Introduction

Pakistan ranks sixth among wheat-producing countries, with approximately 24 million tons cultivated in 2010 (Iqbal et al., 2013). Although wheat is grown throughout the country, maximum yields are achieved in Punjab and Sindh. Because of outdated agricultural methods and the leakage of toxins from unsuitable storage, wheat becomes infected and contaminated with mycotoxins, rendering it unfit for human consumption (Nada et al., 2022). According to Asif I. (2024), the average annual wheat production in Pakistan, which is a staple food, amounts to 24 million tons, which is sufficient to satisfy the domestic demand. The contamination of wheat and other food products with mycotoxins is a major public health issue because of their high toxicity (Gozzi et al., 2024). Research has been carried out globally on mycotoxins to assess their occurrence in various food items and the extent of their severity (Bouelet et al., 2023). Food entities can be severely contaminated by mycotoxins, as some of these toxins are classified as highly carcinogenic by the International Agency of Research on Cancer (IARC). Mycotoxins can have immunosuppressive, teratogenic, and neurotoxic effects on the human body (Latham et al., 2023). Well-known mycotoxins are aflatoxins (AFs), ochratoxins (OTAs), trichothecene or deoxynivalenol (DON), and fumonisins (FUMs) (Agriopoulou et al., 2020; Aloui et al., 2023; Milani, 2013).

In many developing nations, molds grow and spread as a result of a combination of humid environmental conditions and inadequate drying, that is, because of poor pre-harvest and post-harvest conditions (Feldman and Levy, 2023). This leads to unacceptable concentrations of different mycotoxins, especially AFs, in corn, wheat, rice, red chili, tree nuts, turmeric, groundnuts, and various other agricultural products. The presence of mycotoxins in diverse food items has been linked to prolonged health effects, such as instances of cancers and immune deficiency. Currently, numerous mycotoxins have been recognized, which have attracted the attention of scientists because of their severe impacts on human health and presence in food (Patriarca and Pinto, 2017; Sadef et al., 2023).

Fungal species such as Aspergillus and Fusarium, which produce mycotoxins, can develop on various crops and food items (Nazir et al., 2021). They have the ability to penetrate deep into food, rather than being limited to growth on the outer surface. Depending on their types and quantities, mycotoxin contamination can manifest as acute or chronic venoms, even at very low to high doses. Food-borne mycotoxins have adverse effects, which lead to serious illnesses that manifest as acute symptoms shortly after the ingestion of contaminated food. The allowable concentration of AFs in wheat is 4 µg/kg (Nisa et al., 2016). However, according to the European Commission Regulation (EC, 2010) and Codex Alimentarius Commission (2012), the permissible levels for DON, FUMs, and OTAs are, respectively, 1000 µg/kg, 1000 µg/kg, and 5 µg/kg.

It is of utmost importance to create sensitive, rapid, and reproducible methods for identifying the occurrence of considerable mycotoxins in staple crops. The thin layer chromatography (TLC) method can be used for the qualitative analysis of mycotoxins in different food products. It is necessary to validate the method and develop a rapid detection method for mycotoxins. Reliable methods like HPLC and ELISA are extremely useful for quantifying mycotoxins. These tools will undeniably assist the wheat, rice, and corn sectors in identifying mycotoxins for the quality assurance of food commodities (Jaimez et al., 2000; Turner et al., 2015). The present study involves identifying different mycotoxins in wheat collected from local stores in Lahore in the summer and winter seasons. The purpose of this study is to create an awareness about mycotoxin contamination in wheat in local shops that have improper storage. This could help in preventing health hazards.

Materials and Methods

Wheat varieties were chosen for mycotoxin assessment based on their nutritional and economic significance. The following four district types of mycotoxins were chosen for investigation:

1. Total AFs (B1+B2+G1+G2)

2. OTAs (A+B+C)

3. DON (Vomitoxin)

4. FUMs (FB₁+FB₂+FB₃)

Study of seasonal variations

The production of mycotoxins relies on climate, which is considered a primary driving factor in the development of fungi and mycotoxin contamination. From this perspective, nations such as Pakistan are more vulnerable to climatic influences such as temperature changes, drought, flooding, and humidity (Magan et al., 2011). Soil fertilizers, various pesticides, contamination by harmful heavy metals, and numerous potential causes for concern in developing nations are also accountable for mycotoxin contamination. The research was categorized into the following two primary seasonal groups for sample collection:

• Summer/humid/rainy seasons: When temperature may rise from 25ºC to as high as 45°C.

• Winter/foggy/meek weather: Temperature may go down from 20ºC to 4°C.

Lahore is situated on the western side of the flat floodplain along the River Ravi. Lahore is located at a longitude range of 74°-01’ to 74°-39’ east and a latitude range of 31°-15’ to 31°-42’ north. It is the second largest and the second most densely populated district in Pakistan (Rana and Bhatti, 2018). The average range of the lowest and highest temperatures throughout the year were from 5.9°C to 22°C. The summer season has lower relative humidity compared to the winter season. The months of April, May, and June are extremely hot and arid in Lahore, often accompanied by dust storms. The monsoon season begins in late June or early July, bringing humid and muggy conditions along with intense rainstorms. In Lahore, July, August, and September are primarily hot and humid. Cold and dry conditions persist from October to March (Khan et al., 2021).

Collection of samples

The Lahore district has been chosen for wheat collection because of its high consumption in this region of Punjab, Pakistan. Twenty wheat samples (n=20) were obtained from local stores in Lahore, where the storage conditions were observed to be extremely inadequate and unhygienic. Ten samples were collected during summer and ten during winter. All samples were collected by assessing their physical characteristics, storage environments, hygiene, and contamination. The temperature recorded during sample collection ranged from 25 to 40ºC in summer and 13 to 22ºC in winter, with humidity levels at 45–69% in summer and 40–55% in winter. Since mycotoxins are not evenly distributed and wheat grains can contain areas with elevated levels of mycotoxins, an appropriate sampling strategy was implemented. The sampling method outlined in AOAC no. 977.16 was adhered to (Trucksess, 2005). A 1 kg sample was obtained using a probe from two to three different diagonal locations within the container. Subsequently, the gathered wheat samples were processed through a sample divider to 200 g after thoroughly homogenizing the sample. Each sample was once more thoroughly mixed and ground into a fine powder prior to analysis (Nisa et al., 2016; Yi et al., 2022).

Storage conditions

The wheat samples were stored in airtight polyethylene bags and kept at 4°C prior to analysis (Cheng et al., 2022).

Chemicals used in the study

All chemicals utilized in the current study were of analytical grade. Acetonitrile (sourced from Merck, Darmstadt, Germany) was used for the extraction of mycotoxins and also as a mobile phase.

Preparation of standard solutions

Various stock solutions for AFs (50 μg/kg), OTA (25 μg/kg), DON (6 mg/kg), and FUMs (6 mg/kg) were freshly prepared. The corresponding standards were diluted in a mixture of acetonitrile and water (20:80, v/v) and kept at −20°C (Irakli et al., 2017).

Specifications of equipment

The Agilent 1200 system (Agilent Technologies, Urdorf, Switzerland) employed reversed phase-HPLC for quantifying various mycotoxins. The HPLC was outfitted with a Rheodyne injector valve (Irakli et al., 2017) featuring a 20 µL loop and a pump. The diode array and fluorescent detectors were linked in series within the HPLC setup. Agilent Chemstation Software of Agilent Technologies, version B.04.01 was utilized for the capture and assessment of various chromatograms.

Protocol followed

Multiple mycotoxins were analyzed in wheat samples using HPLC equipped with a diode array detector (DAD). One kilogram of every wheat sample was collected and mixed thoroughly (Santos et al., 2022) for homogenizing the sample. The sample was crushed in a grinding mill (Romer Labs). A conical flask received 5 g of the homogenized ground wheat sample, to which 50 mL of 70% acetonitrile was added. The sample solution was agitated on a shaker for 30 min. The sample solution underwent filtration, and the collected extract was filtered again using filter paper (Whatman 4). The sample extracts were subsequently filtered one more time using 0.5-micron syringe filters prior to injection into the HPLC C18 column. Twenty microliters of the extract was injected into the HPLC system. The mobile phase employed consisted of acetonitrile: water (45:55 v/v). The flow rate was 1 mL/min with a duration of 15 min. The excitation and emission wavelengths were 254 nm and 460 nm, respectively.

Statistical analysis

IBM Statistics Version 22.0 was utilized for the statistical evaluation of mycotoxins in wheat (Zahra et al., 2017). Mean and standard deviation were also calculated through descriptive analysis. A one-way analysis of variance (ANOVA) at α = 0.05 was conducted to assess the impact of seasons on levels of mycotoxin in wheat samples collected from local stores in Lahore, Pakistan.

Results and Discussion

Wheat is widely used as a key ingredient in various food items such as cakes, biscuits, and breads. Various wheat-based food items can become contaminated with mycotoxins, compromising the quality of these products (Hedayati et al., 2016). Various mycotoxins in wheat were measured to assess the highest level of contamination. Mycotoxins (AFs, OTAs, DON, and FUMs) were measured through HPLC. The chromatograms for various mycotoxins are presented in Figures 1–4.

Figure 1. Aflatoxins HPLC chromatogram.

Figure 2. Ochratoxins HPLC chromatogram.

Figure 3. Deoxynivalenol HPLC chromatogram.

Figure 4. Fumonisins HPLC chromatogram.

Contamination levels of total AFs, OTA, DON, and FUMs in wheat samples collected from Lahore were recorded at 0.56–115.19 μg/Kg, 0.12–24.13 μg/kg, 0.01–112.00 mg/kg, and 0.02–2.81mg/kg, respectively (Table 1).

The concentration of mycotoxins satisfied the assumptions of one-way ANOVA. Levene’s test confirmed that the homogeneity of variances was satisfied (Table 2) and indicated that the data are significant for AFs across two seasons. For OTA and DON, it is again notable, whereas for FUM it is not noteworthy.

The outcomes of the ANOVA are presented in Table 3. ANOVA indicated a significant difference among groups with p = 0.046, whereas for OTA, DON, and FUM, the results are nonsignificant. While elevated temperature and humidity are crucial for mycotoxin production, some samples were found to be contaminated during winter at levels exceeding permissible limits because of inadequate storage conditions (Taheri et al., 2012).

Descriptive analysis showed that in summer 115.19 μg/kg AF, 24.13 μg/kg OTA, 112.00 mg/kg DON, and 1.19 mg/kg FUM were present in wheat as maximum concentration while in winter 4.43 μg/kg AF, 6.77 μg/kg OTA, 33.69 mg/kg DON, and 2.81 mg/kg FUM were present in wheat as maximum concentration.

Lutfullah and Hussain (2012) examined the levels of AF contamination in various cereals in Pakistan. Maize samples exhibited the highest contamination at 18.5μg/kg, while wheat, barley, rice, sorghum, and broken rice showed contamination levels of 15.5μg/kg, 12.6μg/kg, 10.8μg/kg, 9.4μg/kg, and 8μg/kg, respectively. Various studies globally have reported the determination of multiple mycotoxins in wheat. In two different studies performed in China, researchers discovered DON, Afs, and FUMs in concentrations of 221.4 μg/kg, 0.1–0.8 μg/kg, and 0–400 μg/kg, respectively (Sun et al., 2011; Sun and Wu, 2016). In similar research carried out in Nigeria by Ezekiel and Sombie (2014), the levels of AFs in analyzed snacks were found. The levels of AF contamination ranged from 0.8 to 3.5 ppb in all selected food samples. In wheat-based pastas, 1.2 ppb of mean AFs were found.

A study in Mazandaran Province, Iran, analyzed 118 wheat samples for the quantification of various Aspergillus species and discovered a 64.6% contamination rate of Aspergillus flavus. According to FAO/UNICEF/WHO and the Standard Administration of Iran, the allowable limit for AFs in wheat is 30 μg/kg (Feyzi et al., 2017). AF B1 and G1 were detected at approximately 2.54% and 3.39%, with averages of 1.53 and 2.75 μg/kg, respectively. The highest level of contamination detected in wheat was 7.10 μg/kg.

The overall findings indicated that the samples gathered in the summer season exhibited higher levels of contamination compared to those collected in winter (Figures 5 and 6). Six samples of summer wheat and four from winter were found to have mycotoxin contamination exceeding acceptable limits. In wheat from the Lahore district, contamination levels of AFs, OTA, DON, and FUMs were found to be 55%, 45%, 40%, and 40%, respectively, while 20%, 25%, 20%, and 15% of the samples exceeded permissible limits for AFs, OTA, DON, and FUMs, respectively (Table 4).

Figure 5. Mycotoxin contamination in wheat samples collected from the Lahore district during the summer season.

Figure 6. Mycotoxin contamination in wheat samples collected from the Lahore district during the winter season.

In Pakistan, a comparable study was carried out by Majeed (2018), in which n=124 wheat samples were gathered from the Punjab Province. Wheat samples contained multiple mycotoxins. The contamination percentages were determined to be 43% AFB1, 41% AFB2, 11% OTA, 21% DON, along with 29% FB1, 18% FB2, and 12% FB3. AF was identified as the most common toxin in wheat, a finding that aligns with the current study. Likewise, a study on AF contamination in wheat kernels was conducted (Asghar et al., 2016), revealing 26% AFB1 and 7% AFB2 contamination in wheat samples. In total, just 2.7% of AFs in wheat exceeded the allowable levels. The contamination percentage of wheat with mycotoxins in the current study is elevated compared to both investigations, potentially because the samples were taken from shops with very inadequate storage conditions.

The season may greatly affect fungal growth as it is clear from the study that wheat samples collected in summer are more contaminated with mycotoxins than the wheat samples collected in winter (Figure 7). In a study, it was found that higher temperatures from 20 to 40ºC are favorable for toxin production (Sousa et al., 2019) while toxin production was minimum at 5ºC. The present study is in accordance with the reported work as contagion of mycotoxins was more in summer than in winter. In another study on feed, the effect of temperature on Aspergillus flavus production was studied, and it was found that in high temperature months, the incidence ranged between 66 and 93% while in cold temperature months, it reduced to 40%. High temperature and moisture may increase fungal contamination (Asif M, 2024; Iram et al., 2019; Tittlemier, et al., 2025).

Figure 7. Presence of mycotoxins in wheat during different seasons.

Wheat consumption is increasing day by day because of the increase in population. This research was basically conducted to check the presence of multiple mycotoxins in wheat available in local shops. The results were discouarging as 10 out of 20 samples were contaminated beyond permissible levels and were declared as unfit on the basis of one toxin beyond acceptable range.

Conclusion

The presence of mycotoxins in different food commodities needs attention as the incidence of liver cancer, allergies, stomach ulcers, etc., is on the rise. Improper handling, poor storage facilities, and adverse environmental conditions may contaminate food entities with various mycotoxins. The dire necessity to protect food commodities has prompted all countries to pay attention to contamination by mycotoxins in different food entities. Wheat and wheat products are among the major crops of Punjab, Pakistan. Special attention is required to avoid mycotoxin contamination in these products before exporting them. Overall, 20%, 25%, 20%, and 15% of wheat samples were contaminated with AFs, OTA, DON, and FUM, respectively, and were unfit according to EU permissible levels for human consumption because of the presence of multiple mycotoxins in wheat samples. Six samples in the summer season and four samples in the winter season were infected with mycotoxins beyond acceptable limits. This is because warm temperature may increase fungal growth. High temperature and high humidity are favorable conditions for the growth of mycotoxins. Five out of 20 (25%) samples were contaminated with multiple mycotoxins. The presence of high levels of mycotoxins in wheat samples is highly alarming. Proper handling, suitable environmental conditions, and careful storage can reduce the chances of mycotoxin contamination in wheat and wheat products. Inspection on a constant basis is needed to detect any hazardous and dangerous health circumstances. The awareness must be given to farmers and people to inspect the quality of wheat and wheat products in order to avoid mycotoxin contamination.