Aflatoxin is a toxic compound produced by Aspergillus flavus and
A. parasiticus. The molds can grow in improperly stored feeds and
feeds with inferior quality of ingredients.
Aflatoxins represent a serious source of contamination in foods and feed
in many parts of the world (Murjani, 2003). Aflatoxin B1 is
known to be the most significant form that causes serious risk to animals
and human health. The carcinogenic effect of aflatoxin B1 has
been studied in fishes such as salmonid, rainbow trout, channel catfish,
tilapia, guppy and Indian major carps (Jantrarotai and Lovell, 1990; Lovell,
2001; Tacon, 1992; Wu, 1998; Chavez et al., 1994; Murjani, 2003)
and Penaeus monodon (Bautista et al., 1994).
Aflatoxicosis is a disease that can affect many species of fish and shellfish
and results when feed contaminated with aflatoxins is eaten by the fish
(Ashley, 1970; Hernández et al., 2005; Bautista et al.,
The first documented incidences of aflatoxicosis affecting fish health
occurred in the 1960s in trout hatcheries. Domesticated rainbow trout
(Oncorhynchus mykiss) that were fed a pelleted feed prepared with
cottonseed meal contaminated with aflatoxins, developed liver tumors (Ashley,
1970). As many as 85% of the fish died in these hatcheries (Taniwaki,
In tropical and subtropical conditions, this potential is further increased
due to storage under humid and hot conditions. International trade in
affected commodities and exposure to aflatoxins are worldwide concerns
and the economic impact due to animal losses can be enormous (Golan and
Four major aflatoxins (AFB1, AFB2, AFG1
and AFG2) are direct contaminants of grains and finished feeds.
Factors that increase the production of aflatoxins in feeds include environmental
temperatures above 27°C (80°F), humidity levels greater than 62%
and moisture levels in the feed above 14%. The extent of contamination
will vary with geographic location, feed storage practices and processing
methods. Improper storage is one of the most important factors favoring
the growth of aflatoxin-producing molds and it is a major element that
can be controlled by the fish producer (Payne et al., 1988).
Rainbow trout and nile tilapia are extremely sensitive to AFB1,
while channel catfish are much less responsive (Jantrarotai and Lovell,
1990; Tuan, 2001).
The Rainbow trout was widespread in the Province of West Azarbajan. This
condition was observed in several farms which administered moldy feeds
to their fish. Interview with farmers indicated that moldy feed was caused
by high moisture content and improper storage of their feeds.
The purpose of this study was to assess the production of aflatoxin-contaminated
feeds in the cold (autumn and winter) and warm (spring and summer) seasons
and effect on fish growth. Results from this research will answer some
of the questions of trout farmers on their experience on the rainbow trout
in the West Azarbajan.
MATERIALS AND METHODS
Twenty four samples of food (from April 2006 to April 2007) were evaluated
during 2 phases warm seasons : spring and summer; cold seasons: autumn
and winter in completely random design were used in this study. The compositions
of basal diet were from different factories and of various kinds (SFT,
FFT, GFT, BFT) and sizes.
These consisted of:
||SFT (Starter Food Trout): containing protein> 45-50%
for fry > 5 g
||FFT (Fingerling Food Trout): containing protein ≈ 45-50% for
fingerling = 5-30 g
||GFT (Grower Food Trout): containing protein = 35-40% for fish =
||BFT (Brood Food Trout): containing protein = 45-45% for adult fish>350
||The concentration of aflatoxin in diet was adjusted according to
aflatoxin B1, B2, G1 and G2
levels as representative mycotoxin
Determination of Aflatoxin Production by HPLC
The AFB1, AFB2, AFG1 and AFG2
concentration was determined by HPLC. All samples were threefold extracted
with chloroform and this was followed by evaporation at 36°C under
nitrogen gas; then the samples were finally dissolved in methanol. The
samples were filtered through a Teflon filter (pore size, 0.2 μm;
Chromafil; Macherey+Nagel, Düren, Germany) before they were used
in HPLC analysis. Forty-microliter aliquots of these filtered extracts
were injected for the quantitative determination of the AFB1
The HPLC system consisted of a model L-7100 HPLC-pump (Merck/Hitachi,
Darmstadt, Germany), a model L-7200 autosampler combined with a Peltier
sample cooler (Merck/Hitachi) and a model HP 1050 diode-array detector
(Hewlett-Packard, Böblingen, Germany). The chromatograms were digitally
processed by the ChemStation software system (Hewlett-Packard).
For analysis of AFB1, a reversed-phase C18 column (LiChroCART
250-4 RP-18 [5.0 μm]; Merck) protected by a guard column (LiChroCART
4-4 RP-18 [5.0 μm]; Merck) was used with an isocratic mobile phase
of acetonitrile-methanol-H2O (25:25:50, vol/vol/vol) at a flow
rate of 1.0 mL min-1. The presence of AFB1 was monitored by
the diode array detector at a wavelength of 365 nm (Móricz et
RESULTS AND DISCUSSION
In the Philippines, the limit of aflatoxin in the feed prescribed by
the Bureau of Animal Industry is less than 20 ppb. According to national
feed legislation in the USA, maize (corn) and peanut (groundnut) products
that are to be used for feeding dairy and immature animals (including
fish cannot contain more than 20 ppb of aflatoxin (Lovell, 2001).
Table 1 and 2 summarizes the final
growth Aspergillus spp. in the different feed. Conditions for all
of feeds were similar in all the treatments (p>0.05). Significant differences
were observed in the mean aflatoxin (ppb) levels among feeds (p>0.05)
(Table 3, 4).
Results showed that aflatoxin concentrations increased as the levels
of Aspergillus sp. contamination increased in the feed. It was
observed that feeds contaminated with Aspergillus sp. gave
higher levels of aflatoxin (6.82 ppb) at warm season and lower levels
of aflatoxin (1.1 ppb) at cold seasons. The decrease in aflatoxin level
may have been the result of the deteriorating growth of A. flavus as
|| Aflatoxin levels in the different feed in warm seasons
(T = 18.4°C ± 5.94)
|| Aflatoxin levels in the different feed in cold seasons
(T = 3.6°C ± 7.5)
|| B1 and B2 levels in the different
|| B1+B2 levels in the different
Aflatoxin production is the consequence of a combination of species,
substarte and environment. The factors affecting aflatoxin production
can br divided into three categories: environment, nutritional and biological
factors. Physical factors include temperature, pH, moisture, light, aeration
and level of atmospheric gases. Aflatoxins are produced only between temperatures
of 12 and 14°C and the optimal temperatures is 25 to 35°C (Asis
et al., 2002).
Result in this study showed in second stage, fall and winter were negative,
but of the samples of the first stage, spring and summer there were 5
positive samples because temperature in the first stage was 3.6°C
± 7.5 but in the second phase was 18.4°C ± 5.94. The
total concentration of toxin (B1, B2, G1,
G2) was between 1.21 to 6.62 ppb. The sample has been concentration
of 6.62 ppb highly exceeded the allowed level. During these examinations,
it was revealed that, the farms which had executed the hygienic principals
of stocking, showed lower levels of toxin in the diet and vice versa.
The toxin levels detected between spring and summer are higher than those
of fall and winter due to the high heat and humidity of the warehouse.
Mycotoxin producing fungi are responsible for significant financial losses
encompassing a broad spectrum of food and farm animals and extending through
the food chain to the consumer. Every year a significant percentage of
the world`s grain and oil seed crops are contaminated with hazardous mycotoxins,
such as aflatoxin. Unfortunately, discontinuing the feeding of aflatoxin
contaminated grain is not always practical, especially when alternative
feedstuffs are not readily available or affordable. Thus, these toxins
frequently are detected in animal feed (Sanders et al., 1968; Koehler,
1938; Kiermeier, 1977; Russo and Yanong, 2002).
Aflatoxins are poisons produced by naturally occurring molds. These molds
can grow in grains and prepared feeds intended for fish production when
storage conditions are suboptimal: temperatures of 27°C (80°F)
or warmer and moisture at levels greater than 14%. These conditions are
frequently seen in tropical and subtropical aquaculture.
To prevent aflatoxicosis, follow manufacturer`s recommendations regarding
shelf life and try to determine the feed manufacture date. Avoid using
feeds that appear discolored, lump together and smell musty. Clean feed
storage bins and automatic feeders regularly.
Aflatoxins lower production efficiency of cultured fish by reducing growth
rates, impairing immunity and in some cases, causing mortality. Storing
feed properly (in a cool, dry area on pallets and at least one foot away
from any walls) can prevent unnecessary economic losses.
The moisture content of the substrate and temperature are the main factors
regulating fungal growth and mycotoxin formation (Jarvis, 2008).
Koehler (1938) established that a moisture content of 18.3% on a wet
weight basis, was the lower limit for the growth of A. flavus in
shelled corn. Extensive studies under precisely controlled conditions
(Sanders et al., 1968; Taniwaki, 2001; Davis and Diener, 1970)
established a moisture content in equilibrium with a relative humidity
of 85% (or water activity (aw) = 0.85) as the lower limit for
growth of A. flavus and for the production of aflatoxins. In starchy,
cereal grain such as wheat, oats, barley, rice, sorghum and maize, the
lower limit is a moisture content of 18.3-18.5% on a wet weight basis
and in groundnuts, Brazil nuts, other nuts, copra and sunflower and safflower
seeds, all of which have a high oil content, it is a moisture content
of 9-10%. The minimum, optimum and maximum temperatures for aflatoxin
production are 12, 27°C and 40-42°C, respectively (Davis and Diener,
1970). Northolt et al. (1976) studied the effect of water activity
and temperature on the growth and aflatoxin production of A. parasiticus
and came to the conclusion that no detectable quantities of aflatoxin
B1 were formed at an aw value below 0.83 and at
temperatures below 10°C. In studies by Strzelecki and Gasiorowska
(1974), aflatoxins occurred in 12.7% of 306 samples of animal feed and
feed components in Poland, 4.2% of the samples containing more than 100
μg kg-1 and 2.6% of the samples containing more than 1000
μg kg-1. Feed components, mainly groundnut meals, were
contaminated by aflatoxins more frequently and with higher levels. On
the other hand, aflatoxin was detected in only one sample (2.7%) of cattle
and sheep feeds (300 μg kg-1) and in one sample (1.7%)
of poultry feeds (30 μg kg-1).
Swine feeds contained aflatoxins in 11.4% of samples, with 6 samples
(5.7%) exceeding 250 μg kg-1. Two recent surveys of mixed
feeds in the Federal Republic of Germany revealed that 1 in 60 samples
contained aflatoxin B1 levels exceeding 20 μg kg-1
(Seibold and Ruch, 1977); 45 out of another 105 samples contained levels
of between 7 and 300 μg kg-1 (Kiermeier, 1977). Similar
results were obtained in the United Kingdom (Patterson, personal communication)
where, 95/172 samples of dairy feed were contaminated with aflatoxin B1
levels of 1-350 μg kg-1 and 92.4% contained no more than
30 μg kg-1.
MANAGEMENT AND CONTROL
Purchase of feeds that have been recently prepared and properly stored
is recommended. Debris must be removed from feed ingredients and grains
should be stored in clean bins or buildings. Where possible, complete
fish feeds should be stored in an air-conditioned building for temperature
and humidity control. Otherwise, feed should be stored off the ground,
on pallets and at least one foot away from any walls (to avoid condensation)
in a cool, dry area and for no longer than three months. If feed is held
in bins outside, storage for longer than two weeks is not recommended.
When feeds are stored for long periods or under poor conditions, fish
health problems may arise, not only from molds, but also from loss of
vitamins and rancidity of oils in the feed. Control of rodents and insects
is also important in maintaining nutrient quality and aflatoxin-free feeds.
Feeds that have the manufacturer`s date stamped on the bags will prevent
the purchasing of old feed. It is also a good idea to be familiar with
when the feed was bought and how the feed was being stored by the feed
supplier prior to purchasing feed.
Feeds stored for a long time and probably contaminated with molds appear
stale, are discolored lump together and smell musty. Stale foods are often
saturated with moisture and appear to sweat. Any containers that are used
to store food (bins, automatic feeders) should be cleaned thoroughly on
a regular basis to prevent mold growth on their surfaces (which may be
hidden by newly placed fresh feed).
Regular testing for aflatoxins is a good idea. Simple on-farm inspection
can be done visually (look for the presence of blue/grey mold on feed)
or with a black light which may cause a bright greenish/yellow fluorescence
if A. flavus is present. While the black light method is a rapid
procedure, it is only a potential indicator of the presence of A. flavus
and it may not work in all cases.