microbial degradation of an organophosphate pesticide, malathion

2013

http://informahealthcare.com/mcbISSN: 1040-841X (print), 1549-7828 (electronic)

Crit Rev Microbiol, Early Online: 1–9! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/1040841X.2013.763222

REVIEW ARTICLE

Microbial degradation of an organophosphate pesticide, malathion

Baljinder Singh1, Jagdeep Kaur2, and Kashmir Singh2

1Punjab Pollution Control Board, Patiala, Punjab, India and 2Department of Biotechnology, Punjab University, Chandigarh, Punjab, India

Abstract

Organophosphorus pesticide, malathion, is used in public health, residential, and agriculturalsettings worldwide to control the pest population. It is proven that exposure to malathionproduce toxic effects in humans and other mammals. Due to high toxicity, studies are going onto design effective methods for removal of malathion and its associated compounds from theenvironment. Among various techniques available, degradation of malathion by microbesproves to be an effective and environment friendly method. Recently, research activities in thisarea have shown that a diverse range of microorganisms are capable of degrading malathion.Therefore, we aimed at providing an overview of research accomplishments on this subject anddiscussed the toxicity of malathion and its metabolites, various microorganisms involved in itsbiodegradation and effect of various environmental parameters on its degradation.

Keywords

Carboxylesterase, malaoxon, malathion,microbial degradation

History

Received 29 October 2012Revised 14 December 2012Accepted 31 December 2012Published online 26 February 2013

Introduction

Malathion [S-(1,2-dicarbethoxyethyl)-O, O-dimethyldithio-

phosphate], also known as carbophos, maldison and mercap-

tothion is a nonsystemic, wide-spectrum organophosphorus

pesticide used in public health, residential, and agricultural

settings (Singh et al., 2012a). Malathion is suited for the

control of sucking and chewing insects of fruits and

vegetables, mosquitoes, flies, household insects, animal

parasites (ectoparasites), and head and body lice. Malathion

is used in veterinary medicine (Osweiler et al., 1984) and also

as an anti-infective agent (Wester & Cashman, 1989) to

control insect vector-borne diseases such as malaria, dengue

and yellow fever.

Organochlorine pesticides are banned in many countries

and therefore organophosphate pesticides (OPs) such as

malathion are largely used for public health and agricultural

purposes. Today, the pesticides industry in India comprises

more than 125 basic producers of large and medium scale and

more than 500 pesticide formulations (Singh et al., 2012a).

Pesticide formulations include dusting powders having a

major share (85%) in the market followed by water-soluble

dispersible powder and emulsification concentrates (Abhilash

& Singh, 2009). Malathion is formulated as an emulsifiable

concentrate (EC), a dust, a wettable powder, a pressurized

liquid, and as ready-to-use liquids used for ultra-low-volume

application. Agrisect, Atrapa, Bonide, Prentox, Clean Crop

malathion, Acme malathion, Black Leaf malathion spray,

Eliminator, Fyfanon and Gowan malathion dust are examples

of common product names of malathion.

Malathion comes in two forms: a pure form of a colorless

liquid and a technical-grade solution (brownish-yellow

liquid), which generally have a specific gravity (1.23 at

25 �C), vapour pressure at 30 �C (3.38� 106 mm Hg) solubil-

ity in water (130 mg L�1) partition coefficient (2.7482).

Technical-grade malathion (the grade that is usually used

for agricultural purposes) may contain up to 11 impurities

formed during its production and/or storage, some of these

impurities, such as isomalathion, have been found to be

significantly more toxic than malathion itself or to potentiate

the toxicity of malathion (Uygun et al., 2007). Malaoxon is an

oxygen analogue of malathion and it can be found either as an

impurity in malathion, or generated during the oxidation of

malathion in water, air or soil (Durkin, 2008; Singh et al.,

2012b). Malaoxon is 60 times more acutely toxic

than malathion but it breaks down quickly than malathion

(http://www.bionity.com/en/encyclopedia/Malathion.html).

Toxicity of malathion

Malathion was recognized as the first organophosphorous

insecticide with highly selective toxicity (Goda et al., 2010;

Shan et al., 2009; Singh et al., 2012a). The Environment

Protection Agency (EPA), has classified malathion as a

toxicity class III pesticide and allowed a maximum amount of

eight parts per million (ppm) of malathion to be present as a

residue in specific crops used as foods (U.S. EPA., Office of

Pesticide Programs 1988). In a green house study malathion

applied at recommended rates was easily detected on plant

surfaces after 9 weeks of spraying (Delmore & Appelhans,

1991).

Malathion is absorbed by practically all routes including

the gastrointestinal tract, skin, mucous membranes, and lungs

(Indeerjeet et al., 1997). Malathion is an organophosphate

Address for correspondence: Dr Kashmir Singh, PhD, Department ofBiotechnology, Panjab University, Sector 14, Chandigarh 160014,Punjab, India. E-mail: [email protected]

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parasympathomimetic (impact the parasympathetic nervous

system). The main target of malathion in animals is the

nervous system and because the nervous system controls

many other organs, malathion indirectly can affect many

additional organs and functions. Malathion irreversibly

inactivates acetylcholinesterase (AChE) enzyme that breaks

down acetylcholine, a chemical essential in transmitting nerve

impulses across junctions between nerves. Inhibition of AChE

results in the accumulation of free acetylcholine in nervous

tissues and prolongs the action potential in nerves, causing

spasms, incoordination, convulsions, paralysis and ultimately

death (Kumar et al., 2010; Pillans et al., 1988; Zweiner &

Ginsberg, 1988). Toxic effects of malathion were observed in

immune system of higher vertebrates, tissues of fishes and

reproductive and adrenal gland of vertebrates (Ahmed et al.,

2007; Budischak et al., 2009; El-Dib et al., 1996; Fahmy,

2011; Galloway & Handy, 2003; Gurushankara et al., 2007;

Kumar et al., 2010; Senanayake & Karalliedde, 1987). Many

scientists reported genotoxic potential of malathion in bone

marrow and liver cells (Abd El-Monem, 2011; Giri et al.,

2011; Moore et al., 2011). Malathion is mitogenic at lower

levels, and cytotoxic at higher levels of exposure and

significant increase in DNA damage occurred at the 24 mM

malathion exposure (Moore et al., 2010). Ruckmani et al.,

(2011) observed that acute exposure to malathion caused

transient hyperglycemia and upon subchronic exposure,

progressive hyperglycemia which can be risk factor for

diabetes and weight loss. Data from recent studies suggest

that malathion is highly toxic to most aquatic organisms

(Azizullah et al., 2011; Giri et al., 2012; Rico et al., 2011).

Kundo et al. (2011) reported sublethal toxicity at malathion

concentration (0.006 ppm) on the intestine of cricket frog

(Fejervarya limnocharis), where the cytoplasm of the cells

disintegrated and the cells became empty and vacuolated.

Apart from the long list of adverse health effects of malathion,

the chemical is also a proven teratogen (Durkin, 2008).

Intensive use of malathion in agricultural fields may

expose nearby water-resources to an excess of this compound

from runoff and drift. This may cause an increased risk for

algal blooms, oxygen depletion and/or toxic effects on non-

target organisms, and alter the functions of ecosystems. This

problem is of global concern as agricultural land use has

increased during the last century, and in the last two decades

the agricultural production per square meter has also

increased due to increased use of pesticides, chemical

fertilizers and mechanical means for sowing. Due to extensive

widespread use of malathion, exposure risk of living organ-

isms including human beings are very high. Highest levels of

malathion exposure are received by those who are involved in

the production, formulation, handling and application of

malathion, as well as farm workers who enter treated fields

prior to the passage of the appropriate restricted entry

intervals. Major route of exposure appears to be the dermal

contact, while ingestion and inhalation may also be an

important route of exposure to malathion. Santodonato (1985)

observed mean dermal exposures during malathion

spraying of 2–67 mg/h, and mean airborne concentrations of

0.6–6 mg/m3, indicating a lower potential for exposure via

inhalation relative to the dermal route. Malathion residues

bind to skin (Menczel et al., 1983; Saleh et al., 2000) and on

7th day the proportion that is removable from the skin is only

0.01–0.02% of the amount applied (Kazen et al., 1974). Like

all OPs, malathion kills insects and other animals, including

humans, through its effect on the nervous system. Oxidative

stress in humans due to malathion toxicity is also reported

(Moore et al., 2010). In patients suffering from malathion

poisoning, decrease in blood glutathione levels and increase

in the activity of several blood enzymes that reduces oxidative

damage were noticed (Banerjee et al., 1999). Similarly, signs

of oxidative stress (decreased RBC, superoxide dismutase and

glutathione peroxidase activities) were observed in mice

exposed to dietary doses of malathion at 100, 500 or

1500 mg/kg/d over periods ranging from 15 to 120 d

(Yarsan et al., 1999). At lower doses (25–150 mg/kg),

malathion is associated with general signs of oxidative

stress in brain tissue and cerebrospinal fluid (Ahmed et al.,

2000; Fortunato et al., 2006). In vitro studies indicated that

malathion may induce apoptosis (by damaging mitochondria)

in fibroblast cultures at concentrations below those associated

with neurological effects (Masoud et al., 2003).

Biodegradation of malathion

For high crop yield per available agricultural land, the use of

pesticides has become indispensable. The OPs, being bio-

degradable, have replaced the organochloride pesticides

(Audus, 1964; Racke & Coats, 1988). Overall, organophos-

phorus compounds account for 38% of total pesticides used

globally (Post, 1998). It has been estimated that only less than

1% of the total applied pesticides reach to the target pests

(Battaglin & Fairchild, 2002; Pimentel, 1983). The excessive

use of OPs in the past due to wide applications has resulted in

inexorable environmental pollution. Hence, malathion is

recognized as recalcitrant and given a status of hazardous

material. Although several conventional pump and treat

cleanup methods are currently in use for the removal of

OPs, none has proved to be sustainable. Recently, remediation

by biological systems has attracted worldwide attention to

decontaminate OPs polluted resources. The incredible versa-

tility inherent in microbes has rendered these compounds as a

part of the biogeochemical cycle. Thus, makes it worth

analyzing the mechanism of biodegradation of these mol-

ecules by microbes in detail. Malathion degrading bacterial

isolates have been reported by many workers (Bourquin,

1977; Foster & Bia, 2004; Goda et al., 2010; Hashmi et al.,

2002, 2004; Kamal et al., 2008; Rosenberg & Alexander,

1979; Singh et al., 2012a,b). First time, Matsumura & Boush

(1966) observed rapid degradation of malathion in cultures of

the soil fungus, Trichoderma viride, and a bacterium,

Pseudomonas sp., isolated from soil. Strains of Trichoderma

viride and Pseudomonas sp. metabolized malathion by the

action of soluble carboxylesterase, as evidenced by the

presence of carboxylic acid derivatives in culture medium

in addition to the other demethylated and hydrolytic products.

Mostafa et al., (1972a) reported that the fungi, Penicillium

rotatum and Aspergillus niger, metabolized 76% and 59% of

the malathion in the medium within 10 d through carbox-

ylesteratic hydrolysis as well as by a demethylation process.

Two species of rhizobium, R. Leguminnosaru, R. Trifolii,

were isolated from the Egyptian soil that showed high

2 B. Singh et al. Crit Rev Microbiol, Early Online: 1–9

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carboxyesterase activity in the presence of malathion

(Mostafa et al., 1972a, 1972b). Walker (1972) reported

malathion degradation by indigenous soil microorganisms and

identified these microorganisms belongs to Arthrobacter

species (Walker & Stojanovic, 1974). The four metabolites

produced were identified as malathion half-ester, malathion

dicarboxylic acid, potassium dimethyl phosphorothioate, and

potassium dimethyl phosphorodithioate. It was reported that

heterogeneous bacterial population (Flavobacterium menin-

gosepticum, Xanthomonas sp, Comamonas terrigeri and

Pseudomonas cepacia) obtained from river water are capable

of degrading malathion (Paris et al., 1975). The major

metabolite was b-malathion monoacid with only 1% of the

malathion was transformed to malathion dicarboxylic acid,

O,O-dimethyl phosphorodithioic acid, and diethyl maleate.

The fungus Aspergilus oryzae was isolated from a freshwater

pond capable of degrading malathion and also produced

b-monoacid and malathion dicarboxylic acid (Lewis et al.,

1975). Bourquin (1977) found 20 bacteria from a salt-marsh

environment that were capable of degrading malathion.

Demethylation of malathion in bacterial (Bourquin, 1977)

were also observed similar to fungal system (Mostafa et al.,

1972a, 1972b). Microorganisms reported to degrade or

transform malathion are listed in Table 1. The predominant

biodegradation pathway for malathion involves formation of

mono- and diacid metabolites through carboxylesterase

activity (Figure 1A). Oxidative desulfurization and demethy-

lation leads to complete mineralization. Abo-Amer (2007)

observed that Pseudomonas aeruginosa AA112 is able to use

malathion as a sole carbon source with the formation of

diethylsuccinate and succinate metabolites (Figure 1B).

Malathion is degraded in the environment through two main

pathways, activation and degradation (Mulla et al., 1981).

Activation of the compound involves oxidative desulfuration,

yielding the degradate malaoxon, a cholinesterase inhibitor

with 40–60% toxic than its parent compound, malathion.

Activation may be achieved by photooxidation, chemical

oxidation or biological activation, the latter of which occurs

enzymatically through the activity of mixed function oxidases

(Mulla et al., 1981). Degradation of malathion involves both

chemical and biological means, with hydrolysis being the

most important step for each (Konrad et al., 1969). Biological

degradation is mainly achieved through the enzymatic activity

of carboxylesterases, phosphatases and to a lesser extent

through the activity of reductase (Goda et al., 2010; Kamal

et al., 2008; Laveglia & Dahm 1997; Mulla et al., 1981; Singh

et al., 2012a,b). Biodegradation studies of malathion in lab

scale experiments suggested that degradation is influenced by

several limiting factors (concentration of cosubstarte and

culture conditions). Therefore, studies on such factors

involving the biodegradation of the malathion are necessary

if soil bioremediation will be applied. Based on a series of

preliminary studies, it has been found that the inoculum size,

amounts of additional co-substrates like yeast extract, glucose,

sodium pyruvate and succinate and pH are major factors that

affected the extent and rate of malathion degradation.

Presence of yeast extract (0.04%) and glucose (0.03%) in

minimal salt medium led to an increase in growth rate of both

the strains KB1 and PU compared to growth in a media

containing only malathion as sole source of carbon and

energy (Singh et al., 2012a). It was proposed that low

concentration of yeast extract and glucose accelerated the

degradation of malathion, while high concentration did not.

The organism ignores malathion in presence of high concen-

tration of yeast extract and glucose, thus malathion degrad-

ation period was delayed. The concentration of the stimulant

such as yeast extract and glucose and that of the compound to

be degraded would, therefore, be of prime importance for

removal of malathion from contaminated sites. Singh & Seth

(1989) observed that among various co-substrates like

glucose, ethanol and succinate in addition to malathion

(150 ppm), ethanol was the best to support the growth rate of

Pseudomonas sp.

Major pathway of malathion disappearance in soil, water,

sediments and salt marsh environment is biologically

mediated (Bourquin, 1977; Guha et al., 1997; Kumari et al.,

1998; Mostafa et al., 1972a, 1972b). Degradation of mala-

thion in water is pH dependant and degrades quickly in water

with pH47.0. Hydrolysis is the main route of degradation in

alkaline aerobic conditions. Metabolites resulting from

hydrolysis include malaoxon, malathion a and b monoacid,

diethyl fumarate, diethyl thiomalate, O,O-dimethylphosphor-

odithioic acid, diethylthiomalate. Degradation of malathion

by microorganisms (Lai et al., 1995), insects (Holwerda &

Morton, 1983) mammals (Abel et al., 2004) and humans

(Buratti et al., 2005) results in four metabolites, malaoxon,

diethylthiomalate, malathion monocarboxylic acid and des-

methyl malathion. Five malathion-degrading bacterial strains,

Pseudomonas sp., P. putida, Micrococcus lylae, P. aureofa-

ciens and Acetobacter liquefaciens were isolated from soil

samples collected from different agricultural sites in Cairo,

Egypt, out of which two species P. sp., P. putida, showed the

highest malathion degrading activity (Goda et al., 2010).

Microbial degradation of malathion has also been studied in

different types of water. It was observed that malathion

degradation in non-sterile seawater/sediment system is more

rapid relative to the sterile seawater system, indicating that

microbial activity or interaction of malathion with the

sediment was a contributing factor to the degradation of the

compound (Bourquin, 1977; Cotham & Bidleman, 1989).

Degradation products observed in the study were malathion

monocarboxylic acid and malathion dicarboxylic acid while

malaoxon was not detected as a major degradation product.

Paris et al. (1975) isolated heterogeneous bacterial population

from river water and b-malathion monoacid was detected

major metabolite and only 1% of the malathion was trans-

formed to malathion dicarboxylic acid, O-dimethyl phosphor-

odithioic acid, and diethyl maleate. Eleven out of twenty

selected bacterial cultures from salt-marsh environment were

able to degrade malathion as a sole carbon source and the

remaining were able to degrade malathion by cometabolism

when 0.2% peptone was added as an additional source of

carbon (Bourquin, 1977). A study was conducted to determine

the ability of fungi to degrade malathion in aquatic environ-

ments using A. Oryzae, isolated from a freshwater pond (Lewis

et al., 1975), where authors stated that the rate of transform-

ation of malathion in the lab could not be extrapolated to the

field. It was determined, based on comparisons with data

obtained previously, that malathion was degraded 5000 times

more rapidly by bacteria versus the fungus.

DOI: 10.3109/1040841X.2013.763222 Degradation of malathion 3

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Tab

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Malathion degrades rapidly in soil, with reported half-lives

in soil ranging from few hours to approximately 1 week

(Gibson & Burns, 1977; Howard, 1991; Konrad et al., 1969).

Bradman et al. (1994) reported a range of half-life values of

51–6 d for malathion and 3–7 d for malaoxon in soil. Konrad

et al. (1969) reported that initial degradation of malathion in

sterile soils than in an inoculated aqueous system in which

malathion did not undergo biodegradation until after a 7 d lag

period, indicating that actual biodegradation of the compound

requires acclimation by the microbial population. According

to previous studies on malathion biodegradation, there are

several limiting factors that influence the rate and extent of

malathion degradation in the soil. Degradation of malathion

in soil is directly related to the adsorption of the compound to

the soil surfaces, which serves to catalyze the degradation

process and allows for almost immediate degradation of the

compound (Gibson & Burns, 1977; Konrad et al., 1969). The

presence of cosubstrates (alkanes and 1-alkenes) increased the

rate of malathion biodegradation in soil from a tobacco field

and sediment from an estuary of the Neuse River in North

Carolina.

Enzymes involved in microbial degradation ofmalathion

Degradation of malathion in plants, as in soil and water,

occurs mainly by means of hydrolysis at the P-S bond;

carboxylesterase-mediated hydrolysis is also of great import-

ance. Carboxylesterases (CES, EC 3.1.1.1) hydrolyze ester,

amide and carbamate bonds found in xenobiotics and

endobiotics and are widely found in animals, plants and

microorganisms. Carboxylesterases are enzymes in the

a/b-hydrolase fold family of enzymes, making individual

nomenclature complicated. This enzymes includes cholin-

esterases, epoxide hydrolases and phosphotriesterases (such

as paraoxonase) as well as other enzymes (Wheelock et al.,

2008). In the nomenclature of standard esterase, carboxyles-

terases are termed B-esterases, which are inhibited by OPs,

and A-esterases, which are not inhibited by OPs or other

acylating inhibitors and are hydrolyzing uncharged esters.

Carboxylesterases are found in most of the tissues in animals

and expression and activity vary with both the tissue and

organism. It has been found enzyme carboxylesterase from

microbes is responsible for degradation of malathion (Goda

et al., 2010; Singh et al., 2012a). However, a great deal of

research is needed to understand the three-dimensional

structure and catalytic activity of this enzyme.

The bacterial organophosphorus hydrolase (OPH) enzyme

hydrolyses and detoxifies a broad range of toxic OPs by

cleaving the various phosphorus-ester (P–S) bonds but with

different efficiencies (Singh, 2009). OPH is a dimer that

consist of 336 amino-acid residues of two identical subunits

have a molecular mass of �72 kDa. OPH variant enzymes

generated by mutagenesis improved ability to hydrolyse and

detoxify organophosphates harbouring the P–S bond

(Schofield & DiNovo, 2010). A dimethoate degrading

enzyme was purified from Aspergillus niger was found to

degrade all compounds containing P–S linkage like malathion

(Liu et al., 2001). It have been found in recent studies that

carboxylesterase enzyme instead of OPH is responsible forPse

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malathion degradation in microbes that are capable of

utilizing malathion as a sole source of carbon and energy.

Applications and perspectives in malathondegradation

Degradation of OPs has attracted the attention of many

scientists in the last decade, probably because environment

protection agencies have declared it a pollutant, thus its

removal is a priority as these compounds are highly toxic to

mammals. For the treatment of malathion, several technolo-

gies have been proposed, e.g. radiolytic degradation

(Mohamed et al., 2009), ozonation (Beduk et al., 2012),

heterogeneous ozonation (Meng et al., 2010), enantioselective

degradation (Sun et al., 2012) and electrochemical

degradation (Abdel-Gawad et al., 2011). In the study by

Mohamed et al. (2009), an absorbed dose of 2 kGy is required

for complete radiolytic degradation of malathion. Beduk et al.

(2012) observed complete decomposition of organophos-

phates with TiO2 particles in combination with ozone (O3)

and UV photolysis (O3/TiO2/UV). When the enantiopure

S-(�)- and R-(þ)-malathion were incubated in soil, the

inactive S-(�)-enantiomer degraded more rapidly than the

active R-(þ)-enantiomer (Sun et al., 2012). Use of micro-

organisms in detoxification decontamination of organophos-

phorus compounds is considered a viable and environment

friendly approach. Bacillus sp. S14 have the capability of

removal of malathion from dilute aqueous solution and

biosorption is potentially an attractive technology for the

treatment of wastewater for removing pesticide molecules

Figure 1. Metabolic pathway for degradation of malathion. (A) Formation of diethylsuccinate and succinate metabolites. (B) Formation of mono- anddiacid metabolites through carboxylesterase activity. The scheme is based on articles cited in the text.


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from dilute solutions (Adhikari et al., 2010). We have recently

applied biologically based technology for the remediation of

malathion-contaminated soils (Singh et al., 2012b).

We have recently isolated a fragment containing part

of a carboxylesterase gene from Bacillus cereus strain PU,

Brevibacillus sp. strain KB2 and Lysinibacillus sp. strain KB2

(Singh et al., 2012a,b) and it remains to be demonstrated that

this enzyme is responsible for the observed degradation of

malathion.

Investigations in microbial ecology, chemical composition,

and geophysical properties at contaminated environments can

be applied for bioremediation of malathion. Knowledge of

catabolic pathways of degradation, optimization of various

parameters for accelerated degradation, and design of

microbe(s) through molecular biology tools, capable of

degrading malathion lead to improvements of both the

qualitative and quantitative performance of bioremediation.

Analysis of malathion degrading gene cluster may shed light

on the mechanism underlying the reaction. The use of gene

probes for studying the distribution of this set of genes in

malathion contaminated soils will be useful in identifying

niches in which these kinds of genes prevail and the

conditions under which the population of microbes bearing

these genes increases. It is essential to identify the ecological

niche, which can shed some insight on the use of gene probes,

which may be useful for profiling of explosive contaminated

ecosystems. Rhizoremediation of malathion by microbes

capable of colonizing the rhizospheres of plants may provide

a cheap, fast and efficient process for the removal of this

pollutant from the upper layers of the soil.

Declaration of interest

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of the paper.

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Figure 1. Continued.


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