Mouldy Groundnut Cake and Hydrated Sodium Calcium Aluminosilicate in Practical Diet for African Catfish Clarias gariepinus (Burchell, 1822)

Feed ingredients contamination with toxins may occur anywhere in the supply chain from the field, manufacturing process, transportation to storage. In the tropics conducive environmental condition has majorly been attributed to production and growth of micro-organisms and toxic substances; particularly fungi. This is exacerbated by the cultural practice and lack of adequate regulatory and control system by government for food and feedstuffs contaminant screening in most developing countries in the humid tropics.


Introduction
Feed ingredients contamination with toxins may occur anywhere in the supply chain from the field, manufacturing process, transportation to storage. In the tropics conducive environmental condition has majorly been attributed to production and growth of micro-organisms and toxic substances; particularly fungi. This is exacerbated by the cultural practice and lack of adequate regulatory and control system by government for food and feedstuffs contaminant screening in most developing countries in the humid tropics.
Fungi, Aspergillus flavus and A. parasiticus contamination are mostly responsible for the production of aflatoxins in the humid tropics [1]. They are found when environmental temperature is above 27°C, humidity levels greater than 65% and moisture levels in feedstuff is above 14%. Also suboptimal handling and storage are considered as important factors favouring the growth of aflatoxin-producing fungi [2]. Aflatoxins are the most potent toxic substances occurring naturally. Large yield of aflatoxins are found in feed ingredients with high carbohydrates concentration as found in cereals and to a lesser extent in oil seeds. But in West Africa aflatoxins are of major concern in groundnut. Four major forms of aflatoxins are found in feedstuffs: B 1 , B 2 , G 1 and G 2 . Aflatoxin B 1 is regarded as carcinogenic, most prevalent and toxic of the four. It has significant economic and health implications in both animal and man. Hence efforts have been focused on reducing the impact of aflatoxin B 1 contamination in livestock and fish, with dearth of information on the other aflatoxins.
Recently studies have reported the co-occurrence or cocontamination of substance(s) with more than one aflatoxins [3,4]. This is because in practical situation contaminants are rarely found individually [5]. Especially when environmental condition is conducive, the probability of multiple or co-contamination is always high. Hence there is need to assess contaminants present naturally in feed raw materials particularly in endemic environment like the humid tropics. Sequel to this, there might be need for the identification of fungi present in such substances which are potential toxins producers. This could provide necessary information for designing strategies in the management of these toxins.
Consequently with the recent trend in increase utilization of plant feedstuffs (which are good substrates for aflatoxins) in warm water fish species, there is need to investigate the implications of natural aflatoxins contamination. Also the practice of inclusion of toxin binder like Hydrated Sodium Calcium Aluminosilicate (HSCAS), which is generally routinely added to suspected contaminated (mouldy) feedstuffs in endemic environment [6].
From the foregoing, this study was designed to detect fungi, quantify microbial load and aflatoxins B 1 , B 2 of the diets formulated with mouldy groundnut cake. Also the toxin binding efficacy of HSCAS on growth performance and aflatoxins B 1 and B 2 levels in the diets and

Dietary Design and Feeding
A 50 kg bag of Mouldy Groundnut Cake (MGC) suspected to be naturally contaminated was obtained from a commercial feed ingredients seller at Ojo, Lagos, Nigeria. MGC contained 185.00 ± 7.00 and 137.50 ± 10.00 µg/kg of aflatoxin B 1 and B 2 , respectively. Three experimental diets were formulated to contain 35% crude protein each. Diet one contained uncontaminated groundnut cake and other feed ingredients which served as the control, diet two contained contaminated groundnut cake and other feed ingredients while diet three contained contaminated groundnut cake, other feed ingredients and toxin binder (HSCAS) at the manufacturer's recommended inclusion rate of 0.3% of the diet. Percentage gross composition of the experimental diets is shown in Table 1. The feed preparation was done at a reputable commercial fish feed manufacturer: Act livestock consult, Badagry Expressway, Lagos-Nigeria, where all ingredients were weighed, ground into fine powder, thoroughly mixed and pelleted (into 2 mm) with a 350 kg/hr table top pelleting machine. The pellets were crushed into pieces and stored in air tight containers. Fish were fed twice daily at 8.00 and 16.00 hours at the rate of 3% body weight for a period of 12 weeks. Feeding was done by hand and spread evenly across the water surface of each aquarium.

Fish management
The experiment was conducted at the indoor of Lagos State University Hatchery, Ojo, Lagos, Nigeria. Nine (9) plastic aquaria tanks with 80 L water capacity and 0.5 m depth were filled with borehole water to three quarter capacity. The water in the experimental tanks was aerated by electric air pump (Shining model; horsepower 50Hz). Clarias gariepinus fingerlings of mean weight of 1.68 ± 0.01 gm were obtained from Lagos State Agricultural Cooperative farm, Ojo, Lagos, Nigeria. The fish were transported early in the morning between the hours of 6.30 and 7.00 from the hatchery and acclimatized for 5 days prior to the start of the experiment. They were randomly distributed to the 9 plastic aquaria at the stocking rate of 60 fingerlings per experimental diet and 20 per replicate making a total of 180 fingerlings. Daily 50% of water in each tank was gently siphoned in exchange for fresh water. This was done to get rid of left over feed and faecal matter.

Fungi isolation and quantification of aflatoxins and microbial loads
Mouldy groundnut cake was subjected to extraction and quantification of aflatoxins B 1 and B 2 before the commencement of the study and other major ingredients were screened against aflatoxins. The coarse ingredients were ground, mixed before samples were collected for analyses. Fungi present in the experimental diets were isolated; the diets were also subjected to extraction and quantification of aflatoxins B 1 , B 2 and microbial load. Twenty fingerlings were randomly picked before the start of the experiment. The fingerlings were washed, crushed under sterile condition for microbial load determination. At the end of the experiment 20 fingerlings (divide into two groups) from each treatment were randomly picked for microbial load and aflatoxin B 1 and B 2 determination. The isolation of fungi, extraction and quantification of aflatoxins and microbial load were done at the Institute of Agricultural Research and Training (IAR&T), Ibadan, Oyo-Nigeria [7][8][9][10].

Growth and feed utilization
Batch weighing of fish in each replicate aquarium was done at the beginning of the experiment, subsequently biweekly, using a MettleR 20110 top-loading balance. The evaluation of experimental diets for growth and feed utilization was carried out as follows.
Time represents difference in days between final and initial body weight.

Economy analysis
A simple analysis was conducted to assess the cost effectiveness of the experimental diets, only diet cost was used for the calculations with the assumption that all other operating costs remained the same. The average prices of each feedstuff ($) during the study period was used to calculate the amounts required to make the different diets, cost per kilogram of each diet was calculated. The economic conversion ratio (ECR) was determined using this equation: ECR=COST OF DIET×FCR.

Water quality parameters
Daily water temperature was measured using a mercury-in-glass

Observation
The fish were observed for behavioural and physical abnormalities that could suggest possibility of disease situation.

Statistical and chemical analyses
Feed proximate composition was done according to the method of Association of Analytical Chemists [11]. Data collected from feeding trial were subjected to one-way analysis of variance (ANOVA). Significance difference in means was evaluated by Duncan's Multiple Range Test using SPSS for windows (version 11). Values are expressed as Means ± SD.

Fungi, aflatoxins content and microbial load of the experimental materials:
The results of the fungi isolated, aflatoxins and microbial load of the experimental diets, fish and carcass are presented in Table 2. Summary of growth performance characteristics and water quality results are presented in Tables 3 and 4, respectively.
Six fungi from two genera comprising of Aspergillus and Penicillum were isolated from the three diets. Aspergillus parasiticus was found in all the experimental diets. In addition diet 2 contained five other fungi; A. niger, A. flavus, A. tamarii, Penicillum citriinum and P. oxalicum. Inclusion of HCSAS to diet 3 reduced the number of fungi isolated to two; A. niger and P. citriinum.
Aflatoxins B 1 and B 2 were not detected in the diet and carcass of African catfish fed diet 1 (uncontaminated). Generally addition of HCSAS significantly (P<0.05) reduced the concentrations of aflatoxins B 1 , B 2 and microbial load in the diets and the carcass of African catfish (Clarias gariepinus) in this study.

Feed utilization, growth and economic analyses:
Inclusion of mouldy groundnut cake in the diet of Clarias gariepinus had no significant (P>0.05) effect besides survival, but significantly (P<0.05) reduced final weight, weight gain, specific growth rate, feed intake, feed conversion ratio and protein efficiency ratio. However, addition of HCSAS to diet 3 slightly but not significantly (P>0.05) improved all growth and feed utilization parameters assessed.
Economic analysis showed that mould contaminated diet (2) had the least cost of production ($ 0.791), but most expensive ($ 2.21) for African catfish to attain a kilogram body weight when fed with this diet.

Water quality analysis:
Inclusion of mouldy groundnut cake to the diet of Clarias gariepinus significantly (P<0.05) reduced dissolved oxygen concentration of catfish fed the contaminated diet.

Observation:
No external changes or unusual behaviour was observed in any of the fish fed the experimental diets throughout the study. Fish in all experimental diets appeared healthy and normal throughout the period of study.

Discussion
There is dearth of information on natural aflatoxins contamination of feedstuffs and diets of the African catfish in spite of their prevalence and the economic importance of the fish in this part of the world. The results of fungi isolated in the three experimental diets showed the presence of two of the three fungi genera (Aspergillus, Penicillum and fusarium) associated with mycotoxins production of agriculture and human health significance. The presence of Aspergillus parasiticus in the three diets suggests potentials for the production of the four forms of aflatoxins. Although aflatoxins B 1 and B 2 were not detected in diet 1; which may be due to insufficient availability of conditions necessary for their production in this study [12,13]. The co-occurrence of A. flavus, A. niger and Penicillium in the contaminated diet could probably encourage the production of ochratoxins and cyclopiazonic acid (CPA), which have been reported to be commonly found with aflatoxins [13][14][15]. Also possibilities exist for the production of citrinin, secalonic and     [16,17]. Their co-occurrence may encourage additive, synergy and or antagonistic reactions among these toxins. The addition of HSCAS (diet 3) reduced the number of fungi isolated, however potentials exit for the production of all toxins listed against MGC diet, except secalonic acid which is produced by P. oxalicum [18]. The increased microbial load observed in the mould contaminated diet could have resulted from the high number of fungi present in the MGC diet compared to other diets. Therefore from the fungi isolated, there might be need to investigate the presence of other mycotoxins when mouldy groundnut cake is used in diet formulation.
The reduced levels of aflatoxins B 1 and B 2 in HSCAS diet further attest to the ability of HSCAS in reducing aflatoxins levels particularly B 1 [19,20]. The reduced aflatoxins levels may have contributed to lower microbial load observed in the diet and carcass of African catfish fingerlings fed diet containing HSCAS. It is however imperative to note that the level of aflatoxin B 1 (2.24 ± 0.01) found in fish carcass was more than 0.05 µg/kg [21]. Consequently, the addition of HSCAS alone may not be effective in combating mould contamination in the diets of African catfish.
The feed utilization parameters measured showed that African catfish fed mouldy GNC diets had lower weight gain compared to uncontaminated diet. The reduced weight gain was slightly but not significantly improved by the addition of HSCAS to the diet. This is in agreement with the findings [20,22,23]. The lower weight attained may have emanated from low feed consumed by fingerlings on these diets, occasioned by mould contamination which is known to have negative effect on feed taste and nutritive quality [6,24,25]. The low feed intake and nutritive quality may have contributed to the lower growth and feed utilization; feed conversion and protein efficiency ratio observed with the aflatoxins contaminated diets. However, the results of this study disagree with the findings of [26,27] who reported no significant change in growth when tilapia and channel catfish were fed 250 and 2150 ppb purified aflatoxin B 1 . The discrepancy might be due to the different species of fish, as well as the nature of toxins used (purified toxin was used in their study as against natural toxins present in feedstuff used in this study). This is besides the fact that more than one toxin was assessed in this this study. These might have aggravated the observed adverse effects. Also the inability of HSCAS to significantly improve the growth, feed utilization and economy of production is in agreement with the findings of [20,28,29] who reported ineffectiveness of HSCAS in diets containing multiple mycotoxins. Consequently supporting the need to determine fungi and all toxins present when mouldy feedstuffs are used in diet formulation. The low dissolved oxygen recorded with contaminated diets could have arisen from the quality of the feed fed. This assumption is based on the fact that aflatoxin is known to reduce nutritive quality of feed [26,30]. The reduced nutritive quality could have reduced fish's appetite resulting in reduced feed intake and increase feed or solid suspension in the culture medium, hence the reduced dissolved oxygen. However, the low dissolved oxygen values were within tolerable levels for catfish production.

Conclusion
The results of this study showed the co-occurrence of six fungi; A. parasiticus, A. niger A. flavus, A. tamarii, P. citriinum and oxalicum in diets formulated with mouldy GNC. These fungi have potentials for production of the four forms of aflatoxins, ochratoxins, CPA, citrinin, secalonic and oxalic acid. The co-occurrence of aflatoxins B 1 and B 2 evaluated in this study adversely affected growth, feed utilization and resulted in deleterious impact on production cost of African catfish (Clarias gariepinus) fingerlings. The addition of HSCAS slightly improved the adverse effects of the contamination with the elimination of potential for secalonic acid production. However, aflatoxin B 1 levels in the carcass of African catfish; Clarias gariepinus is beyond the 21 acceptable level. Consequently, there might be need for identification and quantification of all toxins present in diets formulated with mould contaminated feedstuff (in this study MGC) to proffer needed strategies for the toxins management in catfish diets.