EPA-600/3-78-044
April 1978
Ecological Research Series
INSECTICIDE PERSISTENCE
IN NATURAL SEAWATER AS AFFECTED
BY SALINITY, TEMPERATURE, AND STERILITY
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
EPA-600/3-73-044
Aoril 1978
INSECTICIDE PERSISTENCE IN NATURAL SEAWATER AS
AFFECTED BY SALINITY, TEMPERATURE, AND STERILITY
by
William W. Walker
Microbiology Section
Gulf Coast Research Laboratory
Ocean Springs, Mississippi 39564
Grant No. R803342
Project Officer
Al W. Bourquin
Environmental Research Laboratory
Gulf Breeze, Florida 32561
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
-------
DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
Gulf Breeze, U. S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily reflect
the views and policies of the U. S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or recom-
mendation for use.
ii
-------
FOREWORD
The protection of our estuarine and coastal areas from damage caused by
toxic organic pollutants requires that regulations restricting the introduction
of these compounds into the environment be formulated on a sound scientific
basis. Accurate information describing dose-response relationships for
organisms and ecosystems under varying conditions is required. The Environ-
mental Research Laboratory, Gulf Breeze, contributes to this information
through research programs aimed at determining:
the effects of toxic organic pollutants on individual species and
communities of organisms;
the effects of toxic organics on ecosystem processes and components;
the significance of chemical carcinogens in the estuarine and marine
environments.
Due to the increased use of biodegradable pesticides, it becomes impera-
tive that the fate of these compounds be determined to properly assess their
impact on the environment. Information on the effects of physical, chemical,
and biological factors on toxic organics in estuarine waters, as produced by
this report, will contribute greatly to the scientific assessment of how
biodegradable pollutants affect a marine ecosystem.
homas W. Duke
Director
Environmental Research Laboratory
Gulf Breeze, Florida
iii
-------
ABSTRACT
The effect of temperature, salinity, and sterility on the degradation of
malathion, parathion, methyl parathion, diazinon, and methoxychlor in fresh
and estuarine water has been determined under controlled laboratory
conditions. Surface water samples of 0, 10, 20, and 28 ppt salinity were
amended with the above insecticides and incubated in the dark at 30, 20, and
10°C under sterile and nonsterile conditions. Insecticide abatement was
followed by electron-capture gas-liquid chromatographic techniques.
No significant differences between sterile and nonsterile treatments were
observed for any of the insecticides studied, while the effect of increasing
temperature was highly significant with regard to increased degradation of
malathion, parathion, methyl parathion, and diazinon. Methoxychlor reflected
the recalcitrance characteristic of the chlorinated hydrocarbon insecticides
throughout 84 days of incubation and was not significantly affected by
salinity, temperature, or sterility. Salinity effects were varied among the
four organophosphates, being highly significant for malathion and diazinon,
significant for methyl parathion, and not significant for parathion.
Malathion was the shortest-lived of the insecticides tested, with half-
lives at 30°C varying from approximately 11 days in fresh water to less than
two days at 10, 20, or 28 ppt salinity. The rate of methyl parathion dis-
appearance was second only to malathion and ranged, in terms of half-lives,
from 27 days in fresh water to 16 days at 28 ppt. In fresh water, a 45-day
half-life for diazinon suggested a substantial resistance to degradation,
especially at 30°C. In saline water, however, diazinon abatement was con-
siderably accelerated, as indicated by a half-life of 24 days at 28 ppt
salinity. Parathion, the most persistent of the organophosphate insecticides
tested, reflected a half-life of at least 44 days regardless of salinity.
One bacterium, tentatively identified as Moraxella sp., was isolated from
sediment by enrichment and proved capable of readily degrading malathion
either as a primary carbon source or in the presence of peptone. Of two
bacteria tested for the ability to degrade methyl parathion, one, possibly a
Pseudomonas sp. proved quite capable of utilizing the insecticide with or
without peptone, while the other, a Moraxella species, reflected no degrada-
tion of methyl parathion as the primary carbon source and only limited utiliz-
ation in the presence of peptone. Neither of two bacteria screened for
parathion metabolism were capable of insecticide degradation under the condi-
tions of this evaluation.
. This report was submitted in fulfillment of Grant No. R-803842 by the
Gulf Coast Research Laboratory, Ocean Springs, MS, under sponsorship of the
U.S. Environmental Protection Agency. This report covers the period July 15,
1975 through July 14, 1977, and work was completed September 30, 1977.
iv
-------
CONTENTS
Foreward ill
Abstract iv
Figures vi
Tables vii
Acknowledgements viii
1. Introduction 1
2. Conclusions 2
3. Recommendations 3
4. Materials and Methods 4
Water samples 4
Insecticides and incubation parameters 4
Insecticide extraction and analysis 5
Microbial degradation studies 5
5. Results and Discussion 6
Insecticide loss from natural seawater 6
Malathion 6
Parathion 8
Methyl parathion 8
Diazinon 11
Methoxychlor 11
Insecticide half-life 11
Microbial degradation studies 14
Malathion 14
Parathion 14
Methyl parathion 15
Diazinon 16
References 18
Appendix 20
-------
FIGURES
Number Page
Malathion disappearance from nonsterile water at 0,
10, 20, and 28 ppt salinity and temperatures of
30°, 20°, and 10°C
Parathion disappearance from nonsterile water at 0,
10, 20, and 28 ppt salinity and temperatures of
30°, 20°, and 10°C
Methyl parathion disappearance from nonsterile
water at 0, 10, 20, and 28 ppt salinity and
temperatures of 30°, 20°, and 10°C 10
Diazinon disappearance from nonsterile water at
0, 10, 20, and 28 ppt salinity and temperatures
of 30°, 20°, and 10°C 12
\
Insecticide half-life at 30°C and 0, 10, 20, and
28 ppt salinity 13
vi
-------
TABLES
Number page
1 Malathion Degradation by Isolate M14A1 15
2 Parathion Degradation by Isolates P14B1 and P.25C2 16
3 Methyl Parathion Degradation by Isolates MP15A1 and
MP25C1 17
A-l Percent Loss of Malathion from Natural Seawater as
Affected by Temperature, Salinity, and Sterility 21
A-2 Percent Loss of Parathion from Natural Seawater as
Affected by Temperature, Salinity, and Sterility 22
A-3 Percent Loss of Methyl Parathion from Natural Seawater
as Affected by Temperature, Salinity, and Sterility .... 23
A-4 Percent Loss of Diazinon from Natural Seawater as
Affected by Temperature, Salinity, and Sterility 24
A-5 Percent Loss of Methoxychlor from Natural Seawater as
Affected by Temperature, Salinity, and Sterility 25
vii
-------
ACKNOWLEDGEMENTS
We would like to thank the U. S. Environmental Protection Agency for
partially funding this research effort and the project officer, Dr. Al W.
Bourquin, for his help and guidance throughout this project. Appreciation is
extended to Ms. Diana Woroner, Ms. Dale Shelton, and Ms. Dinah Pugh for their
technical assistance and to Ms. Sandra Lofton for help in species identifica-
tion. Special thanks are extended to Mr. David Boyes for statistical evalua-
tions and to Ms. Lucia Ross for the careful typing of this manuscript.
yiii
-------
SECTION 1
INTRODUCTION
The organophosphorus group of insecticide chemicals currently enjoys wide
usage throughout the United States, due in part to various bans and restric-
tions on the so-called "hard" chlorinated hydrocarbon and related pesticides.
And, while the organophosphorus insecticides are as a whole considered rela-
tively safe, certain individual members of the group are potent acetylcholin-
esterase inhibitors (20), and their role as potential environmental hazards
cannot be overlooked.
Considerable information is available regarding the overall fate of
organophosphorus insecticides in soil (6, 7, 12, 16, 18, 19, 22, 23), their
degradation in terrestrial plants and animals (1, 5, 15), and their toxicities
and effects on freshwater species (4, 8, 9, 12, 17). Comparatively little
information, however, is available concerning the disposition of the organo-
phosphorus insecticides in the estuarine or salt-marsh environment (2, 10, 11,
20). This deficit, coupled with the fact that the use of organophosphorus
insecticides in and around estuarine areas is increasing due to various mos-
quito control programs (3), the research effort described herein was
formulated.
The overall objective of these investigations was (1) to determine the
effect of temperature, salinity, and sterility on the persistence and degrada-
tion of representative organophosphorus and chlorinated hydrocarbon insecti-
cides in natural seawater, and (2) to isolate into pure culture representative
estuarine microorganisms capable of utilizing these materials either as a
primary carbon source or in the presence of an additional energy-rich material.
-------
SECTION 2
CONCLUSIONS
It is concluded from this investigation that the effect of increasing
temperature is highly significant with regard to the breakdown of malathion,
parathion, methyl parathion, and diazinon in natural water throughout the
zero to 28 ppt salinity range of this study. Although not demonstrated by
actual data, this relationship probably exists for the organophosphorus
insecticide group as a whole. The significance of observed salinity effects
on insecticide abatement (99% for malathion and diazinon, 95% for methyl
parathion, 42% for parathion), on the other hand, appears to vary from com-
pound to compound with little uniformity within the group.. Sterility effects
appear minimal, as do treatment interactions. The recalcitrance of methoxy-
chlor observed under all conditions of this investigation seems to reaffirm
the persistent nature of the chlorinated hydrocarbon family of insecticides
over a wide range of environmental conditions.
Of the three compounds tested, malathion and methyl parathion were
degraded microbiologically either as primary carbon sources or in the presence
of an additional energy supply, while parathion remained immune to breakdown
under either of these conditions. This information indicates that while the
organophosphate insecticides may be generally considered non-persistent,
particular numbers of the group may well exhibit a substantial degree of
resistance to chemical or microbial attack.
-------
SECTION 3
RECOMMENDATIONS
It is recommended that as the use of organophosphorus, carbamate, and
related "short-lived" pesticides increases, a concomitant broadening of know-
ledge regardfij^ the fate and effect of these materials in the natural environ-
ment be observed. Investigation along these lines should embrace chemical as
well as biological mechanisms, probably through the microcosm and simulated
ecosystem approach.
-------
SECTION 4
MATERIALS AND METHODS
WATER SAMPLES
Surface water samples of 0, 10, 20, and 28 mg/ml (ppt) salinity were col-
lected as required from the Biloxi Bay - Mississippi Sound estuary system
located geographically near Biloxi and Ocean Springs, Mississippi. Samples
were collected on a transect originating in Fort Bayou, traveling through
Biloxi Bay and into the Mississippi Sound, and terminating approximately one
(1) mile due south of the west end of Horn Island. Water samples were col-
lected by dipping chemically clean 3.79 liter (one gallon) amber glass con-
tainers fitted with Teflon lined caps.
Water samples were returned to our laboratory, filtered through glass
wool, and dispensed in 25 ml aliquots into 0.13 liter (8 oz.) prescription
bottles. Sterilization, where appropriate, was accomplished by autoclaving
for 15 minutes at 121°C and 6.8 kg (15 pounds) pressure, and representative
autoclaved treatments were plated on half-strength marine agar to confirm
sterility.
INSECTICIDES AND INCUBATION PARAMETERS
Insecticides employed in these investigations were malathion (S-[l,2-
Dicarbethoxyethyl]-(),0-dimethyldithiophosph.ate) , parathion (0,0-Diethyl-0,p_-
nitrophenyl phosphorothioate), methyl parathion (0,()-Dimethyl-(),p_-nitrophenyl
phosphorothioate), diazinon (0,£-Diethyl-0-[2-isopropyl-4-methyl-6-
pyrimidinyl] phosphorothioate), and methoxychlor (l,l,l-Trichloro-2,2-bis[|>-
methoxyphenyl] ethanol).
Insecticides were filter-sterilized using standard Millipore filtration
apparatii fitted with 0.4 micron Nuclepore filter pads and added to respective
incubation vessels as 0.5 ml acetone solutions. Insecticide concentrations
employed in these investigations were: malathion, 5.04 mg/1; parathion, 4.48
mg/1; diazinon, 1.81 mg/1; methyl parathion, 6.96 mg/1; and methoxychlor, 1.04
mg/1. All incubations were conducted in the dark at 10°, 20°, and 30°C in
Freas model 815 incubators.
-------
INSECTICIDE EXTRACTION AND ANALYSIS
Following incubation, residual insecticides were extracted with 100 ml of
a 1:1 (v:v) ratio of hexane and acetone, both pesticidequality solvents.
Diazinon (90.62 pg) was added to malathion, parathion, and methyl parathion
incubations as an internal standard immediately prior to extraction.
Parathion (112.06 |Jg) was added as internal standard for diazinon incubations,
and heptachlor (1,4,5,6,7,8,8-Heptachloro-3a.4.7.7a-tetrahydro-4,7-
methanoindene, 30.04 (jg) was used as the internal standard for methoxychlor
incubations. Acetone was subsequently removed by washing with distilled
water, and final traces of water were removed by passage through anhydrous
sodium sulfate. The dried hexane extract was then analyzed for residual
insecticides, using two Tracer MT-220 gas chromatographs, each equipped with
one Ni63 electron-capture and one flame photometric detector. Glass columns,
0.6 cm (0.25 in.) inside diameter, and varying in length from 0.9 to 1.8 m (3
to 6 ft.) and two column packings, 1.5% OV-17 + 1.95% QF-1 and 6% QF-1 + 4%
SE-30, both on chromosorb W, HP, 100/120 mesh, were used throughout these
investigations. Nitrogen served as carrier and purge gas at flow rates of 95
and 20 ml/min, respectively. Operating temperatures were as follows: inlet,
200°C; column oven, 185°C; electron-capture detectors, 275°C; and flame photo-
metric detectors (ignited), 215°C.
MICROBIAL DEGRADATION STUDIES
Estuarine microorganisms capable of degrading malathion, parathion,
methyl parathion, and diazinon were sought by enrichment. Approximately 3 cc
of surface sediment was collected from Davis Bayou, an area of the Mississippi
Sound immediately adjacent to our laboratory, and diluted to 100 ml with
artificial seawater of 15 ppt salinity. Each sediment suspension was then
amended with 7312 |Jg raalathion, 11,206 |jg parathion, or 8697 |Jg methyl
parathion at two-day intervals for a period of 22 days. Temperature of incu-
bation during enrichment was 30°C in all cases. Following enrichment, all
treatments were plated onto half-strength marine agar, and all different
colonies isolated into pure culture. The predominant isolates in each case
were then tested for the ability to utilize its enrichment insecticide both as
the primary carbon source (insecticide added in 0.5 ml acetone) or in the
presence of 1% peptone (Bacto-Peptone, Difco Laboratories, Cat. No. 011801), a
readily available energy source.
Each isolate tested was grown in mass on half-strength marine agar bottle
slants, washed three times with physiological saline (0.85% sodium chloride in
distilled water) to remove "carry-over" nutrients, and added to 25 ml mineral
salts solution (13) containing either insecticide alone or insecticide plus 1%
peptone. All treatments were incubated in the dark at 25°C. Respective
malathion, parathion, and methyl parathion concentrations in these investi-
gations were 5.86, 4.46, and 6.20 mg/1.
-------
SECTION 5
RESULTS AND DISCUSSION
INSECTICIDE LOSS FROM NATURAL SEAWATER
Malathion
Losses of malathion from natural seawater as affected by temperature,
salinity, and sterility are illustrated in Table A-l and Figure 1. It
should be noted at this point that analysis of variance and chi-square
analyses did not indicate that sterility was a significant factor in the dis-
appearance of malathion from natural seawater. Both sterile and nonsterile
data are listed in Table A-l, but only nonsterile results are shown in
Figure 1. At 30°C (Figure 1, top), malathion abatement was quite rapid and in
direct proportion to increasing salinity. At 20 and 28 ppt, malathion could
not be detected after 10 days incubation. At 10 ppt, all of the added mala-
thion had disappeared after 14 days. In fresh water, malathion was more per-
sistent, with losses ranging from 30 percent at three days to 63 percent after
20 days.
At 20°C (Figure 1, center), malathion abatement was similar to that
observed at 30°C except that the rate of disappearance was reduced. At 28 ppt
salinity, malathion degradation was complete after 15 days incubation as com-
pared to 20 days at 20 ppt and 25 days at 10 ppt. Malathion disappearance was
again slowest in fresh water (0 ppt), with losses ranging from 22 percent
after one day to 67 percent after forty days.
Incubation at 10°C greatly reduced the rate of malathion breakdown, as
indicated in Figure 1, bottom. At 28 ppt, insecticide losses ranged from 34
percent at four days to 97 percent after 70 days. At 20 ppt, malathion dis-
appearance ranged from 27 to 94 percent over the same period of time as com-
pared to a range of 26 to 89 percent in the 10 ppt incubations. At 0 ppt,
malathion abatement was extremely slow, with losses ranging from eight percent
at four days to only 27 percent at the end of the 70-day incubation period.
Generally speaking, malathion abatement was rapid under all incubation
conditions except zero salinity and 10°C. Analysis of variance revealed that
both temperature and salinity effects were significant at the 99% level of
probability, while sterility reflected a significance of only 72.8 percent.
Levels of significance for a temperature/salinity interaction, a temperature/
sterility interaction, or a salinity/sterility interaction were 81.5, 29.6,
and 13.6 percent, respectively, revealing that no significant (at the 95%
level of probability) two-way interactions occurred. The three-factor level
of significance was only 8.6 percent.
6
-------
too
90
80
70
60
50
40
30
20
10
100
90
80
70
60
50
40
30
20
10
100
90
80
70
60
SO
40
30
20
10
0
L "/
"a
lOppt-
20ppt
28ppt
10 20 30 40 50 60
TIME IN DAYS
70
Figure 1. Malathion disappearance from nonsterile water at 0, 10, 20, and
28 ppt salinity and temperatures of 30° (top), 20° (center),
and 10°C (bottom).
-------
Parathion
Losses of parathion from natural seawater as affected by temperature,
salinity, and sterility are shown in Table A-2 and in Figure 2. Unlike
malathion, the only effect significant at the 99 percent level was
temperature. The effect of salinity on parathion disappearance was of no
meaningful significance (significance level 46.1 percent), and the level of
significance for sterility was only 22.9 percent. No significant interactions
were observed.
At 30°C (Figure 2, top), parathion losses following 84 days incubation
were 78, 76, 78, and 68 percent in the zero, 10, 20, and 28 ppt salinity
treatments, respectively. At 20°C (Figure 2, center), a similar but retarded
trend of parathion breakdown was observed, with losses after 84 days ranging
from 23 percent in fresh water to 37 percent at 28 ppt salinity. At 10°C
(Figure 2, bottom), parathion degradation was slower still, with a maximum
loss of only 27 percent observed in the 28 ppt treatment after 84 days
incubation.
Generally, parathion disappearance was substantially slower than that
observed for malathion. At 30°C, the temperature at which both insecticides
disappeared most rapidly, malathion could not be detected after only seven
days at 28 ppt salinity. Parathion, after seven days at 28 ppt salinity,
reflected a loss of only 11 percent of added material. Further, the fact that
some 32 percent of added parathion could still be detected after 84 days
incubation at 30°C and at 28 ppt salinity indicates that the parathion mole-
cule may well exhibit considerable recalcitrance in high-salinity seawater
even during the warm summer months.
Methyl Parathion
Methyl parathion losses from natural seawater as affected by temperature,
salinity, and sterility are shown in Table A-3 and Figure 3. Methyl parathion
disappearance was intermediate between malathion and parathion.
At 30°C (Figure 3, top), respective methyl parathion losses after 43 days
incubation at 0, 10, 20, and 28 ppt salinity were 82, 82, 90, and 83 percent.
At 20°C (Figure 3, center), these losses were 64, 63, 75, and 67 percent after
90 days incubation, and at 10°C (Figure 3, bottom), respective losses were 25,
25, 31, and 31 percent after 90 days.
Analysis of variance revealed that for methyl parathion temperature
effects were highly significant (significant at the 99 percent level), while
salinity effects were barely significant at the 95 percent level. The level
of significance for sterility was shown to 97.6 percent, a level that appeared
high after close observation of the data. In this regard, each sterile and
nonsterile mean was compared to each other, using Chi-Square analyses for all
times, temperatures, and salinities, a total of 200 comparisons. Of this
total number, only three loss means were found to be significantly different
-------
100
90
80
70
60
50
40
30
20
10
100
90
80
70
| «
Z »
LJLJ
85 40
Q_
Oppt-
lOppt-.
20ppt-
28ppt-
100
90
80
70
60
50
40
30
20
10
0
30
40 50 60
TIME IN DAYS
70
80
Figure 2. Parathion disappearance from nonsterile water at 0, 10, 20, and
28 ppt salinity and temperatures of 30° (top), 20° (center),
and 10°C (bottom).
-------
100
90
80
70
60
50
40
30
20
10
100
90
80
70
60
50
40
30
20
10
100
90
80
70
60
50
40
30
20
10
0
-I 1 1 1
-I 1 I !_
40 50 60
TIME IN DAYS
70
80
Figure 3. Methyl parathion disappearance from nonsterile water at 0, 10,
and 28 ppt salinity and temperatures of 30° (top), 20° (center),
and 10°C (bottom).
10
-------
due to sterility at the 95 percent confidence level, indicating that, in
reality, sterility is of no statistical consequence regarding the disappear-
ance of methyl parathion from seawater. No significant treatment interactions
were observed for methyl parathion.
Diazinon
Diazinon losses from natural seawater as affected by temperature, salin-
ity, and sterility are shown in Table A-4 and Figure 4. At 30°C (Figure 4,
top), diazinon disappearance was intermediate between parathion and methyl
parathion. Losses ranged from 70 percent in fresh water to 80 percent at 10
ppt salinity to 81 percent at 20 ppt to 87 percent at the highest salinity of
28 ppt. Diazinon dissipation was similar to that observed with malathion in
that the effect of salinity was significant at the 99 percent confidence
level. The rate of breakdown, however, was greatly reduced. At 20°C
(Figure 4, center), respective diazinon losses for 0, 10, 20, and 28 ppt
salinity were 33, 40, 62, and 61 percent, as compared to 15, 18, 24, and 21
percent at 10°C (Figure 4, bottom). As was the case with malathion, para-
thion, and methyl parathion, the effect of temperature on the degradation of
diazinon was significant at the 99 percent level. No significant interactions
were observed.
Methoxychlor
Methoxychlor losses from natural seawater. as affected by temperature,
salinity, and sterility are shown in Table A-5. At 30°C, methoxychlor
losses after 84 days ranged from 12 percent in fresh water to nine percent at
28 ppt. Methoxychlor losses after 84 days at 20°C were in the five to eight
percent range, and losses at 10°C were less than two percent throughout the
84-day incubation period. The recalcitrance displayed by methoxychlor under
all test conditions in these investigations is characteristic of the chlor-
inated hydrocarbon insecticide group in general and clearly delineates the
hazard these materials represent as environmental pollutants.
Insecticide Half-Life
Figure 5 illustrates the times required to achieve 50 percent loss of
each of the four organophosphorus insecticides employed in these studies when
incubated in the dark at 30°C and at 0, 10, 20, and 28 ppt salinity. These
times were taken from Figures 1-4 by interpolation and hence are approximate
in most cases and intended solely for use in comparing the four insecticides
to one another. The height of the bars in Figure 5 is~ indicative of the time
in days, and the numbers above the bars represent the salinity in parts per
thousand.
The half-life of malathion in fresh water is approximately 11 days and is
the shortest of the four. Malathion dissipation is greatly enhanced in saline
water, as evidenced by the drop in half-life to less than two days in 10, 20,
and 28 ppt salinity water.
11
-------
100 i-
90 -
80 -
70 -
60 -
50 -
40
30
20
10
100
90
80
70
(SO
-
-
Onnt
IUPP'
OQ_.-,f
^*£
50
40
30
20
10
100
90
80
70
60
50
40
30
20
10
0
10 20 X 40 50 60
TIME IN DAYS
70
80 90 100
Figure 4. Diazinon disappearance from nonsterile water at 0, 10, 20, and
28 ppt salinity and temperatures of 30° (top), 20° (center),
and 10°C (bottom).
12
-------
fV
60
50
2 40
g
z
111
s
^ 30
20
10
0
»
•
-
^
-
(
* m
)
MMI
1020 28
TTTTTrrn
0
TT
10
MMH
20 28
0
10
20
28
MM
MALATHION PARATHION METHYL
PARATHION
0
MMM
10
MMM
20
28
DIAZINON
Figure 5. Insecticide half-life at 30°C and 0, 10, 20 and 28 ppt salinity.
13
-------
Parathion, on the other hand, was the most persistent insecticide of the
four with a half-life of some 44 days in fresh water. It should be noted here
that the temperature of incubation in this case was 30°C, a high temperature
in the natural environment, and that insecticide abatement is considerably
reduced at lower temperatures. The effect of salinity was not significant in
parathion disappearance, and the half-life of parathion in high-salinity water
was essentially the same as that observed in fresh water.
Methyl parathion was considerably less persistent than parathion and was
significantly affected by salinity in that the half-life for methyl parathion
dropped from 27 days in fresh water to approximately 16 days at 28 ppt.
Diazinon recalcitrance was intermediate between parathion and methyl parathion
and was affected by salinity to a highly significant degree. In fresh water
the half-life for diazinon (45 days) was comparable to parathion (44 days),
indicating that diazinon may well be relatively recalcitrant in natural fresh
water systems. In saline water, however, the disappearance of diazinon is
greatly accelerated, as indicated by a half-life of only 24 days in 28 ppt
salinity water.
MICROBIAL DEGRADATION STUDIES
Malathion
A total of 25 bacterial colonies were selected from the previously des-
cribed malathion enrichments, and the predominant bacterium, isolate M14A1,
tested for the ability to degrade the malathion molecule. Bacterium M14A1
was a non-motile, Gram negative rod, showed no reaction on glucose, was oxi-
dase positive and did not liquify gelation. On the basis of these charac-
teristics, bacterium M14A1 was tentatively placed in the genus Moraxella,
probably species locunata. Malathion degradation by this bacterium is shown
in Table 1.
Malathion breakdown in the unamended mineral salts incubation medium
(Column MS), or in mineral salts amended with peptone but no bacterial cells
(Column MS + P), was in the 27-28 percent range following 28 days incubation.
Bacterium M14A1 effected a 63 percent loss of added malathion during 28 days
incubation in mineral salts alone (Column MS + C), indicating that this bac-
terium was capable of limited metabolism of malathion as a primary carbon
source. In the presence of peptone, however, (Column MS + C + P) malathion
degradation was quite rapid, with none of the added malathion detected after
only five days incubation.
Parathion
Eight individual bacteria were isolated from the parathion enrichments,
and the two predominant organisms (P14B1 and P25C2) tested for the ability to
degrade parathion. Both of these bacteria reflected motility, Gram, glucose,
oxidase, and gelatin reactions identical to bacterium M14A1 and were, hence,
thought to be Moraxella species. Neither of the two cultures tested (Table 2)
reflected any real ability to utilize the parathion molecule, either as the
14
-------
primary carbon source or in the presence of peptone. Although only two of the
eight bacteria available could be tested in the time available, the fact that
the two bacteria that were tested represented the two most abundant species
present in the parathion enrichment and could not appreciably degrade the
parathion molecule certainly indicates a fair amount of resistance on the part
of the parathion molecule to microbial attack.
TABLE 1. MALATHION DEGRADATION BY ISOLATE M14A1
Days
incubation
1
3
5
7
10
15
20
28
Percent loss* of added insecticide
MS
2
4
6
9
14
19
22
28
MS + C
0
6
9
14
26
38
54
63
MS + P
4
4
7
13
16
21
23
27
MS + C + P
11
46
100
100
100
100
100
100
The losses listed represent means of duplicate treatments. Column headings
are as follows: MS, mineral salts plus insecticide; MS + C, mineral salts
plus insecticide plus culture; MS + P, mineral salts plus insecticide plus 1%
peptone; MS + C + P, mineral salts plus insecticide plus culture plus 1%
peptone. Incubation temperature was 25 C in all cases.
Methyl Parathion
Degradation of methyl parathion by two bacteria, MP15A1 and MP25C2, is
illustrated in Table 3. Bacterium MP25C1 was tentatively identified as belong-
ing to the genus Moraxella, while isolate MP15A1, a motile, Gram negative rod
was placed in the genus Pseudomonas. A total of 23 individual bacteria were
isolated from the methyl parathion enrichment, but time permitted testing of
only these two.
MP15A1 appeared able to utilize methyl parathion as the primary carbon
source, as evidenced by a loss of 59 percent of the added methyl parathion in
mineral salts plus bacterium (Column MS + C), as compared to only 21 percent
loss in the unamended mineral salts. This bacterium's ability to degrade
methyl parathion was substantially enhanced in the presence of energy-rich
peptone, as evidenced by the fact that all of the added methyl parathion was
completely degraded after 10 days incubation (Column MS + C + P). Methyl
parathion abatement in the presence of peptone but no cells (Column MS + P)
was not significantly different from that in the unamended mineral salts.
15
-------
TABLE 2. PARATHION DEGRADATION BY ISOLATES P14B1 AND P25C2
Days
incubation
MS
Percent loss of added insecticide
MS + C
MS + P
MS + C + P
1
3
5
7
10
15
20
28
1
3
5
7
10
15
20
28
1
0
3
3
5
8
10
13
0
0
3
3
6
8
12
16
ri^Bi
0
0
3
2
6
10
10
12
1
1
4
4
8
11
14
17
0
2
1
4
4
6
9
11
0
1
0
3
6
10
16
19
0
3
4
6
9
13
15
22
1
0
1
2
5
11
17
22
See footnote, Table 1.
Diazinon
A total of nine bacteria were isolated from the previously described
enrichment treatment, but, unfortunately, time has not permitted the testing
of any of these organisms for their ability to degrade the diazinon molecule.
16
-------
TABLE 3. METHYL PARATHION DEGRADATION BY ISOLATES MP15A1 AM) MP25C1
Days
incubation MS
1 5
3 6
5 8
7 9
10 12
15 14
20 19
28 21
1 4
3 5
5 7
7 9
10 10
15 12
20 14
28 16
.»-
/\
Percent loss of
MS + C
MD 1 (^ A 1 _
fir 1 DAI
3
7
15
22
33
40
46
59
3
6
7
8
9
11
12
16
added insecticide
MS + P
1
5
7
10
11
13
15
17
4
5
6
9
11
14
16
17
MS + C + P
4
10
29
48
69
100
100
100
3
5
8
13
18
29
34
42
See footnote, Table 1.
17
-------
REFERENCES
1. Bourke, J. B., E. J. Broderick, L. R. Hackler, and P. C. Lippold. 1968.
Comparative metabolism of malathion-C14 in plants and animals. J.
Agr. Food Chem. 16(4): 585-589.
2. Bourquin, A. W. 1977., Effects of malathion on microorganisms of an
artificial salt marsh environment. J. Environ. Qual. 6(4): 373-378.
3. Coppage, D. L., and T. W. Duke. 1971. Effects of pesticides in estu-
aries along the Gulf and Southeast Atlantic Coasts, p. 24-31. In C.
H. Schmidt (ed.) Proc. 2nd Gulf Coast Conf. on Mosquito Suppression
and Wildlife Management. 1967. Natl. Mosquito Control - Fish and
Wildlife Manage. Coord. Comm., Washington, D. C.
4. Eaton, J. G. 1970. Chronic malathion toxicity to the blue-gill. Water
Res. 4: 673-684.
5. Gardner, A. M., J. N. Damico, E. A. Hansen, E. Lustig, and R. W.
Storherr. 1969. Previously unreported homolog of malathion found as
residue on crops. J. Agric. Food Chem. 17(6): 1181-1185.
6. Getzin, L. W. and I. Rosefield. 1968. Organophosphorus insecticide
degradation by heat-labile substances in soil. J. Agric. Food Chem.
16(4): 598-601.
7. Getzin, L. W. 1971. Partial purification and properties of a soil
enzyme that degrades the insecticide malathion. Biochem. Biophys.
Acta. 235: 442-453.
8. Gibson, J. R., and J. L. Ludke. 1971. Effect of sesamex on brain acetyl-
cholinesterase inhibition by parathion in fishes. Bull. Environ.
Contam. and Toxicol. 6(2): 97-99.
9. Gibson, J. R. 1973. Effect of SKF-525A on brain acetylcholinesterase
inhibition by parathion in fishes. Bull. Environ. Contam. Toxicol.
9(3): 140-142.
10. Hansen, D. J., E. Matthews, S. L. Nail, and D. P. Dumas. 1972.
Avoidance of pesticides by untrained mosquitofish, Gambusia affinis.
Bull. Environ. Cont. Toxicol. 8(1): 46-51.
11. Hansen, D. J., S. C. Schimmel, and J. M. Keltner, Jr. 1973. Avoidance
of pesticides by grass shrimp (Palaemonetes pugio). Bull. Environ.
Cont. Toxicol. 9(3): 129-133.
18
-------
12. Henderson, C., and Q. H. Pickering. 1957. Toxicity of organophosphorus
insecticides to fish. Trans. Am. Fish Soc. 87: 39-51.
13. Lanzillata, R. P., and D. Pramer. 1970. Herbicide transformation. 1.
Studies with whole cells of Fusarium solani. Appl. Microbiol. 19(2):
301-306.
14. Lichtenstein, E. P., T. W. Fuhremann, and K. R. Schultz. 1968. Effect
of sterilizing agents on the persistence of parathion and diazinon in
soils and water. J. Agric. Food Chem. 16(5): 870873.
15. Lichtenstein, E. P., T. W. Fuhremann, A. A. Hackberg, R. N. Zahlten, and
F. W. Stratman. 197. Metabolism of 14C-parathion and 14C-paraoxon
with fractions and subtractions of rat liver cells. J. Agric. Food
Chem. 21(3): 416-424.
16. Mostafa, I. Y., I. M. I. Fakha, M. R. E. Bahig, and Y. A. El Zawahny.
1972. Metabolism of organophosphorus insecticides. XIII. Degrada-
tion of malathion by Rhizobium spp. Arch. Microbiol. 86: 221-224.
17. Post, G., and T. R. Schroeder. 1971. The toxicity of four insecticides
to four salmonoid species. Bull. Environ. Contam. Toxicol. 6(2):
144-155.
18. Sethunathan, N. 1973. Degradation of parathion in flooded acid soils.
J. Agric. Food Chem. 21(4): 602-604.
19. Sethunathan, N., and T. Yoshida. 1973. Parathion degradation in sub-
merged rice soils in the Philippines. J. Agric. Food Chem. 21(3):
504-506.
20. Solon, J. M., J. L. Lincer, and J. H. Nair, III. 1969. The effect of
sublethal concentrations of LAS on the acute toxicity of various
insecticides to the fathead minnow (Pimephales promelas Rafinesque).
Water Res. 3(10): 767-775.
21. Thomson, W. T. 1970. Agricultural chemicals book I. Insecticides,
acaricides, and ovicides. Thomson Publications, Fresno, CA.
22. Walker, W. W., and B. J. Stojanovic. 1973. Microbial versus chemical
degradation of malathion in soil. J. Environ. Qual. 2: 229-232.
23. Wolfe, H. R., D. C. Staiff, J. F. Armstrong, and S. W. Comer. 1973.
Persistence of parathion in soil. Bull. Environ. Contam. Toxicol.
10(1): 1-9.
19
-------
APPENDIX
TABULATED DATA - INSECTICIDE LOSS VS. TIME
2Q
-------
TABLE A-l. PERCENT LOSS* OF MALATHION FROM NATURAL SEAWATER AS AFFECTED
BY TEMPERATURE, SALINITY, AND STERILITY
Salinity (ppt)
Days Temp .
incubation °C
3
5
7 30
10
14
20
1
6
10
15
20 20
25
30
35
40
4
7
14
21
28 10
35
42
49
56
70
S
28
35
44
56
53
68
18
17
19
36
43
51
46
50
61
7
8
—
10
11
22
17
23
31
28
0
NS
30
33
39
49
54
63
22
21
30
36
40
45
60
69
67
8
9
10
18
13
20
23
23
31
27
10
S
65
77
93
95
98
100
21
67
75
85
94
100
100
100
100
20
21
29
40
41
62
66
69
76
82
NS
66
80
91
97
100
100
20
59
75
85
93
100
100
100
100
26
26
39
46
47
75
69
77
81
89
20
S
74
88
92
97
100
100
35
77
90
96
100
100
100
100
100
23
32
44
52
56
75
78
81
87
88
NS
80
91
98
100
100
100
35
79
90
97
100
100
100
100
100
27
38
56
57
72
80
87
88
91
94
28
S
78
89
96
100
100
100
45
85
94
100
100
100
100
100
100
28
33
54
63
72
81
88
88
92
94
NS
88
96
100
100
100
100
44
84
94
100
100
100
100
100
100
34
40
56
65
80
82
94
92
95
97
*Means of duplicate treatments.
21
-------
TABLE A-2. PERCENT LOSS* OF PARATHION FROM NATURAL SEAWATER AS AFFECTED
BY TEMPERATURE, SALINITY, AND STERILITY
Salinity (ppt)
Days Temp .
incubation °C
1
4
7
14
21 30
28
42
56
70
84
4
7
14
21 20
28
42
56
70
84
1
3
7
14
21 10
28
42
70
84
0
S
3
7
8
13
18
28
39
54
56
58
0
1
3
8
7
12
20
19
31
4
4
1
4
6
8
6
14
21
NS
6
6
8
18
22
39
48
66
75
78
2
5
4
9
9
14
21
22
23
0
0
0
1
1
6
9
9
15
10
S
2
1
4
21
23
31
42
54
60
65
2
4
4
7
11
16
22
22
31
0
2
2
1
4
6
8
12
14
NS
0
2
5
9
22
26
42
50
61
76
2
4
5
9
9
16
17
31
27
0
0
0
0
1
0
4
13
16
20
S
1
3
5
16
25
33
44
56
60-
65
2
4
8
10
8
29
40
48
51
0
0
6
7
3
2
10
17
17
NS
0
2
4
16
26
27
46
63
62
78
0
4
3
4
4
15
23
24
33
1
0
5
0
7
7
10
12
20
28
S
0
3
13
24
34
36
44
66
70
66
2
3
4
6
11
20
26
31
47
0
3
2
0
2
3
9
15
18
NS
0
3
11
18
28
38
46
63
70
68
1
2
5
7
11
20
27
22
37
0
3
3
1
5
4
8
19
27
*Means of duplicate treatments.
22
-------
TABLE A-3. PERCENT LOSS* OF METHYL PARATHION FROM NATURAL SEAWATER AS
AFFECTED BY TEMPERATURE, SALINITY, AND STERILITY
Salinity (ppt)
Days Temp .
incubation °C
3
6
9
12 30
18
32
43
3
14
21
28
35 20
42
56
70
90
7
14
21
28
35 10
42
56
77
90
S
19
23
22
30
33
41
76
0
22
24
26
33
40
35
36
44
4
13
15
17
14
16
19
26
24
0
NS
20
24
26
35
40
56
82
0
19
26
26
36
45
44
48
64
6
14
13
15
14
16
19
23
25
10
S
18
23
31
34
36
51
80
0
21
30
27
36
43
40
46
49
7
17
18
15
14
16
21
23
26
NS
27
34
40
43
46
66
82
0
20
42
22
39
42
45
50
63
1
15
16
15
14
14
20
23
25
20
S
16
23
33
40
40
60
82
0
23
24
33
40
48
43
48
58
5
17
17
16
17
17
20
26
29
NS
19
29
42
55
58
77
90
0
23
32
40
41
54
53
56
75
3
5
14
17
15
17
17
26
31
28
S
18
24
33
40
47
64
82
0
19
24
31
38
46
48
52
61
2
14
15
16
16
16
19
25
26
NS
22
29
36
46
52
71
83
0
25
27
38
43
52
56
61
67
0
12
10
14
14
17
17
25
31
*Means of duplicate treatments.
-------
TABLE A-4. PERCENT LOSS* OF DIAZINON FROM NATURAL SEAWATER AS
AFFECTED BY TEMPERATURE, SALINITY, AND STERILITY
Salinity (ppt)
Days Temp .
incubation °C
3
7
14
21 30
28
35
42
56
90
7
14
21
28 20
35
42
56
84
7
14
21
28
35
42 10
56
70
84
98
0
S
4
10
17
27
32
42
49
59
71
1
8
9
13
12
13
28
53
0
0
0
4
0
3
8
14
15
14
NS
4
10
22
31
40
46
49
61
70
2
8
8
12
12
14
1.7
33
0
4
0
7
2
3
1
5
9
15
10
S
5
13
22
35
44
54
61
67
77
3
8
12
17
20
29
37
53
0
0
9
1
9
6
18
16
22
32
NS
7
16
27
38
45
62
63
69
80
0
3
12
11
17
23
30
40
0
3
0
2
3
6
10
12
13
18
20
S
8
15
28
41
63
75
69
82 -
83
3
7
16
20
22
29
34
57
0
0
2
6
7
6
14
19
20
22
28
NS
14
13
27
43
52
62
67
77
81
1
7
22
23
24
32
40
62
1
3
9
13
17
20
24
24
36
24
S
9
24
31
43
62
66
72
82
86
5
7
13
20
21
25
37
46
0
4
2
4
9
9
18
16
24
27
NS
9
22
35
41
62
68
79
83
87
4
12
26
24
27
40
52
61
0
5
11
20
14
14
13
26
40
21
*Means of duplicate treatments.
24
-------
TABLE A-5. PERCENT LOSS OF METHOXYCHLOR FROM NATURAL SEAWATER AS
AFFECTED BY TEMPERATURE, SALINITY, AND STERILITY
Salinity (ppt)
Days Temp .
incubation °C
1
7
14
21
28 30
35
42
56
70
84
0
S
0
0
3.3
4.4
4.2
1.3
5.8
6.3
5.2
12.1
NS
3.9
1.1
6.2
5.1
2.6
8.4
4.1
9.0
9.2
11.9
10
S
3.3
2.8
0
0
4.0
5.7
4.2
6.1
8.2
12.0
NS
2.5
3.3
2.8
4.6
2.0
3.3
1.4
4.5
3.1
9.0
20
S
4.4
2.1
7.0
9.3
6.6
5.6
6.1
4.4
5.5
11.4
NS
0
2.3
4.2
3.8
7.4
3.6
3.8
5.2
10.4
9.7
28
S
9.7
4.8
5.4
5.2
9.4
6.5
5.4
6.3
10.3
12.2
NS
0
2.5
2.0
3.6
5.3
3.7
2.4
10.3
9.6
14.7
84 2,0 Largest losses observed at 20°C under sterile and nonsterile
conditions were 8.5 and 5.1 percent, respectively.
84 10 Largest losses observed at 10°C under sterile and nonsterile
conditions were 1.1 and 1.2 percent, respectively.
Means of duplicate treatments.
25
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-78-(M
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
INSECTICIDE PERSISTENCE IN NATURAL SEAWATER AS AFFECTED
BY SALINITY, TEMPERATURE, AND STERILITY
5. REPORT DATE
MARCH 1978
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
William W. Walker
Gulf Coast Research Laboratory, Ocean Springs, MS 39564
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Microbiology Section, Gulf Coast Research Laboratory
Ocean Springs, MS 39564
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
1EA714
11. CONTRACT/GRANT NO.
GRANT NO. R-803842
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, FL 32561
13. TYPE OF REPORT AND PERIOD COVERED
FINAL
14. SPONSORING AGENCY CODE
EPA/600/04
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The effect of temperature, salinity, and sterility on the degradation of malathion,
parathion, methyl parathion, diazinon, and methoxychlor in fresh and estuarine water
has been determined under controlled laboratory conditions. Surface water samples
of 1, 10, 20, and 28 ppt salinity were amended with the above insecticides and
incubated in the dark at 30°, 20°, and 10°C under sterile and nonsterile conditions.
Insecticide abatement was followed by electron-capture gas-liquid chromatographic
techniques.
No significant differences between sterile and nonsterile treatments were observed
for any of the insecticides studies, while the effect of increasing temperature was
highly significant with regard to increased degradation of malathion, parathion,
methyl parathion, and diazinon. Methoxychlor reflected the recalcitrance charac-
teristic of the chlorinated hydrocarbon insecticides throughout 84 days of incubation
and was not significantly affected by salinity, temperature, or sterility. Salinity
effects were varied among the four organophosphates, being highly significant for
malathion and diazinon, significant for methyl parathion, and not significant for
parathion.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
Malathion
Parathion
Methyl parathion
Diazinon
Insecticides
Moraxella
Pseudomonas
Chlorohydrocarbons
Microbial degradation
Studies
06/06
06/13
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
25
20. SECURITY CLASS {Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
------- |