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Co-occurrence of aflatoxins and ochratoxin A in dried fruits in Iran: Dietary exposure risk assessment
Accepted Manuscript Co-occurrence of aflatoxins and ochratoxin A in dried fruits in Iran: Dietary exposure risk assessment Ali Heshmati, Tahereh Zohrevand, Amin Mousavi Khaneghah, Amir Sasan Mozaffari Nejad, Anderson S. Sant’Ana PII:
S0278-6915(17)30280-6
DOI:
10.1016/j.fct.2017.05.046
Reference:
FCT 9081
To appear in:
Food and Chemical Toxicology
Received Date: 25 April 2017 Revised Date:
20 May 2017
Accepted Date: 22 May 2017
Please cite this article as: Heshmati, A., Zohrevand, T., Khaneghah, A.M., Mozaffari Nejad, A.S., S. Sant’Ana, A., Co-occurrence of aflatoxins and ochratoxin A in dried fruits in Iran: Dietary exposure risk assessment, Food and Chemical Toxicology (2017), doi: 10.1016/j.fct.2017.05.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Co-occurrence of aflatoxins and ochratoxin A in dried fruits in Iran: dietary exposure risk assessment
Nejad a, Anderson S. Sant’Ana d,*
Nutrition Health Research Center, Hamadan University of Medical Sciences, Hamadan, Iran Department of Nutrition, School of Medicine, Hamadan University of Medical Sciences,
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a
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Ali Heshmati a,b*, Tahereh Zohrevand c, Amin Mousavi Khaneghah d,**, Amir Sasan Mozaffari
Hamadan, Iran c
Laboratory of Food and Drug Analysis, Hamadan University of Medical Sciences, Hamadan, Iran
Department of Food Science, Faculty of Food Engineering, State University of Campinas
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d
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(UNICAMP), Monteiro Lobato, 80. Caixa Postal: 6121. CEP: 13083-862. Campinas. São Paulo.
Corresponding authors: *Anderson S. Sant’Ana. E-mail address: [email protected] **Amin Mousavi Khaneghah. E-mail address: [email protected] 1
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ABSTRACT In this study, the contamination levels of aflatoxins (AFs) and ochratoxin A (OTA) in 88 collected
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samples from Iran's market including dried mulberry, date, fig, and apricot were evaluated. The margin of exposure (MOE) was estimated to assess the risk of dietary intake of aflatoxin B1 (AFB1) and OTA. The incidence of AFB1 in dried mulberry, date, fig and apricot samples was 45.5, 40.9, 59.1, and 81.8%, respectively. Although the mean total AFs content in contaminated
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samples of date (2.61 µg/kg), fig (3.43 µg/kg) and apricot (2.91 µg/kg) was lower than the
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maximum limit set in the European Union (EU) (4 µg/kg), dried mulberry samples showed a higher contamination level (4.12 µg/kg). The co-occurrence of OTA and AFs were noted in 4 (18.9%), 2 (9.1%), 4 (18.2%), and 10 (45.5%) in the dried mulberry, date, fig and apricot samples, respectively. Based on the calculated MOE, the dietary exposure to AFs through the consumption of dried fruit in Iran poses a potential risk to consumer health. OTA was detected in
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45.45%, 22.72%, 45.45%, and 50% of mulberry, date, fig and apricot samples, respectively. However, OTA levels in all types of dried fruit were below recommended level in EU regulation
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(10 µg/kg) and MOE >10000, representing no toxicological concerns for consumers.
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Keywords: Aflatoxins, Ochratoxin A, Risk assessment, Dried fruit, HPLC, Dietary exposure
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1. Introduction Mycotoxins as a large group of low molecular weight compounds (below 1000 Da) can be considered as the secondary metabolites generated by some of the filamentous moulds, including
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genera of Aspergillus, Fusarium, Penicillium and Alternaria (Al-Taher, et al., 2017). The presence of mycotoxins in food products is a grave threat to human health. Therefore, the investigation of their occurrence in foods systems is of extreme importance (Campagnollo et al.
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2016; Oteiza, et al., 2017).
Among mycotoxins, ochratoxin A (OTA) and aflatoxins (AFs) can be accounted as the most
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frequently reported mycotoxin in various agricultural products and human foods (Azaiez et al., 2015; Eslami et al., 2015; Kabak, 2016; Mashak et al., 2016; Toma and Rajab, 2014; Trucksess and Scott, 2008 Rastegar et al., 2017). AFs are compounds with potent carcinogenic, mutagenic, estrogenic, tremorogenic, teratogenic and immunosuppressive effects on the health of animals
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and humans (Huong et al., 2016). The major kinds of AFs are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2). Among them, AFB1 is the most important once it has carcinogenic and genotoxic effects on humans its high potent carcinogenic and
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genotoxic effects on human (Tajik et al., 2016; Van de Perre et al., 2015). Hence, it is classified by International Agency for Research on Cancer (IARC) as a carcinogen of group 1(IARC,
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2012). OTA is another mycotoxin which is mainly produced by species of Penicillium (P. verrucosum and P. nordicum) and Aspergillus (A. carbonarius and A. ochraceus). Penicillium species are more often connected to temperate climates, while Aspergillus species are found in warm climates (Clark and Snedeker, 2006). According to the IARC, OTA has been classified into group 2B, "possible carcinogens in humans" (IARC, 2012).
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Risk assessment is a four-step process, which determines the possible adverse effects of a chemical compound such as mycotoxin on an organism. Exposure assessment is one of the essential elements of risk assessment and takes into consideration the occurrence and
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concentrations of mycotoxin in the diet, the consumption patterns as well as the intake value correlated to foods containing mycotoxin. The estimated values from dietary exposure assessments could be determined to decrease risks for humans (Huong et al., 2016). For some
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mycotoxins, a tolerable daily intake (TDI) or a maximum tolerable daily intake (PMTDI) have been established, while for other mycotoxins, a weekly intake (TWI) or a provisional tolerable
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weekly intake (PTWI) are used. TDI/TWI refers to the level that can be taken daily/weekly over a lifetime without a considerable health risk. There is a risk if the calculated daily/weekly intake is over the recommended TDI/TWI values. However, in the case of OTA, the PTWI of 100 and 120 ng/kg bw/day, were recommended by the Joint FAO/WHO Expert Committee on Food Additives
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the (JECFA) and the European Food Safety Authority (EFSA), respectively (EFSA, 2006; JECFA, 2007). No TDI has been established for genotoxic carcinogens such as AFB1 (ESFA, 2006). One of the common methods of assessing the carcinogenic and genotoxic mycotoxins is
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the estimation of the margin of exposure (MOE) (EFSA, 2006; JECFA, 2007). The MOE can be obtained by dividing the dose, which causes a defined impact (benchmark dose, BMD) in
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animals, by the approximated dietary human ingestion of AFB1. A MOE <10,000 could cause a high risk to public health. OTA has been classified as a non-genotoxic carcinogenic by the JECFA (JECFA, 2007). Nonetheless, the MOE for OTA was calculated for the lowest BMDL10 (benchmark dose lower confidence limit of 10%) (21 µg/kg BW/day) which could increases the renal cancer risk (Huong et al., 2016) Dried fruits could offer high nutritional value in the human diet (Chang et al., 2016). However, these products also provide a suitable medium for mould growth and further mycotoxin 4
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contamination. The European Union set maximum permitted levels of AFB1, total AFs, and OTA in dried fruit at 2, 4 and 10 µg/kg, respectively (CET, 2006 a). Also, the Institute of Standards and Industrial Research of Iran (ISIRI) has regulated the maximal accepted levels of AFB1, total AFs
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and OTA in some dried fruits at 5, 15 and 10 µg/kg, respectively (ISIRI, 2012). There are various reports on the occurrence of mycotoxins such as AFs and OTA in dried fruit around the world (Kabak, 2016; Nayebpoor et al., 2013; Ozer et al., 2012; Scott and Trucksess, 2009; Trucksess
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and Scott, 2008). Although some studies have been conducted to evaluate mycotoxin contamination in dried fruit in Iran (Heshmati and Mozaffari Nejad, 2015; Janati et al., 2012;
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Rahimi and Shakerian, 2013), no dietary exposure assessment has been carried out. In this context, the current study was undertaken to determine the occurrence and co-occurrence of AFs and OTA in dried mulberries, figs, dates, and apricots samples as well as to assess the dietary
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exposure to these mycotoxins.
2. Materials and methods 2.1. Materials
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The obtained analytical grade crystalline forms of AFB1, AFB2, AFG1, and AFG2 from SigmaAldrich (St. Louis, MO, US) were used to prepare standard solutions. The acetonitrile, methanol,
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phosphate buffer saline (PBS), other solvents and chemicals of analytical grade were obtained from Merck (Darmstadt, Germany). For the clean-up step, immunoaffinity columns (IAC) of aflatoxins (Puri-Fast AFLA BG IAC) and ochratoxins (Puri-Fast OTA IAC) from Libios (Pontcharra-Sur-Turdine, France) were utilized.
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2.2. Sample collection Eighty-eight dried fig, mulberry, date and apricot samples (22 samples of each) were collected
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randomly from local markets in Hamadan city, Iran, during 2015–2016. The sampling and preparation procedures for the analysis were conducted according to the described procedure by the Commission of the European Communities No. 401/2006 (CET, 2006b). The samples were poured into polyethylene bags and were stored at -18 °C until the experiment day (maximum 2
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wk).
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2.3. Mycotoxin analysis
Dried fruit samples were thoroughly ground. Five g sodium chloride and 300 mL of water– methanol (8:2 v/v) were added to 50 g of dried ground fig, mulberry, date, and apricot samples including those either spiked or not spiked with a given volume of a stock solution of aflatoxins.
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Then they were blended in a mixer at high speed (2000 RPM) for 5 min, to create homogeneous
slurries suspension and they were passed through filter paper (Whatman No. 4). Then, the extract (10 mL) was diluted with 60 mL of phosphate-buffered saline (PBS) solution and passed through
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IAC. Before clean-up, the column was left at room temperature to stabilize and further conditioned with 10 mL of PBS, at flow rate of approximately 1 mL/min under gravity of
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Methanol (1.5 mL, flow rate of 1mL/min) was utilized to elute aflatoxins from IAC. The methanol was collected in a vial, to be analyzed for aflatoxins through HPLC (Janati et al., 2012).The sample preparation and IAC extraction procedure used in the current study to determine OTA levels had been previously optimized and validated in and were reported in our previous study (Heshmati and Mozaffari Nejad, 2015). 2.4. HPLC analysis 6
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The analysis of AFs and OTA was carried out by a Waters reverse-phase HPLC instrument (Milford, MA, USA) equipped with a binary pump, autosampler, fluorescence detector, and an RP C18 analytical column (250 mm × 4.6 mm, i.d., 5 µm) and a column oven set at 25 °C. The
rate
of
1 mL/min
for
aflatoxins,
and
acetonitrile:
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used mobile phases in isocratic elution were water/methanol/acetonitrile (6:3:2, v/v/v) at a flow water::
methanol:
acetic
acid
acetonitrile/water/methanol/acetic acid (39:30:30:1, v/v/v/v) with a flow rate of 1 mL/min for
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OTA. The wavelengths of excitation and detector emission for AFs and OTA were 365/435 nm
2.6. Validation of the analytical method
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and 333/477 nm, respectively. Volume of injection was 20 µL.
The accuracy, precision (intra-day accuracy and inter-day accuracy), linearity, limit of detection (LOD), and limit of quantification (LOQ) were assessed to validate the used analytical
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method. The accuracy of AFs and OTA measurement was examined by determining their recovery. Samples of dried fig, mulberry, date, and apricot were spiked with concentrations of 2, 4 or 6 µg kg–1 of AFB1, AFB2, AFG1 and AFG2 as well as 4, 8 or 12 µg kg–1 of OTA. Recovery
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was calculated by comparing the mycotoxin concentration in the spiked samples with concentrations in the reference standards (AFs and OTA solutions).
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Analytical precision was determined via triplicate analysis of the spiked samples containing two different levels of AFB1 and AFG1 (2 and 4 µg kg–1), AFB2 and AFG2 (1.5 and µg kg–1), and OTA (4 and 8 µg kg–1); this analysis took place on the same day (i.e., intra-day precision) and on three consecutive days (inter-day precision). The relative standard deviation (%) of replicate measurements also was calculated. To construct an external standard calibration by which to quantify mycotoxins in dried fruit samples, working standard solution of AFs and OTA were
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prepared at concentrations of 0.5-20 ng/Ml and 1-25 ng/Ml respectively. The limit of detection (LOD) was set as three times the signal to blank noise, and the limit of quantification (LOQ) was set at 10
2.7. AFB1 and OTA risk and exposure assessment
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times the signal of blank noise.
A deterministic approach was used to calculate dietary exposure to AFB1 and OTA.
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Aflatoxin intake was determined by considering upper, medium, and lower-bound concentrations of mycotoxin in addition to the mean of the collected data regarding consumption of dried fruit
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by using the of following equation:
Daily dietary exposure of AFB1 or OTA (ng/kg bw) = (dried fruit per-capita (g/day) × AFB1 or OTA concentration (µg/kg))/bw (kg)
In Iran, the per-capita consumption of dried figs, mulberries, dates, and apricots is
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thought to be 1.9, 2, 10 and 2 g/day, respectively (Iran ministry of Health and Medical Education, 2009). To estimate the daily dietary exposure, the average body weight of 70 kg for the Iranian adult population was assumed. In order to facilitate the estimation of the lower, medium, and
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upper bound, not detected (ND) values were replaced with zero; LOD/2, LOD, and unquantified
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values were replaced with LOD, LOQ/2, and LOQ, respectively (Huong et al., 2016) The MOEs were calculated only in the case of AFB1 and OTA in following the
recommendations of EFSA (2006) and JEFCA (2007). In our study and in most cases, the BMDL10 is used; this is the lowest dose with a 95% certainty of not leading to a 10% increase in cancer incidence in rodents. In the current study, the MOE was calculated using a BMDL10 value of 170 ng/kg bw/day, as recommended by the EFSA for AFB1 (EFSA, 2007). In addition, the estimated intake of OTA was compared to PTWI, to evaluate potential risks to consumer health 8
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2.8. Statistical analysis The results were reported as mean ± standard deviation values. Data were analysed by
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using SPSS (version 16:0) software (SPSS Inc.; Chicago, IL, USA). To compare the mean concentrations of AFB1, total AFs, and OTA found in the samples with the maximum levels
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allowed under per ISIRI and EU legislation, a one-sample t-test was used.
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3. Result and discussion 3.1. Validation of AFs and OTA analytical method
HPLC validation parameters such as linearity, LOD and LOQ are presented in Table 1. The recovery of AFB1, AFG1, AFB2, AFG2, and OTA are shown in Table 2. The performance of used
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method can be justified with the suitable linear responses with a high coefficient of determination for the analysed AFs and OTA as well as quite good obtained recoveries (AOAC International, 2002; Codex Alimentarius, 1995; Wei, et al., 2013)
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3.2. Incidence of AFs and OTA in dried fruits
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3.2.1. Dried mulberries
AFB1, AFB2, AFG1, and AFG2 were detected in 10 (45.5%), 7 (31.8%), 2 (9.1%) and 4 (18.18%) of 22 dried mulberry samples, respectively. AFB1 was the most abundant aflatoxin and its level varied from 0.35 to 8.4 µg/kg. Moreover, the concentration of AFB1 in four samples exceeded the limit of European regulations (2 µg/kg). Additionally, the levels of AFB1 in two samples were higher than the recommended level by Iranian standard (5 µg/kg), although the total AFs content was within the allowed limit in Iran (15 µg/kg). In the current study, among 9
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several types of dried fruit that were evaluated, mulberries were found to contain the highest mean value of AFB1 and total AFs.
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The incidence of OTA in mulberry samples was 45.5% (10 of 22 samples), with a mean value of 1.75 µg/kg. The OTA values in all samples were lower than established limits in Iran and the European Union (10 µg/kg).
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There were a few reports regarding the occurrence of AFs and OTA in dried mulberry. A higher level of incidence and mean total AFs contents (45.5% and 4.1 µg/kg, respectively) were
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recorded in the current study in comparison with previous research. For instance, Luttfullah and Hussain (2011) reported that four (26%) out of 15 dried mulberry samples contained 1–3.5 µg/kg of AFs (mean value, 2.22 µg/kg). Besides, Toma and Rajab (2014) reported a mean total AFs of 14.3 ug/kg for total AFs in dried mulberry samples collected from various cities in Iraq; a value
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3.5 times higher than in current investigation. According to Kaya and Tosun (2013), the total levels of aflatoxin, OTA and fumonisn in organic mulberry was 9.45, 12.47 and 691.78 µg/kg, respectively. The Mulberry tissues and structures have high sugar levels and are prone to insect
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attack, conditions that favor mould growth and further mycotoxin production(Kaya and Tosun,
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2013).
3.2.2. Dried dates
A relatively high incidence of AFs contamination was observed in dried dates. Nine (40.9%) of 22 date samples contained AFs, with a mean value of total AFs 2.61 µg/kg. Among them, two date samples were contaminated with levels higher than EU limit for AFB1 (2 µg/kg) and total AFs (4 µg/kg).Although the presence of OTA was recorded in five out of 22 date 10
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samples, the mean OTA value (1.21 µg/kg) was below that recommended by the European Union (10 µg/kg). Also, none of them contained a higher level of contamination than the recommended
mould growth and mycotoxin production (Ozer et al., 2012).
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level. Relatively high temperatures and humidity during date maturation might have resulted in
Two out of twenty date samples analysed by Alghalibi and Shater (2004), were contaminated with AFB1. The prevalence of aflatoxins B1, B2, G1 and G2 and sterigmatocystin in
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fresh date fruit (Phoenix dactylifera) during storage conditions (98% relative humidity and 30 °C
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for 14 days) were studied (Shenasi et al., 2002). Their results showed that, neither aflatoxins and nor their precusor, sterigmatocystin, was detected after adverse storage for 14 days. In another investigation Gherbawy et al. (2012), the presence of aflatoxins and ochratoxin A producer fungis in retail date fruits distributed in different markets at Taif, Saudi Arabia was evaluated. Based on the reported results by them; seven out of subjected eighty isolates of A. flavus for detecting their
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aflatoxigenic potentials, were toxigenic. Moreover, among examined thirty-six isolates of Aspergillus niger for ochratoxin production, only nine isolated were able to produce ochratoxin. Also, in previously conducted study regarding the occurrence of OTA in dates in Brazil, it was
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reported a mean value (<0.13 µg/kg) lower than the one found in current study (Beatriz et al.,
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2006). In another investigation done by Azaiez et al. (2015), 16 types of mycotoxins were detected in 228 samples of dates and dried fruits from Tunisian and the Spanish market. In this case, mycotoxins that occurred most frequently were enniatin B (EnnB) (54%), enniatin A1 (36%), AFs (23%), and OTA (22%). In 18 of 48 Tunisian date samples, OTA level ranged from 0.57 to 3.3 µg/kg (mean value, 1.3 µg/kg). The mean total AFs value in the aforementioned study was higher than in current study. The mean value of total AFs of AFB2, AFG1, and AFG2 in the Tunisian date samples were respectively 1.14, 1.4, and 1.7 µg/kg; meanwhile, AFB1 was not
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detected in any of the samples (Azaiez et al., 2015). The elimination of deteriorated dates prior to drying and final packaging might lead to reduced mycotoxin levels (Ozer et al., 2012).
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3.2.3. Dried figs As shown in Tables 3 and 4, AFB1, AFB2, AFG1, AFG2 and OTA were found in 13 (59.1%), 8 (36.4%), 5 (22.7%), 4 (18.9%) and 10 (45.5%) out of 22 dried fig samples, respectively, with
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average concentration of 2.65, 0.74, 0.36, 0.32 and 3.03 µg/kg, respectively. Only 3 (13.6%) out of 22 dried fig samples were contaminated with levels of AFB1 above the Iranian standard (5
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µg/kg) and 7 (31.2%) samples contained AFB1 at higher levels than the respective regulation of European Union (2 µg/kg). The total AFs in 5 (22.7%) of the fig samples exceeded the accepted maximum level of European legislation (4 µg/kg). The OTA content of one sample (12.16 µg/kg) was greater than the recommended level accepted in the European Union (10 µg/kg).
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The potential risk of contamination by mycotoxins in figs can be correlated to their susceptible nature to growth of mycotoxingenic fungi. The growth of mycotoxin-producing moulds including Aspergillus, Fusarium, and Penicillium in collected fig samples in Turkey was
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recently reported (Heperkan et al., 2012). Ochratoxin-producing black Aspergilli is highly resistant to sunlight and UV light; therefore, it has been recognized as the dominant mould in
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some of dried fruits, such as dried figs(Karbancıoğlu-Güler and Heperkan, 2008).In a study done by Rodrigues et al (2012), aflatoxigenic fungi in almond as dominant mold was domenstrated. The incidence of total AFs in fig samples of Pakistan (30%), with a mean of 0.54 µg/kg, was reported by Asghar et al. (2017). In current investigation the incidence and the mean value (3.3 µg/kg) of total AFs in dried fig samples was higher. Nonetheless, our mean value for AFs was lower than the mean (3.8 µg/kg) reported by Kabak (2016). In present study, it was also observed
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that AFB1 was the predominant type of aflatoxin, contrary to what was seen in Turkey (Kabak, 2016) and Morocco (Juan et al., 2008), where AFG1 was the dominant form in samples. The highest incidence of OTA in fig samples (95%, 65%, and 48%,) were reported by Iamanaka et al.
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(2005) in Brazil; Karbancıoğlu-Güler and Heperkan in Turkey (2008) and Zinedine et al. (2007) in Morocco, respectively.
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3.2.4. Dried apricots
Among the assessed dried fruits, the most frequent incidence of AFB1 was found in
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apricots, as 18 (81.8%) out of the 22 samples were contaminated with levels that ranged between 0.39 and to 7.1 µg/kg with a mean of 2.43 µg/kg. However, the AFB1 concentration in eight samples (36.4%) were over European regulation (2 µg/kg), the reported amounts for the total AFs and AFB1 were acceptable considering the Iranian national standard (5 µg/kg for AFB1 and 15
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µg/kg for total AFs). In addition, apricots had the highest frequency of OTA contamination (50%), with a range of 0.75–5.5 µg/kg and mean of 1.76 µg/kg. Nonetheless, OTA levels in the samples were lower than the permissible concentration of OTA (10 µg/kg). Moreover, no OTA
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contamination in Turkish apricot samples by Aksoy et al (1995) reported. In an investigation done by Janati et al. (2012), AFB1, AFB2, AFG1 and OTA was found in 30, 3.3, 3.3 and 3.3% of apricot
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specimens, with averages of 0.88, 0.32, 0.20 and 2.83 µg/kg, respectively; but, AFG2 was not detected. In mentioned study, all the measured AFs levels were lower than reported concentrations in present research. Although the OTA value (2.83 µg/kg) was higher than in current study (1.76 µg/kg), only one sample (6.7%) out of 15 dried apricot samples contained OTA (2.8 µg/kg) (Rahimi and Shakerian, 2013), OTA was also found in one of 20 dried apricot samples with a value of 0.97 µg/kg (Bircan 2009). The utilization of sulfur dioxide as a preservative substance in some dried fruits, such as apricots, inhibits fungal growth; this might be 13
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a reason for the lower incidence of OTA in the samples analyzed in previous studies. Iamanaka et al., (2005) reported that none of the 14 dried apricot samples analyzed in Brazil were contaminated with OTA. In contrast, in previous studies, our results demonstrated that the
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examined apricots samples had the highest potential for AFs and OTA contamination. Asghar et al. (2017) analyzed 65 dried apricots for total AFs. Twenty-one (32%) of the samples were contaminated, with a mean of 1.02 µg/kg and a range of 0.31–11.11 µg/kg.
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The differences found between the mycotoxin levels of dried fruits evaluated in previous
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studies an in current investigation can be related to several aspects. For instance, these differences may be due to variation in sample sizes, to different sampling methods as well as analytical techniques (HPLC, LC and ELISA), climate and seasonal changes, and to the used agricultural practices (Rahimi and Shakerian, 2013; Trucksess and Scott, 2008).
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3.3. Co-occurrence of AFs and OTA
AFs and OTA were found together in 4 (18.2%), 2 (9.1%), 4 (18.2%) and 10 (45.5%) of the dried mulberry, date, fig and apricot samples, respectively. The co-occurrence of OTA with AFs
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was reported by Bircan (2009) in 2 out of the seven dried fig samples analyzed in Turkey. In another study from Turkey, 2 out of 53 in 2003 and 2 of 41 dried figs samples in 2004 were
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contaminated with both AFB1 and OTA (Senyuva et al., 2005), showing lower co-occurrence of mycotoxin than in the present study. Janati et al., (2012) reported the contamination of apricots and prunes with AFs and OTA independently; but, the number of samples that contained both was not mentioned. Moreover, Co-occurrence of AFs and OTA was reported in 69% of dried fruits collected from Tunisia (Ghali et al., 2008). The presence of AFs and OTA in dried fruits probably also reflects the presence of various species of Aspergillus moulds in these products.
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Sun drying is a traditional technique still applied to dry fruits in Iran. During this process, favourable conditions for mould growth and mycotoxin production are provided. However, good agricultural practices, such as the use of new drying technologies and the removal of damaged
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fruits during processing may decrease mycotoxin levels in dried fruits such as figs. Other measures that will contribute to reduction of fungal contamination and presence of mycotoxins include the reduction of mould microflora using chemicals such as sulphur dioxide, sodium
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metabisulphite and potassium metabisulphite, the application of fungicide during storage and
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gamma radiation (Scott and Trucksess, 2009; Trucksess and Scott, 2008). 3.4. Risk assessment
The estimated average exposure to AFB1 through the consumption of dried mulberries, dates, figs and apricots was found to be 0.04, 0.12, 0.04 and 0.06 ng/kg bw/day, respectively
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(Table 5). The MOE, which was determined using the dietary intake levels of each dried fruit and the BMDL10 of 170 ng/kg bw/day, was 4,250, 1,417, 4,250 and 2,833 for the dried mulberry, date, fig and apricot samples, respectively. A small MOE indicates a higher risk than a large
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MOE. As the obtained MOE values were less than 10,000, it can be said that the consumption of dried fruits in Iran puts consumers at an exposure risk to AFB1. The lowest MOE found in dried
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dates suggests a high daily intake of AFB1. The MOE to OTA is shown in Table 5. The MOE to OTA was found > 10,000.
In the current study, the upper-bound which estimates of the intake of OTA through
mulberries, dates, dried figs, and apricots were 0.03, 0.08, 0.06 and 0.03% of PTWI (100 ng/kg bw/day) (EFSA,2006). Therefore, the intake of OTA through dried fruits does not exceed that of the PTWI.
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Little information regarding the MOE in dried figs, mulberries, dates and apricots was reported previously. However, MOE has been calculated and communicated in other dried fruits and foods in past studies. For example, the MOE to AFB1 through peanut consumption in China was
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reported to be 1,273 (Ding et al., 2012). Besides, the estimated MOE of AFB1 through the intake
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of bakery products and pasta was reported to be 24.6 (Bol et al., 2016).
4. Conclusion
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The occurrence of AFs and OTA of consumed dried fruits in Iran was evaluated. The results showed that the mean total AFs level in all contaminated samples except the dried mulberries was lower than that allowed according to European legislation (4 µg/kg). No samples had OTA contamination above the allowable limit (10 µg/kg). The co-occurrence of AFs and OTA were
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noted in 4 (18.2%), 2 (9.1%), 4 (18.2%), and 10 (45.5%) of the dried samples, respectively. In current study, the derived margin of exposure (MOE) values calculated for AFB1 (range 1,417 to 4,250) and OTA (> 10,000). Since the MOE values of AFB1 were found to be lower than 10,000,
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a potential risk to consumer health exists through the dietary consumption of dried fruits and subsequent exposure to AFs. However, further evaluation through a total diet study (TDS) is
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necessary to ensure a better adjustment; thus, more studies should be conducted to determine Iranian consumers' dietary exposure to AFs, OTA, and other mycotoxins. Although the level of AFB1 and OTA were not so high, efficient methods to provide the safeguard for the consumers against their toxic effects and furthermore to keep the public health should be approached. In this context, the employment of good agricultural practices (GAP) with the aim of reducing fungal
16
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growth in all the processing steps (in the field and during storage) as well as the general
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improvement of quality of all the process are crucial.
Acknowledgements
The authors are thankful to Vice-Chancellor of Research and Technology of Hamadan University
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of Medical Sciences and Health services for the study protocol approval and financial support. Amin Mousavi Khaneghah and Anderson Sant’Ana like to thank the support of CNPq-TWAS
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Postgraduate Fellowship (Grant # 3240274290), and "Conselho Nacional de Desenvolvimento Científico e Tecnologico"(CNPq) (Grant #302763/2014-7), respectively.
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Table 1: Linearity range, limit of detection (LOD) and limit of quantification (LOQ) for different mycotoxins analyzed in dried fruitsa.
0.992 0.997 0.991 0.998 0.996
LOD LOQ LOD LOQ LOD 0.09 0.30 0.06 0.20 0.08 0.07 0.23 0.05 0.17 0.08 0.08 0.27 0.08 0.27 0.05 0.06 0.20 0.02 0.07 0.08 0.07 0.23 0.15 0.50 0.26
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LOD and LOQ values are expressed in µg/kg.
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Date
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Y=12564X+238 Y=10890X+2345 Y=25645X+1390 Y=20123X+1009 Y=16007X+5678
Mulberry 2
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Equation of calibration curve
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Range of Mycotoxin linearity (ng/mL) AFB1 1-22 AFB2 0.8-22 AFG1 0.5-20 AFG2 1-18 OTA 2-50
LOQ 0.27 0.27 0.17 0.27 0.87
Apricot LOD LOQ 0.07 0.23 0.08 0.27 0.06 0.20 0.08 0.27 0.18 0.60
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Table 2: The recovery percentage of mycotoxins in dried fruits.
Date
Spiked level (µg kg -1) 2 4 6
recovery percentage 82.4 87.9 91.0
RSD 1.6 1.0 1.2
recovery percentage 82.8 88.7 91.4
RSD 1.6 1.1 1.2
AFB2
2 4 6
84.9 87.1 91.3
0.9 1.4 1.7
85.4 87.5 91.7
AFG1
2 4 6
79.0 80.7 87.4
0.5 1.3 0.7
79.4 81.2 87.8
AFG2
2 4 6
76.5 74.4 78.5
1.9 0.7 2.0
OTA
4 8 12
84.0 88.4 92.3
1.4 0.6 1.7
RSD 1.6 1.0 1.3
RSD 1.5 0.9 1.2
1.1 1.3 1.8
85.5 87.6 91.8
1.1 1.3 1.6
86.3 88.3 92.5
1.1 1.4 1.6
0.6 1.3 0.8
79.6 81.3 87.9
0.5 1.2 0.9
80.2 81.9 88.6
0.5 1.3 0.7
76.9 74.7 78.9
2.0 0.7 1.8
77.1 74.9 79.0
2.1 0.8 1.9
77.7 75.6 79.7
1.9 0.7 2.0
83.8 88.1 92.1
1.2 0.8 1.6
84.6 88.9 92.9
1.4 0.7 1.7
84.6 88.9 92.9
1.4 0.7 1.7
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recovery percentage 82.9 88.5 91.5
Apricot recovery percentage 83.6 89.1 92.2
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Mycotoxin AFB1
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Commodity Fig
Mulberry
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Table 3: Aflatoxin contamination in dried fruitsa. Aflatoxin type
Contamination status
Commodity Date
Fig
Apricot
10(45.5%) 6(27.3%) 2(9.1%) 2(9.1%) 3.0±2.7 0.4-8.4
9(40.9%) 7(31.8) 1(4.5) 1(4.5) 2.1±1.8 0.6-6
13(59.1%) 6(27.37%) 4(18.2%) 3(13.6%) 2.6±2.5 0.3-7.0
18(81.8%) 10(45.5%) 5(22.72%) 3(13.63%) 2.4±2.2 0.4-7.1
AFB2
No. of contaminated samples Mean±SD Range*
7 (31.8%) 1.21±1.1 0.3-2.5
5(22.7%) 0.7±0.6 0.4-0.7
8(36.4%) 0.7±0.6 0.2-2.1
12(54.5%) 0.6± 0.2 0.3-0.8
AFG1
No. of contaminated samples Mean±SD Range*
2 (9.1%) 0.6 ±0.4 0.3-0.8
3(13.6 %) 0.3±0.1 0.29-0.41
5(22.7 %) 0.4±0.1 0. 3-0.4
4(18.2%) 0.3±0.0 0.3-0.4
AFG2
No. of contaminated samples Mean±SD Range*
4 (18.2%) 0.3±0.0 0.3-0.4
1(4.5%) 0.3 -
4(18.2%) 0.3±0.0 0.3-0.4
3(13.6%) 0.30 ±0.0 0.3-0.3
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AFB1
Contamination level( µg/kg)
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Mulberry No. of contaminated samples 0.08-2 2-5 >5 Mean*±SD Range*
No. of contaminated samples 10 (45.4%) 9 (40.9%) 13 (59.1%) Contamination <4 7 (31.8%) 7 (31.8%) 8 (36.4%) Total AFs level( µg/kg) * 4-15 3 (9.1%) 2 (9.1%) 5 (22.7%) >15 Mean±SD 4.1±3.9 2.6±2.3 3.3 ±3.2 Range* 0.6-11.8 0.9-8.1 0.4-9.2 a Calculation of contamination mean and range was based on positive samples. All values are expressed in µg/kg
18 (81.8%) 13 (59.1%) 5 (22.7%) 2.9 ±2.6 0.4-8.5
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Table 4: Ochratoxin A (OTA) contamination in assayed dried fruitsa.
Commodity
10(45.5%)
Apricot 11(50%)
0.26-5
10(45. 5%)
5 (22.7%)
8(36.4%)
10(45.5%)
Contamination level( µg/kg)
5-10
0
0
1(4.54%)
1(4.5%)
>10
0
0
1(4.54%)
-
1.2±0.6
3.03±3.5
1.7±1. 5
Mean ± SD
1.8±0.78
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Range* 0.4-3.4 0. 5-2.1 0.4-12.2 Calculation of contamination mean and range was based on positive sample. All values are expressed in µg/kg.
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a
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10(45.5%)
Date 5 (22.7%)
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Mulberry No. of contaminated samples
0.8-5.5
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Table 5: Dietary exposure of AFB1 and OTA. AFB1
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OTA Exposure (ng/kg Concentration Exposure (ng/kg Dried Concentration (µg/kg) bw/day)d (µg/kg) bw/day) Fruit type MBa LB-UBb MB LB-UB MOEc MB LB-UB MB LB-UB MOE Mulberry 2 1.4 1.37-1.42 0.04 0.04 4,250 1.05 1.05-1.06 0.03 0.03 >10,000 Date 10 0.87 0.85-0.89 0.12 0.12 1,417 0.51 0.51-0.56 0.07 0.07-0.08 >10,000 Fig 1.9 1.58 1.5-1.6 0.04 0.04 4,250 2.06 2.06-2.11 0.05 0.06 >10,000 Apricot 2 2.11 2.10-2.12 0.06 0.06 2,833 1.17 1.17-1.2 0.03 0.03 >10,000 a MB - Medium bound estimate: ND (not detected) values were replace by LOD/2; the un-quantified values (between LOD and LOQ) were replaced by LOQ/2. bLB - Lower bound estimate: the undetected values were replaced by zero; the un-quantified values were replaced by LOD. b UB - Upper bound estimate: the undetected values were replaced by LOD; the un-quantified values were replaced by LOQ. cMOE, Margin of exposure which is calculated as a ratio of benchmark dose lower limit 10% lower bound of AFB1 (170 ng/kg bw/day) or OTA (21.0 µg/kg bw/day) to MB of exposure. dTo estimate daily dietary exposure, the average body weight of 70 kg for the Iranian adult population was used. All values are expressed in µg/kg. Daily intake (g/day)
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Highlights •
Co-occurrence and aflatoxins (AFs) and ochratoxin A (OTA) in dried fruits were
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evaluated •
The mean AFB1 of mulberry, date, fig and apricot samples varied 2.09-3.03 µg/kg
•
The mean value of total AFs in 9.09- 22.72% of samples were more than established legal limit (4 µg/kg)
The OTA contamination in all samples were below the recommended limit (10 µg/kg)
•
The intake of AFs through the dried fruits in Iran poses a potential risk to human health
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S0278-6915(17)30280-6
DOI:
10.1016/j.fct.2017.05.046
Reference:
FCT 9081
To appear in:
Food and Chemical Toxicology
Received Date: 25 April 2017 Revised Date:
20 May 2017
Accepted Date: 22 May 2017
Please cite this article as: Heshmati, A., Zohrevand, T., Khaneghah, A.M., Mozaffari Nejad, A.S., S. Sant’Ana, A., Co-occurrence of aflatoxins and ochratoxin A in dried fruits in Iran: Dietary exposure risk assessment, Food and Chemical Toxicology (2017), doi: 10.1016/j.fct.2017.05.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Co-occurrence of aflatoxins and ochratoxin A in dried fruits in Iran: dietary exposure risk assessment
Nejad a, Anderson S. Sant’Ana d,*
Nutrition Health Research Center, Hamadan University of Medical Sciences, Hamadan, Iran Department of Nutrition, School of Medicine, Hamadan University of Medical Sciences,
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a
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Ali Heshmati a,b*, Tahereh Zohrevand c, Amin Mousavi Khaneghah d,**, Amir Sasan Mozaffari
Hamadan, Iran c
Laboratory of Food and Drug Analysis, Hamadan University of Medical Sciences, Hamadan, Iran
Department of Food Science, Faculty of Food Engineering, State University of Campinas
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d
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(UNICAMP), Monteiro Lobato, 80. Caixa Postal: 6121. CEP: 13083-862. Campinas. São Paulo.
Corresponding authors: *Anderson S. Sant’Ana. E-mail address: [email protected] **Amin Mousavi Khaneghah. E-mail address: [email protected] 1
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ABSTRACT In this study, the contamination levels of aflatoxins (AFs) and ochratoxin A (OTA) in 88 collected
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samples from Iran's market including dried mulberry, date, fig, and apricot were evaluated. The margin of exposure (MOE) was estimated to assess the risk of dietary intake of aflatoxin B1 (AFB1) and OTA. The incidence of AFB1 in dried mulberry, date, fig and apricot samples was 45.5, 40.9, 59.1, and 81.8%, respectively. Although the mean total AFs content in contaminated
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samples of date (2.61 µg/kg), fig (3.43 µg/kg) and apricot (2.91 µg/kg) was lower than the
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maximum limit set in the European Union (EU) (4 µg/kg), dried mulberry samples showed a higher contamination level (4.12 µg/kg). The co-occurrence of OTA and AFs were noted in 4 (18.9%), 2 (9.1%), 4 (18.2%), and 10 (45.5%) in the dried mulberry, date, fig and apricot samples, respectively. Based on the calculated MOE, the dietary exposure to AFs through the consumption of dried fruit in Iran poses a potential risk to consumer health. OTA was detected in
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45.45%, 22.72%, 45.45%, and 50% of mulberry, date, fig and apricot samples, respectively. However, OTA levels in all types of dried fruit were below recommended level in EU regulation
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(10 µg/kg) and MOE >10000, representing no toxicological concerns for consumers.
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Keywords: Aflatoxins, Ochratoxin A, Risk assessment, Dried fruit, HPLC, Dietary exposure
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1. Introduction Mycotoxins as a large group of low molecular weight compounds (below 1000 Da) can be considered as the secondary metabolites generated by some of the filamentous moulds, including
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genera of Aspergillus, Fusarium, Penicillium and Alternaria (Al-Taher, et al., 2017). The presence of mycotoxins in food products is a grave threat to human health. Therefore, the investigation of their occurrence in foods systems is of extreme importance (Campagnollo et al.
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2016; Oteiza, et al., 2017).
Among mycotoxins, ochratoxin A (OTA) and aflatoxins (AFs) can be accounted as the most
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frequently reported mycotoxin in various agricultural products and human foods (Azaiez et al., 2015; Eslami et al., 2015; Kabak, 2016; Mashak et al., 2016; Toma and Rajab, 2014; Trucksess and Scott, 2008 Rastegar et al., 2017). AFs are compounds with potent carcinogenic, mutagenic, estrogenic, tremorogenic, teratogenic and immunosuppressive effects on the health of animals
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and humans (Huong et al., 2016). The major kinds of AFs are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2). Among them, AFB1 is the most important once it has carcinogenic and genotoxic effects on humans its high potent carcinogenic and
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genotoxic effects on human (Tajik et al., 2016; Van de Perre et al., 2015). Hence, it is classified by International Agency for Research on Cancer (IARC) as a carcinogen of group 1(IARC,
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2012). OTA is another mycotoxin which is mainly produced by species of Penicillium (P. verrucosum and P. nordicum) and Aspergillus (A. carbonarius and A. ochraceus). Penicillium species are more often connected to temperate climates, while Aspergillus species are found in warm climates (Clark and Snedeker, 2006). According to the IARC, OTA has been classified into group 2B, "possible carcinogens in humans" (IARC, 2012).
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Risk assessment is a four-step process, which determines the possible adverse effects of a chemical compound such as mycotoxin on an organism. Exposure assessment is one of the essential elements of risk assessment and takes into consideration the occurrence and
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concentrations of mycotoxin in the diet, the consumption patterns as well as the intake value correlated to foods containing mycotoxin. The estimated values from dietary exposure assessments could be determined to decrease risks for humans (Huong et al., 2016). For some
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mycotoxins, a tolerable daily intake (TDI) or a maximum tolerable daily intake (PMTDI) have been established, while for other mycotoxins, a weekly intake (TWI) or a provisional tolerable
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weekly intake (PTWI) are used. TDI/TWI refers to the level that can be taken daily/weekly over a lifetime without a considerable health risk. There is a risk if the calculated daily/weekly intake is over the recommended TDI/TWI values. However, in the case of OTA, the PTWI of 100 and 120 ng/kg bw/day, were recommended by the Joint FAO/WHO Expert Committee on Food Additives
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the (JECFA) and the European Food Safety Authority (EFSA), respectively (EFSA, 2006; JECFA, 2007). No TDI has been established for genotoxic carcinogens such as AFB1 (ESFA, 2006). One of the common methods of assessing the carcinogenic and genotoxic mycotoxins is
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the estimation of the margin of exposure (MOE) (EFSA, 2006; JECFA, 2007). The MOE can be obtained by dividing the dose, which causes a defined impact (benchmark dose, BMD) in
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animals, by the approximated dietary human ingestion of AFB1. A MOE <10,000 could cause a high risk to public health. OTA has been classified as a non-genotoxic carcinogenic by the JECFA (JECFA, 2007). Nonetheless, the MOE for OTA was calculated for the lowest BMDL10 (benchmark dose lower confidence limit of 10%) (21 µg/kg BW/day) which could increases the renal cancer risk (Huong et al., 2016) Dried fruits could offer high nutritional value in the human diet (Chang et al., 2016). However, these products also provide a suitable medium for mould growth and further mycotoxin 4
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contamination. The European Union set maximum permitted levels of AFB1, total AFs, and OTA in dried fruit at 2, 4 and 10 µg/kg, respectively (CET, 2006 a). Also, the Institute of Standards and Industrial Research of Iran (ISIRI) has regulated the maximal accepted levels of AFB1, total AFs
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and OTA in some dried fruits at 5, 15 and 10 µg/kg, respectively (ISIRI, 2012). There are various reports on the occurrence of mycotoxins such as AFs and OTA in dried fruit around the world (Kabak, 2016; Nayebpoor et al., 2013; Ozer et al., 2012; Scott and Trucksess, 2009; Trucksess
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and Scott, 2008). Although some studies have been conducted to evaluate mycotoxin contamination in dried fruit in Iran (Heshmati and Mozaffari Nejad, 2015; Janati et al., 2012;
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Rahimi and Shakerian, 2013), no dietary exposure assessment has been carried out. In this context, the current study was undertaken to determine the occurrence and co-occurrence of AFs and OTA in dried mulberries, figs, dates, and apricots samples as well as to assess the dietary
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exposure to these mycotoxins.
2. Materials and methods 2.1. Materials
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The obtained analytical grade crystalline forms of AFB1, AFB2, AFG1, and AFG2 from SigmaAldrich (St. Louis, MO, US) were used to prepare standard solutions. The acetonitrile, methanol,
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phosphate buffer saline (PBS), other solvents and chemicals of analytical grade were obtained from Merck (Darmstadt, Germany). For the clean-up step, immunoaffinity columns (IAC) of aflatoxins (Puri-Fast AFLA BG IAC) and ochratoxins (Puri-Fast OTA IAC) from Libios (Pontcharra-Sur-Turdine, France) were utilized.
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2.2. Sample collection Eighty-eight dried fig, mulberry, date and apricot samples (22 samples of each) were collected
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randomly from local markets in Hamadan city, Iran, during 2015–2016. The sampling and preparation procedures for the analysis were conducted according to the described procedure by the Commission of the European Communities No. 401/2006 (CET, 2006b). The samples were poured into polyethylene bags and were stored at -18 °C until the experiment day (maximum 2
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wk).
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2.3. Mycotoxin analysis
Dried fruit samples were thoroughly ground. Five g sodium chloride and 300 mL of water– methanol (8:2 v/v) were added to 50 g of dried ground fig, mulberry, date, and apricot samples including those either spiked or not spiked with a given volume of a stock solution of aflatoxins.
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Then they were blended in a mixer at high speed (2000 RPM) for 5 min, to create homogeneous
slurries suspension and they were passed through filter paper (Whatman No. 4). Then, the extract (10 mL) was diluted with 60 mL of phosphate-buffered saline (PBS) solution and passed through
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IAC. Before clean-up, the column was left at room temperature to stabilize and further conditioned with 10 mL of PBS, at flow rate of approximately 1 mL/min under gravity of
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Methanol (1.5 mL, flow rate of 1mL/min) was utilized to elute aflatoxins from IAC. The methanol was collected in a vial, to be analyzed for aflatoxins through HPLC (Janati et al., 2012).The sample preparation and IAC extraction procedure used in the current study to determine OTA levels had been previously optimized and validated in and were reported in our previous study (Heshmati and Mozaffari Nejad, 2015). 2.4. HPLC analysis 6
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The analysis of AFs and OTA was carried out by a Waters reverse-phase HPLC instrument (Milford, MA, USA) equipped with a binary pump, autosampler, fluorescence detector, and an RP C18 analytical column (250 mm × 4.6 mm, i.d., 5 µm) and a column oven set at 25 °C. The
rate
of
1 mL/min
for
aflatoxins,
and
acetonitrile:
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used mobile phases in isocratic elution were water/methanol/acetonitrile (6:3:2, v/v/v) at a flow water::
methanol:
acetic
acid
acetonitrile/water/methanol/acetic acid (39:30:30:1, v/v/v/v) with a flow rate of 1 mL/min for
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OTA. The wavelengths of excitation and detector emission for AFs and OTA were 365/435 nm
2.6. Validation of the analytical method
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and 333/477 nm, respectively. Volume of injection was 20 µL.
The accuracy, precision (intra-day accuracy and inter-day accuracy), linearity, limit of detection (LOD), and limit of quantification (LOQ) were assessed to validate the used analytical
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method. The accuracy of AFs and OTA measurement was examined by determining their recovery. Samples of dried fig, mulberry, date, and apricot were spiked with concentrations of 2, 4 or 6 µg kg–1 of AFB1, AFB2, AFG1 and AFG2 as well as 4, 8 or 12 µg kg–1 of OTA. Recovery
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was calculated by comparing the mycotoxin concentration in the spiked samples with concentrations in the reference standards (AFs and OTA solutions).
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Analytical precision was determined via triplicate analysis of the spiked samples containing two different levels of AFB1 and AFG1 (2 and 4 µg kg–1), AFB2 and AFG2 (1.5 and µg kg–1), and OTA (4 and 8 µg kg–1); this analysis took place on the same day (i.e., intra-day precision) and on three consecutive days (inter-day precision). The relative standard deviation (%) of replicate measurements also was calculated. To construct an external standard calibration by which to quantify mycotoxins in dried fruit samples, working standard solution of AFs and OTA were
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prepared at concentrations of 0.5-20 ng/Ml and 1-25 ng/Ml respectively. The limit of detection (LOD) was set as three times the signal to blank noise, and the limit of quantification (LOQ) was set at 10
2.7. AFB1 and OTA risk and exposure assessment
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times the signal of blank noise.
A deterministic approach was used to calculate dietary exposure to AFB1 and OTA.
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Aflatoxin intake was determined by considering upper, medium, and lower-bound concentrations of mycotoxin in addition to the mean of the collected data regarding consumption of dried fruit
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by using the of following equation:
Daily dietary exposure of AFB1 or OTA (ng/kg bw) = (dried fruit per-capita (g/day) × AFB1 or OTA concentration (µg/kg))/bw (kg)
In Iran, the per-capita consumption of dried figs, mulberries, dates, and apricots is
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thought to be 1.9, 2, 10 and 2 g/day, respectively (Iran ministry of Health and Medical Education, 2009). To estimate the daily dietary exposure, the average body weight of 70 kg for the Iranian adult population was assumed. In order to facilitate the estimation of the lower, medium, and
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upper bound, not detected (ND) values were replaced with zero; LOD/2, LOD, and unquantified
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values were replaced with LOD, LOQ/2, and LOQ, respectively (Huong et al., 2016) The MOEs were calculated only in the case of AFB1 and OTA in following the
recommendations of EFSA (2006) and JEFCA (2007). In our study and in most cases, the BMDL10 is used; this is the lowest dose with a 95% certainty of not leading to a 10% increase in cancer incidence in rodents. In the current study, the MOE was calculated using a BMDL10 value of 170 ng/kg bw/day, as recommended by the EFSA for AFB1 (EFSA, 2007). In addition, the estimated intake of OTA was compared to PTWI, to evaluate potential risks to consumer health 8
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2.8. Statistical analysis The results were reported as mean ± standard deviation values. Data were analysed by
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using SPSS (version 16:0) software (SPSS Inc.; Chicago, IL, USA). To compare the mean concentrations of AFB1, total AFs, and OTA found in the samples with the maximum levels
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allowed under per ISIRI and EU legislation, a one-sample t-test was used.
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3. Result and discussion 3.1. Validation of AFs and OTA analytical method
HPLC validation parameters such as linearity, LOD and LOQ are presented in Table 1. The recovery of AFB1, AFG1, AFB2, AFG2, and OTA are shown in Table 2. The performance of used
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method can be justified with the suitable linear responses with a high coefficient of determination for the analysed AFs and OTA as well as quite good obtained recoveries (AOAC International, 2002; Codex Alimentarius, 1995; Wei, et al., 2013)
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3.2. Incidence of AFs and OTA in dried fruits
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3.2.1. Dried mulberries
AFB1, AFB2, AFG1, and AFG2 were detected in 10 (45.5%), 7 (31.8%), 2 (9.1%) and 4 (18.18%) of 22 dried mulberry samples, respectively. AFB1 was the most abundant aflatoxin and its level varied from 0.35 to 8.4 µg/kg. Moreover, the concentration of AFB1 in four samples exceeded the limit of European regulations (2 µg/kg). Additionally, the levels of AFB1 in two samples were higher than the recommended level by Iranian standard (5 µg/kg), although the total AFs content was within the allowed limit in Iran (15 µg/kg). In the current study, among 9
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several types of dried fruit that were evaluated, mulberries were found to contain the highest mean value of AFB1 and total AFs.
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The incidence of OTA in mulberry samples was 45.5% (10 of 22 samples), with a mean value of 1.75 µg/kg. The OTA values in all samples were lower than established limits in Iran and the European Union (10 µg/kg).
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There were a few reports regarding the occurrence of AFs and OTA in dried mulberry. A higher level of incidence and mean total AFs contents (45.5% and 4.1 µg/kg, respectively) were
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recorded in the current study in comparison with previous research. For instance, Luttfullah and Hussain (2011) reported that four (26%) out of 15 dried mulberry samples contained 1–3.5 µg/kg of AFs (mean value, 2.22 µg/kg). Besides, Toma and Rajab (2014) reported a mean total AFs of 14.3 ug/kg for total AFs in dried mulberry samples collected from various cities in Iraq; a value
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3.5 times higher than in current investigation. According to Kaya and Tosun (2013), the total levels of aflatoxin, OTA and fumonisn in organic mulberry was 9.45, 12.47 and 691.78 µg/kg, respectively. The Mulberry tissues and structures have high sugar levels and are prone to insect
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attack, conditions that favor mould growth and further mycotoxin production(Kaya and Tosun,
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2013).
3.2.2. Dried dates
A relatively high incidence of AFs contamination was observed in dried dates. Nine (40.9%) of 22 date samples contained AFs, with a mean value of total AFs 2.61 µg/kg. Among them, two date samples were contaminated with levels higher than EU limit for AFB1 (2 µg/kg) and total AFs (4 µg/kg).Although the presence of OTA was recorded in five out of 22 date 10
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samples, the mean OTA value (1.21 µg/kg) was below that recommended by the European Union (10 µg/kg). Also, none of them contained a higher level of contamination than the recommended
mould growth and mycotoxin production (Ozer et al., 2012).
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level. Relatively high temperatures and humidity during date maturation might have resulted in
Two out of twenty date samples analysed by Alghalibi and Shater (2004), were contaminated with AFB1. The prevalence of aflatoxins B1, B2, G1 and G2 and sterigmatocystin in
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fresh date fruit (Phoenix dactylifera) during storage conditions (98% relative humidity and 30 °C
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for 14 days) were studied (Shenasi et al., 2002). Their results showed that, neither aflatoxins and nor their precusor, sterigmatocystin, was detected after adverse storage for 14 days. In another investigation Gherbawy et al. (2012), the presence of aflatoxins and ochratoxin A producer fungis in retail date fruits distributed in different markets at Taif, Saudi Arabia was evaluated. Based on the reported results by them; seven out of subjected eighty isolates of A. flavus for detecting their
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aflatoxigenic potentials, were toxigenic. Moreover, among examined thirty-six isolates of Aspergillus niger for ochratoxin production, only nine isolated were able to produce ochratoxin. Also, in previously conducted study regarding the occurrence of OTA in dates in Brazil, it was
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reported a mean value (<0.13 µg/kg) lower than the one found in current study (Beatriz et al.,
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2006). In another investigation done by Azaiez et al. (2015), 16 types of mycotoxins were detected in 228 samples of dates and dried fruits from Tunisian and the Spanish market. In this case, mycotoxins that occurred most frequently were enniatin B (EnnB) (54%), enniatin A1 (36%), AFs (23%), and OTA (22%). In 18 of 48 Tunisian date samples, OTA level ranged from 0.57 to 3.3 µg/kg (mean value, 1.3 µg/kg). The mean total AFs value in the aforementioned study was higher than in current study. The mean value of total AFs of AFB2, AFG1, and AFG2 in the Tunisian date samples were respectively 1.14, 1.4, and 1.7 µg/kg; meanwhile, AFB1 was not
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detected in any of the samples (Azaiez et al., 2015). The elimination of deteriorated dates prior to drying and final packaging might lead to reduced mycotoxin levels (Ozer et al., 2012).
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3.2.3. Dried figs As shown in Tables 3 and 4, AFB1, AFB2, AFG1, AFG2 and OTA were found in 13 (59.1%), 8 (36.4%), 5 (22.7%), 4 (18.9%) and 10 (45.5%) out of 22 dried fig samples, respectively, with
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average concentration of 2.65, 0.74, 0.36, 0.32 and 3.03 µg/kg, respectively. Only 3 (13.6%) out of 22 dried fig samples were contaminated with levels of AFB1 above the Iranian standard (5
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µg/kg) and 7 (31.2%) samples contained AFB1 at higher levels than the respective regulation of European Union (2 µg/kg). The total AFs in 5 (22.7%) of the fig samples exceeded the accepted maximum level of European legislation (4 µg/kg). The OTA content of one sample (12.16 µg/kg) was greater than the recommended level accepted in the European Union (10 µg/kg).
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The potential risk of contamination by mycotoxins in figs can be correlated to their susceptible nature to growth of mycotoxingenic fungi. The growth of mycotoxin-producing moulds including Aspergillus, Fusarium, and Penicillium in collected fig samples in Turkey was
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recently reported (Heperkan et al., 2012). Ochratoxin-producing black Aspergilli is highly resistant to sunlight and UV light; therefore, it has been recognized as the dominant mould in
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some of dried fruits, such as dried figs(Karbancıoğlu-Güler and Heperkan, 2008).In a study done by Rodrigues et al (2012), aflatoxigenic fungi in almond as dominant mold was domenstrated. The incidence of total AFs in fig samples of Pakistan (30%), with a mean of 0.54 µg/kg, was reported by Asghar et al. (2017). In current investigation the incidence and the mean value (3.3 µg/kg) of total AFs in dried fig samples was higher. Nonetheless, our mean value for AFs was lower than the mean (3.8 µg/kg) reported by Kabak (2016). In present study, it was also observed
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that AFB1 was the predominant type of aflatoxin, contrary to what was seen in Turkey (Kabak, 2016) and Morocco (Juan et al., 2008), where AFG1 was the dominant form in samples. The highest incidence of OTA in fig samples (95%, 65%, and 48%,) were reported by Iamanaka et al.
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(2005) in Brazil; Karbancıoğlu-Güler and Heperkan in Turkey (2008) and Zinedine et al. (2007) in Morocco, respectively.
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3.2.4. Dried apricots
Among the assessed dried fruits, the most frequent incidence of AFB1 was found in
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apricots, as 18 (81.8%) out of the 22 samples were contaminated with levels that ranged between 0.39 and to 7.1 µg/kg with a mean of 2.43 µg/kg. However, the AFB1 concentration in eight samples (36.4%) were over European regulation (2 µg/kg), the reported amounts for the total AFs and AFB1 were acceptable considering the Iranian national standard (5 µg/kg for AFB1 and 15
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µg/kg for total AFs). In addition, apricots had the highest frequency of OTA contamination (50%), with a range of 0.75–5.5 µg/kg and mean of 1.76 µg/kg. Nonetheless, OTA levels in the samples were lower than the permissible concentration of OTA (10 µg/kg). Moreover, no OTA
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contamination in Turkish apricot samples by Aksoy et al (1995) reported. In an investigation done by Janati et al. (2012), AFB1, AFB2, AFG1 and OTA was found in 30, 3.3, 3.3 and 3.3% of apricot
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specimens, with averages of 0.88, 0.32, 0.20 and 2.83 µg/kg, respectively; but, AFG2 was not detected. In mentioned study, all the measured AFs levels were lower than reported concentrations in present research. Although the OTA value (2.83 µg/kg) was higher than in current study (1.76 µg/kg), only one sample (6.7%) out of 15 dried apricot samples contained OTA (2.8 µg/kg) (Rahimi and Shakerian, 2013), OTA was also found in one of 20 dried apricot samples with a value of 0.97 µg/kg (Bircan 2009). The utilization of sulfur dioxide as a preservative substance in some dried fruits, such as apricots, inhibits fungal growth; this might be 13
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a reason for the lower incidence of OTA in the samples analyzed in previous studies. Iamanaka et al., (2005) reported that none of the 14 dried apricot samples analyzed in Brazil were contaminated with OTA. In contrast, in previous studies, our results demonstrated that the
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examined apricots samples had the highest potential for AFs and OTA contamination. Asghar et al. (2017) analyzed 65 dried apricots for total AFs. Twenty-one (32%) of the samples were contaminated, with a mean of 1.02 µg/kg and a range of 0.31–11.11 µg/kg.
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The differences found between the mycotoxin levels of dried fruits evaluated in previous
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studies an in current investigation can be related to several aspects. For instance, these differences may be due to variation in sample sizes, to different sampling methods as well as analytical techniques (HPLC, LC and ELISA), climate and seasonal changes, and to the used agricultural practices (Rahimi and Shakerian, 2013; Trucksess and Scott, 2008).
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3.3. Co-occurrence of AFs and OTA
AFs and OTA were found together in 4 (18.2%), 2 (9.1%), 4 (18.2%) and 10 (45.5%) of the dried mulberry, date, fig and apricot samples, respectively. The co-occurrence of OTA with AFs
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was reported by Bircan (2009) in 2 out of the seven dried fig samples analyzed in Turkey. In another study from Turkey, 2 out of 53 in 2003 and 2 of 41 dried figs samples in 2004 were
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contaminated with both AFB1 and OTA (Senyuva et al., 2005), showing lower co-occurrence of mycotoxin than in the present study. Janati et al., (2012) reported the contamination of apricots and prunes with AFs and OTA independently; but, the number of samples that contained both was not mentioned. Moreover, Co-occurrence of AFs and OTA was reported in 69% of dried fruits collected from Tunisia (Ghali et al., 2008). The presence of AFs and OTA in dried fruits probably also reflects the presence of various species of Aspergillus moulds in these products.
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Sun drying is a traditional technique still applied to dry fruits in Iran. During this process, favourable conditions for mould growth and mycotoxin production are provided. However, good agricultural practices, such as the use of new drying technologies and the removal of damaged
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fruits during processing may decrease mycotoxin levels in dried fruits such as figs. Other measures that will contribute to reduction of fungal contamination and presence of mycotoxins include the reduction of mould microflora using chemicals such as sulphur dioxide, sodium
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metabisulphite and potassium metabisulphite, the application of fungicide during storage and
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gamma radiation (Scott and Trucksess, 2009; Trucksess and Scott, 2008). 3.4. Risk assessment
The estimated average exposure to AFB1 through the consumption of dried mulberries, dates, figs and apricots was found to be 0.04, 0.12, 0.04 and 0.06 ng/kg bw/day, respectively
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(Table 5). The MOE, which was determined using the dietary intake levels of each dried fruit and the BMDL10 of 170 ng/kg bw/day, was 4,250, 1,417, 4,250 and 2,833 for the dried mulberry, date, fig and apricot samples, respectively. A small MOE indicates a higher risk than a large
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MOE. As the obtained MOE values were less than 10,000, it can be said that the consumption of dried fruits in Iran puts consumers at an exposure risk to AFB1. The lowest MOE found in dried
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dates suggests a high daily intake of AFB1. The MOE to OTA is shown in Table 5. The MOE to OTA was found > 10,000.
In the current study, the upper-bound which estimates of the intake of OTA through
mulberries, dates, dried figs, and apricots were 0.03, 0.08, 0.06 and 0.03% of PTWI (100 ng/kg bw/day) (EFSA,2006). Therefore, the intake of OTA through dried fruits does not exceed that of the PTWI.
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Little information regarding the MOE in dried figs, mulberries, dates and apricots was reported previously. However, MOE has been calculated and communicated in other dried fruits and foods in past studies. For example, the MOE to AFB1 through peanut consumption in China was
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reported to be 1,273 (Ding et al., 2012). Besides, the estimated MOE of AFB1 through the intake
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of bakery products and pasta was reported to be 24.6 (Bol et al., 2016).
4. Conclusion
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The occurrence of AFs and OTA of consumed dried fruits in Iran was evaluated. The results showed that the mean total AFs level in all contaminated samples except the dried mulberries was lower than that allowed according to European legislation (4 µg/kg). No samples had OTA contamination above the allowable limit (10 µg/kg). The co-occurrence of AFs and OTA were
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noted in 4 (18.2%), 2 (9.1%), 4 (18.2%), and 10 (45.5%) of the dried samples, respectively. In current study, the derived margin of exposure (MOE) values calculated for AFB1 (range 1,417 to 4,250) and OTA (> 10,000). Since the MOE values of AFB1 were found to be lower than 10,000,
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a potential risk to consumer health exists through the dietary consumption of dried fruits and subsequent exposure to AFs. However, further evaluation through a total diet study (TDS) is
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necessary to ensure a better adjustment; thus, more studies should be conducted to determine Iranian consumers' dietary exposure to AFs, OTA, and other mycotoxins. Although the level of AFB1 and OTA were not so high, efficient methods to provide the safeguard for the consumers against their toxic effects and furthermore to keep the public health should be approached. In this context, the employment of good agricultural practices (GAP) with the aim of reducing fungal
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growth in all the processing steps (in the field and during storage) as well as the general
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improvement of quality of all the process are crucial.
Acknowledgements
The authors are thankful to Vice-Chancellor of Research and Technology of Hamadan University
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of Medical Sciences and Health services for the study protocol approval and financial support. Amin Mousavi Khaneghah and Anderson Sant’Ana like to thank the support of CNPq-TWAS
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Postgraduate Fellowship (Grant # 3240274290), and "Conselho Nacional de Desenvolvimento Científico e Tecnologico"(CNPq) (Grant #302763/2014-7), respectively.
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Table 1: Linearity range, limit of detection (LOD) and limit of quantification (LOQ) for different mycotoxins analyzed in dried fruitsa.
0.992 0.997 0.991 0.998 0.996
LOD LOQ LOD LOQ LOD 0.09 0.30 0.06 0.20 0.08 0.07 0.23 0.05 0.17 0.08 0.08 0.27 0.08 0.27 0.05 0.06 0.20 0.02 0.07 0.08 0.07 0.23 0.15 0.50 0.26
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LOD and LOQ values are expressed in µg/kg.
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Y=12564X+238 Y=10890X+2345 Y=25645X+1390 Y=20123X+1009 Y=16007X+5678
Mulberry 2
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Equation of calibration curve
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Range of Mycotoxin linearity (ng/mL) AFB1 1-22 AFB2 0.8-22 AFG1 0.5-20 AFG2 1-18 OTA 2-50
LOQ 0.27 0.27 0.17 0.27 0.87
Apricot LOD LOQ 0.07 0.23 0.08 0.27 0.06 0.20 0.08 0.27 0.18 0.60
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Table 2: The recovery percentage of mycotoxins in dried fruits.
Date
Spiked level (µg kg -1) 2 4 6
recovery percentage 82.4 87.9 91.0
RSD 1.6 1.0 1.2
recovery percentage 82.8 88.7 91.4
RSD 1.6 1.1 1.2
AFB2
2 4 6
84.9 87.1 91.3
0.9 1.4 1.7
85.4 87.5 91.7
AFG1
2 4 6
79.0 80.7 87.4
0.5 1.3 0.7
79.4 81.2 87.8
AFG2
2 4 6
76.5 74.4 78.5
1.9 0.7 2.0
OTA
4 8 12
84.0 88.4 92.3
1.4 0.6 1.7
RSD 1.6 1.0 1.3
RSD 1.5 0.9 1.2
1.1 1.3 1.8
85.5 87.6 91.8
1.1 1.3 1.6
86.3 88.3 92.5
1.1 1.4 1.6
0.6 1.3 0.8
79.6 81.3 87.9
0.5 1.2 0.9
80.2 81.9 88.6
0.5 1.3 0.7
76.9 74.7 78.9
2.0 0.7 1.8
77.1 74.9 79.0
2.1 0.8 1.9
77.7 75.6 79.7
1.9 0.7 2.0
83.8 88.1 92.1
1.2 0.8 1.6
84.6 88.9 92.9
1.4 0.7 1.7
84.6 88.9 92.9
1.4 0.7 1.7
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recovery percentage 82.9 88.5 91.5
Apricot recovery percentage 83.6 89.1 92.2
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Commodity Fig
Mulberry
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Table 3: Aflatoxin contamination in dried fruitsa. Aflatoxin type
Contamination status
Commodity Date
Fig
Apricot
10(45.5%) 6(27.3%) 2(9.1%) 2(9.1%) 3.0±2.7 0.4-8.4
9(40.9%) 7(31.8) 1(4.5) 1(4.5) 2.1±1.8 0.6-6
13(59.1%) 6(27.37%) 4(18.2%) 3(13.6%) 2.6±2.5 0.3-7.0
18(81.8%) 10(45.5%) 5(22.72%) 3(13.63%) 2.4±2.2 0.4-7.1
AFB2
No. of contaminated samples Mean±SD Range*
7 (31.8%) 1.21±1.1 0.3-2.5
5(22.7%) 0.7±0.6 0.4-0.7
8(36.4%) 0.7±0.6 0.2-2.1
12(54.5%) 0.6± 0.2 0.3-0.8
AFG1
No. of contaminated samples Mean±SD Range*
2 (9.1%) 0.6 ±0.4 0.3-0.8
3(13.6 %) 0.3±0.1 0.29-0.41
5(22.7 %) 0.4±0.1 0. 3-0.4
4(18.2%) 0.3±0.0 0.3-0.4
AFG2
No. of contaminated samples Mean±SD Range*
4 (18.2%) 0.3±0.0 0.3-0.4
1(4.5%) 0.3 -
4(18.2%) 0.3±0.0 0.3-0.4
3(13.6%) 0.30 ±0.0 0.3-0.3
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AFB1
Contamination level( µg/kg)
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Mulberry No. of contaminated samples 0.08-2 2-5 >5 Mean*±SD Range*
No. of contaminated samples 10 (45.4%) 9 (40.9%) 13 (59.1%) Contamination <4 7 (31.8%) 7 (31.8%) 8 (36.4%) Total AFs level( µg/kg) * 4-15 3 (9.1%) 2 (9.1%) 5 (22.7%) >15 Mean±SD 4.1±3.9 2.6±2.3 3.3 ±3.2 Range* 0.6-11.8 0.9-8.1 0.4-9.2 a Calculation of contamination mean and range was based on positive samples. All values are expressed in µg/kg
18 (81.8%) 13 (59.1%) 5 (22.7%) 2.9 ±2.6 0.4-8.5
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Table 4: Ochratoxin A (OTA) contamination in assayed dried fruitsa.
Commodity
10(45.5%)
Apricot 11(50%)
0.26-5
10(45. 5%)
5 (22.7%)
8(36.4%)
10(45.5%)
Contamination level( µg/kg)
5-10
0
0
1(4.54%)
1(4.5%)
>10
0
0
1(4.54%)
-
1.2±0.6
3.03±3.5
1.7±1. 5
Mean ± SD
1.8±0.78
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Range* 0.4-3.4 0. 5-2.1 0.4-12.2 Calculation of contamination mean and range was based on positive sample. All values are expressed in µg/kg.
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a
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10(45.5%)
Date 5 (22.7%)
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Mulberry No. of contaminated samples
0.8-5.5
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Table 5: Dietary exposure of AFB1 and OTA. AFB1
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OTA Exposure (ng/kg Concentration Exposure (ng/kg Dried Concentration (µg/kg) bw/day)d (µg/kg) bw/day) Fruit type MBa LB-UBb MB LB-UB MOEc MB LB-UB MB LB-UB MOE Mulberry 2 1.4 1.37-1.42 0.04 0.04 4,250 1.05 1.05-1.06 0.03 0.03 >10,000 Date 10 0.87 0.85-0.89 0.12 0.12 1,417 0.51 0.51-0.56 0.07 0.07-0.08 >10,000 Fig 1.9 1.58 1.5-1.6 0.04 0.04 4,250 2.06 2.06-2.11 0.05 0.06 >10,000 Apricot 2 2.11 2.10-2.12 0.06 0.06 2,833 1.17 1.17-1.2 0.03 0.03 >10,000 a MB - Medium bound estimate: ND (not detected) values were replace by LOD/2; the un-quantified values (between LOD and LOQ) were replaced by LOQ/2. bLB - Lower bound estimate: the undetected values were replaced by zero; the un-quantified values were replaced by LOD. b UB - Upper bound estimate: the undetected values were replaced by LOD; the un-quantified values were replaced by LOQ. cMOE, Margin of exposure which is calculated as a ratio of benchmark dose lower limit 10% lower bound of AFB1 (170 ng/kg bw/day) or OTA (21.0 µg/kg bw/day) to MB of exposure. dTo estimate daily dietary exposure, the average body weight of 70 kg for the Iranian adult population was used. All values are expressed in µg/kg. Daily intake (g/day)
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Highlights •
Co-occurrence and aflatoxins (AFs) and ochratoxin A (OTA) in dried fruits were
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evaluated •
The mean AFB1 of mulberry, date, fig and apricot samples varied 2.09-3.03 µg/kg
•
The mean value of total AFs in 9.09- 22.72% of samples were more than established legal limit (4 µg/kg)
The OTA contamination in all samples were below the recommended limit (10 µg/kg)
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The intake of AFs through the dried fruits in Iran poses a potential risk to human health
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•