international journal of research in environmental science ...affecting cellular membrane, and (d)...

100 International Journal of Research in Environmental Science and Technology 2013; 3(4): 100-112

ISSN 2249–9695

Original Article

EFFECT OF FOUR HERBICIDES ON SOIL ORGANIC CARBON,

MICROBIAL BIOMASS-C, ENZYME ACTIVITY AND MICROBIAL

POPULATIONS IN AGRICULTURAL SOIL

Mayeetreyee Baboo, Mamata Pasayat, Alka Samal, Monty Kujur, Jitesh Kumar Maharana and Amiya Kumar Patel*

School of Life Sciences, Sambalpur University

At/po- Jyoti Vihar, Burla; Pin- 768019

Dist- Sambalpur, Odisha, India.

E-mail: [email protected]

Received 06 September 2013; accepted 09 October 2013

Abstract

Herbicides are biologically active compounds, and an unintended consequence of its application may lead to significant

changes in microbial populations and activities influencing microbial ecological balance affecting soil fertility. The fate of

herbicides applied in agricultural ecosystems is governed by the transfer and degradation processes, and their interaction

with soil microorganisms. The increasing reliance of sustainable agriculture on herbicides has led to concern about their

ecotoxicological effects influencing microbial populations and enzyme activities, which may serve as indicators of soil

quality. The effects of herbicides (butachlor, pyrozosulfuran, paraquat and glyphosate) on soil organic carbon, microbial

biomass-C, enzyme activities (amylase, invertase, protease, urease and dehydrogenase) were assessed over a period of four

weeks. There was significant reduction in organic carbon with time. Herbicides treatment resulted an initial increase upto

14th

day followed by a significant drop in microbial biomass-C. Herbicide treatments resulted variation in enzyme

activities, while highest activity was recorded for control soil. The gradual increase in microbial counts may be attributed

to their ability to temporarily mineralize and use herbicides as energy source. The study suggested that the herbicides cause

transient impact on microbial populations and enzyme activities associated with the type of herbicides at recommended

field application rate.

© 2013 Universal Research Publications. All rights reserved

Keywords: herbicides, microbial biomass C, enzyme activity, microbial population.

1. INTRODUCTION

The sustainable agriculture involves optimizing agricultural

resources and at the same time maintaining the quality of

environment and sustaining natural resources. In achieving

this optimization, the soil microbial community

composition is of great importance, because they play a

crucial role in carbon flow, nutrient cycling and litter

decomposition, which in turn affect soil fertility and plant

growth (Chauhan et al., 2006; Tripathi et al., 2006; Pandey

et al., 2007), and hence occupy a unique position in

biological cycles in terrestrial habitat. The soil microbial

biomass is considered as active nutrient pool to plants and

plays an important role in nutrient cycling and

decomposition in ecosystem (De-Lorenzo et al., 2001). A

healthy population of soil microorganisms can stabilize the

ecological system in soil (Chauhan et al., 2006) due to their

ability to regenerate nutrients to support plant growth. Any

change in their population and activity may affect nutrient

cycling as well as availability of nutrients, which indirectly

affect productivity and other soil functions (Wang et al.,

2008).

Natural and anthropogenic factors may affect the soil

enzyme activities directly or indirectly (Gainfreda and

Bollag, 1996). Among anthropogenic factors, pesticides are

of primary importance due to their continuous entry into

the soil environment. Herbicides are one of the major

groups of pesticides, which include substances or cultured

biological organism used to kill or suppress the growth of

unwanted plants and vegetation (Cork and Krueger, 1992)

in order to minimize the cultivation cost as well as to

sustain high yield. A number of herbicides have not only

been introduced as pre- or post-emergence weed killer

(Ayansina and Oso, 2006) but also leave unwanted residues

in soil, which are ecologically harmful (Haney et al., 2000;

Derksen et al., 2002; Riaz et al., 2007). Preferred

herbicides should not only have good efficacy, but also

poses minimum adverse effects to crop, ecology and

environment (Constenla et al., 1990; Hoerlein, 1994).

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Herbicides not only affect the target organisms, but also

microbial communities in soil. These non-target effects

may reduce the performance of important soil functions

(Altimirska and Karev, 1994; Perucci and Scarponi, 1994;

Hutsh, 2001) and poses a risk to the entire ecological

system (Kalia and Gupta, 2004) by (a) changing their

biosynthetic mechanism, (b) affecting protein synthesis, (c)

affecting cellular membrane, and (d) affecting plant growth

regulators.

Herbicides are extraneous to soil component pools, and are

expected to affect the catalytic efficiency, behavior of soil

enzymes (Bollag and Liu, 1990; Sannino and Gianfreda,

2001), which contribute to the total biological activity of

the soil-plant environment under different states (Dick,

1995; Dick, 1997). The interaction between herbicides and

soil microorganisms may be of practical significance

because of possible inhibition in microbial activities

contributing to soil fertility. Various studies have revealed

that the herbicides can cause qualitative and quantitative

change in enzyme activity (Sannino and Gianfreda, 2001;

Min et al., 2001; Saeki and Toyota, 2004; Sebiomo et al.,

2011, Xia et al., 2011). Hence, the effects of herbicides on

soil microbial communities addressing the apprehensions

about the environmental impacts of herbicide use.

Butachlor [2-chloro-2, 6-diethyl-N- (butoxymethyl) -

acetanilide] belongs to the family chloroacetanilide group

of herbicides, which inhibits protein synthesis, and is

largely used during pre-emergence and/or early post-

emergence (Yu et al., 2003). Butachlor not only inhibits

cell division by blocking protein synthesis, but also inhibit

synthesis of long chain fatty acids. Paraquat [1,1-dimethyl-

4,4-bipyridinium dichloride] is total vegetative control

herbicide that is strongly adsorbed to soil (Terry et al.,

2002). It also undergoes metabolism and degradation under

a range of conditions. Paraquat is stable in acidic or neutral

solutions, but is hydrolyzed at pH>12. It undergoes

photolysis in aqueous solution to form N- methybetaine of

isonicotinic acid, and subsequently methylamine

hydrochloride (Slade, 1965). Pyrazosulfuron-ethyl [ethyl 5-

(4,6-dimethoxypyrimidin-2-1,carbamoyl) sulfamoyl)

methylpyrazole-4-carboxylate] belongs to sulfonylureas,

which are extensively used to control a wide range of

weeds inhibiting acetolactate synthase (key enzyme in

protein synthesis of plants). It exhibits extremely low, acute

and chronic toxicity in comparison to other herbicides

(Brown, 1990). Glyphosate [N-(phosphonomethyl)glycine]

is a broad-spectrum, non-selective, post-emergence

herbicide that control most of the annual and perennial

weeds by inhibiting aromatic amino acids biosynthesis

(tyrosine, tryptophan, and phenylalanine) involved in

protein synthesis (Araujo et al., 2003; Battaglin et al.,

2005). Besides, it also inhibits 5-enolpyruvylshikimic acid

synthase via the shikimic acid pathway (Franz et al., 1997),

which is ubiquitous in microorganisms (Bentley, 1990) that

link primary and secondary metabolism (Carlisle and

Trevors, 1988). Most of the living organisms (excluding

plants) lack this pathway, and are thus unaffected directly

by glyphosate. Glyphosate also acts as a competitive

inhibitor of phosphoenolpyruvate, which is one of the

precursors to aromatic amino acid synthesis.

Several scientific investigations have suggested the

importance of preserving soil fertility and quality, and

consequently soil microbial population. As the soil

microorganisms are very sensitive to low concentrations of

contaminants and rapidly response to soil perturbation, they

are considered as an indicator of soil pollution (Shen et al.,

2005). The enzyme activities are considered to be sensitive

to chemical pollutants/agrochemicals and have been

proposed as potential indicators for measuring the degree of

pollution of contaminated soil (Aoyama and Nagumo,

1995; Insam et al., 1996; Kuperman and Margret, 1997),

and referred to as markers of soil environmental purity

(Aon and Colaneri, 2001). The evaluation of soil enzyme

activities may provide useful information on microbial

activity and be helpful in establishing the effects of soil

specific environmental conditions (Andreoni et al., 2004).

An alternation in soil microorganisms, their number,

activity and diversity may serve as indicators of soil

fertility (Milosevia et al., 1997) and reflect the soil quality

(Schloter et al., 2003). However, most of the studies were

focused on single application for a short period, which may

provide a realistic evaluation of the effects of herbicides on

soil microorganism (Haney et al., 2000). However, the

knowledge about the effect of herbicides on soil enzyme

activities and microbial population in long-term

applications has been limited. Therefore, the present study

was designed to elucidate the effects of four herbicides

(butachlor, pyrazosulfuron, paraquat and glyphosate) on

organic carbon, microbial biomass-C and enzyme activities,

which can provide better understanding of the possible

response of soil microorganisms to different herbicides.

2. MATERIALS AND METHODS

2.1 Study Site

The present study was carried out in the agricultural field

located nearby area of Sambalpur University, Sambalpur,

Odisha (Geographical location: 21° 28' 43" North latitude

and 83° 51' 55" East longitude) with no prior herbicide

treatment. The district experiences temperate climate

throughout the year, and experiences a semi-arid climate

(1790 mm rainfall y-1

, annual average temp 26C) with

three distinct seasons i.e. summer, rainy and winter.

2.2 Experimental Set up and Herbicide treatment

The experiment was conducted in plots set-up as

randomized block design, and each plot measured (5 x 20)

m2. The experiment was laid out in five different

agricultural plots [Control (without treatment) and four for

individual herbicide treatment].

The herbicides used in the experiment were commonly

used in the agricultural fields, which were obtained from a

local agricultural store. The herbicides used were:

Butachlor (BUC), Pyrazosulfuron (PYS), Paraquat (PRQ)

and Glyphosate (GLS). The plots were sprayed with four

different herbicides individually (except control) for a

period of four weeks at company recommended rates of 1

kg/ha for Butachlor (Latha and Gopal, 2010), 25g/ha for

Pyrazosulfuron (Latha and Gopal, 2010), 200 g/l for

Paraquat (Wibawa et al., 2009) and 360 g/l for Glyphosate

(Wibawa et al., 2009).

2.3 Soil Sampling and Bioassay

Sampling was done in accordance with the general methods


for soil microbiological study. Top soil sample were

collected from a depth of (0-3) cm from each plot (Saeki

and Toyota, 2004). Each plot was divided into 5 blocks,

and from each block five soil samples were collected

randomly. Samples collected from each block were referred

as ‘sub-samples’, and were thoroughly mixed to form one

‘composite sample’ The composite samples were

homogenized, sieved (0.2 mm) to remove stone and plant

debris, and were analyzed.

The effect of different herbicides in agricultural soil were

analyzed in response to organic carbon, microbial biomass-

C, soil enzyme activity and microbial enumeration with

respect to control soil (without treatment) in triplicates at

regular intervals i.e. 7th

, 14th

, 21st and 28

th day after

treatment for a period of 4 weeks individually.

2.4 Soil Organic Carbon

Soil organic carbon (OC) in different herbicides treated and

control soil samples were determined by partial oxidation

method (Walkley and Black, 1934) through titration against

1N (NH4)2Fe(SO4)2.6H2O using diphenylamine indicator.

2.5 Microbial Biomass-C

Soil samples were stored at (282)ºC for a week to stabilize

respiration, subsequently used for further analysis.

Microbial biomass-C (MB-C) in herbicide treated as well

as control soil sample was determined by fumigation

extraction method (Vance et al., 1987) through back

titration against 0.04N (NH4)2Fe(SO4)2.6H2O using ferroin

indicator.

2.6 Soil Enzyme activity

Amylase activity of different herbicides treated as well as

control soil was determined in adaptation to the procedure

described by Somogyi (1952) and Roberge (1978) by

taking starch as substrate. Invertase activity was determined

by spectrophotometric method (Ross, 1983) by using

sucrose as substrate. Protease activity was also determined

by spectrophotometer (700nm) with sodium caseinate as a

substrate (Ladd and Butler, 1972). Urease activity of

herbicides treated as well as control soil was determined by

titration method using 0.005 N H2SO4 with boric acid

indicator (Tabatabai and Bremner, 1972). Dehydrogenase

activity was measured following reduction of 2,3,5-

triphenylotetrazolium chloride (TTC) to red-coloured

triphenyl formazon (TPF), which were determined

spectrophotometrically (Nannipieri et al., 1990; Alef and

Nannipieri, 1995).

2.7 Microbial enumeration

The microbial populations were enumerated using selective

media by standard spread plate dilution technique.

Azotobacter mannitol agar was used for enumeration of

azotobacter population in different herbicides treated as

well as control soil (ATCC 1992). Arthrobacter populations

were enumerated using Arthrobacter selective media

(Hagedorn and Holt, 1975). Total heterotrophic aerobic

bacteria were enumerated using nutrient agar (Gray 1990).

Actinomycetes isolates were characterized based on

cultural characteristics, staining reactions, biochemical tests

and enumerated using starch-casein agar (Hunter-Cevera

and Eveleigh, 1990). Streptomycin 40μl/ml and

griseofulvin 50μl/ml were used to prevent bacterial and

fungal contaminants (Alharbi et al., 2012). Rose Bengal

agar supplemented with streptomycin 50μl/ml was used for

fungal count (Alef and Nannipieri, 1995).

2.8 Statistical analysis

The data were statistically analyzed using a two-way

analysis of variance (ANOVA) with SPSS Statistics 17.0

software. Principal component analysis was performed in

order to discriminate different treated herbicides treated

soil with respect to control using Statistrix PC DOS

Version-2.0 (NH Analytical software).

3. RESULTS AND DISCUSSION

3.1 Soil Organic Carbon

The variation in OC content was exhibited with respect to

different herbicide treated soil (Figure 1). The OC in

butachlor treated soil was found to be higher after 14th

day

(2.595 ± 0.089) % as compare to 28th

day (2.362 ± 0.068)

% of incubation. The OC in pyrazosulfuron treated soil

indicated decreasing trend from 7th

day (2.49 ± 0.091) % to

28th

day (2.235 ± 0.080) % of incubation. But the OC in

paraquat treated soil increases upto 14th

day (2.467 ±

0.082)% followed by decline on 21st day (2.152 ± 0.064) %

and 28th

day (2.287 ± 0.078)% of incubation. The OC in

glyphosate treated soil was found to be decreased

significantly on 14th

day (1.905 ± 0.063) % as compared to

21st day (2.475 ± 0.086) % and 28

th day (2.325 ± 0.067) %

of incubation respectively. The variation in soil organic

carbon with respect to different herbicides and different

days after treatment was found to be statistically significant

(p<0.001) (Table 2). There was significant reduction in

percentage OC level after the application of herbicides,

although OC increased after continuous application from

the 14th

day of glyphosate treatment (Sebiomo et al., 2011).

However, the herbicides decomposition is frequently faster

in soils with high OC, presumably due to vigorous

microbial activity (Greaves et al., 1976). Further, the fate

of herbicides is greatly affected by the presence of soil

organic matter by aiding their disappearance (Ali, 1990;

Ayansina and Oso 2006).

Figure 1. Effect of herbicides on soil OC % on different

days after treatment.

3.2 Microbial Biomass Carbon

The microbial biomass-C showed a decline trend from 7th

day to 28th

day of incubation in all herbicide treated soil

(Figure 2). The effect of butachlor on MB-C was found to

be highest on 14th

day (492.5 ± 31.219 µg/g soil) as

compared to 28th day (315.5 ± 12.505 µg/g soil) of


incubation. Similar trend was also exhibited in

pyrazosulfuron [(531.5 ± 40.619 µg/g soil) to (295.2 ±

10.673 µg/g soil)]; paraquat [(505.1 ± 29.731 µg/g soil) to

(385.5 ± 19.613 µg/g soil)] and glyphosate [(575.5 ±

45.813 µg/g soil) to (476.5 ± 28.106 µg/g soil)] treated soil

respectively. Further, the variation in microbial biomass-C

with respect to different herbicides and days after treatment

was statistically significant (p<0.001) (Table 2).

Figure 2. Effect of herbicides on Microbial biomass-C on

different days after treatment.

The biomass of microorganisms is one of the important

properties of ecological studies, which can be related to

parameters describing microbial activity and soil health

(Bolter et al., 2006). Short-term effects in response to the

use of herbicides are related to disturbances of chemical

and biological balance in soil, and are known to selectively

suppress the activity of soil microorganisms. The decrease

in MB-C may be due to the adsorption of small amount of

pesticides on organic matter that mask the affects of these

agrochemicals on soil microbial biomass, and subsequently

led to lysis of microbial cells (Perucci and Scarponi, 1994;

Jayamadhuri and Rangaswamy, 2005). Herbicides affect

various soil microbial processes (Johen and Drew, 1977;

Wainwright, 1978), inhibit decomposition (Grossbard and

Wingfield, 1978), which depends upon the type and rate of

application that can alter the microbial biomass

quantitatively and qualitatively in both short-term and long-

term (Anderson and Armstrong, 1981). Besides, herbicides

affect non-target microorganisms by interfering with vital

processes such as respiration, photosynthesis and

biosynthetic reactions as well as cell growth and division

and molecular composition (De Lorenzo et al., 2001).

Increase in MB-C in some herbicide treated soil may be

due to the fact that some of the herbicides acting as the

source of nutrients (Cook and Hutter, 1981; Wardle and

Rahman, 1992), in which case they significantly affect

microbial growth and multiplication. However, the effect of

herbicides is usually short-term and minor, when compared

with natural, spatial and temporal variation in soil

microbial biomass.

3.3 Soil Enzyme activity

The amylase and invertase activity have been selected for

their importance in soil carbon cycle. Besides, the protease

and urease activity was selected for their involvement in

soil nitrogen cycle. However, soil dehydrogenase was

estimated in order to determine overall microbial activity.

The variation in soil enzyme activities (amylase, invertase,

protease, urease and dehydrogenase) in different herbicides

treated soil is evident form Table 1.

Table 1. Effect of herbicides on enzyme activity on different days after treatment.

Enzyme Days Control Butachlor Pyrazosulfuron Paraquat Glyphosate

Amylase

7 44.976 ± 0.935 6.87 ±0.092 3.75 ± 0.145 17.5 ± 0.442 6.25 ± 0.365

14 46.547 ± 1.015 52.73 ± 0.072 42.56 ± 0.268 36.42 ± 0.680 25.32 ± 1.175

21 49.654 ± 1.214 71.25 ± 1.00 61.25 ± 0.757 11.25 ± 0.242 85.0 ±0.153

28 53.874 ± 1.874 12.7 ± 0.256 12.9 ± 0.801 3.5 ± 0.527 10.0 ± 2.096

Inveratse

7 589.246 ± 6.00 4.0 ±0.433 15.0 ± 0.75 11.25 ± 0.566 3.0 ± 0.661

14 624.547 ± 6.128 12.5 ± 2.055 5.0 ± 1.022 84.37 ± 1.453 17.5 ± 0.708

21 657.741 ± 6.984 15.0± 1.561 6.25 ± 1.975 11.25 ± 0.6 3.75 ± 1.515

28 713.754 ± 7.587 23.9 ± 1.247 93 ± 0.981 20.8 ± 2.291 52.0 ± 2.291

Protease

7 122.717 ±5.484 76.452 ± 1.654 78.549 ± 1.547 54.874 ± 0.825 61.54 ± 0.952

14 123.451 ± 5.573 86.021 ± 1.987 87.576 ± 1.994 56.745 ± 0.857 62.475 ± 0.958

21 123.984 ± 5.942 89.418 ± 2.487 91.247 ± 2.547 59.465 ± 0.879 63.852 ± 0.968

28 124.0124 ± 6.019 91.548 ± 2.697 92.784 ± 2.845 61.24 ± 0.893 64.579 ± 0.973

Urease

7 24.656 ± 0.086 5.68 ± 0.023 6.2 ± 0.034 3.8 ± 0.013 4.2 ± 0.025

14 25.015 ± 0.089 6.514 ± 0.028 7.1 ± 0.038 4.2 ± 0.018 5.1 ± 0.029

21 25.745 ± 0.091 8.547 ± 0.031 7.8 ± 0.041 5.3 ± 0.021 6.1 ± 0.032

28 26.216 ± 0.095 9.741 ± 0.035 8.4 ± 0.043 6.4 ± 0.024 6.9 ± 0.038

Dehydrogenase

7 4.568 ± 0.085 2.208 ± 0.170 1.187 ± 0.042 3.062 ± 0.912 2.145 ± 0.043

14 4.987 ± 0.105 6.669 ± 0.587 4.3 ± 0.884 8.3 ± 0.404 5.7 ± 0.671

21 5.124 ± 0.185 9.6 ± 0.35 5.5 ± 0.902 9.5 ± 0.208 10.0 ± 0.304

28 5.984 ± 0.196 11.0 ± 0.276 12.15 ± 0.916 14.25 ± 1.946 17.75 ± 0.892

Amylase activity in butachlor treated soil exhibited an

increasing trend from 7th

day (6.87 ± 0.092 µg glucose/g

soil/hr) to 21st

day (71.25 ± 5.918 µg glucose/g soil/hr), but

decreases on 28th

day (12.7 ± 0.863 µg glucose/g soil/hr)

after treatment. Similarly trend was also exhibited in

pyrazosulfuron and glyphosate treated soil, which varies

from (3.75 ± 0.145 µg glucose/g soil/hr to 61.25 ± 0.757 µg

glucose/g soil/hr) and (6.25 ± 0.365 µg glucose/g soil/hr to

85.0 ± 0.153 µg glucose/g soil/hr) respectively. However,

the amylase activity in paraquat treated soil was found to be

highest on 14th

day (36.42 ± 0.680 µg glucose/g soil/hr),

and gradually decline on 21st day (17.5 ± 0.242 µg


glucose/g soil/hr) and 28th

day (3.5 ± 0.527 µg glucose/g

soil/hr) after treatment respectively. The variation in soil

amylase activity with respect to different herbicides and

different days after treatment was found to be statistically

significant (p<0.001) (Table 2). The differences in amylase

activity in various herbicide treated soil can be explained

due to the herbicide induced changes in starch degrading

enzyme (Achuba, 2006), and the unavailability of nutrients

thus inducing stress (Perucci and Scarponi, 1994; Anigboro

and Tonukari, 2008). Herbicides are decomposed by

enzymes produced by the soil microorganisms, which

subsequently use the metabolite as a source of biogenous

elements (Cerevelli et al., 1978; Milosevic et al., 2001).

Besides, certain groups of microorganisms start to

decompose herbicides a few days after the application

(Milosevic et al., 2002).

Table 2. Two way ANOVA test revealed the level of significance on the interaction between herbicides and between

different days (7th

, 14th

, 21st and 28

th) after treatment.

Parameters A B A x B

Organic Carbon 44.75*** 7.00*** 16.92***

Microbial biomass-C 29.77*** 66.51*** 5.39**

Amylase activity 1339.5*** 11575.4*** 1649.4***

Invertase activity 535.14*** 2518.56*** 1333.93***

Protease activity 50.71*** 2.14 NS

1.36NS

Urease activity 22640.76*** 21030.25*** 582.95***

Dehydrogenase activity 46.91*** 512.11*** 14.68***

ANOVA values are significantly different at NS

= not significant (p>0.05); * = (p<0.05); ** = (p<0.01) and

*** = (p<0.001) level of significance. A = F-value between different herbicides treatment, B = F-value between different

days after treatment, and A x B = F-value of interaction between A and B.

The invertase activity in butachlor treated soil showed an


day (4.0 ± 0.433 µg sucrose/g

soil/hr) to 28th

day (23.9 ± 1.247 µg sucrose/g soil/hr) of

incubation (Table 1). However, the effect of pyrazosulfuron

on soil invertase activity was pronounced on 14th

day (5.0 ±

1.022 µg sucrose/g soil/hr), but gradually showed an

increasing trend and found to be highest on 28th

day (93 ±

0.75 µg sucrose/g soil/hr) after treatment. The invertase

activity in paraquat treated soil showed a consistent

increase from 7th

day (11.25 ± 0.566 µg sucrose/g soil/hr)

to 14th

day (84.37 ± 1.453 µg sucrose/g soil/hr), and

gradually decline on 21st day (11.25 ± 0.6 µg sucrose/g

soil/hr) after treatment. Similar trend was also exhibited in

glyphosate treated soil, where the invertase activity varies

from 7th

day (3.0 ± 0.661 µg sucrose/g soil/hr) to 28th

day

(52.0 ± 2.291 µg sucrose/g soil/hr) after treatment. The

variation in soil invertase activity with respect to different

herbicides and different days after treatment was found to

be statistically significant (p<0.001) (Table 2). Invertase is

known to be very stable and persistent enzyme, and its

association with different soil components is well

documented (Kiss et al., 1978). The possible reason for the

decrease in soil invertase activity may be attributed to the

cellular destruction caused by the toxic substances in

herbicides (Perucci and Scarponi, 1994) accompanied by

the reduction in nutrient mobilization and glucose level

(Anigboro and Tonukari, 2008). Further invertase is an

extracellular origin and the death of some microorganisms

may cause a decline in the production and excretion of

enzyme which leads to the reduction in the soil activity

(Perucci et al., 1999). Recovery of enzyme activities after

the initial inhibition could be due to growth of microbial

population after adaptation or most probably due to

increases availability of nutrients due to the degradation of

herbicides (Ismail et al., 1998).

The protease activity in butachlor treated soil showed an


day (76.452 ± 1.654 µg tyrosine/g

soil/hr) to 28th

day (91.548 ± 2.697 µg tyrosine/g soil/hr) of

incubation (Table 1). Similar trend was exhibited in

pyrozosulfuron, paraquat and glyphosate treated soil, which

varies from (78.549 ± 1.547 to 92.784 ± 2.845) µg

tyrosine/g soil/hr, (54.874 ± 0.825 to 61.24 ± 0.893) µg

tyrosine/g soil/hr and (61.54 ± 0.952 to 64.579 ± 0.973) µg

tyrosine/g soil/hr respectively. The protease activity

depends on the distribution of proteolytic bacteria (Sardans

et al., 2008; Anjaneyulu et al., 2011; Subrahmanyan et al.,

2011), and the amount of proteinaceous substrate

availability, NH4-N accumulation (Sardans and Penuelas,

2005; Tischer, 2005) in soil organic matter.

The urease activity in butachlor treated soil showed an


day (5.68 ± 0.023 µg NH4+/g

soil/hr) to 28th

day (9.741 ± 0.035 µg NH4+//g soil/hr) of

incubation (Table 1). Similar trend was exhibited in

pyrozosulfuron, paraquat and glyphosate treated soil, which

varies from (6.2 ± 0.034 to 8.4 ± 0.043) µg NH4+/g soil/hr,

(3.8 ± 0.013 to 6.4 ± 0.024) µg NH4+/g soil/hr and (4.2 ±

0.025 to 6.9 ± 0.038) µg NH4+/g soil/hr respectively. The

urease activity was found to be lower as compare to other

enzyme activity. It is usually accepted that soils exhibit

appreciable urease activity. These finding led to the

conclusion that native soil urease are mainly extracellular

and are particularly persistent because of their association

with inorganic and organic soil colloids (Gianfreda et al.,

1995). A considerable amount of total activity of an

enzyme (including urease) in soil may be ascribed to an

enzymatic fraction located either within proliferating and

non-proliferating cells or attached to or contained within

cell debris (Nannipieri, 1994). This enzymatic fraction does

not contribute to be measured activity of soil, because it is

not easily detectable in enzyme assays. Therefore, it could

be assumed that such urease fractions predominate in soils.

The soil dehydrogenase activity showed an increasing trend

from 7th

to 28th

day of incubation in case of all herbicide

treated soil (Table 1). The effect of butochlor on soil

dehydrogenase activity showed an increasing trend from 7th

day (2.208 ± 0.170 µg TPF/g soil/hr) to 28th

day (11.0 ±


0.276 µg TPF/g soil/hr) after treatment. Similar trend was

also exhibited in pyrazosulfuron, paraquat and glyphosate

treated soil, which varies from [(1.187 ± 0.042 µg TPF/g

soil/hr) to (12.15 ± 0.916 µg TPF/g soil/hr)], [(3.062 ±

0.912 µg TPF/g soil/hr) to (14.25 ± 1.946 µg TPF/g

soil/hr)] and [(2.145 ± 0.043 µg TPF/g soil/hr) to (17.75 ±

0.892 µg TPF/g soil/hr)] respectively. It is evident from the

data that the soil treated with pyrazosulfuron had the lowest

set of dehydrogenase activities as compared to other

herbicides treated soil. Further, the soil treated with

glyphosate exhibited higher dehydrogenase activity on 28th

day (17.75 ± 0.892 µg TPF/g soil/hr) as compared to

butachlor, pyrazosulfuron and praquat treated soil. Further,

the variation in soil dehydrogenase activity with respect to

different herbicides and different days after treatment was

found to be statistically significant (p<0.001) (Table 2).

The increase in soil dehydrogenase activity in herbicides

treated soil from 7th

to 28th day of incubation might be due

to the increase in microbial community composition with

the capability of utilizing the herbicides as carbon source

(Sebiomo et al., 2011;Vandana et al., 2012).

Dehydrogenase is an intracellular enzyme involved in

microbial O2 metabolism. This activity depends on the

metabolic state of soil-biota and may be a good indicator of

soil microbial activity (Garcia et al., 1994). Besides the

herbicides used at recommended rate were non-inhibitory

on dehydrogenase activity (Rao and Raman, 1998).

Dehydrogenase activity is the most sensitive to the combine

toxic effect of heavy metals and PAHs in soil (Madejon et

al., 2001; Maliszewska-Kordybach et al., 2003; Shen et al.,

2006).

The data suggested that the detracting effect of herbicides

towards soil microbial populations and enzyme activities

decreased with time. This is because of the recovery of

microbial populations and enzyme activities after initial

inhibition due to microbial adaptation to these

chemicals/herbicides or due to their degradation. Besides, it

can also be due to the microbial multiplication on increased

supply of nutrients available in the forms of

microorganisms killed by the herbicides (Latha and Gopal,

2010; Vandana et al., 2012).

Figure 3. Effect of different herbicides on different microbial groups: (a) azotobacter, (b) arthrobacter, (c) heterotrophic

aerobic bacteria, (d) actinomycetes, and (e) fungal population on different days (7th

, 14th

, 21st and 28

th) after treatment with

respect to control (without treatment).


3.4 Microbial enumeration

Unintended consequence of herbicides applications may be

the reduction of sensitive populations and/or stimulation of

certain microbial groups with or without detriment to co-

existing microbial populations that may compete for

available resources (Microorganisms are a highly

heterogeneous group, including aerobes and anaerobes,

heterotrophs and autotrophs or saprophytes, symbionts and

parasites. The fate of the herbicide residues in soil is a

matter of great concern since they would persist on top soil

(Ayansina et al., 2003), accumulate to toxic level and

become harmful to microorganisms (Amakiri, 1982), and

changes in nutrient levels (Taiwo and Oso, 1997; Wang et

al., 2008). Some microorganisms have the ability to

degrade herbicide, while some others were adversely

affected depending upon the application rate/dose and type

of herbicide used (Wikinson and Lucas, 1969; Ayansina

and Oso, 2006; Sebiomo et al., 2011). Thus, the effects of

herbicides on soil microbial population may be either

stimulating or depressive depending on the agrochemicals

(type/formulation and concentration), mode of application,

groups of microorganisms and environmental conditions

(Subhani et al., 2000; Zain et al., 2013). Besides,

herbicides decomposition is frequently faster in soils that

contain high organic matter, presumably because of more

vigorous microbial activity. Use of herbicides can reduce

total microbial populations in soil (Greaves et al., 1976),

where some researchers attribute to reduced input of

organic residues (Wainwright, 1978). Various studies have

revealed that herbicides can cause qualitative and

quantitative change in soil microbial populations (Taiwo

and Oso, 1997; Busse et al., 2001; Chauhan et al., 2006;

Das et al., 2006: Ayansina and Oso, 2006; Pampulha et al.,

2007; Latha and Gopal, 2010).

The abundance and distribution of different groups of soil

microorganisms were enumerated and expressed in terms

of colony forming units per gram soil (CFU/g).

Differences could be observed between the different

herbicide treated soil profiles based on microbial

enumeration. Besides, the variations in microbial

population in different herbicides treated soil were

presented in terns of log10 transformed of CFU/g soil

(Figure 3). Azotobacter count (AZB) in butachlor and

pyrazosulfuron treated soil showed an increasing trend

from 7th

day to 28th

day after treatment, which ranged from

(8.3 x 103 to 15.43 x 10

3) and (9.2 x 10

2 to 15.82 x 10

3)

CFU/g soil respectively. Similar trend in AZB count was

observed in paraquat and glyphosate treated soil, which

varies from (12.8 x 102 to 9.2 x 10

3) and (11.4 x 10

2 to 6.86

x 103) CFU/g soil respectively (Figure 3a). The AZB count

with respect to different herbicides and days after treatment

was found to be significant (p<0.001).

The effect of butachlor on arthrobacter count (ARB)

showed an increasing trend from 7th

day (17.5 x 102

CFU/g

soil) to 21st day (26.4 x 10

2 CFU/g soil) after treatment.

However, the effect of pyrazosulfuron showed a reversed

trend in arthrobacter count upto 14th

day (61.5 x 102 CFU/g

soil), and then gradually increases and found to be

maximum on 28th

day (54.4 x 103 CFU/g soil) after

treatment. Further, the arthrobacter count in paraquat and

glyphosate treat soil exhibited an increasing trend from 7th

day to 28th

day, which varies from (22.3 x 102 to 11.2 x 10

3

CFU/g soil) and (10.6 x 102 to 8.05 x 10

3 CFU/g soil)

respectively (Figure 3b). The control soil exhibited higher

ARB count as compared to treated soil. The variation in

ARB count with respect to different herbicides and days

after treatment was found to be statistically significant

(p<0.001).

The heterotrophic aerobic bacterial count (HAB) in

butachlor treated soil showed an increasing trend from 7th

day (26.5 x 106 CFU/g soil) to 28

th day (19.3 x 10

8 CFU/g

soil) after treatment. Significant differences were also

observed in HAB population at different days after the

application of pyrazosulfuron, which varies from 7th

day

(29.3 x 106 CFU/g soil) to 28

th day (36.2 x 10

8 CFU/g soil).

The data suggested that the application of butachlor has

higher potential in reducing HAB count as compare to

pyrazosulfuron. Paraquat application significantly reduced

HAB count from 7th

day (9.8 x 102 CFU/g soil) to 28

th day

(1.32 x 102 CFU/g soil) after treatment (Sebiomo et al.,

2011). Further, HAB count in glyphosate treated soil

exhibited an increasing trend from 7th

day (6.28 x 105

CFU/g soil) to 28th

day (8.1 x 106 CFU/g soil) (Figure 3c).

The variation in HAB count with respect to different

herbicides and days after treatment was found to be

significant (p<0.001).

Actinomycetes (aerobic, gram-positive bacteria), one of the

major groups of soil population, which are widely

distributed (Kuster, 1968). The number and dominance in a

particular soil would be greatly influenced by geographical

location such as soil temperature, pH, organic carbon

content, aeration and moisture content (Arifuzzaman et al.,

2010). The actinomycetes count (ACT) at different days

after herbicide application showed significant differences,

with the highest on 28th

day and lowest on 7th

day after

herbicide treatment. Actinomycetes count showed an


day to 28th

day in butachlor and

pyrazosulfuron treated soil, which varies from (10.3 x 102

to 14.6 x 103) CFU/g soil and (13.4 x 10

2 to 15.9 x 10

3)

CFU/g soil respectively. Similar trend was also exhibited in

paraquat and glyphosate treated soil, which ranged from

(3.8 x 102 to 7.2 x 10

2) CFU/g soil and (9.2 x 10

2 to 11.3 x

103) CFU/g soil respectively. The ACT count was severely

affected by paraquat treatment as compared to other

herbicides. Further, glyphosate was observed to be less

toxic than paraquat against the actinomycetes (Figure 3d).

The variation in ACT count with respect to different

herbicides and days after treatment was significant

(p<0.001).

The fungal count in butachlor, pyrazosulfuron and paraquat

treated soil exhibited an increasing trend from 7th

day to

28th

day after treatment, which varies from (1.55 x 103 to

2.58 x 103) CFU/g soil; (71.5 x 10

3 to 76.7 x 10

4) CFU/g

soil and (2.1 x 102 to 3.1 x 10

2) CFU/g soil respectively.

The data suggested that the FUN count was moderately

inhibited by paraquat as compared to butachlor and

pyrazosulfuron. However, the application of

pyrazosulfuron resulted in higher FUN count as compared

to other herbicides application as well as control (Figure

3e). The FUN count in glyphosate treated soil was found to


be less on 14th

day (2.4 x 102 CFU/g soil), but gradually

increases after 14th

day. The variation in FUN count with

respect to different herbicides and days was significant

(p<0.001). The fungal count in butachlor, pyrazosulfuron

and paraquat treated soil exhibited an increasing trend from

7th

day to 28th

day after treatment, which varies from (1.55

x 103 to 2.58 x 10

3) CFU/g soil; (71.5 x 10

3 to 76.7 x 10

4)

CFU/g soil and (2.1 x 102 to 3.1 x 10

2) CFU/g soil

respectively. The data suggested that the FUN count was

moderately inhibited by paraquat as compared to butachlor

and pyrazosulfuron. However, the application of

pyrazosulfuron resulted in higher FUN count as compared

to other herbicides application as well as control (Figure

3e). The FUN count in glyphosate treated soil was found to

be less on 14th

day (2.4 x 102 CFU/g soil), but gradually

increases after 14th

day. The variation in FUN count with

respect to different herbicides and days was significant

(p<0.001).

There is gradual rise in azotobacter, arthrobacter,

heterotrophic aerobic bacteria, actinomycetes as well as

fungal counts in different herbicides treated soil in course

of time. The initial rise in microbial counts in herbicides

treated soil may be due to their ability to temporarily

mineralize and use the herbicides as energy source (Kunc et

al., 1985). However, the decline in HAB count was

exhibited in paraquat treated soil and fungal count in

glyphosate treated soil, which may be due to the fact that

microbial populations that were tolerant of the treated

herbicides were susceptible to the products of soil-

herbicide interactions, which could have possibly been

bactericidal or fungicidal (Taiwo and Oso, 1997). There

exists positive correlation between microbial population

and soil organic matter, and the variation in microbial

activity represents the capacity of soil microorganisms to

respond to the inputs of herbicides in soil (Sebiomo et al.,

2011).

An increase in reproductive ability of bacteria with time

after the initial phase of depression, resulting from toxic

effect of butachlor was reported (Kole and Dey 1989b; Yu

et al. 1993; Solomon, 1999). The azotobacter count is

found to be less as compared to total bacteria (Barman et

al., 2009). Unlike total bacteria, azotobacter could not

regain its lost population indicating relatively higher

susceptibility of azotobacter than the total bacterial

population to butachlor. Butachlor decreases the fungal

count as compare to other soil microorganisms (Min et al.,

2007; Xia et al., 2011). Enhancement of soil

microorganisms was probably associated with the

degradation of butachlor. High concentration of butachlor

application decreases the soil microbial count and had

adverse effects on microbial activity (Xia et al., 2011).

However, the fungal population (unlike bacteria) took more

time to recover from the detrimental effect caused by

herbicides (Shukla and Mishra, 1997). Besides, herbicides

can influence fungal count directly or indirectly by

affecting the interaction of fungi with other

microorganisms (Wardle and Parkinson, 1990; Araujo et

al., 2003). The order of inhibition of butachlor on soil

microorganisms is bacteria > actinomycetes > fungi.

Higher rate of pyrazosulfuron application impaired

microbial parameters, enzyme activity to a greater extend

and had a long lasting negative effect on soil fertility

(Perucci et al., 2000). These xenobiotic compounds force

the microbial biomass to direct a large part of its energy

budget into reducing mineralization activity. Paraquat is

also known to be bounded strongly and coherently to soil

components including clay minerals and organic matter,

therefore limits the access of microorganisms to paraquat in

soil (Smith and Mayfield, 1977; Bromilow, 2003; Isenring,

2006). Adsorption of paraquat rapidly decreases the

bioavailability of herbicide in soil, and the capability of

adsorption to deactivate paraquat application (Roberts et

al., 2002). Some microbial species are capable of

metabolizing paraquat as a source of carbon (Tu and Bollen

1968; Imai and Kuwatsuka 1989).

The presence of glyphosate may cause changes in microbial

population as well as overall microbial activity (Wardle and

Parkinson, 1990). Glyophosate is degraded primarily by

microbial metabolism. The degradation of glyphosate is

slower in soil with a higher adsorption capacity.

Degradation rate was also affected by specific soil

microbial community (Carlisle and Trevors 1988), and also

vary considerably in different soils. Some microbes may

use herbicides as a source of carbon and energy

(Radosevich et al., 1995). Glyphosate is an

organophosphonate that can be used as a source of P, C and

N by either gram-positive or gram-negative bacteria (van

Eerd et al., 2003), and hence increase in bacterial

abundance and biomass (Zabaloy et al., 2008) and fungal

count (Araujo et al., 2003; Ratcliff et al., 2006). The

increases population of actinomycetes and fungi after

glyphosate treatment was observed (Araujo et al., 2003).

Certain microbes (fungi and actinomycetes) are able to

metabolize xenobiotics like pesticides, and thus have the

ability to flourish and multiply following an initial transient

decrease in number. Actinomycetes showed a significant

increase in glyphosate treated soil with time, which

indicated that actinomycetes may use glyphosate as nutrient

and energy source (Araujo et al., 2003).

Figure 4. Principal component analysis discriminate

different soil profiles with herbicide treatment as well as

control soil.

Further, principal component analysis was performed in

order to discriminate the four different herbicides

(butachlor, pyrazosulfuron, paraquat and glyphosate)

treated soil profiles with respect to control soil (Ludwig and

Reynolds, 1988) on the basis of organic carbon content,


microbial biomass-C, enzyme activities and microbial

population on different days (7th

, 14th

, 21st and 28

th day)

after herbicide treatment. The analysis indicated that the Z1

and Z2 components explained the maximum variance

(99%) and well segregated into five independent clusters

(Figure 4). Microbial activity increased as an adaptation to

the stress caused by the herbicides over weeks of treatment.

Besides, the effect of herbicides on soil microbial

populations and enzyme activities, which may be due to the

recovery of different microbial populations and enzyme

activities after the initial inhibition due to the microbial

adaptation to these chemicals/herbicides or due to their

degradation.

4. CONCLUSION

The indiscriminate use of herbicides has increasingly

become a matter of environmental concern altering the soil

fertility status, because of their adverse effects on soil

microorganisms as well as on physico-chemical properties

of soil. Although the efficacy of herbicides in controlling

the weeds is important, its residual impact should also be

considered for environmental safety. The herbicides are

used either as pre-emergence or as post-emergence; a high

proportion of herbicides reaches the soil and accumulates in

the microbiologically active top soil altering microbial

populations, enzyme activities and biodiversity, which are

good indicators of the balance in the agro-ecological

system. The study confirmed that the herbicides (butachlor,

pyrazosulfuran, paraquat and glyphosate) may alter the

microbial populations with respect to different days after

treatment, and thereby affects the different soil enzyme

activities. Since the investigations were performed in vitro,

and the effects of herbicides are highly transitory, it is

particularly difficult to explain a change of soil enzyme

activities in response to certain factors or to establish the

cause-effect relationships between the herbicide treatments

and the various components contributing to the variation in

overall soil enzyme activities. However, the nutrient

management practices like application of organic manures

and mineral fertilizers caused an increase in the abundance

of soil microorganisms and enzyme activities. The soil

fertility status may also be enhanced by microbial processes

including degradation of agrochemicals/herbicides, nutrient

cycling and carbon sequestration. Therefore, there is a need

for the advent and use of cheaper, eco-friendly alternatives

that result in increased crop production along with the

judicious use of the known arsenal of agrochemicals as

suggested by the integrated pest and nutrient management

protocols. Further, it is necessary to strengthen the

scientific basis of modern agriculture, because herbicides

may be advantageously used only if their persistence,

bioaccumulation, and toxicity in agro-ecosystem are strictly

controlled.

5. ACKNOWLEDGEMENTS

The authors are thankful to the Coordinator, Biotechnology

Unit, School of Life Sciences, Sambalpur University,

Odisha for providing the necessary laboratory facilities.

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Source of support: Nil; Conflict of interest: The author declares there is no conflict of interests.

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