**3.2. The occurrence of T-2/HT-2 toxin in cereals**

Data published insofar have revealed that the exposure to T-2 and HT-2 toxins primarily comes as a result of the consumption of cereal grains and cereal by-products, wherein the levels of these toxins found in forages and oilseed meals are generally low. It has been established that T-2 and HT-2 toxins occur together, HT-2 thereby representing approximately two-thirds of the summary T-2/HT-2 concentration. The highest mean T-2/HT-2 concentrations were determined in grains and milled grain products, in particular in oat and oat-based


a Three replicates per concentration level per day; analyses were performed by spiking the negative material (before validation, confirmed by the LC-MS/MS).

LOD: limit of detection; LOQ: limit of quantification; CV: coefficient of variation.

**Table 2.** Validation of the ELISA method used for the determination of summary concentrations of T-2/HT-2 toxins in cereals.

products, the aforementioned applying equally for food, feed and raw cereals. Higher toxin concentrations established in unprocessed vs. processed grains indicate that grain processing succeeds in lowering, at least to some point, both T-2 and HT-2 concentrations [16].

In this study, the levels of T-2 and HT-2 were analysed in different unprocessed cereals (maize, wheat, oat, barley and triticale) sampled from Croatian households during a two-year period. In the first study step, summary concentrations of these toxins were established using the ELISA method. The results obtained in unprocessed cereal samples are shown in **Table 4**.

The study results show a significantly higher (p < 0.05) incidence of T-2/HT-2 toxins in oats (56.9%) in comparison to other unprocessed cereals. However, the maximal summary concentration was determined in maize sample (420 μg/kg), but the maximal mean value was observed in oat (136 ± 55.6 μg/kg). The results also show that in two samples, one maize (mentioned above) and one triticale (169 μg/kg), summary concentrations of T-2/HT-2 were higher than stipulated indicative levels from which onwards/above investigations should be performed in case of repetitive findings according to the Commission Recommendation [18]. Such a finding imposes the need for further sampling and investigations of the causes of such a high level of mycotoxin contamination. The lowest percentage of positive samples was

**Figure 1.** A typical LC-MS/MS-MRM chromatogram of the certified reference material (CRM, oat flour) with values of 77.9 μg/kg determined for T-2 and 75.6 μg/kg determined for HT-2; TIC; HT-2 MRM 447.2 → 285.1; HT-2 MRM

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447.2 → 345.1; T-2 MRM 489.2 → 245.1; and T-2 MRM 489.2 → 387.1.

products, the aforementioned applying equally for food, feed and raw cereals. Higher toxin concentrations established in unprocessed vs. processed grains indicate that grain processing

**Table 2.** Validation of the ELISA method used for the determination of summary concentrations of T-2/HT-2 toxins in

Three replicates per concentration level per day; analyses were performed by spiking the negative material (before

In this study, the levels of T-2 and HT-2 were analysed in different unprocessed cereals (maize, wheat, oat, barley and triticale) sampled from Croatian households during a two-year period. In the first study step, summary concentrations of these toxins were established using the ELISA method. The results obtained in unprocessed cereal samples are shown in **Table 4**. The study results show a significantly higher (p < 0.05) incidence of T-2/HT-2 toxins in oats (56.9%) in comparison to other unprocessed cereals. However, the maximal summary concentration was determined in maize sample (420 μg/kg), but the maximal mean value was observed in oat (136 ± 55.6 μg/kg). The results also show that in two samples, one maize (mentioned above) and one triticale (169 μg/kg), summary concentrations of T-2/HT-2 were higher than stipulated indicative levels from which onwards/above investigations should be performed in case of repetitive findings according to the Commission Recommendation [18]. Such a finding imposes the need for further sampling and investigations of the causes of such a high level of mycotoxin contamination. The lowest percentage of positive samples was

succeeds in lowering, at least to some point, both T-2 and HT-2 concentrations [16].

LOD: limit of detection; LOQ: limit of quantification; CV: coefficient of variation.

**Cereals LOD (μg/kg) LOQ (μg/kg) Level of fortification (μg/kg) Mean recoverya**

48 Fusarium - Plant Diseases, Pathogen Diversity, Genetic Diversity, Resistance and Molecular Markers

Maize 20.6 26.7 50 94.3 6.3

Wheat 27.3 33.6 50 90.7 7.2

Oat 25.8 31.9 50 72.2 6.1

Barley 30.1 37.4 50 88.3 7.4

Triticale 26.2 34.7 50 90.7 8.6

a

cereals.

validation, confirmed by the LC-MS/MS).

100 97.8 5.8 200 100.5 9.2

100 89.3 8.3 200 95.8 6.4

100 75.3 7.6 200 78.5 7.9

100 92.6 8.9 200 94.8 10.3

100 94.1 7.7 200 96.2 9.3

 **(%) CV (%)**

**Figure 1.** A typical LC-MS/MS-MRM chromatogram of the certified reference material (CRM, oat flour) with values of 77.9 μg/kg determined for T-2 and 75.6 μg/kg determined for HT-2; TIC; HT-2 MRM 447.2 → 285.1; HT-2 MRM 447.2 → 345.1; T-2 MRM 489.2 → 245.1; and T-2 MRM 489.2 → 387.1.


the maximal concentration of 84 and 2406 μg/kg, respectively [31–33]. In a study performed in Germany, toxin concentrations found in oats varied from 14 to 214 μg/kg [34]. Within the 2005–2008 timeframe, T-2 toxin was detected in 50% of different barley cultivars coming from the Czech Republic, in the mean concentration of 30 μg/kg [35]. In Serbia, the toxin concentrations determined in 54 analysed samples were lower than the LOD of 0.3 μg/kg [36]. The most frequently *Fusarium-*contaminated T-2-positive food items are maize (28%), wheat (21%) and oats (21%); when it comes to the HT-2 toxin, the most frequently contaminated foodstuffs are oat (41%), corn (24%) and rye (17%) [37]. In a study by the JECFA [8], the T-2 contamination rate was found to be 11% on the overall, with annual variations dependent on the cereal type. In the largescale study by Pettersson et al. [38] on 243 raw oat samples, 529 oat flake samples and 105 oat meal samples, T-2 contamination was corroborated in 73%, 24% and 17% of samples, respectively, the mean concentrations of the toxin thereby being 32 μg/kg, 5 μg/kg and 4 μg/kg, respectively.

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Data published earlier in Croatia showed the T-2 toxin presence in 57% of maize, 25% of wheat, 32% of barley and 18% of oat samples [15]. The highest detected concentration of T-2 toxin in maize was 210 μg/kg, with the pertaining mean value of 110 μg/kg pointing towards a *Fusarium-*induced maize contamination that occurred after heavy rainy periods [14]. In a study performed on different cereals harvested in 2011, the maximal level of T-2 toxin was 42 μg/ kg, with the mean value of 24 ± 27 μg/kg found in maize the maximal and the mean T-2 level found in oat on the same occasion was 10 μg/kg and 7 ± 2 μg/kg, respectively [15]. In the study by Pleadin et al. [17], the highest mean summary concentration of T-2/HT-2 toxins was, as also in this study, found in oats (102.2 ± 73.6 μg/kg), followed by maize (63.1 ± 36.7 μg/kg) and barley (51.7 ± 18.6 μg/kg), while the lowest concentration was found in wheat (34.3 ± 11.2 μg/kg). The maximal toxin level was determined in oats (304.1 μg/kg), but all of the obtained summary T-2/HT-2 concentrations were below the indicative levels necessitating further investi-

In this study, after the implementation of the ELISA method, which was used for the determination of summary values of T-2/HT-2 toxins in all samples under investigation, the LC-MS/ MS method was implemented for the determination and confirmation of each mycotoxin in positive samples (>LOD). **Table 5** shows individual levels of T-2 and HT-2 toxin determined

**Cereals T-2 toxin (μg/kg) HT-2 toxin (μg/kg) Share T-2:HT-2a Mean SD Min Max Mean SD Min Max**

**Table 5.** Levels of T-2 and HT-2 toxin determined in positive unprocessed cereal samples using the LC-MS/MS method.

Maize (n = 25) 27.6 10.7 23.1 128 66.3 21.9 30.9 256 1:2.4 Wheat (n = 12) 23.1 6.8 18.2 42.4 39.0 16.1 28.4 89.4 1:1.7 Oat (n = 44) 45.2 21.1 32.4 107 89.1 38.3 31.6 160 1:2.0 Barley (n = 10) 22.4 8.7 18.5 50.3 37.6 15.9 28.0 80.4 1:1.7 Triticale (n = 11) 26.4 6.8 21.0 47.0 47.6 24.9 27.7 104 1:1.8

gation as advised under the Commission Recommendation 2013/165/EU [18].

using the LC-MS/MS method.

the share of mean values of T-2 and HT-2.

a

a Samples in which T-2/HT-2 summary concentrations were higher than the LOD of the ELISA method. b Six blank maize samples spiked at 50 μg/kg per day.

c Six oat flour CRM replicates with the assigned reference values of 85.3 ± 13.7 μg/kg for T-2 and 86.9 ± 11.9 μg/kg for HT-2 toxin.

LOD: limit of detection; LOQ: limit of quantification; CV: coefficient of variation.

**Table 3.** Validation of the LC-MS/MS method used for the determination of T-2 and HT-2 toxin in positive cereal samplesa .

established in the wheat (21.4%) and barley sample pool (22.7%), in which the lowest mean T-2/HT-2 concentrations were determined, as well.

Literature data mainly address individual T-2 toxin concentrations rather than the summary T-2/HT-2 concentrations. Of note, the level of contamination greatly varies depending on the geographical region (country), the period of investigation and the type of cereal and/or final product investigated [25–27]. It is known that T-2/HT-2 toxins are produced by moulds of the *Fusarium* genus in high-temperature environments, with the maximum productivity at temperatures lower than 15°C accompanied by high relative humidity (of 60–95%) [3, 5]. Higher T-2/HT-2 concentrations ascertained in certain samples (although below the indicative levels) come, first and foremost, as a consequence of the plenitude of rain that facilitated the production of *Fusarium* moulds and therefore also the production of T-2/HT-2 toxins. The occurrence of these toxins in higher concentrations is also linked to other factors that could cause their formation independent of climate conditions.

T-2 toxin levels in different food components retrieved from different parts of the world were found to range from 6 to 2406 μg/kg [28–33]. Investigations performed in eight European countries on over 3000 samples showed 20% of T-2-positive and 14% of HT-2-positive samples. In the United Kingdom, T-2 was found in 16% of wheat and 12% of barley samples, the LOD thereby being 10 μg/kg. When it comes to oat, T-2 was identified in 84% of samples, with the mean and


a Indicative levels of the summary T-2/HT-2 concentrations from which onwards/above investigations should be performed in case of repetitive findings according to the Commission Recommendation 2013/165/EU [18]. b Mean concentration found in positive samples (>LOD).

**Table 4.** Summary concentrations of T-2/HT-2 toxins determined in unprocessed cereal samples using the ELISA method.

the maximal concentration of 84 and 2406 μg/kg, respectively [31–33]. In a study performed in Germany, toxin concentrations found in oats varied from 14 to 214 μg/kg [34]. Within the 2005–2008 timeframe, T-2 toxin was detected in 50% of different barley cultivars coming from the Czech Republic, in the mean concentration of 30 μg/kg [35]. In Serbia, the toxin concentrations determined in 54 analysed samples were lower than the LOD of 0.3 μg/kg [36]. The most frequently *Fusarium-*contaminated T-2-positive food items are maize (28%), wheat (21%) and oats (21%); when it comes to the HT-2 toxin, the most frequently contaminated foodstuffs are oat (41%), corn (24%) and rye (17%) [37]. In a study by the JECFA [8], the T-2 contamination rate was found to be 11% on the overall, with annual variations dependent on the cereal type. In the largescale study by Pettersson et al. [38] on 243 raw oat samples, 529 oat flake samples and 105 oat meal samples, T-2 contamination was corroborated in 73%, 24% and 17% of samples, respectively, the mean concentrations of the toxin thereby being 32 μg/kg, 5 μg/kg and 4 μg/kg, respectively.

established in the wheat (21.4%) and barley sample pool (22.7%), in which the lowest mean

**Table 3.** Validation of the LC-MS/MS method used for the determination of T-2 and HT-2 toxin in positive cereal samplesa

Six oat flour CRM replicates with the assigned reference values of 85.3 ± 13.7 μg/kg for T-2 and 86.9 ± 11.9 μg/kg for

**Analyte LOD (μg/kg) LOQ (μg/kg) Correlation coefficient Mean recoveryb (%) CV (%) Truenessc**

T-2 toxin 5.5 18.2 0.998 89.6 5.1 91.3 HT-2 toxin 8.3 27.5 0.995 77.0 8.9 86.9

50 Fusarium - Plant Diseases, Pathogen Diversity, Genetic Diversity, Resistance and Molecular Markers

Samples in which T-2/HT-2 summary concentrations were higher than the LOD of the ELISA method.

 **(%)**

.

Literature data mainly address individual T-2 toxin concentrations rather than the summary T-2/HT-2 concentrations. Of note, the level of contamination greatly varies depending on the geographical region (country), the period of investigation and the type of cereal and/or final product investigated [25–27]. It is known that T-2/HT-2 toxins are produced by moulds of the *Fusarium* genus in high-temperature environments, with the maximum productivity at temperatures lower than 15°C accompanied by high relative humidity (of 60–95%) [3, 5]. Higher T-2/HT-2 concentrations ascertained in certain samples (although below the indicative levels) come, first and foremost, as a consequence of the plenitude of rain that facilitated the production of *Fusarium* moulds and therefore also the production of T-2/HT-2 toxins. The occurrence of these toxins in higher concentrations is also linked to other factors that could cause their

T-2 toxin levels in different food components retrieved from different parts of the world were found to range from 6 to 2406 μg/kg [28–33]. Investigations performed in eight European countries on over 3000 samples showed 20% of T-2-positive and 14% of HT-2-positive samples. In the United Kingdom, T-2 was found in 16% of wheat and 12% of barley samples, the LOD thereby being 10 μg/kg. When it comes to oat, T-2 was identified in 84% of samples, with the mean and

Maize (n = 84) 200 32.1 94.8 63.7 26.9 420 Wheat (n = 56) 100 21.4 65.6 25.2 29.4 50.6 Oat (n = 72) 1000 56.9 136 55.6 32.4 273 Barley (n = 44) 200 22.7 61.3 20.6 31.6 70.2 Triticale (n = 29) 100 34.5 76.6 30.4 36.0 169

performed in case of repetitive findings according to the Commission Recommendation 2013/165/EU [18].

Indicative levels of the summary T-2/HT-2 concentrations from which onwards/above investigations should be

**Table 4.** Summary concentrations of T-2/HT-2 toxins determined in unprocessed cereal samples using the ELISA method.

 **(μg/kg) Positive (%) Meanb (μg/kg) SD (μg/kg) Min (μg/kg) Max (μg/kg)**

T-2/HT-2 concentrations were determined, as well.

LOD: limit of detection; LOQ: limit of quantification; CV: coefficient of variation.

Six blank maize samples spiked at 50 μg/kg per day.

formation independent of climate conditions.

Mean concentration found in positive samples (>LOD).

**Cereals Indicative levela**

a

a

b

c

HT-2 toxin.

b

Data published earlier in Croatia showed the T-2 toxin presence in 57% of maize, 25% of wheat, 32% of barley and 18% of oat samples [15]. The highest detected concentration of T-2 toxin in maize was 210 μg/kg, with the pertaining mean value of 110 μg/kg pointing towards a *Fusarium-*induced maize contamination that occurred after heavy rainy periods [14]. In a study performed on different cereals harvested in 2011, the maximal level of T-2 toxin was 42 μg/ kg, with the mean value of 24 ± 27 μg/kg found in maize the maximal and the mean T-2 level found in oat on the same occasion was 10 μg/kg and 7 ± 2 μg/kg, respectively [15]. In the study by Pleadin et al. [17], the highest mean summary concentration of T-2/HT-2 toxins was, as also in this study, found in oats (102.2 ± 73.6 μg/kg), followed by maize (63.1 ± 36.7 μg/kg) and barley (51.7 ± 18.6 μg/kg), while the lowest concentration was found in wheat (34.3 ± 11.2 μg/kg). The maximal toxin level was determined in oats (304.1 μg/kg), but all of the obtained summary T-2/HT-2 concentrations were below the indicative levels necessitating further investigation as advised under the Commission Recommendation 2013/165/EU [18].

In this study, after the implementation of the ELISA method, which was used for the determination of summary values of T-2/HT-2 toxins in all samples under investigation, the LC-MS/ MS method was implemented for the determination and confirmation of each mycotoxin in positive samples (>LOD). **Table 5** shows individual levels of T-2 and HT-2 toxin determined using the LC-MS/MS method.


**Table 5.** Levels of T-2 and HT-2 toxin determined in positive unprocessed cereal samples using the LC-MS/MS method.

When the individual concentrations of T-2 and HT-2 toxin obtained by the LC-MS/MS method were summed up, it was established that the latter sum slightly differs from, and is mainly lower than, the summary concentrations of these mycotoxins determined using the ELISA method. This can be explained by the cross-reactivity and a lower specificity of the ELISA method. It is known that the ELISA represents an easy-to-use purification technique with a lesser need for an extensive clean-up, but it may suffer from undesired cross-reactivity with other trichothecenes that give rise to metric uncertainty [16].

**Figure 2** presents the LC-MS/MS-MRM chromatogram of the most contaminated sample (maize), in which the concentration of T-2 toxin of 128 μg/kg and the concentration of HT-2 toxin of 256 μg/kg was determined (summary T-2/HT-2 toxins concentration, 384 μg/kg). Together with the most contaminated oat and the most contaminated triticale sample, this sample was further subjected to thermal processing.

Based on the comparison of the mean T-2 and HT-2 toxin concentrations, the shares of T-2:HT-2 were established to be in the range from 1:1.7 in wheat and barley to 1:2.4 in maize, with the mean share value of 1:1.9 in all investigated cereal samples. The determined share values are comparable to those stated by other literature sources, which show that HT-2 is present in cereals and their products in levels higher than those of T-2 toxin, representing approximately two-thirds (1:2) of the summary T-2/HT-2 concentrations [16].
