Frequency (%)		77.50	78.13	75.00
				1

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

Acknowledgements

We thank Richard Davies for editorial assistance. This work was supported by grants from the Spanish Ministry of Health (FIS 02/1314, 02/309, C03/081 and G03/176), European Union Commission (QLK4- CT-2002-00603 and CASCADE FOOD-CT-2003-506319) and Organización de Consumidores y Usuarios (OCU) de Compramaestra, Spain.

Appendices

Abbreviations: BPA, bisphenol A, CAS nº: 3253-39-2;