Free and Modified Mycotoxins in Organic and Conventional Oats (Avena sativa L.) Grown in Scotland

Small grain cereals are frequently infected with mycotoxigenic Fusarium fungi. Oats have a particularly high risk of contamination with type A trichothecene mycotoxins; their glucoside conjugates have also been reported. Agronomy practices, cereal variety and weather conditions have been suggested to play a role in Fusarium infection in oats. The current study investigates concentrations of free and conjugated Fusarium mycotoxins in organic and conventional oats grown in Scotland. In 2019, 33 milling oat samples (12 organic, 21 conventional) were collected from farmers across Scotland, together with sample questionnaires. Samples were analysed for 12 mycotoxins (type A trichothecenes T-2-toxin, HT-2-toxin, diacetoxyscirpenol; type B trichothecenes deoxynivalenol, nivalenol; zearalenone and their respective glucosides) using LC-MS/MS. The prevalence of type A trichothecenes T-2/HT-2 was very high (100% of conventional oats, 83% of organic oats), whereas type B trichothecenes were less prevalent, and zearalenone was rarely found. T-2-glucoside and deoxynivalenol-glucoside were the most prevalent conjugated mycotoxins (36 and 33%), and co-occurrence between type A and B trichothecenes were frequently observed (66% of samples). Organic oats were contaminated at significantly lower average concentrations than conventional oats, whereas the effect of weather parameters were not statistically significant. Our results clearly indicate that free and conjugated T-2- and HT-2-toxins pose a major risk to Scottish oat production and that organic production and crop rotation offer potential mitigation strategies.


Introduction
Fungal infection is a major problem in global cereal production and results in subsequent contamination of grains with a wide range of mycotoxins. In temperate regions, Fusarium is the predominant mycotoxigenic genus found to infect small grain cereals in the field pre-harvest [1,2]. Prominent strains within the genus Fusarium include F. graminearum, F. culmorum, F. langsethiae and F. poae, all of which have been shown to produce a range of mycotoxins including trichothecenes (type A and B) and zearalenone in small grain Bread, pastries, biscuits, cereal snacks and breakfast cereals 50 1 for T-2 + HT-2 indicative levels are set by EC recommendation 2013/165/EU [20]. 2 for DON and ZEN maximum permitted levels are set by EC regulation 1881/2006 [21].
Recent UK surveys have identified T-2/HT-2 to occur commonly in food oats, although exceedances of European Commission (EC) indicative levels are rare [22,23]. In addition to the free fungal mycotoxins, plant-derived modified mycotoxins such as sugar-conjugated forms DON-glucoside and ZEN-glucoside have also been reported in wheat [12,[24][25][26] at proportions of 4-69% of the free parent mycotoxins. Conjugated glucoside forms of T-2 and HT-2 have also been identified [27], but less information is available on their natural occurrence in cereal grains. These conjugated mycotoxins are released by the activity of the intestinal microbiota in vitro [27][28][29][30][31][32][33] and have been found to contribute to human exposure to free mycotoxins in vivo [34]. Hence the presence of free and modified mycotoxins in cereals warrants further investigation.
Previous studies have identified a range of agronomy practices that might decrease the risk of fungal infection and mycotoxin contamination in cereals. These include spring rather than winter sowing, varietal selection (for wheat) and cereal rotation [35][36][37][38]. Furthermore, some studies suggest that organic production systems may lower mycotoxin contamination in some cereals [39]. Hence, the current paper presents a detailed profiling of free and sugar-conjugated Fusarium mycotoxins in oat samples grown in conventional or organic systems in Scotland.

Prevalence of Free and Modified Mycotoxins in Organic and Conventional Oats
Type A trichothecenes T-2 and HT-2 were highly prevalent in Scottish oat samples, with higher prevalence observed in conventional oats (95.2 and 100%, respectively), compared to organic oats (58.3 and 83.3%, respectively. In contrast, DAS and DAS-Glc were not detected ( Figure 1).

Prevalence of Free and Modified Mycotoxins in Organic and Conventional Oats
Type A trichothecenes T-2 and HT-2 were highly prevalent in Scottish oat sampl with higher prevalence observed in conventional oats (95.2 and 100%, respectively), co pared to organic oats (58.3 and 83.3%, respectively. In contrast, DAS and DAS-Glc w not detected (Figure 1). Overall, type B trichothecenes DON and NIV were less prevalent in oats than type trichothecenes. The difference between organic and conventional oats was less p nounced (DON 33.3 and 47.6; NIV 41.7 and 38.1%). T-2-Glc and DON-Glc were the m prevalent modified mycotoxins, especially in conventional oats (61.9 and 42.8%, resp tively). ZEN was not frequently detected, and no ZEN-Glc was found in any sample.

Concentrations of Free and Modified Mycotoxins in Organic and Conventional Oats
HT-2 in conventional oats was the highest mycotoxin concentration found in a sample group in this study (average 670.8 µ g/kg, Figure 2). In addition, T-2 and HT toxins were frequently found in the same sample resulting in 19% of conventional o samples exceeding the EC indicative level of 1000 µ g/kg for T-2 + HT-2. However, conce trations of T-2 and HT-2 in organic oat samples were significantly lower (21.1 and 14 µ g/kg mean concentration, p = 0.0023 and p = 0.0043, respectively), with no organic o exceeding the EC indicative level. Overall, type B trichothecenes DON and NIV were less prevalent in oats than type A trichothecenes. The difference between organic and conventional oats was less pronounced (DON 33.3 and 47.6; NIV 41.7 and 38.1%). T-2-Glc and DON-Glc were the most prevalent modified mycotoxins, especially in conventional oats (61.9 and 42.8%, respectively). ZEN was not frequently detected, and no ZEN-Glc was found in any sample.

Concentrations of Free and Modified Mycotoxins in Organic and Conventional Oats
HT-2 in conventional oats was the highest mycotoxin concentration found in any sample group in this study (average 670.8 µg/kg, Figure 2). In addition, T-2 and HT-2 toxins were frequently found in the same sample resulting in 19% of conventional oat samples exceeding the EC indicative level of 1000 µg/kg for T-2 + HT-2. However, concentrations of T-2 and HT-2 in organic oat samples were significantly lower (21.1 and 148.9 µg/kg mean concentration, p = 0.0023 and p = 0.0043, respectively), with no organic oats exceeding the EC indicative level. DON concentrations were not significantly different (p = 0.9828) between organic and conventional oats (mean 327.3 and 204.0 µg/kg, respectively), with 1/12 organic and 1/21 conventional samples exceeding the EC maximum permitted level for DON (1750 µg/kg). T-2-Glc was the most frequently detected modified mycotoxin in conventional oats (61.9% prevalence, Figure 1) at ratios ranging from 6-154% of T-2 ( Table 2). Ratios of HT-2-Glc ranged from 34-174% of HT-2, whereas DON-Glc was found at lower ratios (18-130% of DON). DON concentrations were not significantly different (p = 0.9828) between organic and conventional oats (mean 327.3 and 204.0 µ g/kg, respectively), with 1/12 organic and 1/21 conventional samples exceeding the EC maximum permitted level for DON (1750 µ g/kg). T-2-Glc was the most frequently detected modified mycotoxin in conventional oats (61.9% prevalence, Figure 1) at ratios ranging from 6-154% of T-2 ( Table 2). Ratios of HT-2-Glc ranged from 34-174% of HT-2, whereas DON-Glc was found at lower ratios (18-130% of DON).

Co-Occurrence of Free Mycotoxins in Organic and Conventional Oats
Oat samples were frequently contaminated with numerous mycotoxins in different combinations. Co-occurrence is defined here as the presence of type A trichothecenes (T-2/HT-2), type B trichothecenes (DON or NIV) and ZEN. Modified mycotoxins are not included in these figures as they represent plant metabolites of the parent mycotoxins produced by fungi. One conventional oat sample (4.8%) was co-contaminated with all four mycotoxins, while two or more mycotoxins co-occurred in 50.0% organic oats and 61.9% conventional oat samples ( Table 3). None of the samples in this survey were free of all mycotoxins tested (i.e., all mycotoxins < LOQ). Table 3. Co-occurrence of free mycotoxins in organic and conventional oats.

Number (%) of Samples Organic
Number (%) of Samples Conventional The highest concentrations of T-2/HT-2 (>100% of EC indicative levels) were found in four conventional oats, but these samples were not heavily co-contaminated with type B trichothecenes or ZEN ( Figure 3). Conversely, samples with the highest levels of DON were also co-contaminated with ZEN.

Effect of Other Agronomy Factors on Mycotoxin Concentrations in Oats
In addition to organic versus conventional oat production, the effect of cereal rotation intensity and weather conditions, such as average monthly rainfall and average monthly

Effect of Other Agronomy Factors on Mycotoxin Concentrations in Oats
In addition to organic versus conventional oat production, the effect of cereal rotation intensity and weather conditions, such as average monthly rainfall and average monthly temperature one month and two months prior to harvest date, were investigated for their potential impact on mycotoxin concentrations. Cropping history was examined, and a cereal intensity score was calculated [40] as the number of years (over the previous 4 years) during which the previous crop was a small grain cereal (wheat, oats or barley). A significant positive relationship (p = 0.0426) was found between the cereal intensity score and the levels of T-2/HT-2 across all oat samples in this survey (Figure 4). No other significant relationships were found between mycotoxin levels and other factors in this dataset.

Discussion
Type A trichothecene mycotoxins T-2/HT-2 are well-recognised as major contaminants in oat production [17,[39][40][41]. Our study confirms that these mycotoxins occur at the highest prevalence and concentration in this Scottish sample set. Additionally, our survey demonstrates the high prevalence of the modified mycotoxins T-2-Glc and HT-2-Glc in oat samples, further increasing the overall contamination levels. In a longitudinal survey of mycotoxins in UK cereal production [42], authors report a prevalence of T-2/HT-2 of 86-100% (29 food oat samples each year) with mean concentrations of 313-458 µ g/kg sample. The prevalence of contamination is comparable to our study (100% prevalence in conventional oats), but mean concentrations are higher in our Scottish survey (793 µ g/kg). There are currently no maximum regulatory levels set for T-2/HT-2 in oats, but indicative levels can be used to benchmark contamination levels. In our survey, 19% of conventional oats and no organic oats exceeded the EC indicative level for T-2/HT-2, resulting in overall 12% exceedances across all 33 oat samples, which are comparable to other studies reporting 1-30% exceedances in conventional oats in the UK [40] and 7.4% exceedances in organic and conventional oats in Ireland [43].
The prevalence and mean concentration of T-2-Glc reported in the AHDB survey [42] (59-79%, 37.1-67.4 µ g/kg) are also similar to our results (61.9%, 21.9 µ g/kg), but we also detected HT-2-Glc in 24% of samples (mean 39.2 µ g/kg) which were not assessed in previous studies. Furthermore, despite the low mean concentration across the samples, we observed that two conventional oat samples contained high levels of HT-2-Glc (462 and 155 µ g/kg), which significantly contributes to the overall mycotoxin contamination of

Discussion
Type A trichothecene mycotoxins T-2/HT-2 are well-recognised as major contaminants in oat production [17,[39][40][41]. Our study confirms that these mycotoxins occur at the highest prevalence and concentration in this Scottish sample set. Additionally, our survey demonstrates the high prevalence of the modified mycotoxins T-2-Glc and HT-2-Glc in oat samples, further increasing the overall contamination levels. In a longitudinal survey of mycotoxins in UK cereal production [42], authors report a prevalence of T-2/HT-2 of 86-100% (29 food oat samples each year) with mean concentrations of 313-458 µg/kg sample. The prevalence of contamination is comparable to our study (100% prevalence in conventional oats), but mean concentrations are higher in our Scottish survey (793 µg/kg). There are currently no maximum regulatory levels set for T-2/HT-2 in oats, but indicative levels can be used to benchmark contamination levels. In our survey, 19% of conventional oats and no organic oats exceeded the EC indicative level for T-2/HT-2, resulting in overall 12% exceedances across all 33 oat samples, which are comparable to other studies reporting 1-30% exceedances in conventional oats in the UK [40] and 7.4% exceedances in organic and conventional oats in Ireland [43]. The prevalence and mean concentration of T-2-Glc reported in the AHDB survey [42] (59-79%, 37.1-67.4 µg/kg) are also similar to our results (61.9%, 21.9 µg/kg), but we also detected HT-2-Glc in 24% of samples (mean 39.2 µg/kg) which were not assessed in previous studies. Furthermore, despite the low mean concentration across the samples, we observed that two conventional oat samples contained high levels of HT-2-Glc (462 and 155 µg/kg), which significantly contributes to the overall mycotoxin contamination of these samples. Previous in vitro studies have clearly shown that T-2-Glc and HT-2-Glc are rapidly hydrolysed to free T-2 and HT-2 by the microbial activity of the human gut microbiota [27,31] and can therefore contribute to overall exposure to these potent mycotoxins in humans. Hence further investigations into the levels of modified forms of T-2/HT-2 in unprocessed cereals and their carry-over into food products are needed.
Organic oats have previously been found to be contaminated with lower levels of T-2/HT-2 compared to conventional oats in studies conducted in the UK [17,40], Ireland [43], Norway, Poland and Germany [39], while no such consistent differences were found in other cereals [39]. Similarly, we found the T-2/HT-2 levels to be significantly lower in organic oat samples compared to conventional oats. Furthermore, we also found T-2-Glc + HT-2-Glc to be significantly lower in organic oats, further supporting the notion that organic production can decrease the risk of mycotoxin contamination in oats.
Other agronomic factors have also been identified to impact the risk of fungal infection and mycotoxin contamination in cereals. Among them, cereal rotations, ploughing and sowing dates (winter versus spring sowing) have been found to be important factors affecting oat mycotoxin concentrations [40,43,44]. In the present study, we could also confirm that cereal intensity increased the risk of T-2/HT-2 contamination but not other mycotoxins. Similarly, Kolawole et al. (2021) report that previous crops have a stronger impact on T-2/HT-2 than on DON and ZEN contamination in oats [43], and Edwards (2017) reports that cereal intensity was significantly related to T-2/HT-2 levels. Still, DON and ZEN were not investigated as they were detected less frequently in UK oats [40]. These published studies also demonstrate that crop growth season is an important factor, with spring-sown oats containing significantly lower concentrations of mycotoxins than wintersown [40,43]. However, we were unable to assess the effect of crop growth season as only one sample in the current survey was winter-sown.
In summary, this study clearly demonstrates the high prevalence of type A trichothecenes in Scottish oat samples and the frequent co-contamination with type B trichothecenes and zearalenone. In addition, the study indicates the protective effect of organic agronomy against high mycotoxin contamination and points towards the potential benefits of low-intensity cereal rotations.

Study Design
This study was carried out in collaboration with SOPA, Farmton Farm, WN Lindsay and Hamlyns of Scotland. The collaborators designed a detailed sample questionnaire (Supplementary Materials) approved by the Rowett Institute Human Studies Ethics Committee (16 July 2019). Farmers were approached through links with the project collaborators and were asked to provide a 1 kg aggregate sample of unprocessed, dried (<14% moisture content) milling oats. Farmers were asked to complete the sample questionnaire as paper copies or using the online tool https://tinyurl.com (accessed on 20 August 2019) In total, 33 oat samples and corresponding questionnaires were obtained, including 12 samples from organic farms and 21 from conventional farms and were stored at room temperature at the Rowett Institute. Fewer organic than conventional samples reflects the balance between organic and conventional cropping in Scotland. Information on organic status, fungicide use, crop rotation practices, oat variety and harvest date were obtained from sample questionnaires. Total monthly rainfall (mm) and average monthly temperature (degrees centigrade) for the (1 month prior = pre-harvest period, 2 months prior = flowering) period prior to harvest were obtained from the Met Office weather survey for the area of each farm (https://www.metoffice.gov.uk/research/climate/maps-and-data/uk-actualand-anomaly-maps, accessed on 29 October 2020).  (Table 4).

Extraction of Oat Samples
Oat samples were freeze-milled by using a 6870 large freezer/Mill (SPEX SamplePrep, Metuchen, NJ, USA) into fine powder. Next, 0.5 g milled and homogenised oat samples were extracted with 2 mL of extraction solvent (79% ACN, 20% H 2 O, 1% acetic acid; HAc) [48] for 90 min at 1200 rpm on an orbital shaker (IKA ® VXR basic, Thomson Scientific, Aberdeen, UK). Samples were centrifuged at room temperature (2000× g for 5 min), and supernatants were dried under nitrogen stream and reconstituted to achieve 10% of ACN in sample extracts. Prior to LC-MS/MS analysis, sample extracts were combined with 13 C 22 -HT-2, 13 C 15 -DON and 13 C 18 -ZEN to facilitate the quantification of parent mycotoxins using a stable isotope dilution approach (SIDA) [49].

LC-MS/MS Analysis of Mycotoxins
The detection and the quantification of all mycotoxins and the [ 13 C]-labelled standards were performed on a Shimadzu Nexera X2 LC Quaternary pump coupled to a Shimadzu 8060 mass spectrometer fitted with an electrospray ionisation (ESI) source (Shimadzu, Kyoto, Japan). The liquid chromatography separation was performed on a Phenomenex Gemini C18 column, 150 mm × 3 mm, particle size 3 µm. Mobile phase solvents were (A) 0.1% HAc and (B) methanol; after 2 min at 100% A, the proportion of B was increased linearly to 100% within 12 min, followed by a hold time of 3 min at 100% B and 4 min column re-equilibration at 100% A. The flow rate was 800 µL/min, and the injection volume was 15 µL. The LC eluent was directed into the ESI source without splitting. The mass spectrometer was run in positive and negative ion mode with the following settings: interface temperature 300 • C, desolvation temperature 250 • C, heating block temperature 300 • C, and gases 1 and 2 set at 15 and 5 L/min, respectively. Argon gas was used as the collision gas in the collision cell for the fragmentation of the mycotoxin metabolites. Ion transition parameters and precursors used for each mycotoxin are summarised in Table 4. Mycotoxins were quantified using the multiple reaction monitoring (MRM) technique. Standard solutions of approximately 1 ng/µL concentration were prepared and put into the LC auto sampler, where the mass spectrometer sampled from them automatically to optimise the MRM conditions of the individual mycotoxin metabolites.
LOQ was determined in oat matrix by a signal-to-noise ratio of 10/1.

Data calculations and Statistical Analysis
All results were corrected for recovery. For prevalence (% of positive samples), only values > LOQ for each mycotoxin were included. For calculation of mean concentration of mycotoxins and statistical analysis, all values < LOQ for each mycotoxin were replaced by 1 ⁄2 LOQ [40] Mycotoxin concentrations were log-transformed, and for each mycotoxin (single toxins T-2, HT-2, T-2-Glc, HT-2-Glc, DON, DON-Glc, NIV, NIV-Glc as well as T-2 + HT-2 and T-2 + HT-2 + T-2-Glc + HT-2Glc) were fitted to covariates (organic production, cereal intensity score, cereal variety, temperature, rainfall and harvest date) using linear models. Due to sample size, models with one or two covariates were fitted at a time. Analysis of variance was used to test the statistical significance of the covariates in each model, with p < 0.05 considered significant. Diagnostics were carried out to assess the assumptions of the tests. All analyses were carried out using the statistical software "R", version 4.2.2 (R Core Team, 2021. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria).