Livestock Research for Rural Development 30 (10) 2018 Guide for preparation of papers LRRD Newsletter

Citation of this paper

Potential of Thai bentonite to ameliorate aflatoxin B1 contaminated in the diet of the Pacific white shrimp (Litopenaeus vannamei)

L Neeratanaphan and B Tengjaroenkul

Khon Kaen University, Khon Kaen 40000, Thailand


The purpose of this study was to determine potential of Thai bentonite (TB) as toxin binder to ameliorate aflatoxicosis effects in terms of weight gain (WG), survival rate (SR), haemocyte count (H) and blood alanine amino transferase (ALT) of the juvenile pacific white shrimp ( Litopenaeus vannamei) (weighed averagely 2.46 g) after fed 150 ppb aflatoxin B1 (AFB1) contaminated diet for 6 weeks. Four diet treatments were prepared as follows: 1) control diet without containing both AFB1 and the TB; 2) diet containing 150 ppb AFB 1; 3) diet containing 150 ppb AFB1 and 0.25% TB w/w; and 4) diet containing 150 ppb AFB1 and 0.5% TB w/w. At the end of the experiment, average WG and SR were examined, whereas H and ALT as immunity and biochemical biomarkers were investigated in shrimp blood. Results of the study demonstrated that WG and SR were greatest in the control, and they were lower significantly in shrimps fed AFB1 diet mixed with TB, and lowest in those fed AFB1 diet without TB (p<0.05). Levels of H and ALT were increased significantly as the concentrations of AFB1 increased, and decreased significantly when fed AFB1 diets mixed with TB (p<0.05). The results of this study can be concluded that TB, particlularly at 0.5% w/w is capable to ameliorate afltoxicosis effects in the juvenile white shrimp.

Keywords: binder, feed, growth, immunity, mycotoxin, shrimp


Major problems found in culture of the Pacific white shrimp, Litopenaeus vannamei in Thailand are related to the widespread incidences of infectious diseases. However, poor feed quality from mold contamination is reported as another potential threat to the success of the shrimp culture industry (Schatzmayr and Streit 2013). Thailand is located in the tropical rain region tending to encounter mycotoxin problems in feedstuffs, especially aflatoxin (Anklam et al 2002, Suppadit et al 2005; Bbosa et al 2013; Tengjaroenkul et al 2018). Aflatoxin B1 (AFB 1) is generally known as one of the most potent mycotoxin in animals, including crustacean (Divakaran and Tacon 2000; Jayasree et al 2001; Gopinath and Paul Raj 2009). Effects of dietary AFB1 on penaeid shrimps were reported on several aspects with well-description, including survival, growth, feed efficiency and pathology (Boonyaratpalin et al 2001; Bintvihok et al 2003; Tapia-Salazar et al 2012; Ghaednia et al 2013; Zeng et al 2015).

A number of strategies have been evaluated to deal with aflatoxin contaminated in shrimp feed. One worldwide acceptable approach to detoxify the AF is to combine toxin binder to AF contaminated feed to reduce availability of the toxin absorbed through the body (Watts et al 2003; Soonngam 2005; Tapia-Salazar et al 2017). This approach provides enhancing evidences of growth performance, survival, feed efficiency, productive capacities, physical mechanisms, blood parameters, immunity, pathological alterations, and mortality of the shrimp (Lightner 1988; Bautista et al 1994; Boonyaratpalin et al 2000; Ghaednia et al 2013). Several studies have shown that binders, such as zeolite, bentonite and aluminosilicate effectively adsorb AFB1 in vivo (Wu et al 2011; Wielogoska wt al 2016; Tian and Chun 2017). Each binder having distinctive molecular structures can bind AFB1 differently (Anater et al 2016; Tapia-Salazar et al 2017; Tian and Chun 2017). Tengjaroenkul et al (2013) discovered that Thai bentonite (TB) demonstrated ability to adsorb AFB1 into their structure due to crystal lattice of the binder and its electrochemical property. Though, efficiencies of toxin binders to reduce negative consequences of aflatoxin in crustacean were revealed (Miazzo 2000; Soonngam 2005; Suppadit et al 2006; Tapia-Salazar et al 2017). However, efficiency of the TB to enhance performance and health after the shrimp fed AFB1 is lacking, therefore this study aimed to determine potential of the TB to ameliorate aflatoxicosis effects on Weight gain (WG), survival rate (SR), haemocyte count (H) and alanine amino transferase (ALT) as biological parameters in the juvenile white shrimp.

Materials and methods

Aflatoxin preparation

Aspergillus parasiticus strain NRRL 1999 was spiked in autoclaved Jasmin rice, and incubated at 25șC for 7 days. Then, the dried contaminated rice weighed 20 g was ground, and mixed with 10 ml distilled water, 10 g hyflo-supercel and 100 ml chloroform using electric stirrer at 150 rpm for 30 min. The mixture was filtered through cellulose filter paper No. 4. For toxin extraction, the filtered solution was poured into glass column contained with 2.5 g florisil, later consecutively poured with 5 ml chloroform, 20 ml methanol and the mixture of acetonitril and distilled water (ratio 49:1). The final fraction of the solution was collected to detect AFB1 concentration using ELISA test kit (ScreenEZź, Siam Inter Quality Co., Ltd, Thailand)(Srikacha et al 2018). AFB1 solution was dried on warm hot plate, later the AFB1 was dissolved in ethanol before spraying on the AFB1 experimental diets.

Basal and experimental diets

Basal diet in g/kg diet as dry matter as the control diet consisted of fish meal (350), soybean meal (150), wheat flour (100), wheat gluten (70), squid meal (50), shrimp head meal (50), fish oil (20), lecithin (20), calcium lactate (10), mono potassium phosphate (5), vitamin mix (4), mono sodium phosphate (2), vitamin E (1.5), vitamin C (l), cellulose (1), choline chloride (3), yeast (5), butylated hydroxytoluene (BHT)(0.20), potassium chloride (2.50) and broken rice (154.8). The rod-shaped diet was prepared by putting the mixed ingredients of the basal diet in oscillating siever (Erweka AR400, Germany), and then drying the finished rod diets in hot air oven (WTB binder ED400, Germany) at 50°C for 12 hours. For the TB diets, TB was mixed in the basal diet at 0.25% (treatment 3), and 0.5% (treatment 4) w/w before making rod shaped diet using oscillating siever. Additionally, to make AFB1 diets, 150 ppb AFB1 solution sprayed on the basal diet (treatment 2) and the TB diets (treatments 3-4). All experimental diets were later coated with fish oil, and kept in refrigerator upon the feeding time. The complete diets contained dry matter 93.6%; protein 41.5%; fat 7.3%; fiber 18.0%; ash 16.2% and moisture 95.7%, and the content analysis of metabolizable energy was 3.84 Mcal/kg of feed (modified from Robertson et al 1993; Pongpiachan 1996; Suppadit et al 2006).

Experimental design

The experiment was designed using 480 juvenile shrimps Litopenaeus vannamei obtained from a commercial hatchery at Chonburi province, Thailand. The shrimp weighed approximately 2.46 g were allotted to 4 diet treatments as: 1) the basal diet (control) without both AFB1 and TB; 2) diet containing 150 ppb AFB1; 3) diet containing 150 ppb AFB1 and 0.25% TB; and 4) diet containing 150 ppb AFB1 and 0.5% TB. All shrimps were fed experimental diets three times a day (7:00 am, 1:00 pm and 7:00 pm) with feeding rate 7% body weight. The experiment was conducted with 3 replications (40 shrimps per replication) in 400 L fiber glass tank with re-circulating water system. Water quality parameters were controlled under aquaculture standard.

Data collection and analysis

Weight gain and survival rate of shrimps were recorded. Haemocyte count and alanine amino transferase following Goarant and Boglio (2000) and Boonyaratpalin et al (2001) were determined from shrimp blood collected from thoracic sinus. Data were analyzed using analysis of variance (ANOVA), and the differences among treatment were compared using the Duncan New Multiple Range Tests (DNMRT) of Statistical Analysis System at p<0.05 (SAS 2004).

Results and discussion

Growth and survival

Aflatoxin can induce deleterious influences on production, morbidity, mortality, immunity, pathology and health of aquatic creatures. In the present study, the juvenile shrimp fed 150 ppb AFB1 contaminated feed granule demonstrated a significant decrease in WG to approximately 71.78% as compared to the shrimp fed the control diet (p<0.05), and to approximately 89.16% and 82.21% as compared to those fed the AFB 1 diet mixed with TB at 0.25% and 0.5%, respectively. The shrimp fed 150 ppb toxin granules mixed with 0.25% TB demonstrated the lower WG and SR greater than of those at 0.5% TB (Table 1). Furthermore, it was not significantly different (p>0.05) on WG comparing the 0.25% and 0.5% TB in AFB1 diets. These results were in accordance with several studies. Sindermann and Lightner (1988) revealed that AFB1 could decrease in growth, low apparent digestibility, physiological disorders and alterations of hepatopancreatic tissues of Penaeus stylirostris. Lavilla-Pitogo et al (1994) reported that shrimp fed AFB 1 diets with more than 50 ppb exhibited hemolytic infiltration and fibrosis in the sinuses of the hepatopancreas, and stated that the tissue alterations were correlated with the reduction of growth, particularly at the higher doses of AFB1. Ostrowski-Meissner et al (1995) demonstrated that feed efficiency of juvenile P. vannamei varied directly with AFBl levels from 50 ppb to 15 ppm, and the growth rate showed inverse relation with toxin concentrations. The shrimp fed 400 ppb AFB1 for 8 weeks showing 17% reduction in final weight, 9% reduction in digestibility coefficient and a 23% relatively increase in FCR compared with the control treatment. Boonyaratpalin et al (2000) showed that feed efficiency of the P. monodon was greater with increment of the AFB1 in the contaminated diets. Miazzo (2000) found that feed efficiency was lower due to adsorption property of the toxin binder added in mycotoxin contaminated feed. Soonngam (2005) reported in P. monodon that the growth of the shrimp fed with AF diet without adsorbent had the lowest as compared with the adsorbent diets. Suppadit et al (2011) demonstrated that average weight gain of P. monodon decreased with the increase of the toxin concentrations, and when fed AFB1 diet without silicate adsorbent, they showed the lowest growth performance as compared with an addition of adsorbent in the toxin contaminated feed. Ghaednia et al (2013) found that Indian white shrimps fed 400-1600 ppb AFB1 feed demonstrated negative effects on growth and survival. Tapia-Salazar et al (2017) reported that juvenile white shrimp fed aflatoxin at 75 ppb for 6 weeks showed a significantly lower feed consumption and growth rate than those fed the toxin diet mixed with the binder at 0.25% w/w.

Table 1. Average weight gain, survival, haemocyte count and serum biochemistry of the white shrimp fed aflatoxin B1 mixed with Thai bentonite for 6 weeks.


Weight Gain (g)
(Mean± SD)

Survival (%)

Haemocyte (104 cells /ml)


1) Control feed





2) AFB1 150 ppb





3) AFB1 150 ppb + 0.25% TB





4) AFB1 150 ppb + 0.5% TB










a,b,c,d Different superscripts in the same column are significantly different (p<0.05); ALT (Alanine amino transferase; Thai bentonite (TB).

Aflatoxin as well as other mycotoxins can do harm to aquatic tissue structures and its physiology which consequently affect to animals’ health and mortality. In the present study, the juvenile shrimp fed AFB 1 contaminated feed granule demonstrated a significant decrease in SR to approximately 76.74% as compared to the shrimp fed the control diet (p<0.05), and to approximately 93.31% and 87.06% as compared to those fed AFB1 contaminated diet mixed with TB at 0.25% and 0.5%, respectively (p<0.05)(Table 1). These results were similar to several research works. Wiseman et al (1983) reported that juvenile P. vannamei were induced mortality after fed diets containing 50-300 ppm AFB1. Lightner et al (1982) reported that lethal and sub-lethal effects of juveniles of P. vannamei within 14 days after exposed to 15 ppm AFB1 . Furthermore, the acute and sub-acute toxic effects of AFB1 in the marine shrimp P. vannamei and P. stylirostris were conducted. P. vannamei weighed approximately 0.5 g were fed various concentrations of AFB1 (53-300 ppm), and P. stylirostris weighed approximately 3 g were injected intramuscularly of the toxin (2-160 ”g AFB1/g body weight). After 24 hours of toxin exposure, lethal dose at 50% (LD50) was reported from 90 to 200 ppm for P. vannamei, and the single dose of LD50 was found to be approximately 25 ppm for P. stylirostris. Cruz and Tendencia (1989) demonstrated histological alterations, particularly severe damage to the hepatopancreas involving in metabolism, immunity and detoxification abilities of the aquatic animals. These can be factors responsible for the weakness, disease outbreak, and death of the shrimp. Bautista et al (1994) showed that the penaeid shrimps fed AFB1 on concentrations greater than 100 ppb showing inflammation, necrosis, infiltration of haemocytes and severe degeneration of liver tubules. Lavilla-Pitogo et al (1994) revealed histopathological changes of organs in juvenile P. monodon fed AFBl diets 26.5-202.8 ppb for 60 days. Shrimp fed diets with more than 50 ppb AFB1 exhibited hemolytic infiltration and fibrosis in the inter-tubular sinuses of the hepatopancreas, and these negative occurrences were more severe in shrimp fed at the higher doses of AFB1. Jayasree et al (2001) found mass mortality after fungal infection on carapace, appendages and gill with reduction of feed intake in cultured P. monodon. Suppadit et al (2011) reported that SR of the P. monodon fed AFB 1 diet without adding silicate minerals as toxin binder was the lowest as compared with those fed mycotoxin feed in addition with the binder.

Haemocyte and Alanine Amino Transferase

In general, haemocyte is one of defend mechanisms to eliminate foreign substances and toxins in body of crustacean (Soderhall and Cerenius 1992). The results of the present study demonstrated that H of the shrimp exposed to AFB1 was greater as compared to those in the control and in the treatment fed AFB1 mixed with TB in the diet. The shrimp fed 150 ppb toxin granules mixed with 0.25% TB demonstrated greater H and ALT than of those at 0.5% TB. It was not significantly different (p>0.05) on both H and ALT comparing between the shrimp fed 0.25% and 0.5% TB in 150 ppb AFB1 diets (Table 1). Additionally, it was found that ALT, the hepatic cytosol enzyme involving the breaking down amino acid, increased in the blood of the shrimp fed AFB1 compared with the control diet, and these H and ALT were lower in the shrimp fed the toxin mixed TB as compared with the AFB1 contaminated diet solely. These occurred probably due to the damage of liver cellular membrane following the exposure to the AFB1 as reported by several researchers. Ghaednia et al (2013) found that the Indian white shrimps fed the 400-1600 ppb AFB1 showed negative consequences on blood parameters and hepatopancreas structures. Boonyaratpalin et al (2000) described that immune function and blood components changed in the P. monodon weighed averagely 1.17 g after fed AFB l less than 220 ppb for 8 weeks. The total H, phenol oxidase activity as well as plasma glutamic transaminase enzymes of the shrimp showed increasing trends with increasing concentrations of the toxin. These implied that the shrimp were in a poor health status by aflatoxicosis.

Contaminations of AFB1 have been reported worldwide. Several strategies are available for reducing adverse effects in aquatic creatures. Managements at pre-, during- and post-harvesting processes for quality control of feed ingredients are such important, which consequently prevention myco-toxigenic effects in aquatic animals. Addition of adsorbent or toxin binder in AFB1 contaminated diet is one of the most acceptable approaches to ameliorate negative mycotoxin effects in animals, including shrimp (Surai and Dvorska 2005; Gowda et al 2013). Adsorbent can adsorb AFB1 into its structure, therefore, can prevent an absorption of the toxin through the wall of gastrointestinal tract, i.e., having less amount of the aflatoxin metabolites in the shrimp body tissues (Chou et al 1994; Boonyaratpalin et al 2001; Soonngam 2005). From the above information and the results of the present study, TB is capable to adsorb aflatoxin effectively (Tengjaroenkul et al 2013), this can influence on feed intake, WG, feed efficiency, SR, H and ALT enzyme of the shrimp. Furthermore, this can reduce economic loss in shrimp aquaculture as well as potential risk for aflatoxin B from edible shrimp tissues to humans leading to a more safety food source in the food chain. The findings in this study were similar to several reports. Miazzo et al (2000) revealed that addition of adsorbents improved average WG and decreased feed conversion efficiency in the shrimp fed aflatoxin. Soonngam (2005) reported that AFB1 would be adsorbed by adsorbents in the gastrointestinal tract, and excreted less toxin in shrimp body. Suppadit et al (2006) showed that hydrated sodium calcium alumino silicate and vermiculite could counteract adsorption of 500 ppb AFB1 in the white shrimp diet. These two adsorbents had ability to help increase average WG, feed effciency, SR as well as the toxin residue in the shrimp tissues by their adsorption properties in the tetrahedral framework. Tapia-Salazar et al (2017) demonstrated that commercial toxin binders (0.2-0.25% w/w) could elevate growth performance the white shrimp fed 75 ppb AFB1 for 6 weeks. In addition, efficacy of the mycotoxin adsorbent is generally related to several factors, including species and concentration of the toxin, ability of the adsorbent to bind toxins as well as breed, age, and health status of the shrimp (Supamattaya 2004; Gopinath and Paul Raj 2009; Ghaednia et al 2013; Tapia-Salazar et al 2017).



The study was supported by Research Group on Toxic Substances in Livestock and Aquatic Animals, Khon Kaen University, Khon Kaen, Thailand.


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Received 21 August 2018; Accepted 30 August 2018; Published 1 October 2018

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