Triazole fungicides residues and their inhibitory effect on some trichothecenes mycotoxins excretion in wheat grains

: Wheat is one of the global strategic crops and ranks third in terms of cereals production. Wheat crop is exposed to many fungal infections during the cultivation stages, some of which have the ability to secrete numbers of toxic secondary metabolites that threaten the quality of grains, consumer health, producer economics and global trade exchange. Fifty-four random samples were collected from wheat which originated from different countries. The samples included 14 of soft wheat to study the extent of their contamination with Deoxynivalenol (DON) and T-2 toxin by auto-ELISA technology and r-biopharm Microtiter plate. All samples were contaminated with DON toxin except one sample, and the contamination average ranged between 40.7 and 1018.8 µg kg -1 . The highest contamination rates were in Lithuanian and the lowest in Indian wheat. Meanwhile the highest average level of T-2 toxin contamination was in Lithuanian wheat grains with a rate of 377.4µg kg -1 and the lowest in Polish wheat with an average of 115.3µg kg - 1 . GC-MS/MS and multiple reaction monitoring mode (MRM), was used to detect 15 triazole derivatives in collected samples, which may be used to combat fungal diseases on wheat during the growing season. Only 9 derivatives were found; Simeconazole, Penconazole, Hexaconazole, Cyproconazole, Diniconazole, Tebuconazole, Metconazole, Fenbuconazole and Difenoconazole. These derivatives varied according to the origin of wheat samples as well as their concentration whereas, another 6 derivatives were not detected in all samples. A direct inverse relationship was found between the DON concentration in the samples and the residues of Simeconazole, Penconazole, Diniconazole, Tebuconazole, Metconazole, Fenbuconazole and Difenoconazole and the T-2 toxin showed the same relationship except for Tebuconazole. The safe and rational use of some triazole derivatives may be a new approach and a promising strategy not only to reduce plant diseases and their problems, but also to get rid of some mycotoxins as grains contaminates.


Introduction
The wheat is one of the strategic crops in the world to feed both humans and animals. The edible wheat was classified to common wheat (Triticum aestivum), club wheat (T. compactum), durum wheat (T. durum) and many other species [1]. The wheat is the third most important cereal crop after maize and rice in the food security basket, and the global wheat production reached to 762.2 metric tons in 2019/2020 [2,3].
Wheat plants mostly are attacked by several phyto-pathogenic fungi during cultivation, harvesting and storage, which is associated closely with reduction in production and quality. It may extend to consumer's health as well as economic value of crop [4,5]. Fusarium pathogens is the most important seed borne fungi which have been correlated with wheat seedling blight and root rot. However, different species of Fusarium were found in both husks and grains by ratio 3:1, respectively [6,7]. Fusarium graminearum, F. avenaceum, F. culmorum, F. poae were associated with wheat kernels and other cereals grains, meanwhile, F. cerealis, F. equiseti, F. sporotrichoides, F. tricinctum were less frequent [8]. The most species of Fusarium fungi had ability to secret various types of mycotoxins as Fumonisins, Zearalenone and Trichothecenes [9]. Trichothecenes have over 150 toxins produced by various fungi and are considered incurring general cytotoxic effects, had ability to inhibit protein synthesis at ribosomes during all three stages of protein synthesis [10].
There are multiple protocols for fungal disease management but the fungicides are still at the forefront of these treatments and have the ability to control pathogenic fungi and their undesirable effects. Fungicide groups differ in active ingredients and most of them reduce the growth and prevent sporulation as triazoles. The mode of action of triazoles depends on preventing production of sterols which is are considered the main components of fungal cell membranes [11,12]. Triazoles group is one of the largest groups of fungicides and the Bayer Company produced first of the triazoles in 1973. The newer triazoles are intrinsically more active than the previous ones and their effectiveness is related back to their original ED 50 values. Triazoles moiety group is one of the most important systemic fungicides that includes a variety of compounds and contains 1,2,4-triazole moiety, which control a wide range of fungal diseases on wide range of crops especially grains [13]. The study aims to investigate the level of deoxynivalenol and T-2 toxins that are contaminate imported wheat grains, along with the remaining residue of various derivatives of triazole fungicides, which is widely used over the world to reduce the fungal diseases that attack the wheat crop during the growing season. Also, the possibility of predicting generally or indicating the form of the correlational relationship between them, which may be one of the effective directions for reducing mycotoxins that affect the quality of agricultural products, especially grains, during post-harvest and storage periods.

Samples collection
Fifty-four samples, 2 kg each, of wheat imported by Saudi Arabia from the countries of different origin from the harvest season of 2017 /2018, were collected. Random representative samples were prepared according to the procedure described in SANCO/12571/2013 document [14]. Samples were kept in freezing at-20ºC upon arrival to laboratory; samples were finely homogenized, soaked in liquid nitrogen and grinded prior to analysis process. Wheat samples free from pesticides were used in validation studies and matrix-matched calibration standards preparation. Validation method was performed at three fortification levels 0.50, 0.05 and 0.005 mg/Kg with three replicates for each, and results as shown in Table 2. Working standard solutions at 1000 mg/L containing all the pesticides used for the validation method was prepared in acetonitrile. Six matrix-matched calibration standards at 3, 5, 10, 50, 100 and 250 ng/g were prepared at five replicates for each. Values of the limit of detection (LOD) and limit of quantitation (LOQ) of the analytical method used were estimated from the following equations as clarified in ICH (2005) [15]. LOD = 3.3 SD/b LOQ = 10 SD/b LOD: limit of detection, LOQ: limit of quantitation, SD: The residual standard deviation of the regression line or standard deviation of y-intercepts of the regression line.

Extraction and cleaning-up method
Buffered QuEChERS procedure was used in extraction and cleaning up of fungicide residues [16]. As wheat is a dry matrix and a little bit high in fat, steps mentioned by Mastovska et al., (2010) were used to raise method sensitivity and output [17]. Five grams of fine ground wheat sample in 50 ml Teflon centrifuge tube, 10 ml of cold D.I. water was added, shaken carefully and let it to swell and settle for 20 min. Ten ml of 1% HAc (glacial) in MeCN (V/V), were added and mixed well. TPP (Tri Phenyl Phosphate) as internal standard (IS) at rate of (200) ng/g sample was added and mixed well. Dispersive clean-up was done by adding six grams of activated anhydrous MgSO4 and 1.5 grams of anhydrous NaOAc together and hand shaking was started vigorously as fast as possible for one min, then it was shaken with orbital shaker for 60 min. Tubes were centrifuged at 5000 rpm for 10 min. Five ml of the upper layer were precisely transferred in 15 ml centrifuge tube and kept in deep-freezer for 30 min., then 750 mg activated anhydrous MgSO4, 250 mg PSA and 250 mg C18 were added to the cold extract and were mixed well for one min. Tubes were centrifuged at 5000 rpm for 5 min. Four ml were transferred from cleaned extract, were evaporated with Turbovap evaporator under N2 at 40ᵒC till lowest volume (0.2-0.3 ml), then volume was adjusted to one ml with toluene.

Triazoles determination
GC-MS/MS system of Agilent model 7890B-7000C were operated in multiple reaction monitoring mode (MRM) with two ion transitions to obtain the maximum sensitivity for detection of the target molecules, mass transitions used are presented in Table 2. HP-5MS capillary column (30 m × 250 µm × 0.25 µm) from J&W Scientific was used for pesticide residue analysis, multi-mode inlet at 280 °C, splitless mode. Oven programmed at 150 °C for two min, ramped at three °C min -1 to 200 °C then ramped to 280 °C at eight °C min-1 and then held for 10 min., carrier gas Helium (He) at flow rate one ml min-1, retention time (Rt) as shown in Table 2. MS operated in electron impact ionization mode (EI), transfer line, MS source, quad1 and quad2 temperatures were 280, 300, 180 and 180 °C respectively. Helium (He) quenching gas and N2 collision gas at 2.25 and 1.5 ml min-1 respectively. System was back flushed at 300 °C at 50 psi for five min. and method retention time was locked to chlorpyrifos-methyl at 13.093 min, Rt of TTP used as IS was 24.162 min.

Trichothecenes determination
Trichothecenes toxins estimated according to Tima et al., (2016) [18]. 5 gram of ground samples were individually was weighted in suitable container with 25 ml of methanol 75% HPLC grade. The sample was shaken vigorously for 3 min and then the extract was filtrated through Whatman filter No.1. One ml of filtrate extract was diluted with 1 ml of distilled deionized water.
Auto-ELISA (ChemWell, Awareness Technology Inc., USA) was used to conduct the procedures of DON toxin determination. 50 µl of standers? (50, 100, 200 and 400 µl L -1 ) and prepared samples was were injected into separate wells of Microtiter plate No. R5902 (r-biopharm, Germany) accredited from AOAC. 50 µl of enzyme conjugate was added to the bottom of each well and then 50 µl of Anti DON toxin antibody solution was added to each well with gently mixing by shaking the plate and incubating e it for 10 min at 20-20°C. After incubation all the remaining liquid was removed from the wells and was refilled with 250µl distilled deionized water per well. The well was emptied again by removing all remaining liquid and this step was repeated two more times. 100 µl of substrate/chromogen was added to each well, mixed gently and incubated for 5 min at 20-20°C. Finally, 100 µl of stop solution was added to each well, mixed gently by shaking and measuring the absorbance at 450 nm. The measuring was done through 10 min of stop solution addition as well as the measuring range was is 0.2-6.0 ppm. Special software (RIDA®SOFT Win. Net) was used to evaluate the RIDASCREEN®Enzyme immunoassays. T-2 toxin estimation was similar with DON protocol with replacement the kit by No. R5302 (r-biopharm, Germany) which has a special Microtiter plate, enzyme conjugate, antibody solution and substrate/chromogen for T-2 toxin.

Results
Fifty-four samples of wheat of different origins were analyzed for triazoles residues, which is considered one of the most important groups of fungicides used on wheat to control fungal infection. Validation method was performed at three fortification levels of 0.50, 0.05 and 0.005 mg/kg with three replicates for each, recovery values ranged from 85-110, 86-108 and 71-96 % respectively as shown in (Table 2) and average of RSD% ranged from 5-17%. Values of the limit of detection (LOD) and limit of quantitation (LOQ) were statistically calculated and ranged from 0.002-0.004 and 0.006-0.012 ppm respectively. MRL varied from one compound to another. The compounds that had no MRL done yet, such as Simeconazole, Hexaconazole, Azaconazole, Diniconazole and Etaconazole considered the 0.01 ppm level as the lowest determination and MRL level while for Penconazole and Propiconazole 0.05 ppm as the lowest determination and MRL level as reported by the European commission in Table (2).
All samples were analyzed in two replicates and final results as are shown in Table  (3). RSD % of results ranged from 8-22%, and average of contaminated sample (What). Tetraconazole, Azaconazole, Etaconazole, Propiconazole, Epoxiconazole and Bromuoconazole were below the limit of quantitation in all samples, while Simeconazole, Penconazole, Hexaconazole, Cyproconazole, Diniconazole, Tebuconazole, Metconazole, Fenbuconazole and Difenoconazole were detected in 4, 27, 24, 10, 11, 24, 4, 7 and 4 samples of all the 54 samples analyzed. The most of analyzed samples did not exceed the MRL individually, but average may exceed it, that samples have been collected from different shipments during the season and it vary from shipment to another according to area variation and distribution at country of origin. Results exceeded the MRL as reported by EC for hexaconazole and diniconazole in samples of all origins but comparing them with Japanese MRL levels database by (The Japan Food Chemical Research Foundation), it was 0.1 ppm for hexaconazole, so it falls in safe limit.
The results of the Table (        Etaconazole

Discussion
Wheat crop is exposed to several fungal pathogens attack especially on the shoots and spikes, causing blight, spots, streaking, rust and smuts. The triazole compounds are considered one of the many available and recommended fungicides for controlling diseases in most wheat-growing areas and used on a large scale to combat fungal pathogens as Fusarium head blight, Septoria tritici blotch, leaf rust, wheat blast and these are in a harmony with recorded by (Machado et al., 2017;Dorigan et al., 2019) [19,20].
Fifty four samples of imported wheat grains were collected in Saudi Arabia, of which 14 samples of soft wheat and 40 samples of hard wheat were used for estimating qualitatively and quantitatively derivatives of triazole fungicide. The results showed that neither Tetraconazole, Azaconazole, Etaconazole, Propiconazole, Epoxiconazole nor Bromuconazole were detected in all the tested samples. Meanwhile, there were residues of nine of the triazole derivatives, namely Simeconazole, Penconazole, Hexaconazole, Cyproconazole, Diniconazole, Tebuconazole, Metaconazole, Fenbuconazole and Difenoconazole. The detected triazole derivatives varied in retention time (RT), Ion transitions, and Collision cell energy (C.E) as well as the % Recovery at levels of 0.5, 0.05, and 0.005. Meanwhile the M maximum residue level (MRL) ranged between 0.01 and 0.6 according to allowable European limits [21].
The wheat samples of different origins were all contaminated with DON toxin, except one sample of Brazilian origin. The contamination level of the tested samples with DON toxin varied and it ranged between 40.7 to 1018.8 µg kg -1 . The contamination average of DON toxin per different origins was less than the regulation limits, which are estimated at 1250 µg kg -1 , according to European Commission, 2006; but individually there were some of the samples the DON toxin of which were more than the limits, such as Canada, Lithuania and Brasilia [22]. The variation of in DON toxin contamination may have been due return to different factors such as environmental, agricultural treatments, and fungicides application during growing; which may have a role in reducing fungal infection and doing what is known as mycotoxins decontamination [23,24,25]. Also, the tested wheat samples tested were all contaminated with T-2 toxin. The contamination level with T-2 toxin varied and ranged between 98.1 to 1107.0 µg kg -1 whereas, the permissible EU limits are from 400 to 1000 for cereals [25].