Figure 3. Extraction schematic for malathion metabolites.

Aqueous Cell-free

Centrifugate

Extract 2X with
Petroleum ether

Aqueous Pet. Ether

Acidify to pH 1.5 with TCA
GLC

Extract with a) Pet. ether: acetone (1:1)
b) Diethyl ether

Aqueous Ether

Concentrate to 30 ml
under vacuum at 35 C

Add 5 ml Benzene:Methanol
Concentrate to 5 ml or less

TLC Separation

100/120 mesh Gas Chrom Q and the other column 0.75% OV-17 and 0.85%

OV-210 on 100/120 Gas Chrom Q, were employed. Both columns were 0.64
cm x 1.8 m. Column, detector, and inlet temperatures were 200, 250, and
250 C, respectively. The carrier gas was nitrogen, used at a flow rate
of 25 m@/min. For the flame photometric analyses, the detector was
operated in the phosphorus mode. A 0.32 cm x 1.8 m column containing
2% OV-101 on 80/100 mesh Gas-Chrom Q was used for all analyses. Respec-
tive column, detector (ignited), and injector temperatures were 180,
160, and 220 C, and gas flow rates for 02, air, H2, and N2 (carrier)
were 20, 50, 200, and 60 m/min, respectively.

12


10¢

| ee

105

MASTER PLATE (5)

1 1 10
1 1
: -
om Os
° 5 10 i) 20 25 30
DAYS
Figure 15. Heterotrophic (solid line) and mirex-tolerant bacteria

(shaded) from water column of artificial salt-marsh
ecosystems presented as heterotrophs/ml and percentage
of "replica-master-plate", respectively.

33

uw / SHdOULONILIH


there is some evidence which indicates the spots are diethyl-maleate and
maleic acid, respectively. At least one of these compounds, diethyl
maleate, has been identified previously as a malathion metabolite (12).
The other, maleic acid, can be obtained by carboxyesterase activity from
the former. No direct evidence can be obtained from these samples to
identify these metabolites positively at the low concentrations found
here.

Major degradation products have been identified and other metabolites
tentatively identified. 1 believe that the low concentration of the
latter metabolites is a consequence of the nature of the compound itself;
most of these metabolites will serve as good carbon sources for bacteria.

ARTIFICIAL ECOSYSTEMS STUDIES

Aerobic heterotrophic bacterial numbers of 6 artificial salt-marsh
environments are represented in Figures 13, 14, 15, and 16. The
average results of duplicate systems that received (a) malathion, 10X
field application rate (top), (b) malathion field application rate
(middle), and (c) no malathion (bottom, control) are presented in
Figure 13. The data represent heterotrophic counts plated in trip-
licate and the percentage numbers of colonies from nutrient medium
which grew on replica-test medium, as compared to a representative
master plate.

Essentially no differences in total heterotrophic activity (solid line)
were noted between control and experimental cultures of either water
(Figure 13) or sediment (Figure 14) samples. However, numbers of mal-
athion-degrading bacteria in the treated water (Figure 13) increased

over the control in water during the 30 day treatment period for both
sole-carbon-degrading bacteria (SCD = 7 and 13%, dark areas) and mal-
athion co-metabolizing bacteria (MCM = 83 and 84%, shaded area). Sta-
tistically, MCM's were significantly different(s= 0.01) among treatment
levels (10X, 1X, and the control). SCD's in the water increased with
treatment frequency (0, 10, 20 days; a = 0.05) but were not significant
between treatment levels. At 10X application rate only 7% of the hetero-
trophic bacteria on the master plate were SCD's, whereas at 1X application
rate, 13% were SCD's.

Figure 14 represents similar treatment of data from the sediment samples.
Fluctuations in numbers of bacteria were greater in all sediment samples
but the results were similar to those of the water samples. SCD's
increased significantly (a= 0.10) among treatment levels (control, IX
and 10X) and among individual treatment periods (0, 10 and 20 days; a =
0.05), 1.e., frequency of treatment. Numbers of SCD's, 4% of the
heterotrophic bacteria on the master plate in the untreated sediment,
were greater for 1X and 10X application rates of malathion, 8 and 10%

respectively.

29


10.

SECTION VI
REFERENCES

Aaronson, S. Procedures for the Enrichment and/or Isolation of
Microorganisms. In: Experimental Microbial Ecology, New York,
Academic Press, 1970. p. 65-171.

Alexander, M. Microbial Degradation of DDT. Cornell University.
Ithaca, New York. NTIS No. AD-781 903/OWP Office of Naval Research.
Contract No. NO014-67-A-0077-0027. July 1973. 24 p.

American Cyanamid Company. Technical Information on Cythion Insecti-
cide "The Premium Grade Malathion and Malathion ULV Concentrate In-
secticide." Princeton, New Jersey. p. 1-26.

Asheio, J., J. H. Ruzicka and B. B. Wheals. A General Method for the
Determination of Organophosphorus Pesticide Residues in River Waters
and Effluents by Gas, Thin-layer and Gel Chromatography. Analyst.
94: 275-283, 1969.

Butler, P. A. Pesticides & Wildlife Studies. U. S. Fish Wildl.
Serv. Cire. 167:11-24, 1963.

Colwell, R. R. and W. J. Wiebe. "Core" Characteristics for Use in
Classifying Aerobic, Heterotrophic Bacteria by Numerical Taxonomy.
Bull. Ga. Acad. Sci. 28:165-185, Sept. 1970.

Conte, F. S. and J. C. Parker. Ecological Aspects of Selected
Crustacea of Two Marsh Embayments of the Texas Coast. Texas A&M

University Sea Grant Program. College Station, Texas. TAMU-SG-71-
211. June 1971. 184 p.

Coppage, D. L. and T. W. Duke. Effects of Pesticides in Estuaries
Along the Gulf & Southeast Atlantic Coasts. In: Proc. 2nd Gulf
Coast Conf. on Mosquito Suppression & Wildlife Management.

Schmidt, C. H. (ed.). Washington D. C., National Mosquito Control-
Fish & Wildlife Management Coordinating Committee, 1971. p. 24-31.

Culley, D. D. and H. G. Applegate. Residues in Fish, Wildlife &
Estuaries. Pestic. Monit. J. 1:21-28, 1967.

Fleck, E. F. Chemistry of Insecticides. In: Pesticides and Their
Effects on Soils & Water. American Society of Agronomy (ed.)
Madison Wis., SSSA, Inc., Special Publication Number 8, 1966.

p- 18-24.

38


Figure 1. Enzymatic cleavage of malathion. Products of phosphatase
(P'ase) and carboxyesterase (C'ase). Products of malathion
cleavage are given in Table 2.

P

&
Oo $+CH—COO+C)H;

CH.-COO C,H;

H.c-O 5$
ew /7
%

HC

3 p
P’ase C’ase


percentage of added malathion degraded. Controls consisted of malathion
media without added cells. Most isolates were not able to use malathion
effectively as a sole carbon source. Forty-percent of the isolates
tested showed 50% or greater utilization of malathion. However, in the
presence of peptone, malathion was rapidly degraded by all isolates
tested.

Bacterial cultures were grown on 14¢-malathion with peptone and
analyzed by liquid-scintillation counting to determine the extent of
malathion-carbon incorporation in the microbial cell-mass. Most of

the activity was associated with the cell fractions; however, 8-28%
remained in the supernate (Table 5). This fraction probably represented
water-soluble metabolites of malathion. For assessment of radioactive
malathion metabolites, spent medium from 14¢_malathion incubation as

a sole carbon source was assayed for carboxyesterase phosphatase
products by differential extraction and liquid scintillation counting,
and confirmed by chemical analysis (Table 6). The carboxyesterase
products, MCA and DCA, were identified by GLC retention times and
quantitated. There is good agreement between chemical residue analysis
and isotopic analysis for malathion. In most cases, MCA is the pre-
dominate degradation product produced by biological as well as chemical
reactions, as indicated in the control data. The data indicate a greater
portion of phosphatase products due to biological breakdown (cultures
44, 45, 47), with lesser production of similar products due to chemical
breakdown (control). The "mixture" indicated in Table 6 represents equal
proportions of the aforementioned cultures. The degrading activity of
the mixture was greater than the individual cultures, as evidenced by
the lower recovery and higher DCA concentration. The microbial systems
shown here have an effective carboxyesterase system that causes rapid
breakdown of malathion to the acids, with a delayed demethylation
reaction to produce demethyl-malathion. Some microbial systems apparently
catalyze demethylation earlier, resulting in fast release of C02 from
the malathion molecule. Data from cultures 1 through 12, not shown
here, indicated that cultures 1, 4 and 9 affected the release of 4coo
within 2 days, whereas others required incubation for 7 days before
appreciable release of coy was detected. In the sterile control, 20%
of the + C-methoxymalathion label was released as M4co9 after 10 days
incubation.

16


MASTER PLATE ()

8

ot

%
rr ELSES OO

°

DAYS

Figure 16. Heterotrophic (solid line) and mirex-tolerant bacteria
(shaded) from sediments of artificial salt-marsh
ecosystems.

34

wB /SHdOULONIL3H

107

_
() 5 10 15 20 25 30


Section

I

IL

TIL

Iv

VI

VIL

CONTENTS

CONCLUSIONS
INTRODUCTION

MATERIALS AND METHODS
Materials
Experimental Methods
Analytical Methods
Statistical Analysis

RESULTS
Physico-Chemical Degradation
Microbiological Degradation
Isolation and Identification of

Microbial Metabolites

Artificial Ecosystem Studies

DISCUSSION

REFERENCES

ABBREVIATIONS AND SYMBOLS

iii

29

35

38

Al


L2@

2000

WAVE NUMBER (cm)

oo

Figure 11.

4 n +
+ +t + + +

WAVE NUMBER (cmt)

Infrared spectral tracings of (a) extract from

spot 7 and (b) extract from spot 8.



Amyl derivatives of malathion degradation products were prepared

for gas chromatography analyses by previously described methods (23).
All samples were quantified by comparing the peak height with those
standards of known concentration.

Thin-Layer Chromatography (TLC)- Extracts from biodegradation studies
were separated on 250 u-thick Silicargel H, 20 cm X 20cm, thin-layer
glass plates prepared by Quanta/Gram (A. H. Thomas Co., Phil., PA.).
Plates were spotted with 10-20 u% of concentrated extract and devel-
oped in the appropriate solvents. For qualitative analyses, two-dimen-
sional chromatography was employed using the following solvents: (1)
Benzene:Hexane:Acetic Acid (40:40:20) and (2) Hexane:Acetic Acid:Ether
(75:15:10) (18). After allowing the plates to dry, they were sprayed
with 0.5% (wt/vol) N, 2, 6-Trichloro-p-benzoquinoneimine (TCQ, Eastman
Kodak Co., Rochester, N. Y.) freshly prepared in nanograde acetone. The
plates were then developed at 110 C for 10 minutes (16). Spots repre-
senting malathion and its degradation products appear as dark, reddish-
pink on a light background.

Infrared Spectroscopy (IR)- To prepare metabolites for infrared spec-
tral analyses, 1-2 m&% of the concentrated acetone extract was streaked
on a TLC plate and developed with benzene:glacial acetic acid (4:1)

in one direction. After allowing the plate to dry, it was covered
with another glass plate that allowed only about two cm of the TLC
plate to be exposed, then sprayed with TCQ (29). Areas correspond-
ing to metabolite bands were scraped from the plate and extracted with
50 m2 acetone. This extract was concentrated to 1-2 mi under vacuum at
35 C and an appropriate aliquot added to dried potassium bromide
(Harshaw) for analysis on a Perkin-Elmer Model 621 Grating Infrared
Spectrophotometer. Whenever appropriate, the samples were analyzed
by operating the instrument in a 5X expanded ordinate scale. Spectral
tracings for malathion degradation products were compared to standards
supplied by American Cyanamid Co. and EPA's Southeast Environmental
Research Laboratory.

STATISTICAL ANALYSIS

Data obtained from the artificial ecosystem studies were treated by
analysis of variance to determine existence of significant differences
(1) between control and experimental cultures over a 30-day treatment
period, (2) during a single treatment period (every 10 days) and (3)
at each treatment level.

13


61

MALATHION (%)

100 | nena
a o |
() CONTROL
80 e ORIGINAL (%)
[e)
sof
e@
|
40r
‘0 ‘a
20} —~——_ 7
0 (100) (92) (70) 5) \u
es ce ee oe ee ne Shs ee
0 2 4 6 8 10 12 14

INCUBATION (DAYS)

Figure 5. Bacterial degradation of malathion in seawater.



ST

MALATHION (*%)

Figure 4.

°
wl

~
. NY 10%. SW
ss aN 20

ss ; 20 %o

< oe
6 10. 14 18 22. 26 30
INCUBATION TIME (DAYS)

Effect of salinity and temperature on malathion stability. Malathion 1 yg/m2 added in
acetone to sterile seawater at varied salinities, 0 °/oo distilled water. Dashed line

represents microbial degradation.


82

'
me-€-O-Cahg

We- 6 oka.
NovTeass

2000
WAVE NUMBER (CH)

Figure 12.

3080 200 WAVE NUMBER (cH) 1000

Infrared spectral tracings of (a) diethyl maleate and extract from spot 9 and (b) extract
from spot 10, with possible structure in brackets.


The actual rates of malathion degradation were determined by incubation
in seawater medium containing 46 ug/ml malathion and sampling at two-day
intervals for malathion residues. Sterile peptone was added to cul-
tures showing no greater reduction in malathion than control cultures
(malathion-seawater but no cells). These cultures were monitored for
malathion degradation after an additional four days of incubation.

Several bacterial isolates that represented both sole-carbon degraders

and co-metabolizers were inoculated into 10 mé sea-sal-0.2% peptone

medium containing 0.1 uci-l4c-malathion (46 ug/ml). After 10 days
incubation, residual malathion was extracted with petroleum ether. The
cells were removed by centrifugation at 12,000 xg for 10 minutes, washed
with. 79 mf sterile seawater, dried for 24 hours at 85 C, and analyzed

for t C-activity by liquid scintillation counting. The cell-free supernate
was analyzed for soluble degradation products by assaying for residual
1c_activity.

Cultures grown on marine 2216 broth and marine broth plus 200 ug
malathion/m2 were analyzed for contribution of malathion to cell mass.
After 10 days incubation, the cells were separated by centrifugation and
transferred to tared aluminum weighing dishes. The cells were dried for
16 h at 110 C and weighed.

To determine malathion metabolites, Pseudomonas sp. 45 was incubated in
seawater medium and Pseudomonas sp. 8 was incubated in seawater medium

with 2% peptone, both containing 100 ug/mf malathion. (In earlier

screening studies, these organisms degraded malathion as a sole carbon

source and as a co-substrate.) Cultures were incubated 10 days at 28 C

in the dark. Cells and medium were then separated by centrifugation and

the cell-free medium was extracted, with petroleum ether to remove

malathion. The aqueous fraction was (see Fig. 3) extracted again with
petroleum ether: acetone (1:1) and with diethyl ether. The concentrated
extract was analyzed for malathion and metabolites by thin-layer chromatography

Preparation of Laboratory Ecosystems- Sediment and water collected from
a salt marsh on Santa Rosa Island, near Gulf Breeze, Florida (Figure 2)
was transported to the laboratory for initial planting of a laboratory
salt-marsh ecosystem. Sediment (sand) from the Range Point salt-marsh
area was sieved through a No. 7 mesh screen to remove larger particles
and facilitate sampling procedures. Sieved sand (7.62 cm deep) was then
placed into acetone-cleaned battery jars (16 x 16 x 26 cm) and 6.5
liters of marsh water added. A Pasteur pipette was fixed about 2/3
distance down in the water column, and air was slowly bubbled through

to prevent anaerobiosis or surface slick formation. The laboratory
micro-ecosystem was allowed to settle for 72 hours before sampling.
Water temperature and salinity were maintained at 28 C (+ 1) and 20 °/oo
(+ 2 °/oo) for the duration of the experiment. The artificial ecosystems
were maintained under a 12 hour cycling light-dark system (6,000 lux).

10


SECTION II
INTRODUCTION

Estuarine environments are composed of highly complex communities of
organisms, some of which are inseparably linked to coastal marshlands.
The most productive parts of the estuary are the intertidal and adjacent
shallow-water zones (23). Coastal nekton frequently use estuaries as
nursery grounds where larval through adolescent growth stages can take
advantage of the protection and abundant food. Because early life
stages of many important commercial and sport fisheries depend on estuaries,
it is important economically to protect these habitats. These saline
marshlands are also prime breeding sites for mosquitoes. Recently,
massive mosquito control programs have been established to control the
adult mosquito in municipal areas and, in many instances, effective
treatment has necessitated insecticide application on or near marshes
which serve as nursery grounds for a variety of marine species (7).
Concern for possible hazards to non-target species in the salt-marsh has
prompted studies to determine the fate and effects of these chemical
toxicants.

Malathion, S-(1,2-Dicarbethoxyethyl)-0, O-dimethyl dithiophosphate, is

an organophosphate insecticide used extensively to control adult mosquitoes.
It was the single most widely used insecticide in, the United States in
1971, estimated annual production being 1.36 x 10 kg (30 million lbs.,
Table 1). In a single operation in 1971, the U. S. Air Force applied

15.3 x 10 & (40,335 gal.) of technical grade malathion (active ingre-
dient unknown) to 2,016,060 acres in counties

Table 1. ESTIMATED U. S. INSECTICIDE PRODUCTION VOLUME, 1971¢

Chemical Number
group compounds Production
(Mill. lbs. A.I.)
Chlorinated hydrocarbons 13 158
Carbamate 5 64
b b
Organophosphate 23 148
Others 51 23

Total, all synthetic organic

insecticides, miticides, nematocides 393
“From U.S. EPA Pesticide Study Series, 1971 (26)

bMalathion = 24% of total organophosphate production



A.

_ - _ _ a
MALATHION DICARBOXYLIC ACID
SPOT a3
CHs-0, ,S
Py a
(H3-0 “S~CH-C-OR
CHy¢-0H
| 0
' oF0
| 1 pe a . f 4.
ne t000. , 3100 2080 ‘ 1000 200
B. WAVE NUMBER (cM")

i ]

=i

SPOT not

NN

POTRSSIUM DESMETHYL MALATHION

CHy-0. 0S
Pg
K So HE=C-O- Co Hy

Hil =(-O-CrHs

de — 20bo ; aah to he P|

WAVE NUMBER = (cM)

Figure 9, Infrared spectral tracings of (a) malathion-dicarboxylic acid and extract from spot 3 and
(b) potassium desmethyl-malathion with extract from spot 4,


cM)-0. 8
an °
© =O “$-eH-€0- cams
Cita 6 O- Cay
°

Figure 7,

3000 2000

WAVE NUMBER = (cH"')

| ! 1 | | 4
3000 2000 wooo

WAVE NUMBER (cm")

Infrared spectral tracings of (a) malathion and extract from band corresponding to spot 1
and (b) malathion-half-ester with extract from spot 2 (Figure 6).


Table

DUwW N

~

LIST OF TABLES

Estimated U. S. Insecticide Production, 1971
Structural Formulae of Malathion, Metabolites
and By-Products

Microbial Growth on Malathion Plus Nutrients
Microbial Degradation of lathion
Cell-Medium Distribution C from Malathion
Radiometri¢, and Chemical Analyses of Microbial
Growth on C-Malthion

Rg Values (Thin-Layer Chromatography) for
Malathion and Metabolites

Malathion Degradation Products

Page

nN

4
17

18

20

36


LIST OF FIGURES

Figure Page
i Enzymatic cleavage of malathion 6
2 Range Point Salt~Marsh on Santa Rosa Island, Florida 8
3 Schematic for extraction of malathion metabolites 12
4 Effect of salinity and temperature on malathion 15
stability

5 Bacterial degradation of malathion in seawater 19

6 Thin-layer chromatogram of microbial and chemical 21
degradation products

7 Infrared spectral tracings of (a) malathion and TLC 22
spot 1, (b) malathion-half-ester and TLC spot 2

8 Infrared spectral tracing of malaoxon with repre- 23
sentative adsorption bands indicated

9 Infrared spectral tracings of (a) malathion-dicar- 25

boxylic acid and TLC spot 3, (b) K-desmethyl-mal-
athion and TLC spot 4

10 Infrared spectral tracings of (a) TLC spot 5, (b) 26
TLC spot 6

iL Infrared spectral tracings of (a) TLC spot 7, (b) 27
TLC spot 8

12 Infrared spectral tracings of (a) diethyl maleate 28
and TLC spot 9 (b) TLC spot 10

13 Bacteria from water colum of artificial salt-marsh 30
ecosystem treated with malathion

14 Bacteria from sediments of artificial salt-marsh 31
ecosystems treated with malathion

15 Bacteria from water column of artificial salt-marsh 33
ecosystems treated with mirex

16 Bacteria from sediments of artificial salt-marsh 34

ecosystems treated with mirex

iv


Malathion was added in 0.5 m£ acetone to the water surface of two
battery jars at 420 g/ha (field application rate) and 4.2 kg/ha (10X
field rate). Since previous investigation indicated no significant
effects on indigenous microflora resulted from the addition of 0.5m
acetone to similar experimental ecosystems, no acetone controls were
included in this experiment. The artificial ecosystems were treated
every 10 days during two 30-day experiments. This cycle was chosen to
simulate a mosquito-control program.

After the initial 72-hour settling period, water and sediment

samples were assayed microbiologically to insure that microbial numbers
had returned to nominal environmental levels. Once stabilization was
ascertained, malathion was added and the test period begun. Ten-milli-
liter water samples, taken from the middle of the water column, were
added to 90 mf sterile seawater blanks and diluted appropriately for
plate counts. Ten-gram (wet-weight) sediment samples were taken with
a large-bore sterile pipette and diluted for plate counts. Sediment
and water samples were taken immediately before treatment, at "0" time
(immediately after treatment) and at 1, 3, 7 and 10 day intervals
thereafter. Also, pH of the water was checked and samples were collected
for analysis of residual malathion and total organic carbon.

The effects of malathion treatment on numbers of heterotrophic bac-
teria, malathion co-metabolizing bacteria, and chitinoclastic bacteria
were determined by plating appropriately diluted water and sediment
samples (yields 30-100 colonies/plate) on marine agar, on marine agar
plus 50 yg/mf malathion, and on 1% chitin-agar (15), respectively.

After 3 days incubation at 28 C, the heterotrophic plates were checked
for hydrolysis of starch, lipid, and casein by the replica-plating
technique. This technique allows the screening of a large number of
colonies for numerous biochemical activities with a minimum of transfers.
A selected plate, the master plate, is pressed onto a sterile velveteen
pad stretched over a post. The pad is then used to replicate the master
plate colonies onto various media to check growth or biochemical activity
The master plates were chosen from appropriate dilutions such that they
contained 50-100 colonies per plate. Replica plates were incubated at

28 C for 4 days, at which time amylase, lipase, and casein producers
were enumerated. Media were prepared according to the methods of Colwell

& Wiebe (6).

ANALYTICAL METHODS

Gas-Liquid Chromatography (GLC)- Residual malathion and malathion break-
down products extracted from the various physio-chemical and microbial
degradation studies were quantitated by gas-liquid chromatographic
methods. Two instruments, a Varian-Aerograph Model 2100 equipped with a
tritium source electron-capture detector and a Tracor Model MTI-220 with
a Melpar flame-photometric detector, were employed. For the electron-
capture GLC determinations, two columns, one containing 2% OV-101 on

a


SECTION I
CONCLUSIONS

Malathion is rapidly degraded in vitro by salt-marsh bacteria to mala-
thion-monocarboxylic acid, malathion-dicarboxylic acid and various
phosphothionates as a result of carboxyesterase cleavage. In addition,
some expected phosphatase activity produces desmethyl-malathion, phos-
phomono- or -dithionates, and various 4-carbon dicarboxylic acids, as
well as corresponding ethyl esters.

In a simulated salt-marsh environment, malathion is degraded by the
indigenous bacterial community. Numbers of bacteria capable of degrad-
ing malathion in the presence of additional nutrients increase in the
sediments with increasing frequency of application and in the water
column with the increasing level of application. Numbers of bacteria
which degrade malathion as a sole carbon source appear to be linked to
the level of malathion treatment in sediments and the frequency of mala-
thion treatment in the water column. Malathion sole-carbon-degrading
bacteria do not appear to play a significant role in the dissipation of
malathion, comprising only about 10% of the portion of bacteria which
degrades malathion. It is believed that due to increased frequency of
treatment, increased numbers of malathion co-metabolizing bacteria cata-
lyze a more rapid dissipation of the compound, resulting in less selec-
tion of the sole carbon degraders.

The disappearence of malathion in the salt-marsh environment is influ-
enced by both chemical and biological degradation. Chemical hydrolysis
increases with increasing temperature and salinity, but at temperatures
below 26 C and salinities below 20 °/oo, these mechanisms are of lesser
importance than biological degradation.


Table 4. MICROBIAL DEGRADATION OF MALATHION.

Culture Degraded® Degraded”
number Morphology & gram reaction as S.C.S. with 0.2% Peptone
(4) (4)
1 (-) short rod 48 91
2 (-) coecoid rod 2 81
3 (-) medium rod 32 83
4 (-) medium rod 66 100
5 (+) short rod 36 94
6 (-) medium rod 2 82
7 (-) medium rod 1 83
8 (-) short rod 28 90
9 fungus 1 77
10 fungus 50 91
11 (-) slender rod 24 92
12 (-) medium rod 71 100
44 (+) slender rod 90 73
45 (-) short rod 72 100
47 (-) large ovoid rod 87 100

after 10 days incubation as sole carbon source. PAfter 5 days incubation
with 0.2% Peptone.

Table 5. CELL-MEDIUM DISTRIBUTION OF 14c FROM MALATHION®

Culture Percentage of total radioactivity
number Wet cells Supernatant

1 76 24

2 92 8

3 87 13

4 72 28

5 89 11

6 90 10

7 81 19

8 93 7

9 80 20
10 83 17
11 87 13
12 91

@[ncubation medium was seawater medium containing (46 ug/ml) !c-mala—
thion plus 0.2% peptone in all cases.

17


Ecological Research Series



23.

24,

25.

26.

27.

28.

29.

30.

Shafik, M. T, and H. F. Enos. Organophosphorus Pesticides Chemicals
in Human Blood and Urine. J. Agr. and Food Chem. 17(6):1186-1189,
Nov-Dec 1969.

Tagatz, M. E., P. W. Borthwick, G. H. Cook and D. L. Coppage.
Studies on Effects of Ground Applications of Malathion on Salt-
Marsh Environments in Northwestern Florida. Mosquito News.
34(3):38-42, Sept. 1974,

Tiedje, J. M. and M. Alexander. Microbial Degradation of Organophos—
phorus Insecticides and Alkyl Phosphates. Cornell University.
(Presented at Annual American Society of Agronomy Meeting, Washington,
D. C. 1967) 1 p.

U. S. Environmental Protection Agency Pesticide Study Series. The
Pollution Potential in Pesticide Manufacturing. Environmental
Protection Agency, Office of Water Programs. Washington, D. C.
TS-00-72-04, June 1972. p. 41-51.

Walker, W. W. Degradation of Malathion by Indigenous Soil Micro-
organisms. Ph.D. Dissertation, State College, Mississippi State
University. May 1972. 98 p.

Walker, W. W. and B. J. Stojanovic. Microbial Versus Chemical Degra-
dation of Malathion in Soil. J. Environ. Quality. 2(2):229-232.
1973a.

Walker, W. W. and B. J. Stojanovic. Acetylcholinesterase Toxicity of
Malathion and Its Metabolites. J. Environ. Quality. 2(4):474-
475, 1973b. ~

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

Arthrobacter Species. J. Environ. Quality. 3(1):4-10, 1974.

40


Table 2.

STRUCTURAL FORMULAE OF MALATHION, METABOLITES, AND B¥PRODUCTS

Name Chemical structure Name Chemical structure
Malathion CH3 - Q Zs Dimethyl phosphorodithioate, CH3 0. Zs
va potassium salt AY
CH, - 0 “s ct ~ COOC2Hs5 CH3- 0 “S-K
CH2- COC 2H
Malaoxon CH — oO yo Dimethyl phosphorothioate, CH3 - oO oe
Pp potassium salt mA
CH - 07 SS - CH - COO2HS cH3- 0 ‘o-xK
CHy- COO 7H5
Malathion
half-ester CH3 - Qo 3 Diethyl-maleate HO - CH ~ COOC2Hs5
(monocarboxylic ok |
acid, MCA) CH; - 0 Ss 7 - COOH CH2- COOC2H5
CH2- COOC2H5,
Malathion CH3 - Oo Mercaptosuccinate HS - CH - COOH
dicarboxylic aR
acid (DCA) CH3 - O s “ - COOH CH 2- COOH
CHy- COOH
O-Desmethyl CH3 - OL 23 Diethyl-succinate HO - CH - COOC2H5
malathion, Ax
potassium K -O S CH - COOC Hs, CH2- COOC2H5
salt (KDM) |
CHy- COOC2Hs



along the coast of the Gulf of Mexico to control mosquitoes carrying
Venezulan Equine Encephalomyelitis virus.

Malathion has been reported toxic to shrimp (Penaeus duorarum) at a
concentration of 0.5 ppm (EC 50° 48 hours) (5). Also, chronic expo-
sures of spot (Leiostomus xanthurus) to malathion (10 ug/L flowing
seawater for 26 weeks) significantly reduced brain acetylcholinesterase
activity (14). Field studies in marsh embayments of the Texas coast
tested the effects of aerial application of malathion in a concentration
(6 oz/acre) normally used for mosquito control on juvenile commercial
shrimp, Penaeus azteus (Ives) and Penaeus setiferus (Linn) (7). Shrimp
suffered mortalities ranging from 14 to 80 percent, whereas shrimp from
control areas suffered no deaths attributable to the pesticide. Reported
malathion residues were as high as 2.39 ppm in dead shrimp and 2.61

ppm in live shrimp and malathion was found at most test sites. Mala-
thion concentrations in water.samples from test sites ranged from 0.00
‘to 3.20 ppm (7). In another field test (24), no apparent adverse effects
of malathion on resident or confined animals were reported when mala-
thion was applied either as a thermal fog or as ultra-low-volume mist
(ULV) spray on a Florida salt-marsh.

The mode of action of malathion in insects, as well as other.animals, is
inhibition of acetylcholinesterase systems (8, 13). Blockage of this
enzyme system in mammals results in respiratory failure and in insects,
death probably results from the organisms's inability to move or to
feed due to loss of voluntary muscular coordination (13).

Malathion is an organophosphate insecticide (see Table 2 for structural
formula) synthesized primarily by the addition of dimethyl-dithiophosphoric
acid to the diethyl-ester of maleic acid (21). It contains 36.35%

carbon, 5.80% hydrogen, 9.38% phosphorous, 19.41% sulfur and 29.06%

oxygen and has a molecular weight of 330.36. Malathion has a solubility
of 145 mg/2 in distilled water and is miscible with many organic solvents
(3).

Like most organophosphates, malathion is relatively unstable in the
environment. Degradation of malathion occurs non-enzymatically, chiefly
by hydrolysis (10), and enzymatically, by way of two systems--phospha-
tases and carboxyesterases singly or in combination (Figure 1).

The two modes of degradation may be complementary in the breakdown of
the insecticide in soil (25). Seventy-two hours following application
in an estuary, malathion could not be detected at temperatures above
15.6 C (60F); hence, was reported to be completely degraded; however, no
mechanism of dissipation was proposed (9). Immediately after applica-
tion to a salt-marsh in another study, a high concentration of


An understanding of the interactions of microorganisms and malathion
required that we isolate and identify the principal metabolites. We
employed two cultures, which effectively utilized malathion: a sole-
carbon-degrader and a co-metabolizer. Both incubation mixtures containing
bacterial cells yielded 10 TLC-spots which were separated by the methods
described (Figure 6), as opposed to 4 or 5 spots (chemical-degradation
products) produced by the uninoculated control medium. By comparison
with standards, these metabolites were identified tentatively as (1)
monocarboxylic acid malathion, (2) dicarboxylic acid malathion (both
chemical and biological degradation products), (3) K-desmethylmalathion
(small amount produced by chemical degradation), and (4) a number of
compounds believed to be phosphodithionates (found only as metabolites),
for which no standards were available (Table 8).

Table 8. MALATHION DEGRADATION PRODUCTS

Incubation Time Products

Chemical Degradation:

2 days Malthion Monoester (MCA)
Malaoxon
7 days Malathion Diacid (DCA)

Desmethyl Malathion (KDM)

Bacterial Degradation:

2 days MCA, DCA, KDM
10 days Other unidentified products
(Phosphothionates)

Confirmation of the identification of these metabolites was made by

infrared analysis. Comparisons of the IR spectra with reference stan-

dards confirmed the identification of spot 1 as malathion (Figure 7)

spot 2 as malathion monocarboxylic acid (Figure 9) spot 3 as malathion-
dicarboxylic acid and 4 as 0-desmethyl-malathion (potassium salt, Figure

9). Identification of some major adsorption peaks are presented and, in

2 cases, possible compound structures were presented for spots 5-10

(Figures 10, 11, and 12). However, conclusive identification could not

be achieved with the small quantities of samples obtained. It should be
noted, however, that detoxification (loss of ability to inhibit acetylcholi-
nesterase) occurs only after at least 2 metabolic steps, i.e., to the
dicarboxylic acid. Walker and Stojanovic (29) reported acetylcholinesterase
inhibition by the monocarboxylic acid metabolite.

36


that spot 1 is malathion. The spectral tracing of malaoxon differs

in several ways from those of all other metabolites. Malaoxon

(Figure 8) was not present as a metabolite in this culture fluid.

The spectral tracing of TLC spot 2 is identical to that of malathion
half-ester (Figure 7), and it must be concluded that spot 2 was malathion

half-ester.

The spectral tracings of malathion dicarboxylic-acid and spot 3 are
compared in Figure 9. All peaks are identical, indicating the spot
represents that compound.

The IR spectral tracing of TLC spot 4 is illustrated in Figure 9. A
comparison of this spectrum with that of standard K-desmethyl malathion
indicates most peaks are accountable. The dissimilarities of the spec-

tra are probably due to a sparcity of sample and possible water inter-
ference, as indicated by the broad band at 3400 cm-l, The additional
adsorption peaks located between 200-1000 cmleannot be attributed

to desmethyl-malathion. The similarity of bands in the two spectra,

when combined with the thin-layer data, indicates that this metabolite is K-
desmethyl malathion.

Other metabolites eluted from spots on the thin-layer plates are shown

in Figures 10, 11, and 12. These compounds were found only in very

low concentrations and IR spectra were obtained by using an expanded
ordinate scale. Comparisons with available standards showed little similar-
ity to these metabolites; therefore, the compounds cannot be conclusively
identified.

Figure 10 shows the IR spectra of compounds from TLC Spots 5 and 6.
Some major adsorption bands were identified, and from these data, a
possible metabolite structure, "phosphorodithiosuccinate", was proposed
for spot 5. However, it is highly probably that the sample is not pure
and the compound is mercaptosuccinate, previously identified as a mal-
athion metabolite (18). No structure is proposed for TLC spot 6.

Figure 11 shows the IR spectral tracings for compounds from TLC spots
7 and 8. Most major bands were identified, but no structure could

be proposed. I believe that these samples contain phosphorous contam-
ination, rather than the phosphorous bonds indicated on the tracings.
Since the sample is in relatively low concentration, small amounts of
contaminants would be greatly exaggerated by the expanded mode on the
IR instrument. From previous experience (18, 20, 21), I would expect
a number of thioate derivatives from phosphatase and demethylation
reactions but not all phosphorothioates as the spectra indicate.

Figure 12 shows the IR spectra for compounds from TLC spots 9 and 10.

Although there is less evidence for phosphorous bonds, some evidence
is present (490-515 cml). Assuming this band to be contamination,

24


Clase
DCA
DQC
DEE
DDT
EC

GLC
IR
KDM
MCA
MCM
M. lb. AI.
nd
Plase
ppb
ppm
TLC
ULV
°/oo

SECTION VII
ABBREVIATIONS AND SYMBOLS

Carboxyesterase

Dicarboxylic-acid-malathion
Dibromoquinone chloride

Diethyl ether
Dichlorodiphenyltrichloroethane
Effective concentration required to affect
50% of the experimental population
Gas liquid chromatography

Infrared spectroscopy

Potassium desmethyl malathion

Monocarboxylic acid malathion

Malathion co-metabolizers

Million pounds active ingredient
Not detectable

Phosphatase

Parts per billion (ig/kg, 1g/liter)
Parts per million
Thin-layer-chromatography

Ultra-low volume

Parts per thousand

41


€@

MALAOXON

CHy- 0. 10 sym
LPS Q astn
CH3-0 = S- CH- C-0-C,Hs CH
1
CH,-C-0-C,Hs
o
0

_ abo nr en - ie +4

waAVE NUMBER (n*)

Figure 8. Infrared spectral tracing of malaoxon, with representative
adsorption bands indicated.


ACKNOWLEDGEMENT

Aid of the following GBERL personnel is gratefully acknowledged:

Ms. Lynda Kiefer, for assistance in culture work and Mr. Scott Cassidy
in artificial ecosystem studies; Mrs. Chiara Shanika for assistance in
chemical analyses and Ms. Pamela Bahner for careful typing of the manu-
script. Dr. William W. Walker, Gulf Coast Research Laboratory, Ocean

Springs, Mississippi provided stimulating discussions and reviewed the
manuscript.

vi


SECTION IV
RESULTS

PHYSICO-CHEMICAL DEGRADATION

As shown in Figure 4, the hydrolytic degradation of malathion increases
with increasing salinities. The data are presented as ranges and
averages of residual malathion detected at various lengths of incu-
bation. Four replications were employed for each salinity treatment,
one light- and one dark- incubated sample from each temperature.
Although malathion degradation in distilled water was observed to

a limited extent, the rate of degradation was much slower than in
seawater. No effect of light was observed under the test conditions,
but temperatures above 26.7 C (80 F) increased the rate of degradation.
Breakdown products from the chemical degradation of malathion were
identified as malathion monocarboxylic acid (detected at two days)

and malathion dicarboxylic acid (detected at seven days) by flame
photometric gas chromatography by comparing their retention times

with those of standards. Although malaoxon was detected, it did not
increase in quantity over that found in the stock malathion.

MICROBIOLOGICAL DEGRADATION

Indirect evidence for malathion utilization as a carbon source by
bacterial species was shown by growth studies using marine broth 2216
plus malathion. Dry-weight determinations were performed as indicated
in the methods section of this report and growth stimulation by mala-
thion was apparent from the greater cell mass produced (Table 3).
However, the increased cell mass could not be due to malathion carbon
alone, since the total carbon available was only about 8 mg. No
explanation for the increased cell mass can be given at this time.

Table 3. MICROBIAL GROWTH ON MALATHION PLUS NUTRIENTS.

Dry weights (mg) after 10 days incubation

Culture b
number Inoculum Marine® Marine + Malathion
1 40 127 134
12 32 115 108
44 58 145 150
45 35 125 165
47 c 65 155 185
Mixture 70 250 200

Marine 2216 broth. Marine 2216 broth plus 200 yg Malathion/ml medium.
Five isolates in equal proportions.

Estuarine bacteria isolated from Range Point salt-marsh sediment by
malathion- enrichment techniques were tested for biodegradative ability
by incubating a washed- cell suspension containing approximately 10
cells/m£ in sea-salts medium containing 46 ug/m? malathion and in the
same medium with 2% peptone as a supplementary energy source. Residues
analyzed by GLC after 10-days incubation are expressed in Table 4 as

14


MASTER PLATE (%)

Figure 14.

108

z
m
3
| | “ 3
z
10° S
z=
w
N
a
3
; 8%
' 108
1 '
4 + |
dios
wan nn own ne ef a
ee Le

10 5 20 25 30
DAYS

Heterotrophic bacteria from sediments of artificial salt-
marsh ecosystems presented as heterotrophs/gm of wet
sediment. Numbers of heterotrophic bacteria which are
malathion sole-carbon—degraders (dark) or malathion co-
metabolizer (shaded) given as a percent of the "master-
replica-plate". Dashed-line indicates the 30-day ayer-
age for each category. Times of malathion treatment
indicated by arrows; controls are untreated.

31


Numbers of MCM's increased significantly with frequency of treatment
(0, 10, 20 days; o= 0.1) at both treatment levels. Although MCM
bacterial numbers were not significantly different at increased levels
of treatment (control, 1X and 10X; a>0.1); in treated vs. control
systems, MCM's increased from 49% on the control to 72% and 73% of the
heterotrophic bacteria on the master plate from the 1X and 10X treated
systems, respectively.

Malathion increased the number of MCM's and SCD's in the water column
at increased treatment levels but not with frequency of treatment. On
the other hand, malathion application to artificial environments in-
creased both MCM's and SCD's in the sediments with frequency of appli-
cation levels. No significant changes in heterotrophic activities were
noted with respect to amylase, lipase, chitinase, or proteinase pro-
duction by the bacteria in the malathion-treated systems at either
application rate, as compared to the untreated system.

To compare the effects of a recalcitrant insecticide with those of a
biodegradable insecticide (malathion) on the microbial ecosysten,
similar artificial environments were challenged with mirex. The data
are presented in Figures 15 and 16. No differences among control and
treated systems were noted for heterotrophic bacterial numbers in

water or sediment samples. However, some decrease in numbers of "mirex-
tolerant" bacteria (i.e., bacteria which grew on nutrient medium plus

10 wg mirex/m2 shaded area) was noted, especially at the higher treatment
level. This decrease was more pronounced after the initial treatment and
appeared to recover and be unaffected by the latter treatment. No
mirex-degrading bacteria were selected by the replica-plating procedure.
The overall effect of mirex on the microbial ecosystem of an artificial
salt-marsh environment bears little resemblance to that of the "softer"
organophosphate insecticide malathion. No stimulatory or inhibitory
effect, other than the initial treatment, was noted when the artificial
ecosystem was treated with mirex.

32


Good separation of the malathion and its metabolites was achieved

on a two-dimensional TLC. Figure 6 shows typical chromatograms

from microbial degradation and control media (malathion but no cells).
Four compounds - the two carboxylic acids, malathion, and K-desmethyl-
malathion were tentatively identified by comparison with known com-
pounds. The values shown in Table 7, match reference materials well.
Other metabolites, indicated by numbers 5, 6, 7 and 8, were probably
phosphothionates, judging from published R{ values (18), but no standards
were available for comparison. With thé exception of spot number 8,
most of the latter compounds were not present on the control plate and,
therefore, are probably microbial metabolites. No differences in
metabolites could be detected between the two cultures since all com-
pounds visible in culture 8 extracts were also visible in those of
culture No. 45.

Table 7. TLC — R{ VALUES FOR MALATHION AND METABOLITES.

Compound Solvent2 Solventb
Malathion 0.93 0.95
Malaoxon 0.80 0.77
KDM 0.63 0.23
MCA 0.71 0.68
DCA 0.28 0.31

aHexane : Acetic Ac : DEE (2: 2: 1)
bpenzene : Acetic Ac : DEE (75 : 15 : 10)

Figures 7 and 8 show the infrared spectral tracings of analytical

grade malathion and malaoxon with major adsorption bands indicated.
Explanations of infrared spectral analysis of malathion and

metabolites are taken from Walker and Stojanovic, (29), and Jones (17).
Adsorption peaks at 2960 and 1450 reciprocal centrimeters (cm-l) repre-
sent asymmetrical C-H stretches, whereas those at 2940 and 1375 cm! re-
present symmetrical C-H stretches. Bands at 2550 cm-l represent S-H
stretch bonds. A strong band at 1730 cm7! represents a C=0, whereas

a band 1000-1200 cm7! indicates C-0 (1170 cm-l), Methyl and C=C groups
adsorb at 1380 and 1640 cml, respectively. The band at 1010 cm! indicates
P-0-C bonding and C-C stretch bonds are indicated at 1100 cm-l, The
band at 655 cm-l represents P=S bond and the weak peaks at 515 and 490
cm-l possibly represent P-S.

The spectral tracing of TLC spot 1, when compared with that of malathion
in Figure 7, appears to be the same compound. Therefore, it was concluded

20


ABSTRACT

Malathion is rapidly degraded in vitro by salt-marsh bacteria

to malathion-monocarboxylic acid, malathion-dicarboxylic acid and
various phosphothionates as a result of carboxyesterase cleavage.
In addition, some expected phosphatase activity produces desmethyl-
malathion, phosphothionates, 4-carbon dicarboxylic acids, and cor-
responding ethyl esters.

In a simulated salt-marsh environment, malathion is degraded by

the indigenous bacterial community. Numbers of bacteria capable of
degrading malathion in the presence of additional nutrients increase
in the sediments with increasing frequency of application and in the
water column with the increasing level of treatment. Numbers of
bacteria which degrade malathion as a sole carbon source are linked to
the level of treatment in sediments and the frequency of treatment in
the water column; however, these bacteria do not appear to play a
significant role in the dissipation of malathion. I believe that
frequency of treatment, increases numbers of malathion co-metabolizing
bacteria which catalyze a more rapid dissipation of the compound,
which results in fewer sole carbon degraders.

The disappearance of malathion in the salt-marsh environment is
influenced by both chemical and biological degradation; however, at
temperatures below 26 C and salinities below 20 °/oo, chemical mech-
anisms appear to be of less importance than biological degradation.

This report was submitted in fulfillment of ROAP 10AKC, Task 006.by
the Gulf Breeze Environmental Research Laboratory under the sponsor-
ship of the Environmental Protection Agency. Work was completed as
of December 1973,

di


11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

Guerrant, G. 0., L. E. Fetzer, Jr. and J. W. Miles. Pesticide
Residues in Hale County, Texas Before and After Ultra-Low Volume
Aerial Application of Malathion. Pestic. Monit. J. 4(1): 14-20,
June 1970. ~

Gunther, F., A. and R. C. Blinn. Persisting Insecticide Residues in
Plant Materials. Ann. Rev. Entomol. 1:167-180, 1956.

Hassall, K. A. Pesticides. In: Vol II, World Crop Protection.
Cleveland, Ohio. Chemical Rubber Co., 1969. 250 p.

Holland, H. T. and J. I. Lowe. Malathion: Chronic Effects on
Estuarine Fish. Mosquito News. 26 (3):383-385,September 1966.

Hood, M. A. and S. P. Meyers. Biodegradation of Chitin in Louisiana
Salt Marshes. Louisiana State University. Abstracts Ann. Meeting
Amer. Soc. Microbiol., Miami Beach, May 6-11, 1973 p. 49.

Jaglan, P- S. and F. A. Gunther. A Thin-Layer Chromatographic
Procedure for Separating Desmethyl Methyl Parathion (0-methyl
0-p-nitrophenyl Phosphorothionate) and its S-isomer (S-methyl
O0-p-nitrophenyl Phosphorothionate). Bull. Environ. Contam. Toxicol.
5(1):47-49, 1970.

Jones, R. N. Infrared Spectra of Organic Compounds: Summary
Charts of Principal Group Frequencies. National Research Council,
Ottawa, Canada. NRS Bulletin No. 6. 1959.

Kadoum, A. M. Thin-Layer Chromatographic Separation and Colorime-
tric Detection of Malathion and Some of Its Metabolites from Stored
Grains. .L. Agr. Food Chem. 18(3):542-543. 1970.

Konrad, J. G., G. Chesters and D. E. Armstrong. Soil Degradation
of Malathion, A Phosphorodithioate Insecticide. Soil Sci. Soc.
Amer. Proc. 33(2):259-262, 1969.

Matsumura, F. and G. M. Boush. Malathion Degradation by Trichoderma
Niride and by a Pseudomonas Species. Science. 153(3741):1278-1280,
1966.

Melnikov, N. N. Chemistiry of Pesticides. Residue Reviews, 36:1-480,
1971.

Odum, E. P. Fundamentals of Ecology. Third Edition. Philadelphia,
W. B. Saunders Co., 1971. p. 352-363.

39


Malathion was rapidly degraded in vitro by salt-marsh bacteria.

When applied to a simulated salt-marsh environment, malathion was
degraded by the indigenous bacterial community. In a simulated
environment, numbers of bacteria capable of degrading malathion in
the presence of additional nutrients increased in the sediments with
the increasing frequency of application, and in the water column
increased, with the level of treatment (Figures 13 and 14). On the
other hand, numbers of bacteria which degrade malathion as ‘a sole-
carbon-source (SCD's) were linked to the level of treatment in
sediments and the frequency of treatment in the water column. In
either water or sediments, SCD's did not appear to play a significant
role in the degradation of malathion, comprising only about 10% of
the portion of bacteria which degrade malathion. It is possible
that bacteria which co-metabolize malathion (MCM's), which increased
with treatments, catalyzed a more rapid degradation of the compound,
resulting in decreased selection of SCD's. At the same time, the
increase of SCD's in the sediment may have been a result of more
malathion reaching the sediments when higher levels were applied,
probably due to precipitation of the chemical to the sediments.

Malathion, when applied to salt~marsh environments, should rapidly
degrade by chemical and biological mechanisms. Chemical hydrolysis

is stimulated by increasing temperature and salinity, but would appear

to be of lesser importance in overall degradation. Microbial degradation
is more rapid than chemical degradation, and is apparently mediated by

a large proportion of salt-marsh bacteria. In vitro, salt-marsh bacteria
metabolize malathion to malathion half-ester, malathion dicarboxylic
acid, K-desmethyl malathion, and several other unidentified metabolites.
Biological degradation activity appears to be associated with carboxy-
esterase activity, with delayed demethylation and with phosphatase
activity. According to one previous publication (29), malathion half-
ester (MCA) is the only acetylcholinesterase inhibitor among the meta-
bolites. I expect, that, in a complex salt-marsh environment, MCA

would be further degraded rapidly to other non-toxic metabolites. It
appears that MCM’s assume the major role in degradation of malathion

and SCD's are active only when application rates are excessive. I
conclude that malathion, when applied to salt-marsh environments, is
rapidly degraded by the indigenous bacterial community, with no

apparent adverse effect on the microbial community.

37


SECTION III
MATERIALS AND METHODS

MATERIALS

Malathion, malaoxon, and the carboxylic acid products were obtained from
American Cyanamid Co., Princeton, N. J. 14¢-methoxy-malathion was
received from Mallinckrodt, St. Louis, MO. Analytical-grade standards
for chromatography were obtained from EPA, Pesticides Reference Standards
Section, Chemistry Branch, Washington, D.C; standards for IR analyses
were synthesized by the EPA, Southeast Environmental Research Laboratory,
Athens, GA.

Microbiological media used were purchased from Baltimore Biological
Laboratory, Baltimore, Md. All organic solvents used for pesticide
extraction were of nanograde quality (Mallinckrodt Chemical Works, St.
Louis, MO) and others were of spectroanalyzed grade (Fisher Scientific
Co., Fair Lawn, N. J.).

EXPERIMENTAL METHODS

Preparation of malathion-utilizing bacteria- To obtain bacteria for
study, sediment and water samples were collected from a salt-marsh on
Santa Rosa Island, Florida (Fig. 2). The area was considered free of
malathion contamination (not previously sprayed by Escambia County
Mosquito Control Department), the water temperature was 28°C, and the
salinity was 20 °/oo (parts per thousand). Duplicate sets of both water
(10 m 2) and sediment (10 g) samples were inoculated into 250 m2
Erlenmeyer flasks that contained 90 mf Zobell's Marine 2216 Broth (1)

or aged seawater (Rila Marine Mix, Teaneck, N. J. of 20 °/oo salinity
and malathion was added at the rate of 100 mg/£ in 1.0 mé acetone. The
250 mf Erlenmeyer flasks were incubated on a rotary shaker at 28°C for
30 days. Ten-milligram aliquots of malathion in 0.5 mf acetone were
added to the flasks every 7 days to maintain a high pesticide concentration
and adequately large carbon source for malathion-utilizing microbes.
Samples from each flask were streaked on seawater agar and Marine 2216
agar enriched with malathion (100 mg/liter). Colonies were picked from
these plates for pesticide-utilization tests (See Microbial Degradation
Studies).

Physico-Chemical Degradation- The physico-chemical stability of
malathion was tested in sterile seawater solutions. The ranges

of light, temperature, and salinity were selected to cover

only those employed in microbial degradation tests. Flasks that
contained 50 m2 sterile Rila Sea-Salts adjusted with distilled water
to salinities of 0, 10, 20 and 30 °/oo were inoculated with sterile



malathion (filter-sterilized, 0.2u) at a concentration of 1.0 mg/L.
Duplicate flasks of each salinity were incubated at 20 C or 28 C in
continuous darkness (wrapped in aluminum foil) under 6,000 lux illumi-
nation from Growlux” fluorescent tubes with alternating 12-h periods
of light and darkness. Duplicate flasks of each kind were removed and
extracted every two days. Malathion concentration was determined as
described in Gas-Liquid Chromatography (see below).

Microbial Degradation Studies- Selected isolates from enrichment cultures
were screened qualitatively for biodegradation of malathion. Two-tenths
milliliter of an 18-hour culture grown in 10 mf of marine 2216 broth
was inoculated into 10 mt of sterile sea-salts medium containing
malathion (100 ug/mf) and incubated for 5 days at 28 C on a rotary
shaker. Cells and medium were extracted without separation with 10 mf
petroleum ether in the incubation tube and analyzed for residual
insecticide by gas-liquid chromatography. Ability of each culture

to degrade malathion was then compared with that of the control (sterile
seasalt medium, malathion, but no cells). For quantitative determina-
tions, isolates which exhibited greatest ability to degrade malathion
were tested by inoculating approximately 10° cells, washed with sterile
sea-salts solution from an 18-hour agar-slant culture, into sea-salts
medium containing malathion (46 ug/ml), with or without 0.2% peptone.
The cultures were incubated for 10 days at 28 C on a rotary shaker
before extracting malathion for analysis by gas-liquid chromatography.

Bacteria shown by the quantitative tests to be most efficient in the
utilization of malathion as a sole-carbon source were tested for the
ability to incorporate “*C derived from +*C-methoxymalathion.

Cultures grown in marine broth were diluted 1:100 into fresh seawater
medium containing 200 ug/mé malathion and 0.1 uci-l4¢ in Biometer

flasks (Bellco, Inc.). Reaction vessels were incubated for 14 days at
28 C and 14coy sampled daily for !4c0y evolution. Radioactive C0,

was recovered by adding 1.0 mf alcoholic-hyamine solution (1M, Packard
Instruments) to the side-arm of the Biometer flask, incubating 5 minutes,
and removing the COj-hyamine solution with a syringe. The hyamine
solution was added to toluene-scintillation cocktail (10 m) and analyzed
on a liquid-scintillation counter.

After 14 days, the culture medium was treated with 0.5 mt of 0.2%
trichloracetic acid (final pH = 2) and immediately extracted twice with
petroleum ether. The solvent phase was washed with 20 mé 0.5 M potas-
sium phosphate buffer, pH 7. All fractions were assayed radiometrically
to determine extent of malathion breakdown. The ether phase contained
malathion and some degradation products. The aqueous fractions at pH

2 contained the carboxyesterase products and those at pH 7 contained
phosphatase and other hydrolysis products (20). The ether phase and
ether:acetone (1:1) extracts of the pH 2 water were analyzed further by
gas-liquid chromatography.


EPA~660/3-75-035
JUNE 1975

MICROBIAL-MALATHION INTERACTION IN ARTIFICIAL
SALT-MARSH ECOSYSTEMS

Effect and Degradation

by

Al W. Bourquin
National Environmental Research Center
Gulf Breeze Environmental Research Laboratory
Sabine Island
Gulf Breeze, Florida 32561

Program Element Number 1EA077
ROAP Number 10AKC
Task Number 006

NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97730

For sale by the Superintendent of Documents, U.S. Government
Printing Office, Washington, D.C. 20402


MASTER PLATE (s)

0 a eel tir ietieiieetierlinglies cheer

Jos

83%
ee ee ee ee eh es
ee

%

——K— Se

es

Ww / S$HdOwLOUaLIH

Figure 13.

5 10 8 20 25 30
DAYS

Heterotrophic bacteria from water column of artificial salt-
marsh ecosystem presented as heterotrophs/ml (line),

and number of heterotrophic bacteria which are malathion
sole-carbon-degraders (dark) or malathion co-metabolizers
(shaded), given as a percentage of the "master-replica-plate".
Times of malathion treatment are indicated by arrows; con-
trols are untreated. Dashed-line indicates the 30-day aver-
age for each category.

30


Table 6. RADIOMETRIC AND CHEMICAL ANALYSES OF MICROBIAL GROWTH ON
14¢ - MALATHION.

Radiometric analysis - Percentage of total radioactivity remaining in
expended medium

Products of

Culture No. Malathion Carboxyesterase Phosphatase

44 25.7 36.8 37.5

45 8.3 48.2 43.5

47 6.5 61.3 32.2
Mixture 7.4 87.2, 5.4
Control 10.5 82.4 7.13

Residue Analyses- flame photometric GLC analysis - ppm

Recovery (ppm) Percentage
Culture No. Malathion MCA DCA Total recovery
44 19 85 40 144 72
45 75 159 8 172 86
47 75 80 23 108 54
Mixture 75 il 55 75 38
Control* 9 168% 7? 184 92

'Sterile medium plus 14C - malathion, products are due to chemical
breakdown.

Figure 5 shows the results of four different cultures incubated with
seawater-malathion media. The results are plotted as percentage
malathion remaining at time of sampling. Parent compound remaining in
control flasks at each sampling time (i.e., percentage remaining after
chemical degradation) shown as percentage of the original concentration,
are presented in parentheses above each sampling day. Two cultures

(No. 1 and 12) readily utilized malathion, whereas two cultures (No. 6
and 9) incubated for 10 days showed little degradation of the molecule
beyond that of chemical degradation. However, the latter cultures
readily metabolized the insecticide when 0.2% peptone was added.
Microbial degradation, with or without additional nutrient, is dependent
upon the type of microbe utilized, but is faster than chemical deg-
radation, as observed in these cultures.

ISOLATION AND IDENTIFICATION OF MICROBIAL METABOLITES
Spent sea-water malathion medium from cultures 8 and 45, with and without

added nutrient, was extracted, concentrated, and assayed by thin-layer
chromatography to separate and tentatively identify degradation products.

18


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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:

Environmental Health Effects Research
Environmental Protection Technology
Ecological Research

Environmental Monitoring
Socioeconomic Environmental Studies

afWN-

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.

EPA REVIEW NOTICE

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.


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

3. RECIPIENT'S ACCESSION NO.

1, REPORT NO, 2.
EPA-660/3-75-035

4. TITLE AND SUBTITLE 5. REPORT DATE
Microbial-Malathion Interactions in Artificial Salt- March 1975 (Issue)

Marsh Ecosystems: Effect and Degradation 6. PERFORMING ORGANIZATION CODE

I. PERFORMING ORGANIZATION REPORT NO.

7, AUTHOR(S)
Al W. Bourquin GBERL 236

i9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT NO,
U.S. Environmental Protection Agency 1EA077
Gulf Breeze Environmental Research Lab 11, CONTRACT/GRANT NO.

Sabine Island, Gulf Breeze, FL 32561

12, SPONSORING AGENCY NAME AND ADDRESS 13, TYPE OF REPORT AND PERIOD COVERED
U.S. Environmental Protection Agency Interium; FY-1974

National Environmental Research Center 14, SPONSORING AGENCY CODE
Office of Research and Development
Corvallis, Oregon 97730

75. SUPPLEMENTARY NOTES

16, ABSTRACT

Malathion is rapidly degraded in vitro by salt-marsh bacteria to malathion-mono-
carboxylic acid, malathion-dicarboxylic acid and various phosphothionates as a result
of carboxyesterase cleavage. In addition, some expected phosphatase activity pro-
duces desmethyl-malathion, phosphotionates, 4-carbon dicarboxylic acids, and cor-
responding ethyl esters.

In a simulated salt-marsh environment, malathion is degraded by the indigenous
bacterial community. Numbers of bacterial capable of degrading malathion in the
presence of additional nutrients increase in the sediments with increasing frequency
of application and in the water column with the increasing level of treatment.
Numbers of bacteria which degrade malathion as a sole carbon source are linked to
the level of treatment in sediments and the frequency of treatment in the water
column; however, these bacteria do not appear to play a significant role in the
dissipation of malathion. I believe that frequency of treatment, increases numbers
of malathion co-metabolizing bacteria which catalyze a more rapid dissipation of the
compound, which results in fewer sole carbon degraders.

The disappearance of malathion in the salt-marsh environment is influenced by both
chemical and biological degradation; however, at temperatures below 26 C and salini-
ties below 20 °/oo, chemical mechanisms appear to be of less importance than biolo-
gical degradation.

17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS, Ib. IOENTIFIERS/OPEN ENDED TERMS |c. COSATI Field/Group
Microbial Ecology Artificial Ecosystems/
Pesticide - Microbial Interaction Malathion-Microbial Int¢r-
Pesticide Degradation actions
Malathion Degradation Malathion Degradation

Environmental Microbiology

18. DISTRIBUTION STATEMENT 19. SECURITY CLASS /This Report) 21, NO. OF PAGES
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EPA Form 2220-1 (9-73) # U.S. GOVERNMENT PRINTING OFFICE: 1975~699-033 113 REGION 10



9@

4000 —— a Sab + a
2000 1000 ~~ 200
B. WAVE NUMBER = (cM)
i
SPOT 0.6 |
CH ty
(200 CC POC pg
} + ri } ‘a +
4000 3000 2000 1000 m
WAVE NUMBER (cM)
Figure 10. Infrared spectral tracings of (a) extract from spot 5 with possible structure in

brackets (b) extract from spot 6.


SECTION V
DISCUSSION

Malathion, when applied to aqueous environments, was rapidly dissipated
by both chemical and biological mechanisms. Like most organophosphate
insecticides, malathion did not persist in aqueous environments, except
under special conditions; i.e., adsorbed to Juncus grass. Even then
malathion would be degraded within a short time.

Previous reports have indicated that malathion was quite stable under
neutral or acid pH conditions and susceptibility to hydrolysis in-
creased with increasing alkalinity (11, 28). Likewise, malathion is
thermostable at temperatures of 21 C (70 F) and below, but rapidly
dissipates at temperatures of 27-32 C (80-90 F) (11). The extent of
chemical degradation in vitro was extensive (Figure 5), malathion
having a half-life of approximately 92-96 h. I believe that chemical
degradation was influenced by both temperature (27 C) and alkalinity.
Malathion degradation increased with increasing alkalinity of seawater.
The stability of most organophosphate insecticides in sterile seawater
was a factor previously overlooked by investigators of persistence of
chemicals in the estuary. Dr. W. W. Walker (personal communication
Gulf Coast Research Lab., Ocean Springs, MS.) reported that a differ-
ential stability exists among organophosphates in sterile seawater
environments. Further investigations into the relationship of mech-
anisms of chemical degradation to biological mechanisms are needed.

Although chemical degradation is important in understanding the extent
and rate of malathion dissipation from the environment, biological
degradation appears to be as significant in salt-marsh environments
as that reported in soils (28).

We isolated 15 bacterial cultures from salt-water environments which
degraded malathion in vitro within 10 days. Eleven cultures degraded
the insecticide as a sole-carbon-source, whereas all 15 isolates de-
graded the compound within 5 days when an additional carbon source was
added. Little malathion carbon contributed to the cell mass of the
cultures tested, as evidenced by radioactive-carbon assay (Table 6).
However, such contribution could have occurred only by heterotrophic
CO,-fixation or by single-carbon metabolism, because the 14c-tabel

was on the methoxy-group of the malathion molecule. Other cell-mass
contribution could have been from the ethyl-group, due to carboxy-
esterase cleavage to form the malathion-carboxylic acids; however, the
ethyl-radical was not radio-labelled and the resulting cell-mass would
not have been radioactive. Most biological activity appears to be
associated with an effective carboxyesterase system that causes early
breakdown to the acids, with little contribution to the cell's carbon

skeleton.

35


14

DIMENSION

FIRST
(Hexane: Benzene: Acetic Acid) (40:40

20)

STANDARDS

MCA-M

Malathion

Malaoxon

CULTURE EXTRACT

2 Malathion

‘@ MCA-M

GONTROL-NO CELLS

Qoca-m (secam
8
@QKom : ok KOM
Origin TT Origit oN Qin
SECOND DIMENSION
(Hexane: Acetic Acid: Ether) (75:15:10)

Figure 6,

Thin-layer chromatogram of malathion-seawater medium with

(culture extract) and without (control) an inoculum of

bacterium # 45.

parison of spots.

Reference standards are included for com-



malathion was observed, but progressively lower concentrations were
observed at 24-, 33-, and 48-hours after application (7). Similarly,
in another study in which malathion was applied to a marsh (24), insec-
ticide concentrations in marsh water immediately after fogging and ULV
application were 5.2 and 0.49 ppb, respectively, but only trace amounts
(0.1 - 0.3 ppb) persisted as long as one day. This rapid dissipation
of parent compound is probably a consequence of both chemical and bio-
logical degradation. Although malathion is readily degraded chemically,
it also serves as a substrate for microbial degradation (18, 28), and
several species of soil fungi and bacteria capable of attaching mala-
thion and some of its breakdown products have been isolated (20). Sim-
ilarly, in a study designed to assess the relative importance of chemical,
as opposed to microbiological, degradation of malathion in several
Mississippi soils, Walker and Stojanovic (1973a) found microbial degra-
dation to be the chief mechanism of insecticide dissipation in all soils
tested.

Despite numerous studies of the fate and effects of malathion in

estuarine salt-marsh ecosystems or in soil systems, little information

is known of its microbiological fate or its effects on estuarine bacteria
and fungi. The study reported here had three primary objectives: (1) to
isolate by enrichment culture bacteria capable of readily metabolizing
malathion, (2) to isolate and identify the major metabolites of malathion
resulting from this microbial degradation, and (3) to determine fate of
malathion in a simulated salt-marsh environment and its effect on bacteria.


SANTA ROSA SOUND

E.PA.
Laboratory

Range Pt.
~

SANTA ROSA ISLAND

GULF OF MEXICO

STATUTE MILES

os oe oe

y
1 2 ie) 1 2

Figure 2. Santa Rosa Island, near Gulf Breeze, Florida. Arrow
indicates Range Point salt~marsh.