Ecological Research  Series
Microbial-Malathion  Interaction  in
Artificial  Salt-Marsh  Ecosystems
Effect and  Degradation
Effect and Degradation
                                   National Environmental Research Center
                                     Office of Research and Development
                                     U.S. Environmental Protection Agency
                                            Corvallis, Oregon 97330

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                      RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development,
U.S. Environmental Protection Agency, have been grouped into
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          1.   Environmental Health Effects Research
          2.   Environmental Protection Technology
          3.   Ecological Research
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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
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                         EPA REVIEW NOTICE

This report has been reviewed by the National Environmental
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of trade names or commercial products does not constitute endorsement
or recommendation for use.

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                                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

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                               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.
                                  11

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                            CONTENTS


Section

I       CONCLUSIONS

II      INTRODUCTION

III     MATERIALS AND METHODS
           Materials
           Experimental Methods
           Analytical Methods
           Statistical Analysis

IV      RESULTS
           Physico-Chemical Degradation
           Microbiological Degradation
           Isolation and Identification of
               Microbial Metabolites
           Artificial Ecosystem Studies

V       DISCUSSION

VI      REFERENCES

VII     ABBREVIATIONS AND SYMBOLS
                                lii

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                            LIST OF FIGURES
Figure                                                       Page


  1      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
 11      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 column 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

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                            LIST OF TABLES
Table
  1       Estimated U. S. Insecticide Production, 1971        2
  2       Structural Formulae of Malathion, Metabolites       4
          and By-Products
  3       Microbial Growth on Malathion Plus Nutrients       14
  4       Microbial Degradation of Malathion                 17
  5       Cell-Medium Distribution   C from Malathion        17
  6       Radiometric,and Chemical Analyses of Microbial     18
          Growth on   C-Malthion
  7       R(
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                             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 Banner 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

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                                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.

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                                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-(l,2-Dicarbethoxyethyl)-0, 0-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 Ibs.,
 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,  1971a
    Chemical                     Number
     group                     compounds                  Production
	(Mill.  Ibs. A.I.)

Chlorinated hydrocarbons           13                          158

Carbamate                           5                           64

Organophosphate                    23b                         148b

Others                             51                           23

Total, all synthetic organic
insecticides, miticides, nematocides	    393	
aFrom U.S. EPA Pesticide Study Series, 1971  (26)
°Malathion = 24% of total organophosphate production

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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.-Q, 48 hours) (5).  Also, chronic expo-
sures of spot (Leiostomus xanthurus) to malathion (10 yg/£ 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/£ 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

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         Table 2.  STRUCTURAL FORMULAE OF MALATHION, METABOLITES, AND BYPRODUCTS
Name           Chemical structure          Name                         Chemical structure


Malathion      CH3 - 0  ^S                 Dimethyl phosphorodithioate, CH3   0  /S
                       P                   potassium salt                       P
               CH3 - 0  XS - CH - COOC2H5                               CH3 - 0  XS - K

                             CH2- COOC2H5

Malaoxon       CH3 - 0  /O                 Dimethyl phosphorothioate,   CH3 - 0  S
                       P                   potassium salt                       P
               CH3 - 0/XS - CH - C002H5                                CH3 - 0  X0 - K
                             CH2- C002H5

Malathion
half-ester     CH3 - 0  ^S                 Diethyl-maleate              HO - CH - COOC2H5
(monocarboxylic        P                                                      |
acid, MCA)     CH3 - Q" XS - CH - COOH                                       CH2- COOC2H5

                             CH2- COOC2H5

Malathion      CHo - 0  /S                 Mercaptosuccinate            HS - CH - COOH
                      \ ,<''                                                     i
dicarboxylic           P                                                      |
acid (DCA)     CHo - 0  XS - CH - COOH                                       CHo- COOH
                              I
                             CH2- COOH

0-Desmethyl    CH3 - 0  /S                 Diethyl-succinate                 HO - CH - COOC2H5
malathion,             P                                                           |
potassium       K  - 0   S - CH - COOC2H5                                         CH2- COOC2H5
salt (KDM)                    |
                             CH2- COOC2H5

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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.

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Figure 1.  Enzymatic cleavage of malathion.  Products of phosphatase

         (P'ase) and carboxyesterase (C'ase).  Products of malathion

         cleavage are given in Table 2.
     H,C-O      S
       3       -V  //
     HC-0
           P'ase                      C'

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                                SECTION  III
                          MATERIALS AND METHODS
MATERIALS
Malathion, malaoxon, and  the  carboxylic acid products were obtained from
American  Cyanamid  Co., Princeton, N. J.   l^C-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 £) and sediment (10 g)  samples were inoculated into 250 m£
Erlenmeyer flasks  that contained 90 m£ 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 m£ acetone.  The
250 m£ Erlenmeyer  flasks were incubated on a rotary shaker at 28°C for
30 days.  Ten-milligram aliquots of malathion in 0.5 m£ 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 m£ sterile Rila Sea-Salts adjusted with distilled water
to salinities of 0, 10, 20 and 30 °/oo were inoculated with sterile

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               SANTA   ROSA    SOUND
                       SANTA   ROSA   ISLAND
       GULF     OF     MEXICO
    STATUTE MILES
 i_r
Figure 2.  Santa Rosa Island, near Gulf Breeze,  Florida.
          indicates Range Point salt-marsh.
Arrow

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malathion  (filter-sterilized,  0.2y)  at  a  concentration of  1.0 mg/t.
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 m£ of marine  2216  broth
was inoculated into  10 mt of  sterile sea-salts  medium containing
malathion  (100 yg/m-O  and incubated  for 5 days  at 28 C on  a  rotary
shaker.  Cells and medium were extracted  without  separation  with 10 m-t
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  yg/m£), 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 yg/m£ malathion and 0.1 pCi- ^C in Biometer
flasks  (Bellco, Inc.).  Reaction vessels  were incubated for  14 days at
28 C  and ^CC^ sampled daily for 1^C02  evolution.  Radioactive C02
was recovered by adding 1.0 m£ alcoholic-hyamine  solution  (1M, Packard
Instruments)  to the  side-arm of  the  Biometer flask,  incubating 5 minutes,
and removing the -^CC^-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 m£  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.

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The actual rates of malathion degradation were determined by incubation
in seawater medium containing 46 yg/m£ 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 m£ sea-sal-0.2% peptone
medium containing 0.1 yCi-  C-malathion (46 yg/m£).  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 10 m£ sterile seawater, dried for 24 hours at 85 C, and analyzed
for   C-activity by liquid scintillation counting.  The cell-free supernate
was analyzed for soluble degradation products by assaying for residual
14C-activity.

Cultures grown on marine 2216 broth and marine broth plus 200 yg
malathion/m£ 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 yg/m£ 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 C) 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

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Malathion was added in 0.5 m-t 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 0.5 m
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 m£ 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 pg/m£ 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  Tracer Model MT-220 with
a Melpar flame-photometric detector, were employed.   For the electron-
capture GLC determinations, two columns, one containing  2%  OV-101 on

                                   11

-------
Figure 3.  Extraction schematic for malathion metabolites.
                          Aqueous Cell—free
                            Centrifugate
                                Extract 2X with
                                Petroleum ether
Aqueous

Acidify to pH 1.5 with TCA
                                      Pet. Ether
                                             GLC
       Extract with a) Pet. ether:  acetone  (1:1)
                    b) Diethyl ether
Aqueous
                          Ether
                                   Concentrate to 30 ml
                                   under vacuum at 35 C
                                     I
                                   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

-------
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 y-thick Silica=gel 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 y£ 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 mi 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 m& acetone.  This extract was concentrated to 1-2 m£ 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

-------
                              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
number
1
12
44
45
47
Mixture
-fi — — — — 	
Inoculum
40
32
58
35
65
70
Marine
127
115
145
125
155
250
Marine + Malathion
134
108
150
165
185
200
   irine 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 yg/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

-------
    1OO
             2       6       1O      14      18     22     26
                  INCUBATION   TIME    (DAYS)
30
Figure 4.  Effect of salinity and temperature on malathion stability.  Malathion 1 yg/m£ added in
         acetone to sterile seawater at varied salinities, 0 °/oo distilled water.  Dashed line
         represents microbial degradation.

-------
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 -^C-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   C-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 C0£ from
the malathion molecule.  Data from cultures 1 through 12, not shown
here, indicated that cultures 1, 4 and 9 affected the release of   C02
within 2 days, whereas others required incubation for 7 days before
appreciable release of   CC^ was detected.  In the sterile control, 20%
of the   C-methoxymalathion label was released as   C02 after 10 days
incubation.
                                   16

-------
               Table 4.  MICROBIAL DEGRADATION OF MALATHION.

Culture
number
1
2
3
4
5
6
7
8
9
10
11
12
44
45
47

Morphology & gram reaction
(-) short rod
(-) coccoid rod
(-) medium rod
(-) medium rod
(+) short rod
(-) medium rod
(-) medium rod
(-) short rod
fungus
fungus
(-) slender rod
(-) medium rod
(+) slender rod
(-) short rod
(-) large ovoid rod

Degraded3
as S.C.S.
48
2
32
66
36
2
1
28
1
50
24
71
90
72
87
. , 	 	 	 - 	 	 — »- 	
Degraded0
with 0.2% Peptone
91
81
83
100
94
82
83
90
77
91
92
100
73
100
100
aAfter 10 days incubation as sole carbon source.  DAfter 5 days incubation
 with 0.2% Peptone.


        Table 5.  CELL-MEDIUM DISTRIBUTION OF 14C FROM MALATHIONa

Culture
number
1
2
3
4
5
6
7
8
9
10
11
12
alncubation
thion plus
Percentage of total
Wet cells
76
92
87
72
89
90
81
93
80
83
87
91
medium was seawater medium containing (46
0.2% peptone in all cases.
radioactivity
Supernatant
24
8
13
28
11
10
19
7
20
17
13
9
yg/m£) ^C-mala-
                                   17

-------
Table 6.   RADIOMETRIC AND CHEMICAL ANALYSES OF MICROBIAL GROWTH ON
          14C - MALATHION.
Radiometric analysis - Percentage of total radioactivity remaining in
                       expended medium
Culture No.
44
45
47
Mixture
0
Control
Malathion
25.7
8.3
6.5
7.4
10.5
Products
Carboxyesterase
36.8
48.2
61.3
87.2
82. 4a
of
Phosphatase
37.5
43.5
32.2
5.4
7.1a

Residue Analyses- flame photometric GLC analysis - ppm
Culture No.
44
45
47
Mixture
Control3

Malathion
19
75
75
75
9
Recovery
MCA
85
159
80
11
168a
(ppm)
DCA
40
8
23
55
7a

Total
144
172
108
75
184
Percentage
recovery
72
86
54
38
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

-------
0
                                    j-PEPTONE
                                           ( ) CONTROL

                                           ORIGINAL
                                            J51)\(49)
                                                   n§
4       6      8       10     12
  INCUBATION   (DAYS)
14
          Figure 5.  Bacterial degradation of malathion in seawater,

-------
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 Rfj values (18), but no standards
were available for comparison.  With th£ 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 -
                           VALUES FOR MALATHION AND METABOLITES.
Compound
                     Solvent^
                                                        Solvent^3
Malathion
Malaoxon
KDM
MCA
DCA
0.93
0.80
0.63
0.71
0.28
0.95
0.77
0.23
0.68
0.31
aHexane : Acetic Ac : DEE  (2  : 2 : 1)
bBenzene : 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~l re-
present symmetrical C-H stretches.  Bands at 2550 cm~l represent S-H
stretch bonds.  A strong band at 1730 cm~l represents a C=0, whereas
a band 1000-1200 cm~l indicates C-0  (1170 cm~l) .  Methyl and C=C groups
adsorb at 1380 and 1640 cm~l, respectively.  The band at 1010 cm~l  indicates
P-O-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

-------
  o
  C*
   • •

  O
2!
ui <
   O

   4)
   N
   c
   I
   0)

  Z






STANDARDS
§
Malathion
t;j MCA-M
Malaoxon
^j DCA-M


Origin
CULTURE EXTRACT
f 1
-




1
1 Malathion
',_ \ J3 MCA-M

,^ r\A DCA-M
" (.-!7
\ /8 10
U'-
Drigin





CONTROL -NO CELLS
/?H\
i




,^ Malathion
-MCA-M
Malaoxon
DCA-M

S
<.KDM
Origin ^
                                    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.  Reference standards are included for com-

                             parison of  spots.

-------
                                                            2OOO

                                                        WflVE  NUMBER   (CH~l)

4000
                              XXX)
                                                           XXX)
                                                     WAV!  NUMSIR  (CM"1)
                                                                                         WOO
-i
        Figure 1.  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).

-------
l-o
to
       MflLflOKON
       CH3-0X ,0
            P*      0
       CH3-0  5- CH-C-0-CjHs
                 i
  KMO
                                                           WflVE   NUMBER  (W)
                        Figure 8.  Infrared spectral tracing  of  malaoxon, with representative

                                   adsorption bands indicated.

-------
 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"-'-.  The additional
 adsorption peaks located between 200-1000 cm~l-cannot 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 cm~l).  Assuming this band to be contamination,
                                   24

-------
      MflLflTHWN  DICflRBOXYLIC  flCID
N>  MOO
       B.
3fiOO
    2000
WflVE  NUMBER  (CM'1)
1000
200


 1
       SPOT na4
     I
      POTHSSIUM  DESHETHYL  MflLflfHION
                                                                  2000
                                                              WflVE  NUMBER   (CM'1)
       Figure  90   Infrared spectral tracings  of (a) malathion-dicarboxylic acid  and extract  from spot  3 and
                   (b) potassium desmethyl-malathion with  extract from spot 40

-------
•WOO
     SPOT  no. 6
                                C-H
4000
                               3000
                                                              -f-
    2000
WflVE  NUMBER  (0-Tl)
                                                                                            1000
       Figure  10.   infrared spectral tracings  of  (a) extract  fro, spot 5 with  possible structure  in
                    brackets (b)  extract from spot 6.
                                                           200

-------
Figure 11.  Infrared spectral tracings of (a) extract from spot 7 and (b) extract from spot 8,

-------
to
00
                        I	4—      4
      SPOT  nalO
          HC-C-OH

          HC-C-OH
             it
             0
                                           2000
                                                   WflVE  NUMBER  (CM'1)
                                                            1000
          Figure  12,
Infrared spectral tracings  of  (a)  diethyl tnaleate and extract  from spot  9 and (b) extract
from spot 10, with possible structure in brackets.

-------
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.   I 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(&= 0.01) among treatment
levels (10X, IX, 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 IX 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), i.e., frequency of treatment.  Numbers of  SCD's, 4% of the
heterotrophic bacteria on the master  plate in the untreated sediment,
were greater for IX and  10X application rates of  malathion,  8 and 10%
respectively.
                                  29

-------
                                      1OX  FIELD RATE
   XX>r
        B.
                     FIELD RATE
   KX>r
JJ SO

                                                                                13*
        C.
                                                                              -I to*
                                                                              3O
      Figure 13.
Heterotrophic bacteria from water column, of artificial salt-
marsh ecosystem presented as heterotrophs/m£ (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

-------
      A.
1OX
FIELD RATE
1OO
 so
                                                                                10s
                                                                                10*
                                                                               iof
KX>
                                                                               10*
                                                                               49%
      Figure 14.  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 aver-
                  age for each category.  Tijnes of malathion treatment
                  indicated by arrows; controls are untreated.
                                       31

-------
Numbers of MCM's increased significantly with frequency of treatment
 (0, 10, 20 days; a= 0.1) at both treatment levels.  Although MCM
bacterial numbers were not significantly different at increased levels
of treatment  (control, IX 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 IX 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 ecosystem,
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 pg mirex/m£ 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

-------
    A.
                                   lOOOjig/ LITER
                                                                                10*
                                                                               Jio*
lOOr
 5O
                                                                            79*
    B.
                                    lOOjig/LITER
                                                                                10*  O
                                                                                10*
                                                                           87*
                                     CONTROL
                                                        i
                                                        i
                                                        •*•
KX)r
 5O
                                                       86*
                            10
                     15

                   DAYS
2O
25
       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-mastex-plate",  respectively.
3O
                                      33

-------
100
 so
                                                                          78*
     c.
                                                                             JK>»
1OO
 50
                                                                         88*
   O
               10
  15
DAYS
20
                                                                25
                                                                30
Figure 16.
Heterotrophic (solid  line) and mirex-tolerant bacteria
(shaded)  from sediments of artificial salt-marsh
ecosystems.
                                    34

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                              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,-,-f ixation or by single-carbon metabolism, because the   C-label
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

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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 ()-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

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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

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                              SECTION VI
                              REFERENCES

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

 2.   Alexander, M.  Microbial Degradation of DDT.  Cornell University.
     Ithaca, New York.  NTIS No. AD-781 903/OWP Office of Naval Research.
     Contract No. N0014-67-A-0077-0027.  July 1973.  24 p.

 3.   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.

 4.   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.

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

 6.   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.

 7.   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.

8.   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.

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

10.   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

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11.  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.

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

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

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

15.  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.

16.  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
     0-p-nitrophenyl Phosphorothionate).  Bull. Environ. Contain. Toxicol.
     5(l):47-49, 1970.

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

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

19.  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.

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

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

22.  Odum, E. P.  Fundamentals of Ecology.  Third Edition.  Philadelphia,
     W. B. Saunders Co., 1971.  p. 352-363.
                                    39

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23.  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.

24.  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.

25.  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.

26.  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.

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

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

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

30.  Walker, W. W. and B. J. Stojanovic.  Malathion  Degradation by an
     Arthrobacter Species.  J.'Environ.  Quality.   3(1):4-10,  1974.
                                   40

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                               SECTION  VII
                         ABBREVIATIONS AND SYMBOLS
C'ase                         Carboxyesterase
DCA                           Dicarboxylic-acid-malathion
DQC                           Dibromoquinone chloride
DEE                           Diethyl ether
DDT                           Dichlorodiphenyltrichloroethane
EC                            Effective concentration required to affect
                               50% of the experimental population
GLC                           Gas liquid chromatography
IR                            Infrared spectroscopy
KDM                           Potassium desmethyl malathion
MCA                           Monocarboxylic acid malathion
MCM                           Malathion co-metabolizers
M. Ib. A.I.                   Million pounds active ingredient
nd                            Not detectable
P'ase                         Phosphatase
ppb                           Parts per billion (pg/kg, ug/liter)
ppm                           Parts per million
TLC                           Thin-layer-chromatography
ULV                           Ultra-low volume
°/oo                          Parts per thousand
                                   41

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
 1. REPORT NO.
  EPA-660/3-75-035
                                                           3. RECIPIENT'S ACCESSION" NO.
 4. TITLE AND SUBTITLE
   Microbial-Malathion  Interactions in Artificial Salt-
   Marsh Ecosystems:  Effect  and Degradation
             5. REPORT DATE
               March 1975  (Issue)
             6. PERFORMING ORGANIZATION CODE
 7. AUTHOR(S)
   Al W. Bourquin
                                                           8. PERFORMING ORGANIZATION REPORT NO
               GBERL 236
 9. PERFORMING ORGANIZATION NAME AND ADDRESS
   U.S.  Environmental  Protection Agency
   Gulf  Breeze Environmental  Research Lab
   Sabine Island, Gulf Breeze,  FL  32561
             10. PROGRAM ELEMENT NO.

               1EA077
             11. CONTRACT/GRANT NO.
 12. SPONSORING AGENCY NAME AND ADDRESS
   U.S.  Environmental Protection Agency
   National Environmental  Research Center
   Office of Research and  Development
   Corvallis,  Oregon  97730
             13. TYPE OF REPORT AND PERIOD COVERED
                Interium; FY-1974	
             14. SPONSORING AGENCY CODE
 15. 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
                  DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS  C.  COSATI Field/Group
  Microbial Ecology
  Pesticide - Microbial Interaction
  Pesticide Degradation
  Malathion Degradation
  Environmental Microbiology
  Artificial Ecosystems/
  Malathion-Microbial  Infr-
  actions
  Malathion Degradation
 3. DISTRIBUTION STATEMENT
  Release unlimited,  available from OPA,
  NERC, Corvallis,  OR.   97330
19. SECURITY CLASS (This Report)
	Unclassified
20. SECURITY CLASS (This page)
      Unclassified
21. NO. OF PAGES
      A2	
22. PRICE
EPA Form 2220-1 (9-73)
                             t, U. S. GOVERNMENT PRINTING OFFICE- I975-699-033 /I3 REGION 10

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