Food Control 30 (2013) 411e417

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Food Control
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Aatoxin M1 in marketed milk in Portugal: Assessment of human and animal exposure

S.C. Duarte a, b, *, A.M. Almeida a, c, A.S. Teixeira a, A.L. Pereira a, A.C. Falco c, A. Pena b, C.M. Lino b
a

Department of Veterinary Medicine, Escola Universitria Vasco da Gama, 3040-714 Coimbra, Portugal Group of Health Surveillance, Center of Pharmaceutical Studies, University of Coimbra e Polo III, 3000-548 Coimbra, Portugal c Center for Neuroscience and Cell Biology, University of Coimbra, Largo Marqus de Pombal, 3004-517 Coimbra, Portugal
b

a r t i c l e i n f o
Article history: Received 9 March 2012 Received in revised form 26 July 2012 Accepted 21 August 2012 Keywords: Mycotoxin Aatoxin Aatoxin M1 Aatoxin B1 Milk ELISA

a b s t r a c t
From all the currently known mycotoxins, aatoxin B1 (AFB1) presents the greatest public health and agro-economic signicance. Its metabolite, aatoxin M1 (AFM1) has toxicological properties comparable to those of AFB1, albeit a lower carcinogenic potency. The occurrence of AFM1 in milk marketed in Portugal and the evaluation of the exposure degree to the toxin through its consumption in an average citizen were studied. Estimation of the corresponding concentration of AFB1 in feedstuffs was also aimed. Forty samples representing the totality of the pasteurized and UHT half-skimmed milk brands marketed in the country were surveyed. Determination of AFM1 was carried out by means of a commercial competitive ELISA. Eleven samples (27.5%) featured a contamination above the detection limit (mean 23.4 24.0 ng/L). Two milk samples (5%) both produced in Azores presented AFM1 values that surpassed the legal maximum limit (50 ng/L). A third sample, also from the Azores Islands, presented a very high value of contamination. These results are interesting given that dairy production in Azores is traditionally pasture-based which is considered as low risk system regarding AFs contamination. Adult average dietary exposure to the fungal toxin through milk consumption was estimated at 0.08 ng/kg bw/day, which is inevitably higher for infants, considered the main risk group. The concentration of AFB1 in the feeds consumed by the producing cows was estimated as 1.46 mg/kg. The results call for further studies in an attempt to identify and thus control potential inuencing factors in the only region where milk samples contaminated above the legal limit were produced. 2012 Elsevier Ltd. All rights reserved.

1. Introduction
Abbreviations: AFB1, Aatoxin B1; AFM1, Aatoxin M1; AFs, Aatoxins; ALARA, As Low As Reasonably Achievable; CAC, Codex Alimentarius Commission; EC, European Commission; EDI, Estimated daily intake; EFSA, European Food Safety Agency; EFTA, European Free Trade Association; ELISA, Enzyme Linked ImmunoSorbent Assay; EU, European Union; FAO, Food and Agriculture Organization of the United Nations; FDA, Food and Drug Administration; GEMS/Food, Global Environment Monitoring System/Food Contamination Monitoring and Assessment Programme; HPLC-FD, High Performance Liquid Chromatography coupled to Fluorescence Detection; IARC, International Agency for Research on Cancer; IM, Instituto de Meteorologia; JECFA, Joint FAO/WHO Expert Committee on Food Additives; LC-MS/MS, Liquid Chromatography Tandem Mass Spectrometry; LOD, Limit of detection; ML, Maximum level; N.a., non-applicable; N.g., not given; TDI, Tolerable Daily Intake; TLC, Thin Layer Chromatography; UHT, Ultra-High Temperature; WHO, World Health Organization. * Corresponding author. Department of Veterinary Medicine, Escola Universitria Vasco da Gama, 3040-714 Coimbra, Portugal. Tel.: 351 239 444 444; fax: 351 239 437 627. E-mail address: soacanceladuarte@gmail.com (S.C. Duarte). 0956-7135/$ e see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2012.08.002

Aatoxin B1 (AFB1; Fig. 1) is the most common AF in food and amongst the most potent genotoxic and carcinogenic mycotoxins, classied as Group 1 by the International Agency for Research on Cancer (IARC, 1993). It is produced both by Aspergillus avus and Aspergillus parasiticus (JECFA, 2001). Aatoxin M1 (AFM1; Fig. 1) is the 4-hydroxy derivative of AFB1 in humans and animals, which may be present in milk from animals fed with AFB1 contaminated feed. The extent of transfer from feed to milk (carry-over) in dairy cows is inuenced by various nutritional and physiological factors, including feeding regimens, rate of ingestion, rate of digestion, health of the animal, hepatic biotransformation capacity, and actual milk production. This implies that the rate of absorption of AFs, and the excretion of AFM1 in milk, varies between individual animals, from day to day, and from one milking to the next. In high-yielding cows, the consumption of signicantly higher amounts of

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AFB1
H O H O OCH3 O

AFM1
OH O O O

2. Experimental 2.1. Sampling A total of 40 samples representing the half-skimmed milk brands commercialized in Portugal were surveyed. The milk packages were purchased in markets and supermarkets between March and April 2011 as available to the nal consumers. The samples included pasteurized milk (n 4), and ultra-high temperature (UHT) treated milk (n 36), ve of which organic (n 5). Regarding origin, most samples originated from Portugal (mainland n 24 and Azores islands n 3), with 32.5% of the milk being imported from abroad (Spain n 6; Germany n 4; France n 2; Belgium n 1). All the samples were analysed before their expiry date. 2.2. Materials and test procedure Determination of AFM1 was carried out by means of a commercial competitive ELISA (RIDASCREEN, R-Biopharm, Germany), according to the test kit instructions. Briey, 10 mL of each milk sample was centrifuged (Sigma 3K15 centrifuge, Reagente 5, Porto) for degreasing during 10 min at 3500 g and 10  C. The ensuing upper cream layer of the tube was completely removed, through aspiration with a Pasteur pipette. The remaining milk (defatted) was directly used in the test. All samples and standards were tested in separated duplicated wells. The volume of 100 mL of each standard and prepared sample was added to the corresponding wells. The plate was gently shacked manually before incubate during 30 min at room temperature in the dark. Afterwards, the liquid was poured out of the wells. To ensure complete removal of liquid from the wells the microwell holder was tapped upside down three times vigorously against absorbent paper. At the end, the wells were lled with 250 mL of washing buffer and then the liquid was poured out again. This washing step was repeated three times. One hundred microlitres of diluted enzyme conjugate was added to each well. The plate was gently shacked manually and incubated for 15 min at room temperature in the dark. After the incubation period, the liquid was poured out of the wells and the microwell holder was tapped upside down three vigorous times to ensure complete removal of liquid from the wells. All wells were then lled with 250 mL washing buffer and the liquid poured out again. The washing procedure was repeated two times. One hundred microlitres of substrate/chromogen was added to each well. The plate was shaken manually to mix gently all the reagents and then incubated for 15 min at room temperature in the dark. Stop solution (100 mL) was added to each well, and the plate was gently shaken. Within 15 min after addition of stop solution, the absorbance at 450 nm was read. 2.3. Quantication of AFM1 A standard curve was drawn, by means of six concentrations levels (0, 0.5, 10, 20, 40, and 80 ng/L) with two determinations each. Subsequently, AFM1 quantication in milk samples was determined through the formula: [absorbance standard (or sample)/ absorbance zero standard] 100 % absorbance. The absorptions were inversely proportional to the AFM1 concentration. The zero standard was thus made equal to 100% and the absorbance values are quoted in percentages. The values calculated for the standards are entered in a system of coordinates on semi-logarithmic graph against the AFM1 concentration (ng/L). According to the manufacturers description, the cut-off value was 5 ng/L.

OCH3

Fig. 1. Chemical structures of AFB1 and AFM1.

concentrated feeds might result in carry-over percentages as high as 6.2% (EFSA, 2004; Fink-Gremmels, 2008; Veldman, Meijst, Borggreve, & Heeres-van der Tol, 1992). If contaminated foodstuffs are used, AFM1 will appear in the milk 2e3 days following ingestion. Likewise two-three days are the time necessary to reduce to zero the AFM1 level in milk, when a diet without aatoxins is fed (Prandini et al., 2009). Although AFM1 is usually considered to be a detoxication byproduct of AFB1, it is still a question of public concern given the exposure route that consumption of milk can embody. Indeed, even small amounts of this metabolite in milk are of importance for consumers of large quantities of milk, like children, for whom proportionally, milk can represent an important portion of AFs intake (Gonzlez-Osnaya, Soriano, Molt, & Maes, 2008). Furthermore, the acute toxicity of AFM1 seems to be similar or slightly less than that of AFB1 although its carcinogenic potency is probably one or even two orders of magnitude lower than that of AFB1 (EFSA, 2004). AFM1 is classied as a possible human carcinogen (group 2B) (IARC, 1993). Milk has the greatest demonstrated potential for introducing AFM1 into the human diet (Roussi, Govaris, Varagouli, & Botsoglou, 2002). AFM1 is not destroyed by pasteurization of milk, and can thus be transferred into powdered milk, yoghurt and other milkbased products (JECFA, 2001). Jointly with the toxicological prole, these are the reasons for the stringent control of these mycotoxins in the food supply. Regarding liquid milk, maximum level range from 50 ng/L in the European Union (EU) (EC, 2006), in Codex Alimentarius Commission (CAC, 2001), and in Morocco (FAO, 2004) to 500 ng/L, as in Iran (Rahimi, Bonyadian, Rafei, & Kazemeini, 2010), Southern Common Market e Mercosul (MERCOSUL/GMC, 2002), and United States of America (FDA, 2005). In-between regulatory levels include the one from Syria, set at 200 ng/L (FAO, 2004). Thus, the maximum permitted level of AFM1 in milk in the EU is among the lowest in the world, and is based on the ALARA (As Low As Reasonably Achievable) principle (EFSA, 2004). Europe also takes the lead in feedstuffs supply by means of a strict control. A limit of 5 mg/kg feed for dairy cattle is currently applied in the EU countries and in the new member states as well as in EFTA countries (EC, 2002), but only in few countries outside Europe. This level is below the no-effect level in target animals (EFSA, 2004). There is a scarcity of studies regarding AFM1 occurrence in milk in Portugal. Only two were published (Martins, Guerra, & Bernardo, 2005; Martins & Martins, 2000) reporting AFM1 occurrence in raw and UHT milk, although neither one assessed human nor dairy cattle exposure. As a result the present manuscript describes the estimation of exposure of human milk consumers to AFM1 by means of a survey of nearly all the milk brands marketed in Portugal. In addition, the AFM1 milk content is used as a biomarker of exposure to AFB1 in the feeds of dairy cows. Thus ultimately, the purpose of this work goes beyond the scope of a Public Health perspective, as it draw attention to the exposure of dairy cows to this mycotoxin.

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2.4. Calculation of estimated daily intake Estimated daily intake (EDI; ng/kg body weight (bw)/day) was calculated through a deterministic method (IPCS, 2009, chap. 6) combining the sum of AFM1 concentration in the analysed samples (Sc), the mean annual intake estimated (C), the total number of analysed samples (N), the number of days in a year (D), and the mean body weight (K) as follows: EDI (Sc) (C N1 D1 K1). Only samples higher than the cut-off limit of the test (5 ng/L) where considered for calculating Sc and N. The latest assessment of the mean annual intake of milk, corresponding to the period between 2006 and 2008, is of 87 kg/year (FENALAC, 2011). Mean body weight for the adult Portuguese population was considered 69 kg, from data retrieved from Arezes, Barroso, Cordeiro, Costa, and Miguel (2006). 2.5. Calculation of extrapolated values of AFB1 concentration in cattle feeds The values of AFB1 in cattle feeds were extrapolated from back calculation of the values of AFM1 obtained from analysis of milk samples. The calculation was performed considering that 1.6% of ingested AFB1 is converted to AFM1 by lactating cattle (Price, Paulson, Lough, Ginng & Kurtz, 1985), as estimated by using the following formula: AFB1 (mg/kg) [AFM1 (ng/kg) 100]/1.6 1000 (Rastogi, Dwivedi, Khanna, & Das, 2004). 3. Results & discussion 3.1. Analytical performance of the method An ELISA technique was employed in this survey aiming for a reduction in assay time, a simplied sample extraction, specicity for the toxin in question, comparable low limits of detection, in addition to a high throughput (Velasco, Delso, & Escudero, 2003). The ensuing standard curve was obtained by two determinations of six concentrations levels, between 0 and 80 ng/L. An exponential equation (y 84.721e0.012x) was used to calculate the content of AFM1 in the analysed samples. The correlation coefcient (r2) was 0.9855. 3.2. Occurrence of AFM1 in milk samples Of the forty pasteurized and UHT milk samples analysed, only eleven samples (27.5%) surpassed the analytical cut-off limit of the test (5 ng/L). These results are shown in Fig. 2. This cut-off limit was

Fig. 2. Distribution of AFM1 concentration values of the analysed milk samples above than the cut-off value (5 ng/L). EU maximum level (50 ng/L) is represented with a horizontal line.

exactly 10 times lower than the EU maximum permitted level (50 ng/L) (EC, 2006). It is noteworthy that the three UHT milk samples that featured the highest levels of contamination corresponded precisely to the samples produced in the Azores islands. Furthermore, two of these values surpassed the EU maximum permitted level. Azorean milk currently represents 30% of the total national production of milk (Rego, 2010). Considering this increasing importance of Azorean milk, as well as the unpredictability of climatic and environmental conditions, allied to the inability of most agricultural systems to face and manage mycotoxin prevention or contamination (Prandini et al., 2009), the results of the present study suggest the need to further scrutinize the potential determinant factors. The localization of the Azores archipelago in a high pressure area, results in a slightly different climate to that of mainland Europe. According to the Kppen original classication the climate in the East islands (where dairy farming is localized) is a type Cfb, i.e. an oceanic climate, also known as maritime temperate climate, with a humid temperate climate and a temperate summer that occurs in regions distant from the large continental masses (IM, 2011). As a result Azorean edafo-climate conditions allow the pasture production and grazing feeding regime whole year round, in contrast to the conventional mainland milk production system where cows are conned and fed with diets based in conserved forages and concentrates which may feature up to 70% of the daily feed ration (Fink-Gremmels, 2008). Nevertheless, in Azores, supplementation with conserved forage, especially silage from maize and grass is needed given the energetic needs of a high yield herd and the seasonal pasture shortage during summer in lower areas and winter in the higher parts (Rego, 2010). It is considered that pasture-based beef, sheep and dairy production systems present low risk (FinkGremmels, 2008; Motawee, Bauer, & McMahon, 2009; Roussi et al., 2002), because AFs are not produced in pasture, are rarely found in forages and are usually not present in high enough concentrations in corn silage to be of concern. Indeed, AFs risk is believed to be largely conned to feeding of grain-based concentrates. A seasonal trend in milk contamination may therefore be expected, given that when cows are grazing, they receive less concentrated feed. Results also suggest the need to monitor the manufacturing practices of potentially home-grown cereals fed to the animals as well as the storage practices and conditions of the feedstuffs in general (JECFA, 2001; EFSA, 2004). Importantly, local weather conditions may disclose a potential risk factor for the occurrence of AFs in animal feeds, given the enduring humidity and warmth. Contamination may occur at the pre-harvest stage, but it is exacerbated by inadequate storage conditions (CAC, 2001). Regarding UHT milk, besides the three Azorean milk samples, AFM1 was detected in ve other regular UHT milk samples (ranging from 6.9 to 12.8), and one organic milk sample (7.3 ng/L). The latter was in fact the only analysed milk produced in a foreign country (Spain) that presented detectable levels of AFM1. Regarding pasteurized milk, commonly known as day milk, two marketed brands presented detectable levels, at 7.9 and 15.6 ng/L. Previous studies in Portugal concerning UHT milk, as available to the nal consumers, date back 12 years (Martins & Martins, 2000). During such survey the authors reported very high incidences, which cannot be simply ascribed to the detection limit of the technique employed, since it is similar to the one of the present work (5 ng/L). The percentage of samples surpassing the maximum limit was similar. After that Martins et al. (2005) surveyed raw milk sampled from individual farms. With a detection limit of 5 ng/L, about 66% of the raw milk samples were positive, with some of them surpassing the EU maximum limit. However, these samples were not subjected to the pasteurization or UHT process, and were not necessarily destined to milk production.

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Table 1 Worldwide occurrence and levels of contamination of AFM1 in milk. Milk sample category Pasteurized and UHT milk Raw milk Cow Water buffalo Camel Sheep Goat UHT milk Raw bulk cow milk Infant formula milk Pasteurized milk UHT milk Raw milk Pasteurized milk Raw cow milk Summer Rainy season Winter Buffalo Cow Goat Sheep Camel Pasteurized cow milk Pasteurized cow milk Milk (raw, sterilized, pasteurized) Spring Summer Autumn Winter Buffalo Cow Goat Camel Milk Powdera Powderb Pasteurized UHT Raw cow milk Raw sheep milk Raw goat milk Pasteurized milk Milk powder Pasteurized milk Raw milk Pasteurized and UHT milk UHT milk Raw milk January February June Pasteurized milk Pasteurized milk Infant formula Goat milk Pasteurized UHT Powder Country (year) Portugal (2011) Analytical (LOD, ng/L) ELISA (5) Incidence rate (%) 11/40 (27.5%): Mean (ng/L) 23.4 24.0 % exceeding EU MLc 5% Reference Present study

Ahvaz, Iran (2007e2008)

ELISA (5)

Catalonia, Spain (2008) Villa Maria, Argentina (2007) Italy (2006e2007) Central Iran (2008e2009) Sanandaj, Iran (2006e2007)

ELISA (5) LC-MS/MS (10) HPLC-FD (2.5) ELISA (5) ELISA (5)

59/75 29/75 5/40 19/51 19/60 68/72

(78.7%) (38.7%) (12.5%) (37.3%) (31.7%) (94.4%)

60.1 31.9 19.0 28.1 30.1 9.69

57.4 24.6 7.5 13.7 18.3 2.07

36% 8% 0% 3.9% 6.7% 0% 11% 0% 26.7% 17.4% 4.17% 6.25%

Rahimi et al., 2010

Cano-Sancho et al., 2010 Alonso et al., 2010 Meucci, Razzuoli, Soldani, & Massart, 2010 Fallah, 2010b Mohammadian, Khezri, Ghasemipour, Mafakheri, & Langroudi, 2010 Ruangwises & Ruangwises, 2009 Hussain, Anwar, Asi, Munawar, & Kashif, 2010

60/94 (64%) 2/185 (1.1%) 83/116 68/109 226/240 31/32 (71.5%) (62.3%) (94.2%) (96.9%)

28 15 13.6 2.47 73.8 11.2 74.3 15.1 12.65 17.76 12.43 17.53

Central Thailand (2006e2007) Punjab, Pakistan (2007)

HPLC-FD (10)

HPLC-FD (40)

Iran (2009) Khorasan, Iran (2008)

TLC (12) ELISA (5)

80/80 80/80 80/80 19/55 15/40 6/30 4/24 0/20 66/91

(100%) (100%) (100%) (34.5%) (37.5%) (20%) (16.7%) (0%) (72.5%)

50 21 71 28 89 34 13 24 14 22 25 24 0 52 6 74.91 23.82

47.5% 66.3% 80% 15.8% 20% 0% 0% 0% 36.2% 80.6%

Fallah, 2010a Sani, Nikpooyan, & Moshiri, 2010

196/196 (100%)

Ardabil, Iran (2006)

ELISA (5)

Ismailia, Egypt (2003e2004)

ELISA

23/23 22/22 22/22 23/23 32/50 26/50 18/50 9/25 62/65 10/10 7/10 40/40 70/74 13/23 7/11 10/10 1/8 50/50 65/113

(100%) (100%) (100%) (100%) (64%) (52%) (36%) (36%) (95.3%) (100%) (70%) (100%) (95%) (57%) (64%) (100%) (13%) (100%) (57.5%)

52.9 17.4 22.3 56.3 N.g.

4.4 3.1 0.9 6.6

33%

Nemati, Mehran, Hamed, & Masoud, 2010

48% 34% 26% 20% 51.5% 50% 0% 2.5% 58.6% 23.1% 14.3% 80% 0% 62% 0% 30.3% 31% 0%

Motawee et al., 2009

S. Paulo, Brazil (2006)

HPLC-FD (3)

N.g.

Shundo et al., 2009

Syria (2005e2006)

ELISA (10)

Tabriz, Iran (2008) Yogyakarta, Indonesia (2006) Kenya (2006) Turkey (2007e2008) Japan (2004)

ELISA (5) ELISA (5) ELISA (10) ELISA (10) HPLC-FD (5)

143 53.22 67 18.43 19 13.8 492 212.56 12 50.55 3.73 8.53 N.g. 67 11 11 3.5 7 2.1 5 1.6 18.6 72.2 23.5 7.3 3.9

Ghanem & Or, 2009

Ghazani, 2009 Nuryono et al., 2009 Kangethe & Langa, 2009 Tekins en & Eken, 2008 Sugiyama, Hiraoka, & Sugita-Konishi, 2008 Zinedine et al., 2007 Oveisi, Jannat, Sadeghi, Hajimahmoodi, & Nikzad, 2007 Oliveira & Ferraz, 2007

87/89 (97.8%) 67/100 (67%) 101 97 101 48/54 (88.8%) 128/128 (100%) 116/120 (96.6%)

Rabat, Morocco (2006) Tehran, Iran (2005)

HPLC-FD (1) ELISA (5)

7.4% 78% 0%

Campinas, Brazil (2004e2005)

HPLC-FD (10)

5/12 (41.7%) 2/12 (16.7%) 4/12 (33.3%)

72 48 58 44 56 31

25% 50% 33.3%

S.C. Duarte et al. / Food Control 30 (2013) 411e417 Table 1 (continued ) Milk sample category Raw milk Country (year) Western France (2003) Anatolia, Turkey S. Paulo, Brazil (2002e2003) Analytical (LOD, ng/L) HPLC-FD (8) Incidence rate (%) 9/264 (3.4%) Mean (ng/L) 14.3 10.1 % exceeding EU MLc 0% Reference Boudra, Barnouin, Dragacci, & Morgavi, 2007 Unusan, 2006 Shundo & Sabino, 2006

415

UHT milk Raw milk Pasteurized milk UHT Pasteurized milk

ELISA (5) TLC (20)

75/129 (58.1%) 13/22 (59.1%) 32/43 (74.4%) 34/42 (80.9%) 624/624 (100%)

108.17 13 32 34 N.g.

47% 9% 7.2% 7.1% 17.8%

Shiraz, Iran (2003)

ELISA

Alborzi, Pourabbas, Rashidi, & Astaneh, 2006 Elgerbi, Aidoo, Candlish, & Tester, 2004

Raw cow milk Zuwarah & Sabratha Az zawiyha Az iziyah Tripoli Raw milk (1999e2004) Packed powder milk (2004) Infant formula milk Liquid milk Raw milk Raw milk UHT whole milk UHT-semi-skimmed milk UHT-skimmed milk
a

Northwest Libya (2002)

HPLC-FD (10)

7/9 (77.8%) 12/17 8/12 8/11 394/598 (70.6%) (66.7%) (72.7%) (65.8%)

310 290 120 50 490 1070 720 910 N.g. N.a. 326 45 86 35 N.g. N.g. N.g. N.g. N.g.

66.7% 70.6% 66.7% 72.7% 8.2% N.a. 94.4% 25% 0% 0% 5.6% 4.5% 0%

Portugal

HPLC-FD (5)

Martins et al., 2005

0/25% (0%) Lucknow, India ELISA 17/18 (94.4%) 4/12 (33.3%) 5/92 (5.4%) 25/31 (80.6%) 17/18 (94.4%) 20/22 (90.9%) 23/30 (76.7%)

Rastogi et al., 2004

Lon, Spain (2000e2001) Lisbon, Portugal (1999)

ELISA (10) HPLC-FD (5)

Velasco et al., 2003 Martins & Martins, 2000

Powder milk taken from municipal day-care centres and elementary schools. b Powder milk purchased from supermarkets. c 50 ng/L in milk and 25 ng/L in milk destined to children; ELISA, Enzyme Linked Immuno-Sorbent Assay; HPLC-FD, High Performance Liquid Chromatography coupled to Fluorescence Detection; LC-MS/MS, Liquid Chromatography Tandem Mass Spectrometry; LOD, Limit of detection; ML, Maximum level; N.a., non-applicable; N.g., not given; TLC, Thin Layer Chromatography.

Comparison of the present results with the ones published up until now for UHT milk worldwide (Table 1) indicates that Portuguese marked UHT milk presents one of the lowest AFM1 incidence rate and average level. Such results are comparable given the resembling detection limits of the analytical methods applied in most studies. 3.3. Estimated daily intake of AFM1 in human consumers Based on the data described before (Section 2.5) estimated intake of AFM1 through milk consumption of a Portuguese average adult citizen was estimated at 0.08 ng/kg bw per day. This value resembles the one reported for the adult population of Brazil (0.08 ng/kg bw/day) (Shundo, Navas, Lamardo, Ruvieri, & Sabino, 2009) and is higher than the one of the French adult population (15 years and older), estimated at 0.01 ng/kg bw/day by Leblanc, Tard, Volatier, and Verger (2005). However, higher values of daily intake of AFM1 were previously reported for the adult population of Spain (0.305 ng/kg bw/day) (Cano-Sancho, Marin, Ramos, PerisVicente, & Sanchis, 2010), and Morocco (3.26 ng/kg bw/day) (Zinedine et al., 2007). At the international level, on the basis of the mean concentrations of AFM1 in milk and the milk consumption in the GEMS/Food regional diets (WHO, 1998), JECFA (2001) estimated the daily intake of AFM1 through milk consumption. EDI for the European diet was calculated as 0.11, for Latin American 0.058, for Far Eastern 0.20, for Middle Eastern 0.10, and for African diet as 0.002 ng/kg bw/day. Because of the carcinogenic potential of AFs, international expert committees (JECFA, 2001) did not specify a numerical tolerable daily intake (TDI) for AFs and concluded that daily

exposure, even <1 ng/kg bw, contributed to the risk of liver cancer. It was therefore recommended that levels should be reduced to being as low as reasonably achievable. Although in Portugal milk consumption data does not exist for other age groups, beyond average adults, results from foreign studies suggest that infants are the main risk group exposed to AFM1. In France milk consumption contributed to an AFM1 ingestion of 0.04 ng/kg bw/day in the case of children, aged between 3 and 14 years old (Leblanc et al., 2005). In Brazil, Shundo et al. (2009) estimated a value of 0.23 ng/kg bw for children. In Spain higher exposures were estimated for infants (4e9 years; ranging 0.487 for males and 0.572 for females) and teenagers (10e 19 years; ranging 0.390 for females and 0.423 for males) (CanoSancho et al., 2010). Infants are considered a vulnerable population due, in part, to physiology, a fairly restricted diet and a higher consumption relative to body weight (Lombaert et al., 2003). Infant formulas and milk-based products destined to children should be therefore studied for potential AFM1 contamination. 3.4. Extrapolated intake of AFB1 in feed Based on the AFM1 contamination of milk, it was possible to calculate the concentration of AFB1 in feeds consumed by the producing cows as 1.46 mg/kg. This value is lower than the maximum limit of AFB1 permitted in feeds for dairy cattle within the EU (5 mg/kg) as established by Directive 2002/32/EC (EC, 2002) and amending Directive 2003/100/EC (EC, 2003), which conrms the safety of the feeds provided for lactating cows in Portugal concerning AFs.

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Nevertheless, it is important to highlight that such extrapolated intake was done considering a carry-over rate of 1.6% AFB1. However reported carry-over rates range from 2% (assumed average level) to 6% (reported level for high yielding cows), being inuenced by the factors pointed out before (EFSA, 2004). The same back calculation of the average values of AFM1 obtained from analysis of UHT and pasteurized cows milk in the studies included in Table 1 would result in higher values, some of them exceeding the statutory levels. 4. Conclusion The highest levels of AFM1 contamination of milk commercialized in Portugal were found in milk produced in the Portuguese Azorean islands which suggest the local existence of inuencing factors related to weather and feed storage conditions that merit further attention and analysis. In fact two of the Azores-produced milk samples surpassed the EU maximum level. Based on the obtained results milk should be regularly monitored. First for the unpredictability as well as unavoidability of AFs occurrence in feed, allied to the conrmed carry-over phenomena. Second because average levels of AFs contamination in animal derived products may still have a variable effect between individuals, justied for instance by different dietary habits. An evident example is the infants and childrens restricted diet based on milk. It is therefore encouraged a regular testing to monitor AFM1 in milk. Acknowledgements The authors gratefully acknowledge the nancial support provided by Escola Universitria Vasco da Gama and are thankful to Anlia do Carmo for making the microtiter plate spectrophotometer available. References
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