oEPA
United States
Environmental Protection
Agency
Robert S. Kerr Environmental
Research Laboratory
Ada, OK 74820
Research and Development
EPA/600/S-92/001 February 1992
ENVIRONMENTAL
RESEARCH BRIEF
Biodegradation of Atrazine in Subsurface Environments
James L. Sinclair* and Tony R. Lee*
Abstract
The pesticide atrazine is frequently detected in ground water,
including ground water used as drinking water. Little information
is available on the fate of atrazine in the subsurface, including its
biodegradability. The objectives of this study were to evaluate the
biodegradability of atrazine under differing conditions (oxygen
status, prior exposure to atrazine, subsurface sediment texture,
unsaturated and saturated zone sediments) that would be
commonly encountered in the subsurface. Samples of soil and
sediment were taken from a borehole drilled at a location beside
a highway near Stratford, Oklahoma which had received
applications of atrazine annually for 12 years. A second borehole
was drilled 66 feet away in a field that had not received atrazine
applications. Samples were taken from different depths with
respecttothewatertableandsedimenttypes. Core material from
the boreholes was used to make microcosms to study atrazine
biodegradation. Microcosms from Stratford, OK sediment were
incubated aerobically. A samplef rom the Norman, OK landfill was
used to make microcosms that were incubated anaerobically.
Identical microcosms that had been sterilized by autoclaving were
used as controls to differentiate biodegradation from abiotic
processes. Most or all of the atrazine spiked into active and sterile
microcosms made of surface soil from both Stratford locations
had disappeared by 105 days of incubation as determined by
HPLC analysis. No disappearance of atrazine was observed in
either the active or sterile treatment of any of the subsurface
samples from either Stratford borehole. A small amount (3.8%)
of MC ring labeled atrazine was mineralized to "CO2 by 161 days
ManTech Environmental Technology, Inc., Roberts. Kerr
Environmental Research Laboratory, Ada, OK.
in the active treatment of the surface soil from the Stratford
roadside. Little or no atrazine was mineralized to CO in
microcosms of the surface soil from the Stratford field oMhe
subsurface samples from either borehole. A slow decline of
atrazine was noted in the active treatment of the Norman landfill
sediments that were incubated anaerobically. Therefore, some
decline of atrazine concentration was noted in anaerobic
subsurface microcosms of Norman landfill sediments, but no
decline was observed in the aerobic Stratford subsurface sediment
microcosms. Factors responsible for the lack of atrazine
degradation in the Stratford microcosms may have included the
usually small bacterial populations in these samples and the
resistant nature of atrazine.
introduction
Within the last decade, there have been many reports of the
occurrence of pesticides in ground water (Younos and Weigmann,
1988). One of the pesticides which occurs most commonly in
ground water is atrazine. Because of health concerns, there is
interest in factors which would affect the occurrence and fate of
atrazine in ground water. Typically, when atrazine is found in the
subsurface it is present in concentrations of 0.3 to 3 ppb (Younos
and Weigmann, 1988), which is at least an order of magnitude
lower than levels commonly found in surface runoff waters
(Thurman et al., 1991). The amounts of atrazine which occur in
the subsurface are controlled by factors which are discussed in
Cheng and Koskinen (1986) and Helling and Gish (1986).
Predicting the fate of atrazine in the subsurface is difficult because
little work on the behavior of atrazine has been done in the
subsurface or with samples of subsurface material. Mostworkon
atrazine degradation was done in the 1960s and 1970s and was
^gA) Printed on Recycled Paper
-------
dona either with surface soil, water or with pure cultures of
microorganisms. These studies indicated that atrazine was
moderately resistant to degradation in surface soils although
degradation rates varied considerably in different soils (Skipper
and Volk, 1972; Roeth et al., 1969). Enough atrazine often
survived into the next growing season so that inhibition of sensitive
plants occurred. Mineralization of atrazine to CO2 was found to
proceed very slowly with only a few percent of added atrazine
being mineralized in several months time (Skipper and Volk,
1972, and Dao et al, 1979).
A limited number of studies have been done on atrazine
biodegradation in the subsurface. Roeth et al. (1969) reported
that atrazine was degraded 2 to 3 times faster in surface soil than
In subsurface soil from 14 to 24 inches and 36 to 48 inches deep.
Lavy et al. (1973) found that phytotoxic amounts of atrazine were
gone after 5 months at 15 cm, 17 months at 40 cm, and were still
present after41 months at 90 cm after atrazine amended soil from
these depths was reburied at these same depths in a soil pit in
Nebraska Obenhuber (1988) reported that atrazine had a hatf-
lifo of 556 days in aerobic subsurface microcosms, and 2632 days
in anaerobic microcosms based on "CO2 data. This report also
demonstrated that alternating aerobic-anaerobic treatments
speeded up the rate of degradation. These studies suggest that
atrazine degradation proceeds at a slower rate in the subsurface
than in surface soil. Recent work on microbial populations and
activities in the subsurface has shown that microbial abundances
(Sinclair and Ghiorse, 1989, Sinclair era/. 1990) and activities
(Hicks and Fredrickson, 1989) differ between different sediment
types. Thus, H is likely that pesticide biodegradation will proceed
at different rates in different types of subsurface sediment.
This study was designed to provide more information on the
biodegradation of atrazine in the subsurface. The specific
objectives were to determine if atrazine biodegradation occurs in
the subsurface, and if so, at what rates in different parts of the
profile and in different sediment types.
Materials and Methods
Sites and Sampling
A search was conducted for a site where atrazine had been
regularly applied and which had other desired characteristics.
These features were that the site was reasonably level, had
permeable sediments which would permit rapid downward
movement of water, and had a nearby location with similar
geology but had not received applications of atrazine, and was
near Kerr Lab. The only site that met these selection criteria was
the roadside by highway 177 which had been sprayed with
atrazine, glyphosate and sulfometuron methyl once a year for
weed control. Atrazine had been applied annuallyforapproximately
12years. The location was 4.2 miles north of Stratford, OKon the
west side of Highway 177. Surface soil collected January 2,1989
from the roadside was found to contain atrazine. A drilling was
conducted June 9,1989 at the roadside to evaluate whether this
s'rte would be suitable for this study. The subsurface strata were
determined to be permeable to water; the water table level was
found to be somewhat deep but still at an acceptable level for the
migration of atrazine to ground water (10.59 m); and samples
were taken for atrazine analysis. A drilling to obtain samples for
experiments was conducted on July 25,1989 in the roadside and
July 26, 1989, 66 feet west of the roadside borehole in a field
where atrazine had not been applied. Samples were taken from
a number of depths where sediments having different textures
were found. Samples from each borehole that will be discussed
in this paper are the following:
Description
Surface
Unsaturated
Saturated
Roadside
Depth (m)
0.0- 0.1
4.2- 4.5
11.8-12.0
Field
Depth (m)
0.0- 0.1
3.8- 4.1
12.0-12.3
The subsurface samples used in the anaerobic treatment were
from the Norman, OK landfill (Beeman and Suflita, 1990). A hole
was dug with a backhoe to below the water table (1.5 to 2.0 M
deep). Samples were scooped into jars from at or slightly below
thewatertable. Sediments were anaerobic at thiss'rte, and rapidly
reduced resazurin.
The average annual temperature in central Oklahoma (including
Stratford and Norman) is 16 to 17 °C. The temperature of shallow
sediments varies with the seasons, howeverthe deeper sediments
are the average annual temperature. Seasonal variations were
noted in the temperature of the Norman landfill sediments due to
their shallow depth (S.A. Gibson, personal communication).
Microcosms
Microcosms were set up by aseptically weighing out 10 g of
sediment and transferring this to a steriie 60 ml serum bottle. The
bottles were stoppered with sterile 1 cm thick butyl rubber stoppers.
Sediments were adjusted to 30% water content with sterile Milli-
Q water. Sterile microcosms were made by autoclaving for 8
hours at 122 °C, after which, all manipulations of the microcosms
were done in a laminarflow hood using aseptic technique. Sterile
and active (nonsterile) microcosms were made from all samples.
Three replicate microcosms were made for each treatment. The
anaerobic Norman landfill sediments were weighed out for
microcosms in an anaerobic glovebox. One ml of a 1/100,000
dilution of resazurin and a 0.1 ml of a 100 u,m solution of
Na^S^H O were added to each microcosm. The microcosms
were incubated at 22°C in the dark. All microcosms were
amended with 100 ppb of atrazine.
Sample Characterization
The following properties of samples were determined: water
content, particle size distribution, total organic carbon (TOG), pH,
total nitrogen, ammonium nitrogen, total phosphorus, phosphate
phosphorus, and 26 metals.
Bacteria in samples were enumerated by plate counts using dilute
peptone, trypticase, yeast extract, glucose (PTYG) medium
(Balkwill and Ghiorse, 1985). Directcountsof bacteria were made
using acridine orange direct counting (AODC) procedures
described in Balkwill and Ghiorse (1985).
Extraction and Analyses
Atrazine standards were obtained from EPA and ManTech
standards repositories in Cincinnati, OH and Research Triangle
Park, NC, respectively. Standards were also made up from pure
atrazine obtained from Chem Service (West Chester, PA). Little
difference was noted between these standards when they were
compared.
-------
The atrazine extraction method of Muir and Baker (1978) was
tested to determine its efficiency for atrazine extraction from soil.
Soil was spiked with 1 ppm of atrazine which was allowed to
remain in the soil for 1 week before extraction. Twenty ml of
absolute methanol were added to 10 g of soil and were shaken for
one hour. The methanol was poured into aTeflon centrifuge tube
and centrifuged on a Sorvall RC2B centrifuge at 10,000 rpm using
an SS34 rotor for 10 minutes. The supernatant was analyzed by
high pressure liquid chromatography (HPLC) analysis and showed
a95% recovery of atrazine. This method was adopted foratrazine
extraction.
Bond Elute C18 cartridges (Analytichem International, Harbor
City, CA) were investigated as a method to concentrate atrazine
and remove soil organic matter from methanol extracts of soil. To
increase the polarity of the extract so that atrazine would be
retained by the C18 material, the extract was diluted 10:1 with
Milli-Q water. Two tests were run to determine how efficient the
cartridges were at retaining the atrazine while the extract was
being passed th roug h the cartridge. The first test was to run a 100
parts per billion (ppb) solution of 10% methanol: 90% water
through a C18 cartridge. The second method was to stack two
018 cartridges and measure the amount of atrazine which passed
through the first cartridge into the second cartridge. After the
extract was passed through the cartridges, 2.0 ml of absolute
methanol was used to elute the atrazine from the C18 material.
Analysis of the eluate indicated that for the first method of
checking the extraction efficiency of the C18 cartridges, essentially
all of the atrazine in the initial solution was recovered in the
methanol eluate. For the second method, it was found that 1.7%
of the total amount of atrazine had passed through the first
cartridge into the second cartridge. Therefore, the C18 cartridge
method of concentrating the extracts was found to be very
efficient and was selected as the method of concentration to be
used.
HPLC analysis was done using a Scientific Systems Inc. (SSI)
isocratic HPLC system. Samples dissolved in methanol were
diluted 50:50 with Milli-Q water; and if any turbidity was noted, they
were centrifuged in a Microfuge (Fotodyne, Gosheim, F.R. Germany)
for 10 minutes. Samples were injected into a 50 u,l sample loop.
A mobile phase of 28% acetonitrile, 68% water, 4% methanol and
0.0002% acetic acid (v/V) at pH 4.6 gave the best results for
separating atrazine from interfering peaks. Some samples were
analyzed by increasing the methanol concentration stepwise up to
7% with a corresponding decrease in the percentage of water. An
Alftech C8150 X 4.6 mm column and an Alttech C8 guard column,
both with 5 urn packing material were used. A flow rate of 2.0 ml
per minute was used initially and then reduced to 1.5 ml per minute
later to reduce pressure and stress on the column. A UV-VIS
detector was used at 222 nm. The extraction method used in this
study mobilized a considerable amount of soil organic matter. To
prevent this organic matter from degrading the performance of the
column and other parts of the system, regularf lushes and cleanings
of the column and other parts were necessary including a 10
minute methanol flush between each sample analysis followed by
a 20 minute baseline re-equilibration flush with the mobile phase.
Mineralization of the atrazine ring was determined by measuring
MCO2 production from 14C ring-labeled atrazine added to the
microcosms. The amount of 14C labeled atrazine added to the
microcosms was 50,000 DPM with enough nonlabeled atrazine
added to make up a total of 100 ppb. CO was trapped in 0.25 ml
of 1 N NaOH in a centerwell apparatus (Kontes Glass, Vineland,
NJ) suspended from the stopper of the bottle. One day before the
bottles were sampled t% H2SO, was added to the bottles to drive
off carbpnate-CO2. A pilot study using this method indicated that
CO2 inside the bottles was almost completely removed by the CO
trap after 1 hour and therefore this method is satisfactory for
determining CO2 evolution in this type of microcosm (Ghiorse et
al., 1989 and Madsen et al., 1991). A search was conducted for
a suitable scintillation cocktail which would give a high counting
efficiency in the presence of the 1 N NaOH CO absorbent. A 6:l
ratio of Beckman Ready-Sblv EP cocktail (Beckman Instruments
Inc., Fullerton, CA) to 1 N NaOH was found to give equal counts
to a 6:1 ratio of the same cocktail with water even when a
precipitate formed in the NaOH and cocktail mixture. Therefore
this cocktail was used in the experiments.
Results
The physical, chemical and biological properties of the Stratford
samples recovered from the boreholes are listed in Table 1. The
bacterial populations in all of the samples used in this experiment
including those from the sandy strata were lower than usual
(Sinclair and Ghiorse, 1989) with even the highest plate counts
from these samples being less than 105 per gram of sediment.
The 12.0 m deep sample from the field had <50 platable bacteria
per g. The other characteristics showed differences in the
samples as would be expected. Nitrogen and phosphorus showed
a general trend toward lower values with depth. Samples used for
microcosms were analyzed for 26 metals (data not shown). There
was little relation between the metals content of the samples and
biological populations.
Atrazine assays of the samples taken January 2,1989 from the
roadside indicated that there was about 1 part per million (ppm)
of atrazine in the surface soil. Another surface soil sample taken
from the roadside March 13, 1989 had 9.3 ppb indicating that
there was a loss of 99% of the atrazine over a period of 2 months
and 11 days. The temperatures during this time were cold so
biological activity would have been minimal. There had been
substantial rain and snow melt during this period and there was
evidence of the flow of water in the ditch. Therefore, it appears
that most of the atrazine loss occurred as a result of surface runoff.
At the time of the July, 1989 drilling 26 ppb atrazine was found in
the surface soil and 0.46 ppb was found 4.2 m deep in the
unsaturated zone (Table 1).
The results of the atrazine disappearance experiment are shown
in Figures 1 and 2. Atrazine loss occurred at a steady rate in
microcosms of both the active and the sterile treatments of the
surface soil of the roadside. By the end of the experiment at 161
days, more than 80% of the atrazine originally spiked into the
microcosms had been degraded or bound in both the active and
sterile treatments in the roadside surface soil. In the surface soil
of the field where atrazine had not been applied there was a sharp
decline in concentration up to 77 days, after which atrazine had
declined to below the limit of detection in both active and sterile
treatments. Data shown for microcosms of subsurface material
(Figure 1) indicate that there was no atrazine loss from any of the
samples. No differences were noted between the active and
sterile treatments of the subsurface sample microcosms, and not
even abiotic hydrolysis was observed in the other subsurface
samples regardless of the differing characteristics of the samples.
By contrast, in subsurface samples taken from the Norman landfill
(Figure 2), a steady decline in the concentration of atrazine was
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Tfme(days) Time (days)
a" uysis T. Sh°wn for mfcw«»m» <>' *<>» and sediments from the Stratford, OK site. Panel
A ho mf .K u . , .
from ih» fii^ft T T Pro'''* o^epth* from the roadside where atrazine had been sprayed. Panel B shows microcosms from a profile
from the field 66 ft. from the roadside where atrazine had not been sprayed. The error bars are ± 1 S.D.
-------
70
60
60
I
& 40
20
10
Saturated Zone of Norman Landfill
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D Active
»-_ Starila
\
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Time (days)
Figure 2. Alrazlne level* a* determined by HPLC analysis are shown
formIcroco*m»framthe*aUiraledzoneof theNorman Landfill. The
error bar* are * 1 S.D.
noted in the active treatment throughout the experiment. By the
end of the experiment very little of the added atrazine was
detectable. In the sterile treatment, it was not possible to
determinewhethertherewas any degradation of atrazine because
of interferences in the samples.
The results of the atrazine mineralization experiment (Figure 3)
indicated that there was a slow release of MC labeled CO, from
labeled atrazine added to the surface soil of the roadside. By 161
days of incubation, 3.8% of the activity originally added as labeled
atrazine was released as "CO,,. Avery small amount of 14CO2 was
released from the surface soil of the field in the nonsterile
.treatment as compared to the sterile treatment. By 161 days, this
amounted to 0.4% of the total amount of activity added. Very little
activity appeared in the CO2 traps of the microcosms of the
subsurface samples. Most of the activity which was trapped
appeared at 133 or 161 days and often appeared equally in the
nonsterile and the sterile microcosms. Examples of nearly equal
amounts of activity found in nonsterile and sterile treatments can
be seen in the roadside sample from 11.8 m deep and the field
samples from 3.8 and 12.0 m deep. No clear example was
observed where microcosms of the nonsterile treatment of a
subsurface sample had more activity in the CO2 trap than could be
seen in the sterile treatment from the same subsurface sample.
Discussion
Most of the atrazine applied to the roadside appeared to be lost
to surface runoff, howeverthere was some penetration of atrazine
into the upper part of the unsaturated zone. The amount of
atrazine which penetrated into the 4.2 m deep sample (0.46 ppb)
was below the EPA's health advisory limit of 3 ppb of atrazine in
ground water (USEPA, 1989) and detectable levels of atrazine
were not found in deeper samples. Therefore, the indigenous
microbial populations were exposed to atrazine in the surface soil
and upper parts of the unsaturated zone of the roadside. Despite
this prfor exposure in at least part of the profile, there was no
evidence of microbial acclimation to- atrazine in the surface or
subsurface of the roadside in the atrazine disappearance
experiment. In the roadside and the field, abiotic hydrolysis
seemed to be responsible for almost all of the atrazine breakdown
since nearly equal amounts of loss were observed in the active
and sterile treatments. There seemed to be a more rapid rate of
hydrolysis in the surface soil of the field than in the roadside
atthoughthe reason forthis is not clear. The lack of biodegradation
of atrazine in the subsurface samples may not have been
unexpected, however the lack of abiotic hydrolysis was unusual
and the reason for this is not evident.
An objective of this work was to determine how the rate of
biodegradation of atrazine differed in sediments having different
characteristics, such as texture or position with respect to the
water table. Regardless of differing characteristics, no atrazine
degradation was observed in any of the subsurface samples. A
similar observation about atrazine biodegradation was made by
Konopka and Turco (1991). Atrazine was apparently resistant
enough to degradation to mask any differences in biodegradation
which might beobserved in sediments which haddifferent microbial
population sizes or types during the 161 -day time course of the
experiment. Degradation of atrazine was observed in the Norman
landfill sediments which were incubated anaerobically. This
result illustrates that atrazine may degrade in the subsurface
under some conditions.
Very little complete mineralization of atrazine to CO2 was observed
in any sample. These results are similar to those of Skipper and
Volk (1972) and others who also observed little atrazine
mineralization in surface or subsurface soils. There did appear to
be a microbial acclimation effect in the roadside surface soil as
compared to the field surface soil for atrazine mineralization.
Atrazine mineralization to CO2 appeared to be abiological process
because it was only observed in the active treatment. About 10
times as much atrazine mineralization occurred in the roadside
surface soil microcosms as occurred in the field surface soil
microcosms. No evidence could be seenfor atrazine mineralization
in any of the subsurface sediment samples, ft is not clear why 14C
activity was found in the CO2trap cups of the sterile treatments of
several of the subsurface sediment microcosms but not in the
sterile treatment surface soil microcosms, ft is unlikely that the
activity was due to MCO2formed by microbial contaminants in the
sterile subsurface sediment microcosms because this activity
was noted in all of the replicates of these microcosms.
It is difficult to judge how representative the results of these
experiments are of atrazine biodegradation at other subsurface
sites because of the lower than usual bacterial populations at the
Stratford site (compared to Sinclair and Ghiorse, 1989 or Sinclair
era/., 1990). Nonetheless, the microbial populations did have the
. ability to degrade alachlor even though alachlor had not been
applied to this site. Dr. T.B. Moorman of the USDA in Stoneville,
MS (1991 Abstr. Afner. Soc. Microbiol. Ann. Meeting Q94, p. 292)
has reported that several percent of alachlor added to microcosms
of some subsurface sediments from the Stratford site was
mineralized. Alachlor did degrade differently in samples having
different textural characteristics or position with respect to the
water table. The alachlor degradation in the Stratford samples
was reported to be more rapid than that found in subsurface
samples from Plains, GA (T.B. Moorman, personal communication,
and Pothuluri et al, 1990). Therefore the slow rate of atrazine
biodegradation in the subsurface of this site may be a reflection
of the resistance of atrazine to degradation and its decreasing rate
of degradation with depth in soil (Lavy et al., 1973). Nonetheless,
because of the sparse bacterial populations and the lack of abiotic
hydrolysis in the subsurface samples from this site, other sites
-------
10
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Surface Soil
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Active
Sterile
SO 100
Time (days)
10
Saturated Zone (12.0 m)
jk o Active
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50 100
Time (days)
Figure 3. The percent of MC evolved as «CO2-C from added "C ring-labeled atrazine is shown. Panel A shows the results from microcosms
of the roadside where atrazine had been sprayed and panel B shows the results from the field 66 ft. from the roadside where atrazine had
not been sprayed.
-------
should be studied to determine if atrazine degrades more readily
in the subsurface at other locations.
References
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subsurface bacteria associated with two shallow aquifers in
Oklahoma. Appl. Environ. Microbiol. 50:580-588.
Seaman, R.E., and J.M. Sufl'rta. 1990. Environmental factors
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Ghiorse, W.C., J.L Sinclair, K. Malachowsky, and E.L. Madsen.
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Muir, D.C.G., and B.E. Baker. 1978. The dissapearance and
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Disclaimer
The information in this document has been funded wholly or in
part by the United States Environmental Protection Agency. This
document has been subjected to the Agency's peer and
administrative review and has been approved for publication as
an EPA document.
Quality Assurance Statement
All research projects making conclusions or recommendations
based on environmentally related measurements and funded by
the Environmental Protection Agency are required to participate
in the Agency Quality Assurance Program. This project was
conducted under an approved Quality Assurance Program Plan.
The procedures specified in this plan were used without exception.
Informationontheplan and documentation of thequalrty assurance
activities and results are available from the Project Officer.
ttU.S. GOVERNMENT PRINTING OFFICE: 1991 - 648-080/40I4S
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