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Degradation of trichothecene mycotoxins by aqueous ozone
Food and Chemical Toxicology 44 (2006) 417–424 www.elsevier.com/locate/foodchemtox
Degradation of trichothecene mycotoxins by aqueous ozone
q
J. Christopher Young *, Honghui Zhu, Ting Zhou Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ont., Canada N1G 5C9 Received 24 January 2005; accepted 16 August 2005
Abstract The degradation of ten trichothecene mycotoxins by aqueous ozone was monitored by liquid chromatography–ultraviolet–mass spectrometry (LC–UV–MS). Saturated aqueous ozone (25 ppm) degraded these mycotoxins to materials that were not detected by UV or MS. At lower levels (0.25 ppm) of aqueous ozone, intermediate products were observed. On the basis of UV and MS data, it is proposed that the degradation begins with attack of ozone at the C9–10 double bond with the net addition of two atoms of oxygen. The remainder of the molecule appears to have been left unaltered. The oxidation state at the allylic carbon 8 position had a significant effect on the ease of reaction, as determined by moles of ozone required to effect oxidation. The amount of ozone required to effect oxidation to intermediate products and subsequent degradation followed the series allylic methylene (no oxygen) < hydroxyl (or ester) < keto. Ozonation was also sensitive to pH. At pH 4–6, all mycotoxins studied degraded readily; at pH 7–8 the degree of reactivity was dependent upon the carbon 8 oxidation state; at pH 9, there was little or no reaction. Structures for some of the intermediate products are proposed. Crown Copyright 2005 Published by Elsevier Ltd. All rights reserved. Keywords: Mycotoxins; Trichothecenes; Degradation; Ozonation; pH
1. Introduction The trichothecene class of mycotoxins consists of naturally occurring metabolites produced by Fusarium spp. fungi on a variety of cereal grains and are known to be associated with several diseases in animals and humans (Buck and Coˆte´, 1991; Miller and Trenholm, 1994; Pittet, 1998; Placinta et al., 1999; DeVries et al., 2002; Anon, 2003). Because of potential losses to the farmer and toxicological hazards to the consumer, developing cost effective methods to detoxify mycotoxin-contaminated grains and
Abbreviations: 3ADON, 3-acetyldeoxynivalenol; 15ADON, 15-acetyldeoxynivalenol; APCI, atmospheric pressure chemical ionization; DAS, diacetoxyscirpenol; DON, 4-deoxynivalenol; FUS, fusarenon X; HT2, HT-2 toxin; LC, liquid chromatography; MAS, 15-monoacetoxyscirpenol; MS, mass spectrometry; NEO, neosolaniol; RSD, relative standard deviations; T2T, T-2 triol; UV, ultraviolet; VER, verrucarol. q This is Scientific Publication S206 of the Food Research Program, Agriculture and Agri-Food Canada, Guelph. * Corresponding author. Tel.: +519 780 8033; fax: +519 829 2600. E-mail address: [email protected] (J.C. Young).
foods is a priority. A variety of chemical, physical, and biological treatments have been tested for their ability to reduce or eliminate these mycotoxins in contaminated feeds and foods (Charmley and Prelusky, 1994; Karlovsky, 1999; Trigo-Stockli, 2002; Anon, 2003). One of the chemical treatments of deoxynivalenol (DON) contaminated wheat (Young et al., 1986) and corn (Young, 1986) previously investigated by us was with ozone in air. When dry whole wheat kernels naturally contaminated (ca. 1 lg/g DON) were treated with dry ozone, there was no reduction in DON levels. When vacuum oven dried ground unhusked corn artificially inoculated by Fusarium graminearum (ca. 1000 lg/g DON) was treated with dry ozone, the half life of DON disappearance was 2.5 h. However, when the corn was not dried and the ozone stream was moistened by being bubbled through water, the half life of DON disappearance was reduced to about 15 min. Thus moisture appears to be essential in the reaction between DON and ozone. The availability of saturated aqueous ozone prompted us to revisit this reaction and formed the basis for this paper. The difference between the wheat and corn studies results may also have been
0278-6915/$ - see front matter Crown Copyright 2005 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.08.015
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due to a matrix effect. The corn was ground and thus porous whereas ozone may not have been able to penetrate the whole wheat kernels as readily. More recently, ozone gas was demonstrated as being effective in chemically modifying (McKenzie et al., 1997) a variety of non-trichothecene mycotoxins (aflatoxins B1, B2, G1, and G2, cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, secalonic acid, and zearalenone) and reducing their biological activity in the bioassays used (McKenzie et al., 1998; Lemke et al., 1999). More studies are required to establish the extent of hazard reduction. Ozonation of triacetoxyscirpenol was conducted as part of a structural determination (Sigg et al., 1965). Ozone is an unstable and a strong oxidant, capable of reacting with a wide variety of contaminants in water, which makes it suitable for the treatment of waste and drinking water to remove undesirable contaminants (Hoigne´, 1998). Two primary oxidation pathways in water have been proposed (Hoigne´, 1982; Staehelin and Hoigne´, 1982; Staehelin and Hoigne´, 1985): direct oxidation by molecular ozone and indirect oxidation by free radicals, which are formed during the ozone decomposition in water. The relative importance between these two pathways is affected by pH, UV light, ozone concentration, and the presence of radical scavengers. The later pathway can be further divided into three steps: initiation, propagation, and termination to form various free radicals. Among them is hydroxyl free radical, which is much more powerful an oxidant than ozone molecule itself. Thus, the reactions between these free radicals and organic contaminants are often considered to be much faster but less selective. Since the only previous study of the reactivity of ozone with trichothecenes was with DON and ozone in air, the purpose of this study was to investigate the reaction between ozone and DON in an aqueous medium and to determine if this is a general reaction with other trichothecenes. Furthermore, since the reactive species of aqueous ozone is dependant upon pH, we also studied the effect of pH on the course of trichothecene ozonation. 2. Materials and methods
10
O
9
O
3 OH
OH O
O
8
O 15
4
O
OH
O
O
O
O
Diacetoxyscirpenol (DAS)
15-Monoacetoxyscirpenol (MAS)
O
O
HO O
OH
OH
O
O
O Neosolaniol (NEO)
Verrucarol (VER)
O
O
OH
O
O
OH O
O O
O
O
OH
HO
OH
O
O
OH
O T-2 Triol (T2T)
HT-2 Toxin (HT2)
OH
O
OH
O
O
O
O
O HO
HO
OH
O
Fusarenon X (FUS)
Deoxynivalenol (DON) O
O
OH
OH
O
O O
O
O O
O HO
HO OH
O O
3-Acetyldeoxynivalenol (3ADON)
15-Acetyldeoxynivalenol (15ADON)
Fig. 1. Structures of the trichothecenes studied.
stead) water and chilled in an ice bath. For safety reasons, excess ozone was neutralized by passage through saturated aqueous sodium thiosulfate. The concentration of ozone in water was determined by the indigo colorimetric method of Bard and Hoigne´ (1981), which involves spectrophotometric (Milton Spectronic 1000 plus) measurement at 600 nm.
2.1. Mycotoxin samples and reagents 2.3. Ozonation of pure mycotoxins The mycotoxins 3-acetyldeoxynivalenol (3ADON), 15-acetyldeoxynivalenol (15ADON), diacetoxyscirpenol (DAS), 4-deoxynivalenol (DON), fusarenon X (FUS), HT-2 toxin (HT2), 15-monoacetoxyscirpenol (MAS), neosolaniol (NEO), T-2 triol (T2T), and verrucarol (VER) were obtained from Sigma-Aldrich (Oakville, ON). The structures of the various mycotoxins are shown in Fig. 1. Methanol was obtained from Caledon (Georgetown, ON) and oxygen was obtained from Praxair (Kitchener, ON).
In a typical experiment with each of the mycotoxins, about 2.5 lg of mycotoxin was placed into a 1.8 mL glass vial and dissolved in water. Ozone saturated water of known concentration was then added in a ratio such that the total aqueous volume was 1.0 mL and gave the desired final concentration of ozone. The reaction was repeated with DON, MAS and T2T in 0.1 M ammonium acetate buffered solutions at pH 4–9.
2.4. Analysis of mycotoxins and reaction products 2.2. Ozone in water Water saturated with ozone was prepared by bubbling the gas, generated by passage of purified extra dry oxygen through a Triogen Model LAB2B generator (Ozonia North America, Elmwood Park, NJ), for 30 min through a 500 mL gas wash bottle containing NANOpure (Barn-
The reaction mixtures were then analyzed directly without any work up by taking a 20 lL aliquot and injecting onto a 4.6 · 150 mm liquid chromatographic (LC) column packed with Agilent Zorbax Eclipse XDBC18, 3.5 lm particle size. The column was eluted at 1 mL/min with a linear gradient of methanol–water changing from 1:3 to 3:1 over 15 min. Starting
J.C. Young et al. / Food and Chemical Toxicology 44 (2006) 417–424
The treatments were repeated except that molar ratios of ozone to trichothecenes ranging from 0.3 to 5.0 (equivalent to 0.14–1.8 ppm ozone in water) were chosen. The solutions were unbuffered and had a pH of 6.5. Under these conditions, the disappearance of the trichothecenes and appearance of products could be followed. Several phenomena were immediately apparent. Reaction with ozone reached its endpoint quickly; sequential reanalyses of several reaction mixtures revealed that there were no further changes over time when the product mixtures were compared with the initial analysis made shortly after mixing of reagents. In all instances, the appearance of products was transitory; they too disappeared as the ozone concentration increased. A final observation was that some mycotoxins disappeared more readily than others. Fig. 2 shows the disappearance profiles between the various trichothecenes for different amounts of ozone. The median relative standard deviations (RSD) of these determinations was 20.6%. The reaction between ozone and a trichothecene required less ozone for those compounds (DAS, MAS, and VER) lacking an oxygen at the carbon 8 position (Fig. 2a). Those trichothecenes with an allylic oxygen at C8 required more ozone (Fig. 2b and c).
materials and products were detected with a Finnigan SpectraSystem UV6000LP ultraviolet (UV) detector and a Finnigan LCQ Deca ion trap mass spectrometer (MS) operated in the atmospheric pressure chemical ionization (APCI) positive ion mode. The mass spectrometer was tuned for maximum response for DON. Machine operating conditions were as follows: shear gas and auxiliary flow rates were set at 80 and 0 (arbitrary units); voltages on the capillary, tube lens offset, multipole 1 offset, multipole 2 offset, lens, and entrance lens were set at 15.00, 30.00, 5.00, 7.00, 16.00, and 60.00 V, respectively; capillary and vaporizer temperatures were set at 200 and 450 C, respectively; and the discharge needle current was set at 10 lA. Compounds were quantified on the basis of integrated peak areas using absorbance units (UV) or ion counts (MS). All reactions were run in triplicate and the results averaged.
3. Results and discussion 3.1. Unbuffered ozonation At the outset, the mycotoxins were treated with saturated aqueous ozone (concentration typically about 25– 30 ppm). The LC–UV–MS analyses showed that all of the starting material had been destroyed and no products could be detected. McKenzie et al. (1997) also remarked that no products were observed when their series of nontrichothecene mycotoxins were treated with ozone.
Trichothecene Remaining (%)
100
100
a
100
b
80
80
80
60
60
60
50
50
50
40
40
40
20
20
20
0
1
2
3
4
5
c
0
0
0
419
0
1
2
3
4
5
0
1
2
3
4
5
Ozone/Trichothecene Ratio (mol/mol) Fig. 2. Reductions, relative to starting material, in amounts of trichothecenes upon reaction with varying amounts of aqueous ozone. Data points represent averages of reactions run in triplicate and quantified by liquid chromatography–mass spectrometry. (a) Trichothecenes lacking oxygenation at C8: d = verrucarol; j = 15-monoacetoxyscirpenol; m = diacetoxyscirpenol; (b) trichothecenes with free or esterified hydroxyl at C8: d = neosolaniol; j = T-2 triol; m = HT-2 toxin; (c) trichothecenes with a keto group at C8: = deoxynivalenol; d = fusarenon X; j = 3-acetyldeoxynivalenol; m = 15-acetyldeoxynivalenol.
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tion with ozone when the allylic hydroxyl in hex-1-ene3-ol was moved further away from the double bond (hex-1-ene-4-ol). These results are consistent with the well established electrophilic nature of attack of ozone on
Table 1 Molar ratios of ozone/trichothecene required to effect a 50% reduction in trichothecene levels Groups
Trichothecenes
Meana ± std err
Group meansb,c
8-methylene
VER MAS DAS
0.65 ± 0.09 0.75 ± 0.05 0.93 ± 0.16
0.77 ± 0.06a
8-hydroxy
NEO HT2 T2T
1.14 ± 0.12 1.36 ± 0.23 1.67 ± 0.28
1.40 ± 0.11b
8-keto
DON FUS 3ADON 15ADON
1.44 ± 0.05 1.50 ± 0.10 1.62 ± 0.16 2.44 ± 0.26
1.75 ± 0.13c
Table 2 Products from reaction of trichothecenes with unbuffered aqueous ozone Trichothecene
Ozonation products
DON 15ADON
2.79a (100)b; 2.92 (44); 3.21 (8); 3.59 (6); 4.16 (15) 3.10 (64); 4.05 (100); 4.52 (34); 5.39 (23); 6.44 (43); 7.08 (82) 6.38 (8); 6.77 (26); 7.03 (100) 3.20 (7); 3.48 (100); 3.90 (3); 4.29 (4); 4.96 (5) 4.25 (15); 5.14 (7); 5.45 (57); 6.10 (100) 8.18 (35); 8.34 (53); 9.54 (83); 9.98 (32); 10.80 (47); 11.47 (100) 10.80 (48); 11.11(100); 11.90 (31); 12.54 (25); 12.90 (27); 13.47 (50) 6.17 (95); 6.32 (100); 6.71 (21); 8.58 (26); 9.02 (24); 10.74 (34) 2.63 (79); 2.88 (39); 3.09 (100), 3.38 (10); 3.89 (7) 2.11 (14); 2.50 (83); 2.76 (13); 3.09 (100); 3.35 (40); 3.46 (40); 3.69 (32); 4.13 (7)
Means of three replicates of each compound and their standard errors. Averages of means of each group and their standard errors. c Means with different letter in the column are significantly different according to the FisherÕs protected LSD test at P = 0.05 level (UNISTAT 5.5, London, UK).
3ADON FUS NEO T2T
Table 1 shows the relative amounts of ozone required to effect a 50% reduction (R50) in each trichothecene and confirms this effect. The mean R50 values depended upon the oxidation state of C8 and increased from methylene (no oxygen) < hydroxyl (free or esterified) < keto. Hoigne´ and Bader (1983) observed a 1.8-fold increase in rate of reac-
DAS
a
b
Total Product Remaining (% Relative to Starting Trichothecene)
140
140
a
HT2
MAS VER a b
Retention times in minutes. Relative amounts based on peak heights.
140
b
120
120
120
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0
0 0
1
2
3
4
5
6
c
0
1
2
3
4
5
6
0
1
2
3
4
5
6
Ozone/Trichothecene Ratio (mol/mol) Fig. 3. Variation in total amounts of products from reaction of trichothecenes with varying amounts of aqueous ozone. Values are relative to responses for the starting trichothecene taken as 100. Data points represent averages of reactions run in triplicate and quantified by liquid chromatography–mass spectrometry. (a) trichothecenes lacking oxygenation at C8: d = verrucarol; j = 15-monoacetoxyscirpenol; m = diacetoxyscirpenol; (b) trichothecenes with free or esterified hydroxide at C8: d = neosolaniol; j = T-2 triol; m = HT-2 toxin; (c) trichothecenes with a keto group at C8: = deoxynivalenol; d = fusarenon X; j = 3-acetyldeoxynivalenol; m = 15-acetyldeoxynivalenol.
Table 3 Atmospheric pressure chemical ionization positive ion fragmentation patterns and explanation of mass losses for trichothecenes and their major ozonation reaction products (those products listed as (100) in Table 2) Trichothecenea
Source of mass spectral fragment M+1
–H2O
–2H2O –HOAc
DON DON + O2c
297 329
279 311
3ADON 3ADON + O2
339 371
321 353
15ADON 15ADON + O2
339 371
321 353
FUS FUS + O2
355 387
NEO NEO + O2
–2H2O –HOAc
–2HOAc
–H2O –2HOAc
–2H2O –2HOAc
–H2O
–2H2O
–iValOH
–iValOH
–iValOH
281 313
263 295
245 277
323 355
305 337
287 319
–HOAc –iValOH
–H2O –HOAc –iValOH
–2H2O –HOAc –iValOH
263 295
245 277
227 259
261 293 279 311
261 293
303 (335)d
279 311
261 293
243
337 369
319 351
295 327
277 309
259 291
383 415
365 397
347 379
323 355
305 337
287 319
T2T T2T + O2
383 (415)
383 397
347 379
HT2 HT2 + O2
425 (457)
407 439
359 421
VER VER + O2
267 (299)
249 281
231 (263)
MAS MAS + O2
325 357
307 339
289 321
DAS DAS + O2
367 (399)
349 381
365 397
347 379
265 297
247 279
307 339
289 321
263 295
245 277
227 259
229 261 247 279
229 261
J.C. Young et al. / Food and Chemical Toxicology 44 (2006) 417–424
b
–H2O –HOAc
a DON = 4-deoxynivalenol; 3ADON = 3-acetyldeoxynivalenol; 15ADON = 15-acetyldeoxynivalenol; FUS = fusarenon X; NEO = neosolaniol; T2T = T-2 triol; HT2 = HT-2 toxin; VER = verrucarol; MAS = 15-acetoxyscirpenol; DAS = diacetoxyscirpenol. b m/z. c +O2 = product from net addition of two oxygen atoms. d Numbers in parenthesis represent expected ions not observed.
421
422
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olefins and the rate reducing effects of nearby electronwithdrawing groups (Bailey, 1978). At the lower ozone concentrations, the appearance of transitory products could be followed. Fig. 3 shows the relative amounts of the sum of all intermediate products for a given trichothecene as they are formed and subsequently degraded in unbuffered solution. The median RSD of these determinations was 21.2%. The oxidation state at C8 had an effect on the ratio of ozone required to form the intermediate products as well as result in their degradation. Trichothecenes lacking oxygen at C8 required less ozone to afford the intermediate products, which appeared in proportionally higher yield and then disappeared. The requirement for more ozone to reach maximum yield and then effect the disappearance of intermediates increased with allylic hydroxide and was even greater for a ketone at C8. The amounts of ozone needed for total destruction of identifiable products were also in the order methylene < hydroxyl < keto at C8 (data not shown). Table 2 shows that the mix of intermediate products at maximum yield for each of the trichothecenes studied was quite complex with three to eight compounds observed. The reactions with ozone did not terminate at the stage shown in Fig. 3. Additional ozone reacted with the intermediate products and presumably degraded them to much simpler molecules (such as acids, aldehydes, ketones, CO2) (Hoigne´, 1998) that were not detected by UV or MS. 3.2. Site of ozonation The olefinic position is one of the most reactive sites for reaction of ozone with organic compounds (Bailey, 1978; Razumovskii and Zaikov, 1984 Hoigne´, 1998; Rakovsky and Zaikov, 1998). There is an olefinic double bond at the C9–10 position of trichothecenes. Due to extended conjugation, the 8-keto trichothecenes (3ADON, 15ADON, DON, and FUS) are the only ones studied that exhibited significant UV absorption (at 217 nm). When monitored by either LC–MS or LC–UV, the disappearance profiles of these compounds in the presence of ozone were virtually superimposable, which suggests that reaction occurs at the C9–10 double bond. Furthermore, none of the products from these compounds exhibited any significant UV absorption. This site of reaction was also supported by MS data. Under the APCI +ve ion conditions employed, all the trichothecenes showed MS fragmentation patterns consistent with sequential loss of hydroxyl (as water) and/or acyl groups. Table 3 summarizes the observed fragmentation patterns. Reaction of ozone with olefins has been well studied (Bailey, 1978; Razumovskii and Zaikov, 1984; Rakovsky and Zaikov, 1998) and leads to the proposed mechanism for reaction with trichothecenes illustrated in Fig. 4. Two of the expected keto aldehyde products can be explained by isomerization at C11 during or immediately after formation of the aldehyde at C10. The final postulated product(s) arises from the net addition of two
H
16 9 8
R1
H
O
10
11 13 2 6
7
5
R5 O
O3
3
15
R1 R4
14
R2
R4
R3
R3
Trichothecene
H O
Trichothecene molozonide
O O
R5 O
12 4
R2
O O H H O O
H
R5
H2O
O
O
H
O
O
O R1
H
R5 O
R1 R4
R2 R3
R4
R2 R3
Trichothecene ozonide Fig. 4. Proposed mechanism for the addition of ozone to trichothecenes. R1 = H, OH, OAcyl; R2 = H, OH; R3 = OH, OAcetyl; R4 = H, OH, OAcetyl; R5 = OH, OAcetyl.
atoms of oxygen to the molecule. When the MS of the major product from each of the trichothecenes was offset by 32 mass units and compared with the MS of the starting compounds, there was virtually complete matching of the fragmentation patterns. See Table 3 for complete MS patterns of the major products. The combined UV and MS data suggest that aside for the change in oxidation state at C9–10, the remainder of the molecules was left intact at this stage of the reaction. This data is consistent with that reported for the reaction of pure ozone with triacetoxyscirpenol at 70 C (Sigg et al., 1965), which resulted in the cleavage of the C9–10 double bond and enabled isolation of the intermediary ozonide. Catalytic hydrogenation over Pd subsequently afforded the proposed keto aldehyde characterized by infra red and proton nuclear magnetic resonance spectroscopy. 3.3. Buffered ozonation The above discussion is predicated on the mechanism of reaction being direct oxidation by molecular ozone. To test the possible indirect oxidation by free radicals formed during the ozone decomposition in water at elevated pH, the ozonation reaction of representative C8 methylene, hydroxyl, and keto trichothecenes (MAS, T2T, and DON, respectively) was studied over the range pH 4–9. Fig. 5 shows the disappearance of the trichothecenes at the different pH values. The median RSD of these determinations was 6.6%. Each of these mycotoxins was rapidly degraded by ozone at or below pH 6. The R50 values at pH 6 were each about 30% higher than the corresponding values given in Table 1. The oxidation state at C8 influenced the outcome of the reaction. MAS, with a methylene at this
J.C. Young et al. / Food and Chemical Toxicology 44 (2006) 417–424
Trichothecene Remaining (%)
150
150
a
150
b
120
120
120
90
90
90
60
60
60
30
30
30
0
0 0
1
2
3
4
5
6
423
c
0 0
1
2
3
4
5
6
0
1
2
3
4
5
6
Ozone/Trichothecene Ratio (mol/mol) Fig. 5. Reductions, relative to starting material, in amounts of trichothecenes upon reaction with varying amounts of aqueous ozone at different pH. Data points represent averages of reactions run in triplicate and quantified by liquid chromatography–mass spectrometry. = pH 4; = pH 5; d = pH 6; s = pH 7; j = pH 8; h = pH 9 (a) monoacetoxyscirpenol; (b) T-2 triol; (c) deoxynivalenol.
so perhaps the buffer reagent scavenged the radicals before they could react with DON. Fig. 6 shows the relative amounts of the sum of all intermediate products. The median RSD of these determinations was 6.1%. The highest yields of total products were observed at pH 4 and 5, and essentially no products observed above pH 7. pH also influenced the mix of products: MAS afforded more polar products (earlier eluting) at pH 6
position, was the most prone to degradation at pH 7 and 8. Surprisingly, at pH 9, where OH radicals might be expected to be present (Hoigne´ and Bader, 1978; Hoigne´, 1982), there was little or no reaction with any of the trichothecenes. Because the reactions were analyzed by LC–MS, the reaction solution could only be buffered with relatively volatile ammonium acetate. OH radicals are known to react with ammonia (Hoigne´ and Bader, 1978),
Total Product Remaining (% Relative to Starting Trichothecene)
120
120
a
120
b
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0 0
1
2
3
4
5
6
c
0 0
1
2
3
4
5
6
0
1
2
3
4
5
6
Ozone/Trichothecene Ratio (mol/mol) Fig. 6. Reductions, relative to starting material, in amounts of trichothecenes upon reaction with varying amounts of aqueous ozone at different pH. Data points represent averages of reactions run in triplicate and quantified by liquid chromatography–mass spectrometry. = pH 4; = pH 5; d = pH 6; s = pH 7; j = pH 8; h = pH 9 (a) monoacetoxyscirpenol; (b) T-2 triol; (c) deoxynivalenol.
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J.C. Young et al. / Food and Chemical Toxicology 44 (2006) 417–424
and fewer polar products at pH 8; DON yielded different major products at pH 4 and 7. Several studies (see Sundstøl Eriksen et al., 2004 and references cited therein) have shown that de-epoxy trichothecenes are markedly less toxic than their parents. Since the epoxy moiety appears to have been retained in the intermediary degradation products, it is possible that there may be some residual toxicity. However, it is likely that under the more drastic conditions, the epoxide ring is also degraded. In summary, aqueous ozone has been shown to degrade a wide variety of trichothecenes to presumably simple products. The identity of the breakdown products under the different conditions used in the study remain to be confirmed and their toxicity evaluated. Since aqueous ozone reacts quickly and leaves no residue, it shows promise as a reagent for decontamination of trichothecene-contaminated grains. Studies to determine the efficacy of this technology to such matrices are underway. 4. Safety note Ozone is a toxic gas, so all preparations were conducted in a fume hood. The trichothecenes are also toxic and appropriate personal protective equipment was worn when these materials were handled. Acknowledgments The authors thank Kuanji Wei for technical assistance and Dr. Hongde Zhou (University of Guelph) for helpful discussions. References Anon, 2003. Mycotoxins: Risks in plant, animal, and human systems. Task Force Report No. 139, Council for Agricultural Science and Technology, Ames, Iowa. Bailey, P.S., 1978. Ozonation in Organic Chemistry Volume 1 Olefinic Compounds. Academic Press, New York. Bard, H., Hoigne´, J., 1981. Determination of ozone in water by the indigo method. Water Res. 15, 449–456. Buck, W.B., Coˆte´, L.M., 1991. Handbook of Natural Toxins. In: Keeler, R.F., Tu, A.T. (Eds.), Toxicology of Plant and Fungal Compounds, Vol. 6. Marcel Dekker, Inc., New York, pp. 523–555. Charmley, L.L., Prelusky, D.B., 1994. Decontamination of Fusarium mycotoxins. In: Miller, J.D., Trenholm, H.L. (Eds.), Mycotoxins in Grain: Compounds Other Than Aflatoxin. Eagan, St. Paul, MN. DeVries, J.W., Trucksess, M.W., Jackson, L.S., 2002. Mycotoxins and Food Safety: Proceedings of an American Chemical Society Symposium held in Washington, DC, USA, on 21–23 August 2000. Kluwer Academic Publishers, Dordrecht, Netherlands, p. 295. Hoigne´, J., 1982. Mechanisms, rates and selectivities of oxidations of organic compounds initiated by ozonation in water. In: Rice, R.G., Netzer, A. (Eds.), Handbook of Ozone Technology and Applications. Ann Arbor Science Publishers, Ann Arbor, Mich.
Hoigne´, J., 1998. Chemistry of aqueous ozone and transformation of pollutants by ozonation and advanced oxidation processes. In: Hrubec, J. (Ed.), Quality and Treatment of Drinking Water II. Springer-Verlag, Berlin, pp. 83–141. Hoigne´, J., Bader, H., 1978. Ozonation of water: kinetics of oxidation of ammonia by ozone and hydroxyl radicals. Environ. Sci. Technol. 12, 79–84. Hoigne´, J., Bader, H., 1983. Rate constants of reactions of ozone and inorganic compounds in water—I Non-dissociating organic compounds. Water Res. 17, 173–183. Karlovsky, P., 1999. Biological detoxification of fungal toxins and its use in plant breeding, feed and food production. Nat. Toxins 7, 1–23. Lemke, S.L., Mayura, K., Ottinger, S.E., McKenzie, K.S., Wang, N., Fickey, C., Kubena, L.F., Phillips, T.D., 1999. Assessment of the estrogenic effects of zearalenone after treatment with ozone utilizing the mouse uterine weight bioassay. J. Toxicol. Environ. Health Part A 56, 283–295. McKenzie, K.S., Kubena, L.F., Denvir, A.J., Rogers, T.D., Hitchems, G.D., Bailey, R.H., Harvey, R.B., Buckley, S.A., Phillips, T.D., 1998. Aflatoxicosis in turkey poults is prevented by treatment of naturally contaminated corn with ozone generated by electrolysis. Poultry Sci. 77, 1094–1102. McKenzie, K.S., Sarr, A.B., Mayura, K., Bailey, R.H., Miller, D.R., Rogers, T.D., Norred, W.P., Voss, K.A., Plattner, R.D., Kubena, L.F., Phillips, T.D., 1997. Oxidative degradation and detoxification of mycotoxins using a novel source of ozone. Food Chem. Toxicol. 35, 807–820. Miller, J.D., Trenholm, H.L. (Eds.), 1994. Mycotoxins in Grain: Compounds Other Than Aflatoxin. Eagan, St. Paul, MN. Pittet, A., 1998. Natural occurrence on mycotoxins in foods and feeds—an updated review. Revue de Me´decine Ve´te´rinaire 149, 479–492. Placinta, C.M., DÕMello, J.P.F., Macdonald, A.M.C., 1999. A review of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxins. Anim. Feed Sci. Technol. 78, 21–37. Rakovsky, S., Zaikov, G., 1998. Kinetics and Mechanism of Ozone Reactions with Organic and Polymeric Compounds in Liquid Phase. Nova Science Publishers Inc., Commack, NY. Razumovskii, S.D., Zaikov, G.E., 1984. Ozone and its Reactions with Organic Compounds. Elsevier, New York. Sigg, H.P., Mauli, R., Flury, E., Hauser, D., 1965. Die Konstitution von Diacetoxyscirpenol. Helv. Chim. Acta 48, 962–988. Staehelin, J., Hoigne´, J., 1982. Decomposition of ozone in water: rate of initiation by hydroxide ions and hydrogen peroxide. Environ. Sci. Technol. 16, 676–681. Staehelin, J., Hoigne´, J., 1985. Decomposition of ozone in water in presence of organic solutes acting as promoters and inhibitors of radical chain reactions. Environ. Sci. Technol. 19, 120–126. Sundstøl Eriksen, G., Pettersson, H., Lundh, T., 2004. Comparative cytotoxicity of deoxynivalenol, nivalenol, their acetylated derivatives and de-epoxy metabolites. Food Chem. Toxicol. 42, 619–624. Trigo-Stockli, D.M., 2002. Effect of processing deoxynivalenol and other trichothecenes. In: DeVries, J.W., Trucksess, M.W., Jackson, L.S. (Eds.), Mycotoxins and Food Safety: Proceedings of an American Chemical Society Symposium held in Washington, DC, USA, on 21– 23 August 2000. Kluwer Academic Publishers, Dordrecht, Netherlands, pp. 181–188. Young, J.C., 1986. Reduction in levels of deoxynivalenol in contaminated corn by chemical and physical treatment. J. Agric. Food Chem. 34, 465–467. Young, J.C., Subryan, L.M., Potts, D., McLaren, M.E., Gobran, F.H., 1986. Reduction in levels of deoxynivalenol in contaminated wheat by chemical and physical treatment. J. Agric. Food Chem. 34, 461–465.
Degradation of trichothecene mycotoxins by aqueous ozone
q
J. Christopher Young *, Honghui Zhu, Ting Zhou Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ont., Canada N1G 5C9 Received 24 January 2005; accepted 16 August 2005
Abstract The degradation of ten trichothecene mycotoxins by aqueous ozone was monitored by liquid chromatography–ultraviolet–mass spectrometry (LC–UV–MS). Saturated aqueous ozone (25 ppm) degraded these mycotoxins to materials that were not detected by UV or MS. At lower levels (0.25 ppm) of aqueous ozone, intermediate products were observed. On the basis of UV and MS data, it is proposed that the degradation begins with attack of ozone at the C9–10 double bond with the net addition of two atoms of oxygen. The remainder of the molecule appears to have been left unaltered. The oxidation state at the allylic carbon 8 position had a significant effect on the ease of reaction, as determined by moles of ozone required to effect oxidation. The amount of ozone required to effect oxidation to intermediate products and subsequent degradation followed the series allylic methylene (no oxygen) < hydroxyl (or ester) < keto. Ozonation was also sensitive to pH. At pH 4–6, all mycotoxins studied degraded readily; at pH 7–8 the degree of reactivity was dependent upon the carbon 8 oxidation state; at pH 9, there was little or no reaction. Structures for some of the intermediate products are proposed. Crown Copyright 2005 Published by Elsevier Ltd. All rights reserved. Keywords: Mycotoxins; Trichothecenes; Degradation; Ozonation; pH
1. Introduction The trichothecene class of mycotoxins consists of naturally occurring metabolites produced by Fusarium spp. fungi on a variety of cereal grains and are known to be associated with several diseases in animals and humans (Buck and Coˆte´, 1991; Miller and Trenholm, 1994; Pittet, 1998; Placinta et al., 1999; DeVries et al., 2002; Anon, 2003). Because of potential losses to the farmer and toxicological hazards to the consumer, developing cost effective methods to detoxify mycotoxin-contaminated grains and
Abbreviations: 3ADON, 3-acetyldeoxynivalenol; 15ADON, 15-acetyldeoxynivalenol; APCI, atmospheric pressure chemical ionization; DAS, diacetoxyscirpenol; DON, 4-deoxynivalenol; FUS, fusarenon X; HT2, HT-2 toxin; LC, liquid chromatography; MAS, 15-monoacetoxyscirpenol; MS, mass spectrometry; NEO, neosolaniol; RSD, relative standard deviations; T2T, T-2 triol; UV, ultraviolet; VER, verrucarol. q This is Scientific Publication S206 of the Food Research Program, Agriculture and Agri-Food Canada, Guelph. * Corresponding author. Tel.: +519 780 8033; fax: +519 829 2600. E-mail address: [email protected] (J.C. Young).
foods is a priority. A variety of chemical, physical, and biological treatments have been tested for their ability to reduce or eliminate these mycotoxins in contaminated feeds and foods (Charmley and Prelusky, 1994; Karlovsky, 1999; Trigo-Stockli, 2002; Anon, 2003). One of the chemical treatments of deoxynivalenol (DON) contaminated wheat (Young et al., 1986) and corn (Young, 1986) previously investigated by us was with ozone in air. When dry whole wheat kernels naturally contaminated (ca. 1 lg/g DON) were treated with dry ozone, there was no reduction in DON levels. When vacuum oven dried ground unhusked corn artificially inoculated by Fusarium graminearum (ca. 1000 lg/g DON) was treated with dry ozone, the half life of DON disappearance was 2.5 h. However, when the corn was not dried and the ozone stream was moistened by being bubbled through water, the half life of DON disappearance was reduced to about 15 min. Thus moisture appears to be essential in the reaction between DON and ozone. The availability of saturated aqueous ozone prompted us to revisit this reaction and formed the basis for this paper. The difference between the wheat and corn studies results may also have been
0278-6915/$ - see front matter Crown Copyright 2005 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.08.015
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due to a matrix effect. The corn was ground and thus porous whereas ozone may not have been able to penetrate the whole wheat kernels as readily. More recently, ozone gas was demonstrated as being effective in chemically modifying (McKenzie et al., 1997) a variety of non-trichothecene mycotoxins (aflatoxins B1, B2, G1, and G2, cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, secalonic acid, and zearalenone) and reducing their biological activity in the bioassays used (McKenzie et al., 1998; Lemke et al., 1999). More studies are required to establish the extent of hazard reduction. Ozonation of triacetoxyscirpenol was conducted as part of a structural determination (Sigg et al., 1965). Ozone is an unstable and a strong oxidant, capable of reacting with a wide variety of contaminants in water, which makes it suitable for the treatment of waste and drinking water to remove undesirable contaminants (Hoigne´, 1998). Two primary oxidation pathways in water have been proposed (Hoigne´, 1982; Staehelin and Hoigne´, 1982; Staehelin and Hoigne´, 1985): direct oxidation by molecular ozone and indirect oxidation by free radicals, which are formed during the ozone decomposition in water. The relative importance between these two pathways is affected by pH, UV light, ozone concentration, and the presence of radical scavengers. The later pathway can be further divided into three steps: initiation, propagation, and termination to form various free radicals. Among them is hydroxyl free radical, which is much more powerful an oxidant than ozone molecule itself. Thus, the reactions between these free radicals and organic contaminants are often considered to be much faster but less selective. Since the only previous study of the reactivity of ozone with trichothecenes was with DON and ozone in air, the purpose of this study was to investigate the reaction between ozone and DON in an aqueous medium and to determine if this is a general reaction with other trichothecenes. Furthermore, since the reactive species of aqueous ozone is dependant upon pH, we also studied the effect of pH on the course of trichothecene ozonation. 2. Materials and methods
10
O
9
O
3 OH
OH O
O
8
O 15
4
O
OH
O
O
O
O
Diacetoxyscirpenol (DAS)
15-Monoacetoxyscirpenol (MAS)
O
O
HO O
OH
OH
O
O
O Neosolaniol (NEO)
Verrucarol (VER)
O
O
OH
O
O
OH O
O O
O
O
OH
HO
OH
O
O
OH
O T-2 Triol (T2T)
HT-2 Toxin (HT2)
OH
O
OH
O
O
O
O
O HO
HO
OH
O
Fusarenon X (FUS)
Deoxynivalenol (DON) O
O
OH
OH
O
O O
O
O O
O HO
HO OH
O O
3-Acetyldeoxynivalenol (3ADON)
15-Acetyldeoxynivalenol (15ADON)
Fig. 1. Structures of the trichothecenes studied.
stead) water and chilled in an ice bath. For safety reasons, excess ozone was neutralized by passage through saturated aqueous sodium thiosulfate. The concentration of ozone in water was determined by the indigo colorimetric method of Bard and Hoigne´ (1981), which involves spectrophotometric (Milton Spectronic 1000 plus) measurement at 600 nm.
2.1. Mycotoxin samples and reagents 2.3. Ozonation of pure mycotoxins The mycotoxins 3-acetyldeoxynivalenol (3ADON), 15-acetyldeoxynivalenol (15ADON), diacetoxyscirpenol (DAS), 4-deoxynivalenol (DON), fusarenon X (FUS), HT-2 toxin (HT2), 15-monoacetoxyscirpenol (MAS), neosolaniol (NEO), T-2 triol (T2T), and verrucarol (VER) were obtained from Sigma-Aldrich (Oakville, ON). The structures of the various mycotoxins are shown in Fig. 1. Methanol was obtained from Caledon (Georgetown, ON) and oxygen was obtained from Praxair (Kitchener, ON).
In a typical experiment with each of the mycotoxins, about 2.5 lg of mycotoxin was placed into a 1.8 mL glass vial and dissolved in water. Ozone saturated water of known concentration was then added in a ratio such that the total aqueous volume was 1.0 mL and gave the desired final concentration of ozone. The reaction was repeated with DON, MAS and T2T in 0.1 M ammonium acetate buffered solutions at pH 4–9.
2.4. Analysis of mycotoxins and reaction products 2.2. Ozone in water Water saturated with ozone was prepared by bubbling the gas, generated by passage of purified extra dry oxygen through a Triogen Model LAB2B generator (Ozonia North America, Elmwood Park, NJ), for 30 min through a 500 mL gas wash bottle containing NANOpure (Barn-
The reaction mixtures were then analyzed directly without any work up by taking a 20 lL aliquot and injecting onto a 4.6 · 150 mm liquid chromatographic (LC) column packed with Agilent Zorbax Eclipse XDBC18, 3.5 lm particle size. The column was eluted at 1 mL/min with a linear gradient of methanol–water changing from 1:3 to 3:1 over 15 min. Starting
J.C. Young et al. / Food and Chemical Toxicology 44 (2006) 417–424
The treatments were repeated except that molar ratios of ozone to trichothecenes ranging from 0.3 to 5.0 (equivalent to 0.14–1.8 ppm ozone in water) were chosen. The solutions were unbuffered and had a pH of 6.5. Under these conditions, the disappearance of the trichothecenes and appearance of products could be followed. Several phenomena were immediately apparent. Reaction with ozone reached its endpoint quickly; sequential reanalyses of several reaction mixtures revealed that there were no further changes over time when the product mixtures were compared with the initial analysis made shortly after mixing of reagents. In all instances, the appearance of products was transitory; they too disappeared as the ozone concentration increased. A final observation was that some mycotoxins disappeared more readily than others. Fig. 2 shows the disappearance profiles between the various trichothecenes for different amounts of ozone. The median relative standard deviations (RSD) of these determinations was 20.6%. The reaction between ozone and a trichothecene required less ozone for those compounds (DAS, MAS, and VER) lacking an oxygen at the carbon 8 position (Fig. 2a). Those trichothecenes with an allylic oxygen at C8 required more ozone (Fig. 2b and c).
materials and products were detected with a Finnigan SpectraSystem UV6000LP ultraviolet (UV) detector and a Finnigan LCQ Deca ion trap mass spectrometer (MS) operated in the atmospheric pressure chemical ionization (APCI) positive ion mode. The mass spectrometer was tuned for maximum response for DON. Machine operating conditions were as follows: shear gas and auxiliary flow rates were set at 80 and 0 (arbitrary units); voltages on the capillary, tube lens offset, multipole 1 offset, multipole 2 offset, lens, and entrance lens were set at 15.00, 30.00, 5.00, 7.00, 16.00, and 60.00 V, respectively; capillary and vaporizer temperatures were set at 200 and 450 C, respectively; and the discharge needle current was set at 10 lA. Compounds were quantified on the basis of integrated peak areas using absorbance units (UV) or ion counts (MS). All reactions were run in triplicate and the results averaged.
3. Results and discussion 3.1. Unbuffered ozonation At the outset, the mycotoxins were treated with saturated aqueous ozone (concentration typically about 25– 30 ppm). The LC–UV–MS analyses showed that all of the starting material had been destroyed and no products could be detected. McKenzie et al. (1997) also remarked that no products were observed when their series of nontrichothecene mycotoxins were treated with ozone.
Trichothecene Remaining (%)
100
100
a
100
b
80
80
80
60
60
60
50
50
50
40
40
40
20
20
20
0
1
2
3
4
5
c
0
0
0
419
0
1
2
3
4
5
0
1
2
3
4
5
Ozone/Trichothecene Ratio (mol/mol) Fig. 2. Reductions, relative to starting material, in amounts of trichothecenes upon reaction with varying amounts of aqueous ozone. Data points represent averages of reactions run in triplicate and quantified by liquid chromatography–mass spectrometry. (a) Trichothecenes lacking oxygenation at C8: d = verrucarol; j = 15-monoacetoxyscirpenol; m = diacetoxyscirpenol; (b) trichothecenes with free or esterified hydroxyl at C8: d = neosolaniol; j = T-2 triol; m = HT-2 toxin; (c) trichothecenes with a keto group at C8: = deoxynivalenol; d = fusarenon X; j = 3-acetyldeoxynivalenol; m = 15-acetyldeoxynivalenol.
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tion with ozone when the allylic hydroxyl in hex-1-ene3-ol was moved further away from the double bond (hex-1-ene-4-ol). These results are consistent with the well established electrophilic nature of attack of ozone on
Table 1 Molar ratios of ozone/trichothecene required to effect a 50% reduction in trichothecene levels Groups
Trichothecenes
Meana ± std err
Group meansb,c
8-methylene
VER MAS DAS
0.65 ± 0.09 0.75 ± 0.05 0.93 ± 0.16
0.77 ± 0.06a
8-hydroxy
NEO HT2 T2T
1.14 ± 0.12 1.36 ± 0.23 1.67 ± 0.28
1.40 ± 0.11b
8-keto
DON FUS 3ADON 15ADON
1.44 ± 0.05 1.50 ± 0.10 1.62 ± 0.16 2.44 ± 0.26
1.75 ± 0.13c
Table 2 Products from reaction of trichothecenes with unbuffered aqueous ozone Trichothecene
Ozonation products
DON 15ADON
2.79a (100)b; 2.92 (44); 3.21 (8); 3.59 (6); 4.16 (15) 3.10 (64); 4.05 (100); 4.52 (34); 5.39 (23); 6.44 (43); 7.08 (82) 6.38 (8); 6.77 (26); 7.03 (100) 3.20 (7); 3.48 (100); 3.90 (3); 4.29 (4); 4.96 (5) 4.25 (15); 5.14 (7); 5.45 (57); 6.10 (100) 8.18 (35); 8.34 (53); 9.54 (83); 9.98 (32); 10.80 (47); 11.47 (100) 10.80 (48); 11.11(100); 11.90 (31); 12.54 (25); 12.90 (27); 13.47 (50) 6.17 (95); 6.32 (100); 6.71 (21); 8.58 (26); 9.02 (24); 10.74 (34) 2.63 (79); 2.88 (39); 3.09 (100), 3.38 (10); 3.89 (7) 2.11 (14); 2.50 (83); 2.76 (13); 3.09 (100); 3.35 (40); 3.46 (40); 3.69 (32); 4.13 (7)
Means of three replicates of each compound and their standard errors. Averages of means of each group and their standard errors. c Means with different letter in the column are significantly different according to the FisherÕs protected LSD test at P = 0.05 level (UNISTAT 5.5, London, UK).
3ADON FUS NEO T2T
Table 1 shows the relative amounts of ozone required to effect a 50% reduction (R50) in each trichothecene and confirms this effect. The mean R50 values depended upon the oxidation state of C8 and increased from methylene (no oxygen) < hydroxyl (free or esterified) < keto. Hoigne´ and Bader (1983) observed a 1.8-fold increase in rate of reac-
DAS
a
b
Total Product Remaining (% Relative to Starting Trichothecene)
140
140
a
HT2
MAS VER a b
Retention times in minutes. Relative amounts based on peak heights.
140
b
120
120
120
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0
0 0
1
2
3
4
5
6
c
0
1
2
3
4
5
6
0
1
2
3
4
5
6
Ozone/Trichothecene Ratio (mol/mol) Fig. 3. Variation in total amounts of products from reaction of trichothecenes with varying amounts of aqueous ozone. Values are relative to responses for the starting trichothecene taken as 100. Data points represent averages of reactions run in triplicate and quantified by liquid chromatography–mass spectrometry. (a) trichothecenes lacking oxygenation at C8: d = verrucarol; j = 15-monoacetoxyscirpenol; m = diacetoxyscirpenol; (b) trichothecenes with free or esterified hydroxide at C8: d = neosolaniol; j = T-2 triol; m = HT-2 toxin; (c) trichothecenes with a keto group at C8: = deoxynivalenol; d = fusarenon X; j = 3-acetyldeoxynivalenol; m = 15-acetyldeoxynivalenol.
Table 3 Atmospheric pressure chemical ionization positive ion fragmentation patterns and explanation of mass losses for trichothecenes and their major ozonation reaction products (those products listed as (100) in Table 2) Trichothecenea
Source of mass spectral fragment M+1
–H2O
–2H2O –HOAc
DON DON + O2c
297 329
279 311
3ADON 3ADON + O2
339 371
321 353
15ADON 15ADON + O2
339 371
321 353
FUS FUS + O2
355 387
NEO NEO + O2
–2H2O –HOAc
–2HOAc
–H2O –2HOAc
–2H2O –2HOAc
–H2O
–2H2O
–iValOH
–iValOH
–iValOH
281 313
263 295
245 277
323 355
305 337
287 319
–HOAc –iValOH
–H2O –HOAc –iValOH
–2H2O –HOAc –iValOH
263 295
245 277
227 259
261 293 279 311
261 293
303 (335)d
279 311
261 293
243
337 369
319 351
295 327
277 309
259 291
383 415
365 397
347 379
323 355
305 337
287 319
T2T T2T + O2
383 (415)
383 397
347 379
HT2 HT2 + O2
425 (457)
407 439
359 421
VER VER + O2
267 (299)
249 281
231 (263)
MAS MAS + O2
325 357
307 339
289 321
DAS DAS + O2
367 (399)
349 381
365 397
347 379
265 297
247 279
307 339
289 321
263 295
245 277
227 259
229 261 247 279
229 261
J.C. Young et al. / Food and Chemical Toxicology 44 (2006) 417–424
b
–H2O –HOAc
a DON = 4-deoxynivalenol; 3ADON = 3-acetyldeoxynivalenol; 15ADON = 15-acetyldeoxynivalenol; FUS = fusarenon X; NEO = neosolaniol; T2T = T-2 triol; HT2 = HT-2 toxin; VER = verrucarol; MAS = 15-acetoxyscirpenol; DAS = diacetoxyscirpenol. b m/z. c +O2 = product from net addition of two oxygen atoms. d Numbers in parenthesis represent expected ions not observed.
421
422
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olefins and the rate reducing effects of nearby electronwithdrawing groups (Bailey, 1978). At the lower ozone concentrations, the appearance of transitory products could be followed. Fig. 3 shows the relative amounts of the sum of all intermediate products for a given trichothecene as they are formed and subsequently degraded in unbuffered solution. The median RSD of these determinations was 21.2%. The oxidation state at C8 had an effect on the ratio of ozone required to form the intermediate products as well as result in their degradation. Trichothecenes lacking oxygen at C8 required less ozone to afford the intermediate products, which appeared in proportionally higher yield and then disappeared. The requirement for more ozone to reach maximum yield and then effect the disappearance of intermediates increased with allylic hydroxide and was even greater for a ketone at C8. The amounts of ozone needed for total destruction of identifiable products were also in the order methylene < hydroxyl < keto at C8 (data not shown). Table 2 shows that the mix of intermediate products at maximum yield for each of the trichothecenes studied was quite complex with three to eight compounds observed. The reactions with ozone did not terminate at the stage shown in Fig. 3. Additional ozone reacted with the intermediate products and presumably degraded them to much simpler molecules (such as acids, aldehydes, ketones, CO2) (Hoigne´, 1998) that were not detected by UV or MS. 3.2. Site of ozonation The olefinic position is one of the most reactive sites for reaction of ozone with organic compounds (Bailey, 1978; Razumovskii and Zaikov, 1984 Hoigne´, 1998; Rakovsky and Zaikov, 1998). There is an olefinic double bond at the C9–10 position of trichothecenes. Due to extended conjugation, the 8-keto trichothecenes (3ADON, 15ADON, DON, and FUS) are the only ones studied that exhibited significant UV absorption (at 217 nm). When monitored by either LC–MS or LC–UV, the disappearance profiles of these compounds in the presence of ozone were virtually superimposable, which suggests that reaction occurs at the C9–10 double bond. Furthermore, none of the products from these compounds exhibited any significant UV absorption. This site of reaction was also supported by MS data. Under the APCI +ve ion conditions employed, all the trichothecenes showed MS fragmentation patterns consistent with sequential loss of hydroxyl (as water) and/or acyl groups. Table 3 summarizes the observed fragmentation patterns. Reaction of ozone with olefins has been well studied (Bailey, 1978; Razumovskii and Zaikov, 1984; Rakovsky and Zaikov, 1998) and leads to the proposed mechanism for reaction with trichothecenes illustrated in Fig. 4. Two of the expected keto aldehyde products can be explained by isomerization at C11 during or immediately after formation of the aldehyde at C10. The final postulated product(s) arises from the net addition of two
H
16 9 8
R1
H
O
10
11 13 2 6
7
5
R5 O
O3
3
15
R1 R4
14
R2
R4
R3
R3
Trichothecene
H O
Trichothecene molozonide
O O
R5 O
12 4
R2
O O H H O O
H
R5
H2O
O
O
H
O
O
O R1
H
R5 O
R1 R4
R2 R3
R4
R2 R3
Trichothecene ozonide Fig. 4. Proposed mechanism for the addition of ozone to trichothecenes. R1 = H, OH, OAcyl; R2 = H, OH; R3 = OH, OAcetyl; R4 = H, OH, OAcetyl; R5 = OH, OAcetyl.
atoms of oxygen to the molecule. When the MS of the major product from each of the trichothecenes was offset by 32 mass units and compared with the MS of the starting compounds, there was virtually complete matching of the fragmentation patterns. See Table 3 for complete MS patterns of the major products. The combined UV and MS data suggest that aside for the change in oxidation state at C9–10, the remainder of the molecules was left intact at this stage of the reaction. This data is consistent with that reported for the reaction of pure ozone with triacetoxyscirpenol at 70 C (Sigg et al., 1965), which resulted in the cleavage of the C9–10 double bond and enabled isolation of the intermediary ozonide. Catalytic hydrogenation over Pd subsequently afforded the proposed keto aldehyde characterized by infra red and proton nuclear magnetic resonance spectroscopy. 3.3. Buffered ozonation The above discussion is predicated on the mechanism of reaction being direct oxidation by molecular ozone. To test the possible indirect oxidation by free radicals formed during the ozone decomposition in water at elevated pH, the ozonation reaction of representative C8 methylene, hydroxyl, and keto trichothecenes (MAS, T2T, and DON, respectively) was studied over the range pH 4–9. Fig. 5 shows the disappearance of the trichothecenes at the different pH values. The median RSD of these determinations was 6.6%. Each of these mycotoxins was rapidly degraded by ozone at or below pH 6. The R50 values at pH 6 were each about 30% higher than the corresponding values given in Table 1. The oxidation state at C8 influenced the outcome of the reaction. MAS, with a methylene at this
J.C. Young et al. / Food and Chemical Toxicology 44 (2006) 417–424
Trichothecene Remaining (%)
150
150
a
150
b
120
120
120
90
90
90
60
60
60
30
30
30
0
0 0
1
2
3
4
5
6
423
c
0 0
1
2
3
4
5
6
0
1
2
3
4
5
6
Ozone/Trichothecene Ratio (mol/mol) Fig. 5. Reductions, relative to starting material, in amounts of trichothecenes upon reaction with varying amounts of aqueous ozone at different pH. Data points represent averages of reactions run in triplicate and quantified by liquid chromatography–mass spectrometry. = pH 4; = pH 5; d = pH 6; s = pH 7; j = pH 8; h = pH 9 (a) monoacetoxyscirpenol; (b) T-2 triol; (c) deoxynivalenol.
so perhaps the buffer reagent scavenged the radicals before they could react with DON. Fig. 6 shows the relative amounts of the sum of all intermediate products. The median RSD of these determinations was 6.1%. The highest yields of total products were observed at pH 4 and 5, and essentially no products observed above pH 7. pH also influenced the mix of products: MAS afforded more polar products (earlier eluting) at pH 6
position, was the most prone to degradation at pH 7 and 8. Surprisingly, at pH 9, where OH radicals might be expected to be present (Hoigne´ and Bader, 1978; Hoigne´, 1982), there was little or no reaction with any of the trichothecenes. Because the reactions were analyzed by LC–MS, the reaction solution could only be buffered with relatively volatile ammonium acetate. OH radicals are known to react with ammonia (Hoigne´ and Bader, 1978),
Total Product Remaining (% Relative to Starting Trichothecene)
120
120
a
120
b
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0 0
1
2
3
4
5
6
c
0 0
1
2
3
4
5
6
0
1
2
3
4
5
6
Ozone/Trichothecene Ratio (mol/mol) Fig. 6. Reductions, relative to starting material, in amounts of trichothecenes upon reaction with varying amounts of aqueous ozone at different pH. Data points represent averages of reactions run in triplicate and quantified by liquid chromatography–mass spectrometry. = pH 4; = pH 5; d = pH 6; s = pH 7; j = pH 8; h = pH 9 (a) monoacetoxyscirpenol; (b) T-2 triol; (c) deoxynivalenol.
424
J.C. Young et al. / Food and Chemical Toxicology 44 (2006) 417–424
and fewer polar products at pH 8; DON yielded different major products at pH 4 and 7. Several studies (see Sundstøl Eriksen et al., 2004 and references cited therein) have shown that de-epoxy trichothecenes are markedly less toxic than their parents. Since the epoxy moiety appears to have been retained in the intermediary degradation products, it is possible that there may be some residual toxicity. However, it is likely that under the more drastic conditions, the epoxide ring is also degraded. In summary, aqueous ozone has been shown to degrade a wide variety of trichothecenes to presumably simple products. The identity of the breakdown products under the different conditions used in the study remain to be confirmed and their toxicity evaluated. Since aqueous ozone reacts quickly and leaves no residue, it shows promise as a reagent for decontamination of trichothecene-contaminated grains. Studies to determine the efficacy of this technology to such matrices are underway. 4. Safety note Ozone is a toxic gas, so all preparations were conducted in a fume hood. The trichothecenes are also toxic and appropriate personal protective equipment was worn when these materials were handled. Acknowledgments The authors thank Kuanji Wei for technical assistance and Dr. Hongde Zhou (University of Guelph) for helpful discussions. References Anon, 2003. Mycotoxins: Risks in plant, animal, and human systems. Task Force Report No. 139, Council for Agricultural Science and Technology, Ames, Iowa. Bailey, P.S., 1978. Ozonation in Organic Chemistry Volume 1 Olefinic Compounds. Academic Press, New York. Bard, H., Hoigne´, J., 1981. Determination of ozone in water by the indigo method. Water Res. 15, 449–456. Buck, W.B., Coˆte´, L.M., 1991. Handbook of Natural Toxins. In: Keeler, R.F., Tu, A.T. (Eds.), Toxicology of Plant and Fungal Compounds, Vol. 6. Marcel Dekker, Inc., New York, pp. 523–555. Charmley, L.L., Prelusky, D.B., 1994. Decontamination of Fusarium mycotoxins. In: Miller, J.D., Trenholm, H.L. (Eds.), Mycotoxins in Grain: Compounds Other Than Aflatoxin. Eagan, St. Paul, MN. DeVries, J.W., Trucksess, M.W., Jackson, L.S., 2002. Mycotoxins and Food Safety: Proceedings of an American Chemical Society Symposium held in Washington, DC, USA, on 21–23 August 2000. Kluwer Academic Publishers, Dordrecht, Netherlands, p. 295. Hoigne´, J., 1982. Mechanisms, rates and selectivities of oxidations of organic compounds initiated by ozonation in water. In: Rice, R.G., Netzer, A. (Eds.), Handbook of Ozone Technology and Applications. Ann Arbor Science Publishers, Ann Arbor, Mich.
Hoigne´, J., 1998. Chemistry of aqueous ozone and transformation of pollutants by ozonation and advanced oxidation processes. In: Hrubec, J. (Ed.), Quality and Treatment of Drinking Water II. Springer-Verlag, Berlin, pp. 83–141. Hoigne´, J., Bader, H., 1978. Ozonation of water: kinetics of oxidation of ammonia by ozone and hydroxyl radicals. Environ. Sci. Technol. 12, 79–84. Hoigne´, J., Bader, H., 1983. Rate constants of reactions of ozone and inorganic compounds in water—I Non-dissociating organic compounds. Water Res. 17, 173–183. Karlovsky, P., 1999. Biological detoxification of fungal toxins and its use in plant breeding, feed and food production. Nat. Toxins 7, 1–23. Lemke, S.L., Mayura, K., Ottinger, S.E., McKenzie, K.S., Wang, N., Fickey, C., Kubena, L.F., Phillips, T.D., 1999. Assessment of the estrogenic effects of zearalenone after treatment with ozone utilizing the mouse uterine weight bioassay. J. Toxicol. Environ. Health Part A 56, 283–295. McKenzie, K.S., Kubena, L.F., Denvir, A.J., Rogers, T.D., Hitchems, G.D., Bailey, R.H., Harvey, R.B., Buckley, S.A., Phillips, T.D., 1998. Aflatoxicosis in turkey poults is prevented by treatment of naturally contaminated corn with ozone generated by electrolysis. Poultry Sci. 77, 1094–1102. McKenzie, K.S., Sarr, A.B., Mayura, K., Bailey, R.H., Miller, D.R., Rogers, T.D., Norred, W.P., Voss, K.A., Plattner, R.D., Kubena, L.F., Phillips, T.D., 1997. Oxidative degradation and detoxification of mycotoxins using a novel source of ozone. Food Chem. Toxicol. 35, 807–820. Miller, J.D., Trenholm, H.L. (Eds.), 1994. Mycotoxins in Grain: Compounds Other Than Aflatoxin. Eagan, St. Paul, MN. Pittet, A., 1998. Natural occurrence on mycotoxins in foods and feeds—an updated review. Revue de Me´decine Ve´te´rinaire 149, 479–492. Placinta, C.M., DÕMello, J.P.F., Macdonald, A.M.C., 1999. A review of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxins. Anim. Feed Sci. Technol. 78, 21–37. Rakovsky, S., Zaikov, G., 1998. Kinetics and Mechanism of Ozone Reactions with Organic and Polymeric Compounds in Liquid Phase. Nova Science Publishers Inc., Commack, NY. Razumovskii, S.D., Zaikov, G.E., 1984. Ozone and its Reactions with Organic Compounds. Elsevier, New York. Sigg, H.P., Mauli, R., Flury, E., Hauser, D., 1965. Die Konstitution von Diacetoxyscirpenol. Helv. Chim. Acta 48, 962–988. Staehelin, J., Hoigne´, J., 1982. Decomposition of ozone in water: rate of initiation by hydroxide ions and hydrogen peroxide. Environ. Sci. Technol. 16, 676–681. Staehelin, J., Hoigne´, J., 1985. Decomposition of ozone in water in presence of organic solutes acting as promoters and inhibitors of radical chain reactions. Environ. Sci. Technol. 19, 120–126. Sundstøl Eriksen, G., Pettersson, H., Lundh, T., 2004. Comparative cytotoxicity of deoxynivalenol, nivalenol, their acetylated derivatives and de-epoxy metabolites. Food Chem. Toxicol. 42, 619–624. Trigo-Stockli, D.M., 2002. Effect of processing deoxynivalenol and other trichothecenes. In: DeVries, J.W., Trucksess, M.W., Jackson, L.S. (Eds.), Mycotoxins and Food Safety: Proceedings of an American Chemical Society Symposium held in Washington, DC, USA, on 21– 23 August 2000. Kluwer Academic Publishers, Dordrecht, Netherlands, pp. 181–188. Young, J.C., 1986. Reduction in levels of deoxynivalenol in contaminated corn by chemical and physical treatment. J. Agric. Food Chem. 34, 465–467. Young, J.C., Subryan, L.M., Potts, D., McLaren, M.E., Gobran, F.H., 1986. Reduction in levels of deoxynivalenol in contaminated wheat by chemical and physical treatment. J. Agric. Food Chem. 34, 461–465.