Aflatoxins contamination in duck feedstuffs collected from Indramayu District

Aflatoxins are secondary metabolites produced by Aspergillus fungi. Feed exposure to aflatoxins may cause a decreased immune system and low productivity outcomes in poultry. This study conducted a limited survey to determine aflatoxin contaminations in feedstuff. 34 feed samples were analyzed to determine aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2 using High-Performance Liquid Chromatography (HPLC) fluorescent detector. Aflatoxins were extracted from feed samples using acetonitrile : water (84:16,v/v), purified with a solid phase extraction (SPE) column, and detected through a fluorescent detector at wavelengths 365 nm (excitation) and 450 nm (emission). The results show a high concentration of AFB1 contamination in 34 samples. Aflatoxin B1 was found in all samples, ranging from 1.41-42.16 ng/g. Eleven samples were found to contain AFG1 ranging from 4.51-6.39 ng/g. One sample contained AFG2 with a concentration of 7.59 n/g. Also, 16 samples contain AFB2 with a concentration ranging from 1.62-2.3 ng/g. The results above show a high prevalence (100%) of AFB1 contamination. The contamination of aflatoxins especially AFB1 in the feedstuffs were harmful to the animals and should be a concern to prevent any adverse effect on public health.


Introduction
Aflatoxins (AF) are produced by fungi, Aspergillus flavus, and Aspergillus parasiticus [1][2][3].There are fourteen reported aflatoxins.However, the majority of them are endogenous metabolites generated in animals.There are fourteen reported aflatoxins [1], but the well knows of them are aflatoxin (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2) [4,5] are of the most relevance in food safety points of view [6] (structures shown in Figure 1).Aflatoxins B1 and B2 are so characterized due to their blue fluorescence under UV light, whereas aflatoxins G1 and G2 have a greenish-yellow fluorescence [1,2].AFB1 is the most common mycotoxin that contaminates up to 75% of the world's agricultural products used for food and animal feed [7].Indonesia's archipelago, with the presence of a hot and humid climate, has an average temperature range of 28-31 °C and humidity levels of 60-80%, which are favorable to the growth of mycotoxin-producing fungi, are two key factors that contribute to the occurrence of mycotoxins [8].
Aflatoxins can contaminate various agricultural commodities, especially maize, used for animal feed ingredients before harvest or storage [3].Aflatoxin contamination, particularly AFB1, in animal feed may lead to aflatoxicosis [9] and also results in the deposition of aflatoxin residues and their metabolites in meat, milk, and eggs, including aflatoxin M1 (AFM1), aflatoxicol, aflatoxin Q1 (AFQ1), and aflatoxin P1 (AFP1) [11].AFB1 is considered the most toxic among aflatoxins.Aflatoxins cannot detect by smell or taste and are resistant to temperatures above 320 o C without decomposing [2].The presence of aflatoxins in various processed and unprocessed foods is regulated in many nations worldwide due to the adverse consequences of aflatoxins, which include mutagenic, carcinogenic, and teratogenic effects in numerous organisms [12].Therefore, many countries, including Indonesia, set maximum aflatoxin levels for various types of feed, according to the type of livestock (chickens, cows, ducks) through the Indonesian National Standard (SNI), Ministry of Agriculture to ensure the safety of feed to minimize the public health impact.The AFB1 maximum level for maize as feedstuffs is 100 µg/kg [13], poultry feed is 50 µg/kg[14], and duck feed is 20 µg/kg [15].
Each species has different abilities for aflatoxin detoxification.In contrast to other livestock which are resistant to exposure to aflatoxin, ducks are one of the species that is very susceptible to exposure to aflatoxin at low doses (LD50 < 1 ppm).Matter this is suspected due to differences in the rate of aflatoxin biotransformation in the liver of the species, where the ducks have a different path than other avian species.AFB1 contamination on feed ingredients and the low ability of ducks to detoxify aflatoxins are suspected to be a high risk of aflatoxicosis in ducks and aflatoxin residues in their products [16].There have been numerous reports of aflatoxins contaminating animal feed and maize samples from poultry [8] and cattle.However, there has been few research on the occurrence of aflatoxins in duck feedstuffs in Indonesia, particularly in the Indramayu District (West Java Province), which has the highest duck population in Indonesia [17].The objective of this study was to observe the occurrence of aflatoxins contamination using High-Performace Liquid Chromatography (HPLC) fluorescence detector.

Samples
Thirty-four duck's feedstuff of 7 feed mix, 16 commercial feed, three dried rice, two rice bran, and one each of rice hulls, groats, coconut cake, dried fish flour, ebi flour, seaweed samples were collected from different markets and farmers in the Indramayu, West Java Province.All samples were frozen at -20 o C before analysis.

Chemicals
Aflatoxins standards and the chemical were obtained from Sigma Aldrich, Merck (Germany).Deionized water was prepared from a Milli Q Direct 8/16 System (Millipore SAS, 67120 Molsheim, France).The standard stock solutions of AFB1, AFB2, AFG1, and AFG2 were prepared by dissolving them in methanol.These solutions were further diluted to the working standard.All standard solutions were sealed and stored at -20 °C.A mobile phase was made from a mixture of methanol, acetic acid, and deionized water (15:20:65, v/v/v).

Sample extraction and determination of aflatoxins
A 12.5 g of milled feed/feedstuffs sample was extracted with 50 mL acetonitrile: DI water (84:16, v/v) in Erlenmeyer and vortexed for 60 min.The sample was filtered with filter paper, and 1 ml of filtrate was cleaned with a solid phase extraction (SPE) Supel™ Tox Afla-Zea SPE Cartridges (Supelco 55314-U, Sigma Aldrich) in a vacuum chamber.Then the extract was placed into a vial and dried at 40º C in a nitrogen stream.The extract was kept at -20ºC until analysis.A 50 µL trifluoroacetic acid and 200 µL n-hexane were added to the residue from the sample extract or to the aflatoxin working standards, vortexed for 30 s, and kept in the dark for 10 min at room temperature.Sample or standard dried with a nitrogen stream, then reconstitute with 1 mL of the mobile phase.Ten µL of sample solution was then injected into the HPLC-fluorescence detector of Waters Alliance e-2695 (Waters Corp., USA) set at 355 nm and 368 nm.Separation was carried out in a reverse-phase C-18 HPLC column (Sunfire, 250 x 4,6 mm, Ø 5µm) (Waters, USA), with the mobile phase run under the isocratic condition at a flow rate of 1 mL/min.

Data analysis
Empower HPLC software was used to control the system operation and collect and analyze the HPLC data.Raw data from HPLC were then analyzed with Excel.

Optimation of method detection
Due to the complexity of the samples, efficient sorbent materials are required for the cleaning step.The immunoaffinity column is widely utilized due to its high specificity.However, this sorbent is expensive, commercially available only in a single-use format, and has a shorter shelf life than C18 silica-based sorbent materials [18].The need for a quick, simple sample clean-up approach before chromatographic mycotoxin analysis has brought about SPE cartridges that significantly reduce sample preparation time, have good reproducibility, and are more user-friendly than immunoaffinity columns.In addition, using the Supel-Tox Afla-Zea SPE Cartridges (Supelco, Sigma Aldrich) in this study approach requires less equipment and fewer consumables, providing additional cost savings.The SPE Supel Tox Afla-Zea, cartridge extraction method was used without SPE conditioning and elution steps.An HPLC protocol has been developed to separate the aflatoxins with better resolution efficiency and quantitation.The standard chromatogram for the aflatoxins is shown in Figure 2. The retention times of external standards were found to be 6.117, 8.507, 11.127, and 17.177, aflatoxin G1, B1, G2, and B2, respectively.Figure 3 shows the calibration curve for external standard aflatoxins, the concentration of which varied between 3.82 ng/mL to 87.36 ng/mL.The linearity (R 2 ) of 5 different concentrations of aflatoxins were 0.9976, 0.9983,0.9944and 0.991 for AFG1, AFB1, AFG2, and AFB2, respectively.

Aflatoxins contaminant in feedstuff samples
The results of aflatoxin contamination in 34 feeds and feedstuff samples are shown in Table 1.Aflatoxin B1 was found in all samples, ranging from 1.41-42.16ng/g.Eleven samples were found to contain AFG1 ranging from 4.51-6.39ng/g.One sample contained AFG2 with a concentration of 7.59 n/g.Also, 16 samples contain AFB2 with a concentration ranging from 1.62-2.3ng/g.The results above show a high prevalence (100%) of AFB1 contamination.Aflatoxin B1 concentrations with ranging from 1.41-42.16ng/g, where 6 (17.65%) among them showed a concentration of 23.47-42.16ng/g, mainly in feed concentrate and dried rice, were above the maximum level of AFB1 concentration in duck feed.The maximum limit of AFB1 contamination in laying duck feed, according to the EU Codex Alimentarius and SNI No. 3908-2017, is 20 ng/g [15].This study shows a better result than the previous study on South Kalimantan from commercial feed and feed mix ratio samples, which shows a high prevalence (100%) with an average contamination of 32 and 13 ng/g, respectively.Enzyme-Link Immunosorbent Assay, which is the method used, shows high AFB1 contamination in Alabio duck feed (28 ng/g), including in rice brands, dried fish, and rice hulls [16].Positif feed samples (16.67%) were also found in several areas in West Java with an average concentration of 11.03 ng/g [8].A study in India showed the presence of AFB1 within 7 out of 18 samples in the permissible limit (6.25 ng/g to 12.50 ng/g) [19].AFB1 content exceeding the maximum limit was also found in dry rice (45 ng/g).
High AFB1 contamination in dry rice was unexpected because, generally, A. flavus fungus grows on materials with high protein and energy content [16].The aflatoxin contamination is thought to be due to improper storage of dry rice.The traditional method of drying the feedstuffs on the ground in the open air under poor sanitation promotes mold development and the production of mycotoxins [18].A study on the use of zeolite in the high AFB1 contaminated diet suggested that zeolite did not decrease aflatoxins residue in the tissues but could prevent liver alteration of laying duck [20].Additional curcumin as an anti-oxidation and anti-inflammation agent could protect the ileum against acute damage induced by AFB1 administration [21] The results on some samples in this study showed that a high concentration of AFB1 may cause aflatoxicosis to duck, as acknowledged that the toxic level of aflatoxin BI in white Pekin duck was 48 ppb [22].Fortunately, lycopene and silymarin showed benefits to cope with aflatoxicosis in ducks [9], and in vitro study of an extract of iles-iles (Amorphophallus oncophylus) tuber also showed the ability to reduce the aflatoxin content [23].Whereas the additional 0.4% sambiloto (Andrographis paniculata) leaf powder was optimal to increase the economic feed efficiency of Mojosari Duck rearing [24].

Conclusion
The occurrence of aflatoxins shows a high prevalence of aflatoxins contamination (100%) in all samples analyzed, ranging from 1.41-42.16ng/g and 1,61-2.30ng/g, for AFB1 and AFB2, respectively.Some (17.65%) of AFB1 contamination is above the maximum limit of AFB1 in duck feed.Therefore, a monitoring program of the aflatoxin content in duck feed is necessary to prevent adverse effects from the presence of aflatoxin on poultry health.

Table 1 .
Aflatoxins contamination in feedstuffs samples