Mycotoxins in grains (products), Gansu province, China and risk assessment

ABSTRACT This study aimed to estimate the dietary exposure towards mycotoxins of residents in Gansu province, China, from 2014–2020 through surveillance data on mycotoxins in grains and grain products. Fumonisin B1 (FB1), Deoxynivalenol (DON), 3- and 15-Acetyl-deoxynivalenol (3-ADON and 15-ADON), Tentoxin (TEN), Tenuazonic acid (TeA) and Zearalenone (ZEN) in 863 grains and grain products were detected by HPLC-MS and UPLC-MS. DON was the most detected mycotoxin of all samples. For women, the average dietary exposure to DON was 1.49 μg/kg bw/day, with 55.8% of the individuals eating dried noodles exceeding tolerable daily intake. The hazard quotient values were 1.24–12.60, so greater than 1 for DON at the average, 90th percentile, 95th percentile, and maximum levels: 44.6% of the HQ values for men and 45.7% for women were greater than 1.


Introduction
Several filamentous fungi produce secondary metabolites known as mycotoxins (Wu et al. 2014;Mahdjoubi et al. 2020).More than 1,000 different fungi are producing over 4,000 mycotoxins.Deoxynivalenol (DON), zearalenone (ZEN) and fumonisins (FBs) are recognised as being of significant public health concern (Pitt et al. 2012;Ortiz et al. 2013).Mycotoxins are generally present in food and animal feed.According to the Food and Agriculture Organization of the United Nations (FAO), about 25% of the cereals are contaminated with mycotoxins around the world (Bhat and Miller 1991;Adeyeye et al. 2022).They have different biological effects on animals and humans, including liver and kidney toxicity, teratogenicity and carcinogenicity (Gruber-Dorninger et al. 2017;Amirahmadi et al. 2018).
Mycotoxins were obtained through harvesting, drying, processing, packaging, storage and other procedures (Marin et al. 2013;Rodríguez-Carrasco et al. 2013).Not only that, the physical and chemical characteristics of the food itself (pH, water activity, endogenous enzymes and intake) were also associated with the generation of mycotoxins (Reyneri 2006;Rastegar et al. 2017).Practical measures should be taken to avoid mycotoxin contamination of food, such as crop rotation, planting resistant types, control of plant diseases biologically and chemical treatment (Awad et al. 2010;Li et al. 2019;Guo et al. 2020).Mycotoxins were physically eliminated through enzymatic detoxification or degraded through adsorption (Zhu et al. 2017).Still, inappropriate storage conditions, particularly increasing water activity may stimulate fungal growth and the likelihood of mycotoxins production (Adetunji et al. 2014;Ojuri et al. 2018).
Many countries and organisations have established maximum limits (MLs) for mycotoxins in grains and grain products to monitor mycotoxins and assess the risk of exposure.In China the ML for DON in grain and grain products is 1000 μg/kg and the ML for Zearalenone (ZEN) in unprocessed wheat and maize is 60 μg/kg.In the European Union the ML for FBs is 4000 μg/kg in unprocessed maize and the ML for DON is 1250 μg/kg in wheat (Mahdjoubi et al. 2020;Ji et al. 2023).The Scientific Committee on Food of the European Commission established tolerable daily intake (TDI) for DON and FB1 at 1 μg/kg bw/day (Food and Agriculture Organization of the United Nations 2010).
Mycotoxins are commonly found in grains and grain products (Duarte et al. 2010;Society 2011;Pereira et al. 2014).Gansu province is in the northwest region and grains are the traditional staple food for residents.Also, grain consumption data in Gansu province have been increasing from 2014-2020.The level of exposure to mycotoxins in the human body depends on the concentrations of contaminants and the quantity of food consumption.Thus, because of the huge production and consumption of grains and grain products, people could be vulnerable to mycotoxins in Gansu province (Chen, Tan et al. 2022).
Worldwide a lot of studies have evaluated the potential exposure of mycotoxins for the population (Sirot et al. 2013;Kamala et al. 2017;Gilbert-Sandoval et al. 2020;Iqbal et al. 2021;Martins et al. 2021).In China there are also some related studies (Zhang et al. 2021;Chen, Zhang et al. 2022;Ji, Xiao, Lyu et al. 2022).However, few studies have been undertaken in Gansu province in northwestern China, where DON and its derivatives are the major mycotoxins in grains and grain products and FB1, ZEN and two Alternaria toxins have been generally concerned recently (Chen et al. 2021;Eriksen et al. 2021;Chen, Tan et al. 2022).Therefore, this study is intended to evaluate the dietary exposure of residents in Gansu province through the consumption of grains and grain products and to supply support for guiding residents to have a proper diet and building basic information for dietary exposure assessment.

Sample preparation
Monitoring data from 2014 to 2020 in Gansu province were collected in according with the standard method of the China National Manual for Monitoring the Risk of Food Contaminants and Harmful Factors (CFSA 2020).The number of collected samples was 130, 88, 89, 198, 83, 150 and 130, respectively, from 2014 to 2020.A total of 868 samples (142 wheat, 11 flakes of oats, 455 wheat flour, 165 dried noodles, 50 maize flour, 40 fried instant noodles, 5 non-fried instant noodles) of about 1000 g were randomly collected from wholesale food markets, supermarkets and producer markets in Gansu province.The samples were ground to powder using a grinder (Haifuda, Beijing, CHN) and well mixed.Then a 200 g subsample was taken.The milled product was kept in a sealed plastic bag at 4°C prior to analysis.Wheat flour and maize flour subsamples were directly used for mycotoxins extraction, after mixing.

Mycotoxin analysis
All mycotoxin standards were purchased from Fermentek (Qingdao, CHN).Acetonitrile, methanol and ammonium bicarbonate, all of UPLC grade, were supplied by Sigma (Changchun, CHN).Water for the UPLC mobile phase was purified successively by a Millipore Milli-Q system (Thermo Scientific, Beijing, CHN).High-performance liquid chromatography and mass spectrometry (HPLC-MS) were used to assess the concentrations of DON, 3-and 15-Acetyldeoxynivalenol (3-ADON and 15-ADON), FB1 and ZEN.An appropriate amount of internal isotopic standard was added to the samples, followed by extraction with aqueous acetonitrile solution and ultrasonic treatment.After centrifugation the supernatant was purified with Mycosep TM227 (Romer labs, Beijing, CHN), concentrated to a fixed volume and then analysed by the HPLC-MS system.The HPLC was equipped with a HPLC BEH C18 column (100 mm × 2.1 mm × 1.7 µm) at 40°C (Waters, Shanghai, CHN).The injection volume was 5 μl and the flow rate was 0.35 ml/min.A linear gradient program started from 4% B in the first 4 min and kept 20% B constant from 4 to 7 min.Then, B was linearly increase to 100% from 7 to 10 min.Finally, the initial rate of A and B parts was reestablished within 2.2 min.MS/MS analysis was performed from a 9710C mass spectrometer (Agilent, Beijing, CHN).The MS parameters were set as: capillary voltage of 2.5kV, Ionization mode of negative ion mode (ESI-), ion source temperature of 150°C, dysventilation gas temperature of 500°C, Collision gradient (1.5), desolventification gas flow of 900 L/hr, Blowback flow of 30 L/hr, electronic multiplication voltage of 650 V, collision chamber pressure of 3.0E-03 mbar.

Health risk assessment
The estimated daily intake (EDI) of a mycotoxin for a consumer was calculated by , assessed by age and gender, including adults over 18 and young children aged 7 to 18. Young children aged 7 to 18 and adults over 18 were susceptible to varying degrees, whereas 7-18 years old is a critical period of physical development, consuming a large amount of food to satisfy nutritional needs, thus being the most vulnerable group.
Based on the Dietary Guidelines for Chinese Residents of the Bureau of Statistics in China concerning the consumption data of cereals in Gansu province, the average cereal consumption was 0.425 kg/day for adults and 0.300 kg/day for 7-18 year old children.According to the Report on Nutrition and Chronic Diseases of Chinese Residents (2020), the average weight of men and women aged 18 and over in China is 69.6 kg and 59.0 kg, respectively.According to the "Comparative Physical Fitness Report of Chinese and Japanese teenagers in 2017," the average weight of boys and girls between the ages of 7 and 18 were 50.4 kg and 49.5 kg, respectively.
For the majority of the mycotoxins, TDIs were established by the FAO/World Health Organisation Joint Expert Committee on Food Additives (JECFA).The hazard quotient (HQ) was calculated by HQ = EDI/TDI and hazard index (HI), defined by the European FoodSafety Authority (EFSA 2013) as the sum of the individual HQ data for each individual compound in a mixture with no genotoxic or carcinogenic properties, as HI = ΣHQ.

Statistical analysis
All data were organised in Microsoft Excel 2010 and IBM SPSS.25 was performed to address and analyse the statistics, which were expressed in the form of mean, median and percentile.The left-censored data were substituted for half of LOD which was regarded as upper bound (EFSA 2010).

Method validation and quality control
Method validation and quality control were performed according to the National Manual for Risk Monitoring of Food Pollutants and Harmful Factors (CFSA 2020).The limits of detection (LODs) and the limits of quantification (LOQs) were measured according to signal-to-noise ratios (S/N) of 3:1 and 10:1, respectively.The LODs and LOQs were below 0.8 µg/kg and 2.0 µg/kg, respectively, for DON, 3and 15-ADON and ZEN, whereas these were 0.5 µg/kg and 1.5 µg/kg, respectively, for TeA and 0.1 µg/kg and 0.3 µg/kg, respectively, for TEN.Spiked recovery rates were applied for quality control (Table S2).

Mycotoxins in grains and grain products
The results of FB1, DON, 3-and 15-ADON, TEN, TeA and ZEN are shown in Table 1.In addition, concentrations of DON and ZEN in different food categories and concentrations of Alternaria toxins in wheat and wheat flour were depicted in Figure 1.
Figure 2 indicated the contribution of grains and grain products to the exposure of DON and ZEN.From all samples, dried noodles had the highest contribution to ZEN (54.5%) exposure.In contrast, wheat flour had the highest contribution to DON (51.8%).Wheat and rolled oats contributed slightly to the total exposure, with 5.9% and 3.8%, respectively, to DON.

Co-occurrence of mycotoxins
Grains and grain products can be contaminated with various fungi, producing different mycotoxins.The cooccurrence of multiple mycotoxins in grains and their products were displayed in Table 2. From all samples 6.3% (31/493) were contaminated with two mycotoxins.The predominant combination was the co-occurrence of DON and 15-ADON (2.4%).DON and its derivatives coexisted also with TEN, TeA and ZEN.

Dietary exposure in adults and young children
Dietary exposure levels were assessed in men and women over 18 and young children aged 7-18, by gender.The three-line table used MB as the description of EDI and the same age group by gender through the box plots shown in Figures S1 and S2.Regarding the estimated average dietary consumption, grains and grain products, dried noodles and wheat flour were the primary sources of mycotoxin exposure.The EDIs of DON were the highest of all mycotoxins for adults and young children.Table S3 showed the average and 95th percentile exposure to mycotoxins through consumption of grains and grain products for men.The average and 95th percentile of DON intake was 1.24 μg/kg bw/day and 3.99 μg/kg bw/day, respectively, with 44.6% of the individuals exceeding the TDI of 1.0 μg/kg bw/day, whereas 50.0% of the individuals eating dried noodles exceeded the TDI.For women, according to Table S4  the average and 95th percentile of DON intake was 1.49 μg/kg bw/day and 4.71 μg/kg bw/day, respectively, with 55.8% of the individuals who ate dried noodles exceeding the TDI and with 54.7% of the individuals who consumed wheat flour as raw materials exceeding the TDI.The average and 95th percentile of EDIs for FB1 were 0.34 μg/kg bw/day and 3.22 μg/kg bw/day, respectively, with 6.0% of the individuals exceeding the TDI of 2.0 μg/kg bw/day.Young children aged 7-18 are equally exposed to mycotoxins through food intake.For boys, as shown in Table S5, the average and 95th percentile intake of EDIs for TeA were 0.08 μg/kg bw/day and 0.76 μg/kg bw/day, respectively and for ZEN these were 0.03 μg/kg bw/day and 0.31 μg/kg bw/day, respectively, with 1.4% of the individuals exceeding the TDI of 0.25 μg/kg bw/day.For girls, Table S6 shows the average and 95th percentile of the EDIs for DON were 1.41 μg/ kg bw/day and 11.98 μg/kg bw/day, respectively, with 45.6% exceeding the TDI.The average of ZEN, 3-and 15-ADON were 0.04 μg/kg bw/day, 0.05 μg/kg bw/day and 0.04 μg/kg bw/day, respectively and the 95th percentile of ZEN, 3-and 15-ADON were 0.35 μg/kg bw/ day, 0.12 μg/kg bw/day and 1.23 μg/kg bw/day, respectively.

Risk characterization
Consumer's risk was assessed by calculating HQ and HI data for separate mycotoxins and combined exposure to FB1, DON, TeA, 15-ADON, 3-ADON, TEN, TeA and ZEN in grains and grain products in young children and adults.HQ values for 3-ADON, TeA and ZEN were less than 1 in young children and adults.HQ values were higher than 1 for DON (1.24-12.6)at the average, 90th percentile, 95th percentile and maximum levels.HQ values were greater than 1 for 44.6% of men, for 45.7% of women, for 49.8% of boys and for 48.4% of girls.The HQ value of dried noodles was as high as 12.60, resulting in a HQ value above 1 for 57.0% of women.Table 3 shows HI values of DON and 15-ADON were higher than 1 for 45.6% of men, for 52.6% of women, for 47.7% of boys and for 50.0% of girls.In addition, the HI of cumulative DON and 15-ADON exposure was higher than the HQ of individual exposure to DON or 15-ADON.

Analytical results
The number of mycotoxins detected in wheat, wheat flour and dried noodles was relatively high, as well as the detection rate.DON was found most, also at the highest level.According to standard GB2761-2017, only 1 of 86 dried noodles samples exceeded the limit for DON of 1000 μg/kg (1.2%).Compared with 6.1% in Zhejiang Province (Chen, Zhang et al. 2022) the overrun rate of DON in Gansu province was relatively low.The reason for DON being the major mycotoxin of concern might be that the consumption of wheat and its products were relatively high and wheat was susceptible for damage by Fusarium head blight, which may cause high levels of DON.In addition, as DON is produced most common by Fusarium species, less 3-and 15-ADON can be formed (Chen, Zhang et al. 2022).FBs were only detected in maize flour, which is characteristic for maize, particularly when grown in warmer regions.
Alternaria toxins belong to the so-called "emerging" mycotoxins and gather more concern due to their abundance, occurrence and toxicity.Although previous studies have investigated their occurrence in a variety of food matrices, the available data did not accurately assess their risks because of lacking toxicological data such as TDI values (Marin et al. 2013;Ji, Xiao, Jin et al. 2022).In this study, the dietary risk of Alternaria mycotoxins in humans was estimated by using the threshold of toxicological concern (TTC) approach proposed by EFSA (2011).Generally, although other mycotoxins than DON in dried noodles were effectively controlled under governmental supervision, there are certain health risks to consider.
In this study co-occurrence of two mycotoxins was the foremost combination type, mostly in combination with DON (De Ruyck et al. 2015;Smith et al. 2016).Juan et al. (2016) indicated a probability of 34.5% (301/872) for cooccurrence of two mycotoxins, but in our study it was 6.3% (31/493).This can be attributed to the fact that mycotoxigenic fungal growth and the production of secondary metabolites is highly dependent on many ecophysiological and abiotic factors (Gallo et al. 2016;Magan and Medina 2016).Temperature, CO 2 and water activity (a w ) represent the main factors that have a strong influence on fungal development, transcriptional activation of mycotoxin biosynthetic and multiple mycotoxins production (Medina et al. 2015;Lv et al. 2019; Cervini et al. 2021; Aloui et al. 2023).Fusarium species produce a vast number of mycotoxins in infected grains and grain products, with the most relevant ones from a food and food safety perspective being DON (15-ADON, 3-ADON), FBs and ZEN (Ismaiel and Papenbrock 2015;Ferrigo et al. 2016).Mycotoxins produced by Fusarium are more likely to coexist with other mycotoxins, especially under conditions of low a w (Marín et al. 2004).Cooccurrence of mycotoxins is an important issue that needs to be investigated because synergetic effects could increase associated health hazards (Kifer et al. 2021;Lin et al. 2021).

Risk assessment
In this study 45.3% of the boys, 45.5% of the girls, 44.6% of the males and 50.5% of the females exceeded the TDI value for DON from grain and grain products.Han et al. (2014) showed that for Shanghai, China, 11.6% of the population exceeded the TDI value for DON, 3-ADON and 15-ADON from wheat flour.Otherwise, in Anhui Province 94.8% of the population people aged 18-59 exceeded the TDI value (Wang et al. 2019).These studies suggested that DON might not be a severe problem in Gansu Province.However, the EDI of dried noodles was more than ten times the TDI value, indicating that consumption of this product might have to be reduced.FB1 was exclusively found in maize flour, pointing out that people suffering from coeliac disease, which consume corn products instead of wheat, are exposed to higher levels, as shown by Cano-Sancho et al. (2011, 2012) and Dall'asta et al. (2012).
Alternaria toxins include a small percentage of the more than 70 mycotoxins produced by this genus and among them TEN and TeA are the vital types (Meena et al. 2017;Crudo et al. 2019;Aichinger et al. 2021;Chen et al. 2021).Alternaria toxins have been detected in many different food matrices, including fruits, vegetables and beverages.However, the detection rate of TEN and TeA varied between food types.In sunflower seeds TeA had the highest detection rate (Lattanzio et al. 2022) and for TEN the highest detection rate was reported for cereals (Reinholds et al. 2021).Research on natural occurrence of Alternaria toxins in citrusbased products collected from China in 2021 indicated that the TeA had the highest positive rate (76.5%) when compared with TEN (Han et al. 2023).A study on green coffee showed that TeA was the most often detected Alternaria toxin in this raw material and finished products (62%) and at the highest concentrations whatever the food material (Mujahid et al. 2020).According to the European Food Safety Authority (EFSA 2011), one of the contributions of dietary exposure to Alternaria toxins are grain and grain-based products, especially wheat products.In our study the concentrations and EDIs of TEN and TeA due to consumption of grain and grain products were relatively low.
HQ data above 1 indicate a risk for the population.For DON 44.6% of men and 49.8% of women were at  S7-S10).In Shandong province, HQ values higher than 1 for men and women aged 18-59 were found at frequencies of 21.3% and 18.3%, respectively.Because of their co-occurrence, the risks caused by 3and 15-ADON and FB1 to human health must be addressed.Multi-exposure might lead to additive, synergistic or antagonistic toxic effects.For example, ZEN was frequently reported having antagonistic effects on the toxicity of some multi-exposed mycotoxins.Thus, the health risks from this multi-exposure were yet to be further explained (Speijers and Speijers 2004;De Ruyck et al. 2015;Smith et al. 2016).
The methods used throughout this study were based on food consumption data and mycotoxin concentrations to assess dietary exposure.These methods were relatively limited and need to consider individual differences, uncertainties of food consumption data and other factors.Although this study made extensive efforts to assess the main food of dietary exposure in Gansu province, the food categories need to be more diverse, such as including vegetables, fruits and drinks, as to avoid underestimation of the exposure level of the population (Zeng et al. 2015).In addition, more about the health risks related to cumulative exposure could be studied.Regardless these shortcomings, our assessment of the exposure due to grain and grain products in Gansu province could provide a starting point for the relevant institutions to protect people's health better.

Conclusions
Research on mycotoxins in food in Gansu Province has been focused on a few mycotoxins only.Therefore, it is needed to apply multimethod analysis to screen mycotoxins in cereals and cereal products to provide more comprehensive and reliable information on mycotoxin contamination.It was concluded that women were more susceptible to exposure to mycotoxins than men.To reduce mycotoxin exposure it is essential to setup a standardised system for prevention and control from field planting, harvesting and storage to grain processing.Additionally, consumption of food with lower mycotoxin content can be promoted to protect human health.

Figure 1 .
Figure 1.DON and ZEN concentrations in different food categories and concentrations of mycotoxins in wheat and wheat flour.

Figure 2 .
Figure 2. The contribution of different food categories to the exposure by DON and ZEN.

Table 1 .
Incidences of mycotoxins in different food categories.

Table 3 .
Hazard index data for combined exposure to mycotoxins in grains and grain products for the different population groups Gansu province.