DOI: https://doi.org/10.52973/rcfcv-e32090
Received: 24/07/2021 Accepted: 20/10/2021 Published: 06/04/2022
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Revista Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32090, 1 - 9
ABSTRACT
Mycotoxins, even so government regulates to control their levels in
animal balance feeds, they could pose a problem to animal and human
consumption health. Therefore, the aim was to determine the levels
of total aatoxins found in the nisher balanced feeds for broilers and
pigs, produced by balanced feed factories (BFF) in Venezuela. Eleven
BFFs were sampled and evaluated for the presence of aflatoxins:
AFB1, AFB2, AFG1 and AFG2, determined using the analytical chemical
technique HPLC-FLD. Thirty three feed samples for broilers and 33 for
pigs, a total of 66 samples were obtained from BFFs. The presence
of aatoxins was detected in 100% of the nisher balanced feeds
for poultry and pigs. The determined AFB1, AFB2, AFG1 and AFG2
levels in nisher balanced feeds for broilers and pigs, rendered that
the aatoxin with the highest concentration was always AFB1. The
averages of total aatoxins detected from nisher feeds for broilers
and pigs were within the maximum allowed limits (20 micrograms
(µg)/kilograms (kg) of COVENIN standards (Venezuelan regulations).
When compared each aatoxin level (AFB1, AFB2, AFG1 and AFG2)
eight out of eleven BFFs evaluated comply with the standard, but 3
(F1, F2 and F5) BFFs were outside (5 µg/kg). It is pertinent that the
companies that produce final food for poultry and pigs carry out
additional and frequent evaluations of other mycotoxins (Ocratoxins,
fusarium mycotoxins and mycotoxins emerging) present in raw
materials, in order to evaluate the risks in the agri-food chain, in the
way to implement solutions before or after processesment to ensure
public health quality.
Key words: Aatoxins; nisher feeds; broilers; pig; HPLC
RESUMEN
Las micotoxinas, aun cuando son reguladas por el Estado Venezolano
para controlar sus niveles en los alimentos balanceados, podrían
representar un problema para la salud del consumo animal y humano.
El objetivo de la investigación fue determinar los niveles de aatoxinas
totales que se encuentran en los alimentos balanceados nalizadores
para pollos de engorde y cerdos, elaborados por plantas productoras
de alimento balanceado (PPAB) en Venezuela; se muestrearon once
PPAB. Se evaluó la presencia de niveles de aatoxinas: AFB1, AFB2,
AFG1 y AFG2, utilizando la técnica química analítica HPLC – FLD. Con
33 muestras de alimentos para pollos de engorde y 33 para cerdos,
para un total de 66 muestras. Se detectó la presencia de aatoxinas
en el 100% de los alimentos balanceados finalizadores para aves
y cerdos evaluados. Los niveles de AFB1, AFB2, AFG1 y AFG2, en
alimentos balanceados nalizadores para pollos de engorde y cerdos,
se encontró que la aflatoxina en mayor concentración resultó ser
siempre la B1. Los promedios de aatoxinas totales detectadas, de
alimentos nalizadores para pollos de engorde y cerdos estuvieron
dentro de los límites máximos (20 microgramos (µg) / kilogramos
(kg)) permitidos (normas COVENIN). Al realizar la comparación entre
niveles de aflatoxinas (AFB1, AFB2, AFG1 y AFG2), ocho de once
PPAB evaluadas cumplen con la norma, y 3 (F1, F3 y F5) están fuera
de ella (5 µg/kg). Es pertinente que las empresas productoras de
alimentos nalizadores para aves y cerdos realicen evaluaciones de
otras micotoxinas (ocratoxinas, fusarium micotoxinas y micotoxinas
emergentes) presentes en las materias primas y evaluar los riesgos
en la cadena agroalimentaria, para así implementar soluciones antes
y después del procesamiento para garantizar la calidad de la salud
pública.
Palabras clave: Aflatoxinas; alimentos finalizadores; pollo de
engorde; cerdos; HPLC
Aatoxin levels in commercial nisher balanced feeds for broilers and pigs from
Venezuelan factories
Niveles de Aatoxinas en alimentos nalizadores para pollos de engorde y cerdos procedentes de
fábricas de alimentos balanceados comerciales en Venezuela
Ebbis Fernández
1
, José Gregorio Riera-Betancourt
2
, Elena Del Carmen Briceño-Fereira
3
, Simon Gabriel Comerma-Steffensen
3,4
,
Eduard Martínez-Camacaro
5,6
y Darwuin Arrieta-Mendoza
3,7
1
Research and Development Management of ALCONCA farm. Santa Cruz de Aragua, Aragua, Venezuela.
2
SEDICOMVET Laboratory. Maracay, Aragua, Venezuela.
3
Central University of Venezuela, Faculty of Veterinary, Department of Biomedical Sciences, Aragua, Venezuela.
4
Aarhus University, Department of Biomedicine,
Pulmonary and Cardiovascular Pharmacology. Aarhus, Denmark.
5
Centroccidental University “Lisandro Alvarado, Deanery of Veterinary Sciences, Barquisimeto,
Lara, Venezuela.
6
University of Chile, Master student of the Animal Sciences and Veterinary program. Santiago, Chile.
7
University of Chile, PhD student of
Silvoagropecuarian and Veterinary Sciences Program. Santiago, Chile. Email: darwuin@yahoo.com
Aatoxins in nisher balanced feeds for broilers and pigs / Fernandez et al.___________________________________________________________
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INTRODUCTION
According to the Food and Agriculture Organization of the
United Nations (FAO) [33], up to 25% of food crops worldwide are
contaminated with a type of mycotoxin. Among them, aatoxins have
been reported, and dened as secondary metabolites produced by
strains of fungi of the genus Aspergillus, being generally aflatoxin
B1, a highly hepatotoxic compound in poultry and pigs production,
as carcinogenic in pigs and humans [19, 25, 34, 42]. They are
frequently detected in seeds or vegetable raw materials [16, 22],
are thermostable and the pelletization process of animal feed will
not destroy them, if made with contaminated raw materials [44].
Therefore, presence of mycotoxins in food is considered a safety
feeding issue, and only maximum limits are allowed locally in Venezuela
[13, 14] or internationally [18].
Venezuela as a tropical Country is not protected from these types
of mycotoxins and their toxic effects. They are present in different
raw materials used in the production of balanced foods [9, 11, 16,
26], where manufacturing factories (F) located mainly at the central
region of the Country, within the States of Aragua, Carabobo and
Cojedes, as the Western State of Zulia, constitute the main poultry
producers. The previous, with the North of Guárico and Miranda
States have the highest intensive production of pigs (Sus scrofa
domestica)[1, 35].
In view of high production of balanced feed and due to its great
demand within the poultry (Gallus gallus domesticus) or pig production
sector, an attempt has been made to protect human and animal
health from the harmful and agro-economic effects of aatoxins [6,
25, 28, 40, 47]. Agrovigilance organizations in Venezuela through the
COVENIN Standards [14], established that total aatoxins or other
contaminating substances should not be in levels higher than 20 µg/
kg food formulations [14, 36]. However, in Venezuela there is limited
scientic information regarding the amount of aatoxins that could
be present in the balanced feed consumed by pigs and broilers at
their nishing stage. To them being beneted at the agri-food chain,
which represents a public health risk and threatens the safety of these
food products, by not being certain of the contamination levels of this
mycotoxin in these type of food formulations.
As previously described, the research purpose intend to determine
the levels of total aatoxins (AFB1, AFB2, AFG1, AFG2), which are
found in the nisher balanced feeds for broilers and pigs, processed
and marketed by feed-producing F in Venezuela.
MATERIALS AND METHODS
Locations
The collection and assessment of samples were carried out in
a laboratory specialized in toxicology, located in Maracay, Aragua
State, Venezuela (Latitude 10°15’06” N | Longitude 67°36’05” O). The
environmental conditions that the laboratory had were: average
temperatures of 30°C with an average relative humidity of 40%,
of course all analysis were performed over enviromental controlled
conditions.
Methodologic approach
The trial period was 6 months, from January to June 2010,
where 3 randomize samples of nisher feed for broilers and 3 for
pigs in the finishing stage were collected and evaluated at the
laboratory, procedent from each factory (F). A total of 11 Balanced
Food Factories (BFF) were subjected to assessment. Therefore, 33
BFF samples were generated for broilers and 33 for pigs, a total of
sixty-six (66) samples. As they were arriving, the aatoxin extraction
method, AOAC 2005-991 [7], was carried out in order to preserve
the extracted aflatoxin under refrigerated (Revco Scientific. Inc.
ULT 1386-3-A14, EUA) conditions (4°C) for later use in the following
detection technique, AOAC 2005.08-2005 [8].
In all cases, the quality control personnel of the companies or F
carried out the primary and random sampling, directly from their
production process, according to COVENIN 1567:80 standard animal
feed, sampled method refered before [45]. These samples were
homogenized and subsampled until obtaining a 1 kg sample, seeking
to obtain a representative subsample of the sampling. Samples were
delivered to the specialized toxicology laboratory that would carry
out the collection and evaluation of samples.
Sample procedure for samples total aatoxin determination
Chromatographic techniques were used for the extraction of
aatoxins, the ocial method AOAC 2005, 991.31 was applied [7]. A
25 grams (g) sample was placed in a blender, with 5 g of NaCl plus 125
mililiters (mL) of methanol: water (7:3). Mixed at high speed (mixer
Vortex, MIX, Argolab, Spain) for 2 minutes (min). Subsequently, was
ltered with 24 centimeters (cm) Whatman #2 paper. A 15 mL of
the ltered solution was pipetted into a 125 mL balloon. Then, 30
mL of water were added, mixed and ltered with GF/A 9.0cm glass
microfiber paper. Following, a 15 mL of the second filtrate was
transferred through the immunoanity column [28], washed with
10 mL of distilled water. Finally, the aatoxin was extracted with 2.0
mL of High Performance Liquid Chromatography (HPLC) extracted
methanol and placed in a 2.0 mL vial.
To determine aflatoxin levels, the official AOAC 2005, 2005.08
method was used [8], in a reversed phase HPLC chromatograph
coupled to a model 2475 uorescence detector (HPLC-FLD Agilent
1100, Hewlett Packard, USA). The method is based on the fact that the
aatoxins B1, B2, G1 and G2 show natural uorescence, although those
type B1 and G1 have little uorescence, therefore, an attached Kobra
Cell post-column electrochemical cell a (KOBRA® CELL R-BIOPHARM
RHONE Ltd, EUA), to HPLC was used, where the sample reacted with
an derivatizer agent (potassium bromide salt), so that aatoxins B1 and
G1 were derivatized to their bromide derivatives, which gave enhanced
uorescence. It should be noted that the use of the Kobra Cell, apart
from signicantly increasing the uorescence of aatoxins B1 and G1,
allows the detection and quantication of less than 0.5 µg/kg for each
of the four individual toxins [43].
The eluate obtained was evaporated and reconstituted with 2.0mL of
mobile phase. With a ow of 1.0 mL/min of mobile phase, 20 microliters
(µL) of standard and sample were injected. For the determination of the
detection limit and quantication of the HPLC-FLD technique, 20 µL of
total aatoxin standard was injected at different concentrations: 1.0,
1.25, 1.5, 10 and 20 µg/kg. It should be noted that these indicators were
used in order to determine and/or verify the reliability of measurements
obtained from the aatoxins levels. The previous was according to the
HPLC-FLD-electrochemical cell method, which has a detection and
quantication limit that is less than 2 µg/kg of total aatoxin or less
than 0.5 µg/kg for each individual aatoxin (B1, B2, G1, G2). The detection
limits: 1 µg/kg of total aatoxin, and quantication: 1.25µg/kg
of total
aatoxin of the HPLC-FLD technique were established.
________________________________________________________________________Revista Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32090, 1 - 9
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According to the values obtained for the detection limit, the minimum
reliably detectable quantity was 1 µg/kg of total aflatoxin, which is
less than 0.5 µg/kg (0.25 µg/kg) when the four individual toxins are
accounted for, it was possible to verify the validity of the applied
technique, when evaluated foods were analyzed, since it matches
with those declared by the methodology (less than 0.5 µg/kg for
each of the four individual toxins). In the same way, it was fullled for
the quantication limit, where the minimum amount of the analyte
quantiable was 1.25 µg/kg of total aatoxin (0.31 µg/kg for each of
the four individual toxins). The validity of the HPLC-FLD technique
could be veried by evaluating aatoxins in feeding products for
broilers and pigs at the nalizing production stage.
Statistical analysis
Descriptive statistics was used to characterize the variables under
study. Likewise, the experiment was carried out under a randomized
design. Model Type 1: Balanced Fixed Unifactorial Effects. The basic
assumptions were tested and unviable for the variables under study,
therefore the non-parametric test of Kruskal Wallis by ranks under a
one-way analysis of variance [38] was performed and with a degree
of signicance equal to 0.05. Evaluation was applied to each type of
feed: poultry and pigs and its data were analyzed with the statistical
package Statistix Vers. 8.0.
RESULTS AND DISCUSSION
Eleven Venezuelan food F were evaluated, putting emphasis that the
maximum allowed limit for total aatoxin is 20 µg/kg [14] and 5 µg/kg
[13] for each type of aatoxin (AFB1, AFB2, AFG1 and AFG2), established
by COVENIN standards (Venezuelan Standards), which allowed the
comparison of obtained results.
Additionally, it should be appointed that COVENIN regulation it is
currently at revision and to get publish with modication although
it is not available, nevertheless there should be point that most of
the feeding factories investigated are following current guidelines
of animal feeding [14]. F have their own internal quality control of
the production proccess over primary cereal sources, with stronger
level restrictions, such as, the established at the food for human
consumption in Venezuela [13].
The total aatoxins average concentration for each F under study
did not exceed the maximum allowed limit, although aatoxins were
present in all samples obtained. The results from the aatoxin levels
were the following: AFB1, AFB2, AFG1 and AFG2, which were present
in nisher balanced feeds for broilers and pigs. Aatoxin levels and
statistic evaluations were shown at TABLES I-VI.
In TABLE I, all eleven F presented Aatoxin B1, being the most toxic
of this group. Where AFB1 had an average value of 2.95 µg/kg among
the 11 F, with a mean standard error of 0.86, the lowest value of 0.31
µg/kg and the higher of 9.15 µg/kg, followed by aatoxin B2 and G1. The
lowest concentration of aatoxin was on G2, with an average value
of 0.66 µg/kg, the lowest of 0 µg/kg and the highest of 1.83 µg/kg. On
the other hand, it should be noted that the F with the highest level of
aatoxins B1 was F1, with a value of 9.15 µg/kg.
At analysis of these results, aatoxin levels: AFB1, AFB2, AFG1 and
AFG2 in the evaluated feeds samplings, AFB1 was present at all samples,
where two evaluated F (F1 = 9.15 µg/kg and F3 = 6.97 µg/kg) were outside
the maximum allowed limits (5 µg/kg), within a total of 18.18%.
TABLE I
Results of the descriptive statistics for 11 Venezuelan balanced
feed factories, when evaluating Aatoxin levels: AFB1, AFB2,
AFG1 and AFG2, present in nisher balanced feeds for Broilers
FACTORY
Aatoxin levels (µg/kg)
B1 B2 G1 G2
F1 9.15 5.28 2.57 0
F2 1.82 3.80 2.35 1.83
F3 6.97 1.68 1.63 1.30
F4 4.19 1.70 0.31 0.31
F5 0.78 1.58 0.48 0.31
F6 0.63 0.77 0.00 0.00
F7 0.31 0 0 0
F8 1.00 0.42 0.47 0.32
F9 3.98 2.67 0.52 0
F10 1.85 1.75 1.83 1.75
F11 1.78 1.57 1.92 1.45
Average 2.95 1.93 1.10 0.66
SEM 0.86 0.46 0.29 0.23
Lowest 0.31 0 0 0
Higher 9.15 5.28 2.57 1.83
SEM: Standard Error Mean
It should be noted that F1 also is outside maximum limits allowed in
AFB2 (F1 = 5.28 µg/kg), corresponding to 9.09% of the total. Although
the aforementioned F were outside the limit established by the COVENIN
standard, the average of the eleven F did not exceed 5 µg/kg for each
type of aatoxin.
The previous data conrms that it was possible to obtain the total
aatoxin levels for each F under study, presented in TABLE II, which
describes the statistics of the total aflatoxin levels found in the
nisher balanced feed samples for broilers.
TABLE II, in the case of balanced feeds for broilers in the nishing
stage, it can be noted that all the reported evaluated feeds had
presence of total aatoxins. Finding as an average level in eleven F
evaluated the amount of 6.65 µg/kg, with an average standard error
of 2.28 µg/kg, a minimum value of zero (0) µg/kg and a maximum
value of 29.07 µg/kg. In turn, it can be observed that the F that
reports the highest average concentration of total aatoxins is F1
with 17.0 µg/kg, followed by F3 (11.58 µg/kg) and F2 (9.8 µg/kg). The
lowest level of total aatoxins corresponded to F7 (0.37 µg/kg).
The Kruskal-Wallis test was signicant (P<0.05), when compared
concentrations of total aatoxins in the eleven studied F. The test
of means reects in TABLE III that F1 to F2 were homogeneous, F10
to F5 were homogeneous with each other. The F8 and F6 were also
homogeneous, unlike the F1 it is different from the F7.
Aatoxins in nisher balanced feeds for broilers and pigs / Fernandez et al.___________________________________________________________
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TABLE II
Results of the descriptive statistics when evaluating the levels
of total Aatoxin in samples of nisher balanced feeds for
Broilers from eleven factories of balanced feeds in Venezuela
FACTORY
Average
concentrations
of total aatoxin
(µg/kg)
SEM
Lower
(µg/kg)
Higher
(µg/kg)
F1 17.00 6.58 6.43 29.07
F2 9.80 3.58 4.60 16.65
F3 11.58 4.27 3.36 17.70
F4 6.51 2.46 1.60 9.92
F5 3.15 1.05 1.83 5.23
F6 1.4 0.74 0 2.40
F7 0.37 0.23 0 0.80
F8 2.21 1.27 0.93 4.75
F9 7.17 3.54 0.65 12.84
F10 7.18 0.26 6.85 7.70
F11 6.72 1.07 5.10 8.75
Average 6.65 2.28
Lower 0.37 0
Higher 17.00 29.07
SEM: Standard Error Mean
TABLE III
Range means test obtained when evaluating total Aatoxin
levels in samples of nisher balanced feeds for Broilers from
eleven feed factories in Venezuela
FACTORY
Average concentrations of total
aatoxin (µg/kg)
F1 17.00
a
± 0.58
F3 11.58
ab
± 4.27
F2 9.80
abc
± 3.58
F10 7.18
bcd
± 0.26
F9 7.17
bcd
± 3.54
F11 6.72
bcd
± 1.07
F4 6.51
bcd
± 2.46
F5 3.15
bcd
± 1.05
F8 2.21
cd
± 1.27
F6 1.40
cd
± 0.74
F7 0.37
d
± 0.23
Dierent letter within the same column denote signicant dierences
= a - d. n = 3, P<0.05, Standard Error Mean to compare = 4.2098
The data in TABLE IV reects that all the eleven F feeds present all
types of aatoxins (AFB1, AFB2, AFG1 and AFG2). Where the highest
level in AFB1, with an average value of 3.45 µg/kg, a mean standard
error of 1.0, a minimum value of 0.58 µg/kg and a maximum value
of 10.53 µg/kg, followed by AFB2 and AFG2. The lowest average
concentration was AFG1 with 1.3 µg/kg.
TABLE IV
Results of the descriptive statistics by balanced feed processing
factories, at evaluated levels of Aatoxins: AFB1, AFB2, AFG1 AND
AFG2, in nisher feeds for Pigs in Venezuela
FACTORY
Aatoxin level (µg/kg)
B1 B2 G1 G2
F1 9.19 2.93 0.65 0
F2 1.68 2.93 1.59 0.87
F3 10.53 2.87 1.60 1.57
F4 3.98 0.31 0 0
F5 2.81 5.63 0.42 0.45
F6 0.69 0.95 0 0
F7 1.6 1.68 1.48 1.47
F8 0.58 2.72 2.82 3.49
F9 1.97 2.50 2.05 2.13
F10 1.88 1.87 2.00 3.27
F11 3 3.27 2.17 2.31
Average 3.45 2.51 1.34 1.41
SEM 1 0.42 0.28 0.39
Lower 0.58 0.31 0 0
Higher 10.53 5.63 2.82 3.49
SEM: Standard Error Mean
TABLE V presents the total aatoxin levels in pig nishers feed for
each F sampled, it can be seen that all the evaluated feeds samples
reported on average the presence of total aflatoxins. Finding as
the average level of aflatoxins in the total F a value of 8.72 µg/kg,
associated with a mean standard error of 1.92 with a minimum level
of 0.8 µg/kg and a maximum level of 27.2 µg/kg.
Similarly, it can be seen that the F that reports the highest total
aflatoxin level is F3 (16.57 µg/kg), followed by F1 (12.77 µg/kg).
Meanwhile, the F with the lowest level of aatoxins is F6 (1.64 µg/kg).
The Kruskal-Wallis test was not signicant (P>0.05) when comparing
the concentrations of total aatoxins in the eleven F investigated. The
results of the range means tests were reected in TABLE VI, indicating
that there were no significant differences between the eleven F,
although F3 is different from F6 (P<0.05).
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TABLE V
Results of the descriptive statistics to evaluate the levels of
Total Aatoxin, in samples of balanced feeds for Pigs in the
nishing stage, from eleven feed factories in Venezuela
FACTORY
Average
concentrations
of total aatoxin
(µg/kg)
SEM
Lower
(µg/kg)
Higher
(µg/kg)
F1 12.77 7.21 5.39 27.20
F2 7.07 1.25 4.76 9.06
F3 16.57 2.55 12.00 20.80
F4 4.29 3.28 0.80 11.27
F5 9.31 0.76 7.90 10.52
F6 1.64 0.44 2.10 2.60
F7 6.23 0.24 5.95 6.70
F8 9.61 1.55 7.02 12.40
F9 8.65 0.55 7.55 9.20
F10 9.02 1.17 7.45 11.30
F11 10.75 2.63 7.72 15.95
Average 8.72 1.92
Lower 1.64 0.80
Higher 16.57 27.2
SEM: Standard Error Mean
TABLE VI
Range means test obtained when evaluating total Aatoxin
levels in balanced feed samples for Pigs in the nishing stage
and from eleven feed factories in Venezuela
FACTORY
Average concentration of total
aatoxin (µg/kg)
F3 16.57
a
± 2.55
F1 12.77
ab
± 7.21
F11 10.75
ab
± 2.63
F8 9.61
ab
± 1.55
F5 9.31
ab
± 0.76
F10 9.02
ab
± 1.17
F9 8.65
ab
± 0.55
F2 7.07
ab
± 1.25
F7 6.23
ab
± 0.24
F4 4.29
ab
± 3.28
F6 1.64
b
± 0.44
Dierent letter within the same column denote signicant dierences
= a - d. n = 3, P<0.05, Standard Error Mean to compare = 3.8587
Comparison with the maximum allowed limits (COVENIN standard)
and total aflatoxin levels, detected in finisher feed for broilers (33
samples) and finisher feed for pigs (33 samples), were shown in
TABLES VII and VIII.
TABLE VII shows that there is no important difference in the
results of nisher feeds for broilers compared to pigs. Both previous
cases present 18.18% of positive samples (above 5 µg/kg), in AFB1
compared to the total (eleven F evaluated) and 9.09% in AFB2. The
levels of AFG1 and AFG2 were kept below 5 µg/kg (maximum limit
allowed by COVENIN), for this reason the average and the percentage
of positive samples with concentration above the maximum allowed
level were not assigned in TABLE VII.
TABLE VIII shows that the balanced feeds for finisher pigs, from
eleven F evaluated presented slightly higher levels of total aatoxin
(8.72 ± 1.92 µg/kg) than the balanced feed for broilers (6.65 ± 2.28µg/
kg), and that both types of feeding do not exceed the maximum limits
established by the National standard in their average total aatoxins.
On the other hand, it should be noted that the F with the highest
level of AFB1 (with higher toxicity of the group), is F3 (10.53 µg/kg),
followed by F1 (9.19 µg/kg), these were equivalent at 18.18% of samples
outside the maximum allowed limits (5 µg/kg). The one with the highest
concentration in AFB2 is in F5 (5.63 µg/kg), equivalent to 9.09%.
However, when observing the maximum concentration values, it is
distinguished that they are higher than the maximum allowed levels, in
nisher feeds for broilers and pigs (TABLE VIII) in F1 samples. Likewise,
in the detected values, 2 of the F in the case of broilers (F1 and F3),
and three in the case of pigs feeding (F1, F3 and F5), are outside the
limits established by the Venezuelan standard (COVENIN) by its levels
in AFB1 and AFB2 (5 µg/kg).
These results can be attributed to the inadequate handling of raw
materials or the poor control of biotic and abiotic factors that favor
the colonization of cereals by aatoxigenic strains, such the followed
conditions: humidity, temperature, oxygen and the constitution of
the substrate at the time of drying and/or the storage of cereal raw
materials, regional socioeconomic condition, among other intervening
variables that are described by several authors [6, 9, 11, 26, 28, 44].
Without forgetting that the random distribution of mycotoxins affects in
this sense, even if all F buy or are sourced from the same supplier [27].
However, the data from the present research also indicates that
nine of the Venezuelan BFF sampled are carrying out quality controls,
both for raw materials and food, in accordance with national and
international regulations. Possibly emphasizing the good selection
of raw materials. One could even assume the quality of the genetic
content that they possess. In this regard, it should be mentioned that
the Venezuelan BFFs, in order to meet the demand for balanced food
production, they must consider current regulations, the little or no
availability of some raw materials for agroclimatic or socioeconomic
reasons, among others. Having to resort to importation, as is the
case for corn (Zea mays) and soybeans (Glycine max), so they do not
escape the use of transgenic cereals and oilseeds, in the production
of their products [10, 28, 48].
It has been shown that in Bt maize hybrids, there is a reduction of
the vulnerability of grains to fungi that produce mycotoxins compared
to non-transformed hybrids, especially for the control of maize
spoilage caused by Aspergillus and Fusarium genus [37, 48], perhaps
decreasing the aatoxigenic strains of fungi in cereal crops. However,
at the time of sampling in this research, there was no legislation on
Aatoxins in nisher balanced feeds for broilers and pigs / Fernandez et al.___________________________________________________________
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Additionally, experiments in broilers that received levels of 20 µg/
kg of aatoxin B1 in their diet for 5 weeks, suggested a tendency
for the duodenum of these broilers to be more susceptible to
infection by Eimeria acervulina [29]. A prolonged exposure with
low concentrations of aflatoxins in their diet causes important
immunosuppressive effects, characterized by the unexpected
appearance of infectious and parasitic diseases, in addition the
animals do not respond adequately to vaccination programs [32, 41],
these immunotoxic effects will increase susceptibility to infectious
and contagious diseases, with low concentrations of aatoxins in the
diet have also been described in pigs [39, 40, 42, 47].
Other reports, internationally, there was an analysis (chromatographic
methods) of the natural presence of mycotoxins (enniatins, beauvericin,
ochratoxin A, aflatoxins, alternariol monomethyl ether, alternariol,
tentoxin, zearalenone, deoxynivalenol, 3-acetyldeoxynivalenol,
15-acetyl-2 and HT-2), from 122 samples of balanced feeds marketed
in Tunisia, which were destined for poultry (n = 43), found that poultry
feeds were among the most contaminated by other mycotoxins, levels
of aatoxins that did not exceded the established limits by the European
Union in animal feed [24]. Likewise, to evaluate (analytical chemical
methods) mycotoxin contamination in Nigeria, 102 samples of balanced
feed (n = 30) and their raw materials (n = 72) were collected, from poultry
farm mills in 12 states, where the most common mycotoxin in balanced
feed was fumonisin B1, and aatoxin B1, which was detected in 83% of
the feed samples at a concentration of 74 µg/kg, where feed samples
analyzed in this study were contaminated with at least four mycotoxins:
aatoxins and fumonisins coexistent in 80% of the samples [2].
Other authors in South America, have evaluated mycotoxins
(analytical chromatography method) in samples of corn and nished
poultry feeds produced in Brazil, in 119 samples, collected from
farms with balanced feed for poultry: Most of the samples were
contaminated with more than one mycotoxin, where the mean
aflatoxin and trichothecene contamination was low, close to the
allowed values [30].
Others in Europe, collected 228 pig feed samples in Spain to
detect (by analytical chromatography) 19 mycotoxins (aflatoxins
B1, B2, G1 and G2, ochratoxin A, fumonisins B1 and B2, citrinin,
zearalenone, deoxynivalenol, fusarenon X, sterigmatocystin, T2, HT-2
toxin, enniatins A, A1, B, B2 and beauvericin). Most of the samples
(96.9%) agreed with the European Union regulations (which do
TABLE VII
Comparison of Aatoxin levels in nisher balanced feeds for Broilers and Pigs, from eleven food factories in
Venezuela
Toxins
Broiler Pig
R O X
̄
M % R O X
̄
M %
AFB1 0.31 - 9.15 2/11 8.06 18.18 0.58 - 10.5 2/11 9.86 18.18
AFB2 0 - 5.28 1/11 5.28 9.09 0.31 - 5.63 1/11 5.63 9.09
AFG1 0 - 2.57 0/11 - - 0 - 2.82 0/11 - -
AFG2 0 - 1.83 0/11 - - 0 - 3.49 0/11 - -
AFB1= Aatoxin B1; AFB2= Aatoxin B2; AFG1= Aatoxin G1; AFG2= Aatoxin G2. R= aatoxin concentration ranges (lower
and higher µg/kg). O = Occurrence of positive samples/total plants sampled. X
̄
= average concentration of positive samples
(µg/kg). M % = percentage of positive samples with concentration above the maximum allowed level (5 µg/kg allowed
COVENIN standard).
TABLE VIII
Averages of total Aatoxins in samples of nisher balanced
feeds for Broilers and Pigs from eleven factories in Venezuela
Balanced
feed
Average of
total Aatoxins
(µg/kg)
Higher
value
(µg/kg)
Higher allowed
limits of total
Aatoxins (µg/kg)
Broilers 6.65 ± 2.28 29.07 (F1) 20
Pigs 8.72 ± 1.92 27.2 (F1) 20
F1: balanced feed factory where the maximum values were detected
the production and import of transgenics products in the Country, so
imports of transgenic corn and soybeans from Countries, such as the
United States of America (USA), Brazil and Argentina, were probably
carried out [10, 37, 48].
This previous idea could contribute to justify the low levels of
aatoxins detected in this research and that most of the BFF sampled
are within the allowed levels of national regulation. Additionally, the
detected values ratify the results obtained in similar investigations
and samplings on dates close to the present evaluation [28], where
aatoxin levels were evaluated in nisher feeds for pigs, and did not
exceed the Venezuelan regulations in farms of the Center States of
Aragua and Carabobo. Likewise, in this previous research similar
reasons are discussed to explain the aatoxin levels found in the
present study. Additionally, total aatoxin levels similar to those of
the present research, but actually higher than those allowed (greater
than 20 µg/kg), were detected in feed for broilers, obtained from
feeders in Venezuelan poultry farms as previously described [6].
Research carried out in poultry have determined that low
concentrations of aatoxins due to periods of prolonged exposure
generally report subclinical effects and not for that reason, less
harmful, which frequently require to be demonstrated by very
sensitive liver tests or examinations that allow detecting the true
severity of aatoxicosis [4, 5, 15, 31]. These situations can also be
similar/translated to pigs and other monogastric species.
________________________________________________________________________Revista Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32090, 1 - 9
7 of 9
not address emerging mycotoxins: enniatin B and beauvericin, or
co-occurrence), the authors highlight that their results show that
for to ensure absence of mycotoxins, emerging mycotoxins should
always be considered [3].
Likewise, this study also allows to see the need to evaluate the
probable synergistic risks, due to interactions between different
mycotoxins, in the presence of low levels of aflatoxins, despite
the fact that their concentrations are within the regulated limits.
However, although in the present study one hundred percent (100%)
of feeds sampled, both for poultry and pigs, presented some type
of aatoxin (TABLES VII and VIII), no levels were found that could
cause human or animal deaths. However, despite the sublethal
levels of aflatoxins in this study, it should be noted that these
ocial maximum allowed limits (20 µg/kg) are based on avoiding
the presence of aatoxin residues in foods of animal origin for human
consumption. Taking the human species as the “most susceptible
link” to aflatoxins within the food chain for reasons of agri-food
safety and public health [6]. In the same way, is to say, there is no
safe value of aatoxins due to their cumulative effect [12, 20, 46],
so even when tolerable levels are established for the animal and
even values achieved are low, it must be taken into account that the
meat of these animals will be consumed by the human population
[21]. Threading to a toxicity risk for humans that should be avoid by
stricter limits, and better supervisión by the authorities.
Therefore, it needs to be consider the risk levels of aatoxin residues
that are in the agri-food chain, which do not depend exclusively over
the aatoxin levels in the feeding nisher level, but will be present in
the animal product to human comsuption.
Meaning that the probability of other intervening variables should
also be considered, such as the case of repeated improvements
in animal genetic lines, which modify the consumption and food
conversion in the animals [17], and/or their metabolic rate, being
able to promote that broilers and pigs, eventually have different
toxicokinetics from those expected or usual for aatoxins in farms.
This would depend on several factors to which the broilers or pigs
are exposed, before reaching the slaughterhouse.
Such factors can be extreme management, or interactions
with: nutrients, additives, other mycotoxins, disinfectants, drugs,
agrochemicals and other xenobiotics to which the animals are
exposed. The previous with low concentrations of aflatoxins in
the diet, could generate an interaction effect framed within the
toxicology principle known as additivity and synergism.
Additivity occurs for example when the combined effect of two
mycotoxins is equal to the sum of the effect of each mycotoxin
supplied individually. Synergism occurs, when the total effect of two
mycotoxins is greater than the sum of their individual effects and
the synergistic interaction causes the greatest toxic effects, as has
been observed between aatoxin with ochratoxin A or toxin T-2 [23].
Despite being in low concentrations or at permitted levels in the feed
of animals destined for human consumption, it is not easy to predict
the possible interactions and their level of residues or harmfulness for
the agri-food chain, when there are low concentrations of aatoxins
and other mycotoxins at varying levels or unknown.
CONCLUSIONS
In the 11 Venezuelan balanced feed manufacturing F sampled,
during 2010, the presence of aatoxins was detected in 100% of the
nal balanced feed for poultry and pigs. At the levels of AFB1, AFB2,
AFG1 and AFG2, in nisher balanced feeds for broilers and pigs was
found that the aatoxin in the highest concentration was always B1,
considered the most toxic of the group.
When compared the averages of total aatoxin concentrations,
from samples of 11 Venezuelan F., which produced nisher balanced
feeds for broilers and pigs, with maximum limits (20 µg/kg) allowed
(COVENIN standards), it turned out that standards were followed.
However, when comparing the levels by type of aflatoxins AFB1,
AFB2, AFG1 and AFG2, it is established that by type the maximum
allowed limit is 5 µg/kg, where 8/11 F. evaluated comply with the
standard, and three (F1, F3 and F5) were out of the allowed limits.
RECOMMENDATIONS
Monitoring of the genetic quality of raw materials, and requirements
of the supplier to declare the certicated quality of transgenic or not
(currently it is not declared), in order to correlate with studies such as
the one in this evaluation.
It is pertinent that the companies that produce nishing foods for
the nal production stage of poultry and pigs carry out evaluations
not only in terms of aatoxins, but also of other mycotoxins present in
raw materials of transgenic and non-transgenic cereals, and evaluate
the probable agri-food chain risks according to the present results.
ACKNOWLEDGMENTS
The authors recognized the support to fulll this research from
the following institutions or enterprises: to the authorities and
personnel of ALCONCA farm and to the National fund of Science
and Technology (FONACIT-2012-411), for its advised contributions in
development to perform this research.
CONFLICTS OF INTEREST
The authors declare no conicts at the present research.
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