Aflatoxins and fumonisins in rice and maize staple cereals in Northern Vietnam and dietary exposure in different ethnic groups

Food Control 70 (2016) 191e200

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Aflatoxins and fumonisins in rice and maize staple cereals in Northern Vietnam and dietary exposure in different ethnic groups Bui Thi Mai Huong a, b, Le Danh Tuyen b, Tran Thanh Do b, Henry Madsen a, Leon Brimer a, Anders Dalsgaard a, * a

Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Copenhagen, Denmark National Institute of Nutrition, 48 Tang Bat Ho Street, Hanoi, Vietnam

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 January 2016 Received in revised form 24 May 2016 Accepted 29 May 2016 Available online 30 May 2016

Mycotoxins in food are increasingly a food safety hazard concern in particular in developing countries. This study was performed to determine the occurrence and determinants of aflatoxin and fumonisin contamination in rice and maize and to assess health risks through dietary intake exposure among ethnic minority groups in northern Vietnam. A total of 111 rice and 102 maize samples, were tested for occurrence of fungi and mycotoxins, i.e. aflatoxins (AF’s) and fuminisin B (FB). Results showed that 107 (96.4%) rice and 84 (82.4%) maize samples were contaminated by fungi. Aspergillus flavus was found in 68 (61.3%) rice and 30 (29.4%) maize samples, Aspergilus parasiticus in 40 (36.0%) rice and 27 (26.7%) maize samples. AF’s - were detected in 27 rice (24.3%) and 27 maize samples (26.4%) at minimum and maximum levels in rice of 2.06 and 77.8 ng/g and 20.5 and 110 ng/g in maize, respectively. Nine (8.1%) rice and 24 (23.5%) maize samples contained FB at ranges of 2.3e624 ng/g in rice and 5.6e89.8 ng/g in maize. Data collected through interviews and observations in households showed that type of crop, storage duration and presence of fungi, particularly mycotoxigenic fungi were important risk factors for AF’s and FB contamination. Based on daily food consumption data, the estimated average exposure dose of aflatoxin B1(AFB1) from rice was 21.7 ng/kg bw/day for adults and 33.7 ng/kg bw/day for children. For FB, the rice based average exposure amounted to 536 ng/kg bw/day for adults and 1019 ng/kg bw/day for children. The calculated excess risk of liver cancer incidence by ingestion of cereals containing AFB1 was 1.5 per 100,000 adults and 2.3 per 100,000 children per year. The average intake of FB was calculated to be lower than the tolerable diet intake (TDI). Our findings highlight that rice and maize are contaminated with mycotoxins at levels representing actual health hazards for the ethnic minority groups consuming these stable cereals. Proper drying and storage conditions in households are likely to reduce the mycotoxin contamination. © 2016 Published by Elsevier Ltd.

Keywords: Aspergilus Fusarium Mycotoxins Ethnic minority groups Vietnam Liver cancer risk

1. Introduction Rice (Oryza sativa L.) and maize (Zea mays L.) are two of the most consumed staple foods worldwide (Kennedy, 2002). Rice contributes worldwide with 20% of total consumed staple food and 40% of energy intake (Latham & Routledge, 1998). The Asian and Pacific regions produce and consume about 90% of the world rice and in

* Corresponding author. Department of Veterinary Disease Biology, Groennegaardsvej 15, Faculty of Health and Medical Sciences, University of Copenhagen, DK1870 Frederiksberg C, Copenhagen, Denmark. E-mail address: [email protected] (A. Dalsgaard). http://dx.doi.org/10.1016/j.foodcont.2016.05.052 0956-7135/© 2016 Published by Elsevier Ltd.

several countries, rice contributes with 40e80% of the food energy and protein requirements (Singh, Woodheadb, & Papademetriouc, 2002). Asia contributes about one-third of the world’s total maize production and maize is the second most important cereal crop after rice in this region. Vietnam is among the world’s top five rice producers and consumers (USDA, 2015) and maize is often a substitute in rural and mountainous areas, especially during periods of rice shortage. A nutritional surveillance (NIN, 2011) showed that the diet of people in our study area in North West Vietnam is less diverse than in the rest of the country. The different ethnic groups inhabiting this area are all subsistence farmers and most of their daily food

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intake comes from maize and rice with some ethnic groups having maize as their preferred stable crop (Thanh Ha et al., 2004, p. 42). Thus, any hazardous substances in these stable cereals would represent major food safety and health risks. The farmers in the Northern highlands face different climate conditions, e.g. elevated temperatures and higher relative humidity as compared to most other parts of Vietnam (Pandey, Khiem, Waibel, & Thien, 2006). Furthermore, possible different agricultural practices including means of storage and drying harvested cereals, may provide the basis for differences in fungal infestation and associated food safety hazards as compared to those faced by low-land Vietnamese farmers. Rice and maize are reported as good substrates for fungal growth and production of mycotoxins (Reddy, 2009; Reddy, Saritha, Reddy, & Muralidharan, 2009; Trung, 2008) such as aflatoxins (Reddy et al., 2009; Shephard, 2008) and fumonisins (Domijan, Peraica, Jurjevic, Ivic, & Cvjetkovic, 2005; Nikiema, Worrillow, Traore, Wild, & Turner, 2004). Aspergillus flavus shows optimal production of aflatoxins at 28
C and 8e12% moisture content whereas Fusarium verticillioides produce high levels of fumonisins at temperatures between 20 and 26
C (Folcher et al., 2010; JECFA, 2002; Scott, 1993;; Reid et al., 1999) with toxin often produced preharvest when cereals are stored in the field (JECFA, 2002). The growth of fungi and their toxin production is increased by insect damage and post-harvest storage conditions (FAO, 1989). According to the Food and Agricultural Organization (FAO), 25% of the world’s food crops may be affected by mycotoxins each year (Boutrif & Canet, 1998). Amongst the known mycotoxins, aflatoxins, deoxynivalenol and fumonisins pose the greatest threat to human health worldwide (Reddy, 2009). The main producers of aflatoxins are the fungi Aspergillus flavus and A. parasiticus (FAO, 1989). The International Agency for Research of Cancer (IARC) has classified aflatoxin B1 and natural mixtures of aflatoxins as group 1 carcinogens. IARC has further classified mycotoxins derived by F. verticillioides which mainly are the fumonisins and fusarin C in the group 2B as carcinogenic in animals and possibly also in humans (IARC, 1993). The fumonisins are a group of chemically related mycotoxins mainly produced by F. verticillioides (formely Fusarium moniliforme). Fumonisins are mainly found in maize, but has also been reported in rice products (Richard, 2007). Previous surveys on mycotoxins in foods in Vietnam are few and with small sample sizes, but they do indicate that aflatoxins are common in maize kernel and maize flour at retail markets in Hanoi (Wang et al., 1995). AFB1 was found in 10/15 (77%) of maize samples for human consumption coming from different parts of Vietnam with concentrations ranging from 11.3 ng to 126.5 ng/g dry weight while fumonisins were found in 8/25 (32%) samples ranging from 400 to 3300 ng/g (Trung, 2008). Little is known about the occurrence of mycotoxins in stable cereals (rice and maize) produced and consumed by people in the rural and mountainous areas of Northern Vietnam. The Vietnamese standards for maximum content of total aflatoxin and aflatoxin B1 are 10 mg/kg and 5 mg/kg (Ministry of Health, 2011) as compared to the US standard of 20 mg/ kg for any type of food for human consumption (Food Safety Watch, 2013). The EU has lower maximum levels of 10 mg/kg for total aflatoxin in ready-to-eat rice and corn products and as low as 0.1 mg/kg for processed cereal-based baby and infant food (EU, 2010). Both aflatoxins and fumonisins are resistant to processing resulting in a high retention of those mycotoxins in consumed food (Scott, 1993). WHO recently reported a much higher burden of exposure of aflatoxin and fumonisin in low-income regions and also identified priority issues that needed immediately action that is quantifying human health impacts and burden of disease due to the exposure of the mycotoxins and compiling an intervention strategy to control mycotoxins (Strosnider et al., 2006).

The hypothesis of the study was that exposure to and health risks caused by mycotoxins in staple cereals would be different between ethnic groups as well as to farmers in other parts of Vietnam due to the different climatic conditions and agricultural practices. Thus, our study aimed to determine the occurrence and determinants of aflatoxins and fumonisins contamination in rice and maize and assess health risks through dietary intake exposures among ethnic groups in Northern Vietnam. 2. Materials and methods 2.1. Study site and population The study was conducted in Ta Phoi and Hop Thanh communes in the mountainous province of Lao Cai, Northern Vietnam. These are the poorest communes in Lao Cai district with 40% of households having monthly incomes less than 200,000 Vietnamese Dong (approximately 10 USD per person). Households were mostly located in highland villages and members represented seven ethnic groups, i.e. Dao (18 households), Giay (21), Xapho (20), Tay (18), and Hmong (20) with few Chinese households living there as well €nder, (Department of Health Lao Cai district, 2008; Rheinla Samuelsen, Dalsgaard, & Konradsen, 2010) (Table 1). The Giay and Tay ethnic groups live in the lowland about one km away from the center of Hop Thanh commune and typically grow two rice crops per year (Department of Health Lao Cai district, €nder et al., 2010). In addition, the Giay cultivates 2008; Rheinla two maize crops while one maize crop is produced by the Tay’s annually. The Xapho people reside in a highland area of Hop Thanh commune and grow two rice crops and one maize crop every year. The Dao and Hmong people live in highland areas of Ta Phoi commune. The Dao village is located five km from the commune center and inhabitants grow one rice crop and one maize crop per year. Compared with the other ethnic groups, the mountainous Hmong village is the most isolated and least developed with only one maize crop grown annually as the main staple food. The ethnic Vietnamese Kinh group lives in quite developed households in a lowland village in Ta Phoi commune and has a comparable higher income. In contrast to the other ethnic groups which are almost entirely subsistence farmers, the husbands of Kinh households typically work in the local apatite mines and wives take care of household matters. Most Kinh households buy rice and maize at local markets whereas the other ethnic groups in general are selfsupplied with home grown rice and maize (Department of Health €nder et al., 2010). Lao Cai district, 2008; Rheinla The mean annual temperature is around 20e29
C, annual rainfall about 1400e1700 mm and the average humidity above 80%. 2.2. Collection of maize and rice samples A total of 213 samples of dried maize seed and rice were randomly collected from separate purchased batches in October 2009 (Table 1). The samples were collected from three sources: wholesale, retail and household. The “wholesale samples” represent samples purchased at the Coc Leu market, the largest wholesale market in Lao Cai city. Samples purchased at local markets in Ta Phoi and Hop Thanh communes were catalogued as “retail samples”. The “household samples” were collected during visits to individual households. Only one rice and/or maize sample were collected from each individual household. Samples were collected from all stands in operation at the wholesale market and retail (local) markets. This sampling strategy was chosen to obtain adequate and representative sample numbers for analysis. Similarly, we collected samples from all households in a particular commune with children less than 5 years old resulting in different

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Table 1 Characteristics of rice and maize samples collected for fungi and mycotoxins analyses from ethnic households in two communes in Lao Cai province, Northern Vietnam. Source

Commune

Wholesale Retail Household

Village

Hop Thanh Ta Phoi Hop Thanh

Ethnic group

Kip Tuoc Nam Ria Peng Peng Tram Thai Lap May U xi xung

Ta Phoi

Number of sample

Giay Xa pho Tay Dao Kinh Kinh Hmong

Total

Rice

Maize

8 6 6 21 16 17 18 19

2 0 0 16 20 18 17 9

20 111

0 102

Remarks

Self supplied Self supplied Self supplied Self supplied Purchased at markets and self supplied Self supplied

Table 2 Storage conditions of maize and rice samples among different ethnic groups in Lao Cai province, Vietnam. Crop

Source

Ethnic group

Rice

N

Container

Retail or 20 1 whole sale Household Dao 18 12 Giay 21 4 Kinha 19 8 Tay 17 2 Xa pho 16 2 Total 111 29 Maize Retail or 2 0 whole sale Household Dao 17 0 Giay 16 0 Hmong 20 0 Kinha 9 2 Tay 18 1 Xa pho 20 0 Total 102 3

a

Storage place

Closed Open Separate storage

Storage condition

Roof Shelf Floor Mean ± SEM of moisture content (%)

Mean ± SEM of temperature of samples
( C)

Mean ± SEM of storage duration (days)

No of insect positive samples

19

0

0

0

20

10.9 ± 0.24

25.2 ± 0.34

7 ± 0.6

0

6 17 11 15 14 82 2

0 0 0 0 3 0 0

0 0 0 2 0 2 0

0 2 0 0 4 6 0

18 19 19 15 12 103 2

14.3 ± 0.35 12.3 ± 0.38 14.5 ± 0.36 23.3 ± 0.31 12.9 ± 0.12 14.5 ± 0.28 8.6 ± 0.18

20.9 26.5 25.0 26.1 26.5 25.1 27.6

± ± ± ± ± ± ±

0.22 0.18 0.21 0.16 0.12 0.20 0.32

10 ± 2.1 5 ± 0.8 12 ± 2.0 21 ± 5.2 7 ± 1.8 10 ± 2.9 90 ± 3.2

0 0 0 0 0 0 0

17 16 20 7 17 20 99

0 0 0 0 0 3 6

16 1 3 0 3 0 23

0 0 3 0 1 3 7

1 15 14 9 14 14 69

± ± ± ± ± ± ±

22.3 26.2 24.6 25.0 26.7 27.3 25.5

± ± ± ± ± ± ±

0.21 0.30 0.23 0.28 0.22 0.24 0.26

95 80 45 71 88 45 70

± ± ± ± ± ± ±

2 2 0 2 1 2 9

14.7 12.0 15.8 12.0 11.7 16.6 14.0

0.48 0.58 0.67 0.71 0.6 0.62 0.52

3.3 7.6 10.2 10.3 4.6 7.8 6.4

Most samples purchased.

sample numbers from the different sources (Table 2). Means of storage were different between and within ethnic groups and households, e.g. not all means of storage were practiced by a particular type of household (Table 2). Samples of approximately 1 kg were obtained after a thorough mixing from the different storage units used by the wholesalers, retailers and households; including plastic and wooden containers, bamboo baskets, cloth sacks, and plastic bags. Samples were collected using disposable gloves and placed in sealed sterile plastic bags which were encoded and stored at 20
C for a maximum of five days before transport to the laboratory at the National Institute of Nutrition in Hanoi. Here, samples were divided into five subsamples for examination of the appearance of insects, moisture content analysis, fungi enumeration and mycotoxins detection, respectively, the fifth being stocked. During sampling, a simple check-list and questionnaire was applied to collect information that could potentially be associated with presence of fungi and mycotoxins. The collected data included time and condition of storage as well as type of storage container. Storage time was calculated as the number of days from the day the cereal was husked to the day of sampling. Storage conditions included information on the storage location, e.g. in separate storage containers, on the roof, on a shelf or a platform and on the floor. It was also noted whether the type of storage container was an open or a closed container.

2.3. Determination of temperature and moisture content The temperature of samples was measured in the original storage container before sampling using a digital thermometer (Hana Instrument, Germany). The moisture content was measured by drying 10.0 g in an oven at 105
C for 17 h (Miren, Sonia, & Vicente, 2008). 2.4. Identification and enumeration of fungi Level of fungi contamination was assessed following a procedure recommended by the US Food and Drug Administration (FDA, 2001). Briefly, 10 g of homogenized sample was suspended in 100 ml of 1% sterile agar water (Merck, Germany) at 45
C and used to prepare a 10-fold serial dilution. Each dilution was added to Dichloran Glycerol (DG 18) agar in duplicate (Merck, Germany) by the pour plate method and allowed to solidify before incubation at 25
C in the dark for six days when colonies were enumerated. The appropriate dilution factor was determined as plates that contained 10e30 colonies, and results expressed as colony forming units per gram (cfu/g) of sample. Presumptive fungi isolates were sub-cultured and purified on potato dextrose agar to identify single spores of aflatoxinproducing and fumonisin-producing fungi (FDA, 2001). Both macroscopic characteristics of the colonies as well as

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morphological characteristics (Microscope Olympus, Japan) were used for identification (Samson, , Hoekstra, , and Frisvad, , & eds., 2004).

based on weights of 127 children (40 ± 12 months age) present in households during the interviews (data not shown). 2.8. Risk assessment

2.5. ELISA detection of mycotoxins Direct competitive ELISA-based methods using AFB1 and FB1 standards and commercially available detection kits (AgraQuant®, Romer, USA) was used for total aflatoxins (AFB1 þ B2 þ G1 þ G2; AFT) (limit of detection is 1 ng/g) and FB1 (limit of detection is 0.25 mg/g) analyses according to manufacturer’s instructions. Briefly, mycotoxins extraction was performed by blending 20 g of ground rice or maize in 100 ml methanol: water (70:30 v/v) for 2 min. The suspension was centrifuged for 10 min at 4000 rpm. The supernatant was collected for toxin detection. Optical density was measured using a microtiter plate reader (Bio-Rad Plate Reader, Bio-Rad, Hercules, CA, USA). The concentration of AFT and FB was calculated on a dry weight basis according to the specifications of the manufacturer. For each sample, one extract was produced then duplicate determinations of the toxin were performed. Standard curves were plotted using standard AFT (y ¼ 0.0183x þ 0.2719; R2 ¼ 0.9868) and FB1 (y ¼ 0.54720.6321; R2 ¼ 0.9899). The precision of the two assays was tested by analyzing in duplicate four different concentrations of aflatoxin (1, 2, 4 and 8 ng/g) and fumonisins (0.25, 1, 2.5 and 5 mg/g). The experimental recovery values (%) were 104.7 ± 6.7, 98.7 ± 3.3, 101.8 ± 2.0 and 99.8 ± 0.9 for aflatoxin, respectively and 104.0 ± 2.8, 99.0 ± 4.9, 101.0 ± 4.1 and 102.0 ± 1.8 for fumonisins, respectively.

Risk assessment of AFB1 was performed based on dietary exposure to AFB1 and its potency in the induction of primary liver cancer. Since AFB1 has been found to contribute about 60% of total aflatoxin determined (Garrido, Hern andez Pezzani, & Pacin, 2012; Khatoon et al., 2012; Mohamadi Sani, Azizi, & Naeejic, 2012), we assumed that the contamination level of AFB1 was 60% of that of total aflatoxins found by the analysis of the maize and rice samples. The risk of liver cell cancer for the study population was assessed on the basis of the result of the exposure data and an average potency calculated using the prevalence of individuals being hepatitis B surface antigen- (HbsAg) positive and having a potency of 0.3 cancers per year per 100,000 population per ng AFB1/kg body weight (kg bw) per day and the negative individuals set to have 0.01 cancers per year per 100,000 population per ng AFB1/kg bwper day (Liu & Wu, 2010; WHO, 1998). A previous study reported hepatitis B positive prevalence rates in rural areas of Vietnam at 18.4± 5.0%, 20.5± 5.3% and 18.8± 3.1% in children, adolescent and adult, respectively (David et al., 2003). Therefore, we used a HbsAg þ prevalence rate of 20% in our risk assessment in line with the 25% prevalence rate referred to by WHO (WHO, 1998) for developing countries, giving an average potency for liver cell cancer of 0.068. Population risk was obtained by multiplying dietary exposure with average potency.

2.6. Dietary intake of study population

Population risk ¼ Exposure  average potency

Data on maize and rice dietary intake were collected and estimated to allow an assessment of the dietary exposure to aflatoxins and fumonisins when household members consumed these cereals. The consumptions of rice and maize by adults and children were done in two separate ways: 1) consumption of rice and maize by adults were extracted from a food consumption data set of adults living in Lao Cai city collected in the first week of October 2009 by the National Institute of Nutrition, Hanoi as part of a National Survey on Food Consumption (National Institute of Nutrition, 2011) and 2) the amounts of maize and rice consumed by children less than five years of age were calculated based on information collected from 24-hrs recall food intake interviews conducted by our research group on three consecutive days combined with actual weighing of the reported consumptions (EFSA, 2009; Swindale & Vachaspati, 1999). The national survey and our survey did collect data on dietary intake by household interviews of the mother/grandmother on types of dishes consumed in the last day, including information on all ingredients used for food preparation. Supporting tools such as spoon, table spoon, bowl, and cups were used to activate the household member’s memory and to allow subsequent weighing of the foods. Accordingly, the interviewer was instructed to weigh available foods for confirming weight stated by household members using an electronic scale.

Average potency ¼ 0:3  0:2 þ 0:01  0:8 ¼ 0:068

2.7. Assessment of dietary exposure The dietary exposure to a mycotoxin is determined by mycotoxin contamination level (ng/g food) in the food consumed, the actual amount of this food eaten per day (g food/day) and the individual’s body weight (kg bw). The value of body weight of adults was assigned as 49 kg, equal to the average body weight of adults living in north-east Vietnam (National Institute of Nutrition, 2007) and 11.3 kg was estimated as the mean body weight of children

Fumonisin diet exposure was compared to a Tolerable daily intake (TDI) of 2000 ng per kg bodyweight per day (JECFA, 2002). 2.9. Data analysis Mycotoxin concentrations or presence (detected/not detected) were related to various potential predictors one by one (uni-variable) using linear regression or logistic regression, respectively (model 1). Categorical variables were coded as indicator variables while some continuous factors were log transformed to the base 10. In model 2, presence of the causative agents of mycotoxins (i.e. fungi) was tested simultaneously (multi-variable) with predictors from model 1 using a forward stepwise selection procedure (p for removal and inclusion were set to 0.10 and 0.05, respectively). 3. Results 3.1. Storage condition, temperature and moisture content Only 28/91 (30.8%) of rice samples and 3/100 (3.0%) of maize samples were stored in closed containers (Table 2). With one exception all retailers stored cereals in open containers. Overall maize was more often stored in open containers than rice. With little difference among ethnic groups the vast majority of household’s stored rice (92.8%) and maize (67.6%) on the floor except the Dao households which stored maize under the roof. The moisture content of rice and maize samples ranged from 8.6 to 23.3%, and with a mean of 14.5%, the lowest values were found in samples from retail or wholesales. Temperatures measured ranged from 20.9 to 27.6
C. The mean storage duration of rice samples was 10 days ranging from 5 to 21 days. However, maize samples were stored much longer averaging 70 days. Damage by insects was observed in

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nine maize samples, but not in any rice samples (Table 2). 3.2. Fungal contamination Data on the concentration of potential toxigenic fungi are summarized in Table 3. Amongst the 111 rice and 102 maize samples tested, 107 and 84 were found to be contaminated by fungi, respectively. A. flavus was detected in a total of 68 and 30 rice and maize samples, i.e. in 61.3% and 29.4%, respectively. A. parasiticus contamination occurred in six (5.4%) rice samples and in four (3.9%) maize samples. Fusarium verticillioides was found in 40 (36.0%) rice samples and in 27 (26.7%) maize samples. The mean number of fungi in rice samples was 1.57  104 with a range from 37 to 2.80  105 CFU/g. This was lower than the mean concentration found for maize, i.e. 3.34  105 (range from 1.00  104 to 2.58  105 CFU/g). 3.3. Occurrence of mycotoxins The occurrence and concentrations of mycotoxins are shown in Table 4 and Fig. 1. AF’s were detected in 27 rice (24.3%) and in 27 maize samples (26.4%). Minimum and maximum levels of AFT in rice were 2.06 and 77.8 ng/g, respectively, while the levels in maize were 20.5 and 110 ng/g. Nine rice samples and 24 maize samples were found to contain fumonisin B1 with concentrations in rice ranging from 2.3 to 624 ng/g and between 5.6 and 89.8 ng/g in maize. 3.4. Risk factors for mycotoxins AF’s were reported in four of the 22 samples from retail or wholesales while 54 of 191 samples from households contained aflatoxins. The proportion of samples contaminated with mycotoxins did not differ among the different sources of samples. Most rice and maize samples collected in households were selfproduced, but some Kinh households reported to have purchased their cereals from retail or wholesale dealers. All samples from retail or wholesales as well as samples from households reported to have been purchased from these were excluded from subsequent data analysis. The number of samples with A. parasiticus growth was thereby reduced to three and therefore the counts of A. flavus and A. parasiticus were pooled. In uni-variable analyses, model 1 (variables that could be seen as potential risk factors for fungal growth) showed that AF concentrations were on average 21.26 ng/g greater in maize samples than in rice samples (p < 0.001) and was positively associated with storage duration [log10(days)]. A one unit increase on a log scale (a 10-fold increase in storage time in days) was associated with 19.38 ng/g higher concentration of AFT in rice (p < 0.001) while no other factors were significant. None of the model 1 factors were significantly associated with FB1 concentration. Presence of AF’s (concentration >1.0 ng/g) was not associated with any of the predictors while maize had a higher risk for being contaminated with

195

FB1 than rice (OR ¼ 2.45, p < 0.05) and presence of FB1 was positively associated with storage duration (OR ¼ 2.86, p < 0.01). Presence of AF’s at concentration >10 ng/g was more likely in maize than in rice (OR ¼ 8.34, p < 0.01) and with storage time (odds ratio for a 10-fold increase in storage time was 8.92). Ethnic group did not appear as a significant predictor for mycotoxins presence or concentration. Only duration of storage, however, remained in the multivariable model (Table 5). In the multivariable analysis (model 2), maize, growth of Aspergillus spp. and duration of storage were associated with higher total concentrations of AF’s (Table 5). Only growth of Fusarium spp. was a significant predictor of FB1 concentration (Table 5). Looking at presence/absence of AF’s instead of concentration (Table 5), the multivariable analysis showed that maize and growth of Aspergillus spp. were both associated with higher odds of having AFB1 (Table 4). In the uni-variable tests only, growth of Fusarium spp. was significantly associated with presence of FB1, while in the multivariable model also duration of storage was significant (Table 5). 3.5. Dietary exposure and risk assessment Using mean consumption levels from the national survey of adults (National Institute of Nutrition, 2011) and similar data collected by the lead author for children, the dietary exposure to AFB1 and FB1 were calculated (Tables 6 and 7). The average total AFB1 exposure from both rice and maize was estimated to 21.7 ng/ kg bw/day for the adults and associated with an average risk of excessive liver cancer of 1.5 cases per 100,000 individual per year while for children the exposure was estimated to 33.7 ng/kg bw/ day and associated with an average risk of 2.3 cases per 100,000 individual per year. In our study, the mean FB1 exposure level from intake of cereals was 536 ng/kg bw/day and 1019 ng/kg bw/day equal to 26.8% and 51.0% of TDI of adults and children, respectively. 4. Discussion The current study showed that contamination with fungi is common in rice (96.4%) and maize (82.4%) consumed by ethnic minority groups in Northern Vietnam. Aspergillus flavus and Fusarium verticillioides, were isolated from 61.3% and 36.0% of rice samples, respectively while a total of about 30% of maize samples was contaminated with either one or both of these fungi. This supports previous findings from Vietnam (Tran, Bailly, Querin, Le Bars, & Guerre, 2001; Trung, 2008) and in Thailand (Pitt et al., 1994) that A. flavus and Fusarium spp. are important contaminants of cereals. Traditional methods of drying rice directly along roads or on the edge of fields as practiced by the study communities are important transmission routes for fungal spores associated with soil dust particles (Rogge, Medeiros, & Simoneit, 2007). Maize cups were not dried along roads, but were stored and dried within household perimeters on floors, shelves, and roofs with the Dao ethnic group preferring storage on the roof (Table 2).

Table 3 Potential toxigenic fungi found in rice (n ¼ 111) and maize samples (n ¼ 102). Fungi

Total counts A. flavus A. parasiticus F. moniliforme a

Rice

Maize

Na (%)

Mean (CFU/g)

107 68 6 40

1.57 1.62 5.82 3.35

(96.4) (61.3) (5.4) (36.0)

   

104 103 103 102

N is the number of samples found contaminated with fungi.

Range (CFU/g)

Na (%)

Mean (CFU/g)

3.7  10e2.8  105 1.0  102e1.3  104 2.10e2.8  103 1.0  102e2  103

84 30 4 27

3.34 1.43 7.90 4.55

(82.4) (29.4) (3.9) (26.7)

   

105 105 104 104

Range (CFU/g) 104e2.58  106 3  102e2.3  106 6  103e2  105 102e1.90  105

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Table 4 Contamination level of mycotoxins by crops and ethnic groups. Ethnic

Crop

N

Number of samples contaminated above the maximum levela Aflatoxins (>10 ng/g)

Dao

Fumonisins (>1000 ng/g)

Rice

18

2

0

Maize

17

5

0

Rice

21

0

0

Maize

16

6

0

Hmong

Maize

20

3

0

Kinh

Rice

19

5

0

9

4

0

Rice

17

0

0

Maize

18

3

0

Rice

16

0

0

Maize

20

5

0

Rice Maize

91 100

7 26

0 0

Giay

Maize

Tay

Xa pho

Total a b c

Contamination level (ng/g)

b

Mean Min Max Mean Min Max Meana Min Max Mean Min Max Meana Min Max Meana Min Max Meana Min Max Meana Min Max Meana Min Max Meana Min Max Meana Min Max Mean Mean

Aflatoxins

Fumonisins

3.13 19.06 21.23 26.45 21.23 109.64 1.06 2.06 2.21 33.87 24.86 108.19 11.88 58.91 101.67 12.04 13.26 77.75 48.00 103.12 110.36 1.90 3.21 8.67 8.67 103.12 106.74 1.00 2.06 3.08 18.46 20.51 103.12 3.82 24.51

30.74 182.30 358.42 11.80 5.88 69.03 31.67 2.30 624.48 1.83 5.64 15.01 11.31 65.24 89.79 NDc ND ND 1.64 7.03 7.74 17.92 140.35 164.22 5.77 5.90 11.48 23.02 163.45 193.42 3.90 5.90 28.71 69.37 2.44

Maximum level adopted by Vietnam Government (Ministry of Health, 2011). Mean is less than minimum since including many negative samples. Not detected.

Furthermore, long time storage and storing cereals on humid ground floors or in kitchens at elevated temperatures, as practiced by the ethnic minority groups, may promote growth of fungi and development of mycotoxins (Hettiarachchi, Gooneratne & Hirimburegama, 2001; Noomhorm & Cardona, 1989). In our study, moisture content in rice and maize varied between 10.9% to 23.3% and 8.6%e16.6%, respectively (Table 2). Generally, it is assumed moisture content below 12% protects against fungal growth (Hettiarachchi et al., 2001; Noomhorm & Cardona, 1989). However, our risk factor analysis did not find a significant association between moisture content as well as temperature (overall range from 20.9
C to 27.6
C) and presence and level of the mycotoxins in rice and maize (Table 5). In agreement with other studies, duration of storage (rice: 7e21 days; maize: 45e95 days) was the only parameter significantly associated with aflatoxin concentration as well as risk for presence of mycotoxins (Table 5) (Hettiarachchi et al., 2001; Noomhorm & Cardona, 1989). In contrast to our study hypothesis, ethnic group did not appear as a significant predictor for mycotoxins presence or concentration. The occurrence of AF’s in rice and maize was about 25% (Fig. 1) with concentrations ranging from 2.06 to 77.8 ng/g in rice and from 20.5 to 110 ng/g in maize samples (Table 4). These levels are lower than previously reported for cereals in Vietnam (Nguyen, Tozlovanu, Tran, & Pfohl-Leszkowiczb, 2007; Trung, 2008) and other countries with similar climatic conditions (Reddy et al., 2009; Sugita-Konishi et al., 2010; Wang et al., 1995). However, still many

samples including some rice samples had higher AFT concentrations than allowed in most developed countries and certainly much higher than the maximum value (4 mg/kg) set by the EU (EU, 2010). Fumonisin was found in 8.1% of rice samples and 23.5% of maize samples (Fig. 1) showing mean concentrations from 2.3 to 624 in rice and from 5.6 to 89.8 ng/g in maize (Table 4). Our results show a lower prevalence of fumonisin contamination in the cereals but a higher concentration of the toxin as compared with previous reports from Vietnam and other Southeast Asian countries (Wang et al., 1995; Yamashita et al., 1995; Yoshizawa, Yamashita, & Chokethaworn, 1996). Amongst ethnic groups, Kinh adults were observed to have the highest exposure to AFB1 through consumption of both rice and maize with a mean level of 63.6 ng/kg bw/day while this was 7.6 ng/kg bw/day among Xapho people. The exposure of the other ethnic groups ranged from 10.2 ng/kg bw/day to 18.8 ng/kg bw/day. Furthermore it should be noted, that apart from assigning samples showing analytical results less than the limit of detection to non-detects, a lower bound could have been used to estimate the dietary exposure giving rise to a higher total result. In China, total aflatoxin dietary exposure was estimated at 11.7e2027 ng/kg bw/day, from a diet in which rice contribute with about 30% of recommended energy intake (Kenny, 2001), and in Thailand at 6.5e53 ng/kg bw/day (Williams et al., 2004). Similarly, Sugita-Konishi et al. (2010) stated based on analysis of relative few rice samples from Korea that the level of AFB1 contamination in rice in several Asian countries could be a serious food safety problem,

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197

Fig. 1. Distribution of mycotoxins found in rice (n ¼ 111) and maize samples (n ¼ 102).

Table 5 Risk factor analysis for concentration and occurrence of mycotoxins (aflatoxin or fuminosin) in rice and maize samples. Factors were tested one at the time (univariable) or all together in a forward stepwise selection procedure (multivariable). Factors

Regression coefficient (ng/g)

Exponentiated regression coefficient (odds ratio)

Aflatoxin concentration

Fuminosin concentration

Aflatoxin present

Fumonisin present

Aflatoxin (conc>10 ng/g)

Uni

Uni

Uni

Uni

Uni

Multi

Model 1 variables Commune ns ns Village ns ns Ethnic group ns ns Crop (rice the base group) 21.26c ns Type of container ns ns Storage place ns ns Moisture content (%) ns ns Temperature (
C) ns ns Storage duration (log10[days]) 19.38c 19.38c ns Insect damage ns ns Model 2 variables (including also some variables from model 1) Fungi presence ns ns Aspergillus spp. presence 25.31c 42.33c ns b Fusarium presence ns 1.92 17.89a Crop (rice the base group) 21.26 28.95c ns Storage duration (log10[days]) 19.38 9.85a ns a

Multi

Multi

ns ns ns ns ns ns ns ns ns ns

17.89

a

ns 55.94 ns ns ns

ns ns ns 2.45a ns ns ns ns 2.86b ns

194.37c 8.00b

ns ns 15.46c 2.45 2.86

Multi

2.86b

24.27

c

4.86c

ns ns ns 8.34b ns ns ns ns 8.92c ns ns 20.60c ns 8.34b 8.92c

Multi

8.92c

182.60c 92.10c

,p < 0.05; b, p < 0.01; c, p < 0.001; ns, not significant; uni, uni-variable; multi, multi-variable.

except in Japan where a three year survey demonstrated levels of aflatoxins lower than limit of detection in rice, rice ball, maize and maize products (Sugita-Konishi et al., 2010). Based on available toxicity data, the Joint FAO/WHO Expert Committee on Food Additives set a group TDI for fumonisins (B1 and its analogues B2 and B3), which are considered to be associated with a range of toxic syndromes, at 2 mg per kg body weight per day (JECFA, 2002). The intake of FB1 was different amongst ethnic groups, of which the highest exposure was found in the Giay (245.1 ng/kg bw/day) almost entirely originating from rice consumption (244.6 ng/kg bw/day) whereas the lowest estimated exposure was for the Tay group (140.0 ng/kg bw/day) (Table 6).

It is well recognized that children in particular are vulnerable to foodborne hazards due to their higher food consumption per kg body weight and differences in physiology compared to adults. Due to significant postnatal development of different organ systems during childhood, children are more sensitive to some neurotoxic, endocrine and immunological effects up to four years of age (Boon, Bakker, Van Klaveren & Van Rossum, 2009). We therefore estimated the exposure of children to AFB1 and FB1 separately. Since maize was not fed to children in the study population, toxin exposure level of children was only estimated using the data on rice consumption (Table 7). The mean consumption of children was 166.0 g rice/day. Kinh children consumed the highest amount with

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Table 6 Mycotoxins exposure assessment and risk characterization by crop and ethnic group e assumed adult body weight is 49 kg; population risk is calculated on the assumption of HbsAg þ prevalence 20% (WHO 1998) and mean level of the mycotoxins contamination of all samples. Ethnic

Dao

Crop

Rice

Maize

Giay

Mean of consumption (g/day)

378.5

13.3

Both crops

391.8

Rice

378.5

Maize

13.3

Both crops

391.8

Hmong

Maize

166.4

Kinh

Rice

378.5

Maize

Tay

Both crops

391.8

Rice

378.5

Maize

Xa pho

a b c d

13.3

Both crops

391.8

Rice

378.5

Maize

Total

13.3

13.3

Both crops

391.8

Rice Maize Both crops

378.5 13.3 391.8

Aflatoxin B1a

Fumonisins

Contamination level (ng g)

Exposure (ng/kg bw/day)

Riskb

Contamination level (ng/g)

Exposure (ng/kg bw/day)

Meanc Min Max Mean Min Max Meanc Min Max Meanc Min Max Mean Min Max Mean Min Max Meanc Min Max Meanc Min Max Meanc Min Max Mean Min Max Meanc Min Max Meanc Min Max Mean Min Max Mean Min Max Meanc Min Max Meanc Min Max Mean Mean Mean

14.5 88.3 98.4 4.3 3.5 17.9 18.8 91.8 116.3 4.9 9.5 10.2 5.5 4.0 17.6 10.4 13.6 27.9 24.2 120.0 207.2 55.8 61.5 360.3 7.8 16.8 18.0 63.6 78.2 378.3 8.8 14.9 40.2 1.4 16.8 17.4 10.2 31.7 57.6 4.6 9.5 14.3 3.0 3.3 16.8 7.6 12.9 31.1 17.7 4.0 21.7

1.0 6.0 6.7 0.3 0.2 1.2 1.3 6.2 7.9 0.3 0.6 0.7 0.4 0.3 1.2 0.7 0.9 1.9 1.6 8.2 14.1 3.8 4.2 24.5 0.5 1.1 1.2 4.3 5.3 25.7 0.6 1.0 2.7 0.1 1.1 1.2 0.7 2.2 3.9 0.3 0.6 1.0 0.2 0.2 1.1 0.5 0.9 2.1 1.2 0.3 1.5

Meanc Min Max Mean Min Max Meanc Min Max Mean Min Max Meanc Min Max Meanc Min Max Meanc Min Max Mean Min Max Meanc Min Max Mean Min Max Meanc Min Max Meanc Min Max Meanc Min Max Meanc Min Max Meanc Min Max Meanc Min Max Mean Mean Mean

237.5 1408.2 2768.6 3.2 1.6 18.7 240.7 1504.7 3417.9 244.6 17.8 4823.8 0.5 1.5 4.1 245.1 63.5 5113.3 38.4 221.5 304.9 e e e 0.4 1.9 2.1 e e e 138.4 1084.1 1268.5 1.6 1.6 3.1 140.0 1169.4 1404.9 177.8 1262.6 1494.1 1.1 1.6 7.8 178.9 1354.1 1776.1 535.8 0.7 536.5

1.9 11.4 12.7 15.9 12.7 65.8 17.7 24.2 78.5 0.6 1.2 1.3 20.3 14.9 64.9 21.0 16.2 66.2 7.1 35.3 61.0 7.2 8.0 46.7 28.8 61.9 66.2 36.0 69.8 112.9 1.1 1.9 5.2 5.2 61.9 64.0 6.3 63.8 69.2 0.6 1.2 1.8 11 12 62 12 14 64 2 15 17

30.7 182.3 358.4 11.8 5.9 69.0 42.5 188.2 427.5 31.7 2.5 624.5 1.8 5.6 15.0 33.5 7.9 639.5 11.3 65.2 89.8 NDd ND ND 1.6 7.0 7.7 ND ND ND 17.9 140.4 164.2 5.8 5.9 11.5 23.7 146.3 175.7 23.0 163.5 193.4 3.9 5.9 28.7 26.9 169.4 222.1 69.4 2.4 71.8

Assumed aflatoxin B1 concentration contributes about 60% of total aflatoxins detected in samples. Liver cancer cases per 100,000 population per year. Mean is less than minimum since including many negative samples. Not detected.

about 179 g/day, followed by Giay (171 g/day), Tay (167 g/day), Dao (164 g/day), and Xa pho (155 g/day). Hence, similar to Kinh adults, Kinh children were exposured to a high level of AFB1 from rice (114.4 ng/kg bw/day). The lowest exposure to AFB1 was found in Xa pho children with a level of 8.2 ng/kg bw/day (Table 7). The mean AFB1 exposure amongst all children was 33.7 ng/kg bw/day. The FB1 intake level of children from rice consumption was much higher than in adults with a mean of 1019 ng/day; ranging from 34.8 to 9455.6 ng/day (Table 7). Since AFB1 is the most abundant of the aflatoxins analogues and also the most biologically active, the European Food Safety

Authority has approved to use data on AFB1 exposure for health risk assessments (EFSA, 2007). The assessment of health risk associated with consumption of rice and maize contaminated with aflatoxins showed that adults (4.3 cases per 100,000 individuals per year) and children (7.8 cases per 100,000 individuals per year) of the Kinh ethnic group experienced the highest risk of liver cancer in comparison with the other ethnic groups. In comparison, the mean excess cancer risk for all ethnic groups was calculated as 1.5 cases for adults and 2.3 cases for children per 100,000 individuals per year (Tables 6 and 7). Only few data on liver cancer risk estimation are published from Asian countries with rice as the main staple

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199

Table 7 Mycotoxins exposure assessment and risk characterization by crop and ethnic group e assumed child body weight is 11.3 kg; population risk is calculated on the assumption of HbsAg þ prevalence 20% (WHO 1998) and mean level of the mycotoxins contamination of all samples. Ethnic

Mean of consumption (g/day)

Dao

164.1

Giay

171.1

Kinh

178.9

Tay

167.4

Xa pho

154.6

Total

166

a b c d

Aflatoxin B1a

Fumonisins

Contamination level (ng/g)

Exposure (ng/kg bw/day)

Riskb

Contamination level (ng/g)

Exposure (ng/kg bw/day)

Mean Minc Max Mean Min Max Meanc Min Max Meanc Min Max Meanc Min Max Mean

27.2 166.1 185.0 9.6 18.7 20.1 114.4 126.0 738.6 16.9 28.5 77.1 8.2 16.9 25.3 33.7

1.9 11.3 12.6 0.7 1.3 1.4 7.8 8.6 50.2 1.1 1.9 5.2 0.6 1.1 1.7 2.3

Meanc Min Max Mean Min Max Mean Min Max Meanc Min Max Meanc Min Max Mean

446.4 2647.4 5205.0 479.6 34.8 9455.6 e e e 265.5 2079.2 2432.8 314.9 2236.2 2646.3 1019.1

1.9 11.4 12.7 0.6 1.2 1.3 7.2 8.0 46.7 1.1 1.9 5.2 0.6 1.2 1.8 2.3

30.7 182.3 358.4 31.7 2.3 624.5 NDd ND ND 17.9 140.4 164.2 23.0 163.5 193.4 69.4

Assumed aflatoxin B1 concentration contributes about 60% of total aflatoxins detected in samples. Liver cancer cases per 100,000 population per year. Mean is less than minimum since including many negative samples. Not detected.

food. An excess risk of 0.021 cases per 100,000 individuals of population per year was observed in Japan. There is a strong observed synergy between AFB1 exposure and being a hepatitis B surface antigen-positive individual, i.e. aflatoxin is about 30 times more potent as a liver carcinogen in HBV infected persons (WHO, 1998; Williams et al., 2004). In Vietnam, hepatitis B virus affect about 25% of the population (WHO, 1998) and bio-markers of current and previous infection with the virus (HBsAgþ, anti-HBcþ, or anti-HBsþ) have been observed in 36.4% children and 79.2% adults (David et al., 2003). Consequently, the contamination of cereals with aflatoxins is a serious public health concern, as emphasized by the fact that primary liver cancer is one of the most frequent disease in Vietnam (Thanh Ha et al., 2004, p. 42). In contrast, fumonisin intake from rice and maize are 536 and 1019 ng/kg bw/day equal to only 26.8% and 51.0% of the TDI for adults and children, respectively. Furthermore, the dietary intake of FB1 was found to be lower than TDI level for all ethnic groups. Climate change greatly influence environmental factors such as temperature, humidity, insect attack and drought which are factors associated with fungal growth and mycotoxins production (United Nations, 2011). Prolonged hot and dry periods may cause disease outbreaks associated with Aspergilus spp. and aflatoxins while Fusarium spp. and fumonisins have been associated with both dry weather during grain fill and late seasonal rains (Miraglia et al., 2009). Food safety hazards associated with fungi and their mycotoxins are therefore specifically predicted to become more prevalent with foreseen climate chances. Acknowledgment The study was supported by the Danish International Development Assistance (Danida) through the project SANIVAT “Water supply, sanitation, hygiene promotion and health in Vietnam” (www.sanivat.com.vn; 104.DAN.8.L.711) and the WHO Vietnam (project VNM/09/FOS/005250). Special appreciation goes to the people in Ta Phoi and Hop Thanh communes who agreed to take part of this research. The authors thank Nguyen Lan Phuong, Nguyen Thi Anh Tuyet and Ha Thi Tuong Van for their support in the field and laboratory.

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