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Bioaugmentation of Atrazine and Fenamiphos Impacted Groundwater:

Laboratory Evaluation
Peter D. Franzmann a; Luke R. Zappia a; A. L. Tilbury b; Bradley M. Patterson a; Greg B. Davis a;Raphi
T. Mandelbaum c
a
Centre for Groundwater Studies, CSIRO Land and Water, Floreat Park, WA, Australia b Department
of Chemistry, The University of Western Australia, c Volcani Research Center, Institute of Soil and
Environmental Sciences, Israel

To cite this Article Franzmann, Peter D. , Zappia, Luke R. , Tilbury, A. L. , Patterson, Bradley M. , Davis, Greg B.
andMandelbaum, Raphi T.(2000) 'Bioaugmentation of Atrazine and Fenamiphos Impacted Groundwater: Laboratory
Evaluation', Bioremediation Journal, 4: 3, 237 — 248
To link to this Article: DOI: 10.1080/10588330008951112
URL: http://dx.doi.org/10.1080/10588330008951112

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 Bioaugmentation of Atrazine and Fenamiphos
Impacted Groundwater: Laboratory Evaluation

Peter D. Frenzmenn", Luke R. Zappia 1, A.L. Tilb'ury2, Bradley M.

Patterson 1, Greg B. Davis 1, and Raphi T. Menaeloeum:
'Centre for Groundwater Studies, CSIRO Land and Water, Underwood Ave. Floreat Park WA 6014,
Australia; 2Department of Chemistry, The University of Western Australia; 31nstitute of Soil and
Environmental Sciences, Volcani Research Center, 50250 Beit Dagan, Israel

Abstract: After the failure of a three-month pump-and-treat exercise to clean up an aquifer contaminated with
the pesticides atrazine and fenamiphos, microcosm experiments using 14C-labeled compounds were undertaken to
determine under what conditions bioremediation would be most effective, and to investigate the prospects for the
Downloaded By: [PERI Pakistan] At: 05:09 24 May 2010

use ofbioaugmentation. The calculated half-lives for atrazine and fenamiphos mineralization to carbon dioxide in
unamended, anaerobic aquifer material were 730 and 1,000 years, respectively. Oxygenation, coupled with
bioaugmentation with enrichments of atrazine-mineralizing bacteria obtained from the contaminated site or an
imported, atrazine-mineralizing pure strain, Pseudomonas sp. strain ADP, decreased the half-life of atrazine
mineralization, to <20 days. Although strain ADP does not use atrazine as a source of carbon and energy,
amendment of the aquifer material with citrate, which strain ADP uses as a source of carbon and energy, did not
appreciably stimulate the mineralization rate of atrazine in the microcosms, suggesting that the aquifer contains
enough natural organic carbon for atrazine mineralization. Aerobic enrichments of fenamiphos-degrading bacteria
were prepared; however, oxygenation and bioaugmentation of aquifer material with these strains did not enhance
mineralization of fenamiphos within the time constraints of the experiments. The shortest calculated half-life of
fenamiphos mineralization in the microcosms was 6.8 years, which is exceedingly long compared with the half-
life of fenamiphos in most surface soils.

Introduction have occurred through inappropriate disposal of rins-

The city of Perth, situated on the Swan Coastal Plain
in Western Australia, relies on groundwater for about
60% of its urban water use (Cargeeg et al., 1987).
Organic chemicals are extensively used in urban, in-
dustrial, and transport industries, and households and
councils to control weeds and pests by application of
herbicides and pesticides.
In 1993, contamination of groundwater with up to
2,000 J.1gIL atrazine and 1,000 ug/L fenamiphos was
detected in groundwater from the Perth suburb of
Dianella, after extensive plant death occurred in local
gardens that were irrigated with the groundwater
(Appleyard, 1995). The contamination was thought to
ings from pesticide residues into a sump that was
coupled directly to groundwater. Remediation via
"pump-and-treat" with trapping of pesticides onto ac-
tivated carbon, and reinjection of the treated ground-
water, caused a decrease in atrazine concentration and
an apparent increase in the concentration of fenamiphos
(Appleyard, 1995).
A recent review of the literature (Joll and
Franzmann, 1997) identified both pesticides as candi-
dates for bioremediation under appropriate conditions.
Degradation of fenamiphos in aerobic soils that have
been previously exposed to fenamiphos is often rapid
(t1l2 = 1 to 4 d [Ou et al., 1994]), but can be relatively
slow in aerobic soils that have not experienced pre-

* Tel: +61-8-93336306; Fax +61-8-93336211; E-mail: peterJranzmann@per.clw.csiro.au

1058-8337/00/$.50
© 2000 by Battelle Memorial Institute
Bioremediation Journal 4(3):237-248 (2000) 237
 exposure to fenamiphos (h/2 = 12 to 87 d [Simon et al.,
1992]). Pure cultures of fenamiphos-degrading bacte-
ria have not been isolated, however the addition of a
carbon and energy source such as glucose can enhance
fenamiphos mineralization under aerobic conditions
(Ou, 1991). The rate offenamiphos breakdown in soils
from a bowling green on the Swan Coastal Plain that
had been repeatedly exposed to fenamiphos was 10-
fold the rate of breakdown in unexposed soils (Sumner,
1996).
Degradation or mineralization of atrazine in the
environment is considered much more problematic.
Atrazine is commonly encountered as a contaminant in
groundwater due to its mobility and recalcitrance to
biodegradation (Belluck et al., 1991), and although
half-lives of mineralization may be as short as 40 days
in some agricultural soils (Y anze-Kontchou and
Gschwind, 1994), in poorer soils, half-lives can in-
crease to 3 years, or as long as 32 years in anoxic soils
(Nair and Schnoor, 1992). Reported half-lives of atra-
zine mineralization in vadose zone soils of the Swan
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Coastal Plain range from 1 to 6 years (Franzmann et
al., 1998). Although atrazine wouldseem more recal-
citrant than fenamiphos in environmental samples, pure
cultures of bacteria have been isolated that rapidly
mineralize atrazine to carbon dioxide (C0 2) under
appropriate conditions (Mandelbaum et al., 1995).
Bioaugmentation of atrazine-contaminated environ-
ments with atrazine-degrading bacteria may be an
option to enhance remediation. Atrazine mineraliza-
tion in soils (Grigg et al., 1997) and aquifer material
(Shati et aI., 1996) has been enhanced by
bioaugmentation with atrazine-degrading cultures.
Degradation through microbial activity is the pri-
mary pathway for pesticide detoxification in most ter-
restrial and aquatic ecosystems. The behavior of a
substance has been shown to differ markedly in differ-
ent types of soil (Vollner and Klotz, 1995). In addition
to microbial degradation, apparent loss of a compound
from soil may occur due to volatilization, sorption,
modification, polymerization or other chemical reac-
tions such as humification (incorporation of the com-
pound into soil organic components), or inappropriate
sampling techniques (Pennington et al., 1994; Shan-
non and Unterman, 1993).
The aim of this study was to test the effects of
different amendments and bioaugmentation on the rate
of degradation of fenamiphos and atrazine in aquifer
material collected from the contaminated site. As many
of the metabolites of fenamiphos and atrazine are con-
sidered as toxic as their parent compounds (Belluck et
al., 1991; Waggoner and Khasawinah, 1974), mineral-
ization rates to CO 2 using labeled compounds were
measured in soil microcosms.
238
Materials and Methods
Culture Media
Medium for the growth of atrazine-degrading bacteria
was prepared as described by Mandelbaum et al. (1995)
except that the nitrogen-containing compound
nitrilotriacetic acid was omitted from the medium.
This medium was designated "atrazine-N -free
medium"(ANFM). The medium contained citrate as
the only significant source of energy, and atrazine as
the only source of nitrogen.
Fenamiphos basal medium (FBM) which contains
citrate as the only significant source of energy, and
fenamiphos as the only source of nitrogen and phos-
phorus was prepared by combining the following stock
solutions as described.
Fenamiphos stock solution was prepared by the
addition of 250 mg of technical grade fenamiphos
(kindly provided by Dr. John Anderson of Bayer AG
Germany) to 25 mL of methanol.
To prepare the trace metals solution, "Metals 44"
(Staley, 1981), a few drops of concentrated sulfuric
acid were added to 700 mL of distilled water, and the
following ingredients were added: 0.25 g
ethylenediaminetetraacetic acid (EDTA), 1.1 g
ZnS04 •7H 20, 0.5 g FeS0 4 · 7H 20, 0.154 g MnS04 · HzO,
0.04 g CuS04·5H20, 75 mg Co(N0 3) 2·5H20, and 20
mg Na 2B40 7·1 OHzO. The volume of the solution was
adjusted to 1 L with distilled water.
To prepare the mineral salts solution, 109 of
nitrilotriacetic acid was dissolved in 700 mL distilled
water and the solution was neutralized with KOH. The
following ingredients were then added: 14.45 g
MgS0 4·7H20, 9.25 g (NH 4) 6M07024·4HzO, 50 mg
FeS04·7H20, 50 mg nicotinic acid, 0.5 mg biotin, 25
mg thiamin-Hf.l, and 50 mL trace metal solution. The
pH of the solution was adjusted to 6.8 and the volume
was adjusted to 1 L with distilled water. The solution
was sterilized by filtration (0.22 urn).
For the preparation of FBM, 0.2 g MgS04·7HzO,
1.0 gNaCl, 0.03 g csci, 2.0 g trisodium citrate, and
1.0 g sucrose were added to 1 L of water. The solution
was sterilized at 121°C for 15 min. To the sterile
solution, 20 mL of sterile mineral salts solution and 5
mL of fenamiphos stock solution were added prior to
dispensing the medium aseptically. For solid medium,
the medium was solidified with 15 gIL agar.
Bacterial Cultures
An atrazine-mineralizing pure strain, Pseudomonas sp.
strain ADP (Mandelbaum et al., 1995) was imported
from the Volcani Research Center, Israel.
Atrazine-contaminated groundwater was collected
from the atrazine contamination plume at Dianella, from
Franzmann et al.
 wells situated at 25 or 27 Lancaster Street. Enrichments
were prepared by inoculating 1 mL of freshly collected
groundwater into 100 mL of ANFM with incubation
aerobically at 28°C. The cultures were subcultured ev-
ery 3 days for 9 days and cells from the final bacterial
enrichments (designated mixed cultures L25 and L27)
were used in microcosm studies. Soil (1 g) from the
original contaminated site was also collected and inocu-
lated into 100 mL of ANFM and processed as described
for the groundwater samples. The enrichment culture
from the third enrichment was designated mixed culture
SF04. Cells from SF04 were streaked on ANFM so-
lidified with 1.5% (wt/v) agar and isolated colonies
were subcultured onto fresh ANFMagar. Two pure
strains, designated, SF05 and SF06, were prepared by
successive re-streaking on ANFM.
Soil (1 g) from the original contaminated site
was also collected and inoculated into 100 mL of
FBM, which was incubated aerobically at 28°C for 3
days. This fenamiphos enrichment was subcultured
every 3 days for 9 days and the mixed culture enrich-
ment was given the number SF01. Cells from SF01
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were streaked on agar. Isolated colonies were subcul-
tured onto fresh FBM agar ..Two strains, designated
SF02 and SF03, were purified by successive
restreaking on FBM agar.
Screening of Bacterial Mixed Cultures
and Pure Strains for Pesticide-
Degrading Capability
Mixed cultures L25, L27, and SF04 and pure strains
ADP, SF05, and SF06 were tested for their ability to
degrade atrazine in culture medium. Mixed culture
SF01 and pure strains SF02 and SF03 were tested for
their ability to degrade fenamiphos. Media (ANFM or
FBA) were prepared with the addition of 50 mg/L of
the appropriate pesticide (atrazine or fenamiphos re-
spectively). The media were inoculated with 100 ~L of
a four-day-old culture of the appropriate test mixed
cultures or strains, and the inoculated media and
uninoculated controls were incubated at 28°C for 14
days on a rotary incubator shaker. Each culture or
control was analyzed for pesticide content by the
micro extraction technique as described by Patterson et
al. (1993). The culture or control was amended with
10 j..LL of an internal standard solution (1.0 gIL 4,4'-
dibromobiphenyl in methanol). Two mL of diethyl
ether were then added and the organic compounds
were extracted from the water sample by shaking for
2 min. The diethyl ether layer was then transferred to
a 2 mL crimp top autosampler vial, and 1 mL of the
extract was analyzed by gas chromatography-mass
spectrometry (GC-MS).
Bioaugmentation of Atrazine and Fenamiphos Impacted Groundwater: Laboratory Evaluation
Gas chromatographic analyses of the organic
compounds were performed on a Varian 3400 gas
chromatograph with a Saturn II Mass Spectrometer.
Analytical conditions of the GC-MS were as follows.
A 25 m x 0.22 mm inner diameter (J.D.) BPX70
capillary column (SGB) with a film thickness of 0.25
mm was used. A 1 ~L extract was injected into a
septum programmable injector (SPI) held at 80°C for
0.5 min, followed by temperature programming to
290°C at 100°C/min and held at this temperature for
22 min. The column was held at 80°C for 2.6 min
followed by temperature programming at 25°C/min
to 290°C and held at this temperature for 14 min. The
mass spectrometer and transfer line was held at 220
and 260°C respectively. The mass spectrometer was
operated in full scan mode with a range from 50 to
350 mJz.
Radiochemicals and Preparation of
Labeled-Pesticide Solution
The following radiochemicals were used in this study:
atrazine-ring-UL 14C (100 JlCi at 18.8 j..LCi/mmol, 98%
chemically pure, 100% radiometrically pure, Sigma
Chemical Company)and fenarniphos-[phenyl-14Cl (200
~Ci at 46.8 mCi/mmol in acetonitrile, 98.9% radio-
metrically pure, kindly provided by Dr. John Anderson
of Bayer AG Germany).
The 14C-atrazine (1.2 mg) was dissolved in 50
mL of sterile distilled water which resulted in a
preparation of 4,629,720 dpm/mL. A 10-g soil
sample, inoculated with 100 ~L of the atrazine solu-
tion would deliver ca. 46,000 dpm/g wet weight
(final concentration ca. 1.13 umol/kg or 240 ug/kg),
Atrazine concentrations in the groundwater at the
contaminated site were as high as 2,000 mg/L
(Appleyard, 1995); however, monitoring bores
yielded water containing between 2.5 to 580 ug/L
atrazine in October 1998 (Appleyard; Personal Com-
munication) .
The 14C-fenamiphos, (1.37 mg [in acetoni-
trile]), was evaporated to dryness at 30°C under a
stream of nitrogen. The dried fenamiphos was
dissolved in 5 mL methanol that resulted in a
preparation of 86,582,100 dpm/mL. A 10- g soil
sample, inoculation with 5 ~L of the fenamiphos
solution, would deliver ca. 43,291 dpm/g wet
weight (final concentration ca. 0.45 urnol/kg or
137 ug/kg). Fenamiphos concentrations in ground-
water at the contaminated site were as high as
1,000 ug/L (Appleyard, 1995); however, monitor-
ing bores yielded water containing between 0.3
and 8.0 u.g/L fenamiphos in October 1998
(Appleyard; Personal Communication).
239
 Microcosm Tests
A core of aquifer material was collected in 0.5 m x 47
mm LD. aluminum tubes from a depth of 5.7 to 6.0 m
below the water table at Dianella above the leading edge
of the plume of pesticide contamination. Core material
could not be taken from within the contamination plume
because the consistency of the water-saturated sand did
not allow recovery of intact aquifer material from such
depths. The cores in aluminum tubes were tightly sealed
at the site with rubber bungs and returned to the labora-
tory and transferred to a Coy anaerobic chamber with a
gas phase of hydrogen:nitrogen, 10:90. The material
was the consistency of a slurry and had a water content
of 25.5% by weight.
To test the effects of different amendments on the
mineralization rate of atrazine and fenamiphos, soil
was mixed with the different amendments in the anaero-
bic chamber. To 100 mL of aquifer material, 0.1 g of
KN0 3 was added to supply an alternate electron accep-
tor to some samples. Alternate carbon sources were
supplied as sterile aqueous anaerobic solutions (10%
w/v) in distilled water. For organic carbon amend-
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controls were prepared by adding 200 ~L of 10M
sodium azide to appropriate samples which were then
autoclaved at 121DC for 15 min, prior to cooling and
inoculation with the appropriate isotope preparation.
For the augmentation of microcosms with bacte-
rial mixed cultures or pure bacterial strains, cultures
were grown on appropriate medium (either ANFM or
FBM) for 48 hours at 28 DC. The cells were collected
by centrifugation and resuspended in sterile (auto-
claved at 121DC for 15 min), anaerobic (degassed by
boiling, nitrogen flushing, and sealing under nitrogen
with a butyl-rubber stopper) groundwater from the
Dianella site. Each cell suspension was adjusted to an
optical density at 560 nm of 0.3 (ca. 107 cells/mL). For
each augmentation experiment, 100 ~L of the appro-
priate cell suspension was added to each appropriate
10 g soil sample. For aerobic microcosms, the tubes
were removed from the anaerobic chamber. The types
of treatments used in the preparation of the micro-
cosms so as to test the effects of different electron
donors, electron acceptors, and culture additions are
listed in Table 1. All microcosm treatments were tested

ments, 0.5 mL of the carbon source solution was added in triplicate.

to 10 g of core material to give a final concentration
of 5 g/kg. Carbon source amendments were added to
appropriate tubes.
Core material was mixed, and 10-g amounts were
distributed into 50 mL screw-capped vials. Abiotic
A glass vial containing 1.0 mLof 1 M NaOH was
suspended in each microcosm tube via nylon string
and the tube was tightly sealed. The sodium hydroxide
traps were periodically replaced and the radioactivity
in the traps determined. For determination of radioac-

Table ·1. Experimental matrix for testing the effects of different electron acceptors, donors and bacterial
additions on the mineralization of either 14C-atrazine or 14C-fenamiphos in microcosms that contained soil-
groundwater slurry prepared from Dianella aquifer material

Pesticide(s) Oxygenation Amendments/additions

Atrazine or fenamiphos Aerobic Ste riIized control

Atrazine or fenamiphos Anaerobic No treatment
Atrazine or fenamiphos Anaerobic 0.1 % nitrate
Atrazine or fenamiphos Anaerobic 0.1 % nitrate + 0.5% glucose
Atrazine or fenamiphos Aerobic No treatment
Atrazine or fenamiphos Aerobic 0.5% glucose
Atrazine or fenamiphos Aerobic 0.5% citrate
Atrazine Anaerobic Strain ADP
Atrazine Anaerobic 0.1 % nitrate + Strain ADP
Atrazine Anaerobic 0.1 % nitrate + 0.5% citrate + Strain ADP
Atrazine Aerobic Strain ADP
Atrazine Aerobic 0.5% citrate + Strain ADP
Atrazine Aerobic 0.50/0 citrate + mixed culture L25
Atrazine Aerobic 0.5% citrate + mixed culture L27
Atrazine Aerobic 0.50/0 citrate + Strain SF05
Fenamiphos Aerobic 0.50/0 citrate + Strain SF02
Fenamiphos Aerobic 0.5% citrate + Strain SF03
Fenamiphos Aerobic 0.5% citrate + mixed culture SF01

Note: The term strain refers to a pure strain either imported as in the case of strain ADP or isolated from mixed culture enrichments.

240 Franzmann et ale

 tivity, 0.1 mL from each of the traps was added to
10 mL of scintillant (Starscint'", Packard) and counted
in a 2250CA Tri-carb Packard Liquid Scintillation
Analyzer. Quench was accounted for by the "Auto-
matic Efficiency Control -quench curve", with the
quench curve generated from quenched C-14 stan-
dards (Amersham).
Upon completion of the trapping experiment,
5 mL of diethyl ether and 5 mL of water was added to
each reaction flask which was shaken for 2 min and
left for 5 min for the ether and water to extract 14C_
labeled organic compounds (unmetabolized and in-
completely metabolized), and partition into separate
phases. The radioactivity in each phase was deter-
mined so as to calculate total recovery.
Results
Initial attempts to enrich for atrazine-degrading bacteria
using the medium of Mandelbaum et al. (1995) that
supplies atrazine as the only source of nitrogen in a
medium proved successful (Table 2). Limited success
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was achieved in obtaining fenamiphos-degrading bacte-
ria in a medium in which fenamiphos was supplied as the
only source of nitrogen and phosphorus. In the initial
experiments, degradation was measured as disappear-
ance of the parent compound only, and did not relate to
mineralization. Strain ADP showed good degradation of
atrazine in culture experiments, as did strain SF05 and
enrichmentsL25 and L27. Interestingly, enrichmentSF04,
from which strain SF05 was isolated, showed no signifi-
cant atrazine degradation. Strains SF02 and SF03 caused
minimal disappearanceof fenamiphosfrom cultures,while
culture SFO1, from which the two strains were derived,
showed some (37%) ability to remove fenamiphos from
culture over 14 days (Table 2).
Table 2. Atrazine or fenamiphos degradation by mixed culture enrichments and pure strains grown in either
ANFM medium or FBA medium
Strain/mixed culture % Atrazine remaining
Strain ADP
Mixed culture L25
Mixed culture L27
Strain SF05
Strain SF06
Mixed culture SF04
Mixed culture SF01
Strain SF02
Strain SF03
Note: The percentage of either fenamiphos or atrazine remaining was calculated with reference to sterile controls after 14 days
incubation at 28°C. Cultures enriched on atrazine or fenamiphos were only tested for atrazine or fenamiphos degradation.
Bioaugmentation of Atrazine and Fenamiphos Impacted Groundwater: Laboratory Evaluation
As a result of the initial culture experiments, it
was decided to test the effectiveness of bioaugmentation
with strains ADP and SFOS and mixed cultures L25
and L27 on atrazine mineralization, and mixed culture
SFOI and strains SF02 and SF03 on fenamiphos
mineralization. Although little stimulation of mineral-
ization was expected with bioaugmentation in the case
of fenamiphos given that the half-life of fenamiphos in
most environments is generally short, normal amend-
ments of samples with electron acceptors (oxygen,
nitrate) and alternate sources of carbon and energy
(glucose and citrate) were expected to appreciably
increase the rate of fenamiphos mineralization.
An example (for four of the microcosms) of the
mineralization of 14C-atrazine to 14C02 is given in
Figure 1. Under aerobic conditions, amendment of the
soil with an electron donor (citrate) and augmentation
of the microcosm with a pure strain of atrazine-miner-
alizing bacterium (strain ADP) enhanced the rate and
extent of atrazine mineralization over the 73 day ex-
periment' as did enrichment cultures containing atra-
zine-mineralizing bacteria from Dianella (e.g. mixed
culture L25).
Each microcosm contained an initial atrazine con-
centration of 240 ug/kg soil, so although the mineral-
ization was greatly stimulated by the augmentation of
both cultures, at best only 44% of the atrazine was
recovered as,CO 2, Appropriate data from each micro-
cosm test was fitted to first -order kinetics to determine
the degradation rate (k) and half-life (11/2 =O.639/k) for
the mineralization of atrazine in each microcosm. An
example of data fitted to first-order kinetics for two of
the plots from Figure 1 is given in Figure 2.
Table 3 shows the outcomes of the mineralization
rate experiments, and gives the half-life for atrazine
mineralization in each of the microcosms, and the
0/0 Fenamiphos remaining
0.2
0.7
1.1
3.9
70
97
63
103
89
241
 120

fjj 100

~
0 80
0
:::t
.9 60
...~. --~ + IVID(AClI':'(
-·~~A··- Soil + Strain
.~ • • • • • _ •• c

....• ~ Soil
20

o
o 10 20 30 40 50 60 70 80
Downloaded By: [PERI Pakistan] At: 05:09 24 May 2010

Figure 1. Mean values (n=3, and error bars representing standard errors) of 14C02 produced versus time from aerobic
mineralization of 14C-atrazine in sterile soil, soil amended with citrate, and citrate-soil augmented with either Pseudomonas sp.
strain ADP or mixed culture L25.

0.0 ......... . ,@IF' .,' .... ..+

,'.' ", ,.".,

.'
-0.1

-0.2 lot

-0,3 ..
II Mixed-culture L25
L25 after 9

-0.4
1 j
-0.5

-0.6

0 10 20 30 40 50 60 70
• 80

Figure 2. First-order atrazine mineralization plots of data from aerobic microcosms containing soil with citrate and soil with citrate
augmented with an atrazine-degrading mixed culture L25. The degradation rate (k) was determined from the most appropriate
portion of the plot that gave the best line fit (Days < 9 for culture L25, to Day 73 for the unaugmented soil). Co is the initial dpm
of atrazine added to the microcosms and C, is the dpm of the trapped CO2 , Individual points are the mean of three replicates and
the error bars represent the standard errors.

242 Franzmann et al.

 Table 3. Half-lives (t_> of 14C-ring labeled atrazine mineralization to 14C02 in aquifer core material (with and
without amendments), collected from the suburb of Dianella on the Swan Coastal Plain

Bioaugmentation t1l21
Soil amendment culture (day) S.E. r2 0/0 min 2

Sterile control (aerobic) 267,000 48,300 0.69 0.0

Anoxic
7,480 409 0.96 0.7
N03 1,680 700 0.98 0.2
N03 + Glu 3 1,210 141 0.99 0.4

Strain ADP 48.6 2.0 0.99 32.5

N03 Strain ADP 50.4 1.8 0.99 29.4
N03 + CiP Strain ADP 26.7 2.7 0.93 37.0

Aerobic
1,770 25 1.00 2.8
Cit 2,420 140 0.96 0.9
Glu 804 44 0.97 2.4

Strain ADP 14.9 0.9 0.98 42.7

Cit Strain ADP 15.4 0.1 0.99 40.6
Cit Mixed culture L25 16.7 1.1 0.97 44.1
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Cit Mixed culture L27 24.0 1.0 0.99 44.3

Cit Strain. SF05 18.0 0.7 0.99 49.2

1 Half-lives were determined from first order calculations of the mineralization rate; for each first order plot, the P value was
<0.001; 2 % min equates to the extent of mineralization of atrazine after 73 days; 3Glu and Cit signify microcosms augmented
with either glucose or citrate. S.E. = the standard error (n=3) in the half-life. r2 = the correlation coefficient of the first order plot.

amount of 14C-atrazine recovered as 14C02 • The.. data
shows that ·without augmentation with cultures of
known atrazine-degrading microorganisms, half-lives
are long (>800 days) even with the supply of amend-
ments such as glucose, citrate, and oxygen.
Bioaugmentation decreased half-lives for atrazine min-
eralization, even under anoxic conditions, but espe-
cially under aerobic conditions. The addition of an
alternate electron donor (citrate) with bioaugmentation
by strain ADP did not increase the aerobic mineraliza-
tion rates, or the extent of mineralization, which sug-
gest that there was enough organic matter in the aqui-
fer material to maintain the growth of the strain. Citrate
did stimulate the rate and extent of anaerobic mineral-
ization with nitrate supplied as an electron acceptor
(Table 3).
Mineralization data for labeled fenamiphos was
examined in the same way as for labeled atrazine.
Although two of the cultures used in the augmentation
of microcosms had shown some capability to reduce
fenarniphos concentrations in the experiments with
pure cultures, they showed much less activity when
tested for their capability to mineralize fenamiphos.
Microcosms that showed slight stimulation of
fenarniphos mineralization did so only after a long lag
phase of greater than 60 days (for an example see
Figure J).
Although the augmentation of the fenamiphos
microcosm withmixed culture SF01 did appear to
stimulate fenamiphos mineralization (Figure 3) after a
lag phase of about 60 days, the extent of mineralization
after 90 days was still only 1.2% of the fenamiphos
added. It is possible that longer incubation would have
increased the rate and amount of fenamiphos mineral-
ization. Fenamiphos mineralization data was fitted to
first order kinetics as was done for atrazine, except that
for the bioaugmented microcosms, the data points at
the end of the experiment (after the lag phase) were
used instead of at the beginning as atrazine mineraliza-
tion had shown no appreciable lag phase. Half-lives
for each of the amendments, and amendments with
bioaugmentation, are given in Table 4.
Half-lives for fenamiphos mineralization in the
aquifer slurries were long (minimum was over 6 years)
even with the amendments of added electron acceptors
(oxygen or nitrate) and/or electron donors (citrate or
glucose) (Table 4).
Recovery of the 14C label as either 14C02 . in the
sodium hydroxide traps, or in the water and ether
extracts of the material at the end of the incubation was

Bioaugmentation of Atrazine and Fenamiphos Impacted Groundwater: Laboratory Evaluation 243

 • SF01 + citrate + aerobic
1.8
'0 • Anoxic soil
(J)
en 1.6 l:J. Aerobic soil
~
\l Sterile control
<D 1.4
Q..

o N
1.2
•
o
~
1.0

0.8

0.6

004

~ 0.0
0.2
•
* 0 20 40 60 80 100
Days
Downloaded By: [PERI Pakistan] At: 05:09 24 May 2010

Figure 3. Mean values (n=3, and error bars representing standard errors) of 14C02 produced versus time from the mineralization
of 14C·fenamiphos in sterile soil, anaerobic soil, aerobic soil, and aerobic soil augmented citrate and mixed culture SF01.

Table 4. Half-lives (t1l2) of 14C~ring labeled fenamiphos mineralization to 14C0 2 in aquifer core material (with
and without amendments), collected from the suburb of Dianella on the Swan Coastal Plain

Bioaugmentation P
Soil amendment culture t1/2 1 (year) S.E. r2 value- % min 3

Sterile control 1,016 229 0.58 <0.001 0.01

Anoxic
68.7 4.3 0.95 <0.001 0.18
N03 60.3 2.6 0.97 <0.001 0.21
N03 + Glu4 27.9 1.1 0.98 <0.001 0.26

Aerobic
73.4 2.3 0.98 <0.001 0.18

Cit 40 3.5 0.96 <0.001 0.11

Glu 100 7 0.94 <0.001 0.37

Cit Strain SF02 11.0 2.2 0.92 0.039 0.73

Cit Strain SF03 43.3 9.2 0.80 0.009 0.35
Cit Mixed culture SF01 6.8 0.5 0.98 <0.001 1.24

1 Half-lives
were determined from first order calculations of the mineralization rate; 2For each first order plot, the P value was
<0.001; 3 % min equates to the extent of mineralization of fenamiphos after 73 days, except for the microcosms that were
bioaugmented, which were incubated for 93 days; 4Glu and Cit signify microcosms augmented with either glucose or citrate.
S.E. = the standard error (n=3) in the half-life. r2 = the correlation coefficient of the first order plot.

244 Franzmann et al.

 51.8 ± 13.5% for the atrazine microcosms and 70.7 ±
7.0% for the fenarniphos microcosms.
Discussion
Enrichment and Isolation of Pesticide-
Degrading Bacteria
Attempts to enrich and isolate bacteria from the Dianella
plume that were capable of decreasing atrazine concen-
trations in cultures were successful (Table 2). The mixed
cultures L25 and L27 did almost as well as Pseudomo-
nas sp. strain ADP which had been previously shown to
rapidly degrade atrazine as a sole source of nitrogen if
supplied with an alternative electron donor (citrate) and
acceptor (oxygen or nitrate) (Mandelbaum et al., 1995).
The addition of mixed cultures L25 and L27 induced the
production of CO2 from atrazine in the microcosm ex-
periments (Table 3), confirming the mineralizing capa-
bility of components of these mixed cultures. With fur-
ther experimentation, Tilbury (1998) isolated an axenic
bacterial strain from mixed culture L27, and this pure
Downloaded By: [PERI Pakistan] At: 05:09 24 May 2010
strain (designated strain AT2) was closely related, de-
tennined by 16S rRNA sequencing, to Pseudomonas sp.
strain ADP. Strain AT2 contained the same first three
genes of the atrazine degradation pathway used by strain
ADP (atzA, atzB, and atzC genes [De Souza et aI.,
1998; Tilbury, 1998]).
Attempts to isolate and enrich fenamiphos-degrad-
ing bacteria from the Dianella plume were less suc-
cessful. Mixed culture SPOI and strain SP03 showed
some ability to decrease the concentration of
fenamiphos over two weeks (Table 2); however, they
did not significantly stimulate mineralization to CO 2 in
the microcosm tests (Table 4). The decrease in
fenamiphos in these cultures may have been due to
partial degradation of the fenamiphos to the common
metabolic intermediates of fenamiphos degradation,
fenamiphos sulfoxide, and fenamiphos sulfone (Chung
and au, 1996). These metabolites were not searched
for in the GC-MS method used. To date, pure cultures
of fenamiphos-degrading bacteria have never been iso-
lated, although Ou and Thomas (1994) were able to
maintain a mixed bacterial culture that mineralized
fenamiphos in the presence of soil, although the
fenamiphos-degrading capability of that culture has
been subsequently lost (au, Personal Communication).
Mineralization of Atrazine in
Microcosms
The half-life of atrazine mineralization in unamended,
anoxic aquifer material was long (ca. 20 years; Table
3) and explains the observed persistence of atrazine at
the contaminated site over many years (Appleyard,
Bioaugmentation of Atrazine and Fenamiphos Impacted Groundwater: Laboratory Evaluation
1995). The half life of atrazine mineralization in aer-
ated aquifer material was less (ca. 5 years) and compa-
rable to the half-lives of atrazine mineralization mea-
sured in Swan Coastal Plain vadose zone sands collected
from below the surface soil horizon (3 to 7 years
[Franzmann et aI., 1998]). Oxygen amendment alone
did not sufficiently stimulate atrazine mineralization
in these microcosms which suggests that atrazine in
the plume could not be effectively bioremediated by
delivery of oxygen alone. Although the addition of
glucose and oxygen to aquifer material further in-
creased the mineralization rate of atrazine (11/2 = 2.2
years), it was only in microcosms that were
bioaugmented with atrazine-degrading cultures that
the mineralization rates were sufficiently increased (t1l2
= 15 to 50 days) to contemplate implementation of
bioremediation in the field.
The addition of strain ADP stimulated atrazine
mineralization even in the anaerobic microcosms. This
is consistent with the finding that the enzymatic steps
in the atrazine mineralization pathway (at least by
strain ADP) are hydrolytic ones and do not require
oxygen (De Souza et aI., 1998). The addition of nitrate
and citrate (on which strain ADP can grow
[Mandelbaum et aI., 1995]) with strain ADP to the
anaerobic microcosms did stimulate atrazine mineral-
ization (decrease in 11/2 from 50 to 27 days, Table 3).
The greatest stimulation of atrazine mineralization
occurred in microcosms amended with oxygen (air) and
bioaugmentedwith the atrazine-degrading cultures (Table
3). The additional amendment of citrate or glucose to the
ADP augmented microcosms did not increase the atra-
zine mineralization rate (Table 3) which suggests that an
added carbon and energy source would not be required
for bioremediation in the field. The organic carbon con-
tent in aquifer material from Dianella has been measured
at 0.7 g/kg (unpublished data) and carbon amendments
were added at a concentration of 5.0 g/kg.
Bioaugmentation with cultures derived from the
Dianella plume (strain SF05, mixed culture L25, and
mixed culture L27) all induced atrazine mineralization
rates comparable to the rate achieved by strain ADP,
and the extent of mineralization, 40 to 50% of the
added atrazine (Table 3), was not significantly differ-
ent between the Dianella-derived cultures and strain
ADP. Cultures obtained from the Dianella site could
be used as effectively as strain ADP for bioremediation
in the field without concerns for the quarantine restric-
tions placed by Australian authorities for the use of
imported cultures.
Even in bioaugmented microcosms, only 40 to
50% of the atrazine, added at a concentration of 240
ug/kg, was recovered as CO 2 , Masaphy and
Mandelbaum (1997) recovered varying amounts of
245
 CO 2 from atrazine additions to soil slurries depending
on the slurry treatment. Slurries inoculated with strain
ADP resulted in 40 to 60% mineralization unless the
slurries were preincubated with atrazine for 12 days
prior to the addition of strain ADP, in which case 60 to
80% of the label was recovered in CO 2 , They con-
cluded that "aging" of the herbicide in the soil has a
pronounced effect on the mineralization results. Atra-
zine mineralization in fertile New Zealand soils was
shown to be rapid and ceased after 100 days; however,
less than half of the atrazine was decomposed to CO 2
(Sparling et al., 1998). A range of studies have shown
(cited by Sparling et al., 1998) that a large proportion
of atrazine is incorporated into non-extractable bound
residues, probably as partial degradation products.
Mineralization of Fenamiphos in
Microcosms
The mineralization rate of fenamiphos innon-aug-
mented aquifer material was extremely slow (tI/2 =
68.7 years) when compared with the rate ofloss of the
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combined suite of fenarniphos compounds (fenamiphos,
fenamiphos sulfoxide, and fenamiphos sulfone) in soils
of the Swan Coastal Plain (bl2= 50 days for surface soil
or140 days for subsurface soils [Kookana et al., 1997]).
Half-lives for fenarniphos and fenarniphos sulfoxide in
16 surface soils with no previous exposure to
fenarniphos, collected from 11 different countries,
ranged from 12 to 87 days (au et aI., 1994). The half-
life for mineralization of fenamiphos for soils that
have been continually exposed to fenamiphos for over
15 years can shorten to 1 to 3 days (Ou et al., 1993).
Despite all the literature suggesting that fenamiphos
is readily biodegradable in surface and vadose zone
soils, half lives for fenamiphos mineralization in our
aquifer material with or without a range of amend-
ments (nitrate, oxygen, citrate, and glucose) were all
extremely long by comparison (Table 4). The fact that
fenamiphos is still measured in groundwater 5 years
after cessation of further contamination, and after a
pump-and-treatremediation exercise (Appleyard, 1995)
would support the conclusion that fenamiphos miner-
alization half lives are long in this environment. De-
spite the literature pointing to rapid mineralization of
fenamiphos in surface and vadose zone soils, we are
not aware of any rate data for fenamiphos mineraliza-
tion in anaerobic aquifer material. It is probable that
aquifers, and the microbial communities in aquifers,
are rarely exposed to fenamiphos due to its rapid deg-
radation in surface and vadose zone soils. The con-
tamination of the aquifer at Dianella was via a sump,
which allowed the fenamiphos to by-pass the surface
soils which would normally trap fenamiphos in its
246
organic fractions (Appleyard, 1995). Fenamiphos also
persisted in a column experiment that was associated
with this microcosm study (Unpublished data) which
confirmed a degradation rate (disappearance of the
parent compound) in aquifer material from Dianella
that was much slower than rates measured in surface or
vadose zone soils. In the microcosm experiments, not
only was the rate of mineralization slow, but the extent
of mineralization over the time course was also mini-
mal (Table 4).
Bioaugmentation of microcosms with bacteria
enriched from the aquifer by supplying fenamiphos as
their only source of nitrogen and phosphorus showed
no significant effect on the mineralization of fenamiphos
over the time course of the experiments (up to 93 days;
Table 4). However, there did appear to be some in-
crease in fenamiphos mineralizing capability in two of
the augmented cultures after a considerable lag phase
of about 60 days (in the case of mixed culture SF01,
see Figure 3). Experiments conducted over much longer
time frames than those usually used by researchers
investigating the mineralization of fenamiphos in soil
seem warranted.
Total recovery of the 14C label in the sodium
hydroxide traps and water and ether extracts of the
aquifer material at the conclusion of the experiment
showed that on average, only 51.8% or 70.7% of the
label was recovered for atrazine or fenamiphos respec-
tively. The low recovery of the 14C-Iabel from atrazine
at the completion of the incubation is consistent with
the data of Sparling et al. (1998), who showed that
between 54 and 77% of 14C-atrazine added to New
Zealand soils could not be recovered as either 14C02 or
in solvent extracts of the soils. This non-recovered
label was incorporated into non-extractable bound resi-
dues that were unavailable for further metabolism.
Similarly, Ou et al. (1994) found that between 17 and
40% of the label from 14C_fenamiphos additions could
not be extracted from microcosms of soils from a
Florida golf course. Presumably these non-extractable,
non-available fractions are of limited environmental
concern in contaminated sites.
Conclusions
Microcosm evaluation of the effects of chemical amend-
ment and bioaugmentation of aquifer material from
near a contaminated site at Dianella would suggest that
mineralization rates of both atrazine and fenamiphos
in unamended aquifer material are slow. However the
rate of atrazine mineralization can be greatly enhanced
by augmentation with known atrazine-degrading cul-
tures and is further enhanced by the addition of oxygen
Franzmann et al.
 that acts as an electron acceptor for the growth of the
atrazine-degrading bacteria. Even under anaerobic
condition, atrazine-mineralization rates were still im-
proved through bioaugmentation with known atrazine-
mineralizing strains. Cultures of atrazine-mineralizing
bacteria collected from the local contaminated site
were as effective as Pseudomonas Spa strain ADP in
promoting mineralization of atrazine in soil from the
site.
Additions of electron acceptors or donors did not
significantly enhance the rate of mineralization of
fenamiphos in the microcosms, and we were unable to
obtain fenamiphos-mineralizing bacteria from the con-
taminated site, although some strains did show some
potential at degrading the parent compound. The min-
eralization of fenamiphos seemed to require a long lag
phase in augmented microcosms, and repetition of the
experiment over greater time periods may be war-
ranted.
There would seem to be good prospects for suc-
cessful bioremediation of atrazine contamination at
the field site using a combination of oxygen (air) de-
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livery with bioaugmentation. Prospects for successful
bioremediation of fenamiphos remains equivocal at
this stage; however, further experimentation may re-
veal a potential fenamiphos-degrading microbial popu-
lation.
Acknowledgments
This study was partly funded by the Water and Rivers
Commission of Western Australia and the Centre for
Groundwater Studies. David Briegel, Silvana Fidalgo,
Karen McNally, Terry Power, Cynthia Joll, and Wayne
Hick are thanked for their technical assistance.
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