ENVIRONMENT, WELL-BEING, AND BEHAVIOR. Efficacy of Sodium Bentonite as a Detoxifier of Broiler Feed Contaminated with Aflatoxin and Fumonisin

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1 ENVIRONMENT, WELL-BEING, AND BEHAVIOR Efficacy of Sodium Bentonite as a Detoxifier of Broiler Feed Contaminated with Aflatoxin and Fumonisin R. Miazzo,* M. F. Peralta,* C. Magnoli, M. Salvano, S. Ferrero, S. M. Chiacchiera, E. C. Q. Carvalho,# C. A. R. Rosa,** and A. Dalcero,1 *Departamento de Producción Animal, Facultad de Agronomía y Veterinaria, Departamento de Microbiología e Inmunología, Departamento de Biología Molecular, Departamento de Matemáticas, and Departamento de Química, Facultad de Ciencias Exactas Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, (5800) Río Cuarto, Córdoba, Argentina; #Departamento de Patología, Facultade de Veterinaria, Universidade Federal Fluminense, Brazil; and **Departamento de Microbiologìa e Imunología Instituto de Veterinaria Universidade Federal Rural do Río de Janeiro, Brazil ABSTRACT Sodium bentonite (SB) was evaluated for the relative weights of liver, kidney, and spleen. Addition its ability to reduce the deleterious effects of fumonisin of SB to diets containing AFB 1 and FB 1 only decreased B 1 (FB 1 ) and aflatoxin B 1 (AFB 1 ) in broiler diets. It was liver weights. In relation to the control, lower serum levels incorporated into the diets (0.3%) containing 2.5 mg/kg of total protein, albumin, and globulins were observed AFB 1, 200 mg/kg FB 1, or a combination of 2.5 mg/kg for all AFB 1 containing diets without SB addition, whereas AFB 1 and 200 mg/kg FB 1. Aflatoxin B 1 significantly diminished body weight gain, whereas FB 1 or the combina- all other treatments were not altered. Livers of birds fed diets containing AFB tion of FB 1 and SB had no effect. Addition of SB in the diets 1 and a combination of AFB 1 and FB significantly diminished the inhibitory effects of dietary 1 were enlarged, yellowish, friable, and had rounded AFB 1. Feeding AFB 1 alone caused significant increases in borders. The histopathology of them, stained with hematoxylin and eosin, showed multifocal and varied cyto- the relative weights of most observed organs. Feeding FB 1 alone did not alter relative weights of any organs. In plasmatic vacuolization with perilobular location. Incorporation the combined diet (AFB 1 plus FB 1 ) relative weights of the liver, kidney, gizzard, and spleen were increased. Addition of SB to the diet containing AFB 1 diminished of SB reduced the incidence and severity of the hepatic histopathology changes associated with aflatoxicosis. (Key words: aflatoxin, fumonisin, broiler, bentonite, detoxification) 2005 Poultry Science 84:1 8 INTRODUCTION The adverse effects of naturally occurring mutagens and carcinogens and their possible effects on human and animal health are observed by investigating the biological effects of purified compounds in experimental animals. In contrast very little is known about the interaction of complex mixtures in biological systems. A single compound may affect different reactions within one biological system, whereas it may also exhibit additive, antagonistic, or synergistic interactions with other compounds (Bata et al., 1996). The occurrence of mycotoxins together with various endogenous and exogenous risk factors, such as nutrition or virus infections, may modulate metabolism. Mycotoxins act synergistically to influence the risk of several ani Poultry Science Association, Inc. Received for publication April 15, Accepted for publication September 9, To whom correspondence should be addressed: adalcero@exa. unrc.edu.ar. mal chronic diseases. One of the myriad effects of mycotoxins is the ability to impair the immune system in fowl. Aflatoxin B 1 (AFB 1 ) is the best-known mycotoxin for its ability to affect the primary immune response (Thaxton et al., 1974). Aflatoxins are secondary metabolites produced by species of Aspergillus and are natural contaminants of animal feeds (Edds and Bortell, 1983; Dalcero et al., 1997, 1998; Magnoli et al., 1998). They are potent liver carcinogens in laboratory animals (Wogan, 1973, 1992; Wogan et al., 1974). AFB 1 has been implicated as one of the etiological factors, apart from hepatitis B virus, in the development of human liver cancer (van Rensburg et al., 1985; Peers et al., 1987). Fumonisins are produced by Fusarium spp. (Gelderblom et al., 1988) that contaminate corn worldwide (Rheeder et al., 1992; Chulze et al., 1996). Long-term studies have indicated that fumonisin B 1 (FB 1 ) is hepatocarcinogenic in rats, whereas a recent study reported its nephro- Abbreviation Key: AFB 1 =aflatoxin B 1 ; ALB = albumin; FB 1 = fumonisin B 1 ; GLOB = globulin; SB = sodium bentonite. 1

2 2 MIAZZO ET AL. TABLE 1. Composition (g/kg diet) and proximate analyses of the experimental diets Diet Ingredient Grower Finisher Yellow corn Soybean oil meal Soybeans, heat treated Meat and bone meal Vitamin and mineral mix NaCl Oystershell Sunflower oil DL-Methionine L-Lysine 1.0 Monensin Total 1, ,000.0 Proximate composition (g/kg diet) of the test diets Crude protein Crude fat Crude fiber Calcium Total phosphorus Lysine Methionine Tryptophan ME, kcal.kg 3, , Vitamin and mineral mix provided (mg/kg of feed): vitamin A, 8,325 IU; vitamin D 3, 2,075 IU; vitamin K 3, 1.25 mg; vitamin B 1, 1 mg; vitamin B 2, 2.5 mg; vitamin B 6, 2.5 mg; vitamin B 12, mg; folic acid, 0.25 mg; nicotinic acid, 25 mg; calcium pantothenate, 10 mg; biotin, 0.01 mg; choline chloride, 240 mg; manganese, 87.5 mg; iron, 60 mg; copper, 7.5 mg; zinc, mg; I, 1.0 mg; Se, 0.2 mg; and butylated hydroxytoluene, mg. carcinogenicity and cancer-promoting activity in rats and in the livers of mice (Gelderblom et al., 1988, 1991, 2001). Several studies have reported the co-occurrence of AFB 1, FB 1, and other mycotoxins in feedstuffs (Huff et al., 1992; Dalcero et al., 1997, 1998; Ueno et al., 1997; Magnoli et al., 1999). At present, little is known about the interaction between AFB 1 and FB 1 with regard to their toxic properties. Numerous strategies for detoxification or inactivation of mycotoxin in contaminated feedstuffs have been used. Many techniques are impractical or ineffective (Piva et al., 1995; Ramos and Hernández, 1997). One approach to the detoxification of mycotoxin is use of nonnutritive absorptive materials in the diet to reduce the absorption of mycotoxins from the gastrointestinal tract. In vitro and in vivo studies have indicated that natural sodium bentonite (SB) from southern Argentina has a strong ability to absorb AFB 1 from aqueous solution (Miazzo et al., 2000). However, previous in vitro studies carried out with aluminosilicate, a synthetic NaA zeolite, showed that FB 1 can significantly reduce the efficacy of the sorbent to sequester AFB 1 (Kikot et al., 2002). Therefore, the need of 2 Sigma Chemical Co., St. Louis, MO. 3 Labconco Corp., Kansas City, MO. 4 Castiglioni yacimientos, Pes y Cía S. A. F. I. A. M. I, Bs, As, Argentina. 5 From the FusariumResearch Center, Pennsylvania State University, University Park, PA. carrying out an interaction study was observed. In the present study, the in vivo ability of SB to prevent gastrointestinal absorption of AFB 1,FB 1, or a combination of AFB 1 and FB 1 was assayed. Chemicals MATERIALS AND METHODS The purities of purchased AFB 1 and FB 1 standards 2 were assayed by HPLC. Their purities were confirmed as being greater than 99%. Demineralized water (HPLC grade) was obtained with Labconco equipment (model ). 3 Commercial SB del Lago 4 (SB) was obtained from a mine in Cinco Saltos, Province of Río Negro, Argentina. The chemical composition of the SB was 54.91% SiO 2, 21.41% Al 2 O 3, traces of Fe 2 O 3, 0.01% MnO, 0.1% TiO 2, traces of CaO, 2.81% MgO, 1.70% Na 2 O, 0.16% K 2 O, traces of SO 3, 0.05% P 2 O 5, and 5.59% H 2 O. The mean particle size was 53 µm (99.5%). A 6% SB aqueous solution had ph of 8.5, and the swelling capacity after 24 h was 16 cm 3 /g. The batch of SB was previously assayed in vitro and in vivo for its ability to adsorb AFB 1 (Rosa et al., 2001). AFB 1 and FB 1 Production Aflatoxins were produced via fermentation of milled corn by Aspergillus parasiticus NRRL The sterile substrate, placed in Erlenmeyer flasks, was inoculated with 2 ml of the mold aqueous suspension containing 10 6 spores/ml. Cultures were allowed to grow for 7 d at 25 C in darkness. On the d 7 Erlenmeyer flasks were autoclaved, and culture material was dried at 40 C in a forced-air oven for 48 h. The AFB 1 content in the milled corn was 40.9, 4.99, 2.99, and 0.98 mg/kg of AFB 1, AFG 1, AFB 2, and AFG 2, respectively. The milled corn was incorporated into the basal diet to provide 2.5 mg of AFB 1 / kg of diet. Fumonisins were produced via fermentation of milled corn by Fusarium verticillioides M following the procedure described by Ledoux et al. (1995). Ground culture material containing 4,700 mg of FB 1, 930 mg of FB 2, and 430 mg of FB 3 /kg of corn was incorporated to the basal diet to provide the desired level of FB 1 (200 mg/kg of diet). Mycotoxin Analyses Aflatoxin and fumonisin analyses were performed by HPLC, following the methodology proposed by Trucksess et al. (1994) and Shephard et al. (1990), respectively. Zearalenone and deoxynivalenol were analyzed by thinlayer chromatography according to AOAC (1995). Experimental Design and Chicks Ross male broiler chicks obtained from a commercial hatchery were kept in a poultry house during the experi-

3 AFLATOXIN AND FUMONISIN DETOXIFICATION BY DIETARY BENTONITE 3 ment. Birds were maintained with food and water ad libitum for 22 d under standard management conditions. Treatments started on d 23. A grower diet was given from d 1 to 32, and a finisher diet was provided from d 33 until the end of the experiment. Diets were formulated to meet the levels of critical nutrients recommended by the NRC (1994). The composition and proximate analyses of both experimental diets are presented in Table 1. The starting mean BW at d 23 was 748 ± 28 g. During the experimental period, the control diet was screened for aflatoxins, zearalenone, deoxynivalenol, and fumonisins. Assayed levels of naturally occurring AFB 1 and FB 1 ranged from 14 to 25 ppb and 50 to 134 ppb, respectively. The rest of the toxins were below the detection limits of previously described techniques (Dalcero et al., 1997; Magnoli et al., 2002). Housing The poultry house was divided into 40 pens (1 1m; height: 100 cm) built side by side along a corridor where the pens were accessible. The pens were separated by plywood walls (height: 190 cm). The walls were made of wire mesh and reinforced with plywood up to 0.6 m, which allow auditory contact but no visual contact among the groups. Each pen was littered with pine wood shavings and equipped with a suspended bell-drinker and a feeder. The birds were maintained on a 12L:12D schedule with free access to water and diets. Average daily temperature ranged from 15 to 20 C. A total of 160 chicks was randomly divided into 8 treatments with 4 replicate groups per treatment and 5 chicks per replicate group. From d 23 to 50 of age, birds were kept under ideal conditions and fed with the following experimental diets: 1) control feedstuffs without addition of SB, AFB 1,orFB 1 ; 2) 0.3% SB; 3) 2.5 mg of AFB 1 / kg of feed; 4) 2.5 mg of AFB 1 /kg of feed and 0.3% SB; 5) 200 mg of FB 1 /kg of feed; 6) 200 mg of FB 1 /kg of feed and 0.3% SB; 7) 2.5 mg of AFB 1 /kg and 200 mg of FB 1 / kg of feed; 8) 2.5 mg of AFB 1 /kg, 200 mg of FB 1 /kg of feed and 0.3% SB. Chickens were monitored daily for signs of morbidity and mortality. The effect of SB on mycotoxicoses was determined by measuring BW gain; feed:gain ratio; total protein, albumin (ALB), and globulin (GLOB) concentrations in serum of broilers in each group. The aforementioned serum parameter concentrations were determined with a clinical chemistry analyzer 6 according to the manufacturer s recommended procedure. The serum biochemical values of individual birds within a replicate group were pooled and expressed as mean ± standard errors. The birds were killed by cervical dislocation and were necropsied and examined for gross lesions in liver, heart, kidney, gizzard, and spleen. 6 Metrolab 2100, Express Healthcare Management (expresshealthcare mgmt.com). FIGURE 1. Effects of sodium bentonite (SB) on biochemical indicators of broiler chick feed diets containing 2.5 mg of aflatoxin B 1 (AFB 1 ) and 200 mg of fumonisin B 1 (FB 1 ) per kilogram of feed, singly or in combination. (A) albumin (ALB), (B) globulin (GLOB), and (C) total protein. Treatments: a Control, 0.3% SB, a 2.5 mg of AFB 1 /kg of feed; b 2.5 mg of AFB 1 /kg of feed plus 0.3% SB; a 200 mg of FB 1 / kg of feed; a 200 mg of FB 1 /kg of feed plus 0.3% SB; a 2.5 mg of AFB 1 /kg plus 200 mg of FB 1 /kg of feed; b 2.5 mg of AFB 1 /kg plus 200 mg of FB 1 /kg of feed plus 0.3% SB. a,b Values without a common superscript differ (P < 0.05) according to the Tukey test.

4 4 MIAZZO ET AL. TABLE 2. Effects of aflatoxin B 1 (AFB 1 ), fumonisin B 1 (FB 1 ), sodium bentonite (SB), and their combination on BW gains and feed conversions of broiler chicks fed diets Treatment BW gain 1 Feed:gain SB AFB 1 FB 1 23 to 50 Change from Ratio 2 (%) (mg/kg) (mg/kg) d old (g) control (%) (kg:kg) ,865 ± 29 a ± 0.05 a ,895 ± 26 a ± 0.08 a ,463 ± 23 b ± 0.04 a ,805 ± 21 a ± 0.06 a ,812 ± 23 a ± 0.05 a ,855 ± 29 a ± 0.05 a ,338 ± 29 b ± 0.03 b ,505 ± 21 b ± 0.01 b a,b Means values within columns with no common superscripts are significantly different (P < 0.05) according to the Tukey test. 1 Results are reported as mean ± SD for 20 birds. 2 Results are reported as mean ± SD for 4 lots. Histopathology At the termination of the study, livers were excised, weighed, and fixed in 10% neutral buffered formalin. Fixed tissues were trimmed, embedded in paraffin, and stained with hematoxylin and eosin stain for histopathological examination. Liver sections of all birds were microscopically examined. Statistical Analysis A completely randomized experimental design was used, and the GLM procedure of SAS software (SAS Institute, 1995) was used to evaluate the data. Tukey s significant difference test was applied in the event of significant global effects. Although birds were reared in groups, the average BW gain of each treatment was computed on individual basis with the aim of a better statistical evaluation of the parameter. RESULTS The effects of AFB 1 and FB 1 singly and in combination on BW gains and feed conversions are shown in Table 2. Mortality did not occur. When compared with the control, the results showed that AFB 1 intake diminished to about 22% BW. Addition of 200 mg/kg FB 1 singly or FB 1 plus SB did not significantly alter BW. Addition of 0.3% SB to diets significantly diminished the inhibitory effects of feeding 2.5 mg of AFB1/kg of diet. The feed conversion (kg of feed/kg of gain) increased when AFB 1 was combined with FB 1 with or without SB. Data presented in Table 3 show the effects of dietary treatment on relative organ weights. Feeding AFB 1 alone caused significant increases in the relative weights of most of observed organs (liver, kidney, gizzard, and spleen). None of the treatments induced changes in heart weights. Feeding FB 1 alone did not alter relative weights of any organs. In the combined diet (AFB 1 plus FB 1 ) the relative weights of liver, kidney, gizzard, and spleen were increased. Addition of 0.3% SB to the diet containing AFB 1 alone diminished the toxic effect of AFB 1 on relative weights of some organs (liver, kidney, and spleen). However, SB in diets with AFB 1 plus FB 1 only decreased the weights of livers. The changes in the biochemical parameters for different treatments are shown in Figure 1. Compared with the control, alterations in the serum levels of the total protein, ALB, and GLOB were observed for all diets containing AFB 1 without SB addition. The effect became more remarkable at d 50 and coincided with longer exposure to AFB 1. Other treatments were not altered. Figure 2 shows the macroscopic appearances of livers from chicks that had been fed with the control diet, SB, TABLE 3. Effect of a sodium bentonite (SB) on relative organ weights of broiler chicks fed diets containing aflatoxin B 1 (AFB 1 ) and fumonisin B 1 (FB 1 ) alone or in combination 1 SB AFB 1 FB 1 Liver Kidney Heart Gizzard Spleen (%) (mg/kg) (mg/kg) (g/100 g of BW) (g/100 g of BW) (g/100 g of BW) (g/100 g of BW) (g/100 g of BW) ± 0.05 a 0.41 ± 0.03 a 0.63 ± 0.02 a 2.33 ± 0.05 a 0.06 ± 0.03 a ± 0.03 a 0.43 ± 0.03 a 0.65 ± 0.02 a 2.30 ± 0.03 a 0.07 ± 0.02 a ± 0.04 b 0.68 ± 0.02 b 0.64 ± 0.04 a 2.73 ± 0.04 b 0.13 ± 0.03 b ± 0.05 a 0.40 ± 0.01 a 0.61 ± 0.03 a 2.72 ± 0.05 b 0.08 ± 0.02 a ± 0.02 a 0.42 ± 0.03 a 0.62 ± 0.02 a 2.31 ± 0.02 a 0.06 ± 0.01 a ± 0.03 a 0.41 ± 0.02 a 0.62 ± 0.03 a 2.30 ± 0.03 a 0.05 ± 0.03 a ± 0.02 b 0.72 ± 0.03 b 0.64 ± 0.02 a 2.64 ± 0.02 b 0.15 ± 0.02 b ± 0.04 a 0.71 ± 0.02 b 0.67 ± 0.01 a 2.96 ± 0.04 b 0.13 ± 0.03 b a,b Means values within columns with no common superscripts are significantly different (P < 0.05) according to the Tukey test. 1 Values represent means of 8 groups of 5 chicks each per treatment.

5 AFLATOXIN AND FUMONISIN DETOXIFICATION BY DIETARY BENTONITE 5 FIGURE 2. Representative livers from broiler chickens fed with diets: A) control, B) 0.3% sodium bentonite (SB), C) 2.5 mg of aflatoxin B 1 (AFB 1 ) and 200 mg of fumonisin (FB 1 )/kg of feed, and D) 2.5 mg of AFB 1 /kg of feed. AFB 1, and a combination of AFB 1 and FB 1 diets. Livers from the last 2 treatments were enlarged, yellowish, friable, and with rounded borders. Livers from other treatments were not included because they had normal appearances. When compared with the control (Figure 3A), histopathologies of livers from birds fed with AFB 1 alone or AFB 1 plus FB 1 showed multifocal and varied cytoplasmatic vacuolization with perilobular location when stained with hematoxylin and eosin (Figure 3B and C, respectively). Liver samples of broilers consuming the FB 1 diet or other SB-supplemented diets (SB, AFB 1 + SB, FB 1 + SB, or AFB 1 +FB 1 + SB) all revealed discrete and diffuse cellular swelling as depicted in Figure 3D. DISCUSSION Aflatoxin and fumonisin produce severe economic losses and health problems in the poultry industry because of their toxicity and frequency of occurrence in feedstuffs (Kubena et al., 1993). The current study demonstrated the toxicity of AFB 1 combined with FB 1 in growing broiler chicks and a reduction in the ability of SB to decrease the toxicity of AFB 1 in the presence of FB 1. In vitro studies have shown that SB is strong binder of AFB 1 (Veldman et al., 1992; Rosa et al., 2001). An inert, stable, and insoluble complex between SB and AFB 1 was assumed to be the responsible for preventing toxin absorption in the intestine. Body weight reduction, caused by intake of feed contaminated with AFB 1, has already been reported in previous studies (Rosa et al., 2001). Sodium bentonite was inert and nontoxic. Olver (1997) and Parlat et al. (1999) reported similar results for clinoptilolite, a natural zeolite. The present results agreed with our earlier findings about the ability of SB to ameliorate the adverse effects of AFB 1 in growing broilers (Rosa et al., 2001). However, addition of SB did not have the same effect on birds fed a combined diet (AFB 1 plus FB 1 ). Previous in vitro studies involving NaA zeolite as an adsorbent have shown that the presence of FB 1 could significantly decrease the ability of sorbent to capture AFB 1 (Kikot et al., 2002). At ph 2, a competitive adsorption between both toxins was observed, but FB 1 was released from the surface when the ph was increased to 6, value at which AFB 1 is not able to be absorbed. Kubena et al. (1990a,b) reported additive toxicity in the majority of the mycotoxins when acting in combination. These authors reported that a decrease in serum total protein and ALB in broilers, as a result of aflatoxicosis, was not alleviated by hydrated sodium calcium aluminosilicate.

6 6 MIAZZO ET AL. FIGURE 3. Photomicrographs of hematoxylin and eosin stained liver sections of chicks fed with the following diets: A) control, 0.3% sodium bentonite (SB), B) 2.5 mg of aflatoxin B 1 (AFB 1 )/kg feed, C) 2.5 mg of AFB 1 and 200 mg of fumonisin B 1 (FB 1 )/kg of feed, and D) 0.3% SB. Bars equal 10 µm.

7 AFLATOXIN AND FUMONISIN DETOXIFICATION BY DIETARY BENTONITE 7 Protective effects of the nonnutritive additive (SB) on gross hepatic changes produced by AFB 1 or by a combination of AFB 1 and FB 1 were observed. A relative increase in liver weight was caused by AFB 1 or by a combination of AFB 1 and FB 1. Addition of SB ameliorated the observed effects. Chronic mycotoxicoses may be diagnosed by determining serum biochemical alterations even before major clinical symptoms appear (Oguz et al., 2000). Reductions in concentrations of serum total protein and ALB, as indicators of protein synthesis, and GLOB were observed in chicks consuming diets containing AFB 1 alone and combined with FB 1 (Tung et al., 1975; Huff et al, 1992; Kubena et al., 1994). As expected, all of these values in the present results were sensibly affected by AFB 1. The stronger effect at d 50 might have coincided with longer exposure to AFB 1. Changes in concentrations of serum total protein, ALB, and GLOB were reversed by SB in chicks fed diets containing AFB 1 alone or in combination with FB 1. Increases in serum total protein, ALB, and GLOB observed at d 43 might have been due to dietary changes at d 32. Fumonisin B 1 did not affect any of these parameters. Taking into account that the biochemical parameters for mycotoxicoses seemed to depend on bird age and nutritional status at the time of exposure to contaminated feed, we suggest that further in vivo experiments with this adsorbent should be conducted. Our results agreed with those reported by Kubena et al. (1998), who found significant decreases in these biochemical parameters at exposure levels of AFB 1 ranging from 2.5 to 5 ppm. Kececi et al. (1998) and Oguz et al. (2000) showed that some serum biochemical changes could be ameliorated by 0.5% SB administration to the diet of broiler chickens fed with 2.5 mg of AFB 1 /kg of diet. However, Oguz et al. (2002) reported that the use of 50 and 100 ppb of AFB 1 did not cause any significant change in the total protein and ALB, probably due to the low mycotoxin dose in the assay. In our previous studies the biochemical parameters for broilers fed a diet containing 0.3% SB and 5 mg of AFB 1 / kg of diet did not completely return to normal values, showing an inhibition of protein synthesis (Rosa et al., 2001). In the present report, a complete return to normal biochemical parameters was observed, which could have been due to the lower AFB 1 exposure dose compared with the former. It should be stressed that this research showed that SB was effective to counteract only some of the AFB 1 - promoted effects. In fact, addition of SB to a diet containing a combination of AFB 1 and FB 1 was not able to return BW gain to equal that of the control. This result could be attributed to a competition between FB 1 and AFB 1 for the active surface sites of SB, rendering a greater bioavailability of AFB 1 in the presence of high dose of FB 1. The current data indicate toxicity for chicks of AFB 1 alone or in combination with FB 1. Neither additive nor synergistic toxic interaction between AFB 1 and FB 1 seems to be operating as shown with most of the parameters. ACKNOWLEDGMENTS The authors are grateful to the SECyT (Secretaría de Ciencia y Técnica, Universidad Nacional de Río Cuarto) Res. No. 129/01, 077/03, and CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas) PIP Res. No. 962/01, which supported this study through grants. Carina Magnoli thanks CONICET for fellowship support. Ana María Dalcero and Stella Maris Chiacchiera held positions at CONICET. We thank Castiglioni, Pes y Cía SAFIAMI for providing the sodium bentonite. REFERENCES AOAC Sections , in Official Methods of Analysis. 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