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African Journal of Biotechnology Vol. 10(5), pp. 770-778, 31 January, 2011
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 ©2011 Academic Journals
Full Length Research Paper
Effect of four herbicides on microbial population, soil
organic matter and dehydrogenase activity
A. Sebiomo1*, V. W. Ogundero2 and S. A. Bankole2
1Department of Biological Sciences, Tai Solarin University of Education, Ijagun Ijebu-Ode, Ogun State, Nigeria.
2Department of Microbiology, Olabisi Onabanjo University, Ago-Iwoye, Ogun State, Nigeria.
Accepted 3 September, 2010
The effect of four herbicides (atrazine, primeextra, paraquat and glyphosate) on soil microbial
population, soil organic matter and dehydrogenase activity was assessed over a period of six weeks.
Soil samples from cassava farms were treated with herbicides at company recommended rates. Soil
dehydrogenase activity was measured at four-day sampling intervals up to the 20th day. Bacterial,
fungal and actinomycetes populations decreased upon treatment with herbicides when compared to the
control. There was significant reduction in percentage organic matter after the herbicides were applied
to soils. Soil organic matter then increased after continuous application from the second to the sixth
week of treatment. Herbicide treatment resulted in a significant drop in dehydrogenase activity when
compared to the control soil samples. Obtained results indicated that soils treated with primeextra had
the lowest dehydrogenase activity of 16.09 µg (g-1min-1) after the sixth week of treatment, while soils
treated with glyphosate had the highest dehydrogenase activity of 20.16 µg (g-1min-1) when compared to
other herbicides used for treatment. Dehydrogenase activity increased from the second to the sixth
week of treatment. This study indicated significant response of soil microbial activity to herbicide
treatment and increased adaptation of the microbial community to the stress caused by increase in
concentration of the herbicides over weeks of treatment.
Key words: Herbicides, soil organic matter, dehydrogenase activity, treatment.
INTRODUCTION
The increased use of pesticides in agricultural soils
causes the contamination of the soil with toxic chemicals.
When pesticides are applied, the possibilities exist that
these chemicals may exert certain effects on non-target
organisms, including soil microorganisms (Wardle and
Parkinson, 1990; Simon-Sylvestre and Fournier, 1979).
The microbial biomass plays an important role in the soil
ecosystem where they fulfill a crucial role in nutrient
cycling and decomposition (De-Lorenzo et al., 2001).
During the past four decades, a large number of
herbicides have been introduced as pre and post-emergent
weed killers in many countries of the world. In Nigeria,
*Corresponding author. E-mail: rev20032002@yahoo.com. Tel:
+2348077675121 or +2347039334401.
Abbreviations: NA, Nutrient agar; PDA, potato dextrose agar;
INT, iodonitrotetrazolium chloride; DH, dehydrogenase; INTF,
iodonitrotetrazolium formazan.
herbicides have since effectively been used to control
weeds in agricultural systems (Adenikinju and Folarin,
1976). As farmers continue to realize the usefulness of
herbicides, larger quantities are applied to the soil. But
the fate of these compounds in the soils is becoming
increasingly important since they could be leached, in
which case groundwater is contaminated or immobile,
and persist on the top soil (Ayansina et al., 2003). These
herbicides could then accumulate to toxic levels in the
soil and become harmful to microorganisms, plant, wild
life and man (Amakiri, 1982). There is an increasing con-
cern that herbicides not only affect the target organisms
(weeds) but also the microbial communities present in
soils, and these non-target effects may reduce the
performance of important soil functions. These critical soil
functions include organic matter degradation, the nitrogen
cycle and methane oxidation (Hutsch, 2001).
Both glyphosate and paraquat have been reported to
cause activation in soil urease and invertase soil enzy-
mes (Sannino and Gianfreda, 2001), while diquat and
paraquat increased fungal populations (Mewatankarn and
Sivasithamparam, 1987). A degradative microbial popul-
ation that has adapted to the introduced compounds may
exist in many contaminated locations, therefore, it is
necessary to search for various microorganisms which
would be able to reduce water or soil pollution.
All the transformations of nutrients occurring in soil are
stimulated by the enzymes that condition their conversion
into forms available to plants and microorganisms.
Enzymes are frequently referred to as markers of soil
environment purity (Aon and Colaneri, 2001). Microbial
activity measurements appear as good indicators of the
degree of pollution of contaminated soils (Nordgren et al.,
1988; Aoyama and Nagumo, 1995; Insam et al., 1996;
Kuperman and Margret, 1997). Dehydrogenase is
thought to be an indicator of overall microbial activity,
because it occurs intercellularlly in all living microbial
cells and is linked with microbial oxydoreduction proce-
sses (Quilchano and Maranon, 2002; Stepniewska and
Wolinska, 2005). It is a specific kind of enzyme which
plays significant role in the biological oxidation of soil
organic matter by transferring protons and electrons from
substrates to acceptors. Soil dehydrogenase activity is
considered to be a valuable parameter for assessing the
side effects of herbicides treatments on the soil microbial
biomass.
This study was designed to investigate the effect of four
types of herbicides on microbial population, soil organic
matter and soil microbial activity, using soil dehydro-
genase as an indicator of soil microbial activity.
MATERIALS AND METHODS
Soil sampling
Top soil (up to 5 cm depth) samples were collected from cassava
farm in Ijebu-Ode (Ogun State, Nigeria), with no prior pesticide
treatment. The soil samples were sieved through a 2.0 mm mesh
size to remove stones and plant debris.
Herbicides
The herbicides used in this study were obtained from a local
agricultural dealer store in Ibadan. The herbicides used were: Atry-
lone 80WP, trademark of Insis Limited (atrazine); primextra, a
product of Syngenta (a combination of atrazine and metolachlor);
Glysate, Nantong Ji Angshan Agrochemicals (glyphosate); and
Gramoxone, a product of Syngenta (paraquat).
Soil treatments
The treatments were carried out for a period of 6 weeks at company
recommended rates of 4l/h (at 350 ml in 15 L sprayer) for paraquat,
glyphosate and primeextra, while recommended rate of 3 kg/h
(atrazine powder) was used for atrazine treatment (soil treatments
were carried out in triplicates).
Microbial enumeration
Nutrient agar (NA) was used for the enumeration of total hetero-
Sebiomo et al. 771
trophic bacteria by the pour plate method. Incubation was done at
30°C for 24 - 48h. Potato dextrose agar (PDA) was used for
enumeration and isolation of fungi. Incubation was at 25°C for 48 h.
Bacterial and actinomycetes isolates were characterized based on
cultural characteristics, staining reactions and biochemical react-
ions. Identification was thereafter made with reference to Bergey’s
manual of systemic bacteriology (1984). Starch Casein Agar was
used for the enumeration of total actinomycetes counts. Fungal
isolates were characterized as described by Barnett and Hunter
(1972).
Determination of organic matter in soil
The percentage organic matter was determined by the method
described by FAO (1974). Soil samples were collected and ground
to pass through 0.5 mm sieve. One gram of each soil sample was
weighed into 250 ml Erlenmeyer flasks and 10 ml of K2Cr2O7
solution was dissolved into each flask and swirled gently to
disperse soil. Twenty milliliters of concentrated H2SO4 was rapidly
added using automatic pipette and swirled gently until the soil and
reagents were mixed, then the mixture was swirled more vigorously
for one minute, the flasks were then rotated and allowed to stand in
a sheet of asbestos for about 30 min. One hundred milliliters of
distilled water was added to each flask, then 3 - 4 drops of indicator
(ferroin) was added and filterated with 0.5 N ferrous sulphate
solution to the end point, from greenish or dark green to red
(maroon colour) and in reflected light against a white background.
The organic matter was calculated according to the following
formula:
%Organic matter = (me
K
2
SO
4
– me FeSO
4
) x 0.003 x 100 x f x1.729
Weight of air-dried soil
% Organic matter =
Where, correlation factor “ƒ” = 1.33, me = normality of solution ×
milliliter of solution used and 1.729 = conversion.
Soil dehydrogenase activity
Soil microbial activity was estimated by soil dehydrogenase activity
(DH). Soil DH-activity was measured at four-day sampling intervals
up to the 20th day. Measurements were made according to the
iodonitrotetrazolium chloride (INT) method (von Merci and Schinner,
1991). Sub samples of 5 g (air-dried basis) were weighed into test
tubes and mixed with a 10 ml aqueous solution of INT (10 mgl-1).
Test tubes were sealed and incubated in the dark at 40 ± 0.5°C for
2 h and slightly shaken. Developed iodonitrotetrazolium formazan
(INTF) was extracted by keeping the test tubes in the dark for 1 h,
shaking vigorously every 20 min and finally, filtering the solution.
Soil moisture content was maintained at 60% water holding
capacity by weighing and correcting for any weight loss, using
sterile ultra pure water. The INTF was measured spectroph-
otometrically at 464 nm, after extraction with 10 ml of N, N-
dimethylformamide/ethanol solution. Soil DH-activity and all soil
treatments were done in triplicates. Dehydrogenase activity was
determined using the following equation:
CfV106W
Mfmt
A =
Where, A = Soil dehydrogenase activity, Cƒ = concentration of
iodonitrotetrazolium formazan (mg ml-1), V = volume of added
solutions, Mƒ = molar mass of INTF, m = soil mass, W = dampness
coefficient and t = time.
772 Afr. J. Biotechnol.
Table 1. Effect of herbicide treatment on
actinomycetes count (ANOVA).
Factors P-value
Herbicide treatment 0.201
Weeks of herbicide treatment 0.007
Herbicide × weeks of treatment 0.354
Table 2. Effect of herbicide treatment on
bacterial count (ANOVA).
Factors P-value
Herbicide treatment <0.001
Weeks of herbicide treatment <0.001
Herbicide × weeks of treatment <0.001
Statistical analysis
Data generated from this study was expressed in bar charts, line
graphs and subjected to analysis of variance (ANOVA) and
correlation coefficient analysis.
RESULTS
Presented in Table 2 is the effect of herbicide treatment
on bacterial count. Herbicide treatment had high signi-
ficant effect on bacterial count (P < 0.001). The inter-
action between the herbicides and the weeks of herbicide
treatment also resulted to high significant effect on
bacterial count (P < 0.001). The weeks of herbicide treat-
ment followed similar trend as stated above (P < 0.001).
In Figure 1, soil bacterial populations in the cont-rol
samples were found to be significantly higher than those
of herbicide treated soils. The bacterial populations for all
soil samples increased from 2nd to the 6th week of
treatment (including control samples). After the second
and fourth weeks of treatment, soils treated with glypho-
sate had the highest bacterial populations of 3.9 x 104
and 5.67 x 104 cfu/g of all treated soils. The glyphosate
treated soil also after the 6th week of treatment had the
highest bacterial population of 5.9 x 104 cfu/g, while soil
samples treated with paraquat had the lowest bacterial
populations of 2.63 x 104, 3.67 x 104 and 4.57 x 104 cfu/g
after the second, fourth and sixth weeks of treatment,
respectively (Figure 1).
Analysis of variance results presented in Table 1 shows
that only the weeks of herbicide treatment had significant
effect on actinomycetes count (P < 0.007), meanwhile
herbicide treatment and the interaction between herbicide
and weeks of herbicide treatment had little effect on
actinomycetes population (P = 0.201 and P = 0.354).
Figure 2 shows the effect of herbicide treated soils on
actinomycetes populations. Similar to Figure 1, the
control soil samples had the highest actinomycetes
populations of 8.53 x 103, 5.23x104 and 8.47 x 104 cfu/g
after the second, fourth and sixth weeks of treatment,
respectively. Soil samples treated with primeextra had
the highest actinomycetes population of 2.67 x 104 cfu/g
after two weeks of treatment, while paraquat treated soils
had the highest actinomycetes population of 3.93 x 104
cfu/g after six weeks of treatment (Figure 2).
In Figure 3, the fungal populations fluctuated between
the second and sitxh weeks, while the control samples
had the highest fungal populations of 1.9 x 104 and 1.6 x
104 cfu/g on the second and sixth week, respectively. The
soils treated with primeextra had the highest fungal
population of 1.8 x 104 cfu/g. Paraquat treated soils had
the lowest fungal population of 8.7 x 103 cfu/g after six
weeks of treatment. The ANOVA results presented in
Table 3 followed similar trends with the bacterial counts
presented in Table 2.
The herbicide treatments had significant effect on
percentage organic matter of the soil (P < 0.001) (Table 4
and Figure 4). Weeks of herbicide treatment and the
interaction between herbicides and weeks of treatment
had similar effect (P < 0.001) (Table 4). The percentage
organic matter of the soils treated with herbicides
reduced significantly when compared with the control.
Soil samples treated with glyphosate had the lowest
percentage organic matter contents of 1.25, 2.35 and
3.17 of all herbicide treated soils after the second, fourth
and sixth week of treatment, respectively (Figure 3).
Paraquat treated soils had the highest percentage
organic matter contents of 4.68, 5.03 and 5.20 after the
second, fourth and sixth week of treatment, respectively.
Meanwhile, percentage organic matter of herbicide
treated soil samples as well as those of control samples
increased from the second to the sixth week of treatment.
The results obtained in correlation coefficient analysis
between soil organic matter and actinomycetes counts
shows positive correlation (correlation coeficient value =
0.376). There was also positive correlation between soil
organic matter and fungi count (correlation coeficient
value = 0.462). Meanwhile, the corrrelation coefficient
analysis between soil organic matter and bacterial counts
showed the strongest positive correlation (correlation
coeficient value = 0.672).
All the herbicides used for treatment in this study
resulted in significant (P < 0.001) (Table 5) reductions in
soil dehydrogenase activity when compared to the
control. Soils treated with primeextra had the lowest set
of soil dehydrogenase activities of 9.02, 12.55 and 16.09
µg (g-1min-1) after the second, fourth and sixth week of
treatment, respectively (Figures 5, 6 and 7). Meanwhile,
soils treated with glyphosate had the highest set of soil
dehydrogenase activities after the second and sixth week
of treatment (Figures 5 and 7) when compared to other
herbicide treatments, while soils treated with atrazine had
the highest dehydrogenase activity of 14.32 µg (g-1min-1)
after the fourth week of treatment. In Figure 7, after the
sixth week of treatment, glyphosate treated soils had the
Sebiomo et al. 773
Herb
i
cides treated soils
Cfu/g (x10
3
)
Figure 1. Effect of herbicides on soil bacterial populations. CON = Control; ATR = atrazine treated soil; GLS= glyphosate
treated soils; GRZ = paraquat treated soils; PM = primeextra treated soils. For herbicide treatment, weeks of herbicide
treatment and herbicide x week of treatment, P-value <0.001.
Cfu/g (x10
3
)
Herbicides treated soil
Figure 2. Effect of herbicides on actinomycetes populations. P-values for herbicide treatment, weeks of herbicide
treatment and herbicide x week of treatment were 0.201, 0.007 and 0.354, respectively.
highest dehydrogenase activity of 20.16 µg (g-1min-1),
while soils treated with primeextra had the lowest
dehydrogenase activity of 16.09 µg (g-1min-1) after the
sixth week of treatment when compared to other herbi-
cide treatments. In Table 5, weeks of herbicide treatment
had significant (P < 0.002) effect on soil dehydrogenase
activity. All soil dehydrogenase activities increased from
the second to the sixth week.
774 Afr. J. Biotechnol.
Herbicides treated soil
Cfu/g (x10
3
)
Figure 3. Effect of herbicides on soil fungal populations. For herbicide treatment, weeks of herbicide treatment and
herbicide x week of treatment, P-value <0.001.
Table 3. Effect of herbicide treatment on fungal
count (ANOVA).
Factors P-value
Herbicide treatment <0.001
Weeks of herbicide treatment <0.001
Herbicide × weeks of treatment <0.001
The days of incubation had significant effect (P 0.001)
(Table 5) on soil dehydrogenase activities. The results in
Figures 5, 6 and 7 shows that as the days of incubation
increased, the soil dehydrogenase activities also in-
creased concomitantly to the 20th day. The results of the
control samples showed higher increases than those of
the herbicide treated soils which dropped upon treatment
with herbicides. After the 20th day of incubation,
glyphosate treated soils had the highest dehydrogenase
activities of 11.49 and 20.16 µg (g-1min-1) at the second
and sixth week, respectively, while prime-extra treated
soils had the lowest after 20th day of incubation with
values of 9.02, 12.55 and 16.09 µg (g-1min-1) after the
second, fourth and sixth week, respectively.
DISCUSSION
In this study, the observed trends in microbial population
were similar to observations made by Korpraditskul et al.
(1988). Ayansina and Oso (2006) discovered that higher
Table 4. Effect of herbicide treatment on soil
organic matter (ANOVA).
Factors P-value
Herbicide treatment <0.001
Weeks of herbicide treatment <0.001
Herbicide × weeks of treatment <0.001
concentrations of herbicides treatments resulted in much
lower microbial counts when compared to soils treated
with recommended doses. Experiments have shown that
microbes may use herbicides as a source of carbon
(Radosevich et al., 1995). This may consequently explain
the increase in microbial populations obtained in this
study from the second to the sixth week of application of
the herbicides. Some studies report increased popula-
tions of actinomycetes and fungi after treatment with
glyphosate (Araujo et al., 2003), increased soil microbial
biomass (Hanley et al., 2002) or no long-term change in
microbial populations (Busse et al., 2001)
There was significant reduction in percentage organic
matter after the herbicides were applied to soils, although
organic matter increased after continuous application
from the second to the sixth week of treatment. Ayansina
and Oso (2006) reported that soil treatment with atrazine
resulted in significant changes in percentage organic
matter measurements. Ali (1990) had shown that the fate
of pesticides in soils is greatly affected by the presence of
organic matter in the soil by aiding their disappearance.
Sebiomo et al. 775
% Organic matter
Herbicide treated soils
Figure 4. Percentage organic matter content of soil samples. For herbicide treatment, weeks of herbicide treatment and herbicide x week
of treatment, P-value <0.001.
Table 5. Effect of herbicide treatment on soil
dehydrogenase activity (ANOVA).
Factors P-value
Herbicide treatment <0.001
Weeks of herbicide treatment <0.002
Days of incubation <0.001
The application of herbicides to the soils led to a
significant drop in dehydrogenase activity with respect to
untreated control soil samples. Obtained results indicated
that soils treated with primeextra had the lowest dehydro-
genase activity, while soils treated with glyphosate had
the highest dehydrogenase activity when compared to
other herbicides used in this study. In literature, opposite
effects on several soil enzymes are reported (Gianfreda
et al., 1994, 2005; Quilchano and Maranon 2002).
Glyphosate was found to inhibit dehydrogenase activities
in sandy loam soil (Dzantor and Felsot, 1991). No effects
on soil dehydrogenase activity were detected by
Lethbridge et al. (1981) and Nakamura et al. (1990).
Reduced enzymatic activities were also found by Perucci
and Scarponi (1990) and Dzantor and Felsot (1991) in
studies on the interference of atrazine with phosphatase,
dehydrogenase and esterase activity of soil.
In this study, dehydrogenase activity increased from
the second to the sixth week of treatment. This might be
due to increase in microbial populations with the
capability of utilizing the herbicides as carbon source.
Under laboratory conditions, a normal dose of glyphosate
inhibited dehydrogenase activity by 5 - 10% (3 weeks
after herbicide application) (Nada and Mitar, 2002). A
tenfold dose of glyphosate affected negatively, the activity
of this oxide-reducing enzyme by 5% (11 weeks after
herbicide application) (Schuster and Schroder, 1990).
Dehydrogenase activity increased from the 4th to the
20th day of incubation in the present study. Moreno et al.
(2007) reported that an increase in metabolic activity with
atrazine concentration and with incubation time can be
deduced from their work. Similar results were presented
by Rossel et al. (1997).
This study has shown that there exists positive corre-
lation between microbial population and soil organic
matter and that the variation in soil microbial activity
represents the capacity of microorganisms to respond to
inputs of herbicides. Microbial activity increased as an
adaptation to the stress caused by increase in concen-
tration of the herbicides over weeks of treatment. The
results obtained demonstrate a potential capacity for
adaptation of the microorganisms in soils when large
amounts of herbicides are added. Dehydrogenase activity
776 Afr. J. Biotechnol.
Day
4th
8
th
12
th
16
th
20
th
Dehydrogenase activity
(
µ
g g
-1
min
-1
)
Figure 5. Effect of herbicide treatment on soil dehydrogenase activity after two weeks of treatment. For herbicide treatment, weeks of herbicide
treatment and days of incubation, P-value <0.001, 0.002 and 0.001, respectively.
20
th
16
th
4
th
8
th
12
th
Day
Dehydrogenase activity
(
µ
g g
-1
min
-1
)
Figure 6. Effect of herbicide treatment on soil dehydrogenase activity after four weeks of treatment. For herbicide treatment, weeks of herbicide
treatment and days of incubation, P-value <0.001, 0.002 and 0.001, respectively.
Sebiomo et al. 777
Dehydrogenase activity
(
µ
g g
-1
min
-1
)
4th 8th 12th 16th 20th
Day
Figure 7. Effect of herbicide treatment on soil dehydrogenase activity after six weeks of treatment. For herbicide treatment, weeks of
herbicide treatment and days of incubation, P-value <0.001, 0.002 and 0.001, respectively.
is a sensitive bioindicator of the microbial activity resp-
onse to herbicide inputs.
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