Agriculture Science Developments, 2(2) February 2013, Pages: 8-13

TI Journals
ISSN
2306-7527

Agriculture Science Developments

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Effect of Malathion Toxicity on Dry Matter Degradability,

Fermentation Parameters and Cumulative Gas Production by
Using the in Vitro Technique
S. Asadzadeh *1, A. M. Tahmasbi 2, A. A. Naserian 3
1

Ph.D Student of Ruminant Nutrition, Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran.
Faculty member of agriculture, Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran.

2,3

AR TIC LE INF O

AB STR AC T

Keywords:

A study was conducted to investigate the effects of toxicity of malathion in different levels (0, 100,
250, 500, 750 and 1000 ppm) on fermentation parameters (rumen pH, rumen ammonia, gas
production and in vitro dry matter disappearance) was carried out according to in vitro situation.
The study indicated that all the levels of malathion significantly (p<0.001) inhibited the in vitro dry
matter degradability (IVDMD) for 24 and 96h after incubation, but the effect of charcoal on
IVDMD was insignificant after 24h incubation exception of 96h incubation. Also NH3-N decreased
after 24 and 96h incubation compared to the control (P<0.0001). The pH of rumen fluid
significantly (p<0.05) increased after 24h incubation, but there was not any significant effect for
this factor after 96h incubation. With increasing the levels of malathion, cumulative gas production
for 24, 48 and 96h after incubation decreased (p<0.01). With applying the charcoal, some
fermentation parameters such as cumulative gas production for 24, 48 and 96h incubation
increased, but there was a high significant effect for the NH3-N and pH at different times. The
results showed that malathion generally had a toxic effect on fermentation parameters (exception of
pH for 96h incubation) but to some extent charcoal as a toxin binder could be effective for
decreasing of these toxic effects in this experiment.

Malathion
rumen fluid
in vitro
activated charcoal

2013 Agric. sci. dev. All rights reserved for TI Journals.

1.

Introduction

Organophosphorus pesticides (OPs) have been widely used for a few decades in agriculture for crop protection and pest control. Thousands
of these compounds have been screened and over one hundred of them have been marketed for these purposes (Mogda et al 2009).
Pesticides are commonly used for controlling undesirable insects and plants in agriculture and domestic ruminants. The use of OPs has
increased considerably due to their low potency and durability as compared to organochlorine pesticides. Between OPs, malathion is the
most commonly used which is the main cause of most acute pesticide poisonings. Malathion was first registered for use in the United States
in 1956 by the United States Department of Agriculture (USDA), and it is now regulated by the United States Environmental Protection
Agency (U.S. EPA) (EPA 2006). Malathion and other OPs bind to the enzyme Acetylcholinesterase (AChE) at nerve endings throughout
the bodies of insects and other organisms (Reigart and Roberts 1999). Exposure to OPs can lead to additive toxicity, however there was
extremely different for the toxicity between all of them. The use of OPs on man, animals or in his immediate environment has caused
potential hazards that seriously are poisonous for public health (Kazemi et al 2012b). There has been increasing awareness and concern on
the part of food and health officials and of the dairy industry about the presence OPs in milk and other milk products (Kazemi et al 2012c).
Also animal feeds and milk may serve as a transition factor of OPs substances with extrinsic origin which can be potentially toxic to the
consumer. These toxins may originate in cow's milk from the ingestion of plants known to contain toxic methods have been used for the
removal of OPs from substances or feeds contaminated with OPs (Kazemi et al 2012a). Cook (1957) was perhaps the first to suggest that
rumen liquor played an active role in hydrolyzing OPs. Any deleterious influence, even of a temporary nature, could have a profound effect
on the bacterial activity and subsequently upon the host animal. Kutches et al (1970) observed that, toxaphene had the inhibitoriest of the
pesticides tested and lead to more decreases of the in vitro dry matter disappearance, volatile fatty acids and rumen protozoa. Dauterman et
al (1959) found, there was a serious of derivations of dimethoate in treated rats and cattle with radioactive dimethoate for their blood,
tissue, milk, and excreta, although the derivations were rapidly metabolized and excreted. Plapp and Casida (1958) incubated an OP
pesticide, Trolene, with bovine rumen fluid and showed hydrolysis at the phosphorus-oxygen methyl group to yield phenyl phosphoric
acids. Ahmed et al (1958) reported that OPs are degraded by the some microorganisms in the rumen. Some additive material such as
charcoal or bentonite is effective for removing of the some toxic effects of OPs in the ration (Kazemi et al 2012b). Studies show that there
was not any general information about OPs influences on rumen function (Kazemi et al 2012c), so the objective of this experiment was
evaluation of the malathion toxicity on fermentation parameters basis of an in vitro batch culture and whether activated charcoal can be
effective for reduce the toxicity of malathion or not.
* Corresponding author.
Email address: kmohsen88@yahoo.com

Effect of Malathion Toxicity on Dry Matter Degradability, Fermentation Parameters and Cumulative Gas Production by

Agri culture Scienc e Developments , 2(2) February 2013

2.

Materials and Methods

2.1. Sampling method

The applied experimental diet was a mixture of alfalfa hay and concentrate which was ground to pass through 1.5 mm screen. The chemical
composition of experimental diet is shown in Table 1. A sample of 500 mg (250mg alfalfa and 250 mg concentrate) was used for each
replication.

2.2. Collection of ruminal fluid

Ruminal fluid was collected at 6 am from 4 fistulated male cow (600 50 kg LW) normally fed with alfalfa and concentrate. Rumen
fluid was removed before the morning feeding and was strained through three layers of gauze. Immediately ruminal fluid was transported to
the laboratory.

2.3. In vitro gas production and DM digestibility

Procedure of in vitro gas production was performed according to the Menke and Steingass (1988). The sample of 500 mg (TMR) was
introduced to each 120-Ml glass. In an anaerobic condition, 50 ml of buffered rumen fluid [ratio of buffer to rumen fluid was 2:1, buffer
were prepared as proposed by Menke and Steingass (1988)] was dispensed with pipetor pump into a 125 ml serum bottle containing 0.5 g
DM of the experimental diet. Then, each bottle was sealed with rubber stopper and aluminum cap and placed in a shaking water bath for 24
h at 38.6C. To prevent accumulation of gas produced, head space gas pressure of each bottle was recorded using a pressure transducer
(Theodorou et al 1994) at 6, 8, 10, 12 and 24 h of the incubation and then gas released. After 24 h of the incubation, the bottles were
respectively transferred to an ice bath to stop fermentation and then opened to measure medium pH using a pH meter (Metrhom pH meter,
Model 691). Then, each bottle content was filtered (42 m pore size) and a 5 ml sample of each filtrate bottle was taken and acidified with
5 ml of 0.2 N HCl and frozen at -20C. The filtrated residual was oven dried (60C for 48 h) and used to calculate in vitro dry matter and
crude protein disappearances (IDMD and ICPD, respectively).

2.4.Calculations and statistical analysis

NH3 -N concentration of the bottle content was determined using kjeldahl method (Kjeltec 2300 Auto analyzer, Foss Tecator AB, Hoganas,
Sweden). Cumulative gas production data were fitted to the exponential equation y = b (1 e-ct) (Osuji et al 1993), where b is the gas
production from the readily soluble fraction and the insoluble fraction (ml); c is the gas production rate constant (ml/h); t is the incubation
time (h) and y is the gas production at time of t (ml). The data was analyzed with a simple 62 factorial in a completely randomized design
(SAS, 9.1). Significant means were compared; using the Duncans multiple range tests. Mean differences were considered significant at P <
0.05.
Table1. Ingredients and chemical composition of experimental diet
Composition
Amount
Ingredients (% of DM)
Alfalfa Silage

50

Milled Barley

13.50

Corn Ground

9.70

Soybean meal

9.10

Wheat Bran

4.25

Cottonseed meal

6.50

Cottonseed

6.95

Chemical composition (% of DM)

Crude Protein

22.40

Crude Fat

3.50

Neutral detergent fiber

34.90

Non fiber carbohydrates

34.80

ME (M cal/kg DM)

2.54

S. Asadzadeh et al.

10

Agricult ure Sci ence Developments , 2( 2) February 2013

3.

Results and discussion

Cumulative gas production

Table 2 shows the effects of different levels of malathion with different levels of charcoal on gas production. A comparison of cumulative
gas production indicated that there was a significant difference between treatments (p<0.05). The rate of gas production decreased when the
amounts of malathion increased. Analysis of the main effect for malathion was shown that there was a decreasing effect at 250 to 1000 ppm
for all parameters of gas production. Supplementing of charcoal caused to increase all of the gas production parameters. Some OPs were
shown by Williams et al (1963) to stimulate gas production in vitro by rumen holotrich protozoa, whereas these compounds had no
appreciable effect when rumen bacteria served as the inoculums source. Data about toxic effects of malathion on rumen microorganisms is
scarce, so it is obvious that, the high level of malathion (750, 1000 ppm) had most negative effect for cumulative gas production at different
incubation times. Although malathion had an inhibitory effect on cumulative gas production parameters, but charcoal had a positive effect
on these parameters. Some researcher reported that rumen microorganisms can be decrease the toxic effects of OPs (cook 1957 and Kazemi
et al 2012a).
Table 2. Effects of different levels of malathion with different levels of charcoal on some parameters of gas production in a TMR ration

Malathion levels (ppm

Rate of gas
production
0.057 abc
0.059 ab
0.055 abcd
0.057 abc
0.052 bcde
0.053 abcde
0.046 de
0.052 bcde
0.049 cde
0.049 cde
0.043 e
0.043 e
0.003

Gas volume after

24 h
70. 8 ab
70.8 ab
71.2 ab
74. 6 a
67.1 abc
65.9 abc
55.2 ed
63.5 bcd
48.5 e
57.9 cde
38.3 f
54. 8 de
3.39

Gas volume
after 48 h
89.8 a
90.9 a
91.0 a
95.6 a
88.0 a
87.3 ab
75.0 bcd
83.9 abc
60.8 ef
74.9 cd
50.4 f
67.1 de
4.18

Gas volume after

96 h
99.2ab
102 ab
100 ab
106a
98.9 ab
99.2 ab
87.9 bc
96.6 ab
79.9 c
91.5abc
57.7 d
79.5 c
4.52

0
100
250
500
750
1000

0.058 a
0.056 a
0.052 ab
0.049 b
0.049 b
0.053 ab
0.002

70.8 a
72.8 a
66.5 a
59.3 b
53.2 bc
46.5 c
2.28

90.3 a
93.3 a
87.7 ab
79.5 b
67.5 c
58.7 d
3.11

100 ab
103 a
99.1 ab
92.3 bc
85.7 c
68.6 d
3.24

0
1
SEM

0.05 b
0.056 a
0.01

58.5 b
64.6 a
1.11

75.8 b
83.2 a
1.07

87.3 b
95.9 a
1.28

Malathion
Charcoal
CharcoalMalathion

P < 0.01
P < 0.05
P < 0.05

P < 0.01
P < 0.0001
Ns

P < 0.01
P < 0.0001
Ns

P < 0.01
P < 0.0001
Ns

0
0
100
100
250
250
500
500
750
750
1000
1000
SEM

Charcoal levels (%
of DM)
0
1
0
1
0
1
0
1
0
1
0
1

Main effects
Malathion

SEM
charcoal

P value

a, b, c, d, e and f

means in the same column with different superscript differ significantly (P < 0.05).

pH and NH3-N
Effects of different levels of malathion and charcoal on NH 3-N and pH is presented in table 3. There was significantly (P<0.05) different
between treatments after 24 h incubation for the pH. The pH was highest at level of 1000 ppm (malathion), so with increasing the levels of
malathion, pH tended to increasing. After 96 h incubation there were not significantly different between treatments for malathion effect.
However the pH of rumen fluid increased numerically when the amounts of malathion increased. In the main effects analysis, malathion
had an increasing effect by increasing its levels for pH. Also applying the high level of charcoal (1%), lead to increasing the pH.

Effect of Malathion Toxicity on Dry Matter Degradability, Fermentation Parameters and Cumulative Gas Production by

11

Agri culture Scienc e Developments , 2(2) February 2013

Table 3. Effects of different levels of malathion with different levels of charcoal on NH3 -N and pH of a TMR ration after 24 and 96 h incubation

Malathion levels (ppm)

Charcoal levels
(% of DM)

NH3- N after 24 h

NH3-N after 96 h

PH after 24 h

PH after 96 h

0
0
100
100
250
250
500
500
750
750
1000
1000
SEM

0
1
0
1
0
1
0
1
0
1
0
1

17.3 a
17.1 a
16.9ab
16.3 ab
16.1 ab
15.2 b
15.2 bc
14.7 bc
14.5 cd
14.0 d
14.0 d
13.5 e
0.23

17.7 a
17.6 a
17.1 ab
16.5 b
16.4 b
15. 8 bc
15.6 bc
15.2 bc
14.8 c
13.6 d
13.2 d
13.1 d
0.25

6.69 g
6.72def
6.69 g
6.71 efg
6.70 fg
6.71 efg
6.73 cd
6.73 cd
6.77 ab
6.75 bc
6.76 b
6.78 a
0.007

6.74 cd
6.76ab
6.74 cde
6.75 bc
6.74 cdef
6.74 cd
6.72 ef
6.72 f
6.73 def
6.73 def
6.78 a
6.77 ab
0.006

0
100
250
500
750
1000

17.5 a
16.8 b
16.1 c
16.1 c
15.9 c
16.3 c
0.15

17.9 a
17.6 a
16.5 b
16.1 bc
16 bc
16 c
0.16

6.71 c
6.7 c
6.7 c
6.73 b
6.76 a
6.77 a
0.005

6.72 e
6.73 de
6.74 cd
6.75 bc
6.75 b
6.77 a
0.004

0
1

17.4 a
15.9 b
0.8

17.3 a
16.1b
0.9

6.72 b
6.73 a
0.002

6.74 a
6.75 a
0.002

P < 0.0001
P < 0.0001
P < 0.0001

P < 0.05
P < 0.0001
P < 0.01

P < 0.0001

Main effects
Malathion

SEM
charcoal

SEM

P value
Malathion
Charcoal
CharcoalMalathion
a, b, c, d, e, f and g

P < 0.0001
P < 0.0001
P < 0.0001

Ns
Ns

means in the same column with different superscript differ significantly (P < 0.05).

Greeballah et al (2005) reported that all the levels of malathion were not significantly (p< 0.05) effected on pH of rumen fluid. Researchers
found that no significant difference between the pH of rumen fluid treated with different types of insecticides (phosalone and malathion)
(Greeballah et al 2005). OPs did not cause much change in pH of rumen fluid (Roo et al 2010). Repeated oral administration of
monocrotophos in doses of 0.5 and 2.0 mg/kg BW/day resulted significant reduction in rumen pH (Sandhu and Singh 1989). Adding of
charcoal alone, had an increasing effect on pH rather than to control group although these effect had not a rationale process. The results
indicated that malathion significantly (P<0.0001) decreased rumen fluid NH 3-N compared to control treatment. By increasing the
malathion, the NH3-N decreased, so the lowest value was observed at 1000 ppm malathion. Charcoal significantly had a decreasing effect
on NH3-N in the culture medium and it seems that it had an absorbing characteristic on NH 3-N. In the main effects analysis, malathion had
a decreasing effect by increasing its levels for NH3-N. Also applying the high level of charcoal (1%), lead to decreasing the NH3-N.
Greebalah et al (2005) found the NH3-N increased significantly (P<0.05) with increasing the pesticides levels (from 0 to 15 ppm). The
mechanism by which the rumen fluid-NH3 was increased was not clear, but it might be due to some selective effects of these pesticides on
some of the rumen microorganisms and hence the rumen metabolism may be different by using the different pesticides. These results were
in agreed with Alchalabi et al (1988) and Agarwal and Saxena (1990).
Dry matter degradability
The results of the effects of different levels of malathion and charcoal on dry matter digestibility is observed in table 4. In this experiment,
In vitro dry matter degradability (IVDMD) was effective by adding the malathion pesticide, so by increasing the levels of malathion from
500 to 1000 ppm, the IVDMD decreased significantly (p<0.0001) but the difference between 100 and 250 ppm was not significant for 24
and 96h after incubation. Supplementation of the culture medium with charcoal without malathion resulted in decreasing of IVDMD,
although there was not a normal trend between treatment for it. The results showed that generally charcoal had an inhibitory effect for

S. Asadzadeh et al.

12

Agricult ure Sci ence Developments , 2( 2) February 2013

IVDMD and it could not be effective for decreasing of these negative effects by adding malathion. The IVDMD decreased by increasing
the levels of malathion (specially 250 to 1000 ppm). It is observed that with increasing levels of malathion was significantly reduced. Also
by adding the charcoal to the culture media, IVDMD after 96h incubation significantly decreased (P < 0.0001). It had shown that pesticides
have deleterious effects on the rumen fluid (Cook 1969). Cook (1957) was the first researcher that observed rumen microorganisms had an
active function for hydrolyzing the OPs. Additional evidence by Cook (1957) indicated that metabolism of OPs by rumen microorganisms
accounted for its apparent lack of toxicity to cattle. Kutches et al (1970) reported toxaphene were ineffectual in causing a depression of In
vitro dry matter disappearance at the 100, 250, and 500 cg per ml of culture environment (consist of rumen fluid). Ahmed et al (1958)
demonstrated that bovine rumen fluid hydrolyzed many organophosphate insecticides. Oxidation reactions in the rumen fluid were a little
importance, and reduction reactions were a great significance in metabolizing these compounds. Dauterman et al (1959) suggested that
rumen microorganisms dissimilate malathion by phosphatase action to dimethyl phosphate and 0, 0- dimethyl phosphorothioate. Microbial
mediated decomposition is the major, and sometimes the only, mechanism of partial removal or modification of organophosphates in
rumen. Schwartz et al (1973) suggested that a concentration of 1,000 ppm malathion plus 1,000 ppm sevin inhibited IVDMD significantly
(P< .05) while 1,000 ppm sevin or 1,000 ppm malathion did not significantly decrease digestion. This can be explained by the fact that both
sevin and malathion slightly reduced IVDMD, but not significantly. However, when the two pesticides were used in combination, their
additive effects were great enough to reduce IVDMD significantly. The reason for the effects of organophosphate on IVDMD was not
clear. Perhaps the inhibition of IVDMD was reflected on microorganisms deletion from the culture medium.

Table 4. Effects of different levels of malathion with different levels of charcoal on dry matter digestibility of a TMR ration after 24 and 96 h
incubation

Malathion levels (ppm)

0
0
100
100
250
250
500
500
750
750
1000
1000
SEM
Main effects
Malathion

Charcoal levels (%
of DM)
0
1
0
1
0
1
0
1
0
1
0
1

Dry Matter digestibility after 24

h (%)
74.4 a
68.8 ab
72.5 ab
67.9 b
69.3 ab
69.9 ab
60.8 c
60.6 c
54.9 d
54.6 d
53.3 d
55.8 cd
1.79

Dry Matter digestibility after 96

h (%)
79.7 a
76.7 bc
79.5 a
77.8 ab
79.6 a
75.7 bcd
75.9 bcd
74.9 cd
74.2 d
70.6 e
58.1 g
60.9 f
0.76

0
100
250
500
750
1000

71.6 a
70.2 a
69.6 a
60.7 b
54.8 c
54.6 c
1

78.2 a
78.7 a
77.7 a
75.4 b
72.4 c
59.5 d
0.55

0
1

64.2 a
63 a
0.79

74.5 a
72.7 b
0.21

SEM
charcoal

SEM
P value

Malathion
P < 0.0001
Charcoal
Ns
CharcoalMalathion
Ns
a, b, c, d, e, f and g
means in the same column with different superscript differ significantly (P < 0.05).

4.

P < 0.001
P < 0.0001
P < 0.001

Conclusion

Environmental contamination of feed and forages and transition of this contamination to animal products (such as milk, meat and other
tissues) are still a problem for animal health and subsequent human health. Several methods are tested for decreasing of these deleterious
effects in pesticides and subsequently its transition. In this experiment we tested the effect of malathion on some fermentation parameters,

Effect of Malathion Toxicity on Dry Matter Degradability, Fermentation Parameters and Cumulative Gas Production by

13

Agri culture Scienc e Developments , 2(2) February 2013

so the results indicated that there was a toxic effect on some parameters (such as cumulative gas production, IVDMD and NH3-N) specially
at high levels of malathion (500, 750 and 1000 ppm). Also we tested the charcoal as toxin binder, so that the results showed it had a
promoter effect on some fermentation parameters such as cumulative gas production and had a negative effect on IVDMD and NH3-N. For
determination of toxic effect of malathion on animal health and subsequently animal products, more experiment (especially in vivo
experiment) must be tested.

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