Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
ISSN: 2319-7706 Volume 4 Number 5 (2015) pp. 756-771
http://www.ijcmas.com
Original Research Article
Microbial degradation of the fungicide Benomyl in soil as
influenced by addition of NPK
Randa H. Elsalahi1*, Adil A. Elhussein2, Awad G. Osman1 and Ashraf M. Sherif1
1
Biofertilization Department, Environment and Natural Resource and Desertification Research
Institute (ENDRI), National Center for Research, Khartoum, Sudan
2
Botany Department, Faculty of Science, University of Khartoum, Khartoum, Sudan
*Corresponding author
ABSTRACT
Keywords
Biodegradation.
Benomyl.
Soil
microorganisms.
NPK
Benomyl is a broad spectrum fungicide with a long environmental persistence. It is
a suspected human carcinogen and is highly toxic to aquatic and soil dwelling
organisms. A laboratory experiment was carried out to determine the effect of
Benomyl on the main groups of soil microorganisms and to elucidate their role in
the fungicide degradation as affected by NPK addition. NPK was added as 375 mg
N, 187.5 mg P and 187.5 mg K per 600 g soil. Benomyl was applied in three
concentrations; 0.032, 3.2 and 8.0 mg/g soil. Microbial counts of the main groups
of soil microorganisms and Benomyl residue were determined at different intervals
for a period of 360 days. Benomyl has a remarkable stimulating effect on the
growth and multiplication of different microorganisms. The highest percent
increments in microbial counts have always been recorded in Pseudomonas sp.
followed by organic nitrogen users and fungi. Addition of NPK has increased
microbial counts substantially which in turn has been reflected positively on
Benomyl degradation rates. Degradation of more than 90% of Benomyl at 8.0 mg/g
soil was attained in the control and in the NPK amended soil after 360 and 60 days,
respectively.
Introduction
with elements cycles and entering food
chains.
Continuous use of pesticides encompasses
accumulation of appreciable quantities of
them and their degradation products in the
ecosystem. Prevailing data showed that only
2- 3% of the applied chemical pesticides
reach their targets, while the rest remains in
the soil (US.EPA 2005). This raises great
alarm about the heavy contamination burden
the soil is carrying and consequently poses
great risk on soil microbes, hence interfering
Among pesticides used in Sudan, fungicides
rank third after insecticides and herbicides in
terms of quantities used. One of the recently
introduced fungicides in Sudan is Benlate
which is the commercial name for the active
ingredient Benomyl or, Methyl 1(butylcarbamoyl)
benzimidazole-2756
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
ylcarbamate. It belongs to the benzimidazole
class; a member of the carbamate group
(Marrs and Ballantyne 2004). Benomyl is
an endocrine disrupter chemical (European
Commission 2000) a suspected mutagen, a
possible human carcinogen (U.S. EPA 2002)
and a teratogen (Pesticide Action Network
2002).
Nutrients like carbon, nitrogen, and
phosphorus stimulate microbes to produce
the essential enzymes for the break down
contaminants (Vidali 2001). Low nitrogen or
phosphorus availability can inhibit or delay
the degradation of toxic materials (Cosgrove
2010). Addition of mineral fertilizers (NPK)
has been reported to increase the
biodegradation
of
Oxyfluorfen
and
Chlorpyrifos in soils (Mohammed et al.
2011; Tortella et al. 2010).
As
microbes are very helpful to remediate
the contaminated environment, the objective
of this study was to assess the microbial
degradation of the fungicide Benomyl in soil
as influenced by addition of NPK.
It is selectively toxic to microorganisms and
to invertebrates (Kamrin, 2010) and is a
systemic broad spectrum, protective,
curative and iradicant fungicide (Troy
and Beringer 2005; Wang et al. 2009).
Benomyl is used for the control of postharvest rots (Lopez-Garcia et al. 2003;
Valiuskaite et al. 2006) and many plant
fungal diseases such as powdery mildews,
Botrytis rot, Fusarium basal rot, black spot
and blossom rot (Marrs and Ballantyne
2004). In Sudan it is used for the treatment
of powdery mildews mainly in cucurbits and
other vegetables such as okra, tomatoes and
auberjines.
Materials and Methods
Effect of Benomyl on the major groups of
soil microorganisms
Soil samples were collected from Al Geraif
(Blue Nile bank) area North Khartoum
where there is no history of pesticide
application. Samples were randomly taken
with an auger from the top 10 cm from
different parts in the selected site. Large
clods were crushed to a uniform size, mixed
thoroughly to make a composite sample and
air dried at room temperature. Some
characteristics of the soil were determined
according to the methods described by
Rayan et al. (1996).
Pesticides in the environment may be
degraded in soil through chemical, physical
and
biological
mechanisms
(OrtizHernández et al. 2011). Biodegradation of
pesticides in soil is affected by soil
properties (structure, moisture, temperature,
pH and organic matter content) and the
presence of the degrading microbes (Castillo
and Torstensson 2007; Vischetti et al. 2008
and Chowdhury et al. 2008).
Soil was then divided into six 600 g lots and
each was transferred into 1000 ml jar. Three
lot sets were prepared for each of the two
soil treatments (NPK and control). Benomyl
was then added to each of the soil sets in the
concentrations of 0.032, 3.2 or 8.0 (mg/g
soil). Soil in each jar was thoroughly mixed
with Benomyl. NPK was added in a separate
set; soil was then wetted with water to 60%
field capacity and incubated in the dark at
temperature 28°C for 360 days.
Benomyl degrades rapidly by hydrolysis to
Carbendazim
(Methyl-2-benzimidazole
carbamate), or MBC, which is also
registered as an active ingredient (US.EPA
2002).
It has a long environmental
persistence where residues in soil might be
detected even after four years of application
(Lewandowska and Walorczyk 2010).
Benomyl has a half-life of 3 to 6 months and
when applied to bare soil the half-life is 6 to
12 months (Zhang et al. 2005).
757
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
Nitrogen was added in the form of urea at a
rate of 375 mg per 600 g of Benomyl treated
soil (1437 kg/ha) and potassium was added
in the form of KCl at a rate of 187.5
mg/600g soil (718 kg/ha). Phosphate in the
form of super phosphate was added at a rate
of 187.5 mg/600g soil (718 kg/ha). At zero
time and then after 7, 15, 30, 60, 120, 150,
180, 240 and 360 days, soil samples were
taken for the determination of total viable
counts on each of five different media.
declined, due to addition of NPK, reaching
the inhibition zone after 120 days of
incubation at all Benomyl concentrations
Fig. 1 and 2.
Effect
of
different
Benomyl
concentrations on soil microorganisms
growing on meat peptone agar
Counts of organic nitrogen users at the
control exhibited a general trend of
increasing during the first two months then
decreasing towards the end of the incubation
period with the exception of the highest
concentration (8.0 mg Benomyl/g soil)
where populations continued to increase up
to the fourth month Fig. 3. Addition of NPK
showed the highest increments at 180 and
360 days of incubation Fig. 4.
The media used were: Czapek Dox Agar
(CZA), Meat Peptone Agar (MPA), Starch
Ammonium Agar (SAA) and Nitrate Agar
(NA); prepared according to the method
described by Tepper et al. (1993) and
Pseudomonas (CFC) Selective Agar Base
according to (ISO 2000). Viable count
determinations were carried out according to
the procedure of Tepper et al. (1993).
Effect
of
different
Benomyl
concentrations on soil microorganisms
growing on Czapek Dox
Determination of Benomyl residues in soil
Benomyl residue was determined by HPLC
(Sycam, Germany) according to the method
of Zweig and Gao (1983) into a
chromatograph with UV detector, column:
Li-chromosorb RPC18 (25 cm x 3.9-mm-ID
stainless steel), column temperature 28°C,
mobile phase: 70% acetonitrile: 30% water.
Injection volume: 20 l, Mobile phase flow
rate 0.7ml/min, detection: UV light at 286
nm (1-cm cell), retention time: 4.3 min.
Residues of Benomyl were determined at
zero time and then after 7, 15, 30, 60 120,
150, 180, 240 and 360 days.
Fungal counts after the first week underwent
inhibition up to the end of incubation period.
Fig. 5. Increment peak was noticed at day 60
due to NPK addition Fig. 6. Inhibition of
52% was noticed only at day 7 interval, with
the
highest
increment
percentage
(2169.23%) appearing after 60 days of
incubation.
Effect
of
different
Benomyl
concentrations on soil microorganisms
using Mineral Nitrogen
Numbers of mineral nitrogen users showed
increment peaks at 60 and 180 days of
incubation for the control and NPK treated
soil at all Benomyl concentrations.
Actinomycetes
counts
showed
a
contradicting pattern with the other types of
inorganic nitrogen users and for some
intervals they went in the same direction
Table 1.
Results and Discussion
Effect of different Benomyl
concentrations on soil microorganisms
A reasonable increment in Pseudomonas
counts was observed up to two to three
weeks of incubation in both NPK and the
control. Pseudomonas counts have gradually
758
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
at the beginning of the incubation period but
completely disappeared after 120 days of
incubation. This coincided with a
remarkable increase in degradation % of up
to 80% recorded after 60 days. This result
suggested that Pseudomonas sp. play a role
in Benomyl degradation. In line with this,
Fuchs and De Vries (1978) detected high
numbers of
fluorescent Pseudomonas
strains in a mineral medium containing
Benomyl as the sole source of carbon.
Effect
of
different
Benomyl
concentrations on soil microorganisms
growing on Poor Medium
Numbers of microorganisms capable of
growing on poor medium increased
remarkably due to the addition of NPK to
soil treated with Benomyl at seven and 15
days of incubation while the rest of the
counts showed fluctuating increments and
inhibitions compared to control Table 2.
However, at 360 day interval a drastic
decrease in the total count was observed.
Comparatively,
very
low
increment
percentages in numbers of organic nitrogen
users were observed in NPK amended soil at
all Benomyl concentrations up to 150 days.
This could be attributed to the suppression
of such microbes by inorganic nitrogen
addition. Das and Debnath (2006) reported
the stimulatory effect of Oxyfluorfen
herbicide on the growth of non-symbiotic
N2- fixing bacteria in clay soil treated with
NPK fertilizers. Mohammed et al. (2011)
found that addition of NPK fertilizers
increased the population of organic nitrogen
users in Oxyfluorfen treated soils at all
concentrations tested.
Benomyl degradation in soil
In soils treated with 0.032 mg Benomyl/g
soil the control showed higher values (73%)
since the 60 days (Fig. 7). interval while
with NPK, degradation started at lower rates
(34%) (Fig. 8). At day 120 degradation
percentage obtained by the control was 75%
at the same time intensive degradation
percentages of 83% for NPK then the
maximum degradation took place at 360
(Fig. 8). At 3.2 mg Benomyl/g soil,
Benomyl degradation started at lower rates
and gradually increased reaching a
maximum of 97.7% at 180 days interval for
the control (Fig. 7). Whereas for NPK
fertilization a gradual increase in
degradation percentage, and maximum
degradation (99.4%) was attained at 180
days interval (Fig. 8). After 15 days of
incubation at 8.0 mg/g soil Benomyl
degradation was only 13% in the control,
while with the NPK 76% was degraded,
NPK fertilization at this Benomyl
concentration seems to enhance earlier
intensive degradation where maximum
degradation (98.5%) was attained at 150
days interval (Fig. 8) compared to (99.9%)
at 360 days interval for the control (Fig. 7).
On the contrary, Banerjee and Dey (1992)
recorded a significant reduction in the total
number of non-symbiotic N2- fixing bacteria
after 90 days in the rhizosphere of jute after
application of Basalin herbicide and NPK
fertilizers.
Organic
nitrogen
users
might
be
outcompeted by the inorganic nitrogen users
that could have been enhanced by addition
of NPK. Addition of NPK to the fungicide
treated soil produced remarkable increment
in the numbers of fungi up to 180 days of
incubation which indicates a widespread
resistance to Benomy after which
remarkable decline was observed up to the
end of the incubation period.
Counts of Pseudomonas sp. and microbes
growing on meat peptone agar were higher
759
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
Table.1 Effect of Benomyl on Soil Microorganisms Capable of Growing on Mineral Nitrogen
Medium (Starch Ammonium Agar) in clay soil amended with NPK, incubated at room temp.
(28ºC)
Benomyl doses (mg/g soil)
Counts (108)
0.032
Control
8
3.2
NPK
Control
8.0
NPK
Control
NPK
Total (X10 )
10.35
256
46.5
16629
81
250.5
% Inhibition - Increment
0.0
+2373.43
0.0
+35661.3
0.0
+209.26
Actinomycetes (X108)
4.75
0.0
6.45
0.0
1.5
81
% Inhibition - Increment
0.0
100
0.0
100
0.0
5300
% Actinomycetes to total
45.89
0.0
13.87
0.0
1.85
32.34
Total (X108)
15.6
107.5
13.1
57.6
340.45
1997
% Inhibition - Increment
0.0
+589.1
0.0
+339.69
0.0
+486.58
Actinomycetes (X108)
10.05
0.5
7.05
1.1
1.95
0.0
% Inhibition - Increment
0.0
-95.12
0.0
-84.4
0.0
-100
% Actinomycetes to total
64.42
0.47
53.82
1.91
0.57
0.0
Total (X108)
1571.51
635.51
5.56
6820.01
1099.06
10839.51
% Inhibition - Increment
0.0
-59.56
0.0
+122662.05
0.0
+886.25
Actinomycetes (X108)
0.01
1.01
0.0
12.01
0.01
140.51
% Inhibition - Increment
0.0
+10000
0.0
+120000
0.0
+1405000
% Actinomycetes to total
0.000064
0.159
0.0
0.18
0.00091
1.3
Total (X108)
216.85
330
291.35
423.5
11.8
198
% Inhibition - Increment
0.0
+52.18
0.0
+45.36
0.0
+1578
Actinomycetes (X10 )
1.5
1.0
0.05
15.5
0.5
41.5
% Inhibition - Increment
0.0
-33.33
0.0
+30900
0.0
+8200
% Actinomycetes to total
0.69
0.3
0.017
3.66
4.24
20.96
Total (X108)
3.5
2901.5
1950.95
17287.5
2529.05
2588
% Inhibition - Increment
0.0
+82800
0.0
+986.11
0.0
+2.33
Actinomycetes (X108)
0.05
32
0.25
350
2.5
11996.5
% Inhibition - Increment
0.0
+63900
0.0
+139900
0.0
+47660
% Actinomycetes to total
1.43
1.1
0.013
2.02
0.099
46.23
Total (X108)
39550
949.5
149805
80
205850.01
318
% Inhibition - Increment
0.0
-97.6
0.0
-99.95
0.0
-99.85
Actinomycetes (X108)
0.01
57.5
5
2
0.01
27.5
% Inhibition - Increment
0.0
+574500
0.0
-60
0.0
+274900
% Actinomycetes to total
0.000025
6.06
0.0033
2.5
0.0000048
8.65
Total (X108)
9326.5
5032
2517.51
2520.16
11319.01
4288.51
% Inhibition - Increment
0.0
-46.05
0.0
+0.1
0.0
-62.11
Actinomycetes (X108)
60
19.5
0.01
45
0.01
0.01
% Inhibition - Increment
0.0
-67.5
0.0
+449900
0.0
0.0
% Actinomycetes to total
0.64
0.39
0.0004
1.79
0.0001
0.00023
8
760
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
Continue:
Benomyl doses (mg/g soil)
8
Counts (10 )
0.032
Control
Total (X108)
3.2
NPK
8.0
Control
NPK
Control
NPK
3379
14979.15
1834
13812.5
10144
11114
0.0
+343.3
0.0
+653.14
0.0
+9.56
Actinomycetes (X10 )
2437
2.15
8
13.5
5
47
% Inhibition - Increment
0.0
-99.91
0.0
+68.75
0.0
+840
% Actinomycetes to total
72.12
0.014
0.44
0.091
0.049
0.42
Total (X108)
9214.01
27.01
605.01
3701
71.5
16211
% Inhibition - Increment
0.0
-99.71
0.0
+511.73
0.0
+22572.73
Actinomycetes (X10 )
0.01
2.5
0.01
16.5
5
27
% Inhibition - Increment
0.0
+24900
0.0
+164900
0.0
+440
% Actinomycetes to total
0.00011
9.26
0.0017
0.45
6.99
0.17
Total (X108)
333
737.01
422
6508
642.51
117
% Inhibition - Increment
0.0
-121.32
0.0
+1442.12
0.0
-81.79
Actinomycetes (X108)
15
0.01
0.0
0.0
0.01
0.5
% Inhibition - Increment
0.0
9.93
0.0
0.0
0.0
+4900
% Actinomycetes to total
4.5
0.0014
0.0024
0.0015
0.0016
0.43
% Inhibition - Increment
8
8
(%): Percentage of Inhibition (-) and Increment (+)
Table.2 Effect of Benomyl on soil microorganisms capable of growing on poor medium (Nitate
Agar) in clay soil amended with NPK, incubated at room temp (28ºC)
Benomyl
Doses
mg/g
soil
All microorganisms
N. Cells/g
%
dry soil
8
(X10 )
Mycobacterium
N. Cells/g
%
dry soil
8
(X10 )
Micromonospora
N. Cells/g
%
dry soil
8
(X10 )
Nocardia
N.
%
Cells/g
dry soil
(X108)
Arthrobacter
N.
%
Cells/g
dry soil
(X108)
Bactoderma
N.
%
Cells/g
dry soil
(X108)
At zero time
Control
0.032
Control
3.2
Control
8.0
313.56
100
118.46
37.78
170.56
54.4
12.35
3.94
12.22
3.9
0.0
0.0
159.9
51
81.9
51.22
0.0
0.0
0.0
0.0
78
48.78
0.0
0.0
668.33
100
616.33
92.22
13.39
2
14.95
2.24
23.66
3.54
0.0
0.0
1.3
0.19
1.3
100
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
74.49
100
40.3
54.1
4.55
6.11
9.75
13.09
19.76
26.53
0.13
0.17
269.1
361.3
0.0
0.0
0.0
0.0
9.1
3.38
0.0
0.0
260
96.62
At After 7 days
Control
0.032
Control
3.2
Control
8.0
21.32
100
7.93
37.2
0.39
1.83
1.69
7.93
11.18
52.44
0.13
0.61
193.7
908.5
78.1
44.97
0.0
0.0
52
26.85
54.6
28.19
0.0
0.0
98.93
100
15.86
16.03
0.13
0.131
54.73
55.32
28.21
28.52
0.0
0.0
61.1
61.8
16.9
27.66
9.1
14.89
3.9
6.38
31.2
51.06
0.0
0.0
184.47
100
161.72
87.67
1.3
0.7
18.85
10.22
2.6
1.41
0.0
0.0
728
394.6
169
23.21
0.0
0.0
78
10.71
481
66.07
0.0
0.0
Control
0.032
Control
3.2
Control
8.0
108.55
100
26.65
24.55
31.2
28.74
33.8
31.14
16.9
15.57
0.0
0.0
189.8
174.9
123.5
65.07
0.0
0.0
27.3
14.38
39
20.54
0.0
0.0
155.35
100
40.04
25.77
0.0
0.0
29.51
19
85.8
55.23
0.0
0.0
633.1
407.5
500.5
79.06
1.3
0.21
41.6
6.57
89.7
14.17
0.0
0.0
461.76
100
352.56
76.35
10.66
2.31
52.39
11.35
46.15
9.99
0.0
0.0
390
84.5
200.2
51.33
6.5
1.67
7.8
2
175.5
45
0.0
0.0
After 15 days
761
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
After 30 days
Control
0.032
Control
3.2
Control
8.0
49.79
100
43.42
87.21
1.3
2.61
3.25
6.53
1.82
3.66
0.0
0.0
2109.9
4237.6
547.3
25.94
131.3
6.22
288.6
13.68
97.5
4.26
1045.2
49.54
85.41
100
32.63
38.2
0.0
0.0
11.7
13.7
41.08
48.1
0.0
0.0
271.7
318.1
68.9
25.36
39
14.35
57.2
21.05
49.4
18.18
57.2
21.05
12.35
100
4.16
33.68
0.52
4.21
1.17
9.47
6.5
52.63
0.0
0.0
393.9
3189.5
213.2
54.13
50.7
12.87
59.8
15.18
35.1
8.91
35.1
8.91
After 60 days
Control
0.032
Control
3.2
Control
8.0
66.69
100
41.99
62.96
0.0
0.0
20.02
30.02
4.68
7.02
0.0
0.0
32041.1
48044.8
109.2
0.34
30899.7
96.44
40.3
0.13
1.3
0.004
990.6
3.09
36.66
100
18.46
50.35
0.0
0.0
5.33
14.54
12.87
35.11
0.0
0.0
228.8
624.1
54.6
23.86
49.4
21.59
54.6
23.86
70.2
30.68
0.0
0.0
47.45
100
42.64
89.86
0.0
0.0
2.08
4.32
2.73
5.75
0.0
0.0
540.8
1139.7
241.8
44.71
96.2
17.79
70.2
12.98
132.6
24.52
0.0
0.0
After 120 days
Control
0.032
Control
3.2
Control
8.0
2398.5
100
1051.7
43.48
182
7.59
0.0
0.0
1060.8
44.23
104
4.34
262.6
10.9
91
34.65
26
9.9
1.3
0.5
79.3
30.2
65
24.75
3764.8
100
2294.5
60.95
46.8
1.24
187.2
4.97
1050.4
27.9
185.9
4.94
5614.7
149.1
5465.2
97.34
9.1
0.16
27.3
0.49
71.5
1.27
41.6
0.74
709.8
100
275.6
38.83
422.5
59.52
1.3
0.183
10.4
1.47
0.0
0.0
3118.7
439.4
2035.8
65.28
468
15.01
84.5
2.71
455
14.59
75.4
2.42
Continued:
Benomyl
Doses
mg/g
soil
All microorganisms
Mycobacterium
Micromonospora
N.
Cells/g
dry soil
(X108)
N.
Cells/g
dry soil
(X108)
%
N.
Cells/g
dry soil
(X108)
%
%
Nocardia
N.
Cells/g
dry soil
(X108)
%
Arthrobacter
N.
Cells/g
dry
%
soil
(X108)
Bactoderma
N.
Cells/g
dry soil
(X108)
%
After 150 days
Control
0.032
Control
3.2
Control
8.0
25333.1
1684.8
100
6.7
24151.4
894.4
95.34
53.09
1181.7
135.2
4.66
8.02
0.0
169
0.0
10.03
0.0
457.6
0.0
27.16
0.0
28.6
0.0
1.7
3191.5
100
2493.4
78.13
113.1
3.54
559
17.52
26
0.81
0.0
0.0
867.1
27.2
471.9
54.42
57.2
6.6
81.9
9.45
230.1
26.54
26
3.06
672.1
100
516.1
76.79
429
6.38
49.4
7.35
63.7
9.48
0.0
0.0
2164.5
284
1041.3
48.11
5.2
0.24
793
36.64
283.4
13.09
41.6
1.92
Control
0.032
Control
3.2
Control
8.0
1106.43
100
245.7
22.21
0.0
0.0
839.93
75.91
20.8
1.88
0.0
0.0
90896
8215.3
61.1
0.067
26
0.029
26
0.029
10.4
0.011
90772.5
99.86
150.15
100
114.4
76.19
27.3
18.18
7.15
4.76
1.3
0.866
0.0
0.0
88791.3
59135.1
88714.6
99.91
2.6
0.0029
5.2
0.0059
63.7
0.072
5.2
0.0059
20.8
100
1.3
6.25
3.9
18.75
9.1
43.75
6.5
31.25
0.0
0.0
57345.5
275699.5
57048
99.5
6.5
0.01
68.9
0.12
170.1
0.3
52
0.091
Control
0.032
Control
3.2
Control
8.0
15.6
100
0.0
0.0
0.0
0.0
13
83.33
2.6
16.67
0.0
0.0
2870.4
18400
2631.2
91.67
83.2
2.9
49.4
1.72
78
2.72
28.6
0.996
304.2
100
2.6
0.855
1.3
0.43
27.3
8.97
13
4.27
260
85.47
10648.3
3500.4
9128.6
85.73
26
0.24
2.6
0.024
325
3.05
1166.1
10.95
193.7
100
44.2
22.82
39
20.13
27.3
14.09
83.2
42.95
0.0
0.0
67941.9
35075.8
67834
99.84
52
0.077
1.3
0.0019
44.2
0.065
10.4
0.015
Control
0.032
1328.6
100
704.6
53.03
0.0
0.0
546
41.1
78
5.87
0.0
0.0
5929.3
446.3
5816.2
98.09
0.0
0.0
48.1
0.81
65
1.1
0.0
0.0
After 180 days
After 240 days
After 360 days
762
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
Control
3.2
Control
8.0
40.3
100
14.3
35.48
13
32.26
0.0
0.0
13
32.26
0.0
0.0
1831.7
4545.2
1237.6
67.57
26
1.42
28.6
1.56
513.5
28.03
26
1.42
275.6
100
133.9
48.58
26
9.43
0.0
0.0
115.7
41.98
0.0
0.0
68.9
25
1.3
1.89
0.0
0.0
0.0
0.0
41.6
60.38
26
37.74
Fig.1 Inhibition or increment percentages of Pseudomonas sp in Al Geraif soil incubated at 28ºC
by different concentrations of Benomyl (a. 0.032; b. 0.8; c.3.2and d.8.0 mg/g soil)
763
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
Fig.2 Inhibition or increment percentages of Pseudomonas sp in Al Geraif soil amended with
NPK and incubated at 28ºC by different concentrations of Benomyl (a. 0.032; b. 0.8; c.3.2and
d.8.0 mg/g soil)
764
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
Fig.3 Inhibition or increment percentages of organic nitrogen users in Al Geraif soil amended
with NPK and incubated at 28ºC by different concentrations of Benomyl (a. 0.032; b. 0.8;
c.3.2and d.8.0 mg/g soil)
765
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
Fig.4 Inhibition or increment percentages of soil fungi in Al Geraif soil incubated at 28ºC by
different concentrations of Benomyl (a. 0.032; b. 0.8; c.3.2and d.8.0 mg/g soil)
766
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
Fig.5 Inhibition or increment percentages of soil fungi in Al Geraif soil amended with NPK and
incubated at 28ºC by different concentrations of Benomyl (a. 0.032; b. 0.8; c.3.2and d.8.0 mg/g
soil)
767
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
Fig.6 Benomyl degradation rates in Al Geraif soil incubated at 28°C
Fig.8 Benomyl degradation rates in Al Geraif soil amended with NPK and incubated at 28°C
768
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
Generally, higher fungal counts were
observed at higher Benomyl concentrations.
In the same context, Iqbal et al. (2001)
found that application of Monocrotophos
and Menthamidophos insecticides with NPK
fertilizers increased the fungal population in
clay loam soil. Whereas, Banerjee and Dey
(1992) found that Basalin herbicide in
conjunction with NPK fertilizers decreased
the total number of fungi in sandy clay soil.
Effect of NPK amendments on benomyl
degradation
Higher Benomyl degradation rates were
obvious at higher Benomyl concentrations.
In line with this, Wang et al. (2009) reported
higher degradation rates of Carbendazim at
higher than at lower concentrations. The
biodegradation of organic compounds is
often slow because one or more of
inorganic nutrients needed for microbial
growth are in low concentrations in natural
environments (Toolan et al. 1991; Coveney
and Wetzel 1992). It was suggested that the
addition of N and P may increase the
biodegradation of organic compounds
(Pritchard and Costa 1991). Results of
Mohammed et al. (2011) indicated that the
degradation of Oxyfluorfen in soils fertilized
with NPK was higher than in non-fertilized
soil
Counts of microorganisms using inorganic
nitrogen underwent a general suppression at
the first two concentrations as a result of
adding NPK to Benomyl treated soil. In
contrast, Jones and Alexander (1988) found
that adding nitrogen and phosphorus to the
soil enhances microbial growth. Mineral
fertilization of arable land positively affects
and increases the biological productivity of
various ecosystems as well as the microbial
activity in soil (Barabasz et al. 2002).
However, Mohammed et al. (2011) reported
that the addition of NPK fertilizers to
Oxyfluorfen treated soil stimulated the
growth of inorganic nitrogen bacteria
incubated at 40ºC while actinomycetes were
suppressed at the end of incubation period
(45 days).
The most obvious characteristic was that
with the addition of NPK the degradation
percentage for Benomyl was high since after
the first month of incubation for the higher
fungicide concentrations. Degradation was
71% and 94 % for 3.2 and 8.0 mg
Benomyl/g soil, respectively at 60 days
interval. Addition of NPK up to 0.5%
increased Chlorpyrifos degradation during
the first days of incubation (Tortella et al.
2010).
Generally NPK stimulated growth of
microorganisms capable of growing in poor
medium at higher Benomyl concentrations.
Mycobacterium was absolutely dominant
followed by Arthrobacter. Mohammed
(2009) found that the addition of NPK
fertilizers clearly increased the number of
microorganisms
at
all
Oxyfluorfen
concentrations tested and Mycobacterium
was the dominant species throughout the
incubation periods. Elsaid et al. (2009)
found that application of Endosulfan
insecticide and urea + phosphate fertilizers
increased the number of inorganic nitrogen
using microbes in soils after 45 days of
incubation
Addition of NPK has increased microbial
counts substantially which in turn has been
reflected positively on Benomyl degradation
rates. Benomyl has a remarkable stimulating
effect (despite the considerable variations),
on the growth and multiplication of different
microorganisms. The highest percent
increments in microbial counts have always
been recorded for Pseudomonas sp.
followed by organic nitrogen users and
fungi. Maximum degradation percentage in
soil was 99.9% at 8.0 mg Benomyl/g soil
after 360 days of incubation for the control
769
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 756-771
and 99.4% for NPK at 3.2 mg Benomyl/g
soil after 180 days of incubation.
characterization of a new Carbendazimdegrading Ralstonia sp. strain. World J
Microbiol Biotechnol., 21: 265-269.
Zweig, G. and R. Gao, 1983. Determination
of Benomyl by reversed-phase liquid
chromatography. Anal Chem., 55 (8):
1448-1451.
Fuchs, A. and F.W. DeVries, 1978. Bacterial
breakdown of Benomyl. II. Mixed
cultures. Antonie Van Leeuwenhoek.,
44(3-4): 283-292.
Jones, S.H. and M. Alexander, 1988.
Phosphorus
enhancement
of
mineralization of low concentrations of
p-nitrophenol by Flavobacterium sp in
lake water. FEMS Microbiol Lett., 52:
121-126.
Lopez-Garcia, B., A. Veyrat, E. Perez-Paya,
L. Gonzalez-Candelas and J.F. Marcos,
2003. Comparison of the activity of
antifungal hexapeptides and the
fungicides thiabendazole and Imazalil
against postharvest fungal pathogens.
Inter J Food Microbiol., 89: 163 170.
Mohamed, A.T., A.A. Elhussein, M.A.
Elsiddig and A.G. Osman, 2011.
Degradation of Oxyflurofen herbicide
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Mohamed, A.T., 2009. An investigation of
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Elsaid, O.E.G., A.O. Abdelbagi and E.A.E.
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Pesticide Action Network, 2002. Pesticide
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Tepper, E.Z., V.K. Shilnikova and G.I.
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