Published July, 1991
REVIEWS AND ANALYSES
Use of Enzymesto Detoxify Pesticide-ContaminatedSoils and Waters
P. NannipieriandJ.-M. Bollag*
ABSTRACT
Applyingenzymesto transformor degradepesticides is an in-
novative treatmenttechniquefor removalof these chemicals from
polluted environments.Enzyme-catalyzeddegradation of a pollutant
by a parathion hydroinse maybe moreeffective than existing chem-
ical methods.Removalof certain pollutants (for instance phenols
and aromatic amines) fromwastewatermaybe achieved by applying
phenoloxidasesthat can convertthese chemicalsto water-insoluble
polymersthat can then be removedby filtration or sedimentation.
Anotherdecontamination methodinvolves the enzyme-catalyzedin-
corporationof pesticides into organic matter. This procedurereduces
the mountof leachablepesticides as well as the toxicity and bioa-
vailability of the chemicals. Pesticide-detoxifying enzymesmust be
immobilizedbefore they can be used in the environment.In spite of
promisingpotential applications, very few enzymeshavebeentested
as possibletools in the detoxllleationof pesticides. Researchin this
domaincan contribute greatly to developingnewmethodsfor pol-
lution control.
T makestheir
HE TOXICITY OF PESTICIDES and their derivatives
contamination of soil and water a
potential environmental hazard. Although accumu-
lation can occur as a result of deliberate use in agri-
culture, pesticide pollution problemsalso arise due to
accidental spillage, improperdean-up of storage con-
tainers, leaks at pesticide dumpsites, and discharge of
wastesfromproductionfacilities. Thus,it is of interest
to develop methods for degrading and removing the
chemicals not only once they have completed their
function in the soil-plant agricultural system, but also
whenthey accidentally enter soils and waters.
Methodsaimedat controlling pesticide pollution in
terrestrial and aquatic environments must address
both point and nonpoint sources in addition to ex-
tremely low and high concentrations of pesticides. A
variety of methods have been used for the detoxifi-
cation of pesticides because of the diverse chemical
properties of these chemicalsand the multitude of pes-
ticide wastes to be treated. Chemicalmethodsinclud-
ing oxidation, reduction, and hydrolysis--physical
methodsincluding incineration, entrapment, and bur-
ial have been developed(Bretscher, 1981). Biological
methodshave also been utilized such as microbial deg-
radation in activated-sludge-treatment systems or by
microflora and microfauna in soil and water environ-
P. Nannipieri, Dipartimento di Agrobiologia e Agrochimica,Univ-
ersita degli studi della Tuscia, 01100Viterbo,Italy; andJ.-M. Bollag,
Lab. Soil Biochemistry,PennsylvaniaState Univ., University Park,
PA16802. Contribution from the Lab. of Soft Biochemistry, Penn-
sylvania State Univ., University Park. Received22 June 1990. *Cor-
responding author.
Published in J. Environ. Qual. 20:510-517(1991).
510
mentsreceiving dischargedpesticides (Lal et al., 1986;
Lee et al., 1988). Individual physical, chemical, and
biological methodsare generally combinedto produce
the desired environmental result (Munnecke, 1978;
Lee et al., 1988). Conventional clean-up methodsare
not always effective, and are generally cumbersome
and cost prohibitive; therefore, a need exists for low-
cost methodsfor effective on-site pollution manage-
ment (Munnecke,1978; Johnson and Talbot, 1983; Lal
et al., 1986; Bollag and Liu, 1990). The area of bio-
technology involving the use of microorganisms or
their cell-free enzymepreparations to control pesticide
pollution appears to have, at the moment,the most
promising applications.
SUITABILITY OF USING MICROORGANISMS
OR ENZYMES
The idea of using microorganismsor their enzymes
to degrade pesticides in the environment is not new
and is supported by the fact that the degradation of
pesticides is due largely to biodegradative processes
mediated by microorganisms (Alexander, 1980; Kear-
ney et al., 1969, 1987; Bollag and Liu, 1990). Since
Audusfirst suggested in 1951 the use of a microbial
inoculant to enhance the degradation of 2,4-D [(2,4-
dichlorophenoxy)aceticacid] in soil, research has pro-
gressed significantly (Finn, 1983; Lal et al., 1986;
Karnset al., 1989).
Nevertheless, there are several limitations to the
utility of microbial degradationof pesticides. For in-
stance, costly and time-consuming methods may be
necessary to produce microbial cultures. Furthermore,
severe conditions, such as chemical shock, extremes
of pH and temperature, toxins, predators, and high
concentrations of the pesticides or their by-products,
mayirreversibly damageor metabolically inhibit mi-
crobial cells and prevent biodegradationof the pesti-
cide. Difficulty in maintaining active cells during
transportation to the polluted site also limits the use
of whole-cell detoxification technologies (Kearneyet
al., 1987). Other factors that could restrict the use of
microorganismsto detoxify xenobiotics include lim-
ited mobility of the cells within the soil, alternate C
sources, and weakness of the inoculated microorga-
nisms in competition with indigenous populations
(Goldstein et al., 1985). Even if inoculated microor-
ganisms are able to escape predators and succeed in
struggling with the indigenous microbial populations,
nutrient conditions in situ maybe unsuitable for their
growth and degradative activity upon the pesticide.
Althoughthe recent developmentof techniques for ge-
netically manipulatingmicroorganismspresents the ex-
 NANNIPIERI & BOLLAG: PESTICIDE-CONTAMINATED SOILS & WATER

citing possibilityofgenerating microorganisms withim- Kmvalues) at degrading low concentrations of both

proved capacity to degrade anddctoxify pesticides carbaryl and carbofuran.
under a variety of conditions, theuseof suchmanip- Microbial cultures capable of degrading pesticides
ulated cells could potentially bcrestricted bytheun- are generally less effective than the specific enzyme
knowneffectsof thesemicroorganisms on the because, at low pesticide concentrations, other C
environment. sources are generally utilized without cometabolismof
Forthese reasons, theuseofcell-free enzyme prep- the pollutant (Alexander,1985). Therefore, stable, ef-
arations forpesticide dctoxification hasbccnexplored. ficient enzymes, such as the amidase from Acromo-
Although several factors, suchasdifficulty inextract- bacter sp. that can hydrolyzecarbamates, maybe prac-
ingandpurifying theenzyme aswellasthepotential tically used to detoxify low levels of pesticides in the
instability ofthepurified protein, could possibly re- environment.
strict theuseofenzymes, there arcseveral advantages Intracellular enzymepreparations are often unstable
totheapplication of enzyme technology to thedctox- under cell-free conditions. For instance, an amidase
ification ofpesticides (Table I).Forexample, enzymes extracted from Pseudomonasalcaligenes capable of
arcunaffected by inhibitors ofmicrobial metabolism hydrolyzing several phenylcarbamate herbicides and
andcanoftenbc usedunderextreme conditions that propanil [N-(3,4-dichlorophenyl)propionamide] was
would be detrimental toactive microbial cells. Inad- completely inactivated after 10 min at 68 °C (Marty
dition, enzyme technology is nowso sophisticated as and Vouges,1987). In addition to exhibiting such ex-
topcrrnit theuseof cell-free systems under circum- tremes of temperature, soil is an inhospitable envi-
stances inwhich previously active microbial cells have ronment for proteins in that nonbiological denatura-
bccnpreferentially employed (GuandChu,1988). tion, absorption and inactivation, extremesofpH, and
Forinstance, until recently,treatments thatrequire degradation by proteolytic microorganismsall con-
multistcp transformations, involving a number ofdif- tribute to the destruction of the molecule(Bumset al.,
ferent enzymes acting sequentially, and/or requiring 1990; Nannipieri et al., 1990). Cell-free enzymesin
theregeneration of cofactors or cocnzymcs (Mun- soil maythus be short-lived. In this case, anypractical
nccke, 1978; Munncckc ct al.,1982; Johnson andTal- use of the technology would require enhancementof
bot,1983;Honcycutt ct al.,1984) havenecessitatedtheir stability.
theuseof intact cells. However, Gu andChu(1988) One way to increase enzymestability is to immo-
have succeededin encapsulating several different en- bilize it upon a solid support. Enzymesattached or
zymesand a cofactor bound to a soluble dextran and entrapped on/in water-insoluble solid matrices possess
employing the whole multienzyme system to convert other advantages over their free counterparts (Table
urea to L-glutamic acid. The cofactor-dextran complex 2). For instance, an immobilized enzymemolecule is
was free to moveand react with enzymesinside the insoluble in aqueousmedia, maintains catalytic activ-
microcapsules but because of its size could not pass ity, and is physically confined to a certain defined
through the membranesurrounding the system. Similar space. Therefore, immobilized enzymes can be re-
multienzymesystems could be prepared and proved to covered easily from the reaction mixture after com-
be useful in degradingpesticides in the environment. pletion of the catalytic process and used repeatedly to
Because many enzymes that usually are active detoxify wastewaters. More than 100 enzyme immo-
within the cell can also function extracellularly, cell- bilization procedures have been elaborated, including
free enzymesystems maybe used for in situ detoxi- covalent attachment to solid supports, adsorption on
fication of pesticides. Indeed, this bifunctionality is
knownto be a commonoccurrence in an inhospitable Table 2. Advantages of immobilized enzymes over their free coun-
environment such as soil (Bums, 1986; Bumset al., terparts in the detoxification of pesticides.
1990;Nannipieriet al., 1990). For instance, a cell-free 1. Immobilized enzymescan be easily recovered and thus reused in the
extract ofAcromobactersp. was capable ofhydrolyzing detoxificatinnof wastewaters
the carbamatelinkage of carbofuran (2,3-dihydro-2,2- 2. Immobilizedenzymesmaydisplay enhancedstability to extreme
dimethyl-7-benzofuranyl N-methycarbamate)without environmentalconditions
3. Immobilizedenzymescan be used in processes which are operated
any cofactor requirements (Derbyshire et al., 1987). continuouslyin the detoxification of wastewaters
partial purification of the intracellular enzymepro- 4. Immobilizedenzymesare moreresistant to proteolytic degradation; thus
duceda stable preparation that wasvery efficient (low their life in soil is prolonged

Table 1. Advantagesand disadvantages of the use of enzymes to degrade pesticides (as comparedto the use of
Advantages
1. Enzymesare not affected by manyinhibitors of microbial metabolism
2. Enzymescan be used under a wider range of extremeenvironmental
conditions
3. Enzymesmaybe effective at lowpesticide concentrations
4. Enzymesremain active in the presence of microbial predators and
toxins
5. Enzymespreferentially act upon a given substrate while microorganisms
mayprefer more easily degradable compounds
6. Enzymesrequire no uptake mechanismwhereas diffusional and
permeability problemsmayimpair microbial uptake of pesticides and
high-molecular-weishtchemicals
7. Dueto their small size, enzymesmayexhibit greater mobility within the
Soil than microorganisms
microorganisms).
Disadvantages
1. Enzymeextraction and purification are time consumingand expensive
2. Enzymesextracted from cells mayremain unstable even after
immobilization on matrices
3. Enzymesrequiring cofactors maybe dHficolt to apply
4. Interactions between enzymesand pollutants maybe hindered by
diffusional constraints
5. Susceptibility to microbialproteases
512 J. ENVIRON. QUAL., VOL. 20,
solid supports, entrapment in polymeric gels, cross-
linking with bifunctional reagents, and encapsulation
within a solid support (Kennedyand Cabral, 1987).
Immobilization techniques have been refined to the
point at which almost every enzymecan now be im-
mobilizedwith full retention of enzymaticactivity.
For the dctoxification of pesticides by enzymatic
methodsto be feasible, by-products must be less toxic
than the pollutant molecule (Munnecke,1978; John-
son and Talbot, 1983; Honeycuttet al., 1984). Since
the productsof pesticide hydrolysisare frequently less
toxic than the parent compound,and hydrolascs do
not require cofactors or coenzymes, these enzymes
have generally been used as tools for the detoxification
of pesticides. In the case of parathion (O,O-diethyl O-
4-nitrophenyl phosphorothioate) and paraoxon (O,O-
diethyl O-4-nitrophenyl phosphate), hydrolysis cata-
lyzed by a parathion hydrolase produces compounds
that are 60 to 200 times less toxic than the parent
pesticide (Munnecke,1978). Enzymatic hydrolysis
carbamatesalso leads to a significant reduction in tox-
icity. On the other hand, the enzymatichydrolysis of
acylanilides, phenylureas and phenoxyacetate pesti-
cides produces toxic by-products (Munnecke,1978).
Nonetheless, hydrolases maystill be used in these
cases because hydrolysis tends to destroy the biospe-
cificity of the pesticide molecule, formingpotentially
less stable and morebiologically degradableproducts.
Extracellular and intracellular hydrolases such as es-
terases, acylamidases, phosphatases, lyases, etc., are
commonin the microbial metabolism of pesticides
(Table 3) and represent a class of enzymeswith prom-
ising applications in the detoxification of pesticides in
the environment. Commerciallyavailable lipases and
proteases can catalyze numerousnonselective reac-
tions, but no reports were found describing the use of
these enzymesfor transforming pesticides.
Pesticides mayalso be detoxified as a result of syn-
thetic reactions mediated by enzymes; for example,
condensation reactions catalyzed by phenoloxidases
yield oligomeric or polymeric products whichare usu-
Table 3. Pesticides hydrolyzed by esterases and acylamidases.
Esterases References
Dithioates
Malathion Getzin and Rosefield, 1971
Organophosphates
Acephate Munnecke, 1979b
Aspon Munnecke, 1979b
Cyanophos Munnecke, 1976
Diazinon Munneck¢, 1976
Dursban Munnecke, 1976
EPN Mulbry and Karns, 1989
Fenitrothion Mulbry and Karns, 1989
Fensulfothion Munnecke, 1979b
Methyl parathion Munnecke, 1976
Monocrotophos Munnecke, 1979b
Paraoxon Munnecke, 1976
Parathion Munnecke, 1976
Trizophos Munnecke, 1976
Phenoxyacetates
2,4-D Tiedje and Alexander, 1969
MCPA Bollaget al., 1968
JULY-SEPTEMBER
1991
ally less toxic than the substrate. In the enzymatichy-
drolysis of 2,4-dichlorophenoxyacetate(2,4-D), a toxic
compound,2,4-dichlorophenol (2,4-DCP), is formed
(Johnson and Talbot, 1983). However,under aerobic
conditions and in the presence of a laccase and phe-
nolic or quinoid compounds, the dichlorophenol is
short-lived as a result of its oxidative couplingto form
oligomericor polymericproducts (Bollag et al., 1980).
The degradation of manypesticides yields phenol- or
aniline-like intermediates which, in the presence of a
phenoloxidase and phenolic and quinoid compounds
(originating either from organic residues, or synthe-
sized by microorganisms), can be oxidatively coupled
to form polymers resembling humic molecules (Haider
et al., 1975;Felici et al., 1985; Giovannozzi-Sermanni,
1987; Bollag and Bollag, 1990). Thus, these reactions
are thought to play an important role in binding xe-
nobiotics to humic substances. The polymerization of
2,4-DCPby laccase is often accompaniedby the re-
lease of chloride ions as well (Dec and Bollag, 1990).
this dehalogenationalso contributes to the overall de-
toxification of the pesticide, as the dehalogenatedcom-
poundsare usually less toxic and more susceptible to
microbial degradation than the halogenated parent
compound.Thus, as will be discussed further, nu-
merousinvestigations using a variety of microbial en-
zymes and pesticides have shownthat enzymatic re-
movalof pesticides is likely to be a useful methodfor
the decontaminationof pesticide pollution sites.
ENZYMATIC DETOXIFICATION OF
DISSOLVED PESTICIDES AND THEIR
DERIVATIVES IN WATERS
Munnecke(1976) has intensively investigated the
potential use of a particular enzyme, parathion hy-
drolase, for the detoxification of pesticide in waters.
He initially observed that a crude cell-free enzyme
from a mixedculture (consisting mainly of fluorescent
pseudomonads, but also including Brevibacterium,
Azotomonas and Xanthomonas) hydrolyzed parathion
Acylamidases References
Phenylureas
Carboxin Engelhardtet al., 1971
Chlorbromuron Engelhardtet al., 1971
Linuron Engelhardtet al., 1971
Metabromuron Engelhardtet al., 1971
Monalide Engelhardtet al., 1971
Monolinuron Engelhardtet al., 1971
Monuron Walln~fer and Bader, 1970
Pyracarbolid Engelhardtet al., 1971
Acylanilides
Propanil Burge, 1972
Karsil SharabiandBordelau, 1969
Phenyicarbamates
CIPC Marty and Vouges, 1987
IPC Marty and Vouges, 1987
 NANNIPIERI & BOLLAG: PESTICIDE-CONTAMINATED SOILS & WATER 51 3

(at 22 °C) about 2.5 times more rapidly than a chem- bial degradation, adsorption on activated C, and
ical methodbased on the use of sodiumhydroxide (at chemical oxidation, suffer from such serious draw-
40 °C). Seven other organophosphates {triazophos backs as high cost, incompletenessof purification, for-
[O,O-diethyl O-diethyl O-(1-phenyl-l,2,4-triazol-3- mationof toxic by-products, and applicability to a lim-
y/)phosphorothionate], paraoxon, diazinon [O,O-die- ited concentration range. Recent results, however,
thyl O-(2-isopropyl-6-methyl-4-pyrimidi- suggest that enzymetechnologies using phenoloxi-
nyl)phosphorothioate], methyl parathion, dursban dases mayprove useful in the removal of these xe-
(O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphoro- nobiotics from waters (Klibanovet al., 1983).
thioate), fenitrothion [O,O-dimethyl-O-(3-methyl-4- Horseradish peroxidase has been successfully used
nitrophenyl)phosphorothioate], and cyanosphos(0-4- to dephenolize coal-conversion wastewaters (Klibanov
cyanophenyl O,O-dimethyl phosphorothioate)} were et al., 1983). Theenzymeis also effective in removing
also enzymatically hydrolyzed at rates 40 to 1000 chlorophenols, chloroanilines, benzidines, and di-
times more rapid than chemical hydrolysis. Further- phenylanilines. The removalefficiency (defined as the
more, the crude enzymewas not inhibited by the de- percentage of the chemical removedfrom solution) of
tergent and solvent ingredients of such pesticide prep- most of the 31 chemicals studied is greater than 85%
arations (Munnecke,1980). (Table 4) with the exception of 3-chlorophenol, 2,6-
Munnecke and Fischer (1979) also investigated tech- dimethylphenol, aniline, 4-chloroaniline, 4-bromoan-
nical aspects of production of the parathion hydrolase. iline, 4-tluoroaniline, and diphenylamine(Alberti and
Mixed cultures produced parathion hydrolase when Klibanov, 1981). The enzymecatalyzes the generation
growneither on parathion or p-nitrophenol (PNP) of phenolic or aromatic amineradicals whichreact to
sole C and energy source. Dueto its low water solu- produce polymeric substances. Thus, a nearly water-
bility and its high toxicity, parathion wasnot consid- soluble compoundis transformed into a water-insol-
ered feasible as a microbial energy source. Theidea of uble polymerwhichcan be easily removedby filtration
continuous fermentation of PNPwas also discarded or sedimentation(Klibanovet al., 1980). Furthermore,
due to the cost of treating effluents from the system the enzymatic removalof phenols occurs over a broad
(Munneckeand Fischer, 1979). The most economical pH range (from pH 3-12), a potentially important re-
method for the production of parathion hydrolase sult because of the extreme variability in the pH of
used meat extract as a growthsubstrate for P. alcali- wastewaters.
genes. An alternative method is the cloning and Nevertheless, peroxidase has been shownto be in-
expression of the parathion hydrolase glue in a bac-
terial system. Serdar et al. (1989) subclonedthe gene, Table 4. Enzymatic removal of aromatic maines and phenols from
inserted the active portion of it into a PLexpression water by horseradish peroxidase (Alberti and Klibanov, 1981; Kli-
vector, and expressed it in Escherichia coli. Soluble banov and Morris, 1981; Klibanov et al., 1983).
parathion hydrolase can be produced at commercially Removal
adequate quantities. Theseresults suggested that par- Compound efficiency H202 pH
athion hydrolase can be massproduced practically.
Parathion hydrolase has been immobilized on po-
rous glass and further characterized for its ability to Benzidine~" 99.9 1 5.5
3,3’-Dimethoxybenzidine~" 99.9 1 5.5
detoxify parathion-treated waters under laboratory 3,3’-Diaminobenzidine~" 99.6 1 5.5
conditions (Munnecke,1977). The apparent Kmvalue 3,3’-Dichlorobenzidine~" 99.9 1 5.5
3,3’-Dimethylbenzidinet 99.6 1 5.5
of the bound enzymewas 2 times higher than that of l-Naphthylamine 99.7 5 5.5
the free enzyme (Kin = 32uMfor parathion); the 2-Naphthylamine 98.3 5 5.5
boundenzymewasstable (extrapolated half-life of 280 5-Nitro-I -naphthylamine 99.6 5 5.5
d) upon continuous column operation. In an attempt N,N’-Dimethylnaphthylamine 93.2 5 5.5
Phenol 85.3 1 4.0
to simulate more closely a practical application, the 2-Methoxyphenol 98.0 I 5.5
immobilized enzyme was used for the continuous 3-Methoxyphenol 98.6 1 5.5
treatment of parathion-contaminated wastewaters and 4-Methoxyphenol 89.1 1 7.0
was found to remove more than 90%of the parathion 2-Methylphenol 86.2 1 4.0
3-Methylphenol 95.3 1 4.0
(10 t~g/mL) from the wastewater whenpassed through 4-Methylphenol 85.0 1 5.5
a fluidized bed reactor (Munnecke,1980). Parathion 2-Chlorophenol 99.8 1 7.0
hydrolase covalently boundto porous silica beads also 3-Chlorophenol 66.9 1 7.0
exhibited good stability and activity (Munnecke, 4-Chlorophenol 98.7 1 5.5
2,3-Dimethylphenol 99.7 1 4.0
1979b), although the glass- or silica bead-immobilized 2,6-Dimethylphenol 82.3 ! 5.5
enzymewas inactivated whenthe wastewater temper- Aniline 72.9 1 7.0
ature wasgreater than 50 °C or the pHvalue waseither 4-Chloroaniline 62.5 1 5.5
above 10 or below6. These results suggested the po- 4-Bromoaniline 84.5 1 5.5
Fluoroaniline 86.4 1 7.0
tential feasibility of using this enzymesystem to de- 1,3-Diaminophenol 98.6 1 7.0
toxify parathion and related organophosphates. Diphenylamine 80.5 1 7.0
Other compoundspresent in waters as microbio- 1-Naphthol 99.6 1 4.0
logically or industrially generatedby-productsand re- 2-Nitroso-1 -naphthol 98.9 1 4.0
4-Phenylphenol 99.9 1 4.0
quiting removal include phenols and amines that, due 4-Hydroxyquinoline 99.8 1 7.0
to their toxicity, represent a significant health hazard. 100units of peroxidaseper liter;, all other compounds wereincubatedwith
Conventional methods for dephenolization of indus- 1 000 units of enzymeper liter [horseradish peroxidase(EC1.11.1.7; Sigma
trial wastewaters, including solvent extraction, micro- ChemicalCo.) with specific activity of 175 purpurogallin units/mL].
514 J. ENVIRON.QUAL., VOL.20, JULY-SEPTEMBER
effective in removingpolychlorinated biphenyls from
waters. However,high removalefficiencies can be ob-
tained if these nonreactive compoundsare tested in
the presence of enzymaticallyreactive pollutants (Kli-
banovet al., 1983). Presumablyradicals enzymatically
produced from "reactive" compoundscan react with
and coprecipitate "nonreactive" compounds. Al-
though the mechanismof such coprecipitation is not
entirely clear, the practical implicationof this finding
is evident; wastewatersresponsive to peroxidase treat-
ment could be combinedwith those containing "non-
reactive compounds"prior to enzymatic treatment.
Nevertheless, caution is required in using the phen-
oloxidase to treat drinking waters contaminated with
aromatic compoundssince toxic by-products, such as
dioxin or furan derivatives, can be produced during
the peroxidase-induced coupling of chlorinated phen-
ols (Maloneyet al., 1986).
Other phenoloxidasesmayalso be potentially useful
in detoxifying xenobiotics in waters. For instance, a
crude preparation oftyrosinase from Agaricus bisporus
is as effective as a purified commercialenzymein pre-
cipitating phenol and substituted phenols from waters
(Atlowet al., 1984). As with horseradish peroxidase,
in the presence of reactive compounds,pollutants dis-
playing a low susceptibility towards the enzymecan
be enzymatically coprecipitated. Furthermore, the en-
zymatic dephenolization of wastewaters by tyrosinase
is potentially less expensivethan peroxidase-catalyzed
treatment because no hydrogen peroxide is required
for tyrosinase activity.
Theuse of free enzymesto detoxify wastewaterssuf-
fers from shortcomingssuch as the inability to reuse
the enzymeand potential denaturation under extreme
conditions. As discussed previously, immobilization
of the enzymeon/in a solid support can eliminate
these disadvantages. Laccase, an enzymewhich cata-
lyzes the same types of reactions as peroxidase, has
been immobilized to a support and used to remove
various substituted phenols from aqueous solution
(Shuttleworth and Bollag, 1986). The immobilizedlac-
case from Rhizoctonia praticola was very active
against methoxyphenols(removal efficiency 100%)but
relatively inactive towards chlorophenols. Although
the immobilizationof the R. praticola laccase has no
effect on either its activity or stability, the immobili-
zation ofa laccase from Trametesversicolor on porous
glass beads markedlyincreases its thermal stability
(Leonowicz et al., 1988). The immobilized enzyme
also shows broader pH and temperature optima than
the free laccase, an important finding froma practical
point of view, since the pHand temperature of waste-
waters can fluctuate over such a broad range. Thus,
as these examples demonstrate, certain microbial en-
zymescan be used successfully to convert pesticides
or their derivatives to less hazardous chemicals in
waters.
ENZYMEDETOXIFICATION OF PESTICIDES
AND THEIR DERIVATIVES IN SOIL
Detoxification of pesticides in soil can occur as a
result of both inorganic and enzyme-catalyzed reac-
tions (Burns, 1987; Huang, 1990). Enzymesin turn
1991
can exist as both extracellular and intracellular cata-
lysts, being released into the soil environmentupon
cell lysis and death, or during the normallife of cells,
as well as being present in metabolically active cells,
in nonproliferating cells, and within deadcells and cell
debris. Released enzymesmayalso becomeassociated
with humic colloids or clay minerals due to adsorp-
tion, entrapment or copolymerization during humifi-
cation. If the active site is not involved and no enzyme
denaturation occurs in the process, active clay- or hu-
mus-enzymecomplexes can be formed (Burns, 1986;
Burnset al., 1990; Nannipieriet al., 1990).
Stable organo-mineral enzyme complexes have at-
tracted muchattention; they suggest the intriguing
possibility that soil possesses indigenousand persist-
ent enzymatic capacity which is independent of on-
going cell growth. Urease and phosphatase organo-
mineral complexeshave been characterized after ex-
traction from soil and partial purification (Nannipieri
et al., 1982, 1988). Other enzymes, including those
capable of degrading pesticides, have been extracted
from soil as well (Burns and Edwards, 1980; Nanni-
pieri et al., 1980; Ladd, 1985). For example,a phenol-
and aniline-metabolizing peroxidase (Bartha and Bor-
deleau, 1969; Bollag et al., 1987) and an acylamidase
hydrolyzing propanil (Burge, 1972) have been ex-
tracted from soil. Furthermore,an esterase capable of
hydrolyzing malathion to its monoacid has also be
extracted from soil and found to be thermally stable
and resistant to microbial attack (Getzin and Rose-
field, 1971), likely as a result of the protectionprovided
by an attached carbohydrate moiety (Satayanarayana
and Getzin, 1973). This enzymeis particularly attrac-
tive as a potential detoxification agent, becauseof its
remarkable stability to thermal denaturation and pro-
teolytic, activity; the esterase mightbe practically and
successfully used in accelerating malathion conversion
to less toxicby-products in polluted soils.
A parathion hydrolase extracted from Pseudomonas
sp. has also been used to degrade both technical and
formulated diazinon added to an autoclaved soil at
high concentrations (up to 1%w/w) (Batik and Mun-
necke, 1982). Under these simulated pesticide-spill
conditions, more than 98%of the diazinon could be
removedin 24 h at optimal pH (8.0) and temperature
(32 °C). Whenthe enzymewas added to a nonsteril-
ized soil treated with various concentrations of dia-
zinon (from 0.05-0.5%), complete hydrolysis of the
pesticide was observedat all but the highest concen-
tration (0.5%) (Honeycuttet al., 1984). The inability
of the enzymeto hydrolyze the high concentration of
diazinon was attributed to inhibition of the hydrolase
by the pesticide; however,it is likely that proteolytic
degradation of the parathion hydrolase by the endemic
microflora of soil mayhave shortened the enzymelife-
time sufficiently to prevent completehydrolysis of the
highest concentration of diazinon.
This latter possibility again emphasizesthe fact that
in the developmentof a cell-free enzymesystem for
the detoxification of pesticides in soil, the enzyme
must be capable of expressing high activity for a pro-
longed time period. For this reason, procedures based
on the application of free enzymesmaybe inefficient
since various factors, such as nonbiological denatur-
 NANNIPIERI & BOLLAG: PESTICIDE-CONTAMINATED SOILS & WATER
ation, absorption to and inactivation by soil, and deg-
radation by proteolytic microorganisms, combine to
shorten the life of the enzymemolecule(Burns, 1986).
As mentioned previously, one way of increasing the
stability of enzymesin soil is through immobilization
on a solid support. Again, the choice of carder is im-
portant; matrices should be selected so as to minimize
the size of the immobilized enzymeto permit ready
diffusion while increasing its resistance to microbial
degradation; in addition, relative side effects of the
support on nontarget organisms must be negligible.
Enzymesimmobilizedon synthetic humus,clay or soil
appear to meet these requirements because enzymatic
activity following, immobilizationis retained andthese
complexes resemble naturally occurring enzyme-
organomineral complexes(Bums,1987; Sarkar et at.,
1989).
Synthetic humus-enzymecomplexes have been pre-
pared through the oxidative coupling of the selected
enzymewith phenols (Burns, 1986; Burns et at., 1990),
and have proven to be useful in the soil environment.
For instance, synthetic polyphenolic-cellulase com-
plexes were foundto be resistant to degradationin soil
(Sarkar and Burns, 1984), while similarly prepared hu-
mus-phosphatasecomplexeswere used as a seed coat-
ing to accelerate organic phosphate mineralization in
the early stages of seedling establishmentin soil (Burns
and I.add, 1985). Immobilization of several enzymes
(acting sequentially in a degradaiive pathway)
matrices resemblingsoil colloids mayalso be feasible.
For instance, ammoniawas produced from arginine
by the consecutive action of arginase and urease im-
mobilized on smectite organic complexes (Boyd and
Mortland, 1986).
Enzymescapable of degrading pesticides have also
been immobilized on soil and clay with retention of
activity (Sarkar et at., 1989). A laccase fromTrametes
versicolor was immobilized on various clays and to
soil (Ruggieroet al., 1989)andexaminedfor its ability
to transform 2,4-DCP. About 95%of the added 2,4-
DCPwas oxidatively transformed to nonsoluble oli-
gomers by free laccase and by enzyme immobilized
on kaolinite and soil, respectively. Furthermore,the
immobilized laccase was stable toward a proteolytic
enzyme.In addition, their remarkablestability after
prolonged use indicates that these laccase complexes
could be retrieved and used repeatedly for detoxifi-
cation purposes.
The enzyme-catalyzed conversion of 2,4-DCP to
nonsoluble oligomers suggests an additional possible
methodfor the detoxification of pesticides: the en-
zyme-catalyzedcopolymerizationof the pesticide with
humic residues in soil. Phenoloxidase-catalyzed oxi-
dative cross-linking of a numberof xenobiotics, such
as substituted phenols and substituted anilines, to a
variety of naturally occurring humic monomershas
been demonstrated and the reaction mechanismsre-
viewed(Bollag and Bollag, 1990). For example, lac-
case, tyrosinase, and peroxidase can each catalyze the
oxidative coupling of 2,4-DCPto fulvic and humic
acids (Sarkar et al., 1988; Decand Bollag, 1988).
As the xenobiotic is incorporated into humic mol-
ecules by these reactions, its toxicity, that is, the
amountof pesticide available to biota and potentially
515
leachable by water, is reduced. Thus, oxidative cou-
pling of toxic compounds,such as 4-methylphenol, by
a phenoloxidase (peroxidase or laccase from Geotri-
chumcandidum) in the presence of a humic hydro-
lyzate, results in the production of water-unleachable
polymers (Shannon and Bartha, 1988). Even water-
leachable oligomers, such as were generated in the ox-
idative coupling of 2,4-DCPand ferulic acid by the
fungal laccase, are often less toxic than the halogenated
parent compound.
Furthermore, in the aforementionedstudy the poly-
mers could be converted to nonleachable products
whenchitosan was. added to the reaction mixture.
These humic-xenobioticcomplexesare also relatively
stable, as only small amountsof the xenobiotic are
released with time; these small concentrations of re-
leased xenobiotics are almost entirely mineralized by
soil microflora and abiotic factors (Dec and Bollag,
1988). These results indicate that both free and im-
mobilized phenoloxidases can indeed catalyze reac-
tions between xenobiotics and humic molecules. Ox-
idative coupling of xenobiotics to humicmolecules in
soil mayrepresent a detoxification methodand, in this
context, immobilized phenoloxidases maybe used in
situ to enhancethe removalof pesticides from the soil
environment.
CONCLUSIONS AND FUTURE
RESEARCH NEEDS
The application of enzymes which degrade pesti-
cides and their derivatives to less toxic metabolites
mayrepresent an effective methodto control pesticide
pollution. In fact, enzyme-catalyzeddegradation may
be more effective than existing chemical methods as
in the case of the detoxification of several organo-
phosphates by parathion hydrolasc (Munnccke,1976,
1979a).In the detoxification of wastewaterswater-sol-
uble chemicals can be enzymatically converted to
water-insoluble polymerswhichcan then easily be re-
movedby filtration or sedimentation (Klibanov ctal.,
1983). However,a clear identification of the com-
pounds produced by enzymes is required since un-
desirable toxic by-products maybe enzymatically gen-
erated as well (Maloneyet ai., 1986). In addition, the
incorporation of pesticides or other pollutants into
humic molecules by enzyme-induced coupling may
prove useful as a dctoxification procedurein polluted
soils. This reaction not only reduces the amountof
leachable pollutants, but also generally reduces the
toxicity and bioavailability of the chemicals.
In spite of the promisingpotential applications, only
a few enzymeshave been tested as tools in the dctox-
ification of pesticides in waters and soils. Numerous
investigations have documentedthe degradation of
pesticides by soil and water microorganisms;but few
studies have examined the enzymes responsible for
pesticide degradation (Bollag and Liu, 1990). This lack
of investigation concerning practical applications of
pesticide-detoxifying enzymesappears to be related to
the limited knowledgeof the biochemistry of pesticide
degradation, rather than to an inability to overcome
factors which restrict the use of the enzymesthem-
selves. Thus, future research concerning the biochem-
istry of pesticide degradation is greatly needed and
516 J. ENVIRON. QUAL., VOL. 20, JULY-SEPTEMBER 1991

should be aimed not only at denning the type of en-

zyme involved in the various metabolic steps but also
at characterizing their kinetic properties and resistance
to adverse environmental conditions.
Immobilization of pesticide-detoxifying enzymes
seems generally to be an obligatory step preceding their
use in the environment. Particularly in the soil, free
enzymes are rapidly inactivated by adverse environ-
mental conditions. Unlike their soluble counterparts,
immobilized enzymes are often more stable to pro-
teolysis and extremes of temperature. They also can
be reused in wastewater treatments. In addition, en-
zyme immobilization techniques have been refined to
a point where virtually any enzyme can now be im-
mobilized with retention of enzymatic activity, and
multienzyme systems incorporating cofactors have
also been prepared and successfully used for industrial
purposes (Gu and Chu, 1988).
Since the effectiveness of an enzyme depends not
only on its stability but also on its contact with the
substrate, the most efficient means of delivering these
enzyme systems should also be carefully investigated.
The activity of enzyme preparations added to soil in
aqueous solution should be compared with that of cat-
alysts applied at root tips or as seed coatings (Burns,
1987). Effects of these added enzymes on the bio-
chemical and microbiological properties of soils must
also, be critically examined.
Caution is required in predicting pesticide fate in
situ from enzymatic studies under laboratory condi-
tions, and obviously in situ studies of the detoxifica-
tion procedures are necessary as well. Pilot-scale stud-
ies in wastewater treatments and field experiments on
polluted soils are of paramount importance. An eco-
nomic analysis of these applications should be made
and compared with currently available methods for
the detoxification of pesticides. Such experiments and
analyses should allow a determination of the feasibility
of using enzymes to detoxify pesticides in soil and
water and may prove the enzymatic decontamination
method to be of great importance in future attempts
to control environmental pollution.
NANNIPIERI & BOLLAG: PESTICIDE-CONTAMINATED SOILS & WATER 517