Occurrence, Dietary Exposure Scenarios and Risk Assessment of Aflatoxins from Dried Fruits and Chocolates in Armenia

Pipoyan, Davit; Beglaryan, Meline; Arshakyan, Yepraqsya; Harutyunyan, Bagrat

doi:10.3390/foods15081329

Open AccessArticle

Occurrence, Dietary Exposure Scenarios and Risk Assessment of Aflatoxins from Dried Fruits and Chocolates in Armenia

Center for Ecological-Noosphere Studies, Abovyan 68, Yerevan 0025, Armenia

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Foods 2026, 15(8), 1329; https://doi.org/10.3390/foods15081329

Submission received: 9 March 2026 / Revised: 4 April 2026 / Accepted: 7 April 2026 / Published: 10 April 2026

(This article belongs to the Special Issue Advances in Food Toxin Analysis and Risk Assessment)

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Abstract

This study aimed to estimate dietary exposure to aflatoxins (AFs) and characterize its associated risks through the consumption of dried fruits and chocolates among the adult population of Yerevan, the capital city of Armenia. Asflatoxin B1 (AFB1) and total AFs were determined using HPLC in 10 composite samples of widely consumed dried fruits and chocolates, prepared by pooling 100 individual sub-samples into 5 dried fruits and 5 chocolate composites. Individual consumption data were obtained via food frequency questionnaires and were stratified by consumer groups and percentiles. Exposure scenarios (lower-, middle-, upper-bound and detected mean) were applied, and risk was assessed using the margin of exposure (MOE) approach with a BMDL₁₀ of 0.4 μg/kg bw/day. The study findings revealed that dried fruits had higher contamination levels (detected mean content of 10 μg/kg AFB1, 15 μg/kg total AFs) compared to chocolates (detected mean content of 0.5 μg/kg AFB1, and 0.9 μg/kg total AFs), resulting in lower MOE values despite smaller consumption quantities. Detectable AFs in dried fruits from open (street) markets exceeded the EU maximum limits, while Armenia currently lacks national regulatory limits for these products. MOEs were below 10,000 for most consumption groups, indicating a potential public health concern. This research emphasizes the urgent need for continuous monitoring and the establishment of harmonized national regulatory limits for AFs in dried fruits.

Keywords:

mycotoxins; food safety; regulatory gap; health concerns

1. Introduction

Mycotoxin contamination remains a challenge for food safety and public health worldwide [1,2]. Although mycotoxins are a large and diverse group of toxic secondary metabolites, only a limited number have emerged as major public health concerns due to their frequent occurrence in food and potent toxic effects [3]. Among these, aflatoxins (AFs), produced mainly by Aspergillus flavus and Aspergillus parasiticus, are some of the most hazardous compounds [4,5]. The International Agency for Research on Cancer (IARC) has classified naturally occurring mixtures of AFs as carcinogenic to humans and the metabolite aflatoxin M1 (AFM1) in milk as possibly carcinogenic [6]. Aflatoxin B1 (AFB1), the most prevalent and potent in the AFs group, is classified as a Group 1 human carcinogen [7]. Due to its genotoxic and non-threshold carcinogenic properties, regulatory authorities have not established a tolerable daily intake, and international risk assessment frameworks emphasize that exposure should be reduced to the lowest level reasonably achievable to protect public health [8]. In this context, assessment of the health risks associated with aflatoxin contamination cannot rely solely on analytical measurements of hazard levels in food commodities, as consumption patterns critically influence exposure; hence, an integrated exposure-based approach is required [9]. This is particularly important for food categories for which regulatory limits are absent or inconsistent across different countries. Dietary exposure assessment, therefore, provides a scientifically robust framework for estimating population intake, identifying high-risk groups and supporting risk-based public health and food safety interventions.

Recent studies conducted in Armenia have reported the presence of AFs in several food categories, including cereals, nuts, spices and milk, with measurable dietary exposure among the population [10,11]. These findings indicate that aflatoxin contamination is a persistent food safety concern that may contribute to cumulative dietary exposure. Moreover, exposure estimates from these studies highlight risks among population subgroups characterized by higher consumption of specific commodities. Despite these findings, several potentially important dietary contributors remain insufficiently investigated. Dried fruits and chocolates represent two such critical food groups. These commodities are among the food products particularly vulnerable to aflatoxin contamination due to multiple factors throughout the food chain, including pre-harvest conditions, post-harvest handling, drying, storage and processing. Studies from global reviews have reported that the mentioned food items are commonly contaminated with aflatoxigenic species, primarily Aspergillus flavus and Aspergillus parasiticus, as well as other mycotoxigenic fungi, depending on environmental and storage conditions [12,13,14]. Moreover, dried fruits and chocolates are integral components of dietary habits and snacking practices [15,16]. Consequently, these commodities may substantially contribute to cumulative dietary exposure to aflatoxins, yet remain insufficiently considered in exposure-based risk assessment studies in Armenia. From a regulatory perspective, notable differences further highlight the need for such studies. In the European Union (EU), maximum levels have been established for AFB1 and for the sum of AFs (B1, B2, G1 and G2) in dried fruits [17]. In contrast, the Eurasian Economic Union (EAEU), of which Armenia is a member, currently only regulates AFB1 in certain confectionery products, including chocolate [18], and has not established limits for dried fruits or for total AFs in these product categories. In this regulatory context, exposure assessment is essential for estimating potential health risks and guiding consumer protection strategies tailored to national consumption habits and patterns.

Overall, despite the dietary relevance and susceptibility to aflatoxin contamination of dried fruits and chocolates, comprehensive investigations integrating contamination data with dietary exposure assessment in Armenia are currently lacking. Therefore, this study aimed to estimate dietary exposure to aflatoxins (AFs) and characterize the associated health risks through the consumption of dried fruits and chocolates among the adult population of Yerevan, the capital city of Armenia, where approximately one-third of the country’s population resides.

2. Materials and Methods

2.1. Sampling

Random sampling of dried fruits and chocolates was conducted in Yerevan, Armenia, following the principles of the total diet study (TDS) methodology [19,20], with the aim of obtaining representative composite samples reflecting the average dietary exposure of the adult population. Products were selected based on national food consumption data and market availability, prioritizing frequently consumed varieties commonly purchased by consumers. The sampling covered widely consumed dried fruits (major varieties including dried apricots, plums, figs, apples, and raisins) and chocolates (major varieties with different cocoa contents and ingredients). Sampling was carried out from October to November, 2023. A total of 100 individual sub-samples were randomly purchased from major retail markets and online grocery stores, ensuring the representation of leading brands and producers with substantial market presence. In addition, dried fruit samples were collected from open (street) markets, which are outside of official surveillance systems. Following the TDS methodology, individual sub-samples of each food category were pooled to generate 10 composite samples, including 5 dried fruit and 5 chocolate composites. Each composite sample was prepared by pooling at least 10 randomly selected individual sub-samples to ensure representative coverage of the product group. Since AFs can be heterogeneously distributed in food matrices, pooling multiple randomly collected individual sub-samples into composite samples was intended to reduce sampling variability and to provide an estimate of average contamination for the population. Although this approach may average out localized high contamination, it is more suitable for exposure assessment than for lot compliance evaluation.

2.2. AFs’ Quantification

Composite samples of dried fruits and chocolates were analyzed in the “Standard Dialog” laboratory, accredited under the ISO/IEC 17025 international standard for testing and calibration laboratories. The contents of AFB1 and total AFs (sum of B1, B2, G1 and G2) were determined using high-performance liquid chromatography (HPLC) methods following GOST standards [21,22]. Sample preparation, including clean-up, followed the procedures specified in these standards to ensure accurate determination of AFs in complex food matrices. Samples (10 g) were extracted with methanol–water (80:20, v/v), followed by filtration and clean-up using immunoaffinity columns specific for aflatoxins. The analyses were conducted using an HPLC system with a fluorescence detector. The chromatographic conditions were as follows: a C1 reverse-phase column (150 × 4.6 mm, 5 μm), a mobile phase consisting of water–methanol–acetonitrile (60:20:20, v/v/v), a flow rate of 1.0 mL/min, an injection volume of 20 μL and detection with post-column derivatization (excitation of 365 nm and emission of 435 nm). Certified reference standards of AFs (AFB1, AFB2, AFG1, AFG2) were obtained from a commercial supplier Sigma-Aldrich, USA. Standards were prepared in methanol (HPLC grade) and stored at −18 °C in the dark. Quality control procedures included analysis of blank samples, use of spiked recovery samples, and regular calibration verification. Measurement uncertainty was estimated according to EURACHEM guidelines, considering contributions from calibration, repeatability and recovery. The expanded uncertainty (k = 2) was found to be within ±15–25%, depending on the analyte and matrix. Recovery tests were performed using blank matrix samples spiked at three concentration levels for each aflatoxin. The spiking levels for AFB1 were 1.0, 5.0 and 10.0 µg/kg, for AFB2 were 0.5, 2.5 and 5.0 µg/kg, for AFG1 were 1.0, 5.0 and 10.0 µg/kg, and for AFG2 were 0.5, 2.5 and 5.0 µg/kg. These levels were selected to cover the regulatory limits and expected contamination range. The obtained recoveries ranged between 72% and 108%, depending on the matrix and analyte. The analytical method provided a limit of detection (LOD) of 0.1 μg/kg and a limit of quantification (LOQ) of 0.3 μg/kg.

2.3. Dietary Exposure and Risk Assessment

The deterministic risk assessment was conducted through the estimation of daily intake and margin of exposure (MOE) values for both AFB1 and total aflatoxins (AFs).

Dietary exposure to AFB1 and total AFs through the consumption of the studied food items (dried fruits, chocolates) was assessed by calculating the estimated daily intake (EDI):

EDI = C_content × C_food/BW

(1)

where EDI—estimated daily intake of AFs through consumption of the studied food item (μg/kg bw /day), C_content—content of AFs in the food item (μg/kg), C_food—daily consumption of the food item (kg/day), and BW—body weight (kg). In this study, the average body weight (BW) for the studied adult population was 70.48 kg [10].

EDI values were determined under several scenarios considering different approaches to left-censored data treatment using the LOQ value of 0.3 μg/kg [23]. Specifically, in the lower bound (LB) scenario, results below the limit of quantification (LOQ) were assigned a value of 0, in the middle bound (MB) scenario, results below the LOQ were replaced with a value equal to LOQ/2, and in the upper bound (UB) scenario, results below the LOQ were replaced with the LOQ value [23]. In addition, the detected mean (DM) was calculated using only samples with detectable and quantifiable levels.

EDI values were calculated using individual-based consumption data on dried fruits and chocolates derived from a previously conducted food frequency questionnaire (FFQ), an anonymous dietary survey among adult residents of Yerevan (n = 545, including 310 females and 235 males) aged 18–83 years [10]. For the present study, reported portion sizes and frequencies of dried fruit and chocolate consumption were used to estimate daily intake and associated exposure. To address different consumption patterns within the population, consumers of dried fruits and chocolates were classified into three groups using the Visual Binning tool in IBM SPSS Statistics (version 22.0). In addition to this consumer-group-based approach, percentile-based estimations were conducted including the 25th (P25), 50th (P50), 75th (P75) and 95th (P95) consumption percentiles, representing increasing levels of exposure from low to high consumers.

Risk characterization for AFB1 and total AF exposure through dried fruit and chocolate consumption was performed using the margin of exposure (MOE) approach [24]:

MOE = RP/EDI

(2)

The benchmark dose lower confidence limit (BMDL₁₀) of 0.4 μg/kg bw/day for AFB1 was used as the reference point (RP) in Equation (2) to calculate the MOE of AFs. This BMDL₁₀, established by the EFSA, refers to the liver carcinogenicity posed by AFB1 exposure in rats [7]. MOE values were calculated for different exposure scenarios (LB, MB, UB, DM), consumption percentiles (P25, P50, P75, P95) and consumer groups (low, medium and high consumers). According to the EFSA risk assessment framework, and in view of the genotoxic and carcinogenic properties of aflatoxins, MOE values below 10,000 (MOE < 10,000) raise concerns for public health [7].

3. Results

3.1. Aflatoxins in Dried Fruits and Chocolates

Aflatoxins were detected in one composite sample of dried fruits and in three composite samples of chocolate; however, the contamination levels were notably higher in dried fruits than in chocolate (Table 1). The LB, MB and UB substitution scenarios provide a realistic range of possible average contamination levels in the overall sample set by addressing the non-detected and non-quantified results. Figure 1 presents the mean detected contents (DM) of aflatoxin B1 (AFB1) and total aflatoxins (AFs) in the analyzed composite samples. The detected mean (DM) represents the mean calculated considering only positive samples with quantifiable aflatoxin levels, excluding non-detected and non-quantified (<LOQ) results. Because the detected mean for dried fruits was based on a single positive composite sample, calculation of a standard deviation (SD) was not possible for that product category. For chocolates, the AFB1 SEM was 0.1 μg/kg (0.5 ± 0.1 μg/kg), and for total AFs, it was 0.12 μg/kg (0.9 ± 0.12 μg/kg).

3.2. Estimated Daily Intake (EDI) and Margin of Exposure (MOE) of Aflatoxins

Dietary intake of AFB1 (Table 2 and Table 3) and total AFs (Table 4 and Table 5) through the consumption of dried fruits and chocolates was estimated using deterministic approaches, including LB, MB, UB and DM scenarios, to address the uncertainty associated with left-censored data (<LOQ). These approaches provide a comprehensive characterization of exposure, ranging from conservative to realistic and worst-case scenarios. The EDI values of AFB1 and total AFs varied depending on both contamination assumptions (i.e., LB, MB, UB and DM scenarios) and consumption levels.

Due to the absence of a safe threshold for genotoxic carcinogens, risk assessment of aflatoxins relies on the margin of exposure (MOE) approach, as recommended by the EFSA [7]. In the current study, risk characterization based on the MOE approach showed clear differences in aflatoxin-related health concerns associated with dried fruits and chocolates, reflecting consumption levels, consumer groups and percentiles (Table 6, Table 7, Table 8 and Table 9). MOE values below 10,000, derived from the ratio of the BMDL₁₀ to estimated exposure, are considered to represent a possible concern for public health [7].

Table 2. Estimated daily intake (EDI) of AFB1 via consumption of dried fruits.

Studied Food Items	Consumer Groups	Consumption Levels (g/day)	Chronic EDI (μg/kg bw/day) of AFB1
Studied Food Items	Consumer Groups	Consumption Levels (g/day)	LB	MB	UB	DM
Dried Fruits	All (n = 229 consumers)	11.88	3.37 × 10⁻⁴	3.57 × 10⁻⁴	3.78 × 10⁻⁴	1.69 × 10⁻³
	Group 1 (34% of consumers)	2.66 (range 1.23–4.24)	7.55 × 10⁻⁵ (3.49 × 10⁻⁵–1.20 × 10⁻⁴)	8.00 × 10⁻⁵ (3.70 × 10⁻⁵–1.28 × 10⁻⁴)	8.46 × 10⁻⁵ (3.91 × 10⁻⁵–1.35 × 10⁻⁴)	3.78 × 10⁻⁴ (1.75 × 10⁻⁴–6.02 × 10⁻⁴)
	Group 2 (33% of consumers)	7.65 (range 4.28–12.95)	2.17 × 10⁻⁴ (1.21 × 10⁻⁴–3.68 × 10⁻⁴)	2.30 × 10⁻⁴ (1.29 × 10⁻⁴–3.90 × 10⁻⁴)	2.43 × 10⁻⁴ (1.36 × 10⁻⁴–4.12 × 10⁻⁴)	1.09 × 10⁻³ (6.07 × 10⁻⁴–1.84 × 10⁻³)
	Group 3 (33% of consumers)	25.82 (range 13.37–50.4)	7.33 × 10⁻⁴ (3.79 × 10⁻⁴–1.43 × 10⁻³)	7.77 × 10⁻⁴ (4.02 × 10⁻⁴–1.52 × 10⁻³)	8.21 × 10⁻⁴ (4.25 × 10⁻⁴–1.60 × 10⁻³)	3.66 × 10⁻³ (1.90 × 10⁻³–7.15 × 10⁻³)
	P25	3.29	9.34 × 10⁻⁵	9.90 × 10⁻⁵	1.05 × 10⁻⁴	4.6710^-4
	P50	7.25	2.06 × 10⁻⁴	2.18 × 10⁻⁴	2.30 × 10⁻⁴	1.03 × 10⁻³
	P75	16.04	4.55 × 10⁻⁴	4.83 × 10⁻⁴	5.10 × 10⁻⁴	2.28 × 10⁻³
	P95	40.41	1.15 × 10⁻³	1.22 × 10⁻³	1.28 × 10⁻³	5.73 × 10⁻³