Visualization was accomplished by using a uv chromatographic
viewer.

MASS SPECTROMETRY

Pesticide degradation products (malathion monoacid,
diethyl maleate, 0,0-dimethylphosphorodithioic acid, and
methoxych lor-DDE) were identified using gas liquid
chromatography-mass spectrometry. A Varian Aerograph Model
1532-B gas liquid chromatograph, a Finnigan 1055L quadrupole
mass spectrometer having a jet separator, and a Systems
Industry 150 digital computer were used. Sample spectra
were compared with spectra of authentic samples of each
compound.

EXPERIMENTAL PROCEDURES

The following procedures were used for determination of
degradation rates and products, and of distribution
coefficients exhibited by the various organism populations.
Bacteria were grown for 24 hours in nutrient broth diluted
1:10 with water spiked with the test pesticide prior to
harvesting for study. The cultures were then centrifuged,
washed three times with sterile dilution water, suspended in
100 ml of dilution water, and held at room temperature for
an additional 24 hours to allow utilization of endogenous
materials. These cultures were then used as inocula for
media containing the pesticide as a sole carbon source for
determination of degradation rates. The same procedure was
used for yeasts except that Saboraud's medium diluted 1:10
was used rather than nutrient broth.

Inocula for fungi were suspensions prepared by agitating
10 ml of sterile water in a plate containing the sporulating
fungi. A fungal medium was prepared containing basal salts
and 0.278 mmoles glucose per liter. Replicate 500-ml flasks
of the various weights of suspended fungi were prepared by
measuring appropriate portions of an inoculated fungal
medium, incubating on a gyratory shaker at 28°C for three
days, and adjusting to a final volume of 210 ml with a basal
salts solution containing a predetermined amount of
pesticide.

Algal cultures were centrifuged, washed three times, and
suspended in dilution water to use as inocula for sorption
studies. ‘

-12-


indicated no degradation of the organochlorine pesticides
after 27 hours of incubation.

Uninoculated controls were also centrifuged, extracted,
and analyzed in the same manner as were the samples. Any
loss of pesticide in the controls, due to sorption on
glassware or particles, was subtracted from the losses
measured in the microbial cultures.

To determine whether the sorption of the pesticide was
mediated by a metabolic process, the pesticides were also
added to autoclaved cultures. These cells sorbed the
pesticides at least as much as the viable cells. No
metabolic process was therefore involved. Other researchers
(41,42) report similar conclusions from their studies with
bacteria and fungi and organochlorine pesticides.

EQUILIBRATION TIME

The bacterial cultures reached equilibrium with both
methoxychlor and toxaphene within 30 minutes, and no further
change was detected over 24 hours. The algae equilibrated
with the toxaphene within 10 minutes, but required 30
Minutes with methoxychlor. The fungal system took the
longest time to reach equilibrium -- two hours in the
toxaphene medium and 16 hours in the methoxychlor medium.
The fungi formed small clumps while growing. The same
equilibration time was observed when the clump diameter was
5 mm as when it was 1 mm.

EXTENT OF SORPTION

Sorption of pesticides to microorganisms may be
represented by the empirically derived equation of
Freundlich (43)

x 1/n
wake (6)
where x is the amount (mg) of pesticide sorbed to the
microorganisms; m is the dry weight (mg) of the organisms;
c, is the concentration of pesticide in the medium (mg/1) at
equilibrium and k and 1/n are constants. The constant, 1/n,
was determined from the slope of a log-log plot of x/m as a
function of c,. Since in all of our systems 1/n was about
unity, the equation may be simplified to

qk = (7)

-33-


counts. In medium containing 210 pmol/l butoxyethanol as
the carbon source the bacterial population increased 100-
fold in 24 hours.

All four of the fungal populations tested including the
yeast, Rhodotorula glutinis, degraded the ester, 2,4-DBE.
For kinetic studies the yeast was selected because with it,
population increases could be estimated by viable plate
counts, a more convenient procedure than the dry weight
method used for filamentous fungi. Results could therefore
be compared more easily with the bacterial studies. R.
glutinis, although it did degrade the pesticide, did not
divide during the course of an experiment. When the medium
was supplemented with 400 umol/1l butoxyethanol, sufficient
carbon was available to permit the yeast to divide six times
in 72 hours.

The second-order rate constant, k, was found to be (2.6
+ 2.0) x 10-9 liter organism-! hour-! in 2,4-DBE solution
ranging from 0.9 wmol to 20.5 ymol per liter.

The mixed fungal populations (500 mg/l) in a solution
containing 6.2 umol/l 2,4-DBE converted 75-94% of the ester
to 2,4-D within 15 minutes. During the same time period
high concentrations (101! org/l) of the test bacterial
populations converted 50-91% of the ester of 2,4-D. The
short degradation times suggest the presence of a
constitutive enzyme in all the test organisms.

When degradation rates for B. subtilis and R. glutinis
were compared under equivalent conditions with the same
organism concentration, the bacteria hydrolyzed the ester
100 times slower than did the yeast. However, the biomass,
based on dry weight, of a single yeast cell (R. glutinis),
was 100 times greater than that of a single bacterium (B.
subtilis). The seccnd-order rate constants, therefore, are
nearly the same for the bacteria and yeast when compared on
a biomass basis.

Further degradation of the 2,4-D was tested with all
four fungal cultures. After a 48-hour incubation period in
a solution containing 2,4-D as the sole carbon source, no
growth could be detected and 88-99% of the initial acid
remained. After 55 days, however, only 55-60% of the
initial 2,4-D could be recovered. Degradation products were
not identified.

-29-


NATURAL WATERS

We determined k values for microorganisms in natural
waters for comparison with our laboratory data. A water
sample (pH 6.9) was collected from a river near High Shoals,
Georgia, and centrifuged. Half of the supernatant was
removed and replaced with distilled water containing
methoxychlor (0.008 ppm final concentration) . Direct
microscopic examination of the sample showed algae
(Scenedesmus sp. and Chlorella sp.), protozoa, and bacteria.
No fungi were observed. The system equilibrated within 45
minutes. The overall k (average from all species)
calculated from equation 7 was 0.0037, which is similar to
that obtained previously for F. harrisonii and Aspergillus
sp. but is one-tenth that of B. subtilis for the same
pesticide.

Another water sample (pH 6.7) was collected from
Chandler's pond near Athens, Georgia. It was centrifuged
and half of the supernatant was replaced with distilled
water containing toxaphene (final concentration, 0.047 ppm).
The sample contained about twice as many algae as the High
Shoals sample as determined by microscopic and direct
observation. Microorganisms present were green algae
{Arthospira sp., Phytoconis sp., and a few Chlorella sp.),
bacteria, ciliates, and diatoms. The system equilibrated
within one hour and k was determined to be 0.0067- This is
similar to the value obtained for F. harrisonii and B.
subtilis and one-half that observed in the laboratory for
Aspergillus sp. and C. pyrenoidosa.

The algae formed the largest segment of the micro-
organism population in both field samples; total algal
biomass for the two samples were 100 mg/l (High Shoals) and
200 mg/l (Chandler's Pond). The calculated k values for
Chlorella in the laboratory media containing methoxychlor
and toxaphene are in good agreement with those obtained for
the total microorganism populations in the field samples.
Although only two field sites were tested, laboratory data
with isolates appear to give a reasonable approximation of
sorptive behavior in a mixed natural population.

-38-


4,

SECTION Iv

RECOMMENDATIONS

The identification of the major microbial degradation
products of malathion as the f-monoacid should
facilitate an evaluation of the relative significance of
chemical and microbial degradation in the environment.

Compounds naturally occurring in some waters may enhance
microbial metabolism or degradation of pesticides.
Information is needed on the effects of different
concentrations and composition of these naturally
occurring nutrients on microbial pesticide degradation.

Better procedures for determining the degradability of
various pesticides by microorganisms in aquatic systems
are needed to enable us to predict the fate of these
compounds in aquatic systems.

Rates of microbial sorption of pesticides are needed.
Methods for determining the sorption of pollutants by
microorganisms should be developed in order to know more
about the distribution of pollutants in an aquatic
environment.


TECHNICAL REPORT DATA .
(Please read Instructions on the reverse before completing}

1, REPORT NO, 2. 3. RECIPIENT’S ACCESSION NO,
EPA-660/3~75-007
4. TITLE AND SUBTITLE Is. REPORT DATE

MICROBIAL DEGRADATION AND ACCUMULATION OF PESTICIDES January 1975

IN AQUATIC SYSTEMS 6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S) I8. PERFORMING ORGANIZATION REPORT NO.
Doris F. Paris, David L. Lewis, John T. Barnett, Jr. EPA-660/3-75-007

and George L. Baughman

9, PERFORMING ORGANIZATION NAME AND ADDRESS 10, PROGRAM ELEMENT NO,
Southeast Environmental Research Laboratory 1BA023

U. S. Environmental Protection Agency 77. CONTRACT/GRANT NO.

College Station Road
Athens, GA 30601

12, SPONSORING AGENCY NAME AND ADDRESS 13, TYPE OF REPORT AND PERIOD COVERED
Southeast Environmental Research Laboratory Task Milestone Final Report

U. S. Environmental Protection Agency 14, SPONSORING AGENCY CODE
College Station Road
Athens, GA 30601

75. SUPPLEMENTARY NOTES

16. ABSTRACT .
The microbial degradation and sorption of carbaryl, malathion, butoxyethyl ester of

2,4-dichlorophenoxyacetic acid (2,4-DBE), methoxychlor, atrazine, diazinon, captan,
parathion, and toxaphene were investigated. Malathion and 2,4-DBE were found to under-
go transformation readily in both bacterial and fungal cultures. Degradation of mala-
thion and 2,4-DBE at low concentrations (< 1 mg/l) in batch cultures of bacteria
followed second-order kinetics as predicted by the Michaelis-Menten theory. A single
isomer, B-monoacid of malathion, was the primary metabolite in transformation of mala-
thion by both bacterial and fungal populations. The major metabolite found in 2,4-DBE
studies was 2,4-D. Carbaryl underwent chemical hydrolysis to q-naphthol in both
heterogeneous bacterial cultures and uninoculated controls. In the cultures q-naphthol
was metabolized to 1,4-naphthoquinone and two unidentified compounds. Bacterial
degradation of methoxychlor was slower than bacterial degradation of malathion or
2,4-DBE. The insecticide was metabolized to methoxychlor-DDE. Rapid and extensive
sorption of pesticides to fungi, bacteria, and algae was observed with methoxychlor
and toxaphene, but not with any of the other pesticides investigated. Distribution
coefficients for methoxychlor ranged from 1.2 x 10° to 4.8 X 10° for the different
organisms whereas the coefficients for toxaphene ranged from 3.4 X 10° to 1.7 x 10*.
Captan underwent neither microbial degradation nor sorption because of its rapid
hydrolysis in water.

KEY WORDS AND DOCUMENT ANALYSIS,
b.IDENTIFIERS/OPEN ENDED TERMS |c. COSATI Field/Group

7.
a. DESCRIPTORS

Biodegradation*, Sorption*, 2,4-D*, Malathion*, Methoxychlor 06/06
Aquatic microorganisms, Pesticide kinetics | Toxaphene*, Carbaryl*

18. DISTRIBUTION STATEMENT 19. SECURITY CLASS (This Report) 21, NO, OF PAGES
Release Unlimited 46
20. SECURITY CLASS (This page) 22, PRICE

EPA Form 2220-1 (9-73
A Form 2) (9-73) 4 U.S, GOVERNMENT PRINTING OFFICE: 1975-698. 121/112. REGION 10


43

44

45

46

a7

48

Pionke, H. B., and G. Chesters. Pesticide-Sediment-
Water Interactions. J. Environ. Quality. 2(1):29-45
(1973).

Knott, G. D., and 0. K. Reece. MLAB: A Civilized Curve-
Fitting System. In: Proceedings of the ONLINE 1972
International Conference, Vol. 1. Brenel University,
England. September 1972. p. 497-526.

Cummins, C. S. Bacterial Cell Wall Structure. In: CRC

Handbook of Microbiology, Vol. II-Microbial Composition,
Laskin, A. I., and H. A. Lechevalier (eds). Cleveland,

CRC Press, 1973. p. 169.

Shin, Young-Oh, J. J. Choden, and A. A. Wolcott.
Adsorption of DDT by Soils, Soil Fractions, and

Biological Materials. J. Agr. Food Chem. 18(6) 21129-
1133 (1970).

Bailey, G. W., and J. L. White. Factors Influencing the

Adsorption, Desorption, and Movement of Pesticides in
Soil. Res. Rev. 32:329-92 (1970).

Veith, G. D., and G. F. Lee. Water Chemistry of

Toxaphene-~Role of Lake Sediments. Environ. Sci.
Technol. 5:230 (1971).

-44-


where k is a second-order rate constant (liter organism-!
hour-!) for removal of pesticide by bacteria.

Equation 1, the more accurate description of substrate
removal kinetics, requires knowledge of un, Kg, and Y, all
of which may be determined from growth kinetics experiments.
However, the very low solubility of many pesticides in water
often precludes the range of experiments necessary to
determine these parameters. The more simplified equation 4
would be useful if it were found to accurately define the
kinetics for bacterial removal of pesticide. Malathion was
used to establish the reliability of equation 4 because the
insecticide was rapidly degraded by available bacterial
populations and is soluble enough to permit work over a wide
concentration range. We defined the term [up)/Y(Kgt+[S])]
from equation 1 as the second-order rate coefficient (k')
and calculated k' using the kinetic data for Um, Kg, and Y.
The rate constant k in equation 4 was determined
experimentally and compared with the calculated values of
kt.

Values of k and k!* (Table 4) are in agreement at low
bacterial and malathion concentrations. Thus it was
established that the second-order rate expression could be
used for kacterial removal of a pesticide.

Equation 1 may also be used to describe the rate of
pesticide removal by fungi. However, at the low
concentrations of pesticides used in our studies very small
increases in fungal biomass would be expected when the
pesticide was the only external source of carbon. These
small changes in fungal concentration could not be detected
by our method of measurement (dry weight procedure) so the
fungal biomass was assumed to be constant during the
experiments, Values of uy, and K, were not determined.

At low pesticide concentrations, though, equation 4 does
describe the rate of removal of pesticide from solution by
the fungi and can be used to determine the rate constant.
The term (B] (concentration of fungi) is a constant for a
given experiment.

PESTICIDE DEGRADATION

Atrazine, diazinon, and toxaphene were not degraded by
any of the bacteria or fungi tested.

-16-


Arithmetic plots of x/m vs. c, showed a linear relationship
(Figures 8 and 9). The graphs, along with the values of 1/n
and k, (Table 5) were obtained using the least squares
statistical computer program, MLAB (44), developed at the
National Institutes of Health.

The term k, the slope of the arithmetic plot of x/m
versus Ce, is a useful index for comparing the degree of
sorption by the various classes or organisms (Table 5). The
computed t values for the bacterial tests indicate a
significant difference in the slopes at the 95% confidence
level.

In systems in which 1/n is close to unity, k corresponds
to the distribution coefficient, K,, with a correction
factor of 106 to account for the different units used to
obtain k. K, is merely a ratio of the amount of pesticide
sorbed to the microorganisms (mg/mg) to the concentration of
pesticide in water (in mg/mg), whereas k is calculated using
units of mg/l for the concentration of pesticide in water.

The values of k for the four organisms and two
pesticides are within an order of magnitude (Table 5). The
greatest difference observed in ability to sorb the
pesticides was between B. subtilis and F. harrisonii in the
methoxychlor studies (B. subtilis, k = 0.048; F. harrisonii,
k = 0.0012). If we assume B. subtilis to be a typical gram
positive bacterium and F. harrisonii to be a typical gram
negative bacterium, the difference in sorption cannot be
explained by the lipid solubility of methoxychlor. The
lipid content of the gram positive bacterial cell wall's dry
weight is only 0-2%, as compared to 10-20% for the gram
negative bacteria (45), yet the gram positive sorbed more
methoxychlor. Shin et al. (46) studied adsorption of DDT by
soil fractions. In their investigations, treating the soil
with diethyl ether and ethanol for removal of lipoidal
materials increased the adsorption of DDT to the soil,
suggesting that other components of the soil play a larger
role in the sorption of DDT than the lipoidal materials. If
the cell wall of B. subtilis contains fewer polar groups
than that of F. harriso methoxychlor would be a _ more
effective competitor with water for sites on the former.

All of the organisms, except B. subtilis, sorbed
slightly more toxaphene than methoxychlor. However it is
difficult to compare the degree of sorption for toxaphene
itself since it is not a single compound. Toxaphene is a
mixture of polychlorobicyclic terpenes and some components
may be more tightly sorbed to the microorganisms than

-34-


TABLE OF CONTENTS

Sections

III

Iv

vI

INtrOGUCtiON 1. cece cece eee e eee nee c reer eccceces

SUMMALY cee ce cece cece cece cence eee ee ces eeeeeee

CONCLUSIONS 2... cece e eee e erence reer eee ccccees

Recommendations

Materials and Methods .....ee eee e eee cee eee wees

Test MEdia ... ccc cece eee cece cece eee e eee eee

Test OrganiSMS ....... cece eee ee eee ewes

Bacteria .. occ eee eee cece erences

FUNGL Lecce cece cc eee ccc e ec erence eeees

PAs he fa

Gas Liquid Chromatography ...............2..

Thin Layer Chromatography ..................

MaSS SPECtroMetry 2... cece eee eee c cece eee eee

Experimental Procedures .............505200-

Degradation StudieS ...... ee cece eee eee eee eeee

Kinetics .... cc eee ee eee eee eee eee eee ccccces

Pesticide Degradation ....... cece eee ee ee eee

Carbaryl ... eee eee eee eee ee eee enes

Malathion ...eeeeceece ccc cresccerecscecce

Q,4-DBE cece eee ee eee eee eee cece ee ee eee eee

Methoxychlor

CAPtAN Loewe cece cece ccc erc rec cersreecce

Parathion

10

12

14
15
16
18
18
27
30
31

31


dominant degradation pathway. At low concentrations of
malathion~degrading bacteria (2 x 106/1) and low malathion
concentration (3.3 umol/l), the half-life of the pesticide
was calculated to be 41 hours at 28°C. Therefore, in
neutral waters (pH 6.8-7.0) containing little humic acids,
bacterial removal may compete successfully. For the
Malathion degrading fungus to compete with the bacteria the
fungal biomass would have to be 96 mg/liter (dry weight), a
much higher concentration than one would expect in the
environment.

Butoxyethyl Ester of 2,4-Dichlorophenoxyacetic Acid (2, 4-DBE)

Bacillus subtilis grew in culture solution with the
herbicide as a carbon source. Growth rates of B. subtilis
at various concentrations of 2,4-DBE (0.156 to 15.6 mol/l)
were measured; Umax was estimated to be 0.30 hour-1! and Kg
to be 2.47 umol per liter (Figure 7). At low concentrations
of 2,4-DBE (0.1-1.0 mol/l) and of B. subtilis (1 x 108
org/l) the second-order rate expression (equation 4)
describes the rate of removal of the herbicide from solution
by bacteria. The constant k was (4.0 + 1.3) x 10-1! liter
organism?! hour-}!.

The major metabolite of ester degradation is 2,4-D.
After 3 hours, 99% of the 2,4-DBE in a culture (initial
concentrations 9.3 umol/l 2,4-DBE, 1011 bacteria per liter)
could be accounted for in the form of 2,4-D. Further
degradation proceeded slowly. After 264 hours, 20% of the
2,4-D produced remained in the culture. Schwartz (23) and
Aly and Faust (24) in their investigations also found that
2,4-D was persistent in an aqueous environment. Schwartz
(23), following degradation of 2,4-D labelled at the 2-
carbon of the acetic acid moiety, reforted that no more than
37% of the acetic moiety disappeared within six months. Aly
and Faust (24) found that 2,4-D persisted up to 120 days in
lake waters aerobically incubated in the laboratory.

The metabolites of 2,4-D were not identified because the
degradation pathway and products are assumed to be similar
to those reported for soil microorganisms (25-35). In the
studies with soil bacteria the phenoxyacetic acid ring was
cleaved and metabolized to succinic acid.

Since the bacteria grew, but apparently did not use the
2,4-D as a carbon source, growth experiments were carried
out to determine whether the organisms could use the other
breakdown product, butoxyethanol. The bacteria could
utilize the side chain for growth based on viable plate

-27-


Numbers of viable cells of bacteria and yeasts were
estimated by plate counts at zero hour and at each sampling
time (14). Tryptone-glucose-extract agar (Difco) and
Saboraud's agar (Difco) were used as the bacterial and yeast
plating media respectively. Bacteria were incubated
aerobically at 28°C for 48 hours and yeast, at 28°C for 72
hours. Fungi were separated from test cultures for dry
weight determinations by filtering, first through tared pre-
filters, then through tared 0.22 micron Nucleopore filters,
and drying to a _ constant weight at 90°C. Dry weights of
algae and bacteria used in sorption studies were determined
by centrifuging and washing the organisms three times. The
organisms were quantitatively transferred to tared beakers
and dried to a constant weight at 90°C.

Pesticide determinations in degradation studies involved
extraction of a portion of a test culture with isooctane and
subsequent analysis by gas liquid chromatography. The size
of the sample required depended on the concentration of
pesticide, but usually 1 ml was sufficient. In sorption
experiments, algal and bacterial samples were centrifuged
and pesticide concentrations were determined in the
supernatant. The filamentous fungi formed clumps and
quickly settled to the bottom of flasks; they therefore
posed no problem in sampling media without organisms.

-13-


050

CONTROL

SLOPE = -0.61(¢.18) x10°3
040

0.50

10° BACT/}
SLOPE = -0.62(¢.17)x1073

O40F

O50F

log CONCENTRATION CARBARYL (mg/i)

10" BACT/|
oaok SLOPE = -0.80(t.16)x1073 ;
1 = 1 1 1
° 20 40 60 80 100
HOURS

Figure 1. Decrease in carbaryl concentration with time.

~19-


only says that these compounds are not as readily degraded
biologically as a compound such as 2,4-DBE.

KINETICS

The rate of pesticide removal in the bacterial cultures
may be described by the modified Monod expression (15) given
in equation 1 in which [S] is the concentration of pesticide
(umol per liter); uy, is the maximum specific growth rate
(hour-!); (B] is the concentration of bacteria (organisms
per liter); Y is the yield factor or number of bacteria
produced per umol of pesticide; and K, is a constant
numerically equal to the pesticide concentration at which
wee ue

ats} Uy (8) (B)

“ae OY (Ka + 187) @)

To determine Um and Kg for the degradation of pesticides
by bacteria a series of media with varying pesticide
concentrations were inoculated with suspensions of washed
organisms to give viable bacterial concentrations of 106 -
108 organisms per liter. Using a rearranged Monod equation
(16)

($1 (s] Ky
= . (2)
u Mn Um

experimental values [S]/y were plotted as a function of [S].
The slope of the resulting plot, 1/u,,, was determined by
least squares analysis employing a computer program. K, was
determined from the intercept (K,/u,,)-

Equation 1 takes into account the major factors

influencing the rate of substrate utilization by batch
cultures. At high substrate concentrations the equation

reduces to

ae Fy @)

Substrate removal follows pseudo first-order kinetics and is
independent of substrate concentration.

At [S] much less than the value of K,, equation 1 can be
approximated by

ats} ¥
Set ® k1s1 (8) (4)

-15-


100

80

70

60

50

SF

40

30

20

fe) 1 i 1 1 i a)
4 8 12 16 20 24

[s] moles / liter

Figure 7. Lineweaver-Burke plot of specific growth rates and
substrate concentrations for bacteria in butoxyethyl
ester of 2,4-dichlorophenoxyacetic acid studies.

-28-


ml/min; operating temperatures for the inlet port and
detector were 170°C and 260°C. For carbaryl determination,
the detector temperature was set at 225°C.

A short glass column (0.3m x 4mm ID) was used for rapid
analysis of extracts. Columns were packed with 80-100 mesh
Gas Chrom Q containing 3% silicone SE-30 (Applied Science
Laboratories). Column temperatures for the various
compounds are listed in Table 3.

Samples of 2,4-D were methylated with boron
trifluoride/methanol (9) and extracted with isooctane prior
to electron capture gas chromatographic analyses.

Malathion metabolites were methylated for gas liquid
chromatography using a diazomethane procedure outlined in
EPA report #EPA-R2-73-277 (10).

A linear response range was established for each
pesticide and pesticide quantities were determined by peak
height comparison (except for toxaphene) using standards
with closely matched peak heights within the range of
linearity. Toxaphene quantities were determined by peak
area comparisons using a planimeter.

THIN LAYER CHROMATOGRAPHY

Cultures containing malathion were adjusted to pH 2.0
with 1.0 N HCl and extracted with two 100-ml portions of
chloroform. Products in the extract were separated by
preparative thin layer chromatography using plates coated
with silica gel. The developing solvent was hexanezacetic
acidrethyl ether (75:15:10) (11). Products were visualized
by spraying a portion of the plate with the reagent of Menn
et al. (12), 0.5% 2,6-dibromo-N-chloro-p-quinoneimine (DCQ)
in acetone. Rr values of the products were compared with
the Rp values of the standards.

Methoxychlor and products were extracted with hexane and
plates were developed with ethyl ether/hexane (3:1).
Elutions were visualized by spraying the plates with 0.5%
diphenylamine and 0.5% zinc chloride in acetone, heating
them at 110°C for 10 minutes, and exposing them to
ultraviolet light for five minutes (13).

Carbaryl and products were extracted with methylene
chloride and silica gel indicator plates were developed with
benzene : 0.1 N ammonium hydroxide : ethanol (10:5:5).

-10-


Table 1. PESTICIDES SFLECTED FOR

cane’ cme.
5

Cor
%,

0

Caplan
‘OCONHCH,

Carbaryl
CaO,

0. CHO

cate’ CY

CH
Duasinon.
°

catocanocas

a

Bunty ete 0f 24D
como s °
Ls cb -uomcm
cH.0% 1 —C-0-—-CHCH
if
Malethson

ou
ta,
Methcychr
oct

—— —()-r0.

Cate
Parathion

cHi,0- OCH,

PolycMorinated biphenyl
Mixture of isomer chlorinated biphenyls
(e = pomible points of substitution of chlorine)

ie
(omy on
n.c=!

Tozaphene
“Mixture of polychloro bieyeli terpenes with
‘hlorinaled earophene predominating’
Structural formula is representative

STUDY


MALATHION B-MONOAC ID

0.6

LOG CONCENTRATION, Hmoles/1!

0.4

0,2

tl | i i
0 2 4 8 10 12

0,0

6
TIME, hours

Formation of $-malathion monoacid in funqal
cultures.

Figure 5.

-25-


200

180

160

140,

120

Be 100

80

60

40

20

Figure 3. Lineweaver-Burke plot of specific growth rates and
substrate concentrations for bacteria in malathion
studies. [S$] is concentration of malathion (umol/1)
and yp is specific growth rate (hr7!).

-22-


LIST OF FIGURES

Decrease in carbaryl concentration with time

Growth of bacteria and decrease in malathion
concentration

Lineweaver-Burke plot of specific growth rates
and substrate concentrations for bacteria in
malathion studies

Formation of 8-malathion monoacid in bacterial
cultures

Formation of $-malathion monoacid in fungal
cultures

Comparison of chemical and microbial degradation
products of malathion

Lineweaver-Burke plot of specific growth rates
and substrate concentrations for bacteria in
butoxyethyl ester of 2,4-dichlorophenoxyacetic
acid studies

Sorption of methoxychlor by bacteria, fungi, and
algae

Sorption of toxaphene by bacteria, fungi, and
algae

22

23

25

26

28

35

35


Page

VII Sorption of Pesticides to Microorganisms ...... 32
Equilibration Time .....eesceecceeeerceceeee 33

Extent Of Sorption ......cceceeeccecccecceee 33
DESOLPtiOnN 2... cece eee ccccrcceccccscccccecs 37

Natural WaterS ...... eee cece cece nee c eee cece 38

VIII APPENdix .L. icc e cece cece cece cece cree ceecevcees 39
Ix REFETENCES 2. ee cece cece cece c cece cece recscces 40

x Publications 1... . cc eee e cece eee e er eeee eee 45

iv


Table 3. COLUMN TEMPERATURES (°C) FOR THE VARIOUS
PESTICIDES AND METABOLITES INVESTIGATED

Pesticides and Metabolites

Column Temperature (°c)

a-naphthol

2,4-D (methylated)
Atrazine

Captan

8-Malathion Monoacid
(methylated)

Carbaryl
Diazinon
Malathion
Parathion
2,4-DBE
Methoxychlor=-DDE
Methoxychlor=-Dpp
Toxaphene

Methoxychlor

130
149
140
140

150

150
160
170
179
190
199
190
190

210

-ll-


SECTION I

INTRODUCTION

The use of pesticides increases each year as the world
population and demand for food increases. In 1970 alone,
1,034 billion pounds of active ingredients of pesticides and
related products were produced in the United States (1).
For 1972 the National Soils Monitoring Program (2) reports a
significant increase in the use of the most common
pesticides, in particular atrazine, captan, malathion, and
2,4-D, over figures quoted for 1970 (3). This increased
usage of pesticides has raised the amount of these compounds
reaching streams, rivers, and reservoirs by unintentional
(runoff, groundwater) additions or intentional (dumping and
spraying of waters for pests) additions. It has become
important, therefore, to know more about the effects of the
pesticides on the environment. An evaluation of their
environmental impact requires an understanding of their
breakdown processes, both biological and non-biological.

Pesticides may affect the environment in several ways.
A pesticide with a slow rate of degradation will persist in
the environment, stimulating some populations and supressing
others. An imbalance in the ecosystem results. Other
pesticides will degrade rapidly, some to products that are
more toxic than the parent compound and some to harmless
products. Microorganisms are commonly believed to be a key
factor in determining the fate of many pesticides in aquatic
systems; however, a literature review (4) revealed few
studies concerning the rates and products of either
microbial or chemical degradation of some of the most
commonly used pesticides.

Ten pesticides were selected (Table 1) for degradation
studies in 1971. Since then polychlorinated biphenyls
(PCB's) have been excluded from the studies. Our studies
were concerned with the microbial degradation of these
pesticides; a complementary project (5) focused on the
chemical and photochemical degradation.

Caution must be observed in applying laboratory derived
microbial degradation rates to natural systems. Although
the bacterial populations used in the studies approximated
total populations present in natural waters, our cultures
were screened to include only those bacteria that degrade
the pesticide; only a small fraction of the natural
population would be expected to degrade the pesticide.
Also, degradation rates may differ because nutrient

-1-


Table 4. YIELD VALUES AND RATF CONSTANTS

FOR REMOVAL OF MALATHION BY BACTERIA

Malathion Yield (org*/ymol) k k7

(umo1/£) x 101° (2 org7TM! hea!) | (2 orgTM! hr7?)
x 197}? x 10722

0.0273 8.0 2.9 2.1

0.0273 8.0 2.5 2.1

0.21 1.8 1.2 8.6

0.21 2.3 2.2 6.8

0.273 3.0 3.5 5.1

9.273 3.0 1.9 5.1

0.33 4.1 3.4 3.6

0.33 2.6 3.3 5.7

AVERAGE 4.1 + 2,3** 2.6 + 0.8** 4.9 + 2,1**

*org = organism

**Standard Deviation

-17-


Ecological Research Series

Microbial Degradation and
Accumulation of Pesticides
in Aquatic Systems

ED ST
ys 785

7

WV agen

2
Ss
3
%
%

(e)

Y, ~~
7Al pao?

National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency

Corvallis, Oregon 97330


10

SECTION VIII

REFERENCES

The Use of Pesticides in Surburban Homes and Their
Impact on the Aquatic Environment. U.S. Environmental
Protection Agency. Washington, DC. EPA Pesticides
Study Series-2 1 (1972).

Personal communication with Ann E. Carey, National Soils

Monitoring Program, U.S. Environmental Protection
Agency, Washington, DC 20460 (1972).

Crockett, A. B., G. B. Wiersma, H. Tai, W. G. Mitchell,
and P. J. Sand. National Soils Monitoring Program for
Pesticide Residues - FY 1970. Ecological Monitoring
Branch, Technical Services Division, Office of Pesticide
Programs, U.S. Environmental Protection Agency.
Washington, DC. p. 2-85.

Paris, D. F., and D. L. Lewis. Chemical and Microbial
Degradation of Ten Selected Pesticides in Aquatic
Systems. Res. Rev. 45:95-124 (1973).

zepp, R.« G., N- L. Wolfe, J. A. Gordon, R. C. Fincher,
and G. L. Baughman. Chemical and Photochemical
Transformations of Selected Pesticides in Aquatic
Systems. To be published as an EPA Research Report
(1975).

MacRae, I.C., Ke Raghu, and I. F. Castro. Persistence
and Biodegradation of Four Common Isomers of Benzene
Hexachloride in Submerged Soils. J. Agr. Food Chem.
15 (5) 2971-914 (1967).

Payne, W. J., and V. E. Feisal. Bacterial Utilization

of Dodecyl Sulfate and Dodecyl Benzene Sulfonate. Appl.
Microbiol. 11:339-344 (1963).

Hutner, S. H. Some Approaches to the Study of the Role
of Metals in the Metabolism of Microorganisms. Proc.
Amer. Phil. Soc. 94:152-170 (1950).

Metcalfe, L. D., and A. A. Schmitz. The Rapid

Preparation of Fatty-Acid Esters for Gas Chromatographic
Analysis. Anal. Chem. 33: 363-364 (1961).

Webb, R. G., A. We Garrison, L. H. Keith, and J. M.
McGuire. Current Practice in GC-MS Analysis of Organics

-40-


SECTION II

SUMMARY

The microbial degradation and sorption of carbaryl,
malathion, butoxyethyl ester of 2,4-dichlorophenoxyacetic
acid (2,4-DBE), methoxychlor, atrazine, diazinon, captan,
parathion, and toxaphene were investigated.

Malathion and 2,4-DBE were found to undergo
transformation readily in both bacterial and fungal
cultures. Degradation of malathion and 2,4-DBE at low
concentrations (< 1 mg/l) in batch cultures of bacteria
followed second-order kinetics as predicted by the
Michaelis-Menten theory. A single isomer, 8-monoacid of
malathion, was the primary metabolite in transformation of
malathion by both bacterial and fungal populations. The
major metabolite found in 2,4-DBE studies was 2,4-D.

Carbaryl underwent chemical hydrolysis to qg-naphthol in
both heterogeneous bacterial cultures and uninoculated
controls. In the cultures a-naphthol was metabolized to
1,4~naphthoquinone and two unidentified compounds.

Bacterial degradation of methoxychlor was slower than
bacterial degradation of malathion or 2,4-DBE. The
insecticide was metabolized to methoxychlor~DDE.

Rapid and extensive sorption of pesticides to fungi,
bacteria, and algae was observed with methoxychlor and
toxaphene, but not with any of the other pesticides
investigated. Distribution coefficients for methoxychlor
ranged from 1.2 x 103 to 4.8 x 10* for the different
organisms whereas the coefficients for ‘toxaphene ranged from
3.4 x 103 to 1.7 x 104%.

Captan underwent neither microbial degradation nor
sorption because of its rapid hydrolysis in water.


conditions of the laboratory system are not exactly the same
as those of a natural system. Similarly, rates of fungal
degradation observed in the laboratory would be realistic
for only that portion of the fungal population active in the
degradation of pesticides. However, the laboratory
microbial rates provide a basis for comparison with
photochemical and chemical degradation rates, yielding an
insight into the competition of the different processes in
the environment. Laboratory studies also provide
information about the products to be expected.

Sorption of pesticide by aquatic microorganisms affects
the distribution of the compounds within an aquatic system.
Organisms sorb pesticides, die, and become a part of the
sediment. Pesticides in bottom sediments may be recycled to
overlying waters through fall and spring inversions or
through release of pesticides from the sediments. The
sorbed pesticides may also be degraded anaerobically (6) or
they may move up the food chain.

At present sufficient information on microbial sorption
of pesticides in aquatic systems is not available to predict
to what extent these compounds will be sorbed.
Environmental factors and the characteristics of organisms
and pollutants must be studied to determine their effects on
sorption.

The purpose of this research was to study the action of
classes of microorganisms (bacteria, fungi, and algae) on
the selected pesticides. The investigations included two
areas:

e yrates and products of degradation of pesticides by
microorganisms; and

e sorption of pesticides by microorganisms.


-9E-

Table 5. VALUES OF k AND 1/n FOR SORPTION OF METHOXYCHLOR AND

TOXAPHENE TO VARIOUS MICROORGANISMS

Methoxychlor Toxaphene
Organism
1/n k l/n k
Bacillus subtilis 1.2 -948 + .0022 71 -0034 + .00047
Flavobacterium harrisonii 81 290012 + .00015 1.1 29052 + .00016
Aspergillus sp. 91 29952 + .00043 80 -917) + .0016
Chlorella pyrenoidosa 99 20084 + .9N052 79 -017 + .c00088



5)- Also detected were the malathion dicarboxylic acid in
both fungal and bacterial systems and 0, 0-dimethylphosphoro-
dithioic acid and diethyl maleate in the bacterial system
only. These products have been reported previously (21, 22)
for bacterial and fungal degradation of malathion, but no
attempt was made to identify the specific monoacid isomer.
The specificity of the degradation to the f-isomer, as
opposed to the almost exclusive formation of the a-isomer in
chemical systems (5), may represent a typical pathway for
the heterotrophic transformation of malathion.

To determine if the degradation was an extracellular
reaction, filtrates of liquid cultures of bacteria and fungi
containing malathion were incubated and the malathion
concentration was monitored. No change in malathion
concentration could be detected in six hours. Degradation,
therefore, occurs within the cell and is probably catalyzed
by the enzyme carboxyesterase,

The associated product of carboxyesterase activity would
be ethanol. Since the 8-monoacid was apparently not
degraded further, the microorganisms may have used the
ethanol as a carbon source. To determine if the
microorganisms could grow on ethanol, bacteria were
inoculated into basal salts medium containing 90 pmol/l
ethanol, and fungi were introduced into a medium containing
2.1 mmol/l ethanol.

The ethanol concentration in both cultures was higher
than the metabolite would be expected to be in cultures
containing microorganisms and malathion. In both cultures
the organisms increased in biomass within 48 hours.
Apparently the malathion concentration and therefore the
concentration of ethanol produced in the fungal cultures
used for degradation studies was too low to produce a
measurable increase in fungal biomass.

In a natural system physical, chemical, and biological
removal processes (Figure 6) compete and interact and their
rates are controlled by environmental conditions, which are
characteristic of the individual aquatic system. For
example, at pH 6.8-7.0 and 27°C, malathion does not readily
hydrolyze; its half-life is about one month (5). However,
at pH 9.0 and 27°C, the half-life of malathion is about 10
hours. The photolysis rate on the other hand, is dependent
upon the concentration of humic acids. In water containing
no humic acids, the photolysis half-life is 990 hours,
whereas in water containing humic acids, the photolysis
half-life is 15 hours (5). In the absence of humic acids,
therefore, photolysis would not be expected to be the

-24-


VW

12

13

4

15

16

7

18

19

20

in Water. U.S. Environmental Protection Agency.
Washington, DC. EPA-R2-73-277. 1970.

Kadoum, A. M. Thin-Layer Chromatography Sorption and
Colorimetric Detection of Malathion and Some of its
Metabolites from Stored Grains. J. Agr. Food Chem.
18: 542-543 (1970).

Menn, J. J., W. R. Erwin, and H. T. Gordon. Color
Reaction of 2,6-Dibromo-N-Chloro-p-Quinonemimine with
Thiophosphate Insecticides on Paper Chromatograms. J.
Agr. Food Chem. 5(8):601-602 (1957).

Kapoor, I. P., R- L. Metcalf, R. F. Nystrom, and G. K.
Sangha. Comparative Metabolism of Methoxychlor,
Methiochlor and DDT in Mouse, Insects, and in a Model
Ecosystem. J. Agr. Food Chem. 18:1145-1152 (1970).

Standard Methods for the Examination of Water and

Wastewater. 12th Edition. American Public Health
Association, Inc. New York. 1965.

Stumm-Zollinger, E., and R. H. Harris. Kinetics of
Biologically Mediated Aerobic Oxidation of Organic
Compounds in Receiving Waters and in Waste Treatment.
In: Organic Compounds in Aquatic Environments, Faust, S.
J., and J. V. Hunter (eds). New York, Marcel Dekker,
Inc., 1971. p. 555-598.

Lineweaver, H., and D. Burke. The Determination of

Enzyme Dissociation Constants. J. Am. Chem. Soc.
56:658-666 (1934).

Crosby, D. G., E. Leitis, and W. L. Winterlin.

Photodecomposition of Carbamate Insecticides. J. Agr.
Food Chem. 13:204-207 (1965).

Hughes, L. Jr. A Study of the Fate of Carbaryl
Insecticide in Surface Waters. Ph.D. Dissertation,
Purdue University. University Microfilms, High Wycomb,
England (1971).

Bollag, J. M. Biochemical Transformation of Pesticides
by Soil Fungi. CRC Critical Reviews in Microbiology.
2235-58 (1972).

Bollag, J. M., and S. Y. Liu. Hydroxylations of
Carbaryl by Soil Fungi. Nature. 236:177-178 (1972).

-41-


Figure 4.

LOG CONCENTRATION, pmoles/1

1.8
py, L
16 ° a
Ze
lab
a N
Lo e
°
oat MALATHION B-MONOACID
0.6 fF
04
0.2
0.0 -L 1
0 1.0 2.0 3.0
TIME, hours

Formation of f-malathion monoacid in bacterial

cultures.

-23-


LIST OF TABLES

Pesticides selected for study

Microorganisms used in degradation and sorption
studies

Column temperatures for the various pesticides and
metabolites investigated

Yield values and rate constants for removal of
malathion by bacteria

Values of k and 1/n for sorption of methoxychlor and
toxaphene to various microorganisms

vi

ll

17

36


SECTION VI

DEGRADATION STUDIES

Bacterial populations were found that could degrade five
of the nine pesticides investigated: carbaryl, malathion,
2,4-DBE, methoxychlor, and parathion. Fungal populations
were isolated that would degrade malathion and 2,4-DBE. The
ability of bacteria or fungi to degrade captan was
impossible to assess since the pesticide itself hydrolyzed
so rapidly in solution.

Mixed populations were used since they often show a
greater facility to acclimate than single species culture.
A given species may not be able to initiate attack on a
given compound but it may be able to use it for growth if
another species initiates the attack.

The bacterial and fungal populations used in the
degradation studies, although they are mixed, do not contain
the total range of microorganisms present in a natural
system. The initial isolation in the nutrient broth medium
or Rose Bengal plates eliminated those species that could
not grow in these media. The final isolation included
primarily species that could grow in medium containing the
pesticide as the sole carbon source.

The test cultures were monitored for decrease in
pesticide concentration by extraction of whole cultures,
i.e., both the organisms and medium, with isooctane and
subsequent analyses by glc. Results were compared with
those from controls containing pesticides but no
microorganisms. If no change was noted in the pesticide
concentration over a period of time, the pesticide was
assumed to be non-degradable under the prevailing
conditions.

Only a small sampling of natural populations were tested
and a single set of carefully controlled conditions were
used. With different populations or under different
conditions rates may be different. However, testing a
series of samples does give some insight into the general
biodegradability of the compound. For example, in our
system all test cultures rapidly degraded 2,4-DBE to
butoxyethanol and 2,4-D. On the other hand, neither the
bacterial nor the fungal populations tested degraded a
detectable amount of atrazine, diazinon, or toxaphene.
However, this does not say that under other conditions or
with other microbial species degradation will not occur. It

-14-


is more persistent probably because of the slower growth
rate of microorganisms on chloropropham.

Captan

In preliminary experiments with captan and bacteria, the
concentration of captan rapidly decreased in concentration
in uninoculated controls (pH 5.6 to 8.0) until only a trace
was detectable at 19 hours. Only a slight increase in the
degradation rate was noted in the presence of bacteria. No
further attempts were made, therefore, to study the
microbial degradation of the fungicide.

Parathion

One of our bacterial populations degraded parathion;
however, because rate data for the bacterial degradation of
parathion are in the literature, we did not determine
degradation rates. Hsieh and Munnecke (39) assessed the
capacity of microbial cultures to degrade parathion in
water. In a chemostat, at a dilution rate of 0.05 hr-! and
with a sufficient oxygen supply (580 mg per liter per hour)
the bacteria removed parathicn from solution at a continous
rate of 500 ppm per hour. This is about 100 times higher
than the rate of hydrolysis in 1 N sodium hydroxide.

-31-


SECTION vViI

SORPTION OF PESTICIDES TO MICROORGANISMS

Some pesticides have been found to accumulate on or sorb
to microorganisms. Several reasons for this phenomenon have
been postulated. First, the water solubilities of most
organic pesticides are quite low (See Appendix). Second,
microorganisms have a very high surface area to mass ratio
compared to aquatic organisms of higher trophic levels,
e-q., for yeast the ratio is 9,100 cm@ per gram and for
Escherichia coli, a bacterium, 56,000 cm? per gram. Third,
it has been suggested that since most pesticides are
lipophylic, they are partitioned selectively onto surfaces
containing surface lipids (40). The first and third reasons
are only different ways of viewing the same phenomenon. All
three reasons are dependent on the pesticide structure,
which influences sorption of the molecule in microbial and
soil systems.

We screened several microorganisms for their ability to
sorb seven of the nine selected pesticides: a gram positive
bacterium (Bacillus subtilis), a gram negative bacterium
(Elavobacterium harrisonii), and three fungal populations.
Captan was excluded because of its rapid chemical
hydrolysis; 2,4-DBE was also excluded because all organisms
tested converted 50-94% of the ester to 2,4-D within 15
minutes.

Although we did not screen algae along with the bacteria
and fungi for their ability to sorb all the pesticides, we
tested a green alga, Chlorella pyrenoidosa 395, along with
the other microorganisms for extent of sorption of
methoxychlor and toxaphene.

Sorption to the microorganisms was detected only in
cultures containing the organochlorine pesticides --
methoxychlor and toxaphene. Equilibrium was reached within
16 hours. All the fungal populations screened sorbed these
organochlorine compounds; Aspergillus sp. was chosen,
however, for more extensive studies because its active spore
formation made it convenient to transfer and to maintain in
culture. Each culture of bacteria and algae was analyzed at
intervals by centrifuging a sample of a culture, extracting
it with isooctane, and determining the decrease in pesticide
concentration in the supernatant. Fungal cultures were
allowed to settle for one minute and samples of the super-
natant were analyzed. Extraction of whole cultures
accounted for all the pesticide; tle and glc analyses

-32-


0.7

0.5

0.3

0.1

LOG HM MALATHION/ LITER

Figure 2.

b 4 11.0
[*—e, MALATHION

5 10.0
L 9.0
L 8.0
/ 7.0
o
| ee a 1 6.0
0 8 16 24 32 48

TIME, hours
Growth of bacteria and decrease in malathion

concentration.

-20-

LOG BACTERIA CELLS / LITER


PREFACE

The support and assistance of Dr. Walter M. Sanders III,
Chief, Freshwater Ecosystems Branch, Southeast Environmental
Research Laboratory, U. S. Environmental Protection Agency,
Athens, Georgia, are acknowledged with sincere thanks.

We also wish to express our appreciation to the staff of
the Analytical Chemical Branch and the Surveillance and
Analysis Division, Southeast Environmental Research
Laboratory, for their assistance in providing mass spectra
of the metabolites. The assistance of David M. Cline, John
A. Gordon, Carlyn B. Haley, and Henry Patton is also
appreciated.

Appreciation is expressed to the companies donating the
pesticides used in these studies. Special thanks to R. Cc.
Blinn of American Cyanamid Company for standards of
malathion products and to the Center for Disease Control,
Atlanta, Georgia, for identifying bacteria used in malathion
studies.

This report was submitted in partial fulfillment of ROAP
O4AEM, Task 04, by the Freshwater Ecosystems Branch,
Southeast Environmental Research Laboratory, National
Environmental Research Center-Corvallis, U. S. Environmental
Protection Agency. Work was completed as of August 31,
1974,

di


-6€-

Appendix.

IN BASAL SALTS MEDIUM

SOLUBILITIES OF THE SRLFCTED PESTICIDES

ee — <= a
Pesticide Solubility in Clean-up Procedure Source
Basal Salts Medium
(ppm)

Atrazine 30.0 -- Ciba-Geigy

Captan 5.9 -- Matheson, Coleman
and Bell

Carbaryl 43.0 Recryst. with ethyl Union Carhide

ether

2,4-DBE 8.4 Redistilled Amchem Company

Diazinon 36.0 -- Ciba-Geigy

Malathion 100.0 -- American Cyanamid

Methoxychlor 0.05 Recryst. with ETOH Ciba-Geigy

Parathion 19.0 Redistilled Monsanto

Toxaphene 9.2 -- Hercules

*All pesticides except

captan were gifts of

the companies.


others. Studies are underway to determine the degree of
sorption of the various components of the pesticide.

Bailey and White (47) report an inverse relationship
between the water solubility of a pesticide and the extent
of adsorption to soils. However, the phenomenon was
observed only within a family of compounds. We found that
although toxaphene is ten times more water soluble than
methoxychlor it is sorbed to a greater extent than
methoxychlor by all organisms but B. subtilis. Our findings
do not disagree with those of Bailey et al. (47) since these
compounds are not of the same family. However, pesticides
that are much more water soluble than toxaphene, e.g.,
atrazine, carbaryl, diazinon, malathion, and parathion, were
not found to sorb to any detectable extent. This suggests
that large differences in water solubility may affect
microbial sorption.

DESORPTION

When microorganisms that have sorbed pesticides move to
aqueous environments containing little or no pesticide, they
release some of the compound, redistributing it between the
cell surface and the medium. We studied the desorption of
methoxychlor and toxaphene by harvesting bacterial cells
that had reached equilibrium in the pesticide solution and
resuspending them in medium containing no pesticide.
Samples were centrifuged, extracted, and analyzed as before
for pesticide. The sorption was found to be a reversible
process. Desorption equilibrium was achieved within the
same short time as was equilibrium in the sorption studies;
values for k were also the same. This ease of movement of
the pesticide between the organisms and water would affect
the distribution of the insecticides in the aquatic
environment.

Veith and Lee (48) studied the desorption of toxaphene
from Ottman Lake sediments. They suspended flocculent
sediment (134 mg organic carbon per gram sediment) that
contained pesticide in lake water. The pH of the
supernatant was 8.3. After 10 days of leaching, the
toxaphene content of the sediment was essentially unchanged.
When pesticide free sediment was suspended in water
containing toxaphene, sorption increased slowly over 200
days of incubation. The greatly different equilibration
times for sediment and microorganism sorption of pesticides
suggest different mechanisms of sorption.

-37-


SECTION V

MATERIALS AND METHODS

TEST MEDIA

A saturated solution of each pesticide was prepared by
stirring the pesticide into basal salts solution and
sterilized by passing the solution through a sterile 0.22-
micron Millipore filter. The pesticides used in our studies
with their sources, clean-up procedures, and water
solubilities are listed in the Appendix. Replicate
pesticide solutions of various concentrations were prepared
by aseptically diluting the filtrate with sterile basal
salts medium.

Payne and Feisal's basal salts medium (7) was used. All
components of the medium were reagent grade chemicals
purchased from J. T. Baker Company. The pH of the media
used in all studies, except those with carbaryl and captan,
was adjusted to pH 6.8 with 0.1 N HCl as determined by a
Beckman Zeromatic meter. At pH 6.8 no chemical alteration
of atrazine, malathion, diazinon, parathion, methoxychlor,
2,4-DBE, and toxaphene was detected during the course of
experiments. Carbaryl and captan were not stable in the
medium under alkaline or acid conditions.

TEST ORGANISMS

The bacteria, fungi, and algae used in our studies, the
areas from which they were isolated, and the pesticide
studies for which they were used are given in Table 2. The
following procedures were used for isolation and enrichment
of the various classes of organisms studied.

Bacteria

Stock bacterial populations were obtained from water
samples collected from four different aquatic sites and
inoculated into nutrient broth (Difco) diluted 1:10 with
water. These mixed cultures, separated according to their
respective sites of origin, were initially inoculated into
1:10 nutrient broth containing a low concentration of the
test pesticide.

After approximately one week the resultant populations

were transferred to basal salts medium containing 12.6 mmol
glucose and 0.1-1.5 ywmol pesticide per liter and the

-7-


cultures were incubated on a gyratory shaker in an
environmental chamber maintained at 28°C. When bacterial
cultures indicated significant decreases in pesticide
concentrations, an inoculum of the culture was transferred
into fresh medium containing 5.05 mmol glucose and 1.2-3.0
umol pesticide per liter.

Transfers into media containing lower concentrations of
glucose and higher concentrations of pesticide were
continued until a population of bacteria was obtained that
grew in a medium containing the test pesticide as a sole
carbon source. These enriched bacterial populations (Table
2) were lyophilized for degradation and sorption studies.
Bacterial cultures not indicating a decrease in pesticide
concentration after six weeks of transferring were
discarded.

Fungi

Fungi from four field sites were isolated on Rose Bengal
(Difco) plates and maintained on Saboraud's medium (Difco)
slants. Fungal cultures were acclimated to pesticides in a
Manner Similar to that used for bacteria by starting with
high glucose concentration relative to test pesticides and
proceeding to lower glucose concentrations relative to test
pesticides. Fungal cultures were transferred approximately
every two weeks and pesticide concentrations were monitored
regularly for 12 weeks. Cultures not showing a decrease in
pesticide concentration within 12 weeks were discarded.

Algae

Both axenic laboratory cultures of algae and algae
collected from two field sites were used in sorption
studies. The field samples also contained small numbers of
bacteria and protozoa. The axenic laboratory cultures were
grown in Bensen-Fuller medium containing 0.1% Hutner's trace
elements (8) and incubated on a shaker at 15°C under 170 ft-
c¢ of continuous light. No enrichment procedures were
employed.

GAS LIQUID CHROMATOGRAPHY

All quantitative determinations were made using a Tracor
MT-220 gas liquid chromatograph equipped with a nickel-63
high temperature electron capture detector. Pesticides were
extracted from culture samples with Burdick and Jackson
2,2,4-trimethylpentane (distilled in glass) and no sample
clean-up was needed. The nitrogen carrier gas flow was 120

-9~


EPA-660/3-75-007
MARCH 1975

MICROBIAL DEGRADATION AND ACCUMULATION
OF PESTICIDES IN AQUATIC SYSTEMS

by

Doris F. Paris, David L. Lewis,
John T. Barnett, Jr., and George L. Baughman

Southeast Environmental Research Laboratory
National Environmental Research Center-Corvallis
U.S. Environmental Protection Agency

Athens, Georgia 30601

ROAP 04AEM, Task 04
Program Element 1BA023

NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330

‘iy the Soperiatendent of Documents, US. Government Printing Ofice
For sale BY Si cbieuton, D.C. 20402, Stock’ No. 055-001-01010


Methoxychlor

Of the four bacterial populations screened for removal
of methoxychlor, only one isolate, Flavobacterium
harrisonii, caused a decrease in methoxychlor concentration
in the culture within 216 hours. No significant growth of
the bacteria was observed, as determined by viable plate
counts, when methoxychlor was the sole carbon source;
therefore, degradation rates were computed according to
equation 2, assuming a constant bacterial population. The
second-order rate constant, k, was determined to be (1.1 +
0.56) x 10-13 hr-! in cultures containing methoxychlor at
concentrations ranging from 0.006 to 0.15 pmol/l and
bacterial concentrations of 108 to 109 per liter. This rate
of degradation is slow compared to that observed with
malathion and 2,4-DBE. Degradation did not occur in
cultures until after 72 hours of acclimation, and ceased
after 30% of the methoxychlor was degraded. Analyses of
extracts of the cultures showed no further degradation after
192 hours. The rate constant, therefore, is descriptive
only of the period of active degradation.

The main degradation product, identified by tlc, glc,
and mass spectrometry was 2,2-bis(p-methoxyphenyl) -1,1-
dichloroethylene, often referred to as methoxychlor-DDE.
Our rate and product determination are in agreement with
those reported by Mendel et al. (36). In their studies the
bacterium, Aerobacter aerogenes, metabolized 65% of
available methoxychlor to methoxychlor-DDE within 168 hours.

Since the Flavobacterium used in our study could not
utilize methoxychlor as a source of carbon, enrichment of
that organism in the field in the presence of methoxychlor
is not expected. On the other hand, the bacteria in
malathion and 2,4-DBE studies could utilize the pesticides
as carbon sources, and would be expected to exhibit an
enrichment in the presence of those pesticides.

Enrichment of microbial populations that degrade
pesticides in the field has been suggested as an explanation
for the decreasing persistence of certain pesticides
observed upon successive application to field plots. This
enrichment phenomenon can be a function of growth rate
sustained in utilization of pesticides as nutrient sources.
For example, Kearney (37) reports that chloropropham is more
readily hydrolyzed than propham by a purified enzyme from
Pseudomonas striata. However, the intact cells of P.
ta degrade propham more readily and the resultant
population is larger than when P. striata is cultured in the
presence of chloropropham (38). Chloropropham, therefore,

-30-


Carbaryl

Carbaryl has been reported to be hydrolyzed both
chemically (17,18) and biologically (19). Researchers,
however, found it difficult to determine the relative extent
of the two modes of degradation (19).

In our studies bacterial removal of carbaryl was found
to be negligible, even when the bacterial population was
increased from 1 x 109 per liter to 1 x 104! per liter
(Figure 1). Growth of bacteria instead was dependent upon
the rate of chemical hydrolysis of carbaryl to a-naphthol.
The same bacterial population, grown in cultures containing
a-naphthol, used the a-naphthol as a carbon source.
Bacterial concentration increased 10 fold in 24 hours,
removing all of the a-naphthol from the medium. Products of
the bacterial degradation of the a-naphthol were 1,4-
naphthoquinone and two unidentified compounds.

Hughes (18) reports that carbaryl is chemically
hydrolyzed to a-naphthol in pond water under laboratory
conditions. He also found a bacterium (Flavobacterium sp.),
isolated from the pond water, to degrade the a-naphthol
rapidly to o-hydroxycinnamic acid, salicylic acid, and an
unidentified product. In Hughes* work, therefore, the
bacteria cleaved the naphthalene ring.

Researchers working with soil fungi found that the fungi
transformed carbaryl with no ring cleavage. Bollag and Liu
(20) reported the degradation of carbaryl by a large number
of sdéil fungi to naphthyl-N-hydroxy-methylcarbamate, 4-
hydroxyl- 1-naphthylmethylcarbamate, and 5-hydroxy-1-
naphthylmethylcarbamate. The latter products may be the
first step toward ring cleavage since microorganisms usually
hydroxylate a ring prior to cleavage.

Malathion

Both bacteria and fungi were found to degrade malathion.
The bacteria grew in the presence of malathion as the sole
carbon source. As the bacterial concentration increased,
the decrease in concentration of the insecticide was
monitored by gas liquid chromatography of isooctane extracts
of whole cultures, i.e, medium plus bacteria (Figure 2).
All unmetabolized pesticide was therefore detected including
any adsorbed onto the cell surface.

To study the kinetics of degradation, we first
determined the maximum growth rate of bacteria on malathion.

-18-


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development,

U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:

1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research

4. Environmental Monitoring

5. Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL RESEARCH STUDIES
series. This series describes research on the effects of pollution

on humans, plant and animal species, and materials. Problems

are assessed for their long- and short-term influences. Investigations
include formation, transport, and pathway studies to determine

the fate of pollutants and their effects. This work provides

the technical basis for setting standards to minimize undesirable
changes in living organisms in the aquatic, terrestrial and atmospheric
environments.

This report has been reviewed by the National Environmental

Research Center--Corvallis, and approved for publication. Mention
of trade names or commercial products does not constitute endorsement
or recommendation for use.


21

22

23

24

25

26

27

28

29

30

31

Walker, W. W., and B. J. Stojanovic. Malathion
Degradation by an Arthrobacter species. J. Environ.

Quality.

Matsumura, F.,

3(1) 24-10 (1974).

and G. M. Boush. Degradation of
Insecticides by a Soil Fungus, Trichoderma viride.
Journal of Economic Entomology. 6

Schwartz, H. G., Jr.

Pesticides in Aqueous Solution.

Control Federation.

Aly, O. M., and S. D. Faust.

61(3) 2610-612 (1968).

Microbial Degradation of

392 1701-1714

J. Water Pollution

(1967) .

Studies of the Fate of

2,4-D and Ester Derivative in Natural Surface Waters.

J. Agr.

Audus,
Soils.

and G. R. Sagar (eds).

L. J.

Publications,

Bell,

2,4-Dichlorophenoxyacetic Acid.

Food Chem.

12:541-546 (1964).

Microbiological Breakdown of Herbicides in
In: Herbicides in the Soils, Woodford, E. K.,

1960.

Pe 1-19.

Oxford, Blackwell Scientific

G. R. Some Morphological and Biochemical
Characteristics of a Soil Bacterium Which Decomposes

3:821-840 (1957).

Can. J. Microbiol.

Bollag, J. M., G. G. Brigg, J. E. Dawson, and M.

Alexander.
J. Agr.

Food Chem.

Bollag, J. M., and M. Alexander.
Herbicide Detoxication by Bacterial Enzymes. J. Agr.

Food Chem.

15:881-884 (1967).

Enzymatic Degradation of Chlorocatechols.
162829-833 (1968).

Phenoxyacetate

Duxbery, J. M., J. M. Tiedje, M. Alexander, and J. E.

Dawson.

2,4-D Metabolism:

Enzymatic Conversion of

Chloromaleylacetic Acid to Succinic Acid. J. Agr. Food
18: 199-201 (1970).

Chem.

Evans,

W. C.,

and B.

S. W. Smith.

The Photochemical

Inactivation and Microbial Metabolism of the Chiorophen-
oxyacetic Acid Herbicides. Proc.

(1954)

Fernley, H. N., and W. C. Evans.
Dichlorophenoxyacetic Acid by a Soil Pseudomonas:
-Chlorcmuconic Acid as an Intermediate.

Tsolat.
Proc.

ion of
Biochem.

Soc.

73222 (1959).

~42-

Biochem. Soc. 57: xxx

Metabolism of 2,4-


5 5 §
(CH30)p-P-SH + (CH30)p-P-S-CHCOOH + HacooEt + (CHyOh-P-s-CHCOOE
0,0-Dimethylphos- tH,cooet —et00CCH CH,COOH
phorodithioic acid sation a-monoacid Diethyl fumarate Malathion B-monoacid

{ Hydrolysis, pH 8, 27°

i
(CH30)2-P-S-CHCOOEt

CHeCOOEt
Malathion

| Bacteria, 28°

u . +
0,0-Dimethylphos- + (CH30)2-P-S-CHCOOH = + CHCOOEt + Malathion B-monoacid
phorodithioic acid CH_COOH CHCOOEt

Malathion diacid Diethyl! maleate

Figure 6. Comparison of chemical and microhial deqradation
products of malathion.

-26-


080

30) T
E harrisonii B. subtilis °
.
<
Q ‘
x15 ‘
oe €
.
in e rn
03 06 ° 0026 0052
Ce Ce
268 T 208)
C. pyrenordosa Aspergitlus sp.
+ +t . .
fo}
© is) .
«x 234b 4 =ioab 4
£ 5 £ .
x % % ‘
~
‘° 1 n
° O16 032 i) 02 04
Ce Ce

Figure 8. Sorption of methoxychlor by bacteria, fungi, and

algae.
062 + 202,
E harrisonii B subtilis
*o so)
* 03h ~ 101
£ £
v n
° Oe o o3 36
¢ c
833 = 104 <
C pyrenoidoso Aspergillus sp
% .
+ it .
ie} ‘2
* 416) 0 52| <
E £
¢
A
° 025 ° 003 006
Ce Ce

Figure 9. Sorption of toxaphene by bacteria, fungi, and
algae.

-35-


Culture flasks containing malathion at concentrations
ranging from 0.028 to 128 wymol/l were inoculated with
suspensions of washed organisms sufficient to give viable
bacterial concentrations of 106 organisms per liter. The
maximum rate of growth occurred in the medium having an
initial malathion concentration of 13.6 pmol/l. Using
equation 2, experimental values of [S]/y were plotted as a
function of [S] (Figure 3). K, was determined to be 2.17
yumol/1 and Un to be 0.37 hour-1.

At malathion concentrations one-fifth the value of K, or
less, equation 4 describes the rate of bacterial removal of
malathion. The constant k was found to range from 1.2 to
3.5 x 10-12 liter organism-! hour-! (Table 4).

Whereas the bacterial population increased in concentra-
tion, no growth of the fungus, Aspergillus oryzae, was
detected during the laboratory experiments. Fungal biomass
was therfore assumed to be constant. The rate of removal of
malathion fran solution by A. oryzae may be described by
equation 4, the second-order rate expression used in the
bacterial rate studies. The second-order rate constant, k,
for fungal degradation of malathion was (1.10 + .66) x 10-3
liter mg-! hour-!t. As indicated by values for k at very
high organism concentration, based upon dry weight,
malathion is removed by the bacteria approximately 5,000
times faster than by A. oryzae under similar conditions,
i.e, malathion concentration, agitation rate, and
temperature.

To further test these rate data, filter-sterilized river
water containing malathion was inoculated with the fungi and
bacteria found to degrade malathion. Under these
conditions, the microbial half-life of malathion is given by
equation 5. In this expression, k,

0.693

t. = 7_—_—_———
35 k,(B) + kL [F]

and k, are the rate constants previously determined for
bacteria and fungi respectively and [F] is the concentration
of fungi (mg/1) . The half-life measured under the
experimental conditions was 2.2 hours, which is in good
agreement with the calculated value of 2.5 hours.

The major metabolite of malathion degradation (97-99%)
in both the bacterial and fungal systems was found to be the
B-monoacid of malathion (5 )- The monoacid could be
recovered quantitatively from both systems (Figures 4 and

-21-


1.

2.

6.

SECTION III

CONCLUSIONS

Under conditions found in most aquatic environments (pH
5. 6-8. 0) chemical hydrolysis of captan occurs too
rapidly for microbial degradation or accumulation of the
parent compound to be significant.

The butoxyethylester of 2,4-dichlorophenoxyacetic acid
is rapidly degraded to 2,4-D and butoxyethanol by all
bacteria and fungi tested in the laboratory.
Degradation of the resulting 2,4-D is a much slower
process.

Under the conditions of our experiments methoxychlor is
not degraded rapidly by bacteria.

The major metabolite of malathion degradation by the
bacteria and fungi studied is the §-malathion monoacid.

In aqueous solution (pH 6.8-7.0, 27°C) containing low
concentrations of malathion and low concentrations of
malathion degrading bacteria, bacterial degradation can
compete with chemical degradation.

The growth of the bacteria used to study carbaryl
degradation is dependent on the rate of chemical
hydrolysis of carbaryl to a-naphthol. In cultures
containing a-naphthol the bacteria used in the carbaryl
studies utilized a-naphthol as a sole carbon source.

The more water soluble pesticides -- atrazine, carbaryl,
diazinon, malathion, and parathion -- were not sorbed by
any of the bacteria or fungi tested; therefore,
microbial sorption of these compounds would not be
expected under natural conditions.

Sorption of methoxychlor and toxaphene by bacteria,
fungi, and algae can be described by a _ partition
coefficient and the process is rapidly reversible.


32

33

34

35

36

37

38

39

40

44

42

Loos, M. A., J. Ms. Bollag, and M. Alexander.
Phenoxyacetate Herbicide Detoxication by Bacterial
Enzymes. J. Agr. Food Chem. 15:858-860 (1967).

Steenson, T. I., and N. Walker. The Pathway of
Breakdown of 2,4-Dichloro- and 4-Chloro-2-methyl-
Pphenoxyacetic Acid by Bacteria. J. Gen. Microbiol.
16: 146-155 (1957).

Tiedje, J. M., and M. Alexander. Enzymatic Cleavage of
the Ether Bond of 2,4-Dichlorophenoxyacetate. J. Agr.
Food Chem. 17:1080-1083 (1969).

Tiedje, J. M., J. M. Duxbury, M. Alexander, and J. E.
Dawson. 2,4-D Metabolism: Pathway of Degradation of

Chlorocatechols by Arthrobacter sp. J. Agr. Food Chem.
17:21021-1026 (1969).

Mendel, J. L., A. K. Klein, J. T. Chen, and Mae S.
Walton. Metabolism of DDT and Other Chlorinated Organic
Compounds by Aerobacter aerogenes. J. of Association of
official Analytical Chemist. 50(4):897-903 (1967).

Kearney, P. C. Furification and Properties of an Enzyme
Responsible for Hydrolyzing Phenylcarbamates. J. Agr.
Food Chem. 13(6):561-564 (1965).

Kaufman, D. D., and P. C. Kearney. Microbial

Degradation of Isopropyl-N-3-Chlorophenylcarbamate and
2-Chloro-ethyl-N-3-Chlorophenylcarbamate. Appl. Micro.
13 (3) 2443-446 (1965).

Hsieh, D. P. H., and D. M. Munnecke. Accelerated
Microbial Degradation of Concentration Parathion. Proc.
Iv International Fermentation Society: Ferment. Technol.
Today. p. 551-554 (1972).

Ware, G. W., and C. C. Roan. Interaction of Pesticides

with Aquatic Microorganisms and Plankton. Res. Rev.
33: 15-45,.

Ko, W. He, and J. L. Lockwood. Accumulation and
Concentration of Chlorinated Hydrocarbon Pesticides by
Microorganisms in Soils. Can. J. of Microbiol.
14:1075-1078 (1968).

Johnson, B. T., and J. O. Kennedy. Biomagnification of

p,p'-DDT and Methoxychlor by Bacteria. Appl. Microbiol.
26 (1) 266-71 (1973).

~43-


SECTION IX

PUBLICATIONS

Paris, Doris F., and David L. Lewis. Chemical and
Microkial Degradation of Ten Selected Pesticides in
Aquatic Systems. Res. Rev. 45:95-124 (1973).

Lewis, David L., and Doris F. Paris. Direct
Determination of Carbaryl by Gas Liquid Chromatography
Using Electron Capture Detection. J. Agr. Food Chem.
22(1) 2148-149 (1974).

Paris, Doris F., and David L. Lewis. Rates and Products
of Degradation of Malathion by Bacteria and Fungi from
Aquatic Systems. Presented at the Third International
Congress of Pesticide Chemistry. Helsinki. July 3-9,
1974, and to be published in the Journal of
Environmental Quality and Safety.

Paris, Doris F., David L. Lewis, and N. Lee Wolfe.
Rates of Degradation of Malathion by Bacteria Isolated
from an Aquatic System. Environ. Sci. and Tech.

9(2): 135-138 (Feb. 1975).

Lewis, David L., and Doris F. Paris. Transformation of

Malathion by a Fungus, Aspergillus oryzae, Isolated from
a Freshwater Pond. Accepted for publication in the
Bulletin of Environmental Contamination and Toxicology.

Paris, Doris F., and David L. Lewis. Accumulation of
Methoxychlor by Microorganisms Isolated from Aquatic
Systems. In preparation.

Paris, Doris F-, and David L. Lewis. Bioconcentration
of Toxaphene by Microorganisms. In preparation.

-45-


Table 2.

MICROORGANISMS USED IN DEGRADATION AND SORPTION STUDIES

Pesticide

Source

Organisms

Malathion

Potomac River

Pseudomonas cepacia

-8-

Xanthomonas sp.
Commomonas terrigera
Flavohacterium meningosepticum

Malathion Shriner's Pond

Aspergillus oryzae

2,4-DBE, Methoxychlor, Bacillus subtilis

and Toxaphene

Soya Creek

2,4-DBE Shriner's Pond Rhodotorula glutinis

Methoxychlor and Citrus Plant Flavobacterium harrisonii
Toxaphene Effluent

Carbaryl]. Florida Pond Brevibacterium sulfureum

Pseudomonas ovalis

Bacillus megaterium
Flavobacterium lutescans

Toxaphene and Methoxychlor| Starr's collection Chlorella pyrenoidosa 395
Chicken Plant

Effluent

Toxaphene and Methoxychlor Aspergillus sp.