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Digital Re-print - January | February 2007Feature: Feed additives
Feature title: Mycotoxins in Aquaculture feeds: facts & implications
www.aquafeed.co.uk
International Aquafeed is published five times a year by Perendale Publishers Ltd of the United Kingdom.All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies,
the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis ofinformation published.Copyright 2009 Perendale Publishers L td. All rights reserved. No par t of this publication may be reproduced in any formor by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058
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26 | I nternatIonalAquAFeed | Juy-Fbuy 07 -
Mycotoxins are
s e c o n d a r y
metabolites produced
by fungi, commonly
referred as molds. They are
produced by these organisms when
they grow on agricultural products
before or after harvest or during
transportation or storage.
Most of the mycotoxins that have the
potential to reduce growth and health sta-
tus of fish and other farmed animals con-
suming contaminated feed are produced
by Aspergillus, Penicillium and Fusariumsp. These toxic substances are known to
be either carcinogenic (e.g. aflatoxin B1,
ochratoxin A, fumonisin B1), estrogenic
(zearalenone), neurotoxic (fumonisin B1),
nephrotoxic (ochratoxin), dermatotoxic (tri-
chothecenes) or immunosuppressive (afla-
toxin B1, ochratoxin A and T-2 toxin).
Mould toxins vary in their toxicity toward
different animals species and while the effect
of mycotoxins is relatively well known in
most terrestrial farm animals the effect
of mycotoxins on aquaculture species has
not been studied extensively. Nevertheless,
several studies have reported pathological
signs of mycotoxin poisoning in fish and
shrimp species which can cause economic
losses to the industry. These economic
losses can be caused either by unfavorableeffects on the animal themselves caused
by exposure to high contamination levels,
or by an increase potential for detrimental
health effects in animals consuming low or
moderate contaminated products.
Given the trend and the economical need
to replace expensive animal-derived proteins,
such as fish meal, with less expensive plant
proteins sources, the relevance of mycotoxin
contamination in aquaculture feeds have a
tendency to increase since feed ingredients
of plant origin, have higher susceptibility
for mycotoxin contamination. Mycotoxin
contamination is often an additive process,
beginning in the field and increasing during
harvest, drying, and storage. In tropical and
subtropical conditions the potential for
mycotoxin contamination is further increaseddue to storage under humid and hot conditions,
favorable for fungi contamination of stored feed
and grain (CAST, 2003).
tabe 1: occurrence, average and highes eves f mycxins deeced based n cmmdiy ype and cunry f rigin
Mycxin Sampe Size Percen Psiive Average f Highes leve Cmmdiy fund Cunry f originPsiive (g/kg) Deeced (g/kg)
Aflatoxin sTotal 965 18% 39 381 Peanut Meal AustraliaZearalenone 963 35% 409 6,468 Corn ChinaDeoxynival enol 963 45% 866 18,991 Wheat ChinaFumonisin B1 960 46% 664 10,577 Corn ChinaT-2 toxin 748 1% 273 494 Finished Feed ThailandOchratoxin A 128 18% 11.7 143 Corn Malaysia
Mycotoxins in Aquaculture feeds:
facts and implications
by Pedro Encarnao PhD
Biomin Laboratory Singapore Pte. Ltd3791 Jalan Bukit Merah #08-08
E-Center@RedhillSingapore 159471
Email: [email protected]
Feed additives
Effect of different levels of aflatoxin B1 on tilapia growth performance(Source, Dr Jowaman Khajarern)
- Juy-Fbuy 07 | InternatIonalAquAFeed | 27
A recent survey (Chin & Tan, 2006)
conducted in all Asian region analyzed 970
samples of different feed ingredients and
feed samples to determine contamination
levels of the major mycotoxins of interest;
namely, aflatoxins, zearalenone (ZON),
deoxynivalenol (DON), fumonisin (Fum), T2
toxin and ochratoxin A (OTA). In brief, from
the survey results, aflatoxins and ochratoxin
A, accounted for 18 percent of the sample
contamination; 35 percent were positive for
zearalenone, 45 percent for deoxynivalenol
and 46 percent for fumonisin B1 (Table 1).
Though it is impossible to correlate
the occurrence of a specific mycotoxin
to a specific commodity from the data
studied, there is apparent prevalence of
some mycotoxins to some specific sample
types. For instance, 100 percent of the
peanut meal samples analyzed were found
to be contaminated with aflatoxins with the
highest level of 381 g/kg and an average of
202 g/kg (Table 2). For wheat samples, 88
percent were affected by DON with the
highest level found at 18,991 g/kg and an
average contamination level of 1,181 g/kg,
while no aflatoxins were detected in any of
the wheat samples. It was seen that more
than 80 percent of the corn gluten meal
samples were ZON (87 percent) and Fum
(83 percent) positive (Chin & Tan, 2006).
A contaminated ingredient or feed is likely
to contain more than one type of mycotoxin.
Numerous researchers have reported that
mycotoxins act synergistically so that the
negative effects of two mycotoxins are
worse than the effects of each individually
(Manning, 2001). Mycotoxins also appear
to be very heat stable and the pelleting and
extrusion process of fish and shrimp feeds
do not seem to reduce appreciable amounts
of mycotoxins (Manning, 2001).
The contamination of feeds and raw
materials by mycotoxins is a reality and
its increasing on a global basis making it
increasingly likely that any given feedstuff
could contain one or, more likely, several
mycotoxins. They are invisible, odorless and
tasteless toxins with a major impact on
animal health. The awareness on the effect
of mycotoxins in terrestrial livestock is
increasing but still overviewed in aquaculture
species.
Aflatoxins
Aflatoxins are produced by Aspergillus
fungi, which can infect many potential
feedstuffs as corn, peanuts, rice, fish meal,
shrimp and meat meals (Ellis et al., 2000).
Aflatoxin B1 (AFB1) is one of the most
potent, naturally occurring, cancer-causing
agents in animals. Initial findings associated
with aflatoxicosis in fish include pale gills,
impaired blood clotting, anemia, poor growth
rates or lack of weight gain. Prolonged
feeding of low concentrations of AFB1
causes liver tumors, which appear as pale
yellow lesions and which can spread to the
kidney (Manning 2001). These subtle effects
often go unnoticed and profits are lost due
to decreased efficiency in production, such
as slow growth, reduced weights of the
finished product, an increase in the amount
of feed needed to reach market weight, and
increased medical costs.
The extent of disease, caused by
consumption of aflatoxins, depends upon
the age and species of the fish. Fry are more
susceptible to aflatoxicosis than adults and
some species of fish are more sensitive to
aflatoxins than others (Tuan et al., 2002).
Rainbow trout is reported to be one of the
most sensitive animals to aflatoxin poisoning.
In this species, an intake of 1 g AFB1/kg
diet can cause liver tumors and the LD50
(dose causing death in 50 percent of the
subjects) for AFB1 in a 50g trout being 500
1000 ppb (0.51.0 mg/kg) (Lovell, 1989). The
carcinogenic or toxic effects of aflatoxins
in fish seem to be species specific. While
Rainbow trout are extremely sensitive to
AFB1, warm water fish such as channel
catfish (Ictalurus punctatus) are reported
to be less sensitive to aflatoxins (Manning,
2001).
Although less sensitive, warm water
species are still affected by aflatoxin
contamination. Feeding a diet containing
10 ppm AFB1/kg diet to channel catfish
caused reduced growth rate and moderate
internal lesions over a 10-week trial period
(Jantrarotai & Lovell, 1990a). In carp, it
was reported that aflatoxins are potential
immunosuppressors (Sahoo et al. 2001). A
recent study (Manning et al., 2005a) indicated
that feeding diets containing aflatoxins from
moldy corn does not seem to affect channel
catfish weight gain, feed consumption, feed
efficiency, and survival. Studies on the Nile
tilapia (Oreochromis niloticus) showed
reduced growth rates when tilapias were fed
diets containing 1880 ppb AFB1 (Chavez-
Sanches et al., 1994). In addition, tissue
abnormality or lesions in the livers of these
tilapias showed the beginnings of cancer
development. In another study, Nile tilapia
fed diets with 100
ppb AFB1 for 10
weeks had reducedgrowth, and fish
fed diet with 200
ppb AFB1 had 17
percent mortality
(El-Banna et al.,
1992). In a more
recent study, Tuan
et al. (2002) showed
that acute and sub-
chronic effects of AFB1 to Nile tilapia are
unlikely if dietary concentrations are 250
ppb or less. However, diets containing levels
of AFB1 higher than 250 ppb had lower
weight gain and haematocrit count compared
to a control diet. Diets containing 100 ppm
AFB1 caused weight loss and severe hepatic
necrosis in Nile tilapia (Tuan, et al., 2002).
In marine s hrimp, several studies showed
that AFB1 can cause abnormalities such
as poor growth, low apparent digestibility,
physiological disorders and histological
changes, principally in the hepatopancreatic
tissue (Wiseman et al., 1982; Ostrowski-
Meissner, et al., 1995 Bintvihok at al.,
2003; Boonyaratpalin et al., 2001; Burgos-
Hernadez et al., 2005, Supamattaya et al.,
2006). Nevertheless, reports on the effect of
AFB1 on shrimp are inconsistent. Bintvihok
et al. (2003) reported that after just 7
or 10 days of consumption of diets with
AFB1 levels below 20 ppb, mortality rate
table 2: Prevalence of mycooxins in differen commodiies
Prevalence 1s 2nd 3rd 4h 5h 6h
Corn FUM (68%) DON (67%) ZON (40%) OTA (20%) AFLA (19%) NASoybean/Meal ZON (14%) DON(7%) FUM(7%) OTA(5%) AFLA(3%) NAWheat/bran DON (85%) ZON (24%) FUM (5%) T2(
8/14/2019 Mycotoxins in Aquaculture feeds: facts and implications
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28 | I nternatIonalAquAFeed | Juy-Fbuy 07
was slightly higher in AFB1-treated groups
than in the control group. Histopathological
findings indicated hepatopancreatic damage
by AFB1 with biochemical changes of the
haemolymph.
In another study, AFB1 at 50100 ppb
showed no effect on growth in juvenile
shrimps (Boonyaratpalin et al., 2001).
However, growth was reduced when AFB1
concentrations were elevated to 5002500ppb. Survival dropped to 26.32 percent
when 2500 ppb AFB1 was given, whereas
concentrations of 501000 ppb had no
effect on survival (Boonyaratpalin et al.,
2001). There were marked histological
changes in the hepatopancreas of shrimp
fed diet containing AFB1 at a concentration
of 1002500 ppb for 8 weeks, as noted by
atrophic changes, followed by necrosis of the
tubular epithelial cells. Severe degeneration
of hepatopancreatic tubules was common
in shrimp fed high concentrations of AFB1
(Boonyaratpalin et al., 2001). Abnormal
hepatopancreas and antennal gland tissues
were also reported by Ostrowski-Meissner, et
al., 1995 in shrimp fed 50 ppb AF B1/kg after
only 2 weeks. Feed conversion efficiency andgrowth were significantly affected at AFB1
400 ppb. Apparent digestibility coefficients
decreased significantly at AF B1 900 ppb
(Ostrowski-Meissner, et al., 1995).
According to Burgos-Hernadez et al.
(2005), the effect of AFB1 toxicity to shrimp
results in the modification of digestive
processes and abnormal development of
the hepatopancreas due to exposure to
mycotoxins. These effects might be due
to alterations of trypsin and collagenase
activities, among other factors, such as the
possible adverse effect of these mycotoxins
on other digestive enzymes (e.g. lipases
and amylases) (Burgos-Hernadez et al.,
2005). These results show that aflatoxin
contamination in shrimp feed may cause
economic losses by lowering the production
of shrimp.
OchratoxinsOchratoxins are a group of secondary
metabolites produced by fungal organisms
belonging to Aspergillus and Penicillium
genera. Ochratoxin A (OA) is the most
abundant of this group and is more toxic
than other ochratoxins. It contaminates
corn, cereal grains and oilseeds. Ochratoxin
A can adversely affect animal performance.
It primarily attacks the kidneys of affected
animals (CAST, 2003).
Very few studies have been conducted
to determine the effect of ochratoxins in
fish species. In juvenile channel catfish, diets
containing levels of 1 to 8 ppm of OA resulted
in the development of toxic responses.
Significant reduction in body weight gain
were observed after only 2 weeks in fish
fed diets containing 2 ppm of ochratoxin
A or above (Manning et al., 2003a). After
8 weeks body weight gain was significantly
reduced for fish fed diets containing 1 ppmOA or above. Additional toxic responses
included poorer FCR for fish fed diets with
4 or 8 ppm OA, and lower survival and
hematocrit count for fish fed the 8 ppm
OA diet. Severe histopathological lesions of
liver and posterior kidney were observed
after 8 weeks for catfish fed diets containing
levels of OA of 4 and 8 ppm (Manning, et
al., 2003a). In growing rainbow trout the
oral LD50 of ochratoxin
A has been determined to
be 4.67 ppm. Pathological
signs of ochratoxicosis in
trout include liver necrosis,
pale, swollen kidneys and
high mortality (Hendricks,
1994).
Cyclopiazonic acid(CPA)
Cyclopiazonic acid (CPA)
is a mould toxin produced
by several species of
Aspergillus and Penicillium
fungi. Jantrarotai and Lovell
(1990b) found that CPA, a neurotoxin
frequently found in association with aflatoxins,
was more toxic to channel catfish than
aflatoxins and is more frequently found
than aflatoxins in feedstuffs in the southern
United States. A dietary level of 100 ppb
CPA significantly reduced growth, and 10,000
ppb caused necrosis of gastric glands. The
minimum dietary concentration that caused
a reduction in growth rate was 100 ppb for
CPA as compared with 10,000 ppb for AFB1
(Jantrarotai and Lovell, 1990b).
FumonisinsThe fumonisins represent a group of
mycotoxins produced predominantly by
Fusarium moniliforme species. Fumonisin
B1 has been found to be the major toxic
component both in corn culture and in
naturally contaminated corn. Some early
investigations associated this toxin with
a variety of animal diseases. Fumonisins
specifically disrupt sphingolipid metabolism
(Wang et al., 1992). Administration of feed
contaminated with F. moniliforme culture
material was related to certain changes in
some hematological parameters and serum
or plasma chemical concentration and
activities in many animal models (Pepeljnjak
et al., 2002).
The importance of fumonisins as
toxic agents in fish remains still poorly
understood. In one study, channel catfish fed
F. moniliforme culture material containing313 ppm of fumonisin B1 (FB1) for 5 weeks
revealed minimal adverse effects (Brown et
al., 1994). Conversely, Lumlertdacha et al.
(1995) reported that dietary levels of FB1
of 20 ppm or above are toxic to year-1
and year-2 channel catfish. After 10 and
14 weeks, respectively, year-1 and year-
2 catfish fed 20 ppm or more of FB1 in
the diet had lower weight gain compared
to the control, and those fish fed diets
with levels of 80 ppm and above showed
significantly lower hematocrits and red
and white blood cells than those fed
lower doses (Lumlertdacha et al., 1995).
Similarly, Yildirim et al. (2000) found that in
channel catfish, diets containing 20 ppm of
moniliformin (MON) or FB1 significantly
reduced body weight gain after 2 weeks.
According to Yildirim et al. (2000), FB1 is
more toxic than MON to channel catfish.
Adverse effects of fumonisin contaminated
diets have also been reported in tilapia.
Results presented by Tuan et al. (2003)
demonstrated that feeding MON and FB1
at 70 and 40 ppm, respectively, adversely
affected growth performance of Nile
tilapia fingerlings. FB1 is slightly more toxic
than MON to tilapia fingerlings as toxic
symptoms appear earlier in fish exposed
to FB1. Nevertheless, neither MON nor
FB1 caused mortality or histopathological
lesions in Nile tilapia fingerlings. Compared
Feed additives
Hemorrhagic liver affected byaflatoxin B1 contamination
(Source, Dr Jowaman Khajarern)
-
to channel catfish, Nile tilapia appears to be
more resistant to these two mycotoxins in
the diet (Tuan et al., 2003).
Although research studies revealed that
FB1 is toxic to tilapia and channel catfish
by suppressing growth and/or causing
histopathological lesions, this fish survived
mycotoxins levels up to 150 ppm. Reduction
on the percentage of survival of channel
catfish was observed for diets containing240 ppm FB1 (Li et al., 1994). Studies on the
effect of FB1 in carp indicated that long-term
exposure to 0.5 and 5.0 mg per kg body
weight is not lethal to young carp, but can
produce adverse physiological effects. The
primary target organs of FB1 in the carp
are kidney and liver (Pepeljnjak et al., 2002).
Other changes subsequent to fumonisin
exposure that have been reported for carp
include scattered lesions in the exocrine and
endocrine pancreas, and inter-renal tissue,
probably due to ischemia and/or increased
endothelial permeability (Petrinec et al.,
2004).
TrichothecenesTrichothecenes are a group of mycotoxins
produced by certain fungi of the genus
Fusarium that infect the grains, wheat by-
products and oilseed meals used in the
production of animal feeds. The type A-
trichothecene T2-toxin produced by the
fungus Fusarium tricintum proved lethal to
rainbow trout at a dietary concentration
near 6 mg/kg body weight (Marasas et al.,
1967). Poston et al. (1983), however, fed
rainbow trout T2-toxin at 15 ppm of diet and
found that the main effects were reduced
feed consumption, reduced growth, lower
hematocrit, and lower blood hemoglobin.
Results from Manning et al. (2003b)
demonstrated that T2-toxin is toxic to
juvenile channel catfish. Reductions in growth
rate were observed after 8 weeks for fish fed
diets containing levels of T2-toxin ranging
from 0.625-5.0 ppm, compared to a control
diet. Significantly poorer feed conversion
ratio was found only for the highest level
of T2-toxin (5 ppm). The survival of fish fed
T2-toxin at 2.5 and 5 ppm was significantly
lower than that of the control fish (Manning
et al., 2003b).
A recent study with channel catfishindicate that disease resistance of juvenile
channel catfish was reduced when fed
feedborne T-2 t oxin, resulting in significantly
greater mortality when challenged with
Edwardsiella ictaluri compared to a control
group (Manning et al., 2005b). In carp, the
injection of T-2 toxin did not significantly
change the activity of enzymes in carp liver,
although a tendency for reduction was noted
(Kravchenko et al., 1989).
In shrimp, Supamattaya et al. (2006)
reported that in white shrimp growth was
significantly reduced by T-2 toxin at 0.1 ppm
while for black tiger shrimp reduced growth
was observed at levels of 2.0 ppm. The
presence of T-2 toxin at 1.0-2.0 ppm produced
atrophic changes and s evere degeneration of
hepatopancreas tissue, inflamation and loose
contact of hemopoietic tissue and lymphoid
organ on black tiger and white shrimp after
feeding for 10 weeks and 8 week respectively.
The same pathology was
found in shrimp received
1.0 ppm zearalenone
(Supamattaya et al., 2006).
It was concluded by the
authors that white tiger
shrimp are more sensitive
to mycotoxins then black
tiger shrimp.
Deoxynivalenol (DON),
also known as vomitoxin, and
other type B trichothecenes
are produced by Fusarium
sp. and can be an importantcontaminant of wheat.
Deoxynivalenol levels of
0.2, 0.5, and 1.0 ppm in t he diet significantly
reduced body weight and growth rate in
white shrimp Litopenaeus vannamei (Trigo-
Stockli et al., 2000). However, the effects of
0.2 and 0.5 ppm DON were manifested at
later stages of growth, and 0.2 ppm DON
affected only growth rate and not body
weight. Feed conversion ratio and survival of
shrimp fed diets containing 0.2, 0.5, and 1.0
ppm DON were not significantly different
from those of shrimp fed the control diet
(0.0 ppm DON) (Trigo-Stockli et al., 2000).
Reduced weight gain has also been noted
in rainbow trout fed DON-contaminated
feeds and feed refusal has been found to
occur in fish fed with diets containing more
than 20 ppm DON. For rainbow trout,
a dietary level of 112.9 ppm resulted
in reduced growth and feed efficiency
(Hendricks, 1994). Woodward et al. (1983)
showed that rainbow trout had sensitive
taste acuity for DON and reduced theirfeed intake as the concentration of DON
increased from 1 to 13 ppm of diet; the fish
refused to consume the diet with a DON
concentration of 20 ppm.
Combating mycotoxinsAlthough mycotoxin contamination of
feed and feed ingredients represent an
increase threat to aquaculture operations
there are a number of options available to
feed manufacturers and farmers to prevent or
reduce the risk of mycotoxicosis associated
with mycotoxin contamination. These range
from careful selection of raw materials,
maintaining good storage conditions for
feeds and raw materials, and using a good
mycotoxin deactivator to combat the widest
possible range of different mycotoxins thatmay be present.
Binders or adsorbents have been used
to neutralize the effects of mycotoxins
by preventing their absorption from the
animals digestive tract. The most common
binders are clays, bentonites, zeolites silicas
and alumino silicates. Unfortunately, different
mycotoxin groups are completely different
in their chemical structure and therefore
it is impossible to equally deactivate all
mycotoxins by using only one single strategy.
Adsorption works perfectly for aflatoxin
but less- or non-adsorbable mycotoxins
(like ochratoxins, zearalenone and the
whole group of trichothecenes) have to be
deactivated by using a different approach.
MycofixPlus is a mycotoxin deactivatorwhich combines adsorption and bio-
inactivation to break functional groups
of mycotoxins such as trichothecenes,
ochratoxin A and zearalenone, and also
immunostimulation with addition of
selected plant extracts. Biotransformation
is defined as detoxification of mycotoxins
using microorganisms or enzymes which
specifically degrade the toxic structures
to non-toxic metabolites. MycofixPlus
combines different microorganisms, live
bacteria and yeast strains, expressing
specific mycotoxin-degrading enzymes to
successfully counteract all agriculturally
Feed additives
Mold contaminated corn
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relevant mycotoxins in a biological way.
BBSH 797, a Eubacterium species, patented
by Biomin, produces enzymes, so-called de-
epoxidases, which degrade the toxic epoxide
ring of trichothecenes, T. mycotoxinivorans
(vorans lat. degrade, eat), a yeast strain,
successfully counteracts ochratoxin A and
zearalenone by enzymatic cleavage.
Furthermore, all mycotoxins are known
to influence detrimentally toward the liverand cause immunosuppression in animals.
The addition of plant and algae extracts to
the animals diet helps to overcome these
negative influences. Special algae extracts,
tested on their immune enhancing effect,
support the immune system and thus
overcome the immunesuppressive effect of
all mycotoxins. The liver, the main target
organ of mycotoxins, is protected by selected
antiphlogistic plant extracts.
References
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Problem of mycotoxins in fish production. Egyptian
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Bintvihok, A., Ponpornpisit, A., Tangtrongpiros, J.,
Panichkriangkrai, W., Rattanapanee, R., Doi, K.,Kumagai, S., (2003). Aflotoxin contamination in
Shrimp feed and effects of aflotoxin addition to feed
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Boonyaratpalin, M., Supamattaya, K., Verakunpiriya,
V., Suprasert, D., (2001). Effects of aflotoxin B1 on
growth performance, blood components, immune
function and histopatological changes in black tiger
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