Aflatoxin B1 adsorption by clays from water and corn ...

Applied Clay Science 36 (2007) 197 – 205 www.elsevier.com/locate/clay

Aflatoxin B1 adsorption by clays from water and corn meal W.F. Jaynes ⁎, R.E. Zartman, W.H. Hudnall Plant and Soil Science Department, Texas Tech University, Lubbock, Texas 79409, USA Received 27 March 2006; received in revised form 10 June 2006; accepted 10 June 2006 Available online 13 October 2006

Abstract Aflatoxins are toxic compounds found in grains and other food crops infested by Aspergillus fungi. Aflatoxins B1 and M1 are recognized carcinogens for animals and humans. Clay additives have been used to pelletize and improve the flow characteristics of animal feeds. Reduced aflatoxicosis in animals is an extra benefit of clay additives. Clay additive use has also been examined for reducing human aflatoxicosis. In this study, aflatoxin B1 (AfB1) adsorption by reference clays and activated carbon (AC) will be compared to a commercial clay additive, Novasil, that lessens aflatoxicosis in animals. The n-alkylammonium expansion identified Novasil as a low-charge montmorillonite. AC and the montmorillonites, Novasil, SWy-2, and SAz-1 adsorbed ∼ 200 g/kg AfB1 from water, whereas, sepiolite (SepSp-1) adsorbed only ∼ 60 g/kg. For AfB1 adsorption from aqueous corn meal, a 60% methanol extraction was used. Retention of AfB1 from corn meal by all samples was much less (b 1.5 g/kg) than from water and suggests that methanol might remove weakly-adsorbed AfB1. Low-charge montmorillonites, Novasil and SWy-2, retained ∼ 0.7 g AfB1/kg from corn meal, but high-charge montmorillonite (SAz-1) and AC only retained ∼ 0.1 g/kg. SepSp-1 adsorbed less AfB1 from water than AC or montmorillonite, but retained more AfB1 (1.3 g/kg) from corn meal at a lower equilibrium concentration. A plot of AfB1 extracted from corn meal versus % clay suggests SepSp-1 is far more effective than the montmorillonites. Methanol extraction is a more cautious estimate of AfB1 binding than simple aqueous adsorption and might better correlate to reduced aflatoxicosis in animals and humans. © 2006 Elsevier B.V. All rights reserved. Keywords: Feed additives; Clay minerals; Montmorillonite; Sepiolite; Activated carbon; Adsorption; Aflatoxins; Mycotoxins; n-alkylammonium expansion; ELISA

1. Introduction Mycotoxins are toxic compounds that are produced by many species of the Aspergillus, Fusarium, Penicillium, Claviceps, and Alternaria genera of fungi (Huwig et al., 2001). The toxins are secondary metabolites produced by the fungi after infesting grain and other food crops. Crops may be contaminated by mycotoxins in two ways: as parasites on living plants and during storage of harvested ⁎ Corresponding author. E-mail address: [email protected] (W.F. Jaynes). 0169-1317/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.clay.2006.06.012

crops (Huwig et al., 2001). Six groups of mycotoxins are produced by the Aspergillus, Penicillium, and Fusarium genera of fungi (Yiannikouris and Jouany, 2002). Mycotoxins have a diversity of chemical structures which accounts for different biological effects. Mycotoxins can be carcinogenic, mutagenic, teratogenic, oestrogenic, neurotoxic, or immunotoxic. Aflatoxins B1 and M1 have been demonstrated to be carcinogenic to animals and humans (Yiannikouris and Jouany, 2002). Caporael (1976) argued that convulsive ergotism may have been a physiological cause for the Salem witchcraft crisis in 1692. Ergot (Claviceps purpura) is a

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Fig. 1. Chemical structures of aflatoxins.

parasitic fungus that grows on a variety of cereal crops, especially rye. Fusiform sclerotia replace individual grains on the host plant. The scerotia contain large numbers of ergot alkaloids which are potent pharmacologic agents. One of the most potent alkaloids is lysergic acid amide which has 10% of the activity of LSD (lysergic acid diethylamide). The first demonstrated case of mycotoxicosis was in Great Britain in 1960 where more than 100,000 turkeys died of severe liver necrosis and biliary hyperplasia caused by aflatoxins in feed contributed by Aspergilluscontaminated peanut meal imported from Brazil (Sargeant et al., 1961). Hartley et al. (1963) examined fluorescence and other properties of the toxic metabolites of Aspergillus flavus previously identified as aflatoxins B and G. They identified aflatoxins B1, B2, G1, and G2 based on the blue or green fluorescent color of the compounds in ultraviolet light and on differences in melting point (Fig. 1). They noted that naturallyoccurring aflatoxins consisted mainly of B1 (AfB1) with some B2. When cows are fed aflatoxin-contaminated feed, a hydroxylated derivative of AfB1 is found in the milk named aflatoxin M1 (AfM1). Veldman et al. (1992) examined the carry-over of aflatoxin from feed (AfB1) into cow's milk (AfM1) and concluded that maximum levels of AfB1 in feed should be set to limit AfM1 in milk. Food contaminated with very small quantities of aflatoxins can render it unfit for animal or human consumption. Milk is not permitted to contain more than 0.5 μg AfM1/kg (The Texas A&M Aflatoxin

Resource, 2006). According to United States Food and Drug Administration (FDA) guidelines, grain crops for human or animal consumption must generally contain b20 μg aflatoxins/kg (FSRIO, 2005). However, feed for sheep and cattle (not dairy cows) can contain as much as 300 μg aflatoxins/kg. Grain crops containing N1000 μg aflatoxins/kg must be destroyed. Ruminants, such as cattle and sheep, are more resistant to mycotoxins than most animals. This suggests that the microbial population of the rumen plays a role in detoxification. Many bacteria, however, are completely inhibited by b10 μg AfB1/mL and this suggests that the toxin might disturb the growth and metabolic activity of rumen microorganisms (Yiannikouris and Jouany, 2002). Grazing animals probably ingest more soil materials than other animals. The clays present in ingested soil materials might contribute to the reduced risk that mycotoxins pose to ruminants. Although mycotoxins threaten food supplies, these compounds are strongly retained by soil materials and probably do not pose a long-term environmental risk. Dust from grain harvesting equipment, however, can contain high concentrations of mycotoxins. Dust collected near a combine in Georgia contained from 2.03 to 41.2 mg aflatoxins/kg (The Texas A&M Aflatoxin Resource, 2006). Goldberg and Angle (1985) examined the leaching and adsorption potential of aflatoxins in soil and reported that 80 to 92% of total applied aflatoxin was retained in the upper 2.5 cm of soil columns. All of the aflatoxin was retained within the upper 20 cm of all tested soil types and no aflatoxin was detected in any of the soil

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leachates. They concluded that aflatoxin contamination of groundwater would be unlikely unless the soils were extremely sandy or shallow. Clay additives have been used to pelletize animal feeds and as an anti-caking agent to improve the flow characteristics. As an added benefit, clays added to animal feeds were found to reduce the detrimental effects of aflatoxins and other mycotoxins. Because the clays are added to dry feed, aflatoxin adsorption to clays must occur after ingestion. Masimango et al. (1978) determined that bentonite could adsorb 94 to 100% of AfB1 initially present in aqueous solutions. Phillips et al. (1988) evaluated the use of aluminas, silicas, and aluminosilicates for aflatoxin adsorption from water and in reducing the toxicity to chickens. They reported that a sodium calcium bentonite they called “hydrated sodium calcium aluminosilicate” or HSCAS significantly reduced the adverse effects of feed containing 7.5 mg AfB1/kg. Veldman (1992) found that bentonite effectively reduced aflatoxin carry-over in milk, but that a hydrated aluminosilicate was less effective. Schell et al. (1993a) reported that 1% Na+-bentonite (Volclay-90) added to aflatoxin-contaminated feed partially restored the performance and liver function of pigs. Schell et al. (1993b) examined the effects of 0.5 to 2.0% clay (Ca2+-bentonite, HSCA or Novasil, palygorskite, sepiolite, and zeolite) additions to feed in reducing aflatoxicosis in pigs. They determined that all of the clays reduced aflatoxicosis symptoms in pigs, but some of the clays were more effective. Palygorskite was least effective, whereas, Ca2+bentonite and Novasil (i.e. HSCAS) were most effective. Scheideler (1993) examined the effects of various commercial aluminosilicate products (Novasil and Zeobrite) on aflatoxin toxicity to chicks and reported that these additives lessened the growth reduction caused by AfB1. Desheng et al. (2005) measured 0.6 g AfB1/kg sorption to Ca2+-montmorillonite from water and observed that a 0.5% Ca2+-montmorillonite addition to feed contaminated with 200 μg AfB1/kg significantly reduced aflatoxicosis in chickens. Many research studies have demonstrated that clay additions can effectively reduce aflatoxin toxicity to animals. Determining the mechanism of aflatoxin adsorption to clays might facilitate the identification or preparation of more effective adsorbents. Phillips et al. (1995) examined the mechanism of aflatoxin adsorption by HSCAS clay. They suggested that adsorption might involve the β-dicarbonyl system of aflatoxin through the chelation of metal ions at the surface and within the interlayers of the HSCAS phyllosilicate clay. In a review article, Ramos et al. (1996a) noted that activated charcoal, bentonite, zeolite, hydrated sodium calcium

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aluminosilicate (HSCAS), sepiolite, and kaolinite have been shown to be effective feed additives. Grant and Phillips (1998) further examined the adsorption AfB1 to HSCAS clay and concluded that AfB1 was chemisorbed to different sites that might include the interlamellar region, edges, and the basal surfaces of HSCAS particles. Most research on aflatoxin sorption to clays has been conducted by veterinarians and others that likely have a limited mineralogy background. An examination of aflatoxin adsorption by reference clays with known properties might more effectively identify adsorption mechanisms and clay properties that influence adsorption. Rao and Chopra (2001) examined the effects of 1% sodium bentonite and activated charcoal additions to feed that contained 100 μg AfB1/kg on the AfM1 content in goat's milk. Concentrations of AfM1 excreted in the milk were reduced 65% by bentonite and 76% by activated charcoal. Edrington et al. (1996), however, reported that although activated charcoal reduced urinary excretion of AfM1 in turkeys, it did not prevent aflatoxicosis. Similarly, Diaz et al. (2004) found that 0.25% activated carbon had no effect in reducing AfM1 in cow's milk, but several commercial Na+ -bentonite products effectively reduced AfM1 in milk. Aflatoxin exposure in developed countries is mainly a concern for livestock, not humans. In developing countries, however, human populations are at greater risk for aflatoxicosis. Bhat and Vasanthi (2003) noted that mycotoxins are a worldwide problem and that aflatoxins were the most problematic because of widespread occurrence in maize, peanuts, peanut products, cottonseed, chilies, peppers, and pistachio nuts. Similarly, Scholthof (2003) reported that by Food and Agriculture Organization (FAO) estimates, 25% of world food crops are contaminated with mycotoxins. Wang et al. (2001) reported that hepatitus B virus infections were endemic in China and that exposure to AfB1 was correlated with a 60fold increased risk of liver cancer. Pets are also at risk for mycotoxins (Martin, 2003). A commercial pet food contaminated with aflatoxins caused the death of at least 100 dogs in the United States (Laing, 2006). Research supported by the United States Agency for International Development (USAID) is testing the possible use of clays in human diets to prevent aflatoxicosis. Wang et al. (2005) examined the safety of montmorillonite clay (Novasil) additions to human diets. This shortterm study demonstrated the relative safety of clay in human subjects and might justify long-term human trials in populations at high risk for aflatoxicosis. To evaluate possible long-term effects of clay ingestion by humans, Afriyie-Gyawu et al. (2005) examined the chronic

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Nutrition and manufactured by Engelhard Corporation, Chemical Catalysts Group (600 East McDowell Road, Jackson, MS 39204). The b 2 μm clay fractions were separated by centrifugation and the clays freeze-dried (Table 1). Activated carbon (alkaline Norit-A decolorizing carbon) was obtained from Fisher Scientific. Preliminary research showed that decolorizing carbon more effectively adsorbed AfB1 than cocoanut activated carbon. Dispersions of the clays and activated carbon were prepared using an ultrasonic probe and a vortex mixer to aid in dispersion. Corn meal was obtained from a local grocery. Aflatoxin B1, rabbit anti-aflatoxin B1 antibody, aflatoxin B1-BSA conjugate, goat anti-rabbit antibody-horse radish peroxidase conjugate, phosphate buffered saline with 0.05% Tween 20 (PBST), and o-phenylenediamine dihydrochloride (OPD) substrate tablets were obtained from Sigma–Aldrich. Stable AfB1 stock solutions were prepared in 95% toluene/5% acetonitrile and stored in a freezer (AOCS, 1999a). The aflatoxin stock solutions were calibrated by measuring the UV absorbance of AfB1 dissolved in methanol at 340 nm (AOCS, 1999a). Aliquots of known AfB1 content were dried in glass vials and later re-dissolved to prepare standards and AfB1 solutions for the adsorption isotherms.

toxicology of rats fed diets containing montmorillonite clay (Novasil). They concluded that dietary clay additions as high as 2.0% produced no overt toxicity and concluded that the results support dietary clay additions for human populations at high risk for aflatoxicosis. Many potential feed additives effectively remove mycotoxins from water (in vitro), but might not protect animals from the toxicity (in vivo). Huwig et al. (2001) noted that activated carbon efficiently adsorbed aflatoxin and other mycotoxins from water. In feeding studies, however, 0.5% activated carbon produced little or no reduction in toxicity. Only by the use of 10% or more activated carbon in feed was significant toxicity reduction achieved. A batch adsorption isotherm in water might not effectively model mycotoxin adsorption to clays during food digestion. Hence, the aflatoxin adsorption capacity of clays and other additives measured by aqueous adsorption isotherms might not reflect the capacity of feed additives to effectively bind aflatoxins during digestion and prevent or reduce aflatoxicosis. In this study, the adsorption of AfB1 by reference clays and activated carbon will be compared to a commercial clay additive (HSCAS or Novasil) that animal feeding studies have shown to be effective in reducing aflatoxicosis. One objective of this research is to identify clay properties that are important to aflatoxin adsorption. Animal feeding studies are too expensive to effectively examine the great potential of natural and modified clays for reducing or eliminating aflatoxicosis. Hence, another objective is to identify aflatoxin adsorption testing methods that correlate with the actual reductions in animal/human aflatoxicosis measured by feeding studies.

Montmorillonite samples, Novasil, SWy-2, and SAz-1, were expanded with the hydrochloride salts of n-alkylamines (octylamine, dodecylamine, octadecylamine) using the method of Lagaly and Weiss (1976) and the preparation techniques of Rüehliche and Kohler (1981). The amines were obtained from Sigma–Aldrich and treated with dilute HCl to prepare the hydrochloride salts. Oriented mounts of the alkylammonium– montmorillonite complexes were dried on glass slides and X-ray diffraction patterns were collected from 2 to 10°2θ using CuKα radiation and a Philips diffractometer.

2. Materials and methods

2.2. AfB1 adsorption from water

The reference clay samples, SAz-1, SWy-2, and SepSp-1, were obtained from the Source Clay Repository of the Clay Minerals Society now located at Purdue University (West Lafayette, Indiana). A small sample of Novasil clay was provided by another researcher. Novasil is a product of Trouw

Batch adsorption isotherms (6 points in triplicate with 4 blanks) from water were prepared with an initial concentration of 1 μg AfB1/mL in 5-mL aqueous dispersions. The aqueous dispersions contained 10 to 180 μg of clay or activated carbon in 15-mL polypropylene centrifuge tubes. A stock solution

2.1. Alkylammonium expansion

Table 1 Sample composition and layer charge by n-alkylammonium expansion Sample

Composition

Source

XRD basal spacings of n-alkylammonium complexes C8

C12

C18

SAz-1 b2 μm SWy-2 b2 μm Novasil b2 μm Activated carbon SepSp-1b2 μm

High-charge Ca2+-montmorillonite Low-charge Na+-montmorillonite Na+, Ca2+-montmorillonite Alkaline Norit-A decolorizing carbon Sepiolite

CMS Source Clay Repository CMS Source Clay Repository Engelhard Corporation Fisher Scientific CMS Source Clay Repository

1.43 nm 1.37 nm 1.37 nm NA NA

1.77 nm 1.37 nm 1.42 nm NA NA

2.02 nm 1.76 nm 1.76 nm NA NA

NA = not applicable.

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containing 100 μg AfB1/mL in acetonitrile solution was prepared. Aliquots of acetonitrile stock solution were diluted with water and used to deliver aflatoxin to isotherm solutions (50 μL stock + 0.95 mL of water = 5 μg AfB1/mL). Blanks containing only AfB1 and water were prepared. The samples were thoroughly mixed using a vortex mixer. After overnight agitation on a reciprocating shaker, the tubes were centrifuged, and the supernatants were passed through 0.2-μm filters and collected in 20-mL plastic vials. 2.3. AfB1 retention from corn meal Aflatoxin adsorption from aqueous corn meal dispersions was used as a more applied and more conservative measure of aflatoxin retention. Batch adsorption isotherms from aqueous corn meal (6 points in triplicate with 4 blanks) were prepared with an initial concentration of 1.5 μg AfB1/mL (3 μg AfB1/g corn meal) in 2-mL aqueous dispersions containing 1 to 20 mg (0.1 to 2%) clay or activated carbon and 1.00 g of corn meal. Blanks were prepared similarly using only AfB1, corn meal, and water. The 3 μg AfB1/g corn meal is comparable to highly-contaminated (3000 μg aflatoxins/kg) grain. The

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samples were thoroughly mixed using a vortex mixer. After overnight agitation on a reciprocating shaker, 8 mL of a 60% methanol/40% 2 M NaCl extracting solution was mixed with the samples using a vortex mixer. The tubes were centrifuged and the supernatants passed through 0.2-μm filters and collected in 20-mL plastic vials. Aflatoxins are more soluble in methanol than in water. The 60% methanol extraction was modified from the Asis et al. (2002) procedure to extract and measure aflatoxins in peanuts. The AOCS method for aflatoxins in corn, cottonseed, peanuts, and peanut butter similarly uses an 80% methanol/20% water extraction (AOCS, 1999b). 2.4. ELISA AfB1 measurement A modification of the Asis et al. (2002) enzyme-linked immunoassay (ELISA) method was used for measuring aflatoxins. Beaver and James (1991) compared an ELISA technique with liquid chromatography methods for measuring aflatoxins in corn. They concluded that a commerciallyavailable ELISA kit could reliably detect N 5 μg aflatoxins/kg of corn. The American Oil Chemists Society has approved an

Fig. 2. AfB1 adsorption from aqueous dispersions of (a) Novasil, (b) SWy-2, (c) SAz-1, and (d) activated carbon.

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official ELISA method based on commercial ELISA kits for total aflatoxins in corn, cottonseed, peanuts, and peanut butter (AOCS, 1999b). The Asis et al. (2002) method uses commercially-available antibodies and reagents and is less expensive than ELISA kits. The modified ELISA method of Asis et al. (2002) has an AfB1 detection limit of ∼ 0.05 ng/mL. However, AfB1 extracted into 60% methanol/40% 2 M NaCl requires a dilution of at least 1:5 to reduce the methanol content to ∼ 12%. It is a competitive ELISA technique because AfB1 in solution competes with AfB1-BSA bound to the microplate for anti-AfB1 antibodies. In the first step of this method, the dissolved aflatoxin in samples and standards competes with the AfB1-BSA bound to the microplate for rabbit anti-AfB1 antibodies. For samples or standards containing little or no AfB1, rabbit antibodies can only bind to the AfB1-BSA bound to the microplate. For samples or standards with high AfB1 concentrations, most of the rabbit antibodies bind to dissolved AfB1 and are subsequently lost when the plate is washed. In the next step, a secondary antibody to rabbit antibodies (prepared in goats) binds to any rabbit anti-AfB1 antibody that adsorbed to the AfB1-BSA conjugate bound to the microplate.

The secondary antibody has an attached enzyme, horseradish peroxidase (HRP). In the last step, the attached HRP enzyme catalyzes color development in the substrate, OPD. The optical density of the substrate is then measured and is inversely proportional to aflatoxin concentration. Aflatoxin concentrations were measured using 96-well polystyrene microplates that had previously been coated with AfB1-BSA-conjugate. The AfB1-BSA coated plates were prepared as follows: To the wells of new microplates, 100 μL AfB1-BSA-conjugate (5 mg/L) in pH 9.5 carbonate/bicarbonate buffer were added and equilibrated overnight in a refrigerator. The coated plates were subsequently washed 6 times using phosphate buffered saline with 0.05% Tween 20 (PBST) and stored dry in a refrigerator until use. Aliquots (50 μL) of aflatoxin standards containing 0 to 500 ng AfB1/mL in 12% methanol/88% PBST were added to the wells of AfB1-BSA coated plates. Similarly, blanks and sample solutions were diluted using 12% methanol/88% PBST and 50-μL aliquots added to the wells of the microplates. Then, 50-μL aliquots of a 1:2000 dilution of the rabbit antiAfB1 antibody in PBST were added to the microplate wells and the microplates agitated for 2 h. Afterward, the

Fig. 3. AfB1 adsorption from aqueous dispersions of (a) Novasil, (b) SWy-2, (c) SAz-1, and (d) activated carbon in the presence of corn meal.

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Fig. 4. AfB1 adsorption from aqueous dispersions of sepiolite in the absence (a) and presence (b) of corn meal.

microplates were washed with PBST and 100 μL of a 1:5000 dilution of goat anti-rabbit-HRP antibody conjugate were added to the wells and the microplates agitated for 1 h. The microplates were again washed with PBST and 100 μL of OPD substrate solution were added and the microplates incubated at 37 °C for 30 min to allow color development. Finally, 50 μL of 2 M H2SO4 was added to the microplate wells to stop color development. The optical densities of the AfB1 standards and samples in each microplate were subsequently measured using a Bio-Tek ELx800uv microplate reader. Bio-Tek KC-Junior software was used to calculate standard curves and sample concentrations were calculated using an equation fitted to the standard curves. Adsorption isotherms were constructed by plotting equilibrium AfB1 concentrations versus amount adsorbed using all 18 points (6 points × 3 replicates). Sample replicate equilibrium AfB1 concentrations were subtracted from the blank average to calculate the amount of AfB1 adsorbed.

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indicated greatest AfB1 adsorption (∼ 200 g/kg at 0.6 mg/L) by Novasil and SWy-2 (Fig. 2a, b). Adsorption of AfB1 by SAz-1 and AC (∼200 g/kg at 0.6 mg/L) was similar (Fig. 2c, d). In the presence of corn meal, about 100 times less AfB1 was retained by the clays and AC (Fig. 3a–d). The 60% methanol extraction might remove weakly-adsorbed AfB1 and the corn meal might act to reduce adsorption. Retention of AfB1 (∼0.7 g/kg at 0.6 mg/L) by Novasil and SWy-2 was comparable, but AfB1 retention by SAz-1 and AC (∼0.1 g/kg at 0.6 mg/L) from corn meal was much less (Fig. 3a–d). The AfB1 adsorption isotherm of aqueous dispersions of AC indicated adsorption comparable to the montmorillonites, but the corn meal isotherm indicated that most of the AfB1 was weakly retained. Adsorption isotherms from aqueous dispersions suggest that AC is an effective AfB1 adsorbent, but feeding studies suggest otherwise (Huwig et al., 2001). The corn meal adsorption isotherms, because of the 60% methanol extraction, are a more conservative measure of AfB1 retention by clays and might better correlate with the aflatoxicosis reductions observed in feeding studies. Adsorption of AfB1 was similar for Novasil and SWy-2 both from water and from corn meal. Expansion of Novasil and SWy-2 with the hydrochloride salts of the n-alkylamines, octylamine (C8), dodecylamine (C12), and octadecylamine (C18), were similar and indicate that both are low-charge montmorillonites (Table 1). In contrast, the high-charge montmorillonite, SAz-1, was expanded to larger basal spacings by the n-alkylamines, but SAz-1 was a much less effective AfB1 adsorbent. Reference clay, STx-1, is a low- to intermediate-charge Ca2+-montmorillonite mined near Gonzales, Texas (Van Olphen and Fripiat, 1979). Schell et al. (1993b) reported that Astro-Ben 20, a commercial equivalent of STx-1, effectively reduced aflatoxicosis in pigs. This suggests that low-charge montmorillonites are

3. Results and discussion Adsorption isotherms for aqueous dispersions of Novasil, SWy-2, SAz-1, and activated carbon (AC)

Fig. 5. Effect of added clay on extractable AfB1 in corn meal.

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more effective AfB1 adsorbents than high-charge montmorillonites. Greater hydration and interlayer exchangeable cation density in high-charge montmorillonite might inhibit interlayer adsorption of hydrophobic compounds like AfB1 (Jaynes and Boyd, 1991). Adsorption of AfB1 from aqueous dispersions of sepiolite (Fig. 4a) was much lower than the other samples (Fig. 2a–d). AfB1 retention by sepiolite from aqueous corn meal (Fig. 4b), however, was much greater than the other samples (Fig. 3a–d). Sepiolite (SepSp-1) retained a much greater proportion of adsorbed AfB1 after extraction with 60% methanol/40% 2 M NaCl. This suggests that sepiolites might be more effective feed additives than montmorillonites in reducing aflatoxicosis. Different sepiolites probably will vary in aflatoxin adsorption just as has been observed for different bentonites. Most animal feeding studies on the use of clay additives to reduce aflatoxicosis have examined bentonites, such as Novasil (Ramos et al., 1996a; Huwig et al., 2001). Schell et al. (1993b), however, reported that a sepiolite obtained from the Floridin Company (Tallahassee, FL) reduced aflatoxicosis in pigs. Ramos et al. (1996a) noted that sepiolite was generally an effective feed additive. Ramos et al. (1996b) reported that sepiolite effectively adsorbed the mycotoxin zearalenone from water. The structural channels in sepiolite are not large enough to accommodate AfB1 molecules that most likely bind to external sites (Rausell-Colom and Serratosa, 1987). An important difference between sepiolite and montmorillonite that might explain the differences in AfB1 adsorption is the large amount of external Si–OH groups in sepiolite (Rausell-Colom and Serratosa, 1987). Sepiolite samples with shorter fiber lengths would have a greater number of external sites and might have greater AfB1 adsorption. The AfB1 adsorption from aqueous corn meal data can be plotted in a more relevant way for the practical use of clay additives by indicating the percent clay additive needed to reduce extractable AfB1 to acceptable levels. The graph of extractable AfB1 in corn meal versus percent clay (Fig. 5) indicates that the addition of 2.0% Novasil or 2.0% SWy-2 montmorillonite to highly-contaminated corn meal has greatly reduced extractable AfB1. The sepiolite (SepSp-1), however, more effectively reduced extractable AfB1 concentrations in corn meal with less added clay. 4. Summary and conclusions Aflatoxin B1 (AfB1) adsorption from water and from aqueous corn meal by reference clays, activated carbon, and a commercial montmorillonitic clay product, Novasil,

were measured and compared. Animal feeding studies have demonstrated that Novasil reduces aflatoxicosis when added to animal feed. Novasil was identified as a low-charge montmorillonite using n-alkylammonium expansion. AfB1 adsorption from water by Novasil and another low-charge montmorillonite (SWy-2) were about 200 g/kg. AfB1 adsorption from water by high-charge montmorillonite (SAz-1) and activated carbon were also about 200 g/kg. AfB1 adsorption from water by sepiolite (SepSp-1) was only about 60 g/kg. Some feed additives that effectively remove AfB1 from water do not effectively reduce aflatoxicosis in animals. Clays and activated carbon retained 100 times less AfB1 from aqueous corn meal than from water. The 60% methanol extraction used in the aqueous corn meal isotherms apparently extracts weakly-adsorbed AfB1. Due to the methanol extraction, AfB1 retention from aqueous corn meal is a more conservative measure of aflatoxin binding to clays. The high-charge montmorillonite and activated carbon only retained ∼0.1 g AfB1/kg from corn meal, whereas, the low-charge montmorillonites retained ∼0.7 g/kg. The sepiolite retained ∼1.3 g AfB1/kg at a lower equilibrium concentration than the montmorillonites. Aflatoxin retention measured using methanol extraction might better correlate with aflatoxicosis reductions achieved using clays as feed additives. A simple method might be developed to determine extractable aflatoxins in feed before and after clay addition. Acknowledgements We greatly appreciated the initial research funding that was provided for this study by the Texas Corn Producers Board. We thank Dr. J.-S. Wang at The Institute of Environmental and Human Health, Texas Tech University for providing the purified NovaSil sample that originated from Dr. T.D. Phillips of Texas A&M University. Access to Dr. Necip Güven's X-ray diffractometer was helpful. A large sample of Novasil plus kindly donated by Richard A. Davis will be used for future aflatoxin research efforts. References Afriyie-Gyawu, E., Mackie, J., Dash, B., Wiles, M., Taylor, J., Huebner, H., Tang, L., Guan, H., Wang, J.-S., Phillips, T.D., 2005. Long-term toxicological evaluation of Novasil clay in the diet of Sprague-Dawley rats. Food Additives and Contaminants 22, 259–269. AOCS, 1999a. Aflatoxin standards. Official method Aj 0-88. Sampling and Analysis of Vegetable Oil Source Materials. American Oil Chemists Society. AOCS, 1999b. Total aflatoxins (B1, B2, and G1) in corn, cottonseed, peanuts and peanut butter. Official method Aj 6-95. Sampling and

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