Accepted Manuscript

Incidence of aflatoxins contamination in dry fruits and edible nuts collected from
Pakistan

Muhammad Asif Asghar, Aftab Ahmed, Erum Zahir, Muhammad Arif Asgher,
Javed Iqbal, Gavin Walker

PII: S0956-7135(17)30110-X

DOI: 10.1016/j.foodcont.2017.02.058

Reference: JFCO 5493

To appear in: Food Control

Received Date: 13 October 2016

Revised Date: 21 February 2017

Accepted Date: 25 February 2017

Please cite this article as: Muhammad Asif Asghar, Aftab Ahmed, Erum Zahir, Muhammad Arif
Asgher, Javed Iqbal, Gavin Walker, Incidence of aflatoxins contamination in dry fruits and edible
nuts collected from Pakistan, Food Control (2017), doi: 10.1016/j.foodcont.2017.02.058

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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 ACCEPTED MANUSCRIPT

1 Title: Incidence of aflatoxins contamination in dry fruits and edible nuts collected from

2 Pakistan

4 Highlights

6 • The occurrence of AFs in dry fruits and edible nuts from Pakistan were assessed.

7 • Samples 165/624 were found positive with aflatoxins contamination.

8 • The contamination range was 0.22−30.11 µg/kg with a mean level 0.85 ± 0.26 µg/kg.

9 • The seasonal variation of AFs was studied with respect to temperature and RH.

10 • AFs level was compared with reported levels in national and international community.

11
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Aflatoxins contamination in dry fruits and nuts

1 Incidence of aflatoxins contamination in dry fruits and edible nuts collected

2 from Pakistan

4 Muhammad Asif Asghar1,2.*, Aftab Ahmed1, Erum Zahir2, Muhammad Arif Asgher3 and Javed

5 Iqbal4

7 1Food and Feed Safety Laboratory, Food and Marine Resources Research Centre, PCSIR

8 Laboratories Complex, Shahrah-e-Salimuzzaman Siddiqui, Off University Road,

9 Karachi−75280, Sindh−74200, Pakistan.

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11 2Department of Chemistry, University of Karachi, Karachi−75270, Sindh−74200, Pakistan.

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13 3Department of Pharmaceutics, Faculty of Pharmacy, Jinnah Sindh Medical University, Karachi,

14 Sindh-74200, Pakistan.

15 4 Pharmaceutical Manufacturing Technology Centre, Bernal Institute, University of

16 Limerick, Castletroy, Limerick, Ireland.

17

18 Muhammad Asif Asghar1,2 (E-mail: masif345@yahoo.com)

19 Aftab Ahmed1 (E-mail: saftyfood@yahoo.com)

20 Dr. Erum Zahir2 (E-mail: ezahir@uok.edu.pk)

21 Muhammad Arif Asghar3 (E-mail: m.arifasgher@hotmail.com)

22 Gavin Walker4 (E-mail: gavin.walker@ul.ie)

23 Javed Iqbal4 (E-mail: javed.iqbal@ul.ie)

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24 *Corresponding Author (Postal address):

25 * Muhammad Asif Asghar

26 1Food and Feed Safety Laboratory,

27 Food and Marine Resources Research Centre,

28 PCSIR Laboratories Complex, Karachi,

29 Shahrah-e-Salimuzzaman Siddiqui, off University Road,

30 Karachi-75280, Sindh-74200, Pakistan.

31 Tel #: +92-99261920; +92-345-2941505

32 Fax #: +92-213-4641847

33 E-mail: masif345@yahoo.com

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Aflatoxins contamination in dry fruits and nuts

47 Abstract

48 During 2012 to 2015, a total of 624 samples of 13 different types of dry fruits and edible nuts

49 were collected from retail shops and local markets of Pakistan. The collected samples were then

50 studied for the occurrence of total aflatoxins (AFs) after immunoaffinity cleanup followed by

51 HPLC coupled with fluorescence detector and post-column derivatization unit (Kobra Cell™).

52 The seasonal variation of AFs was studied with respect to average temperature and relative

53 humidity in Pakistan. LOD (S/N; 3:1) and LOQ (S/N; 10:1) of the utilized method were in the

54 range of 0.09−0.12 and 0.30−42 µg/kg, respectively. About 165 (26%) samples were found

55 contaminated, ranging from 0.22−30.11 µg/kg with a mean equivalent to 0.85 ± 0.26 µg/kg. In

56 459 (74%) samples, the AFs contamination was found lower than detectable limit corresponding

57 to0.12 µg/kg. In 99 (15%) samples, the contamination range of AFs was 1−4 µg/kg. However, 28

58 (4%) samples exceeded the maximum tolerated limit (MTL) of 4 µg/kg as imposed by EU.

59 However, 6 (1%) samples were found beyond the MTL of20 µg/kg as regulated by USA. Highly

60 contaminated samples were found during the months of July, August and September. Achieved

61 data highlighted the requirement of continuous monitoring and further investigation of AFs

62 contamination in dry fruits / edible nuts under the authority of a definite systematic surveillance

63 program adapted as a food safety measure.

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65 Key words: Aflatoxins, dried fruits, edible nuts, HPLC, Pakistan

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70 1. Introduction

71 Dry fruits and nuts are rich in essential amino acid, vitamins, minerals, dietary fibers and

72 consumed by all age of the people. These are also used as medicines which provide energy and

73 strength to long-term patients seeking out to recover (Emilio, 2010). Dry fruits are extensively

74 cultivated all over the world including Pakistan.

75 Dry fruits and nuts are susceptible for pathogenic fungal attach during their cultivation, fruit

76 growth, ripening, over ripening, handling, drying, storage and transportation. Aspergillus,

77 Fusarium, Penicillium and Alterneria are the most common pathogenic fungi and produce

78 toxigenic compounds known as mycotoxins (Zain, 2011). About more than 400 mycotoxins have

79 already been discovered. However, aflatoxins (AFs) have become the most widely known

80 mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus under favorable

81 conditions (Paterson, 2007; Paterson, & Lima, 2010; Khan, Asghar, Iqbal, Ahmed &

82 Shamsuddin, 2014). Till to date, 20 AFs have been recognized. Among them only 4 AFs, the

83 aflatoxins B1, B2, G1 and G2 (AFB1, AFB2, AFG1 and AFG2), found naturally on a wide variety

84 of agricultural commodities in significant levels (Paterson, 2007; Iqbal, Paterson, Bhatti, & Asi,

85 2010; Zain, 2011; Asghar, Zahir, Rantilal, Ahmed, & Iqbal, 2016). However, AFB1 is the most

86 toxic and has been widely reported in various agricultural commodities (Asghar, Khan, Iqbal,

87 Ahmed, & Shamsuddin, 2014; Bircan, & Koç, 2012). AFs has been reported as mutagenic,

88 carcinogenic, hepatotoxic, immunosuppressive, neoplastic and classified by the International

89 Agency for Research on Cancer (IARC) as Class 1 human carcinogens (Varga, Frisvad, &

90 Samson, 2011; IARC, 2002).AFs could pose a potential risk to the human health because of

91 aflatoxicosis and cancer. AFs are associated with many chronic diseases including immune

92 suppression, digestive, blood and nerve defects, reproductive problems, anemia and jaundice

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93 (Ajani, Chakravarthy, Tanuja, & Pasha, 2014). However, the diseases verity depends on the

94 mechanism of actions for each kind of AFs, the extent of exposure, age and intake of nutritional

95 status.

96 Dry fruits and edible nuts could be infected with AFs contamination due to poor harvesting

97 practices, improper storage, transportation and environmental condition. In addition, existing

98 sub-tropical conditions in Pakistan, such as, high humidity, temperature and the nearness to sea

99 also facilitate a vital role for the fungal growth and production of AFs. A number of monitoring

100 programs and survey have been conducted in different countries to evaluate the a general pattern

101 of the AFs level in different food commodities (Kabak, 2016; Iqbal, Asi, Hanif, Zuber, & Jinap,

102 2015). Due to severe toxicity concerned with AFs, various countries established guidelines for

103 the acceptance level of AFs in dry fruits and nuts. For instance, the European Commission

104 (Regulation no. 165/2010) has set maximum tolerated limits (MTL) 4 and 10 µg/kg for total AFs

105 in dry fruits and nuts, respectively (Commission of the European Communities, 2010).

106 According to the Food and Drug Administration and Food and Agriculture Organization of USA

107 (2009), the MTL for total AFs in dry fruits and nuts is 20 µg/kg. Currently, Pakistan has not yet

108 regularized MTL for mycotoxins in dry fruits and edible nuts. Thereby, followed EU and USA

109 standards. However, Pakistan Standard Quality Control Authority (PSQCA) has been taken

110 many steps to establish the MTL for mycotoxins in food/feed commodities for the protection of

111 human and animal health.

112 On the basis of above mentioned fact, rapid detection and quantification of AFs in dry fruits and

113 nuts is essential for assuring safety, quality and execution of hazard analysis and critical control

114 points (HACCP). Consequently, current study was designed to find out the level of AFs in dry

115 fruits and edible nuts which grown in Pakistan. The seasonal variation of AFs was also studied

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116 with respect to average temperature and relative humidity in Pakistan. Furthermore, the

117 contamination levels were compared with reported levels in the national and international

118 community.

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139 2. Materials and methods

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141 2.1 Sample collection

142 A total of 624 samples of 13 different types dry fruits and edible nuts including almond (n = 10),

143 apricot (n = 65), dates (n = 170), raisins (n = 90), dried mulberry (n = 10), pistachio (n = 126),

144 dried plum (n = 10), dried figs (Injeer) (n = 10), dried melon seeds (n = 5), walnuts (n = 8), pine

145 nuts (n = 95), peanuts (n = 20) and fox nut (makakna) (n = 5) were collected from local markets

146 and retailer shops of Pakistan during 2012 to 2015. It is well documented that AFs are found in

147 the pocket form in high concentration and heterogeneously dispersed throughout the

148 commodities. Hence, the sampling method was adapted according to the AOAC official method

149 no. 977.16 (Latimer, 2012). In brief, about 0.5 to 1 kg of each sample was pulverized using

150 mincer (Westmark, Germany) and then divided as a sub-sample in a final quantity of 100 g. An

151 amount of 25 g of each representative and homogenous sub-sample were taken for the

152 quantification of AFs contamination. The samples were preserved in air tight and opaque

153 polyethylene bags at -20°C in until analysis.

154

155 2.2 Chemical and Reagents

156 Aflatoxin standard of B1 (2 µg/mL; cat. # 002017), B2 (0.5 µg/mL; cat.# 02018), G1 (2 µg/mL;

157 cat. # 002019) and G2 (0.51 µg/mL; cat. # 002020) in acetonitrile were purchased from Biopure

158 (Vienna, Austria). All standards were stored in freezer at -20oC till further use. Analytical grade

159 acetonitrile and methanol were obtained from Merck (Darmstadt, Germany). Potassium bromide

160 and nitric acid were acquired from Sigma-Aldrich (St Louis, Missouri, USA). Phosphate-

161 buffered saline (PBS) tablets (pH 7.4 + 0.2) were purchased from Oxide Ltd. (Hampshire,

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162 England). Highly purified water (18 MΩ•cm) was obtained by processing de-ionized water (DI-

163 H2O) through a Purelab Ultra Option (ELGA, UK) system. All solvents and water were

164 sonicated for 20 min using a Lucos Soniprobe, Lucas Dawe Ultrasonics bath (UK).

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166 2.3 Instrumentation and apparatus

167 The chromatographic HPLC system consisted of a VWR-Hitachi (Darmstadt, Germany) system

168 equipped with pump model no. L-2130, an auto-sampler model no. L-2200 and a fluorescence

169 detector model no. L-2480. A column thermostat was from Jones-Chromatography (UK), a

170 LiChroCART1 100 Å RP-18, (5 µm, 250 × 4.0 mm2) column from Merck (Darmstadt Germany)

171 and an electrochemical bromine derivatization cell (Kobra Cell™) were obtained from R-

172 Biopharm (Glasgow, Scotland). Immunoaffinity AflaStarTM IACs (cat. # COIAC1001) was

173 procured from Romer Labs. (Tulln, Austria).

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175 2.4 Analysis of Aflatoxin (AFs)

176 AFs were analyzed in dry fruits and edible nuts according to the method previously reported by

177 Asghar et al. (2016) with some modifications. Briefly, whole analysis of AFs could be sub-

178 divided in three steps as described below:

179 1. Sample extraction

180 2. Immunoaffinity column clean-up

181 3. Chromatographic analysis

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185 2.4.1 Sample extraction

186 Twenty five gram of each homogenized sample of dry fruits and edible nuts was weighted

187 accurately and blended with 100 ml of methanol/water (80/20; v/v). The sample suspension was

188 then blended at 5000 rpm using an explosion-proof blender (model no. 8018; Ebarch Corp., East

189 Hampton, New York, USA) for 2min, filtered through Whatman filter paper no. 4 and collected

190 in air-tight amber vials..

191

192 2.4.2 Immunoaffinity column clean-up

193 Sample clean-up was carried out using immunoaffinity columns (IACs). In brief, 2 mL of sample

194 extract was mixed with 14 mL of PBS (pH 7.4). The diluted sample extract (16 mL) was applied

195 to IACs at a flow rate of1−2 drop/s. IACs were washed with 20 ml of PBS buffer (pH 7.4) at a

196 flow rate of about 5 mL/min and then dried bypassing air. AFs were then slowly eluted from the

197 IACs with 1.5 mL of methanol followed by 1.5 mL of DI H2O in amber vials.

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199 2.4.3 Chromatographic analysis

200 AFs were quantified using an HPLC system and post column derivatization using Kobra Cell™.

201 AFs were separated on a LiChroCART 100 A° RP-18 column. An aliquot of 99 µL of the AFs

202 standard and sample was injected to the HPLC system through an auto-sampler. The mobile

203 phase composition was acetonitrile/water/acetic acid (22.5/55/22.5; v/v/v) containing 119 mg/L

204 of KBr and 154 µl/L of nitric acid, pumped at a flow rate of 1.0 mL/min. Liquid chromatography

205 was carried out in an isocratic mode. Kobra Cell™ was operated at a constant current of 100 µA

206 throughout the analysis. The chromatogram of AFs was evaluated at 333 nm and 464 nm as

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207 excitation and emission wavelengths, respectively. All four AFs were properly detected within a

208 20 min run time.

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210 2.5 Method performance

211 The analysis of each sample of dry fruits and edible nuts was performed in triplicate to validate

212 the accuracy and extraction performance of the utilized procedure. Validation of the procedure

213 was evaluated in accordance with decision 657/2002/EC (Commission of the European

214 Communities, 2002) and regulation no. 401/2006/EC (Commission of the European

215 Communities, 2006). The overall validation procedure was evaluated in terms of linearity, limit

216 of detection (LOD), limit of quantification (LOQ), recovery and expanded uncertainty of

217 measurement (Table 1). The validation parameters of the utilized method were found

218 satisfactory for the analysis of AFs.

219 Briefly, linearity of the methods was estimated in terms of determination coefficient (R2). A

220 series of working standards in the final concentration of each AFs corresponding to 0.0125,

221 0.025, 0.125, 0.25, 0.625, 1.25, 2.5, 5, 10, 15 and 20 ng/mL was prepared in methanol/water

222 (80/20; v/v). All standards were injected via auto-sampler connected to the HPLC system and

223 chromatograms were recorded. Finally, calibration curves for AFB1, AFB2, AFG1 and AFG2

224 were separately constructed by plotting the mean area under the curve against relevant

225 concentration. The R2 was calculated using the regression equations (Lane, 2015). The

226 calibration curves over the tested range for AFB1, AFB2, AFG1 and AFG2 were found linear

227 assenting in between 0.9990−0.9997. LOD and LOQ values of the utilized method were

228 calculated according to S/N=3 and S/N=10, respectively (Miller, & Miller, 1993). LOD and

229 LOQ values were found in the range of 0.09−0.14 and 0.27−0.42 µg/kg, respectively in the

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230 tested dry fruits and edible nuts. The achieved values satisfy the MTL of AFs as regulated by

231 various international regulated authorities including EU and USA.

232 The efficiency of the HPLC method was evaluated using the standard addition method so called

233 sample spiking. Briefly, 25 grams of uncontaminated sample (verified prior via ELISA) were

234 spiked with three concentration levels (0.5, 5, 10 µg/kg) of each toxin at least 1hour prior to

235 analysis. The spiked samples were extracted and analyzed using the above mentioned procedure.

236 Average recoveries were found in the range of 90.2−92.2%, which were within the tolerable

237 limits as regulated by AOAC International, Codex Alimentarius and EU Standard (Codex, 1995;

238 AOAC International, 2002; Commission of the European Communities, 2006). Measurement

239 uncertainty of the method was calculated as combined uncertainty (Uc) with covering factor k =

240 2 (e.g. 2Uc) at a 95% confidence level. Food and feed safety laboratory is ISO-17025 accredited

241 by the Pakistan National Accreditation Council (PNAC) and is actively participating on regular

242 basis in proficiency testing conducted by Food analysis performance assessment scheme

243 (FAPAS®). General laboratory performance (GLP) was evaluated by participation in FAPAS®

244 via test no. 04188/2012, 04216/2013, 04250/2014 and z-scores were -0.2, 0.3 and 1.6

245 respectively. All z-score values were within the acceptable limits thereby showing good

246 laboratory performance.

247

248 2.6 Statistical analysis

249 The differences in aflatoxin (AFs) concentration in dry fruits and edible nuts were compared by

250 ANOVA using GraphPad Prism 7 software followed by post hoc Tukey's Honest Significant

251 Difference (HSD) Test also called Tukey's Multiple Comparison Test. Mean values were

252 considered significantly different at P ≤ 0.05. All experimental data was reported as the mean ±

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253 standard deviation (SD) of analysis in triplicate using SPSS software (IBM, PASW 117 Statistics

254 19, USA). The value of R2 was determined using regression/correlation analysis.

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276 3. Results and discussion

277 The natural occurrence of AFs contamination in various dried fruits and edible nuts are

278 summarized in Table 2. The results indicated that 165 (26%) samples out of 624 tested samples

279 were perceived infected with AFs at different concentration. The contamination range was in

280 between 0.22−30.11 µg/kg, with an average of 0.85 ± 0.26 µg/kg, which is significantly lower

281 than maximum tolerated limits (MTL = 4 and 10 µg/kg) for dry fry fruits and nuts, respectively

282 as assigned by EU.

283 The overall results revealed that, in 459 (74%) samples, AFs contamination was found below

284 than detectable limit (0.12µg/kg). AFs contamination in 99 (15%) samples was in the range

285 between 1−4 µg/kg. Furthermore, 28 (4.5%) samples exhibited AFs level between 4−10 µg/kg.

286 Only 6 (1%) tested samples contained AFs levels more than 10 µg/kg. It was found that, 610

287 (97.7%) samples retained AFs contamination less than MTL as imposed by EU in dry fruits and

288 nuts. Moreover, remaining 14 (3.3%) samples showed AFs contamination more than the MTL of

289 EU. However, these samples were fit for human utilization with reference to the MTL (20 µg/kg)

290 assigned by the USA (Food and Drug Administration and Food and Agriculture Organization).

291 Only 2 (0.3%) samples were found un–fit for human utilization as per MTL of USA, owing to

292 AFs contamination of 30.11- and 28.98 µg/kg, respectively.

293 It was notified that AFs contamination level in different types of dry fruits and edible nuts was

294 significantly inconsistent. For instance, the highest AFs contamination was found in almond

295 (50.0%) and peanuts (50.0%) with the mean level of 1.22 and 2.37 µg/kg, respectively. However,

296 the lowest AFs mean level found in dates and raisins was 0.24 µg/kg. Similarly, the maximum

297 AFs level in pistachio was 30.11µg/kg. In contrast, dried figs exhibited AFs equivalent to 3.44

298 µg/kg. The variation in AFs contamination in dry fruits and edible nuts was due to diverse

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299 agricultural and harvesting practices, soil nature, microbial flora, variable temperature and

300 humidity all over the country.

301 It was observed that AFs contamination in dried fruits and nuts varied among chronologically

302 due to variable climatic conditions during the cultivation time periods. The variation in the

303 distribution of AFs contamination along with time is illustrated in Table 3. The results indicated

304 that, no significant differences were found during the year 2011-2015. However, the

305 contamination of AFs was slightly higher in the year 2012. During this year, the average

306 temperature and relative humidity (RH) was 27°C and 60%, respectively. Moreover, heavy rain

307 falls were recorded equivalent to 481 mm in the year 2012 (Pakistan Meteorological Department,

308 2016). These conditions are considered favorable for the growth of fungal pathogens and

309 ultimately AFs production.

310 The level of AFs contamination in dry fruits and edible nuts are strongly influenced by climatic

311 conditions throughout the cultivation seasons. During the study, the climatic information such as

312 average temperature and relative humidity data was collected from the Pakistan Meteorological

313 Department (2016). The AFs level in dry fruits and edible nuts was significantly differ among

314 different months (Table 4). During the months of July, August and September, the

315 contamination of AFs was higher in comparison to other months. These months are considered

316 monsoon season in Pakistan. During these months, the average temperature / RH was 33°C /

317 50%, 31°C / 58%and 30°C / 43%, respectively. These climatic conditions are supposed as

318 favorable conditions for the AFs producing fungi. For instance, Pitt, & Hocking (2000)

319 previously mentioned that the suitable temperature for the growth of Aspergillus species in the

320 ranges from a minimum of 10.0–12.8°C and a maximum of 43.0–48.8°C, with an optimal of

321 around 33.8°C. According to Paterson, & Lima (2009), the favorable temperature for the

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322 production of AFs is approximately 28°C and inhibited at 37°C. The achieved results from the

323 present study were found in accordance with the above mention previous studies.

324 Different authors have been reported the contamination levels of AFs in different varieties of dry

325 fruits and nuts from various countries including Pakistan. For instance, Masood, Iqbal, Asi, &

326 Malik (2015) reported from Pakistan that 132 (43%) of dry fruits and edible nuts out of 307

327 samples were contaminated with AFB1 and total AFs. The contamination range was 21.50 µg/kg

328 with an average of 4.90 µg/kg. Luttfullah, & Hussain (2011) also reported from Pakistan that the

329 contamination range of AFs was 20%−50% in different verities of dry fruits and nuts. However,

330 only a single peanut and a lone pistachio sample showed highest AFs contamination

331 corresponding to 14.5-and 14 µg/kg, respectively. Furthermore, in 13 samples the contamination

332 level was above the recommended MTL of EU. On the other hand, the results of the present

333 study showed that AFs content (26%) is relatively lower in comparison with the previous studies.

334 Mphande, Siame, & Taylor (2004) reported from Botswana that 78% of raw peanut were

335 contaminated with AFs concentrations between from 12- to 329 µg/kg. In China, peanut samples

336 were highly infected with AFs. The maximum level was 437.09 µg/kg with an average of 80.27

337 µg/kg (Wang & Liu (2006)). On the other hand, the current study demonstrated fairly low AFs

338 contamination comparatively. For instance, about50% peanuts samples were contaminated

339 ranging from 0.32−28.98 µg/kg with an average level of 2.37±0.36 µg/kg.

340 Several studies have been reported that dried raisins do not exhibit the satisfactory surface or

341 environment for the growth of Aspergillus flavus and AFs production. However, in the present

342 study, small quantity of AFs was found in dried raisins. The achieved results showed that 12

343 (13%) samples out of 90 tested samples were found positive with AFs. The concentrations

344 ranged from 0.24−4.86 µg/kg. Only a single (1%) samples exceeded the MTL of 4 µg/kg set by

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345 EU regulations for AFs (Commission of the European Communities, 2010). Our data regarding

346 AFs in raisins is similar with the study from Iran that have reported an incidence of 19% samples

347 contamination with AFs (0.85−2.14 µg/kg) with a mean of 0.27 µg/kg (Reazai et al., 2014). In

348 addition, the frequency and average contamination level of AFs in dates was higher in

349 comparison to our previous study, which reported AFs contamination in 08/170 samples with an

350 average level of 0.05 ± 0.26 µg/kg (Asghar, Aftab, & Iqbal 2016).

351 About 20%, 10% and 10% samples were found contaminated with AFs in dried mulberry, dried

352 melon seeds and foxnut (makahna), respectively. The contamination range in dried mulberry,

353 dried melons seeds and foxnut (makahna) was1.36−2.22, LOD−1.58 and LOD−1.36 µg/kg with

354 a mean level 0.36, 0.32 and 0.27 µg/kg, respectively. Different authors have been reported the

355 high levels of AFs in dried mulberry and dried melon seeds in comparison to present study. For

356 instance, Luttfullah, & Hussain (2010) reported that 26% samples of dried mulberry were

357 contaminated with AFs. The contamination level was in the range 1−3.5 µg/kg with a mean level

358 2.22µg/kg. Masood, Iqbal, Asi, & Malik (2015) analyzed 13 samples of melon for AFs

359 contamination. About 31% samples showed AFs contamination ranged from LOD−9.52 µg/kg

360 and the mean was 2.91µg/kg. However, still no study is available on the occurrence of AFs

361 contamination in foxnut. In dates samples, the incidence of AFs was 15%, ranging from

362 0.24−5.87 µg/kg and average contamination levels was 0.24 ± 0.10. Out of 170 date samples, 8%

363 exceeded the MTL fixed for AFs by EU. In comparison, analysis of 20 date samples in Yemen

364 Republic revealed that only 02 samples contained AFB1, ranged from 110-180 µg/kg (Alghalibi,

365 & Shater, 2004).

366 In dried figs, assessment reports from Morocco and UK showed that about 30% and 8.6%

367 samples of fig were positive with AFs, respectively (Juan, Zinedine, Molto, Idrissi, & Man˜es,

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368 2008; Sharman, Patey, Bloomfield, & Gilbert, 1991). The results from current study are

369 analogous with these previous studies. About 30% samples were contaminated with AFs, ranged

370 from 0.69−3.44 µg/kg with an average 0.54 ± 0.22 µg/kg. On the other hand, Karaca, & Nas

371 (2006) reported from Turkey, a high incidence of AFs content in dried fig ranged from 117.9–

372 471.9 µg/kg. In pistachio samples, Iranian Ministry of Health published a report indicated that

373 761 (9.6%) samples out of 7926 pistachio contain AFB1 higher than 10 µg/kg (Food and Drug

374 Control Labs, 2002). In Qatar, Abdulkadar, Al-Ali, & Al-Jedah (2000) reported37% prevalence

375 of AFs within the range of 0.53–289 µg/kg. Our results partially agree with the above mentioned

376 finding. The incidence of AFs in pistachio samples was 33% in the range between 0.98−30.11

377 µg/kg.

378 Another study from India reported that 80%, 70%, and 80% samples of peanuts, almonds and

379 pistachios were contaminated with AFB1 and ranged LOD−6.80, LOD−6.2 and LOD−10,

380 respectively (Nagarnaik et al., 2014).In the present study, the occurrence rate of AFs was lower

381 in peanuts (50%), almonds (50%) and pistachios (33%). However, the contamination range of

382 AFs was higher in comparison with the above mentioned study. In another study from Iran,

383 Janati, Beheshti, Asadi, Mihanparast, & Feizy (2012) analyzed30 dried apricots and 15

384 prunes/dried plum for AFs. About 30% and 13.33% samples of apricot and prunes/dried plum

385 contained AFB1 more than 0.2 µg/kg with range from 0.21−5.33 and 0.23−1.17 µg/kg,

386 respectively. The present findings are in accordance with the above mentioned studies.

387 In walnuts, the incidence of AFs contamination has been indicated by various authors around the

388 world. For instance, 74.3% samples of walnuts contaminated with AFs, ranged from 0–112.8

389 µg/kg with an average level of 11.68 µg/kg (Imani, & Farahani, 2012). In India, 21% walnuts

390 samples contained AFB1in the range of 140–1220 µg/kg (Punam, Singh, & Shukla, 2008). The

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391 present data showed that the quality of walnuts is better in comparison with the above mentioned

392 studies. About 37% samples showing AFs contamination, ranged from 0.68−6.66 µg/kg and a

393 mean level of 1.08 µg/kg.

394 As per above mentioned surveys reports, AFs contamination in different verities of dry fruits and

395 edible nuts frequently occur and could be a hazard to the human health. This indicates the fact

396 that the use of poor harvesting and management practices, mechanical damage during the

397 harvesting, limited curing, sub-standard quality materials and inadequate storage and

398 transportation conditions assisted the growth of fungal pathogens. As a result, enhanced the level

399 of AFs are observed. The detection of AFs indicated that there is a strong need for further

400 investigation, routine analysis and to conduct a monitoring program or project as per food quality

401 control standard and procedures. Furthermore, it has been reported that the AFs contamination is

402 associated with the growth of Aspergillus mould at post-harvest level on insufficient stored stuffs

403 (Jay, 1992). Therefore, dry fruits and edible nuts should be stored in sterilize seal bags, dry and

404 cool environment.

405 To enhance the export of dry fruits and edible nuts from Pakistan, it is essential to take measures

406 such as good agricultural practices, appropriate drying, handling, packaging along with adequate

407 storage and transportation. Furthermore, different control measures such as dry heating, roasting

408 and conventional microwave baking, gamma radiation, exposure to UV, hydrogen peroxide

409 treatment and storage at different climatic condition have shown varying degree of destruction in

410 AFs count and could be utilized as food safety measures.

411

412

413

414

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415 4. Conclusion

416 In the present study, a total of 624 samples of dry fruits and edible nuts were collected from

417 Pakistan and investigated for the occurrence of AFs contamination. A total of 165 (26%) samples

418 were positive with AFs contamination ranged from 0.22−30.11 µg/kg with an average level of

419 0.85 ± 0.26 µg/kg. The level of AFs in dry fruits and edible nuts was significantly below than the

420 permissible limits as assigned by the EU and USA and fit for human utilization. Furthermore, it

421 was also concluded that dried fruits and edible nuts are contaminated in different countries and

422 exceeding the regulation levels of EU and USA. Therefore, it is essential for regulatory

423 authorities to develop and apply new and efficient methodology for the control of AFs

424 contamination and investigate on regular basis. It is also essential to develop and apply some

425 food quality control procedures and standard such as good manufacturing practices (GMPs),

426 good agricultural practices (GAPs) and the hazard analysis and critical control point (HACCP)

427 system to minimize the risk of fungal growth and ultimately prevent the formation of AFs.

428

429 Acknowledgement

430 This research work was supported by Food and Feed Safety Laboratory, PCSIR Laboratories

431 Complex, Karachi.

432

433

434

435

436

437

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1 Table Captions and Tables

2
3 Table Captions
4
5 Table 1. Method performance of HPLC method for the quantification of aflatoxins in dry fruits

6 and edible nuts.

7 Table 2. Occurrence of total AFs contamination in different dry fruits and edible nuts samples

8 collected from different areas of Pakistan during 2012 to 2015a.

9 Table 3. Yearly distribution of total AFs contamination in different dry fruits and edible nuts

10 samples collected from different areas of Pakistan during 2012 to 2015a.

11 Table 4. Aflatoxins contamination in dry fruits and edible nuts in different months with respect

12 to average temperature and relative humidity in Pakistan.

13

14

15

16

17

18

19

20

21

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22 Table 1. Method performance of HPLC method for the quantification of aflatoxins in dry fruits

23 and edible nuts.

LOD LOQ Recovery RSD (%) Measurement

Commodities Uncertainty
(µg/kg) (µg/kg) Range (%)a (n=20)
(µg/kg)
Almond 0.09 0.27 94.2–97.2 2.21 0.16

Apricot 0.11 0.33 92.0–96.2 1.58 0.12

Dates 0.12 0.36 95.2–98.2 1.68 0.08

Raisins 0.12 0.36 91.8–96.2 2.11 0.14

Dried mulberry 0.10 0.30 94.2–97.4 1.54 0.18

Pistachio 0.09 0.27 93.2–96.4 2.54 0.36

Dried plum 0.12 0.36 90.2–97.0 1.22 0.54

Dried figs (Injeer) 0.10 0.30 93.5–96.4 1.36 0.14

Dried melonseeds 0.12 0.36 94.1−97.7 2.64 0.36

Walnuts 0.11 0.33 92.2–95.6 2.22 0.14

Pine nuts 0.10 0.30 95.1–98.1 1.64 0.33

Peanuts 0.11 0.33 92.0–96.1 2.44 0.19

Foxnut (Makakna) 0.12 0.36 90.2–95.1 1.98 0.12

24 LOD: limit of detection; LOQ: limit of quantification; RDS: relative standard deviation calculated as
25 standard deviation/mean × 100.
26 aRecovery values are within the acceptable ranges of AOAC, Codex Alimentarius and EU standard: 1–10

27 µg/kg = 70–110%; >10 µg/kg = 80–110%

28

29

30

31

32

33

34

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35 Table 2. Occurrence of total AFs contamination in different dry fruits and edible nuts samples

36 collected from different areas of Pakistan during 2012 to 2015a.

Samples in concentration range, n (%),

Positive
Tested (µg/kg) Mean ± SD Range
Commodities samples
samples (µg/kg) (µg/kg)
n (%)
<LODb <1 1−4 4 to 10 10 to 20d ≥ 20

Almond 10 5 (50) 5 (50) 1 (10) 3 (30) 1 (10)c 0 0 1.22** ± 0.24 0.33−4.98

Dried apricot 65 21 (32) 44 (68) 3 (5) 11 (17) 5 (8)c 2 (3) 0 1.02** ± 0.15 0.31−11.11

Dates 170 25 (15) 145 (85) 12 (7) 10 (6)c 3 (2) 0 0 0.24* ± 0.08 0.24−5.87

Raisins 90 12 (13) 78 (87) 4 (4) 7 (8)c 1 (1) 0 0 0.24* ± 0.10 0.22−4.86

Dried mulberry 10 2 (20) 8 (80) 0 2 (20)c 0 0 0 0.36* ± 0.12 1.36−2.22

Pistachio 126 41 (33) 85 (67) 1 (1) 29 (23) 9 (7)c 1 (1) 1 (1) 1.18** ± 0.38 0.98−30.11

Dried plum 10 3 (30) 7 (70) 0 2 (20)c 1 (10) 0 0 1.31** ± 0.18 2.36−7.41

Dried figs (Injeer) 10 3 (30) 7 (70) 1 (10) 2 (20)c 0 0 0 0.54* ± 0.22 0.69−3.44

Dried melonseeds 5 1 (5) 4 (80) 0 1 (5) 0c 0 0 0.32* ± 0.00 1.58

Walnuts 8 3 (37) 5 (63) 1 (12) 1 (12) 1 (12)c 0 0 1.08** ± 0.32 0.68−6.66

Pine nuts 95 38 (40) 57 (60) 7 (7) 25 (26) 5 (5)c 1 (1) 0 0.96** ± 0.20 0.39−13.64

Peanuts 20 10 (50) 10 (50) 2 (10) 5 (25)c 2 (10) 0 1 (5) 2.37*** ± 0.36 0.32−28.98

Foxnut (Makakna) 5 1 (5) 4 (80) 0 1 (5) 0c 0 0 0.27* ± 0.00 1.36

Total 624 165 (26) 459 (74) 32 (5) 99 (15) 28 (4) 4 (0.6) 2 (0.4) 0.84 ± 0.26 0.22−30.11
37 LOD = limit of detection; SD = standard deviation
38 aAnalysis in triplicate, reported as mean ± SD
39 b Below the limit of detection

40 cSample below European Union standard

41 dSample below USA (FDA and FAO) standard

42 Mean (average) * or ** or *** values along the column are significantly different at p ≤ 0.05
43
44
45
46
47
48
49
50

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51 Table 3. Yearly distribution of total AFs contamination in different dry fruits and edible nuts

52 samples collected from different areas of Pakistan during 2012 to 2015a.

Samples in concentration range, n (%),

Tested Positive
(µg/kg) Mean ± SD* Range
Year samples samples
(µg/kg) (µg/kg)
(n) n (%)
<LODb <1 1−4c 4 to 10 ≥ 10 ≥ 20d

2012 170 53 (31) 117 (69) 9 (5) 36 (21) 5 (3) 1 (0.6) 2 (1) 0.98** ± 0.26 0.66−30.11

2013 150 37 (25) 113 (75) 12 (8) 21 (14) 4 (3) 0 0 0.45* ± 0.18 0.24−5.77

2014 195 47 (24) 148 (76) 2 (1) 34 (17) 10 (5) 1 (0.5) 0 0.62* ± 0.11 0.42−10.56

2015 109 28 (26) 81 74) 9 (8) 8 (7) 9 (8) 2 (2) 0 0.90** ± 0.21 0.36−13.64
Tota
624 165 (26) 459 (74) 32 (5) 99 (15) 28 (4) 4 (0.6) 2 (0.3) 0.84 ± 0.26 0.22−30.11
l
53 LOD = Limit of detection; SD = Standard deviation
54 aAnalysis in triplicate, reported as mean ± SD
55 b Below the limit of detection

56 c Sample below European Union standard

57 d Sample below USA (FDA and FAO) standard

58 * Mean (average) values along the column are significantly different from ** Mean (average) values at p ≤ 0.05
59

60

61

62

63

64

65

66

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67 Table 4. Aflatoxins contamination in dry fruits and edible nuts in different months with respect

68 to average temperature and relative humidity in Pakistan.

Tested
Ta RHb Positive Mean ± SD* Range
Month Samples
(°C) (%) Sample n (%) (µg/kg) (µg/kg)
(n)
Jan 12.5 46 50 10 (20) 0.52* ± 0.16 0.39−3.44

Feb 14.5 41 45 8 (18) 0.46* ± 0.19 0.48−6.66

Mar 20 29 52 10 (19) 0.39* ± 0.10 0.52−2.22

Apr 26 23 35 8 (23) 0.54* ± 0.13 0.32−4.86

May 31 20 46 11 (24) 0.42* ± 0.16 0.22−5.87

Jun 34 22 50 10 (20) 0.69* ± 0.23 0.36−11.11

Jul 33 50 55 21 (38) 1.40** ± 0.38 0.42−13.64

Aug 31 58 62 28 (45) 2.02** ± 0.42 1.08−30.11

Sep 30 43 54 22 (41) 1.81** ± 0.24 0.88−28.98

Oct 25 33 58 15 (26) 0.74* ± 0.27 0.66−7.41

Nov 19 41 54 10 (19) 0.54* ± 0.14 0.43−4.98

Dec 13.5 49 63 12 (19) 0.66* ± 0.21 0.44−6.66

69 aMonthly average temperature in Pakistan
70 bMonthly average relative humidity in Pakistan
71 * Mean (average) values along the column are significantly different from ** Mean (average) values at
72 p ≤ 0.05
73

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