Ecological Research Series
The Effect of Mirex and Carbofuran
on Estuarine Microorganisms
I
55
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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
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH STUDIES
series. This series describes research on the effects of pollution
on humans, plant and animal species, and materials. Problems are
assessed for their long- and short-term influences. Investigations
include formation, transport, and pathway studies to determine the
fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living
organisms in the aquatic, terrestrial and atmospheric environments.
EPA REVIEW NOTICE
This report has been reviewed by the Office of Research and
Development, EPA, and approved for publication. Approval does
not signify that the contents necessarily reflect the views and
policies of the Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or
recommendation for use.
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EPA-660/3-75-024
JUNE 1975
THE EFFECT OF MIREX AND CARBOFURAN
ON ESTUARINE MICROORGANISMS
By
Dr. Lewis R. Brown
Associate Dean Arts and Sciences
and Professor of Microbiology
Mississippi State University
Mississippi State, Mississippi 39762
Dr. Earl G. Alley
Director Research and IAS Division
Mississippi State Chemical Laboratory
Mississippi State, Mississippi 39762
Dr. David W. Cook
Assistant Director and Head
Microbiology Section
Gulf Coast Research Laboratory
Ocean Springs, Mississippi 39564
Contract No. 68-03-0288
Program Element No. 1-EA077
ROAP/TASK No. 10AKC/33
Project Officer
Dr. Al W. Bourquin
Gulf Breeze Environmental Research Laboratory
Sabine Island
Gulf Breeze, Florida 32561
National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Corvalis, Oregon 97330
For sale by the Superintendent of Documents, U.S. Government
Printing Office, Washington, D.C. 20402
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ABSTRACT
The purpose of this investigation was to help establish the chemical, physical and micro-
biological fate of mirex and carbofuran in the estuarine environment and determine the effect(s) on
important estuarine microorganisms and their activities. Chemical studies on the adsorption, fate
and hydrolysis were conducted. The microbiological studies involved the use of both pure cultures and
mixed cultures in a microcosm system and included twelve distinct physiological groups of micro-
organisms.
It was concluded that neither mirex nor carbofuran would have a deleterious effect on estuarine
bacteria under normal conditions, and there was no evidence of bioaccumulation. Degradation
products of both compounds were shown to be toxic to some microorganisms.
This report was submitted in fulfillment of Contract No. 68-03-0288 by Mississippi State Universi-
ty under the sponsorship of the Environmental Protection Agency. Work was completed as of 31
December 1974.
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CONTENTS
Page
I. Conclusions 1
II. Recommendations 2
III. Introduction 3
IV. Materials and Methods 4
V. Results 14
VI. Summary 44
VII. Discussion 45
VIII. References 46
m
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TABLES
No. Page
1 Adsorption of Mirex on Montmorillonite Clay 14
2 Adsorption of Mirex on Kaolinite Clay 14
3 Adsorption Studies of Carbofuran on Montmorillonite Clay 15
4 Carbofuran Adsorption on Kaolinite Clay 15
5 Fate Studies - Mirex 16
6 Fate Studies - Carbofuran 16
7 Hydrolysis of Mirex 17
8 Hydrolysis of Carbofuran in 15 ppt Salinity 17
9 Hydrolysis of Carbofuran in 0 ppt Salinity 17
10 Effect of Varying Concentrations of Mirex on Growth of
Pure Cultures of Bacteria in Broth Medium 19
11 Effect of Varying Concentrations of Carbofuran on Growth of
Pure Cultures of Bacteria in Broth Medium 19
12 Effect of Mirex and Carbofuran on Methane Consumption by
Microflora in Estuarine Sediments 20
13 Effect of Mirex and Carbofuran on Methane Consumption by
Methanomonas methanooxidans 20
14 Effect of Mirex and Carbofuran on Ammonification by
Four Pure Cultures of Bacteria 21
15 Effect of Mirex and Carbofuran on Nitrate-Reduction by Achromobacter (#274) 21
16 Effect of Mirex and Carbofuran on Nitrification by
Microorganisms in Estuarine Sediments 22
17 Effect of Mirex and Carbofuran on the Metabolic Activity and
Primary Productivity of Estuarine Pond Water 22
18 Effect of Mirex and Carbofuran on the Metabolic Activity and Primary
Productivity of Estuarine Pond Water Enriched for Phytoplankton 23
19 Comparison of Chemotactic Response of the Estuarine Bacterium Toward 10 ~6M
Asparagine in the Presence or Absence of 10 ppm Mirex or Carbofuran 23
20 Comparison of Chemotactic Response by Estuarine Bacterium Toward 10 "6M
Asparagine in the Presence or Absence of 50 ppm Mirex or Carbofuran 24
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TABLES (Continuted)
No. Page
21 Comparison of Chemotactic Response of the Estuarine Bacterium Toward 10"4M
Asparagine in the Presence or Absence of 50 ppm Mirex or Carbofuran 25
22 Comparison of Chemotactic Response of the Estuarine Bacterium Toward 10 ppm
Mirex or Carbofuran in the Presence of 0.08% Nutrient Broth 25
23 Comparison of Chemotactic Response of the Estuarine Bacterium Toward 50 ppm
Mirex or Carbofuran (as residue) in the Presence of 0.08% Nutrient Broth 26
24 Microbial Counts on Sediment Samples from Microcosm Tanks
Immediately after Amending with Mirex 26
25 Microbial Counts on Sediment Samples from Microcosm Tanks
One Week after Amending with Mirex 27
26 Microbial Counts on Sediment Samples from Microcosm Tanks
Two Weeks after Amending with Mirex 27
27 Microbial Counts on Sediment Samples from Microcosm Tanks
Four Weeks after Amending with Mirex 28
28 Microbial Counts on Sediment Samples from Microcosm Tanks
Immediately after Amending with Carbofuran 28
29 Microbial Counts on Sediment Samples from Microcosm Tanks
One Week after Amending with Carbofuran 29
30 Microbial Counts on Sediment Samples from Microcosm Tanks
Two Weeks after Amending with Carbofuran 29
31 Microbial Counts on Sediment Samples from Microcosm Tanks
Four Weeks after Amending with Carbofuran 30
32 Mean Bacterial Counts (x 104) in Sediments Amended with 100 ppm Mirex and
Held Under Various Conditions of Salinity and Temperature 31
33 Mean Bacterial Counts (x 104) in Sediments Amended with 100 ppm Carbofuran and
Held Under Various Conditions of Salinity and Temperature 32
34 Dehydrogenase Activity in Sediments Treated with Different Preparations and
Concentrations of Mirex 32
35 Dehydrogenase Activity in Sediments Treated with Different Preparations and
Concentrations of Carbofuran 33
36 Proteolytic Activity of Microorganisms in Sediment Samples
Amended with Carbofuran and Mirex 34
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TABLES (Concluded)
No.
37 Mineralization of Glucose by Microorganisms in Sediments Amended with
Various Concentrations of Carbofuran and Mirex ....................................... 34
38 Oxygen Consumption During Glucose Utilization by Organisms in
Sediments Amended with 1000 ppm Carbofuran ........................................ 35
39 Bioaccumulation Studies Using Carbofuran ............................................ 36
40 Bioaccumulation Studies of Mirex by Culture #280 (Benecka) ............................ 37
41 Results of "Disc Assays" of UV Irradiated Hexachlorobenzene and
Mirex Against Culture #274 (Achromobacter) ........................................... 38
42 Effect of Mirex and Carbofuran Derivatives on Methane Consumption by
Microflora in Estuarine Sediments [[[ 39
43 Effect of Derivatives of Mirex and Carbofuran on
Ammonification by a Pure Culture of an Estuarine Bacterium - #280 (Benecka) .......... 40
44 Effect of Mirex Derivatives and Carbofuran Derivatives on
Nitrate-Reduction by an Estuarine Bacterium - #280 (Benecka) .......................... 40
45 Effect of Mirex and Carbofuran Derivatives on Nitrification by
Microorganisms in Estuarine Sediments ................................................ 41
46 The Effect of Mirex and Carbofuran Derivatives on the Metabolic Activity and
Primary Productivity of Estuarine Pond Water ......................................... 41
47 The Effect of Mirex and Carbofuran Derivatives on the Metabolic Activity and
Primary Productivity of Estuarine Pond Water Enriched for Phytoplankton ............. 42
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ACKNOWLEDGMENTS
The contribution of the following individuals is acknowledged with sincere appreciation: Mr.
Gary W. Childers and Mr. Dinesh D. Vaishnav of the Microbiology Department of Mississippi State
University; Mrs. Terri Gray, Mrs. Ming Ming Fang, Mr. Larry Lane and Dr. B. R. Layton of the Mis-
sissippi State Chemical Laboratory; Miss Sandra Lofton of the Gulf Coast Research Laboratory; and
Dr. C. M. Ladner of the Mississippi State University-National Space Technology Laboratory.
vn
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SECTION I
CONCLUSIONS
It is concluded from this study that: (1) essentially all of the mirex in aqueous solution will be
removed by adsorption to particulate matter (clay and dead bacterial cells) and will resist degrada-
tion; (2) only a small percentage (<15%) of carbofuran will be removed from aqueous solution by ad-
sorption but appreciable hydrolysis will occur; (3) there was no evidence of bio accumulation of either
mirex or carbofuran; (4) a wide variety of physiological types of bacteria studied in both pure culture
and in microcosm systems were not adversely affected by mirex or carbofuran in concentrations up to
100 ppm although both compounds inhibited primary productivity and (5) a variety of degradation
products inhibit a number of different microorganisms and processes (particularly nitrification and
primary productivity) at concentrations as low as 1 ppm, but it is considered unlikely that these com-
pounds would ever reach the 1 ppm concentration in the estuarine environment.
Based upon the data obtained in this investigation it is concluded that mirex and carbofuran in
concentrations below 100 ppm would not have a deleterious effect on estuarine bacteria.
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SECTION II
RECOMMENDATIONS
It is recommended that studies be conducted to identify the microbiologically toxic material(s) oc-
curring in some batches of mirex as well as the products resulting from UV radiation of moist mirex.
The toxicological and chemical behavior of conjugates of carbofuran metabolites should be per-
formed to completely delineate the effect of this pesticide.
Since mirex and probably carbofuran will partition into an organic phase from an aqueous
system and because oil is a common contaminant in estuaries, it is recommended that the impact of
oil-mirex and oil-carbofuran systems should be examined for their deleterious impact on estuarine
organisms, biodegradability and chemical and physical fate.
These investigations should be followed by studies of more complex systems which would include
higher members of the food chain, both in the laboratory and in the field studies.
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SECTION III
INTRODUCTION
The primary utilization of mirex as an insecticide is in the control of the western harvester and
the imported fire-ant. The latter species infest most of the southeastern United States and millions of
acres are treated annually in this area by aerial application of baits containing this toxicant at a rate
of 0.20 to 1.66 g per hectare. There are few areas in the southeast that do not have some history of
mirex application.
Carbofuran is registered for utilization on field corn, rice, peanuts, alfalfa and sugar cane (EPA1).
It is applied to and incorporated into the soil at rates from 45.9 to 1725.5 g per hectare.
With the history and rate of application of these pesticides to the greater part of the southeast, it is
obvious that some portion, both of mirex and carbofuran, will enter the estuaries of the Gulf of Mexico.
In these estuaries, microorganisms are a vital link in the overall ecological system. There are no data
available on the effect of either mirex or carbofuran on this estuarine microbial ecosystem.
In order to fill this gap in our existing knowledge, the Environmental Protection Agency awarded
Mississippi State University Contract No. 68-03-0288, "The Effect of Mirex and Carbofuran on Es-
tuarine Microorganisms".
The objective of this investigation was to supply a sufficient amount of scientifically valid data
on the fate and effects of mirex and carbofuran in the estuary in such a form that these data can be
employed in deriving water quality standards for these pesticides. Specifically, the work was design-
ed to establish the chemical, physical and microbiological fate of these pesticides in the estuarine en-
vironment and determine meaningful effect(s) on important estuarine microorganisms and their ac-
tivities. It is extremely important to maximize the transferability of scientific results to the environ-
ment, and thus the experimental approach described involves the use of microcosm systems as well as
pure culture studies.
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SECTION IV
MATERIALS AND METHODS
MATERIALS
Carbofuran
Technical carbofuran (98% purity) and analytical standards (99% purity) were obtained from
FMC Corporation. Analytical standards of 3-hydroxy- and 3-ketocarbofuran as well as the three
phenol metabolites were obtained from the same source. Technical carbofuran was further purified by
recrystallization from toluene in one case, and ethanol in another. (See Figure 1)
Mirex
One batch of mirex (batch 1) was obtained from the FDA Laboratory at Perrine, Florida. All other
batches of mirex (technical) were obtained from Allied Chemical Company. This material was
purified by chromatography on a silica gel column with cyclohexane as the solvent. Examination of
the technical material and some analytical standards showed the presence of hexachlorobenzene and
other less abundant compounds of similar volatility. Hexachlorocyclopentadiene was usually present
in very small quantities; and, while the other compounds could not be identified, it was shown that
they were not hexachlorofulvene or l,2,3,4,5-pentachloro-5-trichloromethyl cyclopentadiene. The
total of these impurities was estimated to be about 0.1%. The chromatographic purification effectively
removed these impurities when only the early eluting fractions were utilized.
The major product of the pyrolysis of mirex is hexachlorobenzene (Kennedy et al.2), so the
presence of this impurity is not surprising.
Kepone
Technical kepone was obtained from Allied Chemical Company and purified by azeotropic dis-
tillation of water and crystallization from benzene.
Mirex and Kepone Photoproducts
1,2,3,4,5,5,6,7,9,10,10 undecachloro[5.3.0.02-6.03-9.0".8]pentacyclodecane, 1,1,3,4,5,5,6,7,9,10,10-
decachloro [5.3.0.02'6.03. 9.04. 8] pentacyclodecane 2, 1,2,3,4,5,5,6,7,8,9,10 undecachloro
[5.3.0.02-6.03' 9.04-8] pentacyclodecane 3 and 1,2,3,4,5,6,7,8,9,10-decachlora [5.3.0.02' 6.03> 9.04>8] pen-
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tacyclodecane, 4 were prepared from mirex by photochemical methods (Alley et a/.3 4). 1,2,3,4,6,7,8,9,-
10,10 decachloro-5 hydroxy-[5.3.0.02 '6.03. 9.04> 8] pentacyclodecane, 5 was prepared by the method of
Billing5. (See Figure 2)
Medium
Unless otherwise noted, all media used in this study were obtained from Difco Laboratories and
prepared with Rila sea salts mixture to obtain 15 ppt salinity. The mineral salts medium contained 1.0
g KNO3; 0.5 g K2HPO4.3H2O; 0.2 g MgSO4«7H2O; and 0.05 g FeCl3«6H2Oper 1000 ml distilled water
(final pH adjusted to 7.0).
Cultures
Pure cultures of estuarine bacteria were obtained from the stock collection at Gulf Coast Research
Labortory, Ocean Springs, Mississippi. Methanomonas methanooxidans (Brown and Strawinski),
Mycobacterium, and Nocardia were obtained from stock collection at Mississippi State University.
Sediments used for the study of methane consumption and nitrification were obtained from Biloxi
Bay, Mississippi.
Figure 1. Carbofuran and derivatives
Carbofuran, R]_ = R2 H
3-hydroxycarbofuran, R-^ = H, R2 = OH
3-ketocarbofuran, R,, R2 0
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Figure 2. Structural configuration of mirex and derivatives of mirex.
For mirex, R, - R2 = R3 = R4 = R5 = Cl
For kepone, R + R = double bond oxygen; R3R4R5=C1
For 8-hydrogen derivative, 1,R4 = H;R,-R2 = R3 = R4=C1
For 2,8-dihydrogen derivative, 2, R4=R5 = H;R,R2 = R3-C1
For 10-hydrogen derivative, 3, R, = H;R2 = R3 = R4 = R5 = C1
For 5,10-dihydrogen derivative, 4, R, = R3 - H; R2 = R4 - R5 = Cl
For reduced kepone, 5, R3 - OH; R, = R2 = R4 = R5 = Cl
METHODS
Chemical Methods
Analysis of Carbofuran and its Derivatives-
A number of problems was encountered in attempts to develop good analytical methods for these
compounds. Direct analysis by gas chromatography was extensively studied utilizing a conductivity
detector that is specific for nitrogen. Despite numerous approaches that were tried, decomposition
during the analysis turned out to be an insurmountable problem.
Direct determination by ultraviolet absorption (Metcalf, et a/.6) was not suitable for the
hydrolysis studies because it was shown that the absorption observed by these workers was not due to
either the carbofuran or any of its identified derivatives, including the phenol.
Several other methods were tried and failed, but two methods were found to provide satisfactory
results. The gas-liquid chromatography (glc) method is most suitable for low levels and where car-
bofuran and its 3-hydroxy- and 3-keto-derivatives are expected to occur together. The high pressure
liquid chromatography (hplc) method is best suited for higher concentrations and where only the car-
bofuran is expected to occur along with its phenolic hydrolysis product.
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Analysis of Carbofuran in Water by hplc-
Analyses were performed directly on the aqueous solutions by injecting the samples into a 1.8 m
by 1.5 mm i.d. column packed with Corasil II. The packing was deactivated by equilibrating it with
diisopropyl ether saturated with water and the same solvent system was used for the mobile phase.
Carbaryl was used as an internal standard and the quantitation done by measuring peak heights.
Analysis of Carbofuran in Sediments-
Ten grams of sediment and water were shaken with 50 ml of methylene chloride in a centrifuge
bottle. The sediments were removed by centrifugation and filtration through glass wool. The sedi-
ment was treated twice more with 25 ml of methylene chloride. The combined extracts were placed in a
separatory funnel, and the methylene chloride layer was removed. The aqueous phase was washed
with 25 ml of methylene chloride, the combined extracts dried with sodium sulfate and the solvent
removed with a Kuderna-Danish evaporator. The residue was taken to dryness under a stream of
nitrogen and then redissolved in 5 ml of 15% diethyl ether in petroleum ether. The entire sample was
introduced into a 1 cm diameter column containing 4 g qf activated florisil which had been prepared
with 10 ml of petroleum ether. The column was eluted with 70 ml of 15% diethyl ether in petroleum
ether followed by 100 ml of 50% diethyl ether. The 50% fraction was reduced to a small volume and
transferred to a Miniaktor (Applied Science Laboratories, Inc.) and the solvents removed with a
stream of nitrogen. Ethyl acetate (0.2 ml) and heptafluorobutyric anhydride (0.1 ml) were added to the
reactor, and it was tightly capped, wrapped with aluminum foil and heated at 45 C for 16 h. The con-
tents were diluted with 10 ml of petroleum ether, washed three times with water, and the organic layer
dried and made to an exact volume for electron capture gas chromatographic analysis. The
parameters for this analysis were: a 1.5 m by 4 mm i.d. all glass column utilizing on-columninjection
was used; the column temperature was 165 C, the injector 245 C, and the detector 210 C. The column
was packed with 3% diethylene glycol succinate on Gas-Chrom Q. With these conditions, Carbofuran
had a retention time of 1.2 min, 3-hydroxycarbofuran 1.8 min, and 3-ketocarbofuran 2.4 min.
Analysis of Mirex and its Derivatives-
Mirex and the photoproducts described in the materials section can all be quantitated by the
following procedures.
Analysis of Mirex in Water-
A sample of the water to be analyzed was extracted three times with 75 ml portions of petroleum
ether. The combined extracts were dried and passed through a florisil column. Petroleum ether was
used to elute the column and the eluate concentrated to 1-10 ml and then quantitated by electron cap-
ture gas chromatography. This method was shown to produce high recoveries (90-100%) of the com-
pounds.
Analysis of Mirex in Sediments-
Sediments were analyzed in the same manner as waters except that the samples were shaken
with two volumes of 1:1 acetone-petroleum ether. The samples then were centrifuged and the superna-
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tant decanted into a 1 liter separatory funnel. The petroleum ether layer was washed twice with water,
the organic layer dried and introduced into a florisil column. After this point in the procedure, the
water method was followed exactly.
Other Chemical Methods-
Nitrate, nitrite and ammonia were determined by standard procedures (APHA7). Protein-
nitrogen was determined according to the modification of Lowry's procedure (Cook8).
Microbiological Methods
Disc Assay Techniques-
A number of variations of the disc assay procedure was employed to test the effect of mirex on
bacteria. Tests involved dissolving test compounds in acetone and placing the material on number
740E, 12.7-mm paper discs (Scheicher and Schnell, Inc., Keene, N.H.). The saturated discs were kept
overnight to allow the acetone to evaporate. In other tests, solid discs of test compounds were prepared
by using an hydraulic press with a 12.7-mm die and 137,880,000 N/m2 (14,062,000 kg/m2; 1 kg =9,805
newtons) pressure. In other tests, loose particles of test compounds were applied directly on the sur-
face. In all cases, seeded nutrient agar plates were employed with incubation at 30 C for 24 h.
Growth Curve Studies-
Nutrient broth medium was employed and growth was monitored by plate count or changes in op-
tical density of the broth using a B&L Spectronic-20 colorimeter (520 nm). Studies were conducted in
duplicate at 30 C for 57 h.
Qualitative Physiological Studies-
Qualitative observations of test-tube reactions were employed to evaluate the effect of varying
concentrations of test compounds on glucose fermentation and hydrogen sulfide production. Glucose
fermentation studies were conducted in Durham fermentation tubes using 1% glucose broth with
phenol red indicator. Lead acetate semisolid agar was employed to test for hydrogen sulfide produc-
tion. For these tests, the compounds were added aseptically to the test media after sterilization. In-
cubation was at 30 C for 5 days.
Bacto-gelatin agar, Bacto-spirit blue agar with cottonseed oil, Bacto-starch agar, skim milk, and
finely ground purified chitin in Vz strength Bacto-marine agar were employed for the studies on
gelatin-, lipid-, starch-, casein-, and chitin-hydrolysis, respectively. Test compounds were applied to
the plates using three different techniques: (1) test compounds (in acetone) were spread over the en-
tire surface of the agar, the acetone was allowed to evaporate, and the test bacteria were streaked in a
single line across the surface; (2) the test compounds (in acetone) were applied in a single line on the
surface of the agar and the test culture was streaked evenly over the entire plate; and (3) filter paper
strips impregnated with test compounds were placed in the agar for streak plates or on the surface of
pour plates.
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Quantitative Physiological Studies -
Tests on the consumption of methane by estuarine sediments were conducted in Sohngen units as
modified by Hutton and ZoBell9. The inoculum was prepared by making a slurry of 1000 g of sediment
and 3000 ml of 15 ppt salinity mineral salts medium (prepared with Rila sea salts). Aliquots of 50 ml of
the slurry containing 0.0, 0.1,1.0, 10.0, or 100.0 ppm of the test compounds were placed in the "reac-
tors". The air atmosphere of the "reactors" was replaced with a gas mixture consisting of 65% CH4,
30% 02, and 5% CO2. Incubation was carried out at 30 C under static conditions. Gas consumption was
measured daily in terms of millimeter increase in height of the liquid in the "reactors".
Experiments employing Sohngen units for the studies utilizing M. methanooxiddns were con-
ducted in accord with the procedures described by Brown et at10. Appropriate concentrations (same as
in sediments above) of filter-sterilized test compounds in acetone were added to empty "reactor"
bottles, and the acetone was allowed to evaporate prior to the addition of the mineral salts medium.
After gassing, the "reactors" were inoculated with 3 ml of a culture of M. methanooxidans, and the
units were incubated at 30 C under static conditions.
Medium for the studies on ammonification was peptone broth containing Rila sea salts mixture to
yield 15 ppt salinity. The culture vessels were 500 ml Erlenmeyer flasks each containing 300 ml of
medium. Filter-sterilized test compounds were added to the flasks so that the final medium contained
100 ppm. The inoculum consisted of 1 ml of a 24 h broth culture of the test organism, and incubation
was carried out at 30 C for 5 days under static conditions, in duplicate. Optical density and ammonia-
N were measured daily. Protein was determined on the filtered (0.45 ^ membrane filter) medium ini-
tially and at the termination of the experiment.
Experiments on nitrate reduction were conducted in duplicate in 500 ml Erlenmeyer flasks each
containing 300 ml of Bacto-nitrate broth. The concentrations of test compounds in these tests were 0,
1,10, and 100 ppm. Each flask was inoculated with 1ml of a 24 h broth culture of Achromobacter #274
and incubated at 30 C under static conditions. Optical density, NO3-N, and NO2-N were determined
initially and thereafter at 6 to 12 h intervals up to 48 h.
Nitrification studies were performed in 500 ml Erlenmeyer flasks each containing 300 ml of
ammonium-calcium carbonate medium at a salinity of 15 ppt. Test compound concentrations were 0,
1,10, and 100 ppm. The inoculum was 5 ml of a mixture of one part estuarine sediment to three parts
mineral salts medium at a salinity of 15 ppt. All tests were conducted in duplicate, and incubation was
carried out under shake conditions at ambient temperature. Substrate depletion (NH3) and product
formations (NO3, NO2) were determined initially and every 5 days thereafter up to 20 days.
Primary productivity studies were conducted utilizing estuarine pond water directly or after enrich-
ment of the phytoplankton population by incubation in the presence of added nitrate, phosphate and
light for 12 h. Test compounds (contained in acetone) were pipetted into each 300 ml B.O.D. bottle and
10 ml of boiling distilled water added immediately. The bottles then were filled with the test water and
incubated at ambient temperature for 12 h between 2 sets of flourescent lights, each having 8 light
bulbs. Bottles used for the dark reaction were painted black and covered with aluminum foil. Solvent
controls and controls containing 1000 ppm HgCl2 were included in the tests. Dissolved oxygen (D.O.)
was determined with an Oxygen Meter Model YSI54.
Studies on Chemotaxis -
A gram negative rod-shaped motile bacterium was isolated from an estuarine environment (Bay
St. Louis, MS) on nutrient agar increased to 15 ppt salinity.
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The isolate was inoculated into an Erlenmeyer flask containing Bacto-nutrient broth having a
salinity of 7.5 ppt and incubated at ambient temperature under shake conditions for 15 to 18 h. Cells
were harvested by centrifugation at 20 C at 4500 rpm for 10 min, washed 3 times in physiological
saline, and resuspended to the desired concentration in a solution having a salinity of 7.5 ppt. Acetone
containing mirex or carbofuran at 10 ppm or 50 ppm was pulled into capillary tubes by capillary ac-
tion until the tubes were % full. The tubes were then placed in an oven at 80 C, thus leaving a residue of
the pesticide on the internal walls of the capillary tube. Acetone "residue" tubes were prepared in a
similar manner except no pesticide was contained in the acetone.
Chemotaxis was determined by use of the capillary tube technique, and the procedures employed
were a modification of those described by Mitchell, Fogel and Chet11-
Capillary tubes were filled % full with the desired test solution; subsequently the top ends of the
tubes were plugged with silicone grease and the outsides of the tubes were washed in a stream of water
and wiped dry with a sterile Kim wipe®. The tube was inserted into a small vial containing 0.4 ml cell
suspension of approximately 3.0 x 109 viable cells/ml for 12 min. Subsequently, the tube was removed,
washed in a small stream of water and broken into 3 or 4 sections (excluding upper 1A section) into
physiological saline diluent. Dilutions in physiological saline were prepared, plated, and spread on
the surface of nutrient agar plates containing 7.5 ppt salinity.
Microcosm Studies-
Microcosms consisted of all glass vessels (usually 4 liter wide mouth jars) containing a mixture of
estuarine sediment and sea water. This mixture was prepared by homogenizing sediment from the top
11.6 cm of the estuary bottom with artificial sea water in a ratio of 2:1 (w/w) using a large Waring
blender. Within 24 h after the sediment mixture was placed in the vessel, settling occurred and about a
3 cm deep layer of clear water appeared over about 9 cm of sediment, with the top sediment layer ox-
idized and the bottom layer reduced as indicated by the color. The microcosm thus mimiced the es-
tuarine sediment system. Some infauna forms of polychaetes survived the homogenizing and became
active in the microcosms.
Sediments in these studies were obtained from Davis Bay and were generally described as "sandy
course silt" Sediments were checked for mirex and it was not detected.
A single lot of technical grade carbofuran (Niagara Chemical Division, FMC Corporation,
Middleport, N. Y., ME-L514,4717-54-A, 8/9/73) was used for all the sediment microcosm studies. Two
lots of technical grade mirex were used with lot number SN-16-6988-43 (Allied Chemical Corp.,
Baltimore, Md.) being used for studies on the effect of various mirex concentrations on the
heterotrophic bacterial populations and the various biochemical groups of organisms. Lot number
SN-3-100-36 (Mirex Technical, Allied Chemical Corp., Baltimore, Md.) was used for the temperature-
salinity studies and for all other studies. The purified mirex and carbofuran were prepared by
recrystallization, and the water washed carbofuran prepared by placing in a filter funnel and
washing with distilled water until the filtrate was colorless.
Pesticides in concentrations of 1 ppm and greater were added as the dry powder by weight during
the sediment mixing procedure. The amount added thus represented the addition to the whole system.
However, after the microcosm settled, most of the pesticides were probably in the sediment. For a
pesticide concentration of 0.1 ppm, 10 mgof the pesticide were dissolved in 100 ml of acetone and 0.1
ml of this solution added to each 1000 g of the sediment water mixture.
10
-------
Rila sea salts dissolved in distilled water was used in all studies at a salinity of 15 ppt unless
otherwise noted.
Microcosms were incubated in the dark at 25 C unless noted.
To sample the sediment in the microcosm, the entire contents were thoroughly mixed with a
mechanical stirrer. The desired amount of sediment was then removed with a sterile wide tip pipet.
All dilution water blanks were made with artificial sea water diluted to the same salinity as the
water in the microcosms being sampled. Incubation was carried out for 10 days at 25 C. The base
medium was composed of 0.5% peptone and 0.1 % yeast extract prepared with artificial sea water of the
desired salinity and modified by the addition of appropriate materials. Solidified media were
prepared by the addition of 2% agar (Difco). The conventional spread plate technique was employed
with incubation under air for aerobic counts and incubation under an atmosphere of 95% N2-5% CO2
for anaerobic counts. The spot plate technique employed for determining starch-, gelatin-, casein-,
lipid-, and chitin-utilizing populations was conducted as follows: Three 0.01 ml portions of each sam-
ple were spotted on plates of media. After incubation, the plates were developed, and the number of
positive reactions out of the three possible were recorded at each dilution. Numbers of organisms were
calculated using a standard three-tube MPN table.
The standard three-tube MPN procedure was employed for estimating the numbers of sulfate-
reducers, organisms capable of producing H2S from organic sulfur, nitrate-reducers and am-
monifiers.
Tests on dehydrogenase activity were carried out as follows: Two g portions of sediment were
weighed into glass centrifuge tubes. Fifty to 100 mg of CaCO3 powder were added to each tube and,
with the exception of the blank, all tubes received 0.5 ml of a 1% solution of triphenyl tetrazolium
chloride (TTQ prepared in 15 ppt artificial sea water. Samples were mixed, placed in a vacuum desic-
cator, evacuated for 10-15 min, and incubated under vacuum at 30 C. At the end of 22 h the vacuum
was released and 10 ml of methanol added immediately to each tube. Samples were mixed and cen-
trifuged at low speed for 2-3 min. The supernatant then was filtered through a cotton plugged funnel
into a 50 ml volumetric flask and the methanol extraction procedure repeated using 10 ml amounts of
methanol until color could no longer be extracted from the sample. The filtrate was made to volume (50
ml) using methanol. The color intensity was measured at 485 nm on a B&L Spectronic 20 colorimeter
using the blank as a reference. Results were calculated from a standard curve and expressed as .jg
triphenyl tetrazolium formazan (TPF) per g of sediment.
Studies on the oxygen consumption by organisms in sediments were conducted using conven-
tional Warburg manometric techniques with glucose as a substrate. Sediment samples were amended
with 1000 ppm carbofuran and incubated. This mixture was subsampled after 5 and 12 days incuba-
tion and 2.5 g wet weight was used in each Warburg flask. The salinity was 15 ppt and temperature
was 25 C.
For the glucose mineralization experiments, the procedure employed was a modification of
Harrison et a/.12 The major change involved the amount of C-14 glucose added (0.1 ^Ci per bottle) and
the incubation time (1 h).
Proteolytic activity studies were monitored in terms of the change in soluble protein concentra-
tion within a sediment slurry to which peptone had been added as the protein source.
Five g of sediment and ten ml of sea water (15 ppt) containing 10 mg of Bacto-peptone was placed
in 60 ml prescription bottles and incubated with shaking for 22 h at 25 C. The slurry was centrifuged at
high speed to separate the liquid from the cells and sediment. The supernatant was checked for pro-
tein content using the method of Lowry et a/.13
11
-------
It was found that carbofiiran reacted with the reagents used in the protein determination produc-
ing an overestimation of the protein concentration. Blanks without added peptone were used to com-
pensate for this error.
Bioaccumulation-
These studies were conducted with culture #280 (Beneckd) using mineral salts medium (15 ppt)
with static incubation at 30 C. Inoculum consisted of cells from a 24 h, 30 C nutrient broth-grown
culture which had been centrifuged, washed three times in saline and resuspended in the same solu-
tion. The dead cell controls were prepared by heating a portion of the cell suspension for 15 min at 80
C. For the studies utilizing mirex, a saturated solution was obtained by stirring 2 g of mirex in 31 of
mineral salts medium for 3 days. This solution then was filtered through a 0.45n membrane filter and
the clear filtrate was considered to be saturated with mirex (approximately 10"9 g/ml). For studies
utilizing carborfuran, a 150 ppm concentration was obtained in the test flask by dissolving 40 mg of
carbofuran in 1 ml of ethanol and adding 0.75 ml into 100 ml of mineral salts medium.
The tests were conducted as follows: A 5% inoculum of both living and dead cell suspensions was
separately placed in flasks containing mineral salts medium with 1 % glucose and the test compounds.
The test flask along with several control flasks was incubated for 48 h at 30 C under static conditions.
Cells then were removed from the respective media by centrifugation followed by filtration through
0.45.J membrane filter. The clear filtrate was analyzed for the test compound to determine how much
of the material either adsorbed to the cells or was bioaccumulated. Bioaccumulation is defined here as
the difference between the amount of test material adsorbed to the dead cells and the amount of
material found in the live cells. Dry weights were determined initially and after 48 h on the test flask
containing live cells to determine the increase in cell mass.
Isolation of Mirex- and Carbofuran-Utilizing Microorganisms-
Throughout the course of this contract a considerable effort was put forth to isolate mirex- and
carbofuran-utilizing microorganisms. In a number of cases encouraging results were obtained but in
no instance was it proven that a particular culture could use either of these compounds.
In regard to carbofuran, it was not anticipated that problems would arise in the isolation of an
organism capable of utilizing this compound. Most of the work was directed toward obtaining a
culture that was capable of utilizing carbofuran either as a sole carbon and/or nitrogen source. In
most of the work minimal media were employed and it is entirely possible that richer media con-
taining vitamins, accessory growth factors, etc. might have yielded different results. While efforts to
isolate a carbofuran-utilizing microorganism were unsuccessful, this fact is not interpreted to mean
that such organisms do not exist, but different methodology, different inocula, etc. should be
employed in future work.
Similarly, no mirex-utilizing microorganisms were obtained even though a considerable amount
of effort was expended in the search; and, at times results seemed quite encouraging. Cultures
suspected of being mirex-utilizers (demonstrating growth in the presence of mirex and hexane and the
concurrent lack of growth in hexane alone) failed to give any concrete evidence of mirex utilization.
No conclusive verification could be obtained using manometric techniques and in general, results
12
-------
were erratic. There is some evidence to support the hypothesis that some of the growth that was
observed and some of the results obtained through manometry were the action of the organism on
either mirex or a compound formed from mirex. Once again, failure to obtain a mirex-utilizing
organism does not prove the non-existence of an organism possessing this characteristic, but rather
reflects only the fact that under the conditions and materials used in this investigation none was ob-
tained.
13
-------
SECTION V
RESULTS
MIREX AND CARBOFURAN
Adsorption Studies with Mirex
Water was saturated with mirex and the amount of mirex in the aqueous phase determined as a
function of time by extraction with hydrocarbon solvent and analysis made by electron capture gas
chromatography. The saturated solutions were made by stirring mirex hi distilled water for 25 h
followed by removal of the excess mirex with a millipore filter (0.45J. Table 1 summarizes the results
for montmorillonite clay and Table 2 for kaolinite clay.
Table 1. ADSORPTION OF MIREX ON MONTMORILLONITE CLAY
Time
days
Concentration mirex UK/ml
0 pg/ml
clay
4 0
7 0
14 0
30 0
.003
.004
.001
.002
0.9 ug/ml
clay
0
0
0
0
004
002
002
00007
0.45 pg/ral
clay
0
0
0
< 0
002
001
003
00003
0.09 Mg/ml
clay
0
0
0
< 0
003
003
001
00003
0.045 ug/ml
clay
0.
0.
0.
< 0.
002
001
006
00003
No detectable mirex residues were found in the samples containing only clay. Adsorption to the
glass vessels was not a major problem, as shown by the lack of disappearance of mirex in the systems
without added clay. The precision of the data is not high; it is typical of this type of low-level residue
analysis. For the zero clay samples which should have had constant mirex levels, the percent stan-
dard deviation is 52. It is clear that adsorption or slow chemical reaction does occur on the clay sur-
face, ultimately removing all the pesticide from the aqueous phase.
Table 2. ADSORPTION OF MIREX ON KAOLINITE CLAY
Time
days
Concentration mirex pg/ml
0 pg/ml
clay
4 0
7 0
14 0
30 0
.003
.004
.001
.002
0.9 pg/ml
clay
0
0
0
0
002
0007
0008
00008
0.45 pg/ml
clay
0
0
0
0
002
0008
0007
0001
0.09 pg/ml
clay
0
0
0
< 0
003
0008
0005
00003
0.045 Ug/ml
clay
0.
0.
0.
< 0.
002
0008
0004
00003
14
-------
Adsorption Studies with Carbofuran
Two independent studies were made on carbofuran adsorption to clay. In the first, mont-
morillonite clay was employed and the analyses conducted by ultraviolet adsorption at 276 nm. The
apparent adsorption was about 10-15% after 35 days (Table 3). In the second study a solution of 189
Table 3. ADSORPTION STUDIES OF CARBOFURAN ON MONTMORILLONITE CLAY
Time
days
Absorbances
0 pg/ml
clay
1 1
2 0.93
3
7 1+
14 1+
21 1+
28 1+
35 1+
0.06 Mg/ml
clay
-
0.85
0.93
0.92
0.96
0.98
0.95
0.13 Mg/ml
clay
0.84
0.87
0.84
0.84
0.86
0.89
0.82
0.83
0.6 ug/ml
clay
--
0.87
0.87
0.91
1+
0.83
0.83
ppm carbofuran in 0.05 M sodium acetate-acetic acid buffer at pH 5 was treated with acidified
kaolinite clay such that the clay concentration was 1200 mg/1 (Table 4).
Table 4. CARBOFURAN ADSORPTION ON KAOLINITE CLAY
Time
0
36 mln
81 Bin
180 min
240 min
9 h - 46 min
21 h
28 h - 26 min
54 h
% Carbofuran remaining
in solution
100
72
70
86
82
68
68
75
79
Apparently about 25% of the carbofuran is very rapidly adsorbed under this set of conditions, and
then the system is at equilibrium and no further adsorption is observed.
Fate Studies with Mirex
The analytical method employed in this study had a coefficient of variation of about 25%. Table 5
lists the data obtained on disappearance of mirex from an estuarine sediment - water system. To this
system 0.1 ppm mirex was introduced. A blank control consistently had undetectable levels of mirex.
15
-------
The numbers listed for mirex are all the result of averaging three replicates. The recovery by this
method is 70-80%, and thus the data show that no significant decrease in mirex concentration oc-
curred during the duration of the experiment. No indication of the presence of dehalogenated
derivatives was found. The samples were held in gallon jars during the experiment and had normal
exposure to sunlight.
Table 5. FATE STUDIES - MIREX
Spiked 6-10-74 with 0.01 ppm Mirex
Time/days % Mirex recovered
0
1
2
4
8
16
31
40
53
63
70
81
88
100
113
123
130
75
79
88
83
76
79
78
71
68
77
79
72
71
70
62
68.
64
Fate Studies with Carbofuran
The analyses were conducted by the heptafluorobutyric anhydride derivitization procedure
described in the method section. The coefficient of variation for this method is about 50%. Recoveries
were very poor when preliminary hydrolysis to free conjugated residues were employed and so the
reported data are for free carbofurans. No detectable residues of 3-hydroxy- or 3-ketocarbofuran were
found in any of these samples. The phenols would not be detected by this method. Table 6 is a compila-
Table 6 . FATE STUDIES CARBOFURAN
Time/days
0
1/2
1
2
4
6
10
13
16
41
49
% Carbofuran
81
50
86
53
61
55
64
54
67
33
24.
.7
tion of the data for disappearance of carbofuran from an estuarine sediment-water system. The initial
concentration of carbofuran was 10 ppm. The data are more variable than for mirex. Occasionally one
of the replicates showed no detectable carbofuran. These results were sometimes obtained on identical
samples run in the same set on the same day. The reason for this erratic behavior has not been deter-
mined.
16
-------
Hydrolysis and Aqueous Photochemical Studies with Mirex
Hydrolysis of mirex was studied in four systems: Distilled water, saline water 15 ppt, distilled
water with 1 ppm arginine added, and distilled water with 1 ppm arginine added and exposed to
sunlight. Table 7 summarizes the results.
Table 7. HYDROLYSIS OF MIREX
Time
Days
Distilled
H20
0 0.23
30 0.33
60 0.13
90 0.11
Concentrations
15 ppt
Salinity
0.20
0.14
0.06
0.03
of mirex in
1 wg/1
Arginine
0.13
0.12
0.38
0.05
Ug/1
1 Vg/1
Arginine plus
light
0.23
O.AO
0.13
0.04
The numbers in Table 7 are for the most part the average of three replicates. The percent standard
deviation for these data is about 65. While the precision is not high, the data show a definite trend
towards disappearance of mirex with time.
Hydrolysis of Carbofuran
The hydrolysis of carbofuran was studied in 0.005 M sodium carbonate, buffered solution at 0 and
15 ppt, 25 C and pH 10. The initial concentration was about 300 ppm in all cases. Solutions were
prepared by adding the appropriate amount of carbofuran in 30 ml of ethanol to 220 ml of the buffer
solution. Aliquots were removed at various time intervals for analysis by liquid chromatography.
These data are shown in Tables 8 and 9.
Table 9. HYDROLYSIS OF CARBOFURAN
Table 8. HYDROLYSIS OF CARBOFURAN
15 ppt
Time
(min)
4
9
25
35
45
55
65
75
85
95
105
salinity pHIO 0.005M Na2C03
% Carbofuran
remaining
100
94
80
77
72
70
69
65
63
64
60
0 ppt salinity pH 10
Time
(min)
4
10
20
35
45
55
65
85
95
105
115
% Carbofuran
remaining
100
89
83
73
71
64
62
57
55
55
55
17
-------
The rate constants were calculated for both pseudo-first and second order kinetics. The second
order rate expression gives a slightly better fit, but it was discovered that the pH was dropping slight-
ly during the latter part of the reaction. When the experiment was repeated using 0.05 Mbuffer an ex-
cellent fit for first order kinetics was obtained up through 70%reaction. The rate constants in 0 and 15
ppt solution were identical within experimental error. The rate constant was greater in 0.05 M than in
0.005 M buffer as would be expected if the pH was dropping in the 0.005 M buffer. The first order rate
constant was 0.011 sec l. This is about the same as that observed by Metcalf et a/.6 at pH 9 and 37 C.
Our earlier experiments showed that Metcalf s measurements were not based upon disappearance of
carbofuran or appearance of phenol, but apparently were based on an artifact, and our experiments
indicate that the rate constants they obtained were not related to the rate of hydrolysis of carbofuran.
Disc Assay Studies
The disc assay technique was employed as a rapid screening method to test the inhibitory effect of
the mirex on a variety of pure cultures of bacteria isolated from an estuary. The first series of tests
with the mirex produced zones of inhibition on 18,2,1 and 7 of the 20 cultures tested using four separate
batches of mirex. The results were extremely erratic with three of the batches of mirex in that they did
not consistently and/or repeatedly produce zones of inhibition.
Ten new batches of mirex were obtained from Allied Chemical Company and tested by means of
"disc assay" against 20 bacterial cultures. No zones of inhibition were noted even when the experi-
ment was replicated 5 times. Because of the results cited above, and the fact that gas chromatographic
analyses indicated varying degrees of purity for the different batches, laboratory purified mirex was
employed in subsequent tests unless otherwise noted.
Carbofuran failed to inhibit any of the 20 bacterial cultures when tested using the disc assay
technique.
In another study using technical mirex and technical carbofuran, no inhibition was observed
when tested against 596 isolates from estuarine and non-estuarine origin.
Effect of Mirex and Carbofuran on Growth Curves of Selected Cultures
Tests to determine the effect of mirex and carbofuran on pure cultures in liquid media (15 ppt
salinity) were performed using 8 cultures and concentrations of the test compounds of 0 ppm, 0.1 ppm
and 10 ppm. Essentially no reduction in counts occurred after shake incubation for 72 h (Tables 10 and
11). It should be pointed out that these tests were conducted using technical grade mirex rather than
purified mirex.
In another series of tests optical density was employed to monitor growth. Mirex and carbofuran
at concentrations of 10 and 1000 ppm failed to alter the growth response of any of the cultures tested:
two species each of Bacillus (#222 and #275), Achromobacter (#223 and #274), and Vibrio (#221 and
#295), and one species each of Benecka (#242), Pseudomonas (#297), and Acinetobacter (#173).
Results of Qualitative Studies on Effect of Mirex and Carbofuran
Qualitative tests using mirex and carbofuran in concentrations of 0.1,10.0, and 100 ppm failed to
give any evidence of inhibition of growth, glucose fermentation, or H2S production by any of the
cultures tested (7 glucose fermentors and 4 H2S producers). Visual observations on solid media in-
dicated no inhibition of growth or hydrolyzing ability of 16 gelatin-, 7 casein-, 1 lipid-, 6 starch- and 4
chitin-hydrolyzers.
18
-------
Table 10. EFFECT OF VARYING CONCENTRATIONS OF MIREX ON GROWTH OF PURE CUL-
TURES OF BACTERIA IN BROTH MEDIUM.
Culture
Oh
No /ml
0 . 0 ppm
Bacterial
No A
ol
Mirex (technic
0.1 ppm 1.0
counts
No /ml
after
al)
PPm
72
No/m
h
1
10.0
ppm
incubation
No/
ml
Bacillus (#222) 8.0 x 105 2.0 x 109 4.1 x 109 1.2 x 109 2.4 x 10
Achromobacter (#274) 1.6 x 105 1.5 x 10 9.9 x 10 7.8 x 10 1.0 x 10
Vibrio (#276) 5.1 x 1Q5 4.2 x 108 1.4 x 109 7.8 x 108 9.5 ^ 108
Benecka (#242) 3.3 x 105 2.0 ^ 10 7.9 x 10 9.1 x 10 6.2 x 10
Pseudomonas (#297) 1.2 x 105 6.4 A 108 1.2 x 109 1.3 x 109 2.9 x 10
Achromobacter (#223) 2.0 x 105 3.3 x 108 4.8 x 108 1.6 x 108 0.6 * 108
Enterobacter (#189) 2.7 x 104 1.1 x 108 0.5 x 108 1.3 x 108 1.6 x 108
Acinetobacter (l?173) 1.3 x 105 7.3 x 108 4.4 x 108 4.8 x 108 3.1 x 105
Conditions of test: Growth medium was nutrient broth prepared with 15 ppt
salinity seawater; temp of incubation was 25 C. Plate counts were deter-
mined in duplicate.
Table 11. EFFECT OF VARYING CONCENTRATIONS OF CARBOFURAN ON GROWTH OF PURE
CULTURES OF BACTERIA IN BROTH MEDIUM.
Culture
Oh
No /ml
Carbofuran (technical)
0 . 0 ppm 0 . 1 ppm 1 . 0 ppm
10.0 ppm
Bacterial counts after 72 h incubation
No/ml No/ml No/ml
No /ml
Bacillus (#222) 8.0 x 105 2.0 x 109 2.3 x 109 1.0 x 109 2.3 x 109
Achromobacter («74) 1.6 * 105 1.5 x 109 7.1 x 108 8.8 x 108 8.7 x 108
Vibrio (#276) 5.1 x 105 4.2 x 108 2.3 x 108 8.5 x 108
Benecka (#242) 3.3 x 105 2.0 x 109 5.1 x 108 1.1 x 109 3.2 x 108
Pseudomonas (#297) 1.2 x 105 6.4 x 108 4.2 x 108 9.4 x 108 8.2 x 108
Achromobacter (#223) 2.0 x 105 3.3 x 108 2.6 x 108 4.7 x 108 4.8 A 108
Acinetobacter (#173) 1.3 x 105 7.3 x 108 8.5 x 108 2.8 x 108 5.6 x 108
Conditions of tests: Growth medium was nutrient broth prepared with 15 ppt
salinity seawater; temp of incubation was 25 C. Plate counts were deter-
mined in duplicate.
Effect of Mirex and Carbofuran on Methane Utilization
No inhibition by mirex or carbofuran of methane utilization by estuarine sediments was observed
as determined by the total volume of gas consumed (Table 12). Although not of estuarine origin, a pure
culture of M.methanooxidans was employed in a similar study (Table 13). In this case, however, all
concentrations (0.1, 1.0, 10, and 100 ppm mirex and carbofuran) tested retarded gas consumption for
several days, but after 10 days of incubation, the gas consumption in the presence of test compounds
was the same as that in the absence of test compounds.
19
-------
Table 12. EFFECT OF MIREX AND CARBOFURAN ON METHANE CONSUMPTION BY
MICROFLORA IN ESTUAR1NE SEDIMENTS.
Text system
Control
Mirex 0 . 1 ppm
Mirex 1.0 ppm
Mirex 10.0 ppm
Mirex 100.0 ppm
Carbofuran 0.1 ppm
Carbofuran 1.0 ppm
Carbofuran 100.0 ppm
3
7S
18
15
15
20
11
15
11
4
9
26
24
24
29
11
22
11
5
18
40
35
37
37
20
31
22
Time
6
29
48
44
48
48
26
44
37
33
in
7
37
57
51
59
55
33
46
44
42
Days
8
48
64
59
68
66
42
64
55
53
9
59
75
66
75
77
55
68
66
66
10
70
79
73
77
77
66
75
75
73
11
73
77
79
77
77
68
77
75
77
12
73
77
77
77
77
73
75
77
77
ml of gas consumed ,,
Conditions of test: Sohngen unit apparatus, medium 15 ppt salinity with
mineral salts, incubated under gas atmosphere of 65% CH.t 30% 0^ and
5% CO., static conditions. All values are the average of three deter-
minations.
Table 13. EFFECT OF MIREX AND CARBOFURAN ON METHANE CONSUMPTION BY
METHANOMONAS METHANOOXIDANS.
Test system
Control
Mirex 0 . 1 ppm
Mirex 1 . 0 ppm
Mirex 10.0 ppm
Mirex 100.0 ppm
Carbofuran 0.1 ppm
Carbofuran 1.0 ppm
Carbofuran 10.0 ppm
Carbofuran 100.0 ppm
2
4"
4
4
it
7
4
4
7
4
3
15
11
13
15
13
18
15
15
15
4
29
17
20
22
18
29
22
26
24
Time in days
5 6
46
24
29
249
24
44
31
37
35
66
33
42
42
33
62
46
57
53
7
70
44
53
48
42
70
59
62
64
10
73
70
68
64
70
73
75
70
68
12
75
73
75
75
73
75
77
75
70
14
81
77
75
77
77
80
80
81
77
ml of gas consumed ,,
Condition of test: Sohngen unit apparatus, medium - mineral salts, incu-
bated under gas atmosphere of 65£ CH , 30% 0^ and 5% C0?, static condi-
tions. All values are the average or three determinations.
Effect of Mirex and Carbofuran on Ammonification
The data in Table 14 demonstrate that mirex and Carbofuran (100 ppm) did not inhibit the produc-
tion of ammonia from protein by four cultures of bacteria [Benecka (#280), Pseudomonas (#297), and
two species of Achromobacter (#223 and #274)]. In the case of Achromobacter #274, substrate (pro-
tein) disappearance was decreased by the mirex even though the amount of ammonia produced was
not decreased.
20
-------
Table 14. EFFECT OF MIREX AND CARB07URAN ON AMMONIFICATION BY FOUR PURE CULTURES OF BACTERIA.
Test Culture
No.
Blank
Control 223
100 ppm
Mirex "
100 ppm
Carbofuran "
Control 274
100 ppm
Mlrex
100 ppm
Carbofuran "
Control 297
100 ppm
Mlrex
100 ppm
Carbofuran "
Control 280
100 ppm
Mlrex
100 ppm
Carbofuran "
Optical density of culture
Days Incubation
1 Z 3 4
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0,
00
06
06
06
26
25
23
11
10
11
60
,59
.62
0.00
0.12
0.13
0.12
0.32
0.34
0.32
0.20
0.11
0.12
0.95
0.89
0.95
0.00
0.28
0.20
0.27
0.50
0.47
0.42
0.25
0.26
0.24
1.10
1.00
1.10
0.00
0.35
0.31
0.32
0.62
0.56
0.53
0.38
0.35
0.36
1.15
1.10
1.20
NH,-N (mg/1)
Days or Incubation
1234
0
0
0
0
10
10
10
10
10
10
18
14
10
0
0
6.5
0
13
10
10
24
18
18
77
79
78
0
35
20
22
23
37
40
63
49
45
140
139
140
0
37
30
28
61
557
70
58
68
56
125
133
150
Protein-N"
(mg/1)
initial final
1125
1120
1125
1120
1120
1110
1130
1120
1110
1120
1120
1130
1130
1125
643
643
650
275
553
325
558
555
565
593
583
583
a ppm proteln-N of medium after filtration through 0.45 v membrane filter. Conditions of test: medium: 15 ppt
Rila salts + peptone. Incubation: 30 C, static conditions. All values are the average of two determinations.
Effect of Mirex and Carbofuran on Nitrate Reduction
The effect of mirex and Carbofuran on nitrate reduction by Achromobacter #274 showed that
nitrate utilization and nitrite production were essentially uneffected by concentrations of test com-
pounds up to 100 ppm after 18 h of growth (Table 15). The data do suggest, however, that increasing
concentrations of mirex and Carbofuran delay the onset of nitrate reduction.
Table 15. EFFECT OF MIREX AND CARBOFURAN ON NITRATE-REDUCTION BY ACHROMOBACTER (0274) .
Test system
Control
Mirex 1 ppm
Mirex 10 ppm
Mirex 100 ppm
Carbofuran 1 ppm
Carbofuran 10 ppm
Carbofuran 100 ppm
Hours of incubation
6
N03-N N02ON
(mg/1) (mg/1)
101
110
120
130
1 132
128
124
12
N03-N N02-N
(mg/1) (mg/1)
81 0.75
92 0.60
94 1.0
140
137
100 0.77
137
18
N03-N N02-N
(mg/1) (mg/1)
70 1.48
66 1.70
66 1.50
80 1.15
72 1.30
68 1.58
106 0.50
24 36 48
N03-N N02-N
(mg/1) (mg/1)
66 1.66
63 1.75
65 1.65
60 1.58
66 1.50
65 1.58
68 0.75
N03-N N02-N
(mg/1) (mg/1)
60 1.75
60 1.90
63 1.85
63 1.80
70 1.70
65 1.80
66 1.65
N03-N N02-N
(mg/1) (mE/1)
55 1.75
62 1.75
68 1.80
69 l.oO
71 1.60
70 1.75
81 1.50
All values are the average of two determinations.
21
-------
Effect of Mirex and Carbofuran on Nitrification
To determine if mirex and carbofuran would affect nitrification, estuarine sediments were
employed rather than pure cultures. Within the limits of the tests employed, no deleterious effect on
the process was observed in terms of substrate disappearance (ammonia) or product formation (nitrite
and nitrate) even after 20 days of incubation (Table 16).
Table 16. EFFECT OF MIREX AND CARBOFURAN ON NITRIFICATION BY MICROORGANISMS IN ESTUARINE SEDIMENTS.
Blank
Mirex
1 ppm
10 ppm
100 ppm
Carbofuran
1 ppm
10 ppm
100 ppm
0
NH3-N N02-N N03-N
(mg/1) (mg/1) (mg/1)
125 0 0
125 0 0
120 0 0
119 0 0
120 0 0
118 0 0
120 0 0
D
5
NH3-N N02-N N03-N
(mg/1) (mg/1) (mg/1)
110 0 0
93 0 0
102 0 0
105 0 0
96 0 0
103 0 0
105 0 0
ays of incubation
10
NH3-N N02-N N03-N
(mg/1) (mg/1) (mg/1)
110 0 0
91
100
105
91 "
106 "
100 " "
15
NH3-N H02-N HO -N
(mg/1) (mg/1) (mg/1)
113 0 0
83 " -.025
100 " -.025
100 " -.025
88 " <.025
100 " -.025
100 " -.025
20
NH3-N N02-N N03-N
(mg/1) (mg/1) (mg/1)
120 0 0
92 .05 -.025
110 .01 <-5
110 .01 <.5
93 <.01 -.05
105 -.01 -.5
105 -.01 -.5
Conditions of test: Medium: 15 ppt Rila salts + NH^Cl + mineral salts, pH 7.0, Incubation: 30 C, shake
conditions. All values are the average of two determinations.
Effect of Mirex and Carbofuran on Primary Productivity
As shown in Table 17, neither mirex nor carbofuran inhibited the metabolic activity of estuarine
pond water, but did inhibit primary productivity. These experiments were repeated employing pond
water enriched with phytoplankton. The results (Table 18) confirmed the lack of inhibition of
metabolic activity by both compounds, confirmed the inhibition of primary productivity by car-
bofuran, but differed in the fact that mirex failed to inhibit primary productivity.
Table 17. EFFECT OF MIREX AND CARBOFURAN ON THE METABOLIC ACTIVITY AND PRIMARY
PRODUCTIVITY OF ESTUARINE POND WATER.
Test system
Metabolic activity Primary
(mg 0? consumed/1
Control
Acetone control
HgCl2 control
Mirex (1 ppm)
(10 ppm)
Carbofuran (1 ppm)
(10 ppm)
In
0
0
0
1.
1
1
1
12 h)
.95
.95
.00
.00
.05
.20
.05
productivity
(mg 02
in
2.
2.
0.
1.
1.
1.
1.
produced /I
12 h)
35
10
00
30
70
35
15
All values are the average of two determinations.
22
-------
Table 18. EFFECT OF MIREX AND CARBOFURAN ON THE METABOLIC ACTIVITY AND
PRIMARY PRODUCTIVITY OF ESTUARINE POND WATER ENRICHED FOR
PHYTOPLANKTON.
Test system
Control
Acetone control
HgCl2 control
Mirex 1 ppm
10 ppm
Carbofuran 1 ppm
10 ppm
Metabolic
activity
(mg 02 consumed/1
In 12 h)
1.40
1.30
0.00
1.30
1.10
1.25
1.30
Primary
p roductivity
(mg 0. produced/1
in 12 h)
4.
4.
0.
5.
5.
4.
3.
65
85
,00
.00
.00
05
05
All values are the average of two determinations.
Effect of Mirex and Carbofuran on the Chemotactic Response of an Estuarine Bacterium
Preliminary experiments indicated that concentrations of 10 and 50 ppm of mirex and carbofuran
were not toxic to this bacterium while a concentration of 250 ppm was definitely lethal. Additionally,
it was demonstrated that no significant increase in bacterial numbers occurred during an incubation
period of 54 min. With asparagine as the attractant, a positive chemotactic response toward this com-
pound occurred with increasing concentrations from 10 "6 M to 10"2 M. The bacterium failed to
demonstrate a chemotactic response in tests employing 10"6 M asparagine in the presence of 10 ppm
mirex or carbofuran (Table 19), but gave a positive chemotactic response in the presence of 10~6 M
Table 19. COMPARISON OF CHEMOTACTIC RESPONSE OF THE ESTUARINE BACTERIUM TOWARD ICf M ASPARAGINE IN THE
PRESENCE OR ABSENCE OF 10 PPM MIREX OR CARBOFURAN.
Sample
No.
Contents
in vial Cells in Rila
Acetone residue
Contents in
tube
Rila
30
(a) 34 Ave.
36 35
39
No . colonies
on plate 51
at (a) 57 Ave.
10~J 66 60
dilution 68
30
(a) 30 Ave.
50 41
53
2
Cells In Rila
Acetone residue
10~6M
40
50
50
58
79
82
96
99
56
66
66
76
Rila
Asparagine
Ave.
50
Ave.
89
Ave .
66
3
Cells in Rila
Acetone residue
Mirex residue
Rila
10~6M Asparagine
51
62 Ave.
71 65
76
86
109 Ave.
130 114
133
74
83 Ave.
84 82
87
4
Cells in Rila
Acetone residue
Carbofuran residue
Rila
10 M Asparagine
56
66 Ave.
67 70
90
84
118 Ave.
118 112
128
72
87 Ave.
87 86
98
'a) Replicates of separate experiments.
23
-------
asparagine and 50 ppm mirex or carbofuran (Table 20). This positive chemotactic response was
nullified when 10 4 M asparagine was employed along with 50 ppm mirex or carbofuran (Table 21). In
tests employing nutrient broth (0.08%) as the attractant, no chemotactic response was observed in the
presence of 10 ppm mirex or 10 or 50 ppm carbofuran (Table 22 & 23). Apositive chemotactic response
was observed with nutrient broth (0.08%) in the presence of 50 ppm mirex. This positive response to 50
ppm mirex could be neutralized by incubating the mirex and nutrient broth in combination for a
period of 3.5 h.
Microcosm Studies with Mirex and Carbofuran
The systems, termed here as microcosms, were not true microcosms or mini-ecosystems in that
they were static with no input of light energy or detrital organic matter which is constantly fed into
the natural sediment ecosystem. During construction of the microcosms, the sediments were dis-
turbed which undoubtably altered their nature and possibly the niche of some of the microorganisms
present in the system. Even with these drawbacks it was felt that static microcosm studies were im-
portant in establishing the effect of pesticides upon the mixed microbial flora within the sediments.
Effect on Bacterial Populations-
In microcosm studies the numbers of aerobic heterotrophic bacteria, anaerobic heterotrophic
bacteria and nine biochemical groups of microorganisms were followed for a period of four weeks after
the microcosm sediments were amended with various concentrations of mirex or carbofuran. The
results are summarized in Tables 24 through 27 (mirex) and Tables 28 through 31 (carbofuran).
Changes in the microbial populations at the 0.1 ppm level could not necessarily be linked to the
pesticide because acetone was added as a diluent.
Table 20. COMPARISON OF CHEMOTACTIC RESPONSE OF THE ESTUARINE BACTERIUM TOWARD 10"^ ASPARAGINE IN THE
PRESENCE OR ABSENCE OF 50 PPM MIREX OR CARBOFURAN.
Sample No.
Contents
in vial
Contents in
capillary
tube
No. colonies
on plate
at 10~3
dilution
1
Cells in Rila
Acetone residue
Rila
11
14 Ave.
16 15
20
2
Cells in Rila
Acetone residue
Rila
10~6M Asparagine
21
23 Ave.
33 28
34
3
Cells in Rila
Acetone residue
Rila
10"6M Asparagine
63
74 Ave.
79 76
86
4
Cells in Rila
Acetone residue
Rila
10"6M Asparagine
47
54 Ave.
58 55
60
24
-------
-4
Table 21. COMPARISON OF CHEMOTACTIC RESPONSE OF THE ESTUARINE BACTERIUM TOWARD 10 M ASPARAGINE IN THE
PRESENCE OR ABSENCE OF 50 PPM MIREX OR CARBOFURAN.
Sample No.
Contents
in vial
Contents in
f*an4 1 1
tube
(a)
No. colonies
on plate
at 10~3
dilution
(b)
(a)
Overall ave.
1
Cells in Rila
Acetone residue
Rila
36
41 Ave.
44 42
45
36
43 Ave.
48 47
60
11
14 Ave.
15 16
22
35
2
Cells in Rila
Acetone residue
Rila
10~^M Asparaglne
49
58 Ave.
65 60
66
65
65 Ave.
70 70
81
57
62 Ave.
77 69
79
66
3
Cells in Rila
Acetone residue
. . ,
Rila
10~Sl Asparagine
53
62 Ave.
73 66
74
—
—
—
—
76
85 Ave.
86 83
86
74
4
Cells in Rila
Acetone residue
_ , £ J J
Rila
10"4M Asparagine
80
93 Ave.
98 93
102
80
84 Ave.
90 88
96
56
67 Ave.
70 68
79
83
(a) Replicates obtained from experiment in which time sequence began with sample no. 1 followed sequen-
tially by samples no. 2,3, and 4.
(b) Replicates obtained from experiment in which time sequence began with sample no. 4 followed sequen-
tially by samples no. 3,2, and 1.
Table 22. COMPARISON OF CHEMOTACTIC RESPONSE OF THE ESTUARINE BACTERIUM TOWARD 10 PPM MIREX OR CARBOFU-
RAN IN THE PRESENCE OF 0.08% NUTRIENT BROTH.
Sample No.
Contents
in vial
Contents in
capillary
tube
No. colonies
on plate
at 10~3
dilution
1
Cells in Rila
Acetone residue
Rila
69
72 Ave.
73 72
74
2
Cells in Rila
Acetone residue
Rila
Nutrient broth
107
125 Ave.
131 124
131
3
Cells in Rila
Acetone residue
Rila
Nutrient broth
140
153 Ave.
160 156
173
4
Cells in Rila
Acetone residue
Rila
Nutrient broth
96
110 Ave.
Ill 109
118
25
-------
Table 23. COMPARISON OF CHEMOTACTIC RESPONSE OF THE ESTUARINE BACTERIUM TOWARD 50 PPM MIREX OR CARBOFU-
RAN (AS RESIDUE) IN THE PRESENCE OF 0.081? NUTRIENT BROTH.
Sample No.
Contents
in vial
Contents in
capillary
tube
(a)
(b)
No. colonies
on plate
at ID*3
dilution
(a)
Cells
Acetone
in Rila
residue
Rila
34
34
35
38
29
32
38
43
26
34
43
43
Ave.
35
Ave.
36
Ave.
36
Cells In Rila
Acetone
residue
Rila
Nutrient broth
48
60
73
80
95
107
119
121
93
105
115
132
Ave.
65
Ave.
110
Ave.
Ill
Cells
in Rila
Acetone residue
Mlrex residue
Rila
Nutrient broth
131
141
143
144
225
247
251
255
249
283
307
316
Ave.
140
Ave.
244
Ave.
289
Cells in
Rila
Acetone residue
Carbofuran residue
Rila
Nutrient broth
97
97
106
116
134
136
167
175
118
156
156
161
Ave.
104
Ave.
153
Ave.
149
Overall ave.
36
95
224
135
(a) Replicates obtained from experiment in which time sequence began with sample no. 1 followed sequen-
tially by samples no. 2,3, and 4.
(b) Replicates obtained from experiment in which time sequence began with sample no. 4 followed sequen-
tially by samples no. 3, 2, and 1.
Table 24. MICROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS IMMEDIATELY AFTER AMENDING WITH MIREX.
Amended with mirex
Control
0.1 ppm
10 ppn
1000 ppm
10,000 ppm
Microbial group
Nitrate reducers
Ammonifers
H?S producers
Sulfate reducers
Llpid hydrolyzers
Casein hydrolyzers
Gelatin hydrolyzers
Starch hydrolyzers
Chitin hydrolyzers
Aerobic plate count
X
105
105
10*
103
10=
105
105
10=
10*
106
Tank
11
4.3"
4.3
1.5
2.3
4.3
24.0
9.3
2.4
2.4
9.3
4.3
4.3
2.4
2.4
9.3
9.4
2.3
1.2
1.1
Tank
16
9.3
7.4
2.4
9.3
2.3
3.9
43.0
4.3
1.5
2.4
4.3
2.4
24.0
24.0
2.4
7.5
2.3
4.1
1.6
1-3
Tank
12
4.3
9.1
12.0
2,4
15.0
2.3
4.3
2.3
1.2
2.4
2.4
9.3
4.3
24.0
0.43
2.4
4.3
2.1
1.6
1.1
Tank
17
9.3
9.3
7.5
43.0
15.0
15.0
24.0
4.3
2.4
7.5
21.0
15.0
9.3
9.3
24.0
4.3
12.0
1.6
1.3
Tank
13
2.4
7.5
9.3
2,4
4.3
9.3
24.0
9.3
1.5
9.3
9.3
24.0
2.4
0.23
2.4
4RO.O
4.3
9.3
1.4
1.3
Tank
18
2.9
9.3
2.4
2.4
9.3
4.3
15.0
15.0
0.93
4.3
1.2
7.5
4.3
2.4
2.4
4.3
9.3
2.9
1.0
1.0
Tank
1*
2.1
2.4
2.4
15.0
4.3
4.3
9.3
4.3
1.5
4.3
2.4
4.3
9.3
9.3
2.4
4.3
0.91
1.4
0.9
Tank
19
4.3
2.4
4.3
4.3
24.0
15.0
4.3
2.4
4.3
2.4
2.1
4.3
4.1
3.9
4.1
4.3
9.1
1.6
Tank
1.5
n.Qi
2.4
4.3
4.3
2.3
1.5
24.0
7.5
1.5
2.4
0.91
4.3
7.5
2.4
21 .0
9.3
75. n
1.1
Tank
20
43.0
15.0
2.4
9.3
9.3
b
V
2.4
2.4
15.0
4.1
9.3
4.3
2.4
0.91
0.9
1 1
1.6
Anaerobic plate count
Replicate counts
Missing value
26
-------
Table 25. MICROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS ONE WEEK AFTER AMENDING WITH MIREX.
Amended with mirex
Control
Mlcrobial group
Nitrate reducers
Ammonifers
H2S producers
Sulfate reducers
Llpld hydrolyzers
Casein hydrolyzers
Gelatin hydrolyzers
Starch hydrolyzers
Chitin hydrolyzers
Aerobic plate count
Anaerobic plate count
tf/ml
X
io5
105
10*
10*
IO5
IO5
IO5
IO5
10*
!06
io5
Tank
11
9.3s
9.3a
2.4
43.0
4.3
4.3
2.4
2.4
4.3
2.4
2.4
2.4
4.3
9.3
2.4
4.3
2.4
7.5
3.7
3.9
7.0
10.0
Tank
16
9.3
21.0
7.5
9.3
43.0
9.3
2.4
43.0
7.5
4.3
9.3
9.3
15.0
1.5
2.4
2.4
2.3
9.6
9.5
15.0
17.0
0
.1
Tank
12
150.
93.
9.
23.
2.
4.
7.
9.
240.
9.
9.
24.
43.
2.
0.
2.
9.
49.
35.
79.
95.
0
0
3
0
3
3
5
3
0
3
3
0
0
4
93
4
3
0
0
0
0
ppm
Tank
17
43.0
240.0
24.0
93.0
9.3
9.3
0.93
2.4
9.3
24.0
4.3
24.0
93.0
240.0
4.3
4.3
0.04
2.3
18.1
19.6
113.0
138.0
10 ppm
Tank
13
2.1
15.0
7.5
2.4
2.3
9.3
2.4
9.3
9.3
9.3
24.0
4.3
9.3
9.3
4.3
1.5
1.5
0.9
5.1
11.0
11.0
Tank
18
24.0
75.0
4.3
4.3
4.3
2.4
2.4
2.4
4.3
9.3
2.4
0.93
4.3
46.0
0.9
2.4
3.9
2.4
4.2
7.0
11.0
1000
Tank
14
43.0
4.3
93.0
7.5
2.3
9.3
2.3
0.43
4.3
4.3
2.4
2.4
4.3
4.4
4.3
2.4
2.3
1.1
4.4
9.0
8.0
ppm
Tank
19
24.0
4.3
4.3
4.3
2.3
2.3
2.4
4.3
7.5
4.3
2.4
2.1
2.5
21.0
2.4
4.3
0.9
1.5
5.7
8.0
10.0
10,000 ppn
Tank
15
4.3
9.3
9.3
4.3
4.3
15.0
4.3
1.5
9.3
9.3
9.3
7.5
4.3
7.5
9.3
2.4
4.3
2.3
5.2
7.0
7.0
Tank
20
43.0
24.0
24.0
9.3
7.5
4.3
9.3
0.39
4.3
9.3
9.3
4.3
4.3
7.5
2.4
2.4
2.3
1.5
6.9
6.6
15.0
15.0
Replicate counts
Table 26. MICROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS TWO WEEKS AFTER AMENDING WITH MIREX.
Amended with mirex
Control
Microbial group
Nitrate reducers
Anrmnnl f prs
H?S producers
Sulfate reducers
Lipid hydrolyzers
Casein hydrolyzers
Gelatin hydrolyzers
Starch hydrolyzers
Chitin hydrolyzers
Aerobic plate count
Anaerobic plate count
///ml
X
in5
io5
10*
103
105
105
10^
105
10*
IO6
105
Tank
11
240. C^
2in.oa
15.0
4.3
7.5
9.3
43.0
4.3
9.3
4.3
9.3
24.0
9.3
9.3
3.9
2.4
2.3
0.9
18.1
16.1
14.7
15.1
Tank
16
43.0
15.0
4.3
4.3
2.3
24.0
9.3
9.3
4.3
9.3
b
b
15.0
21.0
b
b
0.4
1.1
19.5
20.8
24.7
23.1
0.1 ppm
Tank
12
93.0
240.0
93.0
240.0
7.5
2.4
9.3
15.0
13.0
24.0
4.3
43.0
24.0
43.0
2.4
2.3
0.9
0.36
41.0
30.0
141.0
151.0
Tank
17
150.0
_240.0
93.0
240. 0
43.0
9.3
9.3
4.3
93.0
93.0
b
b
93.0
24.0
b
b
b
b
46.0
38.0
155.0
154.0
10 ppm
Tank
13
15.0
15.0
9.3
2.4
1.5
4.3
4.3
9.3
24.0
24.0
4.3
2,4
24.0
24.0
2.4
1.5
0.9
b
3.9
5.2
9.3
11.9
Tank
18
24.0
4.3
4.3
4.3
24.0
9.3
3.9
24.0
24.0
4.3
b
b
4.3
9.3
b
b
2.3
0.4
5.3
5.4
10.6
10.0
1000 ppm
Tank
14
93.0
15.0
2.4
3.9
2.3
2.3
24.0
43.0
15.0
2.4
b
b
12.0
4.3
2.4
2.4
0.7
2.1
8.5
4.2
22.9.
28.2
Tank
19
24.0
Q.3
2.4
7.4
4.3
9.3
9.3
24.0
24.0
9.3
b
b
9.3
V-
b
b
b
4.3
5.1
12.9
12.2
10,000 ppm
Tank
15
39.0
4.3
4.3
4.3
1.5
2.3
9.3
9.3
9.3
43.0
b
b
9.3
43.0
0.93
4.3
0.9
2.3
4.1
4.6
13.0
11.5
Tank
20
24.0
43 n
9.3
7 q
15.0
9.3
15.0
9.3
4.3
9.3
9.3
9.3
b
b
2.4
4.3
0.4
10.7
9.9
34.0
37.0
Replicate counts
Missing value
27
-------
Table 27. MICROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS FOUR WEEKS AFTER AMENDING WITH MIREX.
Amended with mlrex
Control 0.1 ppm 10 ppm 1000 ppm 10,000 pp»
Microbial group
Nitrate reducers
Ammonlf ere
H^S producers
Sulfate reducers
Llpid hvdrolyzers
Casein hydrolyzers
Gelatin hydrolvzers
Starch hydrolyzers
Chitin hydrolyzers
Aerobic plate count
Anaerobic plate count
If/ml
X
106
106
in*
10*
m5
10=
105
10*
10 4
10 6
10 5
Tank
11
4.6a
4.9a
1.1
1.7
1.7
it. 9
1.3
1.7
4.9
4.9
7.9
4.9
33.0
13.0
2.2
4.9
0.4
0.4
19.8
18.4
20.3
16.1
Tank
16
1.1
2.2
0.8
1.1
2.8
24.0
0.8
0.3
79.0
79.0
3.3
7.9
24.0
13.0
4.9
2.2
0.2
0.2
25.4
23.0
101.0
87.0
Tank
12
13.0
1.8
7.9
7.9
4.9
1.7
1.3
0.7
17.0
13.0
17.0
33.0
130.0
79.0
3.3
3.3
0.2
2.3
47.0
57.0
150.0
135.0
Tank
17
3.2
11.0
4.7
7.0
4.9
7.9
1.1
0.8
17.0
24.0
22.0
17.0
240.0
240.0
0.8
3.3
2.1
1.4
62.0
59.0
158.0
141.0
Tank
13
1.4
7.9
1.3
2.8
3.3
3.3
0.8
0.8
3.3
24.0
7.0
2.4
33.0
33.0
3.3
4.9
0.7
0.2
15.0
16.7
50.0
64.0
Tank
18
2.4
0.5
1.4
1.3
3.3 '
3.3
0.5
0.2
24.0
33.0
0.8
7.9
7.9
7.9
0.8
1.3
1.1
0.8
8.3
6.9
34.0
34.0
Tank
14
7.9
3.3
3.3
1.4
2.4
2.2
0.7
0.5
3.3
4.9
1.4
4.9
3.3
24.0
1.7
1.3
0.7
1.2
8.7
7.6
34.0
32.0
Tank
19
3.3
2.1
0.5
0.3
3.3
4.9
1.3
0.8
33.0
13.0
3.3
4.9
17.0
11.0
0.4
28.0
0.5
9.5
7.8
31.0
24.0
Tuk
15
l.«
;,1.
1.1
0.7
3.3
2.4
1.4
0.8
7.9
7.0
3.3
1.3
4.9
7.9
1.3
2.4
1.2
0.8
7.1
8.7
36.0
Tank
20
7.»
4.9
0.8
1.1
4.9
2.4
0.8
0.5
24.0
79.0
2.4
7.9
24.0
33.0
2.4
17.0
0.5
1.7
11.2
13.4
50.0
57.0
a Replicate counts
b Missing value
Table 28. MICROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS IMMEDIATELY AFTER AMENDING WITH CARBOFURAN.
Amended with carbofuran
Microbial group
Nitrate reducers
Amnioni f ers
H^S p reducers
Sulfate reducers
Lipid hvdrolyzers
Casein hv drolvzers
Gelatin hvdrolvzers
Starch hvdrolvzers
Chitin hvdrolvzers
Aerob ic plate Count
Anaerobic plate count
Control
///ml
x
10 5
105
10 5
io4
105
io5
5
10
Id5
IO4
IO5
io5
Tank
25
3.3
7.9
1.3
1.3
4.9
7.9
0.8,
0.8
1.7
7.3
2.9
Tank
26
4.9
1.7
0.3
0.7
4.9
2.1
7.9
A. 9
0.7
7.6
3. A
0.1 ppm
Tank
27
0.8
1.3
1.3
7.9
4.9
3.5
24.0
3.3
3.3
6. A
2.6
Tank
28
2.1
A. 9
0.5
2.4
2.4
2. A
2.2
1.7
2.3
5.6
2. A
10 ppm 1000
Tank
29
2.1
7.0
0.2
0.8
4.9
3.3
3.3
l.A
2.3
5.2
2.8
Tank Tank
30 31
1.7 l.A
1.7 7.0
0.3 0.5
l.A 1.7
4.9 2. A
2.1 2. A
A. 9 2. A
2. A 2. A
0.9 17.0
6.6 5.9
2.9 3.7
ppm
Tank
32
0.
1
0
0.
4.
2
13
2
4
6
3
.8
.1
.5
.7
.9
.A
.0'
.A
.9
.A
.5
10,000
Tank
33
1.3
1.7
1.1
3.3
3.3
3.3
7.9
l.A
3.3
7.7
2.7
ppm
Tank
3A
2.
11.
0,
0.
1.
1.
2,
1
3
8.
3
4
0
.5
.5
.3
, 7
.8
.3
.A
.7
.A
28
-------
Table 29. MCROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS ONE WEEK AFTER AMENDING WITH CARBOFDRAB.
Amended with carbofuran
Control
Microbial group
Nitrate reducers
Anmonifers
H2S producers
Sulfate reducers
Lipid hydrolyzers
Casein hydrolyzers
Gelatin hydrolyzers
Starch hydrolyzers
Chitin hydrolyzers
Aerobic plate count
Anaerobic plate count
Table 30. MICROBIAL
#/ml Tank
x 25
105 49.0
105 7.9
105 0.5
10* 3.3
105 3.3
105 4.9
105 4.9
105 2.2
10* 4.9
106 10 . 4
106 6.6
COUNTS ON SEDIMENT
Tank
26
1.7
4.9
2.4
3.3
11.0
2.4
7.9
2.2
4.6
11.2
6.2
SAMPLES
0.1
Tank
27
79.0
13.0
1.3
2.2
22.0
3.3
17.0
4.9
3.3
12.6
10.0
ppm
Tank
28
79.0
3.3
1.7
2.8
7.9
3.3
7.9
4.9
2.2
11.7
9.2
FROM MICROCOSM
10
Tank
29
240.0
7.9
7.9
2.4
7.9
2.2
13.0
0.8
2.7
6.7
4.6
ppm
Tank
30
49.0
14.0
1.4
2.8
4.9
1.3
4.9
1.3
2.2
8.5
5.4
TANKS TWO WEEKS
1000
Tank
31
7.0
33.0
0.03
4.9
49.0
11.0
2.4
4.9
1.4
3.8
3.3
ppm
Tank
32
13.0
24.0
0.3
3.3
7.9
13.0
49.0
2.2
0.8
4.7
3.8
AFTER AMENDING
10,000 ppo
Tank
33
2.8
33.0
3.3
0.95
33.0
49.0
24.0
7.9
0.8
5.9
5.8
Tank
34
11.0
14.0
0.5
0.8
33.0
13.0
2.4
7.9
0.5
6.1
4.1
WITH CARBOFURAN.
Amended with carbofuran
Control
Microbial group
Nitrate reducers
Ammonifers
H2S producers
Sulfate reducers
Lipid hydrolyzers
Casein hydrolyzers
Gelatin hydrolyzers
Starch hydrolyzers
Chitin hydrolyzers
Aerobic plate count
Anaerobic plate count
#/ml Tank
x 25
105 79.0
105 1.3
105 .08
10* 3.3
105 3.3
105 4.3
105 3.3
105 1.3
10* 0.9
106 14.0
106 7.8
Tank
26
49.0
4.9
.08
7.9
13.0
3.1
7.9
1.2
3.3
19.2
10.8
0.1
Tank
27
4.9
7.9
0.3
3.3
4.9
4.6
2.4
2.2
0.5
22.4
11.0
ppm
Tank
28
170.0
13.0
0.5
2.4
33.0
7.9
13.0
4.9
4.9
19.5
12.4
10
Tank
29
4.5
4.9
1.3
3.3
22.0
7.0
4.9
1.3
3.3
20.5
10.8
ppm
Tank
30
21.0
14.0
0.5
4.9
7.9
4.9
11.0
1.3
4.9
14.5
9.8
1000
Tank
31
79.0
46.0
0.5
1.3
79.0
17.0
130.0
1.8
0.8
21.9
12.5
ppm
Tank
32
240.0
13.0
0.8
1.3
33.0
13.0
22.0
1.1
0
26.3
14.6
10,000
Tank
33
130.0
17.0
1.3
5.8
79.0
17.0
240.0
4.9
0
35.0
15.1
ppm
Tank
34
240.0
0.8
1.7
0.2
33.0
17.0
130.0
3.3
0.4
32.0
13.2
29
-------
Table 31. MICROBIAL COUNTS ON SEDIMENT SAMPLES FROM MICROCOSM TANKS FOUR WEEKS AFTER AMENDING WITH CARBOFURAN.
Amended with carbofuran
Microbial
Rroup
Nitrate reducers
Ammonifers
H^S Producers
Sulfate reducers
Lipid hvdrolyzers
Casein hvdrolvzers
Gelatin hydrolyzers
Starch hvdrolvzers
Chitin hvdrolvzers
Aerobic plate count
Anaerobic plate count
Control
X
in6
10 5
105
io4
10 5
io5
io5
io5
io4
io6
io6
Tank
25
7.9
1.7
0.3
1.3
2.4
4.6
7.9
1.7
1.1
13.1
8.9
tank
7.0
13.0
0.3
2.8
22.0
3.3
33.0
2.1
1.3
16.7
13.3
0.1
Tank
27
2.6
4.9
1.3
0.8
3.3
9.5
4.9
3.3
1.1
20.7
19.6
ppm
Tank
28
0.4
4.9
0.7
1.3
79.0
18.0
7.9
1.1
1.7
17.2
18.1
10
Tank
29
0.3
3.3
0.3
0.3
22.0
3.S
24.0
1.3
2.6
15.5
13.7
ppm
Tank
30
4.0
3.3
3.3
1.3
2.4
7.0
33.0
2.4
1.3
17.5
7.3
1000 ppm
Tank
31
24.0
33.0
1.1
0.8
24.0
33.0
240.0
2.4
0
34.0
13.1
Tank
32
17.0
140.0
1.1
0.8
79.0
33.0
240.0
1.1
0.7
39.0
20.0
10,000 ppm
Tank.
33
24.0
170.0
0.24
0.22
33.0
49.0
49.0
1.7
0.6
23.4
18.2
Tank
34
28.0
170.0
1.3
0.5
33.0
49.0
22.0
7.9
0.2
27.0
18.4
In the studies with mirex, it was noted that concentrations in excess of 10 ppm caused a slight but
significant reduction in the two and four week aerobic plate counts as compared to the control.
Morphological characteristics of the colonies on plates from all treated samples were similar to those
on plates from the control samples. Anaerobic plate counts were not significantly different from the
control at any concentration of mirex. Statistical analysis of the data gathered on the other nine
biochemical groups of organisms was conducted without showing any difference between treatments
and controls.
After two weeks incubation, the samples amended with 1000 ppm and 10,000 ppm carbofuran
showed an increase in the number of aerobic bacteria, anaerobic bacteria and some of the other
groups of heterotrophic bacteria over numbers found in control sediments. Colonies on the plates from
both treated and control samples were morphologically the same. At a concentration of 10 ppm, pop-
ulations in treated and control samples were about the same.
Since the aerobic heterotrophic bacterial plate counts were easy to perform and could be
replicated with a minimum of effort to produce a direct number rather than a statistically derived one
as produced by the MPN counts, it was decided to use the plate count as the measure of effect of the
pesticide treatments in further studies.
Salinity-Temperature Studies-
The effect of various salinities and incubation temperatures on the bacterial population of the
sediment was measured in the presence and absence of both mirex and carbofuran in a concentration
of 100 ppm. The temperatures and salinities employed were approximately the maximum and
minimum values recorded in the estuary.
Overall, the bacterial populations in mirex-amended sediments did not differ from the controls at
any of the salinities and temperatures tested. Statistical analysis of the data verified this observation
(Table 32). The previous study showed that mirex suppressed the bacterial populations. However, this
difference was attributed to lot-to-lot variability in the mirex since in the previous study a different lot
of mirex was employed.
30
-------
The addition of carbofuran to samples produced significantly higher bacterial counts as com-
pared to the control under all conditions of salinity and temperature tested (Table 33). None of these
temperature-salinity conditions produced results noticeably different from the other.
Table 32. MEAN BACTERIAL COUNTS (x 10^) IN SEDIMENTS AMENDED WITH 100 PPM MIREX AND HELD
UNDER VARIOUS CONDITIONS OF SALINITY AND TERMPERATURE.
Incubation
period
after
pesticide
added
(days)
15 ppt - 10 C 15 ppt -21 C 15 ppt - 30 C
5 ppt - 21 C 30 ppt - 21 C
-H
x
g
0
x
3
0
4
9
16
22
30
44
113a 143C
442 420
743 697
802 792
868 824
680 679
972 926
91 98
662 651
942 843
892 845
1177 1046
980 1136
[8300] 4485
96 168°
722 772
933 878
1155 907b
[3885] [10,136]c
608 645
918 802
54 54
645 648
1066 938
798 755
710 668
745 769
1236 977
111 110
532 487
807 695
748 682
966 952
1080 846b
[5638] 1370°
a Mean of three replicates with five plates counted on each replicate
M Poor agreement among replicates which differed by one log in count.
Treatment counts significantly different from control counts at 95% level as deter-
mined using t-test.
c Treatment counts significantly different from control counts at 99% level as deter-
mined using t-test.
Dehydrogenase Activity-
Dehydrogenase activity is used as an indication of anaerobic metabolism in sediments. Pamat-
mat and Bhagwat14 pointed out that anaerobic metabolism may greatly exceed aerobic metabolism
in sediments. Therefore, a measure of this activity was used to evaluate the effect of pesticides on
microbial activity.
Since previous studies had indicated variability among lots of technical grade pesticides, purified
preparations of the pesticides as well as the technical grade material were employed in these studies.
As shown in Table 34, preparations of mirex added to sediments produced no alteration in the
dehydrogenase activity when compared to untreated sediments. At some incubation times, the
dehydrogenase activity was increased slightly and in others it was decreased slightly when compared
to the control. Differences in levels of mirex did not alter this phenomenon.
All preparations of carbofuran at a concentration of 100 ppm significantly depressed the
dehydrogenase activity (Table 35). At concentrations of 10 and 1 ppm carbofuran, depression of
dehydrogenase activity was noted in only a few cases and in some cases, the activity was higher in
amended sediments than in untreated ones. Overall, there appeared to be little difference among the
different preparations of carbofuran.
31
-------
Table 33. MEAN BACTERIAL COUNTS (x 10 ) IN SEDIMENTS AMENDED WITH 100 PPM CARBOFURAN AND
HELD UNDER VARIOUS CONDITIONS OF SALINITY AND TEMPERATURE.
Incubation 15 ppt - 10 C 15 ppt - 21C 15 ppt - 30 C
period
after
pesticide
5 ppt - 21 C
c
30 ppt - 21 C
c
added 2-52^2^ 2 -g 2 -g
(days) c •£ c •£ c •£ c -g = "S
3 3 <3 3 S 3 3» 35
0
4
9
16
22
30
44
113a 167°
442 638C
743 880b
802 1344°
868 1463°
680 1335C
972 1601°
91 123C
662 636
942 1009
892 1300°
1177 [5086]C
980 1326C
[8300] 2013b
96 147C
722 1075C
933 1416C
1155 1487b
[3885] 1438°
608 1242C
918 620C
54 55
645 589
1066 1227b
798 1359°
710 2116C
745 1498C
1236 1932C
111 105
532 530
807 660
748 2053°
966 1392°
1080 1374C
[5638] [10275]b
- Mean of three replicates with five plates counted on each replicate
{] - Poor agreement among replicates which differed by one log in count.
- Treatment counts significantly different from control counts at 95% level as deter-
mined using t-test.
c - Treatment counts significantly different from control counts at 99% level as deter-
mined using t-test.
Table 34. DEHYDROGENASE ACTIVITY IN SEDIMENTS TREATED WITH DIFFERENT PREPARATIONS AND CONCENTRATIONS
OF MIREX.
Addition
Untreated
Technical mirex
Purified mirex
Untreated
Technical mirex
Purified mirex
Untreated
Technical mirex
Purified mirex
Untreated
Technical mirex
Purified mirex
Incubation period after pesticide added
Initial
22'
20
22
23
24
25
21
23
24
31
43
34
i
(O.lj3
(NS)
(NS)
(NS)
(NS)
(.10)
(.001)
(NS)
7 days
180
182
174
164
165
207
108
119
158
152
181
157
14 days
lUUUppm
190
(NS)
(NS)
(NS)
(.001)
(NS)
(.01)
(.01)
(NS)
174
196
137
211
244
98
105
127
158
162
198
(NS)
(NS)
(.001)
(.001)
(NS)
(.01)
ppm —
(NS)
(.10)
21 days
171
155
169
212
191
231
115
54
139
150
151
148
(NS)
(NS)
(NS)
(NS)
(.001)
(.05)
(NS)
(NS)
28 days
127
106
139
172
181
213
88
102
134
104
158
158
(.05)
(NS)
(NS)
(.001)
(.01)
(.001)
(.001)
(.001)
a Mean dehydrogenase activity (ug of TPF produced per gram of wet sediment) from six replicates.
" Significance level of difference between treated and untreated samples as determined by the t-test.
NS means not significant at the 0.1 level.
32
-------
Table 35. DEHYDROGENASE ACTIVITY IN SEDIMENTS TREATED WITH DIFFERENT PREPARATIONS AND CONCENTRATION OF CARBOFURAN.
Addition to
sediment
Untreated
Technical carbofuran
Purified carbofuran
Water washed carbofuran
Untreated
Technical carbofuran
Purified carbofuran
Water washed carbofuran
Untreated
Technical carbofuran
Purified carbofuran
Water washed carbofuran
Incubation period after pesticide i
Initial ,
113
86
105
124
49
68
47
71
115
121
140
148
a
(.05) b
(NS)
(NS)
(.01)
(NS)
(.001)
(NS)
(.001)
(.001)
6 days
89
23
22
40
36
38
36
37
73
62
77
46
nended with
(.001)
(.001)
(.001)
aended with
(NS)
(NS)
(NS)
nended with
(.05)
(NS)
(.001)
13
57
16
25
24
37
45
41
40
57
43
53
41
added
days
(.001)
(.001)
(.001)
(.01)
(NS)
(NS)
(.001)
(NS)
(.001)
20
62
19
16
29
3A
42
21
59
24
32
32
35
days
(.001)
(.001)
(.001)
(.10)
(.001)
(.001)
(.001)
(.01)
(.001)
• Mean dehydrogenase activity Cug of TPF produced per gram of wet sediments) from six replicates.
Significance level of difference between treated and untreated samples as determined by the t-test. NS means
not significant at the 0.1 level.
The question arose as to why the dehydrogenase activity in carbofuran amended sediments
decreased, while the bacterial numbers increased. To answer this question, seven pure cultures of
bacteria of the types which predominated in sediments were isolated and inoculated into autoclave-
sterilized sediment which had been enriched with 1% glucose. As a positive control, enriched sterile
sediment was inoculated with fresh bay sediment. In all inoculated sediments, the bacteria increased
in number, but only in the sediments inoculated with the fresh sediment did the dehydrogenase activi-
ty increase. This indicated that these aerobic heterotrophic bacteria did not significantly contribute
to the dehydrogenase activity within the sediments, and their increase in numbers would not be ex-
pected to increase dehydrogenase activity.
Proteolytic Activity-
Protein utilization studies were conducted to determine if the pesticides affected the assimilatory
power of the sediment microflora. As shown in Table 36 neither of the pesticides at any of the concen-
trations tested appeared to greatly change the amount of protein utilized.
Glucose Mineralization-
Radioisotopic studies were employed to determine the effect of various concentrations of mirex
and carbofuran on the carbon dioxide released during the mineralization of glucose. Results of these
studies are shown in Table 37.
The addition of carbofuran in concentrations of 1000 and 100 ppm significantly depressed the
rate at which glucose was converted into carbon dioxide. After four weeks incubation with carbofuran
33
-------
Table 36. PROTEOLYTIC ACTIVITY OF MICROORGANISMS IN SEDIMENT SAMPLES AMENDED WITH CARBOFURAN AND MIREX.
Concentration
of pesticide Sediment incubation period after pesticides added
added to
sediment
(ppm)
0
1000
100
10
1
0
100
10
1
Initial
81. Om
79.0
80.0
79.0
73.0
81.7
75.2
80.8
1 week
8S.O
82.0
86.0
87.0
87.5
90.3
71.0
72.6
77.1
2 weeks
amended s<
75.6
77.0
77.1
80.6
83.7
in amended
82.7
81.3
83.7
81.3
3 weeks
82.0
75.0
74.0
78.0
76.0
74.7
69.9
69.9
70.6
4 (reeks
76.0
74.0
72.0
74.0
75. 0
82.0
80.3
83.5
80.0
a - Percent of added peptone protein utilized during 22 hour incubation period.
Average of four replicates.
Table 37. MINERALIZATION OF GLUCOSE BY MICROORGANISMS IN SEDIMENTS AMENDED WITH VARIOUS CONCENTRATIONS OF
CARBOFURAN AND MIREX.
Concentration
of pesticide Sediment incubation p-eriod after pesticide added
added to
sediment
(ppm) Initial
0
1000
100
10
1
0
1000
100
10
1
1158a
922C
1033C
915°
897C
518
225C
37SC
295C
281C
-21. Ob
-10.8
-21.0
-22.5
-56.6
-27.6
-43.1
-45.8
1 week
.
1715
2225C
1690
1818
2 weeks
: amended sedimei
1328
+ 29
- 1
+ 5
1583 - 7
Carbofuran
1165
233C
904C
1149
1261
-80
-22
- 1
+ 8
.7
.5
.9
.7
amended
.3
.4
.4
.2
1277
1317
1246
1369
sedii
1022
230°
763C
987
991
- 3.8
- 0.8
- 6.2
+ 3.1
-77.5
-25.3
- 3.4
- 3.0
3 weeks
941
877
829
855
891
719
169C
S54C
721
603C
- 6.8
-11.9
- 9.1
- 5.3
-76.5
-22.9
+ 0.3
-16.1
4 weeks
1040
890
1123
910
894
1758
219C
1244C
1789
1728
-14.4
+ 7.9
-12.6
-14.0
-87.5
-29.2
+ 1.8
- 1.7
a - Level of radioactivity (CPM) in the C02 produced from the mineralization of
glucose. Average of four replicates.
b- Percent difference between control and treated sediments.
c- Significantly different from control (P>.05). t-test.
34
-------
at the above concentration, carbon dioxide production was reduced by 87.5% and 29.2%, respectively.
No significant alteration in the carbon dioxide evolution rate was detected at lower concentrations of
carbofuran or at any concentration of mirex tested.
Respirometric Studies-
With the rate of glucose mineralization being decreased in sediments amended with carbofuran,
it was expected that the oxygen consumption during glucose utilization may also be affected. As
shown in Table 38, the amount of oxygen consumed during the breakdown of glucose was reduced in
amended sediments. This reduction was comparable percentagewise to the reduction in glucose
mineralization.
Table 38. OXYGEN CONSUMPTION DURING GLUCOSE UTILIZATION BY ORGANISMS IN SEDIMENTS
AMENDED WITH 1000 PPM CARBOFURAN.
Total oxygen Oxygen consumption Reduction
consumption corrected for in oxygen
endogenous consumption
(>jl) respiration of amended
[pi) s edimen t
CO
Five days after sediments amended
Untreated sediment
(endogenous respiration) 224"
Untreated sediment
with glucose 303 79
Amended sediments
(endogenous respiration) 225
Amended sediments
with glucose 236 11 86.1
Twelve days after sediments amended
Untreated sediment
(endogenous respiration) 195
Untreated sediment
with glucose 223 28
Amended sediments
(endogenous respiration) 176
Amended sediments
with glucose 188 12 57.1
d Average of two replicate samples over a six hour period.
Hydrogen Sulfide Production-
Microcosms prepared with beach sand were amended with 1000 ppm mirex, 1000 ppm carbofuran
or 100 ppm carbofuran, and were incubated in the dark. After seven days, the control microcosm and
the one amended with mirex developed a dark gray-colored reducing layer below the sand water inter-
face. This layer was absent in the carbofuran amended samples. Subsequent studies showed a concen-
tration greater than 10 ppm carbofuran was required to produce this effect.
Hydrogen sulfide, which causes the blackening effect, is produced either from organic sulfur com-
pounds or from sulfate reduction. Previous microcosm studies have shown that the addition of car-
bofuran does not reduce the number of organisms capable of making these transformations in
sediments. Thus it is speculated that carbofuran in some way prevents the production of hydrogen
sulfide without killing the organisms.
35
-------
Bioaccumulation Studies on Mirex and Carbofuran-
Table 39 lists the results of a 48 h study on the bioaccumulation of carbofuran by culture #280.
Cells in the test system were removed by centrifugation and the carbofuran in the liquid was analyzed
by liquid chromatography. Peak heights were compared to an internal standard (carbaryl) in each
sample and the carbofuran to internal standard ratio was established for each test. Relative values
were obtained by making sample #2 equal to unity since it should not have a loss of carbofuran. The
relative values found in Table 39 are not significantly different with respect to tests with live cells,
dead cells and test with no cells. Sample #7, which contained the live cells +1% glucose, had the lowest
relative value, but it also has a 27% increase in total solids due to increase in cell mass. These data in-
dicate that the carbofuran was removed from the test system by adsorption to the cells and not by
bioaccumulation.
Table 39. BIOACCUMULATION STUDIES USING CARBOFURAN.
Sample 1
Controls
(no cells)
#1
K
#3
ft
Live cells
#5
#6
#7
Dead cells
#8
#9
#10
Test system Ratio
carbofuran/ internal
s-tandard
100 ml Mineral
100 ml Mineral
100 ml Mineral
100 ml Mineral
100 ml Mineral
100 ml Mineral
100 ml Mineral
+ 1% glucose
100 ml Mineral
100 ml Mineral
100 ml Mineral
+1% glucose
salts
salts + 150 ppjn carbofuran 1.22
salts + 1% glucose
salts + 150 ppm carbofuran + 1% glucose 1.14
salts + 5% cells
salts + 5% cells + 150 ppm carbofuran 1.14
salts +- 5% cells + 150 ppm carbofuran 1.09
salts + 5% cells
salts + 57, cells + 150 ppm carbofuran 1.12
salts + 5% cells + 150 ppm carbofuran 1.15
Relative value
(#2 - 1)
1.0
0.97
0.93
0.89
0.92
0.94
= No carbofuran detected, all values are the average of two determinations.
The protocol for the bioaccumulation studies with mirex was the same as that for the studies with
carbofuran. After 48 h incubation, the cells were removed by centrifugation and the mirex remaining
in the liquid was determined by standard techniques outlined by the official method of the Food and
Drug Administration (Anonymous15) using electron capture gas liquid chromatography. The results
are listed in Table 40. By considering the amount of mirex found in the liquid of samples #7 & 8 to be
100%, it can be noted that the amount adsorbed by the dead cells (samples #9 & 10) was about 95% of
the total present. The live cells (samples #11 & 12 adsorbed only 88% of the mirex even though there
was a 27% increase in total mass of cells due to multiplication. Within the limitations of the test
system used, it is concluded that there was no bioaccumulation of mirex occurring using culture #280.
36
-------
Table 40. BIOACCUMULATION STUDIES OF MIREX BY CULTURE #280 (BENECKA).
Sample It
1
2
3
4
3
6
7
8
9
10
11
12
Test system Total mirex
in liquid
(yg)
200 ml Mineral salts
200 ml Mineral salts
200 ml Mineral salts + 1% glucose
200 ml Mineral salts + 1% glucose
200 ml Mineral salts + 5% cells
200 ml Mineral salts + 5% cells
200 ml Mineral salts + mirex +
17. glucose 20.14
200 ml Mineral salts + mirex +
1% glucose 22.22
200 ml Mineral salts + 57. dead
cells + mirex + 1Z glucose 0.94
200 ml Mineral salts + 5% dead
cells + mirex + 1% glucose 1.31
200 ml Mineral salts + 57. live
cells + mirex + 17. glucose 2.71
200 ml Mineral salts + 57. live
cells + mirex + 1Z glucose 2.71
Percent
remaining
in liquid
100
100
4
6
12
12
DERIVATIVES OF MIREX AND CARBOFURAN
As reported previously, the inhibitory effect of mirex (technical) varied from batch to batch and
was erratic even when multiple tests were conducted using the same batch. Purified mirex samples
never exerted an inhibitory effect when tested using the disc assay technique. There was reason to
believe that contamination arises in aged mirex samples since older samples of mirex were toxic to the
greatest number of bacterial cultures.
Since hexachlorocyclopentadiene is the starting material for mirex synthesis, this compound is a
potential contaminant in the mirex. However, disc assay tests failed to indicate any inhibition with
the 20 bacterial cultures tested when it was added to mirex at a concentration of 1:1000.
Chemical analyses of technical grade mirex demonstrated the presence of hexachlorobenzene
(HCB) in some of the mirex samples. However, this compound was not found to be toxic to any of the
20 bacterial cultures as determined by disc assay tests.
Since Alley et a/.3 have identified 10-hydrogen; 5,10-dihydrogen; 8-hydrogen; and 2,8-dihydrogen
derivatives of mirex as photodegradation products, and Ivie, Borough, and Alley16reported kepone to
be a degradation product of mirex, these compounds along with reduced kepone were tested against a
number of bacterial cultures using the disc assay technique. Neither the 8-hydrogen derivative of
mirex nor the 2,8-dihydrogen derivative of mirex inhibited any of the 10 cultures tested. The 10-
hydrogen derivative of mirex and the 5,10-dihydrogen derivative of mirex inhibited all 5 of the five
cultures tested, while the kepone and reduced kepone both inhibited 10 of the 20 cultures tested. It is in-
teresting to note that neither of the two products with substitutions on the 2 and 8 positions of the
mirex molecule were inhibitory, while the four products with substitutions on the 5 and/or 10
positions of the mirex molecule were highly toxic to the test microorganisms. Whether these com-
pounds were actual contaminants in the mirex employed in this investigation is unknown.
37
-------
All attempts to isolate the specific compound(s) responsible for the inhibitory activity of the
technical mirex failed.
In another series of experiments water-moistened mirex alone and in combination with HCB
were exposed to UV irradiation (20 min under high pressure mercury lamp, 1800-6000 ./?) and
employed in disc assay tests. The data (Table 41) clearly illustrate that irradiation of the mirex
resulted in a material which was toxic to Achromobacter #274. Curiously, irradiated mirex plus HCB
appeared to have a greater toxicity than irradiated mirex alone, even though nonirradiated mirex
plus HCB and irradiated HCB were nontoxic. Unfortunately, no record is available on the storage
conditions under which the batches of mirex were kept, and it could well be that they had a history of
exposure to moisture and UV irradiation over a period of time.
Table 41. RESULTS OP "DISC ASSAYS" OF UV IRRADIATED
HEXACHLOROBENZENE AND MIREX AGAINST CULTURE
#274 (ACHROMOBACTER).
Test system Zone of inhibition
UV No UV
t reatment treatment
Purified mirex
Technical mirex
HCB
HCB + purified mirex (2:100)
HCB + purified mirex (2:10)
a Increase in "+'"s represents an increase in the diameter of
the zone of inhibition
Since no organisms capable of utilizing either mirex or carbofuran were obtained, tests on the im-
pact of metabolites were impossible. Under the circumstances, the above named photoproducts of
mirex and 3-hydroxycarbofuran and 3-ketocarbofuran (known degradation products of carbofuran)
were employed in subsequent tests.
Results of Qualitative Studies on Effect of Mirex and Carbofuran Derivatives
Qualitative studies on the effect of derivatives of mirex and carbofuran on the hydrolysis of
starch, lipid, chitin, casein, and gelatin by several pure cultures of estuarine bacteria were conducted.
Four cultures, #222, #276, #242 and #280, were tested for chitin hydrolysis. Only cultures #242 and #280
showed the ability to hydrolysis chitin and neither of these cultures was affected by any of the test
compounds. Six cultures were utilized for the tests involving starch hydrolysis. Kepone and reduced
kepone inhibited growth of three out of six while the remaining compounds showed no inhibition.
None of the test compounds affected the growth of seven casein hydrolyzers. Growth of two out of six
gelatin hydrolyzers was inhibited by kepone, and one out of six by reduced kepone. Only one culture,
#242, illustrated the ability to hydrolyze lipid and it was not affected by any of the test compounds.
38
-------
Effect of Mirex and Carbofuran Derivatives on Methane Consumption
by Microflora in Estuarine Sediments
Table 42 shows the results of the effect of mirex and carbofuran derivatives on the process of
methane consumption by the microflora in estuarine sediments after 12 days of incubation at 30 C un-
der static conditons. No inhibition was noted with any of the test compounds; instead, there are in-
dications of a stimulatory effect on the rate of methane consumption by several of the derivatives.
Since the test system (sediments) is so complex in nature, it is difficult to properly assess this
phenomenon. One possible explanation could be that these compounds are retarding certain groups
of bacteria that might otherwise compete with the methane comsumers.
Table 42 . EFFECT OF MIREX AND CARBOFURAN DERIVATIVES ON METHANE CONSUMPTION BY MICROFLORA IN
ESTUARINE SEDIMENTS.
Time in
Test system 2
Blank O8
Control 4
3-Hydroxycarbofuran-10 ppm 4
1 ppm 4
3-Ketocarbof uran-10 ppm 4
1 ppm 4
Kepone-10 ppra 2
1 ppm 4
Reduced kepone-10 ppm 4
1 ppm 7
2,8-Dihydrogen mirex-10 ppra 4
1 ppm 4
10-Hydrogen 10 ppm 7
and 5, 10-d Ihydrogen-1 ppm 4
mirex
3
0
9
9
9
11
13
9
9
11
11
11
11
15
9
4
0
11
11
11
15
18
11
13
13
18
13
15
20
13
5
0
18
15
15
24
26
18
22
24
26
22
24
31
20
6
0
24
20
11
31
37
22
29
33
35
29
35
44
29
7
2
31
29
)9
42
48
26
40
44
46
40
48
59
40
days
8
k
35
35
22
51
59
35
46
53
55
48
57
73
46
9
4
42
46
44
59
75
44
57
66
68
59
70
84
59
10
4
53
62
55
70
81
53
73
77
81
73
79
88
73
11
4
64
70
64
79
84
64
81
84
86
79
79
88
81
12
4
75
77
73
86
86
75
86
86
86
84
84
88
86
a ml of gas consumed.
Co
m o gas consume.
nditions of test: Sohngen unit apparatus, medium 15 ppt salinity with mineral salts incuba-
ted under gas atmosphere of 65% CH^, 30% 02 and 57. C02, static conditions. All values are
,
the average of three determinations.
Effect of Mirex and Carbofuran Derivatives on Ammonification
A pure culture, #280, was used to study the effect of mirex and carbofuran derivatives on the
process of ammonification. After 3 days of incubation at 30 C under static conditions no difference
was noted between the tests containing the derivatives and the control when monitoring substrate
disappearance (protein-N) and product formation (NH3-N). These results are illustrated in Table 43.
Effect of Mirex and Carbofuran Derivatives on Nitrate Reduction
The results of the effect of mirex and carbofuran derivatives on nitrate reduction are found in
Table 44. Cultures containing the test compounds (concentration 1 and 10 ppm) showed the same rate
of substrate utilization (NO3-N) and product formation (NO2-N) as did the control.
39
-------
Table 43. EFFECT OF DERIVATIVES OF MIREX AND CARBOFURAN ON AMMONIFICATION BY A PURE
CULTURE OF AN ESTUARINE BACTERIUM - i>280 (BENECKA).
Test Astern
Blank
Control
3-Hydroxycarbofuran 10 ppm
1 ppm
3-Ketocarbofuran 10 ppm
1 ppm
Kepone 10 ppm
1 ppm
Reduced kepone 10 ppm
1 ppm
2,8-Dlhydrogen 10 ppm
mlrex 1 ppm
10-Hydrogen 10 ppm
and 5,10- dlhydrogen- 1 ppm
mirex
NHj-N (mg/1)
Initial Day 1 Day 2 Day 3
0 000
0 119 195 270
0 121 200 272
0 125 230 310
0 125 192 280
0 125 227 292
0 75 100 240
0 127 210 297
0 120 200 282
0 133 235 302
0 125 200 275
0 127 220 300
0 123 202 277
0 123 240 300
Proteln-Na (mg/1)
Initial
1200
1200
1200
1200
1200
1200
1200
1200
1200
1200
1200
1200
1200
1200
Final
1200
698
710
710
Til
710
698
715
690
715
698
710
710
727
Protein-N of medium after filtration through 0.45 P membrane filter:
of test: Medium: 15 ppt Rlla sea salts, mineral salts. Incubation:
tic conditions. All values are the average of two determinations.
Conditions
30 C, sta-
Table 44. EFFECT OF MIREX DERIVATIVES AND CARBOFURAN DERIVATIVES ON NITRATE-REDUCTION BY
AN ESTUARINE BACTERIUM if 280 (BENECKA) .
Test System
Hours o f Incubation
0 6 12 24
" N03 -N N02 -N N03-N NOpN NO^N N02~N NC^-NN02~N
Blank
Control
3-Ketocarbof uran 10 ppm
1 ppm
3-Hydroxycarbof uran 10 ppm
1 ppm
Kepone 10 ppm
1 ppm
Reduced kepone 10 ppm
1 ppm
2,8-Dihydrogen mirex 10 ppm
1 ppm
10~Hydrogen 10 ppra
and 5,10- dltydrogen- 1 ppm
m Lrex
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100
4S
75
64
72
69
88
90
84
73
56
72
93
88
0
0.7
.65
.63
.64
.75
.73
.62
.74
.73
.77
.70
.68
.70
100
7.5
14.0
12.0
5.0
7.5
7.0
24.0
7.0
7.5
10.0
7.5
7.5
6.5
0
2.4
2.4
2.4
2.1
2.1
2.1
2.1
2.4
2.3
2.3
2.1
2.4
2.4
100 0
<1.0 2.8
<1.0 3.2
<1.0 3.2
<1.0 3.1
<1.0 3.2
<1.0 3.0
<1.0 3.2
<1.0 3.1
<1.0 3.2
<1.0 3.0
<1.0 3.0
<1.0 3.0
<1.0 3.0
All values are the average of two determinations.
Effect of Mirex and Carbofuran Derivatives on Nitrification
Three of the test compounds, 3-hydroxycarbofuran, kepone and reduced kepone at concentrations
of 1 and 10 ppm retarded nitrification as illustrated in Table 45 by reduction in both disappearance of
substrate (NH3-N) and product formation (NO2-N and NO3-N). No effect on nitrification was found
with 3-ketocarbofuran, 2,8-dihydrogen mirex and the mixture of 10-hydrogen and 5,10-dihydrogen
mirex.
40
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Table 45. EFFECT OF MIREX AND CARBOFURAN DERIVATIVES ON NITRIFICATION BY MICROORGANISMS IN ESTUARINE SEDIMENTS.
Test system
Blank
Control
~Ketocarbof uran
10 ppm
1 ppm
3-Hydroxycarbofuran
10 ppm
1 ppm
Cepone 10 ppm
1 ppm
Reduced kepone 10
10 ppm
1 ppm
2,8-Dlhydrogen
mirex 10 ppm
1 ppm
10-Hydrogen
and 5,10-dihydroge
nirex 10 ppm
1 ppm
Days of incubation
0
NH -N NO.-N NO,^N
mg/1) (mg/1) (mg/l)
130*
130
130
130
130
130
130
130
130
130
130
130
n-
130
130
5
NH,-N SOj-N ND.-N
!mg7l)(mg7l) (mg/1)
130
128
128
130
130
128
128
130
128
130
130
128
130
130
10
NH3-N ND,-N NO -N
mg/l)(mg/l)(mgh)
130
125 trace trace
124 + +
117 + +
127 + +
115 + +
124 + +
115 + +
125 + +
120 + +
123 + +
123 H- +
120 + +
120 + +
15
NH,-N NO,-N NOj-N
mg/1) (mg/l) (mg/1)
130
100 .02 .6
100 .02 .4
90 .01 .4
105 + <.l
90 + <.l
120 + <.l
110 + <.l
99 .01 <.l
90 .01 <.l
105 .02 <.l
90 .02 <.l
103 .02 <.l
90 .02 <.l
20
NH3-N NO?-N N03~N
(mg7l) (mg7l) (mg7l)
130
90 .12 .9
93 .02 .
-------
this experiment was rerun using a water previously enriched in phytoplankton population. Table 47
lists the results of this experiment. No growth inhibition was observed with the 10-hydrogen mirexor
8-hydrogen mirex. Both concentrations (1 and 10 ppm) of the reduced kepone, kepone, 3-
ketocarbofuran, 3-hydroxycarbofuran reduced the primary productivity. A reduction in the primary
productivity also was noted with the 2,8-dihydrogen mirex, but the reduction was not as great as for
the above compounds.
Effect of "Wing Derivatives" of Mirex (Mixture of 10-hydrogen and
5,10-dihydrogen mirex) on the Dehydrogenase Activity in Sediments
Table 48 lists the results of the effect of a mixture of "wing derivatives" of mirex (10-hydrogen and
5,10-dihydrogen mirex) on dehydrogenase activity in sediments. No reduction in dehydrogenase ac-
tivity was observed with any of the concentrations of the test compounds. On the other hand, several
of the concentrations showed a stimulatory effect on dehydrogenase activity of sediments sampled up
to 28 days after application of the test compound.
Table 47. THE EFFECT OF MIREX AND CARBOFURAN DERIVATIVES ON THE METABOLIC
ACTIVITY AND PRIMARY PRODUCTIVITY OF ESTUARINE POND WATER EN-
RICHED FOR PHYTOPLANKTON.
Test system Metabolic
activity
(mg ©2 consumed/1
in 12 h)
Control
Acetone control
HgCl2 control
Reduced kepone 1 ppm
10 ppm
Kepone 1 ppm
10 ppm
10-Hydrogen mirex 1 ppm
10 ppm
8-Hydrogen mirex 1 ppm
10 ppm
2,8-Dihydrogen mirex 1 ppm
10 ppm
3-Ketocarbof uran 1 ppm
10 ppm
3-Hydroxycarbofuran 1 ppm
10 ppra
1.40
1.30
0.00
1.25
1.00
1.20
1.20
1.20
1.20
1.35
1.35
1.35
1.40
1.45
1.45
1.40
1.10
Primary
productivity
(mg 0~ produced /I
in 12 h)
4.
4.
0.
1.
0.
3.
1.
5.
4.
5.
4.
4,
4.
4.
3.
3,
2
65
85
00
15
30
60
80
.15
,60
,20
,85
,40
.05
,10
.05
.90
.70
All values are the average of two determinations.
42
-------
Table 48. DEHYDROGENASE ACTIVITY IN SEDIMENTS TREATED WITH DERIVATIVES OF MIREX.
Addition
to sediment
Untreated
"wing derivatives1'
of mirex
Untreated
"wing derivatives'1
of mirex
Untreated
"wing derivatives"
of mirex
Untreated
"wing derivatives"
of mirex
Period after pesticide added
Initial
22a
19 (.05)
23
24 (NS)
21
19 (NS)
31
32 (NS)
7 days
180
168 (NS)
164
205 (.001)
108
150 (.001)
152
147 (NS)
14 days
190
184 (NS)
137
222 (.001)
98
129 (.001)
158
205 (.01)
21 days
171
193 (NS)
212
260 (.02)
115
108 (NS)
150
204 (.01)
28
127
166
172
224
88
144
104
176
days
(.01)
(.001)
(.001)
(.001)
a Mean dehydrogenase activity (yg of TPF produced per gram of wet sediment) from six replicates.
NS means not significant at the 0.1 level.
43
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SECTION VI
SUMMARY
At least 95% of the mirex in aqueous solution is removed by adsorption to organic matter (dead
bacterial cells), kaolinite clay and montmorillonite clay within 2,7 and 30 days, respectively. Only 8-
15% of the carbofuran is removed from aqueous solution by adsorption to these materials.
No significant decrease in mirex concentration occurred in a sediment-water system exposed to
sunlight for 130 days, but there was evidence of some disappearance in purely aqueous solutions. Con-
trary wise, approximately 75% of the carbofuran disappears within 49 days in sediment-water system,
but no detectable residues of 3-hydroxy- or 3-ketocarbofuran were found.
In disc assay tests, carbofuran failed to inhibit the growth of any of the 20 bacterial isolates tested
while the results with mirex varied from batch to batch with one batch producing zones of inhibition
on 18 of the 20 cultures. Gas chromatographic analyses demonstrated that different batches of mirex
contained varying amounts of unidentified impurities. For the most part, the remaining studies were
conducted with purified mirex.
In pure culture studies, mirex and carbofuran failed to inhibit gelatin hydrolysis, starch
hydrolysis, lipid hydrolysis, casein hydrolysis, chitin hydrolysis, glucose fermentation, H2S produc-
tion, nitrate reduction, ammonification and methane utilization. These concentrations also failed to
inhibit methane utilization and nitrification by estuarine sediments. Ten ppm mirex and carbofuran
did not inhibit the metabolic activity of estuarine pond water, but did inhibit primary productivity.
Concentrations of 1000 ppm mirex and carbofuran did not effect the growth curves of bacterial
cultures in broth.
Microcosm studies confirmed the lack of inhibition by mirex. A concentration of 1000 ppm did not
significantly alter the mixed microbial populations or the microbial activities of estuarine sediments.
On the other hand, the addition of carbofuran to sediments in concentrations of 100 ppm or greater
caused an increase in the numbers of aerobic heterotrophic bacteria, but did not favor the develop-
ment of any single type of bacteria. Further, the addition of carbofuran to sediments, in concen-
trations of 100 ppm or greater, significantly reduced the sediment dehydrogenase activity, the ability
of the sediment microflora to mineralize glucose, the rate of oxygen utilization during glucose
assimilation by sediment microorganisms, and the production of hydrogen sulfide by sediment
bacteria.
Degradation products of mirex and carbofuran were shown to be far more toxic to
microorganisms than were the parent compounds. Of the mirex degradation products, kepone and
reduced kepone exhibited the greatest toxicity. The 3-hydroxycarbofuran was more toxic than either
carbofuran or 3-ketocarbofuran.
Ultraviolet irradiation of moist mirex resulted in a bacteriologically toxic material.
44
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SECTION VII
DISCUSSION
The following paragraphs describe briefly the rationale whereby the conclusions were drawn
from this study.
Mirex is known to be very insoluble in water; also, it is quickly adsorbed onto particulate matter
and ultimately settles to the bottom where it is not subject to photochemical degradation. It resists
decomposition and is virtually inert under normal environmental conditions. Therefore in nature, in
the aqueous environment, mirex will not remain long in the water column but may have an ap-
preciable lifetime in the sediments.
While carbofuran is not readily adsorbed, it has been shown that more than 75%of the carbofuran
in an aqueous environment disappears in a short time probably due to hydrolysis to the phenol.
Therefore, buildup of this pesticide is not likely.
The only microbiological activity appreciably effected by either mirex or carbofuran at a level
below 100 ppm was the inhibition of primary productivity. As a result of the low water solubility and
the high rate of depletion by adsorption in the case of mirex and the rapid disappearance of car-
bofuran by other mechanisms, neither are expected to have any significant effect on primary produc-
tivity in the environment since the majority of the phytoplankton are in the aqueous phase, rather
than in the sediments and most of the material would be attached to particulate matter prior to en-
trance into the water.
While 3-hydroxycarbofuran has been shown to be toxic in concentrations as low as 1 ppm, this
derivative was not produced from carbofuran in the fate studies. Although degradation products of
mirex have been shown to be toxic at concentrations of 1 ppm, they were not found in detectable quan-
tities in the fate studies. In another study 16 of mirex under environmental conditions, the yields of
degradation products were extremely low. This low conversion, coupled with the probability that the
degradation products may be more biodegradable than the parent compound, leads to the expectation
that they will not cause problems in the environment.
Highly significant was the finding that purified mirex was not toxic to microorganisms while
some batches of technical mirex and UV irradiated moist mirex were toxic. It may be concluded that
other studies on the toxicity of mirex should be re-evaluated. This is especially true in view of the fact
that one of the breakdown compounds studied in this investigation has the same retention time as
mirex and because the concentrations of degradation products that would cause observable toxicity is
such that they would not be detected using normal gas chromatographic procedures. All of these com-
pounds are more polar than mirex and should have higher water solubility than mirex. These facts
may be important factors in toxicity studies in water.
45
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SECTION VIII
REFERENCES
1. EPA. 1972 Summary of Registered Agricultural Pesticide Chemical Uses. III-C.1-4.2, 1972.
2. Kennedy, M., M. Holliman and B. R. Layton. (Personal communication) Identification of the Ma-
jor Thermal Product of the Insecticide Mirex, to be published J. Ag. Food Chem.
3. Alley, E. G., D. A. Dollar, B. R. Layton and J. P. Minyard, Jr. Photochemistry of Mirex. J. Agr.
Food Chem. 21(1):138-139, 1973.
4. Alley, E. G., B. R. Layton and J. P. Minyard, Jr. Identification of the Photoproducts of the Insec-
ticides Mirex and Kepone. J. Agr. Food Chem. 22(3):442-445, 1974.
5. Billing, W. L., H. P. Braendling and E. T. McBee. Pentacyclodecane, Chemistry II. Tetrahedron
23:1211-1224, 1967.
6. Metcalf, R. L,, T. R. Fukato, C. Collins, K. Borck, S. A El-Azie, R. Munoz and C. C. Cassil.
Metabolism of 2,2-dimethyl-2,3-dyhydrobenzofuranyl-7-N-Methylcarbamate (Furadan) in
Plants, Insects, and Mammals. J. Ag. Food Chem. 16:300, 1968.
7. American Public Health Association (APHA). Standard Methods for the Examination of Water
and Wastewater. 13th ed. American Public Health Association, Inc., New York, 1971.
8. Cook, D. W. Metabolism of Carbohydrates by Hydrogenomonas eutropha. Ph.D. Dissertation,
Mississippi State University, 1966.
9. Hutton, W. E. and C. E. ZoBell. The Occurrence and Characteristics of Methane-Oxidizing
Bacteria in Marine Sediments. J. Bacteriol. 58:463-473, 1949.
10. Brown, L. R., R. J. Strawinski and C. S.McCleskey. The Isolation and Characterization of
Methanomonas methanooxidans (Brown and Strawinski). Can. J. Microbiol. 10:792-799,1964.
11. Mitchell, Ralph, S. Fogel and I. Chet. Bacterial Chemoreception: An Important Ecological
Phenomenon Inhibited by Hydrocarbons. Water Res. 6:1137-1140, 1972.
12. Harrison, M. J., R. T. Wright and R. Y. Morita. Method for Measuring Mineralization in Lake
Sediments. Appl. Microbiol. 21:698-702, 1971.
13. Lowry, 0. J., N. J. Rosenbrough, H. L. Farr and B. J. Randall. Protein Measurements with Folin
Phenol Reagent. J. Biol. Chem. 193:265-275, 1951.
46
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14. Pamatmat, M. M. and A. M. Bhagwat. Anaerobic Metabolisms in Lake Washington Sediments.
limn, and Oc. 18:611-623, 1973.
15. Anonymous. U. S. Dept. H.E.W. Washington, B.C. Pesticide Analytical Manual 1:211-212,1973.
16. Ivie, G. W., H. W. Borough and E. G. Alley. Photodecomposition of Mirex on Silica Gel
Chromatoplates Exposed to Natural and Artificial Light. J. Agr. Food Chem. 22(6):933-935,
1974.
47
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-660/3-75-024
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
The Effect of Mirex and Carbofuran on Estuarine
Microorganisms
5. REPORT DATE
March 1975
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Lewis R. Brown, Earl G. Alley, David W. Cook
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Mississippi State University
Mississippi State, Mississippi 39762
10. PROGRAM ELEMENT NO.
1 KA077
. CONTRACT/'
11
F/GRANT NO.
68-03-0288
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
National environmental Research Center
Office of Research and Development
Clm-vaTI is. Orpgnn 97730
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
ROAP/TASK NO. 10AKC/33
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The purpose of this investigation was to help establish the chemical, physical
and micro-biological fate of mirex and carbofuran in the estuarine environment and
determine the effect(s) on important estuarine microorganisms and their activities.
Chemical studies on the adsorption, fate and hydrolysis were conducted. The micro-
biological studies involved the use of both pure cultures and mixed cultures in a
microcosm system and included twelve distinct physiological groups of microorganisms.
It was concluded that neither mirex nor carbofuran would have a deleterious
effect on estuarine bacteria under normal conditions, and there was no evidence of
bioaccumulation. Degradation products of both compounds were shown to be toxic to
some microorganisms.
This report was submitted in fulfillment of Contract No. 68-03-0288 by Missis-
sippi State University under the sponsorship of the Environmental Protection Agency.
Work was completed as of 31 December 1974.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Estuarine Environment
Mirex - Fate
Carbofuran - Fate
Degradation Products
Toxicity
Chemical Studies
Microcosm
Adsorption Fate
Hydrolysis
Physiological Groups
3. DISTRIBUTION STATEMENT
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
69
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
* U S GOVERNMENT PRINTING OFFICE 1975-698.639 /I63 REGION 10
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