The anaerobic degradation of endosulfan by indigenous microorganisms from low-oxygen soils and sediments

Environmental Pollution 106 (1999) 13±21

www.elsevier.com/locate/envpol

The anaerobic degradation of endosulfan by indigenous
microorganisms from low-oxygen soils and sediments
Turlough F. Guerin*
1691 E Green Briars Drive, Suite 3821, Schaumburg, IL 60173, USA
Received 27 July 1998; accepted 24 February 1999

Abstract
Indigenous mixed populations of anaerobic microorganisms from an irrigation tailwater drain and submerged agricultural
chemical waste pit readily biodegraded the major isomer of endosulfan (endosulfan I). Endosulfan I was biodegraded to endosulfan
diol, a low toxicity degradation product, in the presence of organic carbon sources under anaerobic, methanogenic conditions.
While there was extensive degradation (>85%) over the 30 days, there was no signi®cant enhancement of degradation from enriched inocula. This study demonstrates that endosulfan I has the potential to be biodegraded in sediments, in the absence of
enriched microorganisms. This is of particular importance since such sediments are prevalent in cotton-growing areas and are
typically contaminated with endosulfan residues. The importance of minimizing non-biological losses has also been highlighted as a
critical issue in determining anaerobic biodegradation potential. Seals for such incubation vessels must be both oxygen and pollutant impermeable. Te¯on-lined butyl rubber provides such a seal because of its resistance to the absorption of volatiles and in
preventing volatilization. Moreover, including a 100 mM phosphate bu€er in the anaerobic media has reduced non-biological losses
from chemical hydrolysis, allowing biodegradation to be assessed. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Endosulfan; Biodegradation; Anaerobic; Sediment; Cotton; Natural attenuation; Thiodan1

1. Introduction
Endosulfan is a chlorinated cyclodiene insecticide currently used throughout the world for the control of
numerous insects in a wide variety of food and nonfood
crops. Endosulfan (CAS, 115-29-7) comprises two parent isomers, the a-, and b-endosulfan, or endosulfan I
and II, respectively. These isomers have a wide distribution in the environment and have been detected in
soil and sediments at considerable distances from the
point of their original application (Mansingh and Wilson, 1995; Miles and Pfeu€er, 1997).
There is limited information on the mechanisms of
loss of endosulfan in low-oxygen and anaerobic environments (Cotham and Bidleman, 1989; Peterson and
Batley, 1993; Guerin, 1993) and few studies have
demonstrated that related compounds can be anaerobically biodegraded. Liquid culture studies with related

* Tel.: +1-847-240-4247; fax: +1-847-619-9905. 248 Gladstone
Avenue, Coniston, New South Wales, 2500, Australia.
E-mail address: turloughg@hotmail.com (T.F. Guerin)

cyclodienes have demonstrated that microorganisms
may monodechlorinate the hexachloronorbornene ring.
Schuphan and Ballschmitter (1972) ®rst reported the
anaerobic dechlorination of hexachloronorbornene and
its dechlorinated derivatives by pure cultures of anaerobic soil bacteria. Since then, the dechlorination of the
hexachloronorbornene moiety of the cyclodienes aldrin,
dieldrin, and endrin, has been demonstrated (Maule
et al., 1987), and it has been shown that a consortium
of anaerobic microorganisms create conditions for the
monodechlorination. Pure cultures isolated from the
consortium, including those from the genus Clostridium,
were less e€ective in bringing about monodechlorination (Maule et al., 1987).
Recent liquid culture studies have indicated that anaerobic soil microorganisms can metabolize selected
degradation products of endosulfan (Schneider and
Ballschmiter, 1995). There is, however, only limited
information on the anaerobic degradation of the major
parent isomer (Guerin, 1993, 1995). The current paper
describes the biodegradation of endosulfan I in mixed
anaerobic cultures by inocula from low-oxygen soils and
freshwater sediments. Speci®cally, these environments

0269-7491/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0269-7491(99)00067-6

14

T.F. Guerin / Environmental Pollution 106 (1999) 13±21

were irrigation tailwater drains and agricultural chemical evaporation (sullage) pits, where there was only
limited water perturbation and aeration. Such conditions are prevalent in the cotton-growing areas of
northern New South Wales, Australia, but low-oxygen
environments in general (such as sediments), are widely
distributed throughout the environment. The hypothesis
tested in the current research was that endosulfan I can
be biodegraded by indigenous microorganisms from
low-oxygen soils and sediments under strictly anaerobic
conditions.
2. Materials and methods
2.1. Collection of sediment and sullage pit samples and
preliminary anaerobic studies on endosulfan I
biodegradation
A ®eld trip was made to Narrabri, New South Wales,
Australia, to obtain samples from agricultural chemical
sullage pits and tailwater drains near cotton-farming
®elds. These samples were collected during winter when
there was considerable water ponding. Seawater sediment was collected 200 m o€ the shore of Botany Bay,
New South Wales, Australia, by dredging a sample
from the surface of the seabed (approximately 10±20
cm depth). Six subsamples were carefully removed from
the centre of the total mass of sediment that was collected (0.5±1 kg), and these were pooled to give composite samples in duplicate. Tailwater drain sediment
samples were collected from the top 10±15 cm of the
submerged sediment pro®le and sullage pit samples
were collected from 10±15 cm below the surface of the
bulk of the sludge. From each of the locations, six
subsamples were pooled to give composite samples in
duplicate. The duplicate samples from each of the
locations were kept in airtight glass jars at 4 C (Shelton
and Tiedje, 1984) and were then used as separate sour-

ces of inocula for the anaerobic incubation assays. The
samples collected were from anaerobic environments
and this was evident by: (1) their submerged locations,
(2) their colour, and (3) distinct odour of H2S. All the
samples had become black and evolved an H2S odour.
Table 1 describes key properties of the sediments and
soils used.
2.2. Preparation of soil inocula
Soil and sediment samples were stored at 4 C (1±3
weeks) in the absence of oxygen prior to their extraction. The samples were then brie¯y shaken for 15 min
with sterile and degassed minimal salts medium in
a ratio of 1:10 (w/v) (soil:minimal salts medium) in a
sealed ¯ask. A 10À1 dilution of this extract was added to
the anaerobic media to give a ®nal concentration of
0.4% (v/v).
2.3. Preparation of anaerobic enrichment cultures using
technical grade endosulfan and Tween 801
An anacrobic enrichment was prepared using endosulfan (a mix of isomers I and II in a ratio 70:30,
respectively, at 98% purity) and the surfactant, Tween
801 (CAS, 9005-65-6, 99% purity, Sigma Chemical Co.,
Sydney, Australia). The rationale for preparing the
enrichment was to develop a culture potentially capable
of degrading endosulfan I at high rates and to compare
this with an indigenous population (with little or no
previous exposure to endosulfan). The reason for using
Tween 801 was to increase the aqueous solubility (and
bioavailability) of endosulfan so that it may be more
accessible to the growing microorganisms for biodegradation (Rouse et al., 1994). Inocula were introduced
into minimal salts medium containing (in g/l) K2HPO4
(anhydrous) 6.8, KH2PO4 8.3, NH4NO3 1.0, MgSO4
0.05, CaCl2 (anhydrous) 0.02, and FeSO4 0.01. This
medium gave an a€ective bu€ering capacity (100 mM) at

Table 1
A description of selected properties of the samples used in the trial
Sample

Tailwater drain sediment (TD)
Sullage pit (new) (NSP)
Sullage pit (old)
Freshwater sediment
Seabed sediment
a
b
c
d

yga (g/g)

0.460
0.213
0.347
0.497
0.325

OC (%)
[Organic Carbon]

OM (%)
[Organic Matter]

Particle size fraction (w/w%)
Cb

Sc

FSd

CSe

1.62
1.30
1.20
2.03
0.95

2.28
2.85
2.11
3.56
1.67

53.7
18.3
51.1
16.5
15.9

24.2
7.5
15.9
8.8
2.6

19.7
49.5
13.0
42.9
12.5

2.4
24.7
16.0
31.8
69.0

yg is the gravimetric moisture content of the samples in their ®eld condition.
Clay fraction.
Silt fraction.
Fine sand fraction.


pH 7.0. Endosulfan I (98% purity) (1000 mg/l in
methanol) and Tween 801 (1%) (v/v) were added and in
conjunction with the small volume of methanol used for
dissolving the endosulfan, were the only sources of carbon in the liquid medium for microbial growth. In other
treatments, aldrin (98%) and dieldrin (98%) were added
separately at a concentration of 1000 mg/l, instead of
endosulfan I. The rationale for this was to compare
endosulfan with these related compounds, on which
other anaerobic studies have been performed (Maule et
al., 1987). Cultures were kept unshaken in the dark for
10 days in an incubator at 30‹1 C before subculturing.
The resulting mixed populations of anaerobes grown in
this medium containing endosulfan (or aldrin or dieldrin), were subcultured seven times prior to analyzing
their degradative capacity in the routine incubations.
Methane production was measured on the 10th day of
incubation during each of the third to the seventh subcultures. Argon (Ar) was used as the headspace gas in
these enrichment cultures and this provided an environment that was selective for the carbon sources in the
medium only.
2.4. Incubation conditions for routine anaerobic
incubation
A medium was prepared for routine anaerobic biodegradation (Maule et al., 1987) and contained (in g/l)
K2HPO4 (anhydrous) 6.8, KH2PO4 (anhydrous) 8.3,
NH4C1 1.3, MgSO4.7H2O 0.01, FeSO4, yeast extract
(Difco) 2.0, Peptone (Difco) 2.0, sodium formate
(anhydrous) 2.0, biotin 1Â10À6 and pyridoxal phosphate 2Â10À6. A trace element solution that contained
(in g/l) Na2EDTA 15.0, Cu(NO3)2, 1.0, ZnCl2, 2.5,
MnCl2, 1.0, CoC12 0.5, H3BO3 1.0, and Na2SO4, 1.0, was
added at 1.0 ml/l. The pH of the bu€ered medium
was adjusted to 7.0 and then ®ltered with Whatman No.
1 paper prior to autoclaving and adding the heat-sensitive components. The vitamin solution was ®lter sterilized and added when the medium was at room
temperature. The medium was reduced by the aseptic
addition of cysteine hydrochloride (in sterile distilled
water) to give 0.1% (w/v) followed by purging and
degassing with Ar for 15 min while the medium was
cooled rapidly (Miller and Wolin, 1974). Inocula were
then added (as described earlier). All incubations were
kept strictly anaerobic during the course of the enrichments and this was checked with resazurin (7-hydroxy3H-phenoxyazin-3-one-10-oxide). In addition to sterilized medium, a second set of controls were included,
which was medium with an autoclaved 10-day-old culture. Endosulfan I was added to growth medium in a
1000 mg/l methanol solution so that only a minimal
amount of methanol (a potential carbon source) entered
the medium. The ¯asks were made anaerobic by evacuation followed by degassing with Ar 6±8Â. Successful

15

reduction was demonstrated by the colour change of
resazurin from pink to colourless.
2.5. Incubation conditions and endosulfan I
determination
2.5.1. Sealing of the incubation vessels and addition of
incubation atmosphere
Wheaton serum bottles (125 ml) were used in the
non-sterile enrichment and non-enrichment cultures
where aliquots of culture were subsampled. Smaller
bottles were used for the sterile treatments where it was
important that the entire contents (including a solvent
rinse of the incubation vessel) be analyzed. In the preliminary experiments, butyl rubber stoppers were used
to seal the serum bottles and were the same used in
previous anaerobic studies (Miller and Wolin, 1974;
Balch et al., 1979; Shelton and Tiedje, 1984; Maule et
al., 1987; Holliger et al., 1992; Kohring et al., 1992). In
the routine incubations (described later in Materials
and Methods), butyl rubber stoppers were replaced
with PTFE (Polytetra¯uoroethylene)-lined butyl rubber
stoppers, which was reported in the early work by
Bouwer and McCarty (1983). This latter sealing system
was adopted to overcome diculties in retaining pesticide, due to the low absorptive capacity of PTFE (i.e.
Te¯on1) for insoluble and volatile organochlorine
compounds such as endosulfan I, while ensuring oxygen impermeability. Subsamples were taken from the
serum bottles by removing 1±2 ml from the incubation
broth (after homogenizing) using a syringe. Therefore,
over the entire period of the trial, there was a decrease
in the volume of the broth of 1 ml, and an equivalent
increase in headspace. Filter-sterilized gas was introduced to the vessels. Vessels were kept unshaken in the
dark for 30 days in an incubator at 30‹1 C. A mixture
of CO2:H2 (80:20) or Ar alone was used as headspace.
The gas was ®lter sterilized and introduced using 200ml syringes. Prior to sampling when the sampling syringe was used to homogenize the contents of individual
¯asks so that the cell material, and any pesticide residues absorbed into this, would be distributed evenly
throughout the liquor. This ensured that subsamples
taken from the microbiologically active ¯asks were
representative of the total ¯ask contents. In the sterile
treatments, subsamples were not taken, but rather the
entire ¯ask contents were sacri®ced because of adsorption of endosulfan onto glass.
2.5.2. Measurement of methane gas production
The atmospheres of the incubation vessels were subsampled (500±1000 ml) and analyzed immediately using
a Shimadzu gas chromatograph (Model GC-8A) ®tted
with a ¯ame ionisation detector (FID) and a 1 m Â 2.6
mm packed glass column (Poropak T, Waters/Millipore). Temperatures of 130 and 150 C were used for the

16


column and the detector, respectively. The N2 head
pressure was 4.0 kg/m2.
2.5.3. Measurement of microbial growth and pH of
cultures
Microbial growth was measured by determining
OD700 nm on a Beckman spectrophotometer (Model
DU-64). Controls containing the same media, without
added microorganisms, were also analyzed to determine
whether the increases in absorbance were due to microbiological activity.
2.5.4. Analysis of endosulfan
Endosulfan I was analysed by electron capture detector±
gas chromatograph (ECD±GC) and degradation products
were analyzed simultaneously with the parent isomers
(Guerin et al., 1992).
2.5.5. Microscopic examination of the mixed cultures
and enumeration
Gram stains of cultures were prepared. Carbol-fuchsin
and methylene blue were used as the spore stains.
Preparations were examined using a 100Â immersion oil
objective using light transmission microscopy (Olympus
Model BHA). Both non-enriched and routine incubation cultures were examined. Total anaerobic counts
were conducted on solidi®ed media (Balch et al., 1979).

degradation product, endo (ED), was detected in all the
inoculated treatments. No traces of ED were detected in
either of the controls. This was consistent with endosulfan
I absorption into the butyl rubber stoppers, preventing
it from undergoing hydrolytic reaction in the liquor.
3.2. Stability of endosulfan I under routine incubation
conditions
Butyl rubber seals were replaced with PTFE-lined
butyl rubber (with sterilized medium only), to reduce
absorption. When sterile controls were prepared using
PTFE-lined butyl rubber, the rate of endosulfan I dissipation was relatively slow at applied concentrations of
1, 2, and 10 mg/l after 10 days (Table 3). Thus, provided
that incubations in ¯asks were sealed with PTFE, were
sterile, and bu€ered at pH 7, relatively little dissipation
of endosulfan I occurred. The trend of increasing endosulfan I loss at decreasing spiking concentrations, illustrated a solubility e€ect. At higher concentrations,
endosulfan I was adsorbed onto the glass walls and was
less available for degradation reactions. There was only
very limited endosulfan I degradation in the controls
containing sterilized cells, indicating that non-living cell
matter was not catalyzing degradation.
3.3. Biodegradation of endosulfan I in routine
incubations
The inocula used were derived from either sediment
or from an irrigation tailwater drain (TD) or from a

3. Results and discussion
3.1. Preliminary studies
In all ¯asks (using butyl rubber seals to restrict oxygen penetration) endosulfan I decreased to very low
concentrations after 25 days. Controls containing sterile
media alone demonstrated a faster initial rate of dissipation than that from inoculated ¯asks, indicating
that there were nonbiological losses. It was apparent
that the observed losses were due to absorption by the
unlined butyl rubber seals (Table 2). These results highlight the importance of using PTFE-lined butyl rubber
sealed ¯asks in biodegradation studies of volatile or semivolatile organics to prevent absorption. The hydrolytic

Table 3
The stability of endosulfan I in sterilized anaerobic mediaa
Days incubation

Amount remaining (% of originally applied)
1 mg/l

0
2
4
6
8

100‹3
98‹2
95‹4
90‹2
80‹3

2 mg/l
100‹3
97‹2
94‹2
96‹2
90‹3

10 mg/l
100‹4
100‹1
98‹1
97‹1
98‹2

a
Duplicate analyzes conducted and vessels were sealed with PTFElined butyl rubber seals.

Table 2
The anaerobic dissipation of endosulfan I using extracts taken from low-oxygen soils and sediments incubated in ¯asks sealed with butyl rubbera
Inoculum source

Endosulfan 1 (% remaining)

ED (mg/l)

Biomass (OD700 nm)b

10 days
Freshwater sediment
Seabed sediment
Sterile medium
a
b
c

25 days

10 days

25 days

5 days

10 days

0.04
0.02
0.002c

0.5
0.3
0.002c

70
54
7.3

2.0
0.001c
1.0

0.02
0.05
0.01

0.15
0.20
0.01

1 mg/ml of endosulfan I initially applied and incubated at 30 C.
OD, optical density.
Not detected.


new sullage pit (NSP), and were either non-enriched or
enriched. Using NSP inocula, not previously enriched
by subculturing, led to an almost complete dissipation
of endosulfan I (originally applied at 1 mg/l) after 10
days (Fig. 1). Inoculation with a sample of the TD
sediment (that was non-enriched) also led to an almost
total loss of applied endosulfan I after approximately 10
days (Fig. 2). The same inocula applied to the medium
containing 10 mg/l of endosulfan I, resulted in slower
rates of decrease but only slightly higher remaining
endosulfan I concentrations (Figs. 1 and 2).
In the routine anaerobic incubations containing
inocula from enriched NSP and TD sediments, the
amount of endosulfan I remaining after 30 days incubation was less than 2% (Figs. 1 and 2). There was no
signi®cant di€erence between the extent of degradation
of endosulfan I between either the enriched or nonenriched incubations. These results demonstrated that
there was no enhancement of degradation in the enriched incubations. An explanation for the extensive
degradation observed in both the enriched and nonenriched incubations can be attributed to the primary
degradation reaction occurring. The formation of endosulfan diol occurs from opening of the cyclic diester ring

Fig. 1. Endosulfan I degradation and endosulfan diol formation in
enriched (E) and non-enriched (NE) irrigation tailwater drain (TD)
incubations.

Fig. 2. Endosulfan I degradation and endosulfan diol formation
in enriched (E) and non-enriched (NE) new sullage pit (NSP)
incubations.

17

system on endosulfan, and the current ®ndings suggest
that this may be catalyzed by a non-speci®c enzyme.
In the 10 mg/l incubations, there was an apparent
increase in the endosulfan I in the subsampled aliquots.
This was observed 2 days after the addition of inoculum (Figs. 1 and 2). This phenomenon was due to the
partitioning of adsorbed endosulfan I from the glass
surfaces on the inside of the incubation vessels to the
more lipophilic microbial cells. Endosulfan I was distributed to the glass surfaces in the ungrown (or very
young cultures) because 10 mg/l is 4±5Â higher in concentration than its water solubility (Guerin and Kennedy, 1992).
3.4. Formation of endosulfan I degradation products
ED, a hydrolysis product of the parent endosulfan
isomers, was formed in all the non-sterile treatments.
The dissipation of endosulfan I from the non-enriched
and enriched cultures correlated with an increase in ED.
This occurred in treatments containing either 1 or 10
mg/l of applied endosulfan I. The highest concentrations of ED were present in the incubations containing
10 mg/l of applied endosulfan I (Figs. 1 and 2). The
formation of ED was higher in the medium inoculated
with microorganisms from the NSP, compared to TD
sediment. Only traces of ED were reported in the nonsterile treatments, where concentrations did not exceed
3Â detection limit, or 0.006 mg/l.
There were signi®cant increases in the biomass of all
the inoculated treatments. In the sterile treatments, and
in the ®ltrate from biologically active incubations with
endosulfan I added, there were no increases in optical
density (OD 700 nm). This indicated that the turbidity
in the inoculated vessels was due to microbial growth
and not a result of chemical processes (e.g. precipitation) and this was con®rmed by microscopic examinations, and there were no metabolites formed during the
incubations with absorption at 700 nm. Lower maximum biomass values were recorded in the incubations
with 10 mg/l of applied endosulfan I, compared with

Fig. 3. E€ect of endosulfan I concentration on growth rates.

18


incubations of 1 mg/l (Fig 3). Growth rates and maximum biomass values were only marginally higher in the
incubations inoculated with microorganisms from the
enriched treatments (data not shown). The growth rates
in the cultures inoculated with soil extracts from the
sullage pit (both new and old) and TD were similar. In
all the treatments, the maximum growth was reached by
the sixth day of incubation. OD700 nm values in control
media containing inocula from TD and NSP without
endosulfan I (but containing solvent carrier), reached a
maximum of 0.45±0.47 at 2 days. This indicated that
there was only very minor inhibition of biomass production from added endosulfan I. Increased biomass
production did not correspond to higher ED formation
as the most ED was produced when biomass production
was lowest.
In many of the incubations it was noted that there
was evidence of ED dissipation, suggesting further
degradation. There was evidence that degradation to
metabolites, other than ED, was occurring from the
detection of traces of endosulfan hydroxy ether (EHE),
endosulfan ether (EE), and endosulfan lactone (EL) in
selected incubation vessels. In an enriched TD incubation, containing 10 mg/l endosulfan I, in addition to the
4.3 mg/l ED formed, EHE and EE were found at 0.21
and 0.04 mg/l, respectively, at 10 days. In a separate
trial, EL (2.6 mg/l) was detected in a NSP enrichment
culture, containing 10 mg/l of originally applied endosulfan I. EL was not consistently formed in all the
non-sterile treatments, re¯ecting its aqueous instability
(Miles and Moy, 1979). The identity of the degradation
products was con®rmed by the use of synthesized standards and chromatographic behaviour on two gas
chromatography (GC) columns (Guerin et al., 1992).
The formation and subsequent detection of degradation
products (at the end of the incubation period) could not
account for all the originally applied endosulfan I. This
lack of stoichiometric mass balance when endosulfan I
and the identi®ed degradation product concentrations
were considered, indicates that endosulfan I is most
likely being converted to anaerobic degradation products which are escaping detection. These could include
dechlorinated forms of endosulfan.
Endosulfan sulfate (ES), the major oxidation product
of both the parent endosulfan isomers, was not detected
in any of the anaerobic incubations. This is consistent
with the predicted pathway of degradation for this
compound where oxygen is limiting. These include tailwater drain and seabed sediments; freshwater muds and
soils that remain saturated with water for extended
periods of time. Thus from the current study, it appears
that the presence of ES in sediments is most likely
attributable to the transport and deposition of this
degradation product from its site of formation (such as
in the cotton ®eld itself), to the waterways and subsequently to sediments.

3.5. Minimizing the non-biological hydrolysis of
endosulfan
It is generally recommended that the pH of media for
biodegradation studies be in the region of pH 6±8
(Painter, 1992). Also, bu€ered media has been recommended for use in biodegradation assays to o€set the
e€ects microorganisms can have on broth pH and subsequently on any compounds added to the broth (Painter, 1992). The ®ndings of the current study indicated
that using a 100 mM phosphate-bu€ered medium provided pH stability against the growing anaerobic cultures (Fig. 4). The growing cultures did not increase the
pH of the medium more than 0.25 pH units. Klein and
Alexander (1986) have reported that the survival of
microorganisms from lake water was improved if it was
bu€ered with 100 mM phosphate bu€er, indicating that
such an approach is unlikely to a€ect the selection and
growth of microorganisms. The degradation observed
for endosulfan I in the current experiments was due
therefore to the activity of microorganisms and not a
result of nonbiological hydrolysis.
3.6. Analysis of the incubation vessel atmosphere
Methane was detected in the headspace of all the
enrichment cultures as well as those from the routine,
non-enrichment incubations. In the enrichment cultures,
methane production was measured in enrichments 3±7
(Table 4). Methane was also produced in cultures enriched with high concentrations of aldrin and dieldrin as
well as endosulfan, which indicated that these related
organochlorines did not inhibit the growth and proliferation of methanogenic microorganisms. Methane
production was at its peak at the sixth subculture for
both the enriched sullage pit (new and old) and TD
treatments and for each of the cyclodiene enrichments.
Methane production was slightly higher in the endosulfan-enriched, TD sediment. In all of the treatments,
methane concentrations decreased at the fourth and
seventh enrichment indicating that there was a reduction

Fig. 4. pH stability at varying endosulfan I concentration in the nonenriched TD incubation.


19

Table 4
A comparison of methane production from mixed anaerobic cultures enriched with high concentrations of endosulfan, aldrin and dieldrina
Compound

Source

Methane produced at enrichment No. (nmol/25 ml culture)
3

b

4

5

6

7

Endosulfan

NSP
TDc

59.4‹1.6
50.7‹0.4

18.95‹2.3
20.9‹0.5

65.4‹13.9
101.4‹64.8

70.9‹8.0
138.9‹14.2

17.3‹3.5
76.6‹21.0

Aldrin

NSP
TD

74.5‹15.0
63.9‹4.7

26.5‹15.0
15.8‹3.1

44.2‹16.2
36.0‹4.6

52.3‹1.6
79.9‹19.4

28.4‹1.7
27.8‹6.1

Dieldrin

NSP
TD

68.1‹8.2
56.6‹11.9

15.2‹3.0
19.4‹2.9

85.0‹64.8
34.4‹20.9

98.5‹29.6
90.8‹16.5

48.8‹4.4
15.5‹0.9

a
Endosulfan I:II (70:30) parent isomers (98%), aldrin (98%) and dieldrin (85%) were added to anaerobic medium at 1000 mg/l with Tween 801
(1%). Incubations were subcultured every 10 days and kept under Ar.
b
Soil from a new agricultural chemical sullage pit (10±15 cm below surface).
c
Sediment from an irrigation tailwater drain.

in the activity of the methanogenic bacteria at these
times. This suppression of activity, however, was not
maintained. No methanogenesis was detected in the
sterile controls.
In the routine non-enriched incubations, increases in
endosulfan I caused a signi®cant reduction in methane
production. At 20 and 50 days of incubation in the
treatments containing 1 mg/l applied endosulfan I, the
methane production was 136‹12 and 119‹10 nmol/25
ml of growing culture, respectively. The values for the 10
mg/l treatments were 27‹7 and 47‹9, respectively.
These results indicated that endosulfan I may be inhibitory to methanogenic organisms in non-enriched
cultures.
The cells observed in both the enrichment cultures
and routine cultures were predominantly Gram-negative
anaerobic rods and methane-producing bacteria. The
relatively high concentrations of methane produced in
the enrichment cultures indicated that methanogenic
bacteria were not inhibited to any great extent by high
concentrations of endosulfan, aldrin or dieldrin. This is
consistent with the ®ndings of Mancinelli (1982) and
Thomann (1976) who have shown that cyclodiene
pesticides a€ected the activities of Gram-negative soil
bacteria to a much lesser extent than Gram-positive
isolates.
The current ®ndings indicate that methanogens were
active in the endosulfan I degrading cultures. Maule et
al. (1987) similarly found that methane was produced
by enrichment cultures growing in the presence of dieldrin (10 mg/l). These researchers, however, found that
methane production ceased in routine anaerobic cultures that were inoculated with microorganisms grown
initially in these enrichment cultures (containing dieldrin non-enriched at 10 mg/l). In both these enriched
and non-enriched cultures, dieldrin was monodechlorinated. However, in the current study, there was no
evidence for the signi®cant formation of dechlorinated
endosulfan I compounds. Any dechlorinated forms of

endosulfan I would have been extracted, analyzed and
detected by the chromatographic procedure used
(Guerin et al., 1992). In previous studies it has been
found that dechlorination was greatest with the populations of anaerobes that did not produce methane
(Maule et al., 1987). This may indicate the reason why
endosulfan I dechlorination was not observed in the
current study, where all the incubations contained
methanogens.
3.7. E€ect of headspace atmosphere composition on
endosulfan I biodegradation
Endosulfan I was biodegraded in incubations containing TD inoculum and a headspace that was ¯ushed
with Ar instead of the H2:CO2 (80:20) atmosphere that
was routinely used (Fig. 5). Endosulfan I degradation,
was not signi®cantly di€erent between these two treatments. As described in the previous section, the
treatments without any added H2:CO2, i.e. with Ar
headspace, still produced methane. This indicated that
these cultures were not forming methane from CO2, but
from one of the carbon sources in the medium. These
®ndings indicate that the endosulfan-degrading populations studied were not H2, CO2, or H2:CO2 dependent.
3.8. Enumeration and microscopic examination of the
cultures
Microscopic examination of the inoculated anaerobic
medium con®rmed that cell matter increased with time,
indicating that the increase in turbidity was due to
microbial growth. The number of microorganisms able
to grow on Medium No. 1 of Balch et al. (1979) solidi®ed with 1% agar (Oxoid), under strictly anaerobic
conditions, were 105±106 cfu/ml. These were enumerated
in the routine incubations at 10 days. The numbers of
microorganisms in cultures after the seventh enrichment
were 108±109 cfu/ml. Within the non-enriched cultures

20


Acknowledgements

Fig. 5. E€ect of incubation atmosphere on endosulfan I degradation.

and the enriched cultures there was no signi®cant
di€erence in the numbers of microorganisms (at 5%
signi®cance level). No growth was observed in the sterile
incubations and no growth was evident when the same
inocula were incubated under aerobic conditions.
A small proportion of the cells in the enrichment cultures were endospore-forming bacteria (approximately
5% of all ®elds viewed). The majority of the routine
cultures possessed cells that were either free or in small
chains of two to three cells in length. The enriched cultures contained clusters of Gram-negative rods, often
very large in size (50±100 cells each), but were not present in the routine incubations. The predominant Gramnegative rod identi®ed was of the genus Desulfotomaculum (Allen et al., 1985; Dindal, 1991). This genus reduces sulfates to sul®des, and may have been responsible
for the black colour of the media. The methanogenic
bacteria identi®ed in the cultures were those from the
genus Methanobacterium (Allen et al., 1985; Dindal,
1991). This genus is Gram negative which is commonly
rod shaped and in chains, and it is mainly found in
anaerobic muds. Clostridium and Desulfotomaculum
were identi®ed as the genera of the endospore-forming
bacteria present in the enrichment cultures (Allen et al.,
1985).
All the incubations, including those containing 1, 10
and 1000 mg/l endosulfan, possessed predominantly
Gram-negative cells. This was also observed when
enrichments were made with aldrin and dieldrin (1000
mg/l). These ®ndings can be explained from previous
research that has shown that Gram-positive bacteria are
more sensitive to the e€ects of cyclodienes (Thomann,
1976; Mancinelli, 1982). Mancinelli (1982) has suggested that the reason Gram-negative bacteria tend to
predominate in the presence of organochlorine pesticides, is due to the presence of an outer lipopolysaccharide on their cell walls. These compounds have
been postulated to protect the cell from high concentrations of pesticides (Mancinelli, 1982) and this is
likely to have been the case with the anaerobic cultures
in the current study.

To I.R. Kennedy, Department of Agricultural Chemistry and Soil Science, University of Sydney, Australia,
for helpful feedback on early drafts. The ideas of
Andrew Maule, PHLS Laboratories (Salisbury), are
also appreciated, as were the helpful suggestions of Ron
Harris (University of Guelph) and Graeme Batley
(CSIRO). Funding from the Cotton Research Development Corporation, Australia, and support from
Hoechst (Melbourne and Frankfurt) is gratefully
acknowledged.
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