ENVIRONMENT AND HEALTH

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1 ENVIRONMENT AND HEALTH Aflatoxicosis in Turkey Poults is Prevented by Treatment of Naturally Contaminated Corn with Ozone Generated by Electrolysis 1,2 K. S. McKENZIE,* L. F. KUBENA, A. J. DENVIR, T. D. ROGERS, G. D. HITCHENS, R. H. BAILEY, R. B. HARVEY, S. A. BUCKLEY, and T. D. PHILLIPS*,3 *Department of Veterinary Anatomy and Public Health, Faculty of Toxicology, College of Veterinary Medicine, Texas A&M University, College Station, Texas , USDA, Agricultural Research Service, Food Animal Protection Research Laboratory, 2881 F&B Road, College Station, Texas 77845, and Lynntech, Inc., 7610 Eastmark Drive, Suite 105, College Station, Texas, ABSTRACT Previous studies have demonstrated that a novel source of ozone gas (O 3 ) may be used to chemically degrade numerous mycotoxins, including aflatoxin (AF) B 1. Subsequent in vitro analyses demonstrated detoxification of AFB 1, suggesting a potential method to remediate AF-contaminated grain. The objective of this study was to evaluate the capability of electrochemically produced ozone to degrade AFB 1 in naturally contaminated whole kernel corn and confirm detoxification in turkey poults. Corn was procured from the southern coastal area of Texas and HPLC revealed 1,220 ± 73.3 ppb AFB 1. Control and contaminated corn were treated for 92 h with O 3 at 200 mg/min in 30 kg batches; greater than 95% reduction of AFB 1 in contaminated corn was achieved. One-day-old female turkey poults were fed 1) control corn, 2) control corn + O 3,3) INTRODUCTION Mycotoxins are ubiquitous foodborne contaminants possessing the potential to cause a myriad of detrimental effects to humans and animals. These compounds represent a chemical array of fungal-originated toxins that occur naturally in cereal and oilseed-based feed (Council for Agricultural Science and Technology, 1989). The particular group of mycotoxins that encouraged investigation into the toxic, mutagenic, and carcinogenic potentials of fungal toxins were the aflatoxins (AF). These toxins are produced by strains of Aspergillus flavus Received for publication June 23, Accepted for publication March 31, Mention of trade name, proprietary product or specific equipment does not constitute a warranty by USDA or Texas A&M University and does not imply its approval to the exclusion of other products that may be suitable. 2Portions of abstract published in Poultry Sci. 76(Suppl. 1):121. (Abstr.) 3 To whom correspondence should be addressed: tphillips@vetmed.tamu.edu (Key words: aflatoxin, detoxification, ozone, corn, turkey) AFB 1 corn, or 4) AFB 1 corn + O 3 mixed in rations (46% by wt.) and consumed ad libitum for 3 wk. When compared with controls, turkeys fed AFB 1 corn had reduced body weight gain and relative liver weight, whereas turkeys fed control corn + O 3 or AFB 1 corn + O 3 did not differ from controls. Furthermore, alterations in the majority of relative organ weights, liver discoloration, serum enzyme activity, hematological parameters, and blood chemistry caused by AFB 1 were eliminated (no difference from controls) by treatment with O 3. These data demonstrate that treatment of contaminated corn with electrochemically produced O 3 provided protection against AFB 1 in young turkey poults. It is important to note that treatment of control corn with O 3 did not alter the performance of the turkey poults Poultry Science 77: and Aspergillus parasiticus and include four major metabolites: AFB 1, AFB 2, AFG 1, and AFG 2 (CAST 1989). Aflatoxin B 1 and G 1 have been demonstrated to bind covalently to the N7 of guanine in DNA after P-450 mediated activation of the C8 C9 vinyl ether to the highly reactive epoxide (Essigmann et al., 1977). Aflatoxin B 1 is the most potent of the four naturally occurring AF and because of its remarkable hepatotoxicity and carcinogenicity, this feed contaminant has been the focus of considerable research since its discovery (Eaton and Groopman, 1994). The AF have been reported to cause severe economic losses in livestock (CAST, 1989), with poultry being one of the groups most sensitive to aflatoxicosis. (Muller et al., 1970; Smith and Hamilton, 1970; Edds and Bortell, 1983) The effects of AF on broiler chickens have been well documented (Smith and Hamilton, 1970; Tung et al., Abbreviation Key: AF = aflatoxin; CPA = cyclopiazonic acid; FB 1 = fumonisin B 1 ; OA = ochratoxin A; SBM = soybean meal; ZEN = zearalenone. 1094

2 AFLATOXIN TOXICITY IN POULTS PREVENTED BY OZONE ; Campbell et al., 1983), although chicks are less sensitive to AF than turkeys (Muller et al., 1970; Hamilton et al., 1972; Witlock and Wyatt, 1981). The AF are capable of causing various health effects in turkeys, including poor performance, increases in relative pancreas weights, decrease in relative liver weight, and alterations of serum enzyme activities, biochemical values, and hematological values (Witlock and Wyatt, 1981; Giambrone et al., 1985; Kubena et al., 1991, 1995a). The adverse effects of the AF (and other mycotoxins) on human and animal health have led to the investigation and development of practical detoxification or inactivation strategies (Goldblatt and Dollear, 1977; Park et al., 1988; Phillips et al, 1988, 1994; Kubena, et al., 1990; Huff et al., 1992; McKenzie et al., 1997). Contamination of grains and oilseeds with AF can be extensive and also characterized by the presence of potentially additive or synergistic combinations with other mycotoxins (Huff et al., 1986, 1988; Kubena et al., 1993, 1995a,b, 1997). This problem has presented an ardent challenge to the development of uniform methodologies for remediation of contaminated grain. Although a decontamination protocol may effectively reduce the detectable levels of one toxin, another toxin may persist at harmful levels or the procedure may cause the production of biologically active compounds that maintain toxic or mutagenic potential (Park et al., 1988; Marquez-Marquez et al., 1995). Several approaches to detoxification have been utilized, including physical separation of contaminated kernels or seeds, biological degradation, and chemical reaction with acids, bases, organic solvents, and gases (Goldblatt and Dollear, 1977; CAST 1989; Hagler, 1991). Ozone (O 3 ) gas is a powerful oxidant capable of reaction with numerous chemical groups, though it has an affinity for double bonds (Creigee in Bailey, 1958). A chemical method of AF decontamination was developed that reduced levels of the four major AF in ground corn and peanut meal by treatment with O 3 generated by corona discharge (Dwarakanath et al., 1968; Dollear et al., 1968). This ozone production method can generate up to a maximum of 6 wt % O 3 in dried oxygen-fed systems (Foller and Tobias, 1982), although much lower concentrations are usually used. Alternatively, electrochemical O 3 production couples anodic decomposition of water (hydrolysis) at the water/porous anode interface and utilizes a proton exchange membrane in an electrolysis cell to produce up to 20 wt % (242.0 mg/l air) O 3 gas (Rogers et al., 1992; Verostko et al., 1992; Murphy et al., 1994; Hitchens et al., 1994). This concentrated ozone has 4Sigma Chemical Co., St. Louis, MO Waters Corp., Milford, MA Epson America, Inc., Torrance, CA Digital Equipment Corp., Merrimack, NH Vicam Corp., Watertown, MA Burdick and Jackson, Muskegon, MI Lynntech, Inc., College Station, TX been used to degrade and detoxify numerous mycotoxins in vitro, including the four major aflatoxins, cyclopiazonic acid (CPA), ochratoxin A (OA), patulin, secalonic acid D, and zearalenone (ZEA) (McKenzie et al., 1997). Fumonisin B 1 (FB 1 ) was rapidly degraded to a ketosubstituted compound (3k-FB 1 ), although detoxification was not demonstrated by two bioassays. Although numerous studies have been performed to evaluate the efficacy of diverse mycotoxin decontamination methods, few have actually utilized grains that were naturally contaminated. The purpose of this study was to employ electrochemically produced O 3 to degrade AFB 1 in naturally contaminated corn and confirm detoxification of the corn in vivo in turkey poults. MATERIALS AND METHODS Chemical Analyses Aflatoxins B 1, B2, G 1, G2, and CPA were purchased from Sigma Chemical Company.4 The structural identity and purity of all standards were confirmed by GC/MS, UV spectrophotometry, and HPLC. Levels of AF and CPA in corn were determined using a Waters HPLC system,5 which included model 510 pumps, an automatic sampler (WISP Model 710), a variable UV wavelength absorbance detector (Model 481), a fluorescence detector (Model 420), a photodiode array detector (Model 998), an Epson6 Stylus color printer and Digital workstation. Levels of ochratoxin A (OA), zearalenone (ZEN), and fumonisins were determined using methods previously outlined by Vicam8 and a Series-4 fluorometer. All solvents were spectral grade from Burdick and Jackson9 and reagents were of the highest purity that was commercially available. Aflatoxin-Contaminated Corn Dried yellow dent corn ( 900 kg) contaminated with AFB 1 was procured and transported through the Texas State Chemist s Office and stored at 3 C prior to treatment. Sampling of the corn for mycotoxin contamination was performed as a modification of the method reported by Park and Pohland (1989). Ground subsamples were analyzed for AF by a method described by Siraj and coworkers (1981) and for CPA by a previously published method (Urano, et al., 1992). Analyses of ZEN, OA, and fumonisins were performed by antibody affinity chromatography according to the manufacturer s specifications.8 Ozone Treatment/Diet Formulation Ozone was electrochemically generated and used to treat contaminated corn at Lynntech, Inc.10 This method of ozone production has been patented (U.S. patent 5,460,705) and described in detail (Rogers et al., 1992;

3 1096 Verostko et al., 1992; Hitchens et al., 1994; Murphy et al., 1994). The resulting O 3 output used for corn treatment was 14.0 wt % at a flow rate of 200 mg/min. Ozone concentrations were measured spectrophotometrically at 254 nm using a Shimadzu11 model UV-2101PC. One batch (30 kg) each of AF-contaminated corn or control corn was placed in a stainless steel column and the O 3 was allowed to distribute, mix and flow throughout the corn for 92 h. The grain was then removed, placed into flat pans for 18 h and ground (within 7 d of treatment). The ground corn was mixed with a commercial soybean meal (SBM) based concentrate (46:54 by weight, respectively) such that the finished ration contained 27% protein, 2,900 kcal ME/kg diet and met or exceeded levels of other nutrients recommended by the National Research Council (1994). By blending the corn with SBM, the concentration of AFB 1 was effectively reduced by 54%. The SBM based concentrate was free of any detectable mycotoxins and contained no antibiotics, coccidiostats, or growth promoters. Experimental Design 11Shimadzu Scientific Instruments, Inc., Columbia, MD Coulter Electronics, Hialeah, FL Gilford Impact, Ciba Corning Diagnostics Corp., Oberlin, OH McKENZIE ET AL. A total of 120 day-old female British UTA turkey poults (obtained from a commercial hatchery) were individually weighed, wing-banded, and randomly distributed into battery pens with each containing five poults. Six replicates of five poults per pen (n = 30 per treatment) were grouped based on the following four dietary treatments: 1) control feed containing uncontaminated corn, 2) control feed containing uncontaminated corn treated with 10 wt % O 3 at 200 mg/min for 92 h, 3) feed containing corn contaminated with AFB 1 and 4) feed containing corn contaminated with AFB 1 treated with 10 wt % O 3 at 200 mg/min for 92 h. Turkeys were housed in electrically heated batteries under continuous fluorescent illumination with forced ventilation and provided ad libitum access to feed and water. Poults were individually weighed and feed consumption for each replicate was recorded weekly. At 3 wk of age, 18 poults (six replicates of 3 poults each) from each treatment group were bled by cardiac puncture for serum biochemical analyses and 12 samples from the same poults (six replicates of 2 poults each) from each group were used for hematological determinations. The same poults bled by cardiac puncture were killed by cervical dislocation and the liver, kidney, spleen, pancreas, proventriculus, bursa of Fabricius, and heart were dissected and weighed. Erythrocyte count and mean corpuscular volume were determined with a Coulter12 Model ZM Counter equipped with a Model C 256 channelyzer and acucomp software. Hemoglobin was measured as cyanmethemoglobin. Hematocrits were measured by the microhematocrit centrifugation method. Mean corpuscular hemoglobin and mean corpuscular hemoglobin concentrations were calculated. Serum concentrations of uric acid, urea nitrogen, glucose, calcium, inorganic phosphorus, total protein, albumin, cholesterol, triglycerides, and activities of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and creatine kinase were determined on a clinical chemistry analyzer13 according to the manufacturer s specifications. Statistical Analysis Data (pen means) for all response variables were subjected to ANOVA as a 2 2 factorial using the General Linear Models procedure in PC-SAS software version 6.02 (SAS Institute, 1987). Variable means for treatments showing significant differences in the ANOVA were compared using the Fisher s protected least significant difference procedure (Snedecor and Cochran, 1967). Values were judged to be significantly different if P < All other data were represented as mean (n = 3) ± SD. RESULTS The analysis of the corn by HPLC indicated the presence of four mycotoxins, including AFB 1, AFB 2, fumonisin, and CPA. The concentration of AFB 1 was equal to 1,220 ± 73.3 ppb; whereas, the concentration of AFB 2 was less than 100 ppb and no AFG 1 or AFG 2 were detected. Fumonisins were present at a level of 3.6 ± 0.3 ppm of the corn and the concentration of CPA was 193 ± 18.6 ppb. Subsequent analysis by antibody affinity chromatography demonstrated the absence of ZEN and OA. The control corn had no detectable levels of AF, fumonisin, CPA, ZEN, or OA. Batch treatment of 30 kg of the contaminated corn with O 3 for 92 h reduced the level of AFB 1 from 1,220 ± 73.3 ppb to 58.4 ± 18.2 ppb, a reduction of over 95%. Based on the ratio of corn to SBM in the rations, the concentration of AFB 1 in the AFB 1 corn treatment group was 560 ppb, which was reduced to 26.8 ppb by O 3 treatment. The effects of AF-contaminated corn and treatment with O 3 on poult performance are presented in Table 1; no mortalities were recorded (data not shown). At the end of Week 1, there were no differences in weight gain among the four treatment groups. However, by the end of Week 2, body weight gains were reduced in poults fed the AF corn when compared to controls. By the end of Week 3, the poults receiving the AF corn diet had a 23% reduction in weight gain compared to controls (further illustrated in Figure 1a). The decrease in weight gains caused by the AF corn was eliminated by treatment of the corn with O 3. Of importance is the fact that performance was not affected by treatment of control corn with O 3. Feed conversion (feed:body weight gain) was not affected by any of the treatments and no mortalities were recorded.

4 AFLATOXIN TOXICITY IN POULTS PREVENTED BY OZONE 1097 TABLE 1. Effects of alfatoxin B 1 -contaminated corn alone and after treatment with ozone on body weight gain and feed conversion (FC) in turkey poults at 21 d 1 Body weight gain Treatment 1 to 7 d 7 to 14 d 14 to 21 d 1 to 21 d Deviation from control BW gain FC (1 to 21 d) (g) (%) (kg:kg) Control corn 46.8 ab 138 a 247 a 433 a Control corn + ozone 52.5 a 143 a 254 a 450 a Aflatoxin corn 42.5 b 96 b 148 b 288 b Aflatoxin corn + ozone 52.5 a 146 a 258 a 457 a LSD a,bmeans within column with no common superscript differ significantly (P < 0.05). 1Values represent the mean of six groups of five turkeys each per treatment (n = 30). 2LSD = least significant difference as determined by Fisher s protected LSD procedure. When compared to controls, relative weights of the kidney, spleen, pancreas, proventriculus, and bursa of Fabricius increased in poults fed the AF corn diet, whereas the mean relative weight of the liver decreased (Table 2). The liver appeared pale (Figure 1b), although no hemorrhaging was apparent. There was no significant change in the relative weight of the heart (data not shown). There was a total protective effect by treating the AF corn with O 3 for the liver, kidney, spleen, pancreas, and proventriculus and a partial protection for the bursa of Fabricius. The discoloration of the liver due to the contaminated corn was eliminated by O 3 treatment. Additionally, relative organ weights and liver color were not affected by treatment of control corn with O 3. The corn contaminated with AF caused significant changes in serum enzyme activities and hematological values as depicted in Table 3. Significant increases were recorded for creatinine kinase, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in poults receiving the AF corn diet. These effects were completely mitigated by treatment of the AF corn with O 3. Erythrocyte counts, mean corpuscular hemoglobin, and mean corpuscular volume were increased in the aflatoxin corn group. However, birds consuming the diet containing the AF corn treated with O 3 did not differ from controls in these hematological parameters. There were no differences in mean corpuscular hemoglobin concentration, hemoglobin levels, or hematocrit between all four dietary groups (data not shown). In addition, serum enzyme activities and hematological values for poults fed the ration containing control corn treated with O 3 were not different than activities and values for controls. Table 4 shows the results for serum biochemical values. The AF corn caused a significant decrease in serum concentrations of triglycerides, cholesterol, calcium, total protein, and albumin. Ozonation of the AF corn alleviated these changes; poults on diets containing O 3 -treated AF corn had values that were not different than controls. There was no effect of any dietary treatment on urea nitrogen, uric acid, inorganic phosphorous, or glucose. No poults fed control corn treated with O 3 had any serum chemical values different than controls. DISCUSSION FIGURE 1. Representative poults from each of the four dietary treatment groups on Day 21 demonstrating changes in (A) body frame size and (B) changes in liver color and size due to aflatoxin and the protective effect of ozone. From left to right: Control corn, control corn + ozone, aflatoxin corn, aflatoxin corn + ozone. The ability of electrolytically produced O 3 to degrade and detoxify AFB 1 was investigated using naturally contaminated corn with sufficient concentrations of AFB 1 to produce significant toxicity in vivo. The corn also contained CPA and fumonisins, though these mycotoxins were present at concentrations that were not as toxic as the AF and should have a negligible effect on the poults when compared to the effects of 1,220 ppb AFB 1. In poultry studies, levels as high as 200 to 300 ppm FB 1 and 34 to 50 ppm CPA are required for

5 1098 McKENZIE ET AL. TABLE 2. Effects of aflatoxin B 1 -contaminated corn alone and after treatment with ozone on relative organ weights in turkey poults at 21 d 1 Bursa of Treatment Liver Kidney Spleen Pancreas Proventriculus Fabricius (g/100 g BW) Control corn 2.78 a 0.44 b 0.10 b 0.37 b 0.62 a 0.19 b Control corn + ozone 2.72 a 0.44 b 0.10 b 0.34 b 0.60 a 0.19 b Aflatoxin corn 2.02 b 0.57 a 0.12 a 0.47 a 0.54 b 0.22 a Aflatoxin corn + ozone 2.70 a 0.46 b 0.10 b 0.34 b 0.60 a 0.20 ab LSD a cmeans within column with no common superscript differ significantly (P < 0.05). 1Values represent the mean of six groups of three turkeys each per treatment (n = 18). 2LSD = least significant difference as determined by Fisher s protected LSD procedure. significant toxicity (Kubena et al., 1995a, b, 1997; Smith et al., 1992; Kubena, et al., 1994; Dwyer et al., 1997). Also, the potential for significant toxic interactions between these mycotoxins and AFB 1 is lacking (Smith et al., 1992; Kubena et al., 1995a). The presence of AFB 2 in the corn is insignificant, because this compound is approximately 200 times less toxic than AFB 1 in ducklings (Wogan et al., 1971). All of these mycotoxins have been shown to be susceptible to breakdown by electrolytically produced O 3 (McKenzie et al., 1997) and may have actually been degraded after O 3 treatment of the contaminated corn. The successful reduction of AFB 1 from 1,220 ppb by 95% provided a basis for evaluating detoxification of AFB 1 in poults. The growth performance of turkey poults can be modified by the addition of aflatoxin to the diet in a dose-dependent manner (Muller et al., 1970; Giambrone et al., 1985; Kubena et al., 1995a). This study showed a 23% reduction in body weight in poults receiving approximately 0.56 mg AFB 1 /kg (560 ppb) in the diet by the end of the 3rd wk. These results agree with those of Kubena et al. (1991), who noted a 19% reduction in 3-wk BW gain in poults fed 0.5 mg AFB 1 /kg and with Hamilton et al. (1972), who showed a growth rate depression of 18% at 0.5 mg AFB 1 /kg and 25 to 47% at 1.0 mg AFB 1 /kg. The lack of poult mortality in the group fed the AF corn diet is in agreement with previous reports by Kubena et al. (1991), who demonstrated a lack of poult mortality when exposed to 0.5 mg AFB 1 /kg, even though poults receiving 1.0 mg AFB 1 /kg showed a high incidence of mortality (88%). The reduced relative liver weights in poults fed diets containing AF observed in this study concur with the results reported by Hamilton et al. (1972) and Kubena et al. (1991, 1995a). Additionally, these authors showed an increase in relative weights of kidney and pancreas in poults. Weibking et al. (1994) did not observe a decrease in relative liver weights or an increase in pancreas and proventriculus weights. This difference could be due to the much lower concentration of AFB 1 in the rations (200 mg AFB 1 /kg vs 560 mg AFB 1 /kg used in this study). However, these authors did observe an increase in relative spleen weight similar to the results of this study. Treatment of the contaminated corn with O 3 degraded AFB 1 and protected the poults from the significant relative organ weight changes caused by the contaminated corn. Importantly, none of the relative organ weight values of the poults fed control corn treated with O 3 differed from controls. Several serum biochemical values, enzyme activities, and hematological values were either increased or decreased due to AF-contaminated corn. Many of these TABLE 3. Effects of feeding diets containing aflatoxin B 1 -contaminated corn alone and after treatment with ozone on serum enzyme activities 1 and hematological values 2 in turkey poults at 21 d Aspartate Alanine Lactate Mean Mean Creatinine amino- amino- dehydro- Mean red corpuscular corpuscular Treatment kinase transferase transferase genase blood cell hemoglobin volume (IU/L) ( 10 6 mm 3 ) (pg) (mm 3 ) Control corn 458 b 175 b 18.9 b 524 b 1.82 b 46.7 a 160 a Control corn + ozone 450 b 182 b 20.4 b 548 b 1.83 b 47.1 a 162 a Aflatoxin corn 1,568 a 234 a 24.8 a 1,106 a 1.95 a 42.7 b 154 b Aflatoxin corn + ozone 503 b 166 b 18.5 b 496 b 1.84 b 46.7 a 160 a LSD ameans within column with no common superscript differ significantly (P < 0.05). 1Values represent the mean of six groups of three turkeys each per treatment (n = 18). 2Values represent the mean of six groups of two turkeys each per treatment (n = 12). 3LSD = least significant difference as determined by Fisher s protected LSD procedure.

6 AFLATOXIN TOXICITY IN POULTS PREVENTED BY OZONE 1099 TABLE 4. Effects of feeding diets containing aflatoxin B 1 -contaminated corn alone and after treatment with ozone on various blood chemistries in turkey poults at 21 d 1 Blood urea Total Treatment Triglycerides Cholesterol nitrogen Calcium protein Albumin (mg/dl) (g/dl) Control corn 92 a 146 a 1.68 ab ab 2.48 a 1.01 a Control corn + ozone 82 a 172 a 1.98 ab bc 2.56 a 1.04 a Aflatoxin corn 34 b 69 b 2.12 a 9.76 c 1.07 b 0.50 b Aflatoxin corn + ozone 100 a 158 a 1.60 b a 2.51 a 0.99 a LSD a cmeans within column with no common superscript differ significantly (P < 0.05). 1Values represent the mean of six groups of three turkeys each per treatment (n = 18). 2LSD = least significant difference as determined by Fisher s protected LSD procedure. alterations in poults are in general agreement with those reported by Pier and Heddleston (1970), Witlock and Wyatt (1981), Kubena et al. (1991, 1995a), and Weibking et al. (1994). Levels of triglycerides, cholesterol, calcium, protein, and albumin decreased as did mean corpuscular hemoglobin. Activities of creatine kinase, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were elevated in addition to elevations of both RBC counts and mean corpuscular volume. Ozone treatment of the AFB 1 -contaminated corn eliminated all increases in serum enzyme activities and changes in hematological values due to the AFB 1 - contaminated corn. Decreases in body weight gain, relative liver weight, and serum concentrations of albumin and total protein were observed in the poults fed the AF-contaminated corn in their diets and are indicative of inhibition of protein synthesis (Tung et al., 1975) and associated with aflatoxicosis (Kubena et al., 1995b). The increased relative kidney weight in these poults could be due to slight renal damage. The increased level of creatine kinase, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase are most likely associated with tissue damage and leakage of enzymes into the vasculature (Tietz, 1976). Reduction in serum triglycerides and cholesterol may reflect impaired lipid FIGURE 2. Degradation of aflatoxin B 1 by ozone and ammonia. The primary site of attack of ozone is at the C8 C9 double bond on the terminal furan; whereas, ammonia opens the lactone ring in the coumarin and leaves the C8 C9 double bond intact. This site on the molecule has been shown to be responsible for aflatoxin s mutagenicity and carcinogenicity.

7 1100 transport and inhibition of cholesterol biosynthesis (respectively), as suggested by Kubena et al. (1995b). Treatment of the AF corn with O 3 provided complete or partial protection against changes associated with AF toxicity. These results indicate that electrolytically generated O 3 gas was able to degrade AFB 1 in corn that had been naturally contaminated with the mycotoxin and protect turkey poults from the deleterious effects of AFB 1 in corn-based feed. Gaseous O 3 generated via corona discharge has been reported to degrade the AF in ground corn and cottonseed meals (Dollear et al., 1968; Dwarakanath et al., 1968) and degrade and detoxify AFB 1 in aqueous solution (Maeba et al., 1988). Ozone has traditionally been generated via gas-based methods, which involve either passing an electrical discharge through air or UV excitation at 185 nm (Bablon et al., 1991). The conventional gas-based methods have several drawbacks, including the requirement of pre-drying the oxygen or air, low O 3 concentrations, cogeneration of nitric oxide byproducts (air fed units), and large capital/operational costs. Additionally, UV excitation generates only a few thousand parts per million under normal operating conditions. Alternatively, O 3 can be produced by electrolysis, in which water (as the environmentally safe reactant) provides the source of atomic oxygen for the formation of O 3. This aqueous-based method is also attractive because it can produce O 3 up to 20 wt % (Rogers et al., 1992; Verostko et al., 1992; Hitchens et al., 1994; Murphy et al., 1994). Previous studies indicated degradation and detoxification of aflatoxin using O 3 produced by electrolysis (McKenzie et al., 1997) and were predictive of the degradation and detoxification of AFB 1 in whole kernel corn presented in this study. Unlike methods using ammonia to degrade AF in corn, the primary reaction site of ozone in AFB 1 and AFG 1 is the C8 C9 double bond at the terminal furan (Figure 2). This site has been associated with AFB 1 s toxicity, mutagenicity, and carcinogenicity (Eaton and Groopman, 1994). The ammoniation process involves opening of the lactone ring in the coumarin, but leaves the C8 C9 double bond intact (Park et al., 1988), and (because of mutagenicity) retention of this bond in ammoniation products is undesirable (Marquez-Marquez et al., 1995). The reaction of O 3 with aflatoxins lacking a double bond at C8 C9 (i.e., AFB 2 and AFG 2 ) occurs, but is slower (McKenzie et al., 1997). This result is in agreement with previous data describing the degradation of aflatoxins with O 3 (Dwarakanath et al., 1968; Maeba et al., 1988). Schemes to rapidly and effectively degrade AF in grain using O 3 may require concentrations that are only obtainable using this aqueous-based fuel cell technology. Ozone is fairly stable as a gas, but in an aqueous environment, the half life drops to approximately 20 min. It decomposes to form oxygen gas and therefore can be classified as a nonpersistent chemical; however, it must be generated at the location of its intended use. McKENZIE ET AL. Utilizing O 3 generated by this method may make it possible to remediate bulk quantities of corn with minimal destruction of important nutrients for a minimal cost. In conclusion, these findings indicate a novel potential approach to the remediation of unprocessed corn contaminated with AFB 1. ACKNOWLEDGMENTS The authors wish to thank Jeff Ellis, Larry Whitlock, and Richard Johnson at the Texas State Chemists Office for their assistance in obtaining the contaminated grain. The authors would also like to recognize the excellent technical assistance of Maxene Dwyer at Texas A&M University and Maurice Connell, Laura Ripley, and Joel Billke at USDA ARS/FAP. This study was supported in part by USDA , TAES H6215, and ATP REFERENCES Bablon G., W. D. Bellamy, M. M. Bourbigot, F. B. Daniel, M. Dore, F. Erb, G. Gordon, B. Langlais, A. Laplanche, B. Legube, G. Martin, W. J. Masschelein, G. Pacey, D. A. Reckhow, and C. Ventresque, Fundamental aspects. Pages in: Ozone in Water Treatment: Application and Engineering. B. Langlais, D. A. Reckhow, and D. R. Brink, Lewis Publishers, Chelsea, MI. Bailey, P. S., The reactions of ozone with organic compounds. Chem. Rev. 58: Campbell, M. L., J. D. May, W. E. Huff, and J. A. Doerr, Evaluation of immunity of young broiler chickens during simultaneous aflatoxicosis and ochratoxicosis. Poultry Sci. 62: Council for Agricultural Science and Technology, Pages 1 91 in: Mycotoxins: Economic and Health Risks. K. A. Nisi, ed. Task Force Report No Council for Agricultural Science and Technology, Ames, IA. Dollear F. G., G. E. Mann, L. P. Codifer, Jr., H. K. Gardner, Jr., S. P. Koltun, and H.L.E. Vix, Elimination of aflatoxins from peanut meal. J. Am. Oil Chem. Soc. 45: Dwarakanath C. T., E. T. Rayner, G. E. Mann, and F. G. Dollear, Reduction of aflatoxin levels in cottonseed and peanut meals by ozonization. J. Am. Oil Chem. Soc. 45: Dwyer, M. R., L. F. Kubena, R. B. Harvey, K. Mayura, A. B. Sarr, S. Buckley, R. H. Bailey, and T. D. Phillips, Effects of inorganic sorbents and cyclopiazonic acid in broiler chickens. Poultry Sci. 76: Eaton D. L., and J. D. Groopman, The Toxicology of Aflatoxins: Human Health, Veterinary and Agricultural Significance. Academic Press, San Diego, CA. Edds, G. T., and R. A. Bortell, Biological effects of aflatoxins poultry. Pages in: Aflatoxin and Aspergillus flavus in Corn. U. Diener. R. Asquith, and J. Dickens, ed. Southern Cooperative Series Bulletin 279, Auburn University, Auburn, AL. Essigmann, J. M., R. G. Croy, A. M. Nadzan, W. F. Busby, V. N. Reinhold, G. Buchi, and G. N. Wogan, Structural identification of the major DNA adduct formed by aflatoxin B 1 in vitro. Proc. Natl. Acad. Sci. USA 74:

8 AFLATOXIN TOXICITY IN POULTS PREVENTED BY OZONE 1101 Foller, P. C., and C. W. Tobias, 1982 The anodic evolution of ozone. J. Electrochem. Soc. 129: Giambrone, J. J., U. L. Diener, N. D. Davis, V. S. Panangala, and F. J. Hoerr, Effect of purified aflatoxin on turkeys. Poultry Sci. 64: Goldblatt, L. A., and F. G. Dollear, Detoxification of contaminated crops. Pages in: Mycotoxins in Human and Animal Health. J. Rodricks and C. Hesseltine, ed. Pathotox, Park Forest South, IL. Hagler, W. M., Jr., 1991 Potential for detoxification of mycotoxin-contaminated commodities. Pages in: Mycotoxins, Cancer and Health. G. A. Bray and D. H. Ryan, ed., Pennington Center Nutrition Series, Louisiana State University Press, Baton Rouge, LA. Hamilton, P. B., H. T. Tung, J. R. Harris, J. H. Gainer, and W. E. Donaldson, The effect of dietary fat on aflatoxicosis in turkeys. Poultry Sci. 51: Hitchens G. D., T. D. Rogers, C. L. Sheffield, K. C. Anderson, and C. E. Salinas, Wastewater and process water treatment based on electrochemically generated ozone. Page 204 in: Water Purification By Photocatalytic, Photoelectrochemical, and Electrochemical Processes. T. L. Rose, E. Rudd, O. J. Murphy, and B. E. Conway, ed. Electrochemical Society, Pennington, NJ. Huff W. E., R. B. Harvey, L. F. Kubena, W. M. Hagler, and G. E. Rottinghaus, Toxic synergism between aflatoxin and T-2 toxin in broiler chickens. Poultry Sci. 67: Huff W. E., R. B. Harvey, L. F. Kubena, W. M. Hagler, S. P. Swanson, T. D. Phillips, and C. R. Creger, Individual and combined effects of aflatoxin and Deoxynivalenol (DON, Vomitoxin) in broiler chickens. Poultry Sci. 65: Huff W. E., L. F. Kubena, R. B. Harvey, and T. D. Phillips, Efficacy of hydrated sodium calcium aluminosilicate to reduce the combined toxicity of aflatoxin and ochratoxin A. Poultry Sci. 71: Kubena, L. F., T. S. Edrington, C. Kamps-Holtzapple, R. B. Harvey, M. H. Elissalde, and G. E. Rottinghaus, 1995a. Effects of feeding fumonisin B 1 present in Fusarium moniliforme culture material and aflatoxin singly and in combination to turkey poults. Poultry Sci. 74: Kubena, L. F., T. S. Edrington, C. Kamps-Holtzapple, R. B. Harvey, M. H. Elissalde, and G. E. Rottinghaus, 1995b. Influence of fumonisin B 1, present in Fusarium moniliforme culture material, and T-2 toxin on turkey poults. Poultry Sci. 74: Kubena, L. F., T. S. Edrington, R. B. Harvey, T. D. Phillips, A. B. Sarr, and G. E. Rottinghaus, Individual and combined effects of fumonisin B 1 present in Fusarium moniliforme culture material and diacetoxyscirpenol or ochratoxin A in turkey poults. Poultry Sci. 76: Kubena, L. F., R. B. Harvey, T. D. Phillips, D. E. Corrier, and W. E. Huff, Diminuation of aflatoxicosis in growing chickens by dietary addition of a hydrated sodium calcium aluminosilicate. Poultry Sci. 69: Kubena, L. F., R. B. Harvey, W. E. Huff, M. E. Elissalde, A. G. Yersin, T. D. Phillips, and G. E. Rottinghaus, Efficacy of a hydrated sodium calcium aluminosilicate to reduce toxicity of aflatoxin and diacetoxyscirpenol. Poultry Sci. 72: Kubena, L. F., W. E. Huff, R. B. Harvey, A. G. Yersin, M. H. Elissalde, D. A. Witzel, L. E. Giroir, T. D. Phillips, and H. D. Petersen, Effects of a hydrated sodium calcium aluminosilicate on growing turkey poults during aflatoxicosis. Poultry Sci. 70: Kubena, L. F., E. E. Smith, A. Gentles, R. B. Harvey, T. S. Edrington, T. D. Phillips, and G. E. Rottinghaus, Individual and combined toxicity of T-2 toxin and cyclopiazonic acid in broiler chicks. Poultry Sci. 73: Maeba H., Y. Takamoto, M. Kamimura, and T. Miura, Destruction and detoxification of aflatoxins with ozone. J. Food Sci. 53: Marquez-Marquez, R., I. T. de Hernandez, and E. M. Bujaidar, Genotoxicity of aflatoxin B 1 and its ammonium derivatives. Food Add. Contam. 12: McKenzie, K. S., A. B. Sarr, K. Mayura, R. H. Bailey, D. R. Miller, T. D. Rogers, W. P. Norred, K. A. Voss, R. D. Plattner, L. F. Kubena, and T. D. Phillips, Oxidative degradation and detoxification of mycotoxins using a novel source of ozone. Food Chem. Toxicol. 35: Muller, R. D., C. W. Carlson, G. Semeniuk, and G. S. Harshfield, The response of chicks, ducklings, goslings, pheasants and poults to graded levels of aflatoxins. Poultry Sci. 49: Murphy O. J., C. E. Salinas, K. C. Anderson, M. Novak, and G. D. Hitchens, Electrochemical production of ozone for water purification. Page 132 in: Water Purification by Photocatalytic, Photoelectrochemical, and Electrochemical Professes. T. L. Rose, E. Rudd, O. J. Murphy, and B. E. Conway, ed. Electrochemical Society, Pennington, NJ. National Research Council, Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Park, D. L., and A. E. Pohland, Sampling and sample preparation for detection and quantitation of natural toxicants in food and feed. J. Assoc. Off. Anal. Chem. 72: Park, D. L., L. S. Lee, R. L. Price, and A. E. Pohland, Review of the decontamination of aflatoxins by ammoniation: Current status and regulation. J. Assoc. Off. Anal. Chem. 71: Phillips, T. D., L. F. Kubena, R. B. Harvey, D. R. Taylor, and N. D. Heidelbaugh, Hydrated sodium calcium aluminosilicate: a high affinity sorbent for aflatoxin. Poultry Sci. 67: Phillips, T. D., B. A. Clement, and D. L. Park, Approaches to reduction of aflatoxins in foods and feeds. Pages in The Toxicology of Aflatoxins: human health, veterinary and agricultural significance. L. D. Eaton, and J. D. Groopman, ed. Academic Press, New York, NY. Pier, A. C., and K. L. Heddleston, The effect of aflatoxin on immunity in turkeys. I. Impairment of actively acquired resistance to bacterial challenge. Avian Dis. 14: Rogers T. D., G. D. Hitchens, C. E. Salinas, O. J. Murphy, and H. W. Whitford, Water purification, microbiological control, sterilization and organic waste decomposition using an electrochemical advanced ozonation process. SAE Technical Paper No nd International Conference on Environmental Systems, Society of Automotive Engineers, Inc. Warrendale, PA. SAS Institute, SAS/STAT Guide for Personal Computers. 6th ed. SAS Institute Inc., Cary, NC. Siraj, M. Y., A. W. Hayes, P. D. Unger, G. R. Hogan, N. J. Ryan, and B. B. Wray, Analysis of aflatoxin B 1 in human tissues with high pressure liquid chromatography. Toxicol. Appl. Pharmacol. 58:

9 1102 McKENZIE ET AL. Smith, E. E., L. F. Kubena, C. E. Braithwaite, R. B. Harvey, T. D. Phillips, and A. H. Reine, Toxicological evaluation of aflatoxin and cyclopiazonic acid in broiler chickens. Poultry Sci. 71: Smith, J. W., and P. B. Hamilton, Aflatoxicosis in the broiler chicken. Poultry Sci. 49: Snedecor, G. W., and W. G. Cochran, Pages in: Statistical Methods. 6th ed. The Iowa State University Press, Ames, IA. Tietz, N., Fundamentals of Clinical Chemistry. W. B. Saunders, Philadelphia, PA. Tung, H. T., R. D. Wyatt, P. Thaxton, and P. B. Hamilton, Concentrations of serum proteins during aflatoxicosis. Toxicol. Appl. Pharmacol. 34: Urano, J., M. W. Trucksess, R. W. Beaver, D. M. Wilson, J. W. Dorner, and F. E. Dowell, Co-occurrence of cyclopiazonic acid and aflatoxin in corn and peanuts. J. AOAC Int. 75: Verotsko, C. E., O. J. Murphy, G. D. Hitchens, C. E. Salinas, and T. D. Rogers, Post-treatment of reclaimed waste water based on an electrochemical advanced oxidation process. SAE Technical Paper No nd International Conference on Environmental Systems, Society of Automotive Engineers, Inc., Warrendale, PA. Weibking, T. S., D. R. Ledoux, A. J. Bermudez, and G. E. Rottinghaus, Individual and combined effects of feeding Fusarium moniliforme culture material containing known levels of fumonisin B 1 and aflatoxin B 1 in the young turkey. Poultry Sci. 73: Witlock, D. R., and R. D. Wyatt, Effect of dietary aflatoxin on hemostasis of young turkey poults. Poultry Sci. 60: Wogan, G. N., G. S. Edwards, and P. M. Newberne, Structure-activity relationships in toxicity and carcinogenicity of aflatoxin and analogs. Cancer Res. 31: