PB82-242017
Determination of the Environmental Impact of
Several Substitute Chemicals in
Agriculturally Affected Wetlands
Louisiana State Univ.
Baton Rouge
Prepared for
Environmental Research Lab,
Gulf Breeze, FL
Jul 82
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
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?b62-2l2017
EPA 600/4-82-052
July 1982
DETERMINATION OF THE ENVIRONMENTAL IMPACT OF SEVERAL SUBSTITUTE
CHEMICALS IN AGRICULTURALLY AFFECTED WETLANDS
by
S. P. Meyers, R. P. Gambrell,
and J. W. Day, Jr.
Center for Wetland Resources
Louisiana State University
Baton Rouge, LA 70803
Grant No. R-804976
EPA Project Officer
Frank G. Wilkes
Environmental Research Laboratory
Gulf Breeze, FL 32561
Prepared for
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, FL 32561
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NOTICE
THIS DOCUMENT HAS BEEN REPRODUCED
FROM THE BEST COPY FURNISHED US BY
THE SPONSORING AGENCY. ALTHOUGH IT
IS RECOGNIZED THAT CERTAIN PORTIONS
ARE ILLEGIBLE, IT IS BEING RELEASED
IN THE INTEREST OF MAKING AVAILABLE
AS MUCH INFORMATION AS POSSIBLE.
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TECHNICAL REPORT DATA
(Please read Innrjciioni on the reverse before complrrinfl
:. REPORT NO. 2.
EPA-600/4-82-052 ORD Reoort
. . TITLE AND SUBTITLE
Determination of the Environmental Impact of Several
Substitute Chemicals in Agriculturally-Affected Wetlanc
' » u T H O R ( S i
S. P. Meyers, R. P. Gambrell, and J. W. Day, Jr.
P = oFOR.V.:NG ORGANIZATION NAME AND ADDRESS
" Center for Wetland Resources
Louisiana State University
Baton Rouge, LA 70803
is. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Environmental Research Laboratory
Office of Research and Development
Gulf Breeze, FL 32561
3. RECIPIENT'S ACCESSION NO.
<>? 2 A 2 0 1 7
5. REPORT DATE
July 1982
6. PERFORMING ORGANIZATION CODE
S
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
R-804976
13 TYPE OF REPORT AND PERIOD COVERED
Final 9/1/76 - Q/1/80
1«. SPONSORING AGENCY CODE
Procedures have been developed for processing of anaerobic wetland sediments for
pesticide recovery along with formulation of simulation models of anaerobic/aerobic soil
and sediment environments to study pesticide degradation. Redox conditions of soils and
sediment-water systems have a significant effect oh in situ" persistence of svnthetic
organic pesticides. Chemical and microbiological characteristics of wetland sediments
Have equally important consequences on mobility and degradation of toxic compounds
The total invertebrate community of selected backswamp regions has been examined as
affected by Guthion and other pesticides. Effect of xenobiotics on leaf litter de-
composition in pesticide-supplemented field plots has been examined.
A system of continuous-flow and static microcosm systems have been developed for
quantitative analyses of the effect of selected toxic substances, including Guthion
methyl parathion, and Kepone. Decomposition of ecologically-significant substrates such
as chitin is variously affected by different toxic substances as shown in microcosm
investigations. Enzymatic tests, i.e., dehydrogenase and phosphatase, and ATP measure-
ments , are sensitive indicators of biotransformation processes. Significant correla-
tions are seen with microbial diversity indices and specific microbial groups such as
filamentous fungi. Factorial analyses of physicochemical and microbial processes and
xenobiotic interaction have demonstrated the application of the microcosm as a protocol
or tool to simulate pristine and impacted in situ ecosystems.
7- . KEY WORDS AND DOCUMENT ANALYSIS ~"
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20. SECURITY CLASS fTliispafC)
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22. PRICE
PA Form 2220-1 (R«y. <_77) =RCviOu» CC'TiOK IS
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DISCLAIMER
Although the research described in this report has been funded wholly
or in part by the United States Environmental Protection Agency under
Grant agreement number R804976 to S.P, Meyers, Center for Wetland
Resources, Louisiana State University, Baton Rouge, Louisiana, it has not
been subjected to the Agency°s required peer and administrative review
and, therefore, does not necessarily reflect the view of the Agency and no
official endorsement should be inferred.
ii
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FOREWORD
The protection of our estuarine and coastal areas from damage caused by
toxic organic pollutants required that regulations restricting the introduction
of these compounds into the environment be formulated on a sound scientific
basis. Accurate information describing dose-response relationships for
organisms and ecosystems under varying conditions is required. The EPA
Environmental Research Laboratory, Gulf Breeze (ERL.GB), contributes to this
information through research programs aimed at determining:
the effects of toxic organic pollutants on individual species and
communities of organisms;
the effects of toxic organics on ecosystem processes and components;
the significance of chemical carcinogens in the estuarine and marine
environment.
Many agricultural chemicals are applied in close proximity to sensitive
wetland regions. This report describes field and laboratory studies of the
fate and behavior of selected xenobiotics in the coastal wetlands of Louisiana.
Data from these investigations are used by the U.S. Environmental Protection
Agency to develop strategies that will minimize the harmful impact of toxic
substances on the environment.
HenrjL/F. Enos
Director
Environmental Research Laboratory
Gulf Breeze, Florida
111
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ABSTRACT
This research program was developed with the overall objective of exam-
ining toxic substances, especially organophosphorus compounds, in wetland
regions and ascertaining fate and effect in. situ and in controlled laboratory
microcosm systems. Major attention has been given to azinphosraethyl
(Guthion) , including effect of abiotic and biotic factors on compound sta-
bility and behavior under diverse pH and oxidation-reduction conditions as
affected by the microbial biomass.
Procedures have been developed for processing of anaerobic wetland
sediments for pesticide recovery along with formulation of simulation models
of anaerobic/aerobic soil and sediment environments to study pesticide
degradation. Redox conditions of soils and sediment-water systems have a
significant effect on in situ persistence of synthetic organic pesticides.
Analyses indicate that chemical and microbiological characteristics of
wetland sediments have equally important consequences on mobility and degra-
dation of toxic compounds.
The total invertebrate community and leaf litter decomposition of
selected backswamp regions has been examined as affected by Guthion and other
pesticides. Ash-tupelo litter decomposition was not adversely affected by
Guthion. Dissimilar responses of the complex invertebrate biota and its
individual taxa to Guthion and other toxic substances demonstrates the
importance of a total community analysis in terms of xenobiotic impact.
Systems of continuous-flow and static microcosm systems have been
developed for quantitative analyses of the effect of selected toxic sub-
stances, including Guthion, methyl parathion, and Kepone . Laboratory/field
protocol have included formulations of microbial/enzymatic protocols to
analyze xenobiotic effect. A data analysis program has been developed to
demonstrate significant correlations between in situ observations and
microcosm-generated information. Decomposition of ecologically significant
substrates, such as chitin, is variously affected by different toxic sub-
stances as shown in microcosm investigations. Enzymatic tests, i.e.,
dehydrogenase and phosphatase, and ATP measurements, are sensitive indicators
of biotransformation processes. Significant correlations are seen with
microbial diversity indices and specific microbial groups, such as filamen-
tous fungi. Factorial analyses of physicochemical and microbial processes
and xenobiotic interaction have demonstrated the application of the microcosm
as a protocol or "tool" to simulate pristine and impacted in situ ecosystems.
This report was submitted in fulfillment of Grant No. R-804976 by the
Center for Wetland Resources, Louisiana State University, under the sponsor-
ship of the U.S. Environmental Protection Agency. The report covers the
period September 1, 1976 to September 1, 1980, and work was completed as of
December 1, 1980.
iv
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CONTENTS
Foreword iii
Abstract iv
Figures vii
Tables xi
Acknowledgment xiii
Introduction 1
Effect of Oxidation-Reduction Conditions on Pesticide Persistence ... 5
Introduction 5
Materials and Methods 11
Experimental reaction chambers 11
Soil and sediment material 13
Pesticide extraction procedures 13
Results and Discussion 17
Guthion 17
Trifluralin 23
Methyl parathion 27
2,4-D 31
Conclusions 31
References 32
In Situ/Microcosm Investigations and Microcosm Evaluation and
Validation 36
Introduction 36
Experimental Procedures 37
Study site and sample collection 37
Enumeration of microorganism 37
Enzyme assay 38
Gas chromatographic techniques 39
Data analyses 39
Description of the microcosms 40
Results and Discussion 44
Initial In Situ, Static, Continuous Flow, and Carbon
Metabolism Microcosm Studies 44
^n situ enumeration of total heterotrophs and
chitinoclasts 44
Determination of pesticide half-life 47
Additional in situ studies 47
Initial static microcosm studies for total heterotroph/
chitinoclast enumeration 52
Continuous flow microcosm studies for total heterotroph/
chitinoclast enumeration 52
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Changes in sensitivity and utilization of
azinphosmethyl 59
Pesticide accumulation 62
Initial enzymatic studies 62
Initial carbon metabolism studies 65
Determination of pesticide half-life 68
Studies with Guthion, methyl parathion and Kepone 70
Microbial/enzymatic correlations: Guthion and
methyl parathion 70
Continuous flow studies (Part 2) 70
Continuous flow studies (Part 3) and carbon metabolism
studies (Part 2) 76
ATP response 79
Further enzyme responses 81
Radiotracer analysis 82
Axenic flask studies 90
Gas chromatography analysis 92
Conclusions 93
References 96
In Situ Effects of Guthion on Backswamp Aquatic Invertebrates and
Shallow Water Leaf Litter Decomposition 99
Introduction 99
Experimental 101
Short-term effects of single exposure to Guthion 101
Long-term effects of single exposure to Guthion 106
Effects of continuous exposure to Guthion (0.01 ppm) .... 110
Short-term comparison of Guthion with Furadan, methyl
parathion, and Azodrin 112
Conclusions 115
References 119
Project Conclusions 124
Bibliography 127
Appendix 128
VI
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FIGURES
Number page
1 Map of Barataria Bay Hydrologic Unit With Des
Allemands Swamp Sampling Site 2
2 Reaction Chamber Apparatus for Controlling Redox
Potential and pH 12
3 Effects of Controlled Redox Potential on Recovery
of Guthion From a Swamp Forest Soil (Incubated
at pH 6.0) 19
4 Effects of Controlled Redox Potential on Recovery
of Guthion From a Swamp Forest Soil (Incubated
at pH 7.0) 20
5 Effects of Controlled Redox Potential on Recovery
of Guthion From a Swamp Forest Soil (Incubated
at pH 8.0) 21
6 Effects of Controlled Redox Potential on Recovery
of Trifluralin From a Bayou Chevreuil Sediment
(Incubated at pH 7.0) 24
7 Effects of Controlled Redox Potential on Recovery
of Compound E From a Bayou Chevreuil Sediment
(Incubated at pH 7.0) 25
8 Effects of Controlled Redox Potential on Recovery
of Compound Y From a Bayou Chevreuil Sediment
(Incubated at pH 7.0) 26
9 Effects of Controlled Redox Potential on Recovery
of Compound X From a Bayou Chevreuil Sediment
(Incubated at pH 7.0) 28
10 Effects of Controlled Redox Potential on Recovery
of Methyl Parathion From a Bayou Chevreuil
Sediment (Incubated at pH 6.0) 29
11 Effects of Controlled Redox Potential on Recovery
of Methyl Parathion From a Bayou Chevreuil
Sediment (Incubated at pH 8.0) 30
Vll
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Number Page
12 Diagram of Microcosm Units: (A) Continuous Flow;
(B) Carbon Metabolism Unit 42
13 Effect of Guthion on Heterotrophic and Chitino-
clastic Population in Swamp Sediments (Field
Study No. 1) 44
14 Effect of Guthion on Heterotrophic and Chitino-
clastic Population in Swamp Sediments (Field
Study No. 2) 44
15 Effect of Guthion on Heterotrophic and Chitino-
clastic Population in Swamp Sediments (Field
Study No. 3) 46
16 Variations in Microbial Populations and
Enzyme Activities 52
17 Effect of Guthion on Heterotrophs and Chitino-
clasts in Chitin-Amended Soils 54
18 Static Microcosm Study. Treatment Variation of
Total Heterotrophs 55
19 Effect of Guthion on Heterotrophs and Chitino-
clasts in Continuous Flow Microcosms (Aerobic) ... 56
20 Effect of Guthion on Heterotrophs and Chitino-
clasts in Continuous Flow Microcosms (Anaerobic) . . 57
21 Treatment Mean Variations for Heterotrophs and
Chitinoclasts in Guthion Supplemented Con-
tinuous Flow Microcosms 58
22 Effect of Chitin-Addition on Dehydrogenase
Activity In Situ 63
23 Effect of Guthion on Phosphatase Activity in
Three Soil Types (Control, Chitin-Amended,
Starch-Amended) 64
24 Effect of Guthion on 14C02 Release in Carbon
Metabolism Microcosm 66
25 Effect of Guthion on 14C-Glucosamine Uptake in
Carbon Metabolism Microcosm 67
26 Mean Treatment Effect of Guthion in Carbon
Metabolism Microcosms 69
Vlll
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Number page
27 Effect of Methyl Parathion on In Situ Microbial
and Enzymatic Activity 71
28 Variation in Microbial/Enzyme Activity in
Continuous Flow Microcosm (Control and Guthion-
Treated) 73
29 Variation in Microbial/Enzyme Activity in
Continuous Flow Microcosm (Control and Methyl
Parathion-Treated) 74
30 Microbial Response to Methyl Parathion (5 ppm)
in Static Carbon Metabolism Microcosm 76
31 Microbial Response to Kepone (0.5 ppm) in
Static Carbon Metabolism Microcosm 77
32 Microbial Response to Methyl Parathion (5 ppm)
in Flow-through Microcosm 78
33 Microbial Response to Kepone (0.5 ppm) in Flow-
through Microcosm 79
34 Response of ATP to Addition of Methyl Parathion
and Kepone in Static Microcosms 80
35 Response of ATP to Addition of Methyl Parathion
and Kepone in Flow-through Microcosms 80
36 Response of Phosphatase and Dehydrogenase to
Addition of Methyl Parathion and Kepone in
Static Microcosms 81
37 Response of Phosphatase and Dehydrogenase to
Addition of Methyl Parathion and Kepone in
Flow-through Microcosms 82
38 Residual levels of 14C-Methyl Parathion and
14C-Kepone in Static Microcosms 83
39 14C Assimilation Within Colony Forming Units
(CFU) With Methyl Parathion and Kepone in
Static Microcosms 84
40 Effect of Chitin on 14C02 and 14C Uptake in Methyl
Parathion Carbon Metabolism Microcosms 85
41 Chitin Turnover in an Estuary 86
42 Microbial Response to Methyl Parathion in
Chitin-Amended Flow-through Microcosms 87
ix
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Number Page
A3 Microbial Response to Kepone in Chitin-
Amended Flow-through Microcosms 88
44 Residual Levels of 14C-Methyl Parathion and
14C-Kepone in Carbon Metabolism Microcosms 89
45 Effects of Guthion on Settling of Biota of
Floating Vegetation (F.V.) 102
46 Changes in Guthion Concentrations in Sediment
and Water 103
47 Effects of Guthion (1 and 10 ppm) on the
Biota in Floating Vegetation (F.V.) 104
48 Effects of Guthion (25 ppm) on the Biota in
Floating Vegetation (F.V.) 105
49 Effects of Plastic Enclosure on the Biota in
Floating Vegetation (F.V.) 106
50 Effects of Guthion on Leaf, Little Weight Loss
Due to Decomposition Ill
51 Response of Swamp Invertebrates to Methyl Parathion
(MP), Guthion (G), Furadon (F), and Azodrin (A) ... 114
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TABLES
Number page
1 Operating Conditions for Gas Chromatograph for
Pesticide Analysis 18
2 Ratio of Soluble to Adsorbed Guthion at pH 6.0 and
7.0 in a Swamp Soil-Water Suspension 22
3 2,4-D Recovery from Bayou Chevreuil Sediment
Material Incubated Under Controlled pH and Redox
Potential Conditions 31
4 Mean Residual Pesticide Concentration from Field
Application Studies 48
5 Field Sites: Soil Profiles with Depth 49
6 Fluctuation of Microbial Population with Depth ... 50
7 Enzyme Activity at Various Sites with Soil Depth . . 51
8 Changes in Sensitivity to and Utilization of Guthion
in Continuous Flow Aerobic Microcosms 60
9 Growth of Selected Axenic Cultures Isolated from
Continuous Flow Aerobic Microcosms 61
10 Residual Guthion Concentrations in Soil from
Dehydrogenase and Phosphatase Activities Trials ... 65
11 Residual Guthion Concentrations in Soil from
Carbon Metabolism Study 68
12 Mean Treatment Variations and Enzyme Response for
Total Heterotrophs in Continuous Flow Microcosms
(Control and Guthion-Treated) 75
13 Microcosm-Isolated Species Level Response to
Tagged Xenobiotic Addition 91
14 Toxicity of Guthion (ppb) to Aquatic Fauna 101
15 Sampling Schedule for the Test Series II (April-
June, 1978) and III (June, 1978) 108
xi
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Page
Effects of Guthion on Back-Swamp Vertebrates
(Sediment and Floating Vegetation) 108
17 Test Series II. Use of Stovepipe Enclosures
(25 ppm Guthion) 109
18 Test Series II. Use of Stovepipe Enclosures
(Control) 110
19 Average Density, Diversity, and Evenness of
Animals in the Floating Vegetation After
30 Days of Continuous Exposure 113
XII
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ACKNOWLEDGMENTS
Various research personnel have actively participated in the development
of this composite investigation and their contributions are readily acknowl-
edged.
Dr. Gambrell's work on effect of oxidation-reduction conditions on
pesticide persistence and degradation was ably assisted by Ms. Vickie
Collard, Cheryl Hinton, Stephanie Scott, Michele Adamcak, and Barbara Taylor.
The in situ/microcosm investigations involved the dedicated and enthu-
siastic efforts of Mr. Ralph J. Portier who has served throughout the study
as a co-investigator and coordinator. His contributions to the total program
have been invaluable. We are also pleased to recognize the efforts of
Ms. Blair Roy and those of Mr. Nelson May in providing statistical advice.
The in situ analyses of Guthion on aquatic invertebrates were ably
conducted by Mr. Fred H. Sklar who was assisted in his field work by various
Center for Wetland Resources personnel, including Scott McNamara, William
Conner, Paul Kemp, and Ronald Paille. Microscopic assistance was provided by
Ms. Nancy Stevens, Jim Bahr, and Dianne Lindstedt. Walter and Jean Sikora
aided in various species identifications.
xiii
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SECTION 1
INTRODUCTION
Increasing emphasis on use of a variety of chemicals in agricultural
practices presents potential environmental and real problems in terms of land
use and danger to adjacent wetland systems. This is further complicated by
increasing discharge of an array of toxic substances (xenobiotics) from
industrial facilities and chemical disposal areas in close proximity to such
sensitive wetland regions. An example of the complexity of the problem is
evident in the coastal zone of Louisiana, comprising more than seven million
acres of marshes and estuaries and representing approximately 40% of the
total coastal wetland area of the contiguous United States. The significance
of the region as a fishery resource is well established. This ecosystem is
close to major economically significant agricultural and industrial sites in
the fertile Mississippi delta. A wide variety of organic materials, in-
cluding potentially hazardous chemicals, are brought into the region by the
gulfward movement of water. In spite of the widespread use of these chem-
icals, limited data are available on the fate of pesticides and other
chemicals in such agriculturally affected wetlands.
Important chemical changes take place during passage of water through
the aforementioned wetlands. Suspended sediment settles with decreased water
flow, and inorganic nutrients, especially nitrogen and phosphorus, are uti-
lized by plant growth. Major modification of xenobiotics, via abiotic
hydrolysis and microbial degradation, in all likelihood is most active in
this area.
Degradation of xenobiotics in the environment occurs by abiotic as well
as biotic processes. The degree of physical adsorption of compounds to
colloidal mineral and humic materials strongly influences the susceptibility
of soil- and sediment-bound pesticide residues to chemical or biological
attack. Although there is considerable information on persistence and fate
of pesticides in soils, limited data are available on the effects of pH,
oxidation, and salinity levels and the combined interactive effects of
chemical, physical, and biological conditions affecting degradation processes
in wetland soil/sediment ecosystems.
Organophosphorus insecticides, the major class of compounds studied,
generally are known to be less persistent in soils than many of other chem-
ical classes of pesticides, such as chlorinated hydrocarbons. However, some
Organophosphorus compounds have been shown to persist in soils for several
months; furthermore, chemical and biological properties of soils and
sediment-water systems affect such persistence.
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Our research is intended to provide information on the fate and behavior
of selected xenobiotics in the agriculturally affected coastal wetlands of
Louisiana. The major area of study is shown in Figure 1. An understanding
of the biological, chemical, and physical conditions conducive to degradation
of pesticides may enhance the predictive capability for assessment of poten-
tial environmental impact and aid development of management practices that
minimize adverse effects.
rm
Figure 1. Map of Barataria Bay Hydrologic Unit with Des Allemands Swamp
Sampling Site.
The organization of the project involves linkages of research between
three major components of this study, namely, microbiology, in situ investi-
gations, and physicochemical studies. The microbiological portion comprises
both laboratory and in situ approaches involving correlation analyses and
controlled simulation studies. The three field and laboratory approaches
developed are designated as: Effect of Oxidation-Reduction Conditions on
Pesticide Persistence, In Situ/Microcosm Investigations and Microcosm Evalu-
ation and Validation, and In Situ Effects of Guthion on Backswamp Aquatic
Invertebrates and Shallow Water Leaf Litter Decomposition. For clarity,
these major subjects are initially treated in separate sections of this
report, but as noted subsequently, are closely related topics within the
composite coastal zone management area.
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Within this interrelated framework of research several important com-
ponent relationships among the individual projects need to be emphasized, in
order to provide a transition between major blocks of research data and to
attenuate these major project outputs into a concise package. The contribu-
tion of each individual research projects in achieving the research goals
originally proposed are addressed in the final project discussion.
Beginning with physical/chemical studies, the primary research objec-
tive, as noted, was to ascertain the effect of oxidation reduction conditions
on pesticide persistence. An understanding of the underlying physical/
chemical characteristics affecting pesticides in aquatic systems provides a
framework for further studies within the microbiological/biological component
and, in combination, provides an insight into pesticide effects within the
general ecology of the target wetland system, an important consideration for
coastal zone management. Specific objectives for the physical/chemical
studies included:
Development of efficient and reproducible extraction methodologies
which eliminate problems associated with anaerobic sediments.
Development of procedure for processing of anaerobic sediment
samples to recover pesticides without introduction of degradation
artifacts.
Formulation of physical/chemical simulation studies, emphasizing
oxidation reduction conditions, of anaerobic/aerobic soil and
sediment environments to study pesticide degradation.
Microbiological investigations were two-fold in nature. Our overall
objective was to ascertain the effect and fate of a pesticide on the micro-
bial component in an aquatic microenvironment. In doing so, we felt an
important linkage between biotic and abiotic factors impinging on pesticide
effect and fate could be ascertained, providing further insight into pesti-
cide effects in higher trophic communities. To accomplish this, it was
necessary to design and validate a laboratory/field protocol which would
provide quantitative analysis of pesticide fate and effect. To do so, the
following objectives were proposed:
Development of laboratory/field protocol for pesticide analyses
(including microbial/soil enzymatic activity and response, 14C-
respiration and bioaccumulation interaction, and complimentary
residue analysis by GC methods established in physical/chemical
studies).
Development of laboratory/field protocol for in situ/laboratory
analysis of key organic substrates, i.e., chitin and cellulose, as
affected by xenobiotic addition under aerobic and anaerobic condi-
tions .
Development of selective media for in situ/laboratory sampling.
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Our earlier studies have demonstrated the unique microbial composition
of the salt marsh ecosystem and its extremely active chitinoclastic (chitin-
decomposing) heterotrophic population. Significant rates of detrital turn-
over into the trophic food web have demonstrated the important role of the
extant microbial biomass and the impact of stress factors on microbial
ecosystems and specific transformation processes. With the completion of
preliminary studies with the organophosphate, Guthion, two additional
research objectives were proposed:
The analysis of heterotrophic activity/enzyme activity profiles in
several ecologically significant in situ soil microenvironments to
correlate these observations with variations in controlled micro-
cosms due to xenobiotic addition.
Formulation of a statistical and computer-base program for data
analysis of microbial and enzymatic activity variations.
Field studies of indigenous invertebrate populations primarily involved
the analysis of in situ effects of the organophosphate, Guthion, on backswamp
aquatic invertebrates, as well as providing indications of the effect of this
pesticide on shallow water leaf litter decomposition. Specific objectives of
this research group included:
Development of total invertebrate community analyses of population
dynamics with pesticide input.
Development of laboratory cultures of selected wetland inverte-
brates and their possible use as indicators of environmental
stress.
Analysis of sampling errors in studies of in situ effects of pes-
ticides in wetland communities.
Also included in the above research objectives were the analysis of tech-
niques and problems associated with biochemical stress analysis (energy
charge ratio) in pesticide affected food webs in estuarine ecosystems.
In all facets of the study, major attention has been given to the or-
ganophosphorus compound Guthion (0,0-dimethyl S-[4-oxo-l,2,3-benzothriazin-3
(4H)-ylmethyl] phosphorothioate) used in control of the cane borer on sugar-
cane (Saccharum officinarum L.) in Louisiana. Guthion (azinphosmethyl)
represents 80% of the total volume of insecticides applied to sugarcane,
being substituted for Endrin over a decade ago because of problems in fish
toxicity and insect resistancy. Other xenobiotics, such as methyl parathion,
have been used to develop comparative data in terms of methodology and dose
response.
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SECTION 2
EFFECT OF OXIDATION-REDUCTION CONDITIONS
ON PESTICIDE PERSISTENCE
R. P. Gambrell
INTRODUCTION
The objective of our studies was to determine the effect of oxidation-
reduction conditions and variations in the persistence of selected pesticides
in soils and sediments typical of Louisiana wetlands. Soil and sediment
materials were treated with pesticide compounds and incubated in special
laboratory reaction chambers as suspensions under conditions of carefully
controlled pH and redox (oxidation-reduction) potential. Sample aliquots
were withdrawn periodically over a 2 to 3 week incubation period and ex-
tracted to determine how compound concentrations changed with time under the
imposed physicochemical conditions.
The compounds studied included Guthion, as well as Trifluralin (6, 6,
6-trifluoro -2, 6-dinitro-N, N-dipropyl-p-toluidine), methyl parathion (0,
O-dimethyl-0-p-nitrophenyl phosphorothioate), and 2,4-D (2,4-dichloro
phenoxy-acetic acid).
Guthion, as noted previously, is used to control the caneborer in sugar-
cane, an important crop grown adjacent to freshwater wetlands in south
Louisiana. Trifluralin is an important herbicide for weed control in soy-
beans, an increasingly important crop in south Louisiana. Though perhaps not
used extensively near wetlands, methyl parathion was selected because it is
an effective insecticide which has found increasing demand as some of the
more persistent chlorinated hydrocarbon insecticides were phased out because
of environmental problems. The herbicide 2,4-D is widely used to control
broad-leaf weeds. This and its related compounds are also important in
controlling aquatic weeds in many waterways, especially in the southern
U.S.A.
It is well known that pH affects the dominant microbial populations and
their activities in soils and sediments. In general, a soil pH between 6.5
and 8.0 is considered optimal for microbial activity (Alexander 1961). The
oxidation-reduction potential of a soil or sediment-water system also affects
the overall kinetics of microbial populations. As oxygen becomes depleted
and the soil becomes reduced, aerobic organisms are inactivated, and faculta-
tive and obligate anaerobes predominate. As a result, a different array of
microbial biochemical processes occur with substitutes for oxygen as the
terminal electron acceptor, different metabolic pathways, and end products.
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In this way, the microbial bioraass of reduced systems are substantially
altered from those of aerobic systems (Patrick and Mikkelsen 1971). Since
different redox and pH conditions have been shown to strongly affect the
breakdown of some pesticides, it is necessary to study the combined effect of
these physicochemical factors on decomposition of the "target" pesticides.
Changes in oxidation potential may produce both quantitative and quali-
tative alterations in huraic materials (Reddy and Patrick 1975; Stevenson and
Ardakani 1972). Pesticides are reported to be strongly bound with soil and
sediment organics; thus, the availability of a given pesticide to microorgan-
isms, or the fauna of wetland ecosystems, may be regulated to a considerable
extent by the extant physicochemical environment affecting these adsorption
processes (Bailey and White 1970; Pionke and Chesters 1973; White and
Mortland 1970).
Pionke and Chesters (1973) in their extensive review, suggested that
colloidal hydrous oxides of iron may be effective adsorbents for some pesti-
cides. These oxides are thought to form as reduced ferrous iron is trans-
ported into oxidized sediment-water systems. Greater degradation rates for
DDT in reduced environments compared with those in oxidized environments has
been reported by a number of investigators (Fries 1972). Though less work
has been conducted with organophosphorus insecticides, there is some indica-
tion that the aforementioned may also apply to this class of compounds.
Sethunathan (1972) noted that Diazinon persisted for 180 days in unflooded
soils but disappeared in as little as 60 days in flooded soils. Presumably,
this organophosphate was reduced. An effect of pH on the persistence of this
compound also was noted. Walker (1976) reported on the chemical and micro-
biological degradation of malathion and parathion in seawater and sediments.
Malathion disappearance in the systems described was attributed primarily to
chemical rather than to microbiological processes, and its rate of loss was
in direct proportion to increasing salinity. Parathion was more persistent,
with rate of loss less dependent on salinity levels.
Walker and Stojanovic (1973) studied microbial versus chemical degrada-
tion of malathion in three Mississippi soils. Microbial degradation was
found to be the chief mechanism of insecticide dissipation in all soils
tested. Malathion was quite stable under neutral or acid pH conditions, but
was susceptible to hydrolysis under alkaline conditions. Chemical degrada-
tion appeared due to both alkaline hydrolysis and adsorption by the soil
particles.
Bourquin (1975) examined raalathion degradation in a simulated salt-marsh
environment and observed rapid in vitro breakdown by a variety of salt-marsh
bacteria. Bacterial concentrations were correlated with levels and frequency
of malathion treatment, although malathion sole-carbon degrading bacteria
comprised only about 10% of the bacterial population decomposing the pesti-
cide. It was suggested that increased numbers of malathion-co-metabolizing
bacteria catalyzed a more rapid dissipation of the compound. Chemical hydrol-
ysis of malathion was directly correlated with increasing temperature and
salinity. However, at temperatures below 26 C and salinities below 20°/00,
biological mechanisms appear to be of greater significance.
-------
Walker (1976) isolated microorganisms capable of degrading malathion and
parathion from insecticide-enriched sediment and examined in vitro breakdown.
Various degrees of pesticide decomposition were observed. Malathion was
essentially completely degraded during 18 days incubation in sterile and
nonsterile water, while respective parathion losses were from 16 to 23%.
Direct correlation between salinity and compound persistence were reported
during the field studies. Malathion degraded faster in higher salinity
water, while parathion was persistent up to 25°/00.
Sethunathan (1972) reported persistence of Diazinon for different
lengths of time in flooded rice soils depending on whether the soils had been
treated previously. Diazinon persisted for 15 days in previously treated
soils and about 60 da in nontreated soils. A flavobacterium, isolated from
water of a treated rice field had a high capability to metabolize diazinon as
sole carbon source.
Weber (1972) reviewed the interaction of organophosphorus pesticides in
aquatic and soil systems and noted that these compounds do not accumulate in
soil fauna, such as earthworms, and concentrate less in birds and fish, than
the chlorinated hydrocarbons. Most are readily degraded and do not accumulate
for long periods. Organophosphorus compounds readily adsorb to organic and
clay components of the soil. The main routes of disappearance of organophos-
phorus chemicals are via hydrolysis and microbial degradation.
Pionke and Chesters (1973) observed that pesticide persistence in water-
sheds is dependent largely on the stability of soil-applied pesticides. Many
organic phosphorus pesticides can be chemically hydrolyzed by nonmicrobial
means, whereas the longer-lived compounds are degraded by both chemical and
microbial routes. Soil conditions, such as unusual pH levels and high pesti-
cide residues not conducive to microbial activity, increase the persistence
of such compounds in the soil, thereby increasing the potential for aquatic
contamination. Generally, the adsorption of most nonionic pesticides is
correlated with soil organic levels.
Runoff from agricultural land is one of the main mechanisms for intro-
duction of pesticides into aquatic environments (Pionke and Chesters 1973).
Excessive rainfall from storms soon after application can result in losses
within 1 or 2 da greater than that which would normally occur in a year.
This may be especially true in southern Louisiana where sudden heavy rains
are common. Willis et al. (1975), in studies of losses of four herbicides in
surface drainage water in Louisiana, found that seasonal losses ranged from
0.05 to 3.0%. Highest concentrations in runoff were usually associated with
rainstorms that occurred soon after application. Willis and Hamilton (1973),
in investigations of endrin loss in runoff from sugarcane test plots, noted
that on an annual basis, about 0.2% of applied endrin was lost. Rainfall
soon after application increased loss rates. The low rates of loss may be
attributed to substantial losses by volatilization.
Pesticides are readily adsorbed by clay particles and organic matter.
The accumulation of organic matter in the anaerobic zone of lakes and wet-
lands increases the adsorption of pesticides. Some metal complexes of Fe
and Mn formed under these conditions may stabilize pesticides. Adsorption
-------
by algae and aquatic vegetation is rapid, often with concentrations of 100 to
10,000 times that in the aqueous portion of the ecosystem.
Willis et al. (1976) examined pesticide concentrations in runoff from
the Mississippi Delta watershed and noted a linear relationship between
pesticide yields in runoff and sediment yields. Emphasis was placed on the
limited amount of extant information on the role of sediment in pesticide
adsorption, transport, desorption, and degradation.
*
Organophosphorus compounds may be the least hazardous pesticides in
aquatic systems because of their rapid hydrolysis, especially favored at
alkaline pH.
Guthion
Heuer et al. (1974), in studies of the effect of pH and temperature on
half-life of Guthion in aqueous solutions and on glass surfaces, found that
both factors have a significant effect on degradation rates. At pH > 9,
half-life is about one month at temperatures of 6 C and 25 C. At pH > 9.5,
the half-life is about one week, while at 40 C and pH > 9.5, half-life was
less than one day.
Meyer (1965) reported that half-life of a Guthion formulation applied to
farm ponds to be about 2 da with a 90% reduction after 4 da. In a report by
the manufacturer's research staff (Chemagro Division Research Staff 1974) the
half-life in a pond water at 30 C was 1.2 da. The Chemagro paper also re-
ported a 50% reduction in Guthion in a Florida "muck-sand" in one month and
that a three-month period was required for a 50% Guthion loss from a clay
soil in Louisiana.
Yaron et al. (1974) studied the kinetics of Guthion in soil samples in
Israel with a pH of 8.4 and an organic matter content of < 1%. Experiments
were conducted with wet and dry, and sterile and natural soils at tempera-
tures of 6, 25, and 40 C. They found that natural, wet, and warmer soils
gave higher rates of degradation. The half-life in wet, natural soils at 6,
25, and 40 C was 64, 13, and 5 da, respectively. From these data, it can be
postulated that the breakdown of Guthion in warm Louisiana wetland soils
(with a mean annual temperature of 20 to 21 C) will be quite rapid. However,
since the pH of wetland soils is almost always lower than 9 and often acidic,
the half-life of the pesticide may be lengthened.
No breakdown metabolites have been identified for Guthion in studies of
animal tissues (Murphy and DuBois 1957; Nakatsugawa and Dahm 1962). Else-
where, Harvey et al. (1969) examined the decomposition of Guthion in vitro to
elucidate degradation mechanisms involved, and parameters that affect the
decomposition.
Ethyl and Methyl Parathion
The pathway of parathion degradation is likely affected by both avail-
ability of an energy source and the presence and size of microbial popula-
tions with the ability to hydrolyze parathion. Sethunathan (1973) reported
8
-------
on the effect of rice straw on the degradation of parathion in a flooded
soil. Rice straw amendments inhibited parathion hydrolysis to p-nitrophenol
and diethyl thiophosphoric acid in soils inoculated with an enrichment cul-
ture with a high ability to hydrolyze the compound. However, in uninoculated
soils, the addition of rice straw enhanced parathion breakdown by way of
nitro group reduction. By one pathway, p-nitrophenol was a degradation
product that included the aromatic portion of the molecule, while p-amino-
phenol was the corresponding degradation product by the other pathway.
Rajaram and Sethunathan (1976) reported that a factor was present in a
flooded soil amended with glucose or rice straw that inhibited the microbial
hydrolysis of parathion. This factor developed within 36 hr after flooding
the rice straw-amended soil.
Predominant degradation products of parathion are reported to be diethyl
thiophosphate, p-nitrophenol, aminoparathion, and p-aminophenol (Saltzman et
al. 1974; Rajaram and Sethunathan 1976; Rajaram and Sethunathan 1975).
Munnecke and Hsieh (1976) reported p-aminophenol and diethyl thiophosphoric
acid were degradation products under low oxygen conditions while p-nitro-
phenol and either diethylphosphoric acid or diethylthiophosphoric acid
(depending on the pathway) were formed under oxidized conditions. The afore-
mentioned research is for ethyl parathion, commonly called parathion. Con-
siderably less work is published on the degradation of methyl parathion, a
compound included in our study, though the similarity of the two compounds
would suggest a similar environmental chemistry.
Garnas et al. (1977) reported on the fate of methyl parathion in a
marine benthic microcosm. Ring-labelled methyl parathion disappeared from
the water column after 14 da, mostly as a result of transport and binding of
the compound and/or unextractable residues in the sediment. It was shown
that methyl parathion degraded rapidly, primarily by microbial processes, to
a number of more polar products including aminomethyl parathion and p-nitro-
phenol .
Sudhakar-Barik et al. (1976) reported on the metabolism of nitrophenols
by two bacterial isolates from parathion-amended flooded soil. Of the radio-
activity applied as p-nitrophenol, 23% and 80% (for Pseudomonas sp. and
Bacillus sp., respectively) was recovered as 14C02 after 72 hr.
Trifluralin
Trifluralin, a selective pre-emergence soil incorporated herbicide, is a
prominent compound in the dinitroaniline series of herbicides used as a
selective pre-emergence herbicide for the control of broad-leaved weeds and
grasses (Cripps and Roberts 1978). The compound is very sparingly soluble in
water and thus not subject to leaching in most soils. Trifluralin has a
relatively high vapor pressure and some volatilization losses may occur
(Weber 1970). Trifluralin is strongly adsorbed by organic soil colloids as
demonstrated by a greatly reduced effectiveness with increase in the soil
organic matter content (Lambert 1967; Probst et al. 1967).
-------
Parr and Smith (1973) studied trifluralin under various oxidation condi-
tions and reported that aerobic degradation was initially characterized by
the sequential dealkylation of propyl groups while anaerobic degradation
began with reduction of the nitro groups. In a substrate (energy)-amended
soil, degradation was more rapid under anaerobic versus aerobic conditions
(99% and 15%, respectively, after 20 da). Experimental evidence indicated
microbial involvement in initial degradation.
Probst et al. (1967) reported on the effects of soil aeration on tri-
fluralin. Under oxidized conditions, several metabolites were formed as a
result of dealkylation and reduction reactions, but these degradation
products were short-lived and were soon converted to polar products. Under
anaerobic conditions, the rate of degradation was greater. Reduction reac-
tions predominated and were followed by dealkylation.
Willis et al. (1974) reported on trifluralin degradation under controlled
redox potential conditions using a Sharkey clay soil (pH 5.7, 3.4% organic
matter). Flooding the soil to exclude oxygen initiated rapid degradation
only when the redox potential decreased below a potential between +150 and
+50 mv. At +50 mv, the trifluralin recovered decreased to zero in 8 da while
approximately 60% of the initial 1 ppm applied was present by 20 da. Exam-
ining the degradation pathways under aerobic and anaerobic conditions
proposed by Probst et al. (1967), Compound E (one nitro group reduced and one
propyl group dealkylated) was found in trace amounts at +250, +150, and
+50 mv, and no other degradation products were found in treatments better
oxidized than +50 mv. This compound is thought to be an intermediate in both
aerobic and anaerobic degradation. Another compound (one nitro group reduced)
was found at +50 mv, a compound postulated to be part of the anaerobic
pathway.
LaFleur et al. (1978) reported a two-stage process for the loss of
trifluralin from a Congaree soil. The initial disappearance process had a
half-life of 19 da while the slow process had a half-life of 450 da. This
multistage trifluralin disappearance was believed associated with various
adsorption-desorption phenomena.
2,4-Dichlorophenoxyacetic Acid
The herbicide 2,4-D is known to degrade rapidly in soils so that its
persistence is usually reported to be on the order of a few weeks (Brown
1978).
In a review of phenoxyalkanoate pesticides, Kaufman (1974) reported the
major metabolic reactions associated with phenoxyalkanoic acids include ring
hydroxylation, B-oxidation of the long chain aliphatic acid moiety, cleavage
of the ether linkage, dehalogenation, and ring cleavage. From published
information, Kaufman (1974) presented a microbial metabolism chart for 2,4-D
that indicates ring hydroxylation is a common and predominant process in
initial degradation prior to further degradation to "ecologically acceptable
metabolites." Succinic acid is believed to be the final product in 2,4-D
metabolism.
10
-------
Though 2,4-D and related compounds have been extensively studied, we
have been unable to find any information on oxidation-reduction effects on
its degradation.
Gambrell and Patrick (1978), in their review of the literature, con-
cluded that pH and especially oxidation-reduction conditions of soils and
sediment water systems affects the degradation rate of a number of chlori-
nated hydrocarbon pesticides. From the limited information available on
oxidation reduction effects, it may be concluded that if redox potential
conditions influence the degradation rate of pesticide compounds, they de-
grade more quickly under reduced conditions. Recently, Gambrell et al.
(1981) have reported that Permethrin, a synthetic pyrethroid, is degraded
more rapidly under oxidizing rather than reducing conditions.
From this short literature review, it is apparent that the physico-
chemical conditions of soils and sediment-water systems affect the fate of
many pesticide compounds. Because of the wide range of physicochemical
conditions to which residues may be subjected, especially if transported from
the site of application, it is important to understand the effects of param-
eters such as pH and redox potential conditions on the environmental chem-
istry of pesticides.
MATERIALS AND METHODS
Experimental Reaction Chambers
The effect of pH and redox potential (oxidation-reduction) conditions on
the degradation of four pesticide compounds was studied in sediment-water
suspensions. The reaction chamber used is illustrated in Fig. 2 and has been
described previously (Patrick et al. 1973). This apparatus has been used
successfully in our laboratory to study effects of these physicochemical
parameters on nutrient transformations in soils and the geochemical distribu-
tion of trace and toxic metals in sediments as well as their availability to
marsh plants. The sediment-water mixtures were incubated in 2-1, 3-necked
flasks and continuously stirred using a magnetic stirrer. To prevent wear to
the 5.1 cm-long teflon-coated magnetic stirring bar due to abrasion with sand
and silt particles, the stir bar was slipped inside an equal length of flex-
ible polyvinyl chloride tubing. A combination pH electrode was securely
mounted in each flask and the pH was checked twice daily and adjusted as
necessary using a syringe to add IN HC1 or IN NaOH through the serum cap
located in the center rubber stopper.
A thermometer was mounted in one rubber stopper and the temperature
maintained at 28 ± 1°C by inserting thin asbestos sheets as required between
the stirring motor and the flask to regulate heat transfer. Each flask was
fitted with separately valved inlet tubes for air and nitrogen and an outlet
tube which ended in a water trap to reduce oxygen diffusion into the flask.
Redox potential in the suspensions was measured continuously with two
bright platinum electrodes (and a calomel reference electrode connected to
the suspension with a KCl-saturated agar salt bridge) which were connected to
11
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Calomel
Half-cell
rpri Meter
Millivolt
Meter Relay
1 Air Pump
-N2
p H Meter
Magnetic
S tirrer
Stirring bar
pH electrode
Platinum electrodes
Gas outlet
5. KCI salt bridge
6. Air inlet
7. N 2 inlet
8. Thermometer
9. Septum
\
Figure 2. Reaction Chamber Apparatus for Controlling Redox Potential and pH.
a millivolt meter. Automatic redox potential control was achieved by con-
necting the recorder output of the millivolt meter to a meter relay that
switched on a small air pump whenever the redox potential decreased below the
set level for each incubation flask. By metering the air flow to about 2 to
4 ml/min, the suspension would slowly oxidize to the set potential during the
air pumping cycle until the meter relay switched off the pump.
Soil- and sediment-water systems supporting considerable microbial
activity become more reducing in the absence of oxygen. Thus, by regulating
the addition of oxygen to soil and sediment-water systems, redox potential
can be controlled over a wide range. Redox potential was controlled to
within 10 millivolts of the selected potential in this study. Nitrogen gas
was continuously supplied to the suspension at a flow rate of about 5 ml/min.
This addition was useful in preventing an accumulation of gaseous decomposi-
tion products which might adversely affect microbial activity and in purging
residual oxygen from the suspension at the end of each air pumping cycle.
Prior to removal of sediment material for loading into reaction chambers,
each small storage container was thoroughly mixed on a roller-mixer to insure
12
-------
sample homegeneity. Based on moisture content, sufficient wet sediment was
weighed into the incubation flasks to give 300 g of solids (oven dry basis),
following which water was added to give the desired solids to solution ratio
(1:6). Stirring was begun for the preliminary incubation which consisted of
gradually adjusting both pH and redox potential to preselected levels. After
the suspensions were maintained at those levels for one week for the suspen-
sions to equilibrate at the controlled physicochemical conditions before
pesticide addition.
Samples were obtained from the flasks by withdrawing suspension aliquots
through the serum cap using a 50-ml glass syringe fitted with a 12-gauge
stainless steel needle. The samples were stored in a freezer in 112 ml glass
bottles sealed with aluminum foil-lined caps.
Soil and Sediment Material
The soil material for the Guthion degradation studies was from the
surface, approximately 5 cm depth (after the forest litter layer had been
scraped away), of the crawfish farm site described elsewhere in this report.
The material had a medium texture and a dark color indicative of appreciable
organic matter content. The area is subject to periodic flooding. Large
pieces of twigs, roots, and decaying leaves were removed by hand. Sediment
material for use with methyl parathion, trifluralin, and 2-4,D was taken from
Bayou Chevreuil (near the bridge on Louisiana State Road #22), a site located
approximately 3 km from the crawfish farm. The sediment material was fine
textured (predominately clay) and contained an appreciable (~6%) organic
matter content.
Both soil/content materials were batched when returned to the labora-
tory, mixed well, and stored in small containers in a refrigerated room at
their natural moisture condition.
Pesticide Extraction Procedures
Guthion (soil)--
1. Thaw sample in lukewarm water.
2. Centrifuge in original glass storage bottles at 1650 rpm (IEC #266
rotor) for 25 min.
3. Decant water (into separatory funnels if water is to be analyzed).
4. Mix 2 ml of toluene with soil to retard microbial activity during sample
drying; place samples in a forced draft oven at 40 C.
5. Finely grind the dried samples with a mortor and pestle and weigh 2 to
4 g into a tared extraction thimble.
6. Extract for 4 hr with 130 ml of acetone using the Soxhlet extraction
apparatus.
13
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7. Rinse the glass Soxhlet extraction tube and thimble with acetone into
the Soxhlet boiling flask.
8. Pour the acetone extraction solvent through sodium sulfate into 250 ml
flasks to dry the sample.
9. Concentrate as necessary in a water bath heated to 40 C, then quantita-
tively transfer into graduated test tubes with screw caps.
10. Bring to volume and seal sample with aluminum foil-lined caps.
Guthion (water)--
1. To a 50 ml sample in a 250 ml separatory funnel, add 50 ml of a hexane-
methylene chloride (85:15 ratio) and 1 g of sodium chloride and shake
for 2 min venting frequently.
2. After phase separation, drain the aqueous layer (lower) into a second
separatory funnel.
3. Add 50 ml of methylene chloride to aqueous sample in second funnel and
shake for two min.
4. Transfer the hexane-methylene chloride extract of the first separatory
funnel to the second and transfer the aqueous layer from the second
funnel to the first.
5. Add 50 ml of methylene chloride to the aqueous sample now in the first
funnel and shake for 2 min venting often.
6. Drain the aqueous phase into tared containers and determine weight of
the water.
7. Combine methylene chloride extracts, rinsing separatory funnels with
hexane; filter extracts through hexane-washed sodium sulfate into 250 ml
flasks.
8. Add several glass beads and 1 ml of a 10% Nujol (in hexane) solution to
the flasks, rinse Snyder column with 5 ml of hexane, and connect columns
to flask.
9. Under a fume hood in a boiling water bath, concentrate the methylene
chloride extract to 5 ml.
10. Add 100 ml of a 1:1 hexane-acetone mixture and concentrate again to
5 ml. (Note: solvents are changed to protect N.P. detector on GC.)
11. Pour extract from flasks into graduated test tubes and make volume to 5
to 10 ml with acetone.
12. If further concentration is necessary, evaporate some solvent with a
stream of dry nitrogen gas.
14
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Methyl parathion--
1. Thaw sample in lukewarm water.
2. Mix 0.5 g NaCl with the thawed sediment-water mixture and centrifuge at
about 1500 rpm (IEC #266 rotor) for 15 min.
3. Discard the supernatant solution or pour into separatory funnels if
solution phase is to be analyzed.
4. Under a hood, mix 1 to 2 ml of toluene with the wet sediment (to retard
microbial activity) and dry in a forced draft oven at a maximum of 40 C.
5. Grind the sediment samples to a fine powder and weight into tared ex-
traction thimbles.
6. Extract for 4 hr on a Soxhlet extraction apparatus with 130 ml of ace-
tone .
7. Rinse extraction glassware and thimble with acetone into a beaker.
8. Dry the acetone extract by filtering the sample through acetone-rinsed
Na2S04.
9. Collect in 200 or 250 ml voluraetrics and bring to volume with acetone.
10. Concentrate if necessary.
Trifluralin
1. Add an aliquot of the well mixed sediment-water suspension sample (con-
taining 2.5 to 5.0 g solids on a dry weight basis) to preweighed glass
centrifuge tubes.
2. Centrifuge and discard the water (centrifugation speed and time depends
on the type of sediment; 6,000 rpm in a Sorval GS-3 rotor for 25 min was
necessary to remove fine particles from Bayou Chevreuil sediment suspen-
sion. A slower speed probably would have been adequate if NaCl had been
added to enhance flocculation.)
3. Add 75 ml of a hexane.: acetone mixture (41:59 vol:vol) to each sample,
mix well with a Teflon spatula, and shake for 30 min.
4. Centrifuge at 3500 rpm (Sorval GS-3 rotor) for 20 min. Collect the
solvent extract.
5. Repeat steps 3 and 4 and store extract in a dark place or amber bottle;
refrigerate if sample is to be stored overnight.
6. Oven dry centrifuge tubes and obtain dry weight of sediment extracted
(about 24 hr at 50 C or until a constant weight is obtained).
15
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7. Wash the hexane:acetone extract three times with distilled water (vol-
umes of 100, 30, and 30 ml) in 250 ml separatory funnels, discarding the
water:acetone layer (bottom) after each wash.
8. Dry the hexane extract by filtering through hexane saturated anhydrous
sodium sulfate into a volumetric flask.
9. Bring to volume and dilute as necessary with nanograde hexane.
2,4-D--
1. Thaw sample in lukewarm water (1-2 g oven dry solids basis).
2. Add 15 ml of 1% KOH, check pH to insure it is near 11.
3. Shake for 15 min.
4. Let samples stand in a water bath at 45 C for 15 min; transfer to glass
centrifuge tubes.
5. Centrifuge at 3500 rpm (GS-3 rotor) for 30 min.
6. Decant through glass wool into a separatory funnel.
7. Repeat steps 2-6 and combine extracts.
8. Oven dry residual soil or sediment solids and determine dry weight.
9. Extract KOH with 20 ml of diethyl ether in a separatory funnel by
shaking for 1 min and permitting phase separation. Transfer the KOH
(bottom) layer to a second separatory funnel and discard ether.
10. Wash the KOH layer twice more with diethyl ether and discard the ether.
11. Transfer the KOH extract into a glass centrifuge bottle or a separatory
funnel, add 1 ml of an H.SO, (9N) solution, and check to see that the pH
is 3.0 or lower.
12. Add 20 ml of diethyl ether, shake, and allow the solution to clear or
centrifuge to separate layers.
13. Remove ether layer with a pipet and save. Repeat step 12 and combine
ether extracts.
14. Add a small amount of acidified Na SO, to ether extract and evaporate to
5-10 ml under N2> L *
15. Transfer ether to a 15 ml centrifuge tube and evaporate to dryness.
16. Add 1 ml of diazoraethane to each tube, cap tightly, and place in a 45 C
water bath for 15 min.
16
-------
17. Add 1 ml of hexane and evaporate to 1.0 ml.
18. Dilute as necessary with hexane filtered through anhydrous Na SO,.
Gas Chromatograph Analysis
Operating conditions for GC determination of the breakdown products of
the four pesticides evaluated are given in Table 1.
RESULTS AND DISCUSSION
Guthion
Guthion was incubated with a swamp forest soil material in reaction
chambers under controlled pH (6.0, 7.0, and 8.0) and redox potential (-150,
+50, +250, and +450) conditions. Sample aliquots were taken from the micro-
cosms periodically, extracted, and analyzed for Guthion. A decrease in
recovery with time was assumed to be a measure of degradation. Data are
given in Figs. 3, 4, and 5 for pH 6.0, 7.0, and 8.0, respectively. It was
found that with Guthion, a 3-wk incubation was sufficient to determine treat-
ment effects on degradation with the soil material used. The data confirm
what is commonly known about the organophosphate insecticides, namely, they
are not persistent in the environment. Recovery was less than one-third of
initial spiking levels within 3 weeks under physicochemical conditions
resulting in slowest degradation.
Redox potential levels had a marked effect on the rate of degradation.
Guthion levels decreased most rapidly at +450 mv (well-oxidized conditions)
at all pH levels. The degradation rate decreased with redox potential through
+250 and +50 mv to -150 mv (strongly reducing conditions), which resulted in
the slowest degradation rate. Over the range studied, there did not appear
to be a critical potential below which Guthion was stable since the degrada-
tion rate increased sequentially with increasing redox potential. Soil pH
affected degradation less than redox potential, though it appeared, at redox
potential levels of 250 and 450 mv, degradation was slower at pH 6 than at
either of the higher pH levels. This effect, if real, is likely attributed
to pH levels of 7.0 and 8.0 being more favorable for microbial activity than
pH 6.0. Under well-oxidized conditions, Guthion had a half life of about
2 da at each of the pH levels. This increased to about 5 or 6 da under
strongly reducing conditions.
Degradation rates observed in this study under oxidized conditions were
comparable to those reported for Guthion in soil (Yaron et al. 1974) and pond
water (Meyer 1965; Chemagro Division Research Staff 1974), but considerably
faster than the 3-month half life reported for a Louisiana clay soil. Redox
potential conditions and other experimental parameters for these literature
reports are not available, thus, it is not possible to explain the reasons
for differences and similarities in degradation rates between this study and
earlier reports. However, the temperature, moisture conditions, and organic
matter content of the swamp soil were quite favorable for microbial activity,
which would enhance biological breakdown. The small increase in stability of
17
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Table 1. Operating Conditions for Gas Chromatograph.
oo
Compound
Parameter
carrier gas
carrier gas flow
rate, ml/min
column
injector
temperature, C
column
temperature, C
Guthion
zero grade N
70
6'
1.5% OV-17, 1.95% QF-1
230
220
Methyl Parathion
zero grade N
70
61
1.5% OV-17, 1.95% QF-1
230
200
Trifluralin
zero grade N
70
6'
3% OV-1
230
165
2,4-D
zero grade N_
70
6'
3% OV-210
230
170
interface
temperature, C 250 250 250 250
detector Nitrogen/Phosphorus Nitrogen/Phosphorus ECD at 275 C ECD at 275 C
-------
OUTHION, Phi). -liOMV
LKUXItn; A « I OHS, n - i Ot'S, STC.
10
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IS
10
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o !
0 I 2 1 <» 5 o 7 d 9 10 II 12 1] 14 1b 16 17 18 "t9"2o""il"
CUTII10N, !*l(b,
Lf'IKKP: » «
t O.ny, B - ^ NBS. ETC.
Ot2 I « 5 b 7
10 II 12 1J 14 1i It- 11 ta 19 20
DAIS
ClIThlOM, PIIO, iOMV
LECPND: i t OHS. D * 2 HDS, KTC
>) I I \ 4 S 6 7 A S101112131415I61718192021
D*Y5
iIlirilinN, PHh, ^''"HV
LFMUNn: A - I DBS, B * 2 OBJ, ETC.
I
ti 4 10 11 12 II 14 IS 16 17 16 19 20 21
DAI'S
Figure 3. Effects of Controlled Redox Potential on Recovery of Guthion From a Swamp Forest Soil
(Incubated at pH 6.0).
-------
ro
o
0 I 2 1
PHI. -IMJHV
ll): It = I HBS, b
» 1ft II 12
OtTb
GIITllinfl, PHI, iSO
7 a 9 10 I I 12 13 Hi
DATS
16 17 IS 19 2Q 21
LKr.EHD: I 1 DBS, B > i OBS. ETC.
) « 5 b 7 B
9 10 11 12 11 1« !> tb 17 IB 19 20 21
DATS
TIIinH, PU7, 4MJHV
LKCEBD: A 1 ODS, B « 2 DBS, ETC.
8 4 10 11 12 13 Id IS 1b 17 18 19 20 ~2l*
DI!S
Figure 4. Effects of Controlled Redox Potential on Recovery of Guthion From a Swamp Forest Soil
(Incubated at pH 7.0).
-------
fiUllllOS, ?IIH, -ISllMV
LSC.FSP: i = i ons, u - 'i nnr., ETC.
:
u
*
*
; ; ........... :
'i 2 1 « S b f d 9 10 11 t2 13 lU IS 16 11 IB 1-i 20 21
OIKS
CUTHIO1*, PUS, .'MIHV
1<> lb 17 IB 19 20 21
0 I 2 1
GlITIIIOH. Plia. iiWV
LFCEND: I a | ons , B i OUS, PTC.
ti 4 10 II 12 13 in ib 16 17 IB 19 20 21
DAIS
CUTIHON, PIlB, *10MV
LE*iENP; 1 > QOS, I> 2 OUS, ETC.
9 10 11 12 11 11
DftIS
Figure 5. Effects of Controlled Redox Potential on Recovery of Guthion From a Swamp Forest Soil
(Incubated at pH 8.0).
-------
Guthion as pH decreased from 8 to 6 corresponds with the limited pH informa-
tion available for Guthion degradation and reports of pH effects for several
other organophosphate insecticides.
Table 2 indicates concentration ratios of Guthion bound to the soil
phase vs. Guthion extracted from the aqueous phase of the samples on a few
selected sampling dates (jjg Guthion/ml in solution phase -r by (Jg Guthion/g
solids). In these samples, both bound and dissolved Guthion were measured to
Table 2. Ratio of Soluble to Adsorbed Guthion at pH 6.0 and 7.0 in a Swamp
Soil-Water Suspension.*
Incubation
Period (in days) pH Parameter
0 6 water
soil
water/soil
1 6 water
soil
water/soil
3 8 water
soil
water/soil
6 8 water
soil
water/soil
8 8 water
woil
water/soil
Redox Potential, mv
-150
0.158
14.1
0.011
0.025
9.0
0.003
1.290
21.5
0.060
0.813
16.1
0.050
0.259
7.9
0.033
+50
0.098
11.6
0.008
0.024
9.4
0.003
0.920
23.1
0.040
0.270
20.2
0.013
0.110
7.4
0.015
+250
0.103
18.8
0.005
0
21.0
0
0.695
20.0
0.035
0.872
10.9
0.080
0
1.3
0
+450
0.087
18.3
0.005
0
22.4
0
0.123
4.0
0.031
0
0.9
0
0.079
0.1
0.790
^Soluble (|jg/ml solution); Adsorbed (|Jg/g oven dry solids)
22
-------
get preliminary information on the possibility that oxidation-reduction
conditions affect partitioning between the aqueous and solid phases of
sediment-water systems.
Where sufficient Guthion was present in both phases for good measure-
ments, there was an increase in the ratio of solution phase Guthion to solid
as the sediment-water mixture became more oxidizing. This suggests that
reducing conditions may enhance soluble levels of Guthion in interstitial
waters of sediments compared to oxidizing conditions. While the mechanism(s)
accounting for this observation were not investigated, two general possibil-
ities may be considered: 1) properties of the anaerobic solid phase, making
it slightly less effective in binding Guthion than under oxidized conditions;
and 2) some characteristic of the solution phase enhancing soluble levels.
For example, it has been reported that soluble hmnic materials, presumably
fulvic acids, are present in higher concentration in anaerobic sediment-water
systems than in more oxidized materials (DeLaune et al. 1981). The increased
soluble Guthion may be associated with dissolved fulvic acids.
Trifluralin
Trifluralin was incubated at -150, +50, +250, and +500 mv at pH 7.0 in
sediment material from Bayou Chevreuil.
Fig. 6 indicates the effect of redox potential on recovery of triflura-
lin during a 24-da incubation. Under strongly reducing conditions, recovery
dropped from initial spiking levels of 8 ppra to less than 0.5 ppm within
1 da. The rate of loss for the three higher redox potential treatments were
similar in the Bayou Chevreuil such that approximately one-half could not be
recovered after 4 to 5 da. The +50 mv levels were slightly lower with time
(about 1 (Jg/g for most of the incubation) than the two more oxidized treat-
ments which were very nearly identical until the end of the experiment. By
24 da, the +50 mv level was less than 0.1 |Jg/g while the levels in the +250
and +500 mv treatments were around 0.75 to 1.5 (Jg/g. Though while it is not
a persistent herbicide under any oxidation-reduction condition studied, it is
apparent that strongly reducing conditions considerably increase the rate of
trifluralin loss.
Willis et al. (1974) identified 6, 6, 6-trifluoro-5-nitro-N4-propyltol-
uene-3, 4-diamine as a degradation product of Trifluralin in a Sharkey clay
soil incubated under controlled redox potential conditions. This was referred
to as "Compound E," a designation used in the present report. The concentra-
tion of Compound E increased rapidly to around 2.5 M8/8 under strongly re-
ducing conditions by 1 da and then decreased to less than 0.5 |Jg/g by 6 da
(Fig. 7). Comparisons of Figs. 6 and 7 indicate that: Compound E is indeed
a degradation product of the parent molecule; its production and possibly
degradation rate is dependent on redox potential conditions, (none was mea-
sured under well-oxidized conditions); and like Trifluralin, Compound E could
not be considered a persistent molecule under the experimental conditions
studied.
Fig. 8 shows the relative levels of another Compound (Y) associated with
the degradation of Trifluralin. Though present in somewhat lower levels than
23
-------
TRIFI.IIPALIII. - lt>0»V
LEC^nn: * i nns, a = 2 ous, ETC.
LEGEND: » = t OBS. B « 2 OH5, ETC.
N>
-P-
0 1 2 J (i 5 h 7 B 9 10 M 12 11 1« 15 16 17 IB 19 20 H 22 21 20
ftMS
LEGEND: A t UBS, 0*2 UOS, ETC.
0 I 2 1 « 5 h 7
I 9 10 11 I? M 11 15 1b U 16 19 20 21 22 21 20
DATS
TOIFLdHtLlH, SOOnV
LtUKMD: i = t OBS, b » 2 OBU, ETC.
0 1 2 3 1 5 A 7 R t 10 11 U 1J 1H 15 Ifc 17 IB 19 20 21 22 2) 21
DUS
0 1 2 1 4 b l> 7 Q <} 10 II 12 1) m 10 16 IT 18 19 20 21 22 2) 24
DAIS
Figure 6. Effects of Controlled Redox Potential on Recovery of Trifluralin From a Bayou Chevreuil
Sediment (Incubated at pH 7.0).
-------
u: i c i ons. D > 2 ous. ETC.
i.E^KND: i < t OBI;, 8 2 OBS, ere.
0 1 2 I » b 6 1 a 9 10 II 12 11 14 IS Ib 17 18 19 20 21 22 21 21
S (, 7 B 9 10 11 12 13 14 IS 16 17 IB 19 20 21 22 21 2«
1)1(5
recCNt): t - i ons. b » 2 nes, ETC.
i
t*
p
i
t
u
0 *
I
0 1 J 1 0 S fe 1 8 s "lO Vl *12*Vr'ii~'ii~'lfc~l7~'lB~19~20~21~22~21~2i
1*1(5
Figure 7. Effects of Controlled Redox Potential on Recovery of Compound E From a Bayou ChevreuiL
Sediment (Incubated at pH 7.0).
-------
conpniJHD T, - lio.iv
COMPOUND T, bOfll
PLUT OF _->0_r«T,lflE LEGEND: i « I UBS, B = 2 OBS, ETC.
ho
0 1 2 1 Q S t> 7 0 t 10 11 12 11 Id 15 lt> >7 IB 19 20 21 22 21 24
B4TS
HP _2SO.T»TIflE LSUEN3: i » »b3, 8-2 "US. ETC.
"0",""2""l & S (. 7 8 9 10 H 12 13 1H 1b 16 17 18 19 20 21 22 23
D1V5
cOHi'OUNn T , SOOnv
PLOT OK _500_l«TinK LEGEND: It I OBS, U - i OBS, ETC.
1b " 18 " 20 21
I
I 2 1 a , 6 7 « 9 10 II 12 13 l« IS Id 17 18 19 20 21 22 2J 2«
DAIS
Figure 8. Effects of Controlled Redox Potential on Recovery of Compound Y From a Bayou Chevreuil
Sediment (Incubated at pH 7.0).
-------
Compound E, comparison of Fig. 8 with Figs. 6 and 7 indicate the conclusions
given about Compound E in the previous paragraph are applicable to Com-
pound Y.
Fig. 9 shows the relative recovery of another compound in the sediment
suspensions, shown to be a contaminant or impurity associated with Tri-
fluralin. The original dosing solution was checked for this impurity. This
compound, N-nitrosodipropylaraine, initially was present at about 2.5 to 3
(Jg/g for the three highest oxidation treatments. Its low levels or absence
at -150 mv may be due to chemical instability under strongly reduced condi-
tions. Because of the health hazard from nitroso compounds, the environ-
mental chemistry of this impurity may be of equal or greater importance than
that of the primary compound. If this is an impurity, it is apparent from
Fig. 9 that its potential for an adverse environmental impact is negligible
in the strongly reduced sediment material and that under better oxidized
conditions, its loss from the system occurs at approximately the same rate as
Trifluralin.
Methyl Parathion
Methyl parathion was incubated also in the Bayou Chevreuil sediment
material at -150, +50, +250, and +450 mv, at pH 6.0 and 8.0 (Figs. 10 and
11). Recovery of this compound decreased very rapidly in the laboratory
chambers under all pH and redox potential conditions. Methyl parathion was
most persistent under well oxidized conditions, but had decreased to less
than one tenth original levels in only 4 da in the most oxidized sediment.
Under strongly reducing conditions, recovery decreased to undetectable levels
(less than 0.25 ppm as this spiked study was done) at 1 da into the incuba-
tion (Fig. 10). At pH 8.0 where spiking levels were around 15 Mg/g> recovery
had decreased to around 2.5 (Jg/g within 4 hr. This very rapid loss may be
due to factors other than those attributable to biological degradation. It is
possible that under the strongly reducing environment, conditions are favor-
able for rapid chemical or enzymatic decomposition. Based on the similarity
of this compound and information available for parathion (ethyl parathion),
nitro group reduction may be involved in the rapid alteration of the methyl
parathion molecule.
Wahid et al. (1980) reported the almost instantaneous degradation of
parathion (ethyl parathion) when the compound was shaken with a pre-reduced
soil. Aminoparathion was the major degradation product. The parathion
levels decreased to about 44% and 12% of original spiking levels after 5 sec
and 30 min of equilibration, respectively.
It is interesting to note that a change in oxidation-reduction condi-
tions affects the apparent degradation rate of Guthion and methyl parathion
differently. For example, transport of methyl parathion into anaerobic
sediments will enhance the loss of methyl parathion and inhibit the removal
of Guthion.
27
-------
COnPOUND I, - IbOfll
LEGEND: t " 1 OBS, D « 2 DBS, ETC.
Lgii'.ND: it = I on;;, B = i ous. ETC.
ro
00
0~ 1 ? 1 Q 5 6 7 0 9 tO 11 12 13 14 IS 16 17 IS 19 20 21 72 2) 2«
DifS
COflPOUHD I. 2bOH?
LEGEND: ft a 1 OUS, B * 2 OHS, ETC.
~o"r~2~"l~ 1 "» * 7 fl 9 10 11 12 11 1« 15 1b 11 IB 19 30 21 12 it 30
DAIS
COflPOiJND I. 500HV
LKGEHD: » = 1 OBS, U ' 2 OUS, ETC.
0 1 2 1 4 5 6 ? S 9 10 11 12 IJ U IS 16 17 18 19 20 21 22 21 24
DM5
0 1 2 J Q *> t> 7 8 9 Irt 1 1 12 1 J 1«4 IS 16 17 Ifl It 20 21 72 21 21
(UTS
Figure 9. Effects of Controlled Redox Potential on Recovery of Compound X From a Bayou Chevreuil
Sediment (Incubated at pH 7.0).
-------
'lUENt: A - i nns. a - 2 nns. RTC.
LCfipKD: A » 1 OOS, fl « 2 OUS, ETC.
r :t
01 2 i
9 10 It 12
n: A = i DBS. o « 2 OBS, ETC.
0 1 2
*. b 7 8
DftIS
riBKTIIIOH, PHt., »iOf1»
10 It 12
1 II *
0 1 2 1 a >
4 10 11 12
0 1 2 1
10 II 12
Figure 10. Effects of Controlled Redox Potential on Recovery of Methyl Parathion From a Bayou
Chevreuil Sediment (Incubated at pH 6.0).
-------
P Tim. PARATIUON, PUS. bonv
LRGKNP: * " I OnS, D 2 OBS, ETC.
U)
o
0 > 2
"« S b 7 H 4 10 II 12
DAYS
I."Clip; » I ons. il 2 0113. fTC.
0 t 2
10 II 12 13 11
n<:H9: * » I OUS, 0 2 OBS, ETC.
I) I 2
0 I 2 1
Figure 11. Effects of Controlled Redox Potential on Recovery of Methyl Parathion From a Bayou
Chevreuil Sediment (Incubated at pH 8.0).
-------
2,4-D
The herbicide 2,4-D was incubated in a Bayou Chevreuil sediment suspen-
sion under controlled pH and redox potential conditions as described for
Guthion, Trifluralin, and methyl parathion.
Sample aliquots were stored in a freezer until extraction and methyla-
tion procedures were tested to confirm proper reproducible recoveries.
Unfortunately, a power failure occurred during this period for an undeter-
mined amount of time and thus it must be assumed that the sample integrity
was not maintained during the thaw. A very few samples were extracted to
determine if oxidation-reduction effects on degradation were indicated,
though any trends must be interpreted carefully. The data indicate that
2,4-D may be more persistent under strongly reduced conditions (Table 3), but
further studies of the effect of redox potential on the environmental fate of
2,4-D are needed.
Table 3. 2,4-D Recovery from Bayou Chevreuil Sediment Material
Incubated under Controlled pH and Redox Potential
Conditions!.
Time, Days
2
17
26
Redox Potential, mv
-150 +500
,4-L), pg/g
11. if 10.3" ± 0
4.9±1.6 0.04±0
3.7 ± 1.1 .08?
-05
.00
These samples were subject to thawing for an undetermined period
during storage. The data should not be considered reliable
except possibly as an indication of relative redox effects.
(Duplicate sample unavailable.
JL.
Mean and standard deviation.
CONCLUSIONS
This study has demonstrated that oxidation-reduction conditions of soils
and sediment-water systems have an important effect on the persistence of
synthetic organic pesticides in the environment. Of the insecticides studied,
Guthion degradation was enhanced under oxidizing conditions while methyl
parathion, another organophosphate insecticide, degraded more rapidly under
anaerobic conditions.
31
-------
The herbicide Trifluralin degraded more quickly under reducing condi-
tions. Preliminary evidence indicates that oxidation-reduction conditions
affect the persistence of the herbicide 2,4-D, but this cannot be quantified
from the microcosm studies conducted because of problems in storing the
samples before analysis. Most studies on the environmental persistence of
pesticide residues have been with aerobic, agricultural soils. Compared to
typical agricultural soils, sediments, especially those of wetlands, tend to
be weakly to strongly reducing, near neutral in pH, and characterized by a
somewhat higher clay and organic matter content than agricultural soils of
the same area.
The different chemical, and especially microbiological characteristics,
of wetland sediments compared with soils can have important consequences on
mobility and degradation of toxic compounds. For example, Permethrin, a
synthetic pyrethroid, is very toxic to certain aquatic animals and the com-
pound is considerably more stable in anaerobic sediments than in the agri-
cultural soils from which the residue may be transported (Jolley et al. 1978;
Gambrell et al. 1981). Persistence studies based on degradation rates in
aerobic soils may not accurately indicate the residence time and potential
impact of the residues which move into wetlands during periods of runoff.
This was an intensive study of oxidation-reduction effects on a few
selected compounds used adjacent to Louisiana wetlands in a very limited
number of soil and sediment materials. The importance of oxidation-reduction
conditions on the environmental chemistry of pesticide compounds is indi-
cated. However, the different soils and sediments with which residues become
associated differ greatly in their physical and chemical properties, thus
affecting the fate of pesticides. Moisture content, temperature, and con-
tinuous stirring of research materials in laboratory reaction chambers affect
chemical and biological processes in that while the data indicate the rela-
tive effects of oxidation-reduction conditions on degradation, such informa-
tion probably do not reflect the actual rates at which the compounds would
degrade under in situ conditions.
Because noteworthy oxidation-reduction effects have been demonstrated,
additional research is needed to provide information on physicochemical
effects on the degradation rates of pesticides under a variety of natural
field conditions.
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35
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SECTION 3
IN SITU/MICROCOSM INVESTIGATIONS
AND MICROCOSM EVALUATION AND VALIDATION
S. P. Meyers and R. J. Portier
INTRODUCTION
In conjunction with studies on the impact of selected xenobiotics on
microbial ecology and substrate turnover, efforts have been directed toward
development of analytical approaches to simulate in situ activities in con-
trolled laboratory systems that are both accurate and reproducible and can be
subjected to critical program evaluation.
The mechanism used and reported here comprised a microcosm, or micro-
environmental system, to simulate the target field environment under closely
monitored laboratory conditions. The application of the microcosm approach
in assessing effect of toxic chemicals is documented by Hague (1980) and has
been discussed by other investigators (Pritchard et al. 1979; Portier 1979).
In general, use of microcosm technology in analyses of terrestrial ecosystems
and in assessment of fate, transport, and effects of xenobiotics is well
established (Giesy 1980), although there is minimal documentation in wetland
ecosystems.
A variety of model ecosystems or microcosms have been developed in
approaching environmental problems (Gillett and Witt 1977; Bourquin and
Pritchard 1979). Close coupling of field and laboratory (microcosm) infor-
mation, as proposed by Gillett (1980), facilitates development of valid
predictive models and provides useful quantitative data on in situ interac-
tions between xenobiotic(s) and the ecosystem. Furthermore, analyses of
microcosm data allow a better understanding of abiotic and biotic factors
that affect toxic chemical breakdown over a range of salinity conditions.
However, methodologies developed for terrestrial studies may not be appli-
cable to investigations in aquatic regimes. Primary differences involve the
long-term effects of water itself. Thus, microcosm work in wetland or
flooded ecosystems, especially those with diverse salinity gradients, is
generally lacking. These and other factors affecting fate of chemicals have
been described recently (Anon. 1981).
Our studies involved three correlated areas of investigation: the
design and evaluation of continuous flow and static microcosms, presentation
of microcosm-generated information on chitin substrate turnover and pesticide
hazard, and study of data factors and analytical approaches needed to formu-
late predictive models for use in hazard assessment. The microbiological
36
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phase has included monitoring of alterations in total heterotrophs and spe-
cific physiological groups in both in situ and microcosms, to establish
utilization of substrates, such as chitin and enzyme activity related to
pesticide levels. Field studies have been designed to detect in situ changes
in total microbial biomass and its composition (diversity index) and a pre-
liminary indication of compound half-life.
Major attention has been given to Guthion with comparisons made with
another organophosphorus compound, methyl parathion. Additional comparative
studies have been made with the organochlorine compound, Kepone . Use of
chitin in our investigations is based on its abundance in aquatic ecosystems
and its key role in recycling of carbon and nitrogen in aquatic environments
(Seki 1965; Hood 1973; Hood and Meyers 1977, 1977a).
Microcosms for microbiological studies and physicochemical analysis have
been used in the work reported here to determine the behavior of the dif-
ferent pesticides under diverse pH, redox, and chemical conditions. The
experimental approach combines field and microcosm methods, with the latter
employing a variety of determinants, including microbial indexes, enzymatic
activities, respiratory response, and transformation of key substances such
as chitin and cellulose. Ultimate application of this composite information
will be valuable in development of protocols for analyses of xenobiotics, and
evaluation of their impact on critical environmental processes.
EXPERIMENTAL PROCEDURES
Study Site and Sample Collection
Soil and sediment collections were made in the Lac des Allemands envi-
ronment of the Barataria Bay drainage basin, Louisiana (see Map, Fig. 1).
This is an interdistributary region bordered by natural levees of the Mis-
sissippi and Bayou Lafourche, the latter an abandoned distributary of the
Mississippi River. Several distinct vegetation zones are evident. A band of
saline marshes borders the Gulf of Mexico; inland, there are successive zones
of brackish and freshwater marshes and swamp forest. The regions of the
basin most directly affected by runoff are the brackish and fresh areas,
located adjacent to intensively cultivated agricultural fields.
Cores consisting of aluminum cylinders, 60 cm tall x 7.6 cm diameter,
were inserted into the sediment to the water level, capped and sealed, and
stored on ice for transportation to the laboratory for microbial analysis.
In drier sites, an inclined trench to a depth of 60 cm was dug with an undis-
turbed wall at one end for aseptic sampling. All samples were processed
within a 12 hr period. Sediment from the particular sites was used to inocu-
late individual microcosms and to categorize the major microbial groups
present.
Enumeration of Microorganisms
For enumeration of total heterotrophic aerobes and anaerobes, sediment
samples were appropriately diluted with phosphate buffer, pH 7.2. Replicate
0.1 ml samples were added by the pour plate method to standard plate count
37
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agar (Difco) incubated for three days at 26 C, followed by enumeration of
colony forming units (CFU). For anaerobic studies, proper atmospheric con-
ditions were maintained by using a disposable hydrogen/carbon dioxide
envelope (Gaspak) in the presence of a cold catalyst in an anaerobic jar. A
raethylene blue indicator strip was used to insure anaerobiosis. Plates were
incubated at 26 C and examined every 24 hr.
Four groups of microorganisms, i.e., bacteria, actinomycetes, filamen-
tous fungi, and yeasts, were enumerated in terras of biomass present based on
colony forming units (CFU). Appropriate replicate dilutions were made of all
sediment samples using the pour plate method. For enumeration of bacteria
and actinomycetes, replicate 1.0 ml aliquots were added to Jensen's agar
medium. Cycloheximide (Sigma), an antifungal agent, was added at 40 (Jg/ml to
inhibit overgrowth by filamentous fungi. For enumeration of filamentous
fungi and yeasts, Martin's agar medium was used together with streptomycin
(Sigma) and chlortetracycline (Sigma) at 30 ug/ml to retard bacterial growth.
All plates were incubated at 30 C and examined after three days. Colonies
were enumerated using a scan dissection microscope to distinguish the four
major morphological groups.
Chitinoclasts were enumerated using a chitin medium containing 2.5%
precipitated chitin (Sigma) plus mineral salts (Hsu and Lockwood 1975).
Preparation of the precipitated chitin followed the methods of Okutani (1966)
and Hood (1973). Pure chitin, ball-milled for 72 hr at 4 C, was dissolved in
concentrated HC1 in quantities of 10 g/150 ml. The dissolved chitin was
added to a large volume of distilled water. The milky white precipitate was
washed repeatedly to remove acid, and the solution adjusted to give a value
of 10 mg/ml at pH 7.2. The plates incubated at 30 C were inspected after
three days. Colonies exhibiting clearing zones were diagnostic of chitin
utilization.
Enzyme Assay
Chitinase--
The assay for chitinase activity followed the method described by
Okutani (1966) using the N-acetyl-D-glucosamine assay (Reissig et al. 1955).
A crude enzyme extract was obtained by filtering 10 ml of culture broth
aseptically collected through a sterile 0.45 |J cellulose acetate filter. One
milliliter of cell-free liquid was added to an enzyme assay solution (Hood
1973), incubated for seven days at 30 C, and N-acetyl-D-glucosamine deter-
mined spectrophotometrically at 585 run.
Phosphatase
Phosphatase activity was determined using the procedure outlined by
Tabatabai and Bremner (1969), and modified by Atlas et al. (1977). Replicate
2 g aliquots of control and pesticide-treated sediment were placed in 150 ml
Erlenmeyer flasks with 0.1 ml toluene, 4 ml sterile distilled deionized water
and 0.5 rag/ml p-nitrophenol phosphate (Sigma). Flasks were incubated at 30 C
for 100 min. With the addition of 1 ml of 0.05 M calcium chloride, 4 ml of
0.5 M sodium hydroxide and 20 ml of distilled deionized water, each sample
38
-------
was centrifuged at 10,000 x g for 10 min and the resulting yellow solution
assayed at 410 nm for p-nitrophenol production. In subsequent microcosm
studies, replicate 1 ml aliquots were assayed for phosphatase activity using
the procedure outlined above.
Dehydrogenase--
Dehydrogenase determination was based on formation of 2,3,5 triphenyl-
tetrazolium formazan (TPF) from a standard of 2,3,5 triphenyltetrazolium
chloride (TTC). Replicate 10 gm aliquots of control and pesticide-treated
soils were placed in 250 ml Erlenraeyer flasks and brought to 100% water-
holding capacity with addition of a 0.5% solution of 2,3,5-triphenyltetra-
zolium chloride (Sigma), as described by Bartha et al. (1967). Each flask
was flushed with 02-free nitrogen, tightly stoppered, and incubated for six
hrs (Casida 1977). TTC is converted by dehydrogenase enzymes to 2,3,5
triphenyltetrazolium formazan forming a characteristic red color. The latter
was extracted with 50 ml of spectroscopic grade methanol (Baker Chemical),
centrifuged and filtered through No. 5 filter paper (Whatman). Absorption
was measured at 485 nm and dehydrogenase activity units expressed in mg/ml
TPF. In subsequent microcosm studies, replicate 2 ml aliquots were assayed
for dehydrogenase activity using the procedure outlined above.
Adenosine Triphosphate
An adenosine triphosphate (ATP) assay, as advanced by Holm-Hansen (1966)
and further presented by Karl (1980) and Stevenson et al. (1979), was used
for determination of microbial biomass. In our earlier investigations,
releasing reagents from a variety of commercial manufacturers, designed for
specific release and quantification of raicrobial and somatic ATP, were used.
These reagents allow selective permeable release of nucleotides without cell
lysis. Thus, nucleotides are released without being broken or changed by the
enzymes of the cells, and ATP measurement can be achieved with minimal in-
terfering effects as noted by Stevenson et al. (1979). Excellent reproducible
assays were achieved with a short preparation time. Use of selective appli-
cations of these reagents (somatic and microbial) provides good differential
quantifications of ATP. Furthermore, only 200 pi of sample is needed for
easy assay.
Gas Chromatographic Techniques
Gas chromatography methods for determining Guthion concentrations in
soil and water samples are those outlined in the previous section, along with
an outline of these procedures described in detail. The recovery efficiency
for azinphosmethyl extraction is 98 to 102% for spiked swamp surface waters
and 65 to 70% for both oxidized and reduced sediment materials. Identifica-
tions were based on comparisons with known standards of each xenobiotic.
Data Analyses
Statistical methods used have included multivariant analyses to test the
validity and reproducibility of the design features of the microcosm, to-
gether with SAS and Fortran programming approaches. Evaluations and tests of
39
-------
the variables considered, including microbial population composition, enzyme
response, and tagged compound turnover, have all been based on repeated runs
of replicate sets.
Description of the Microcosms
Continuous-Flow Units--
To maintain a more precise controlled environment to study raicrobial/
substrate changes induced by pesticide addition, a continuous flow-through
microcosm (Portier 1979) has been developed and evaluated (Fig. 12A). The
initial unit designed consisted of a 1000 ml tall form beaker (Pyrex brand,
Corning #1060) to which was attached an overflow spout to prevent possible
buildup of inoculum due to blockage in the system. Pesticide was pumped into
the beaker by means of a peristaltic pump (Harvard Aparatus Model #1203)
through a sterile medical grade tubing (Silastic brand, 1/8 ID X 1/4 OD)
having negligible pesticide sorption characteristics. Accurate flow rates as
low as 0.0042 ral/min were obtained.
Upon entering the reaction vessel, the pesticide is uniformly distrib-
uted into the sediment sample as a result of air (or nitrogen) sparging
through a Buchner-type funnel (Corning #6060) attached and replacing the
bottom of the beaker to promote an aerobic (or anaerobic) environment.
Original designs consisted of air sparging of the sample as it was stirred
intermittently by means of a magnetic stirrer.
The peristaltic pump maintained the desired level of water and sediment
by removing excess water from the system at the same rate as pesticide addi-
tion. A sediment trap at the top of the column prevents sediment loss to the
system and allows for the re-introduction of possible sediment accumulation
to the reaction vessel. Excess water was collected in 4000 ml aspirator
bottles (Corning #1220) to monitor pesticide loss to the system and maintain
aseptic conditions. A 60 ml separatory funnel (Corning #6420) placed at the
top of the sedimentation column effectively recovered lost sediment.
A temperature of 28 C was maintained by means of a heat lamp regulated
by a temperature controller (Versa Therm Model No. 2158) with a sensitivity
of ±.06 C. In earlier designs, a water bath heated with standard aquarium
heaters was used, however, a uniform temperature could not be maintained.
The pH of the reaction vessel is maintained at the desired level by a pH
meter/controller (Horizon Model No. 5997) connected to another peristaltic
pump (Masterflex Model No. 7545). Accuracy is within a 0.05 pH unit.
Samples can be aseptically withdrawn from the reaction vessel by means
of a micropipette or syringe. Due to the uniqueness of the surgical grade
tubing used, the entire assembly can be autoclaved and sterile conditions can
be maintained for prolonged periods of time.
The design of this bench top microcosm allows introduction of a variety
of analytical features including: variation in flow rate and sequential
40
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CONTINUOUS FUOW MICROCOSM
A Fril"d °"k VC''^;\v':ZG"-»
cid/Bo.e N*^:-'i;ix 9«odl
npul Line
Sample Line
Syringe Port
Sediment Trap
Export Line
Adjuiiable Temperature
Regulator
Heat Lamp
Membrane Air I N7 Supply -
Filler 9«gulalor
|.45U Poroiity]
CARBON METABOLISM MICROCOSM
T) Syringe Por I
2) oH Probe
B
Heal Lamo
Acid/ Base Solution
Figure 12. Diagram of Microcosm Units: (A) Continuous Flow; (B) Carbon
Metabolism Unit.
41
-------
compound addition; control of pH/Eh, temperature, 02 , and salinity; main-
tenance of various soil/water conditions; generation of controlled quantita-
tive data. The entire apparatus is capable of maintaining the desired
parameters of temperature, flow rate, and pH for a period of five weeks or
longer.
For the controlled microcosm studies, sediment from the Lac des Alle-
mands site was placed into four continuous flow microcosms. Approximately
50 g (wet weight) of sediment was introduced with appropriate amounts of
filter-sterilized (0.45 (J porosity cellulose acetate filter) site water to
give a final volume of 500 ml. Two microcosms served as controls while test
solution of the xenobiotic was introduced into the two test microcosms fol-
lowing equilibration. The test solution, added at varying flow rates
consisted of filter-sterilized site water, followed subsequently by site
water amended with the xenobiotic for test microcosms. A 10 ppm solution of
the pesticide azinphosmethyl (Guthion) was used in preliminary studies. In
tests with methyl parathion (Chem Service) or the organochlorine Kepone
(Applied Science Labs., Inc.), solutions were added from a stock solution to
5 ppm and 0.5 ppm, respectively in a final microcosm volume of 1 L. Temper-
ature was maintained at 29 C with the pH at 7.2.
Carbon Metabolism Studies--
A static microcosm (Fig. 12B) was originally designed for studies in-
volving analyses of chitin breakdown rates by production of N-acetylamino
sugars in chitin-supplemented reaction chambers. The substrate used was
14C-labeled N-acetyl-D-glucosamine, with subsequent analyses of 14C02 pro-
duction via liquid scintillation techniques.
Carbon Metabolism Units
To study effects of pesticide addition on respiration rates and biomass
accumulation of labeled 14C-compounds, a static microcosm (Portier 1979)
equipped to trap 14C02 by liquid scintillation vials was designed (Fig. 12B).
The reaction vessel consisted of a 1000 ml tall form beaker (Corning #1060)
firmly supported and immobile to prevent spillage of radioactive material.
In the initial experiments N-acetyl-D-glucosamine (glucosamine 1-14C) [New
England Nuclear #723], added to non-labeled compound, was introduced into the
sealed reaction vessels by means of syringe. Guthion (non-labeled) and other
test compounds were also added by this method.
Air sparged into each reaction vessel facilitated entrapment of 14CO by
liquid scintillation vials containing a complete oxidizer cocktail (Oxi-Fluor
- C02; New England Nuclear). Samples were aseptically removed by means of
micropipette or syringe. Temperature and pH were controlled by equipment and
methods previously described for continuous flow microcosms.
Sediment from the field site was dispensed in 25 g aliquots into four
static microcosms with 400 ml of sterile distilled deionized water. A labeled
solution and a non-labeled aliquot of N-acetyl-D-glucosamine (NAG) was added
to each microcosm to give a final concentration of 0.1071 |J Ci/500 ml. The
specific activity of labeled N-acetyl-D-glucosamine was 58.18 mCi/mmol. The
42
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final concentration of both labeled and non-labeled NAG was 40 ppm. Tempera-
ture of the vessels was maintained at 28 C with a pH range of 6.8-7.2. Two
microcosms served as controls while the test chambers included a final pesti-
cide concentration of 10 ppm Guthion. Radiolabeled Guthion was not available
for these initial studies.
In subsequent carbon metabolism studies with methyl parathion and Ke-
pone, sediment from the field site was dispensed into replicate series of
four static microcosms with 500 ml of filter-sterilized site water for each
compound. In studies with methyl parathion, a uniformly labeled 14C-methyl
parathion solution (Pathfinder Laboratories) was used with a non-labeled
solution (Chem Service) to give a final solution of 5 ppm (1.0 p Ci/500 ml).
An air-dried ball-milled chitin material obtained from the freshwater red
swamp crawfish (Procambarus clarkii) was added to each microcosm at 0.25 mg/g
sediment wet weight to, monitor substrate effects. For similar studies with
Kepone , a 14C-Kepone solution (Pathfinder Laboratories) was used with a
non-labeled solution (Applied Science Labs, Inc.) to give a final solution of
0.5 ppm (1.0 p Ci/500 ml).
A moderate airflow into each microcosm allowed for entrapment of 14C02
by liquid scintillation vials containing 15 ml of complete oxidizer cocktail
(New England Nuclear). To determineuptake rates of labeled compound, repli-
cate one milliliter aliquots from 10 serial dilutions were filtered through
0.45 (J membrane filters. Each filter was then rinsed with sterile distilled
deionized water (buffered, pH 7.2) to remove excess compound not utilized by
the microbial biomass. Filters were dried and placed in scintillation vials
containing 15 ml of phase combining scintillant (Amersham). Membrane filters
were previously checked for sorption characteristics. Sorption from stan-
dards accounted for less than 0.2% loss.
All scintillation vials were counted for 10 min on a Beckman 250 liquid
scintillator. A present error of 0.2% was used in conjunction with the
external standard ratio mode for quench correction.
Axenic Flask Study--
Representative members of the four morphological groups, from azinphos-
methyl, methyl parathion and kepone microcosms, were isolated using axenic
culture techniques to screen for possible compound sensitivity and/or
utilization. Sensitivity to the xenobiotics methyl parathion and Kepone was
determined by relative changes in ATP levels compared with controls. Assim-
ilation rates of each radiolabeled compound was determined by methods out-
lined in the previous microcosm section. Total biotransformation of each
compound was based on 14C levels following incubation, and was adjusted for
possible abiotic effects using sterile control flasks. Total biotransforma-
tion was considered to be the difference between total 14C added minus 14C
residual levels and 14C assimilation levels.
43
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RESULTS AND DISCUSSIONS
Initial ID Situ, Static, Contiguous Flow
And Carbon Metabolism Microcosm Studies
In Situ Enumeration of Total Heterotrophs and Chitinoclasts--
The effect of Guthion on the total heterotrophic and chitinoclastic
population in swamp sediments for Field Study No. 1 is shown in Fig. 13.
FIELD STUDY 81
HETEROTROPHS
Guthion
; 25 ppm
I . 10 ppm
FIELD STUDY 81
CHITINOCLASTS
1 ppm
control (0 ppm| 33 -
Guthion
/ 25 ppm
j 7
EXPOSURE TIME (in doyi)
10 ppm
'
control (0 ppm)
3 1
EXPOSURE TIME (in days)
Figure 13. Effect of Guthion on Heterotrophic and Chitinoclastic Population
in Swamp Sediments (Field Study No. 1).
Values shown are least square mean values for replicate plots. Statistical
analysis revealed a significant difference (P ^ .01) among the treated plots
for total heterotrophs. An analysis for chitinoclasts, although not statis-
tically significant (P i -06), was strongly indicative of variation within
the population due to both presence and level of pesticide. The data sug-
gested that amendment of swamp sediment with Guthion, at concentrations of 1,
10, and 25 ppm, had a pronounced effect on concentrations of both total and
chitinoclastic bacteria populations. These rather high concentrations were
used based on observations in an earlier study using Guthion, at concentra-
tions of 0.01, 0.1 and 1.0 ppm in which no variations of total heterotrophs
or chitinoclasts between control and pesticide amended sites were noted. GC
44
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residue analysis failed to detect Guthion in these sediments. It was un-
certain as to whether Guthion indeed had no inhibiting effect at these
concentrations or that the concentrations used were too low for our field
methodology to detect any variability.
Since the highest variation in bacteria concentrations for Field Study
No. 1 occurred at levels of 10 and 25 ppm Guthion, the second field applica-
tion consisted of additional replicate plots of 0 ppm (control) and 25 ppm,
respectively. Fig. 14 shows the effect of Guthion on the total heterotrophic
and chitinoclastic populations. Values shown are least square mean values
for replicate plots. Statistical analysis revealed no significant differ-
ences among the treated plots for heterotrophs and chitinoclasts. The data
suggested that pesticide applications at levels of 25 ppm did not contribute
to an increase in the bacteria concentration, in contrast with data observed
in Study No. 1. There appeared to be a lack of statistically significant
increase in bacteria concentrations due to the considerable variation of
counts for total heterotrophic biomass within the control plots (0 ppm).
Fig. 15 shows the effect of Guthion in Field Study No. 3 on total
bacteria and the chitinoclastic population in swamp sediments amended at
levels of 0, 10, 25, and 100 ppm. Values shown are least square mean values
FIELD STUDY »2
HETEROTROPHS
FIELD STUDY «2
CHITINOCLASTS
o
x "0.
:oo
Guthion
control (0 ppm)
25 ppm
control (0 ppm}
25 ppm
5 »
Guthion
25 ppm
control (0 ppm|
control (0 ppm|
25 ppm
EXPOSURE TIME (in days)
EXPOSURE TIME (in days)
Figure 14. Effect of Guthion on Heterotrophic and Chitinoclastic Population
in Swamp Sediments (Field Study No. 2).
45
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FIELD STUDY «3
HETEROTROPHS
FIELD STUDY «3
CHITINOCLASTS
190 ,
110 .
Gulhion
control (0 ppfl
100 ppm
10 ppm
25 ppm
Guthion
ppm
100 ppm IU PPm
V^^** " PPm
"__"'-'^^^^^"^" control (0
ppm)
EXPOSURE TIME (in days)
EXPOSURE TIME (in doy»)
Figure 15. Effect of Guthion on Heterotrophic and Chitinoclastic Population
in Swamp Sediments (Field Study No. 3).
for replicate plots. Statistical analysis revealed a significant difference
among the treated plots for total heterotrophs (P ^ 0.05), but not for
chitinoclasts. The data suggest an inhibitory effect by Guthion in swamp
sediments amended at levels of 10 and 25 ppm but a pronounced stimulation of
microbial growth in sediments amended at levels of 100 ppm.
As noted, the three field application studies present an inconsistent
picture on the effects of Guthion in amended swamp sediments. Field Study
No. 1 indicates significant increases in the microbial population due to
pesticide levels, in contrast with data from Field Studies No. 2 and 3.
Overall, these results are somewhat questionable due to the unknown effects
of ground water seepage and intermittent flooding in the impounded swamp.
Subsequent microcosm studies have indicated that these sediments enhance
breakdown of Guthion, resulting in significant increases in the microbial
population. It is entirely possible that such increases in microbial concen-
tration occurred at a very rapid rate prior to sampling for Field Study No. 2
and 3. Sampling may represent a period of population decline following an
initial peak.
46
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Changes in microbial bioraass as a result of transfer and storage of
samples may have contributed to the variability observed. Kaper et al.
(1978) noted rapid changes in number of organisms on stored water samples
that were not reproducible and did not follow detectable trends. Increases
and decreases in counts occurred in samples regardless of whether they were
maintained at room temperature, refrigerated, or stored in plastic or glass
containers. Immediate sample processing is strongly recommended to minimize
variations in the microbial populations of samples from aquatic environments.
Numerical data from stored samples are, more or less, crude estimates, al-
though, as noted, field samples were placed in ice immediately and trans-
ported to the laboratory for microbial enumeration within 6 hr. for these
studies.
Determination of Pesticide Half-Life--
Table 4 shows the residual pesticide concentrations in amended swamp
sediments for each field application. Linear regression techniques were used
in calculating Ta . Although the small number of samples from each field
application provided a limited estimator of Tl and, hence, reduces the accu-
racy of half-life prediction, when one looks^ at all samples, a very short
half-life, i.e., from 1 to 2 da, can be seen for all treatments. This is
considerably less than the one month half-life for Guthion in water reported
by Huer (1974) and Yaron (1974a). Such a short half-life may be attributable
to the high microbial activity in the swamp sediment or, as mentioned ear-
lier, to the unknown effects of ground water seepage and intermittent
flooding in the swamp forest. Subsequent microcosm studies have demonstrated
the high microbial activity prevalent in swamp sediments.
Additional In Situ Studies
In follow-up iji situ studies, estimates of microbial activity, as ex-
pressed by numerical estimates of the four major groups of microorganisms,
i.e., actinomycetes, bacteria, filamentous fungi, and yeasts, were made at
various depths for five distinctive soil environments reflecting variations
in land use and management. A brief description of these soil profiles with
depth for each sample site are presented in Table 5. It is important to
consider the differentiation of these soils which are historically similar by
agricultural practices and flooding, both natural and controlled. Most of
these soils have considerable concentrations of organic material with varying
levels of clay content as a function of depth.
Table 6 shows variations reflected by dissimilarities in total micro-
biological levels and differences between physiological groups as a function
of depth. Table 7 presents analogous site enzyme activity with depth on a
dry weight basis. Variations in microbial population correlated with enzyme
activity for each soil environment and depth are given in Fig. 16. Soil
phosphatase and dehydrogenase activity was examined in terms of bacterial and
fungal populations with depth and the particular soil environment. Both of
these enzyme levels closely parallel changes in total microbial activity.
Bacterial and fungal populations, reflecting microbial population activity,
are presented for comparison purposes since they represent distinct microbial
groups. However, subsequent analysis of enzyme activity for yeasts and
47
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Table 4. Mean Residual Pesticide
Studies.
Concentrations from Field Application
Field Application
Enclosure
A
B
C
D
Field Application
Enclosure
A
B
C
D
Study No. 1
Initial
Cone .
0
1 ppm
10 ppra
25 ppm
Study No. 2
Initial
Cone.
0
0
25
25
Final
Cone .
0
0
0
2.48
Final
Cone.
0
0
4.44
3.58
Percent Half-Life
Recovery (T^)
0
0
0
9.92 2.099 days
Exposure Time: 7 Days
Percent Half-Life
Recovery (T'j)
17.76 2.005
14.32 1.783
Exposure Time: 5 Days
Field Application Study No. 3
Enclosure
A
B
C
D
Initial
Cone.
0
10
25
100
Final
Cone.
0
1.39
4.16
Percent
Recovery
0
5.56
4.16
Half-Life
1.199
1.089
Exposure Time: 5 Days
actinomycetes show a similar relationship. Analyses showed a high correla-
tion (r = 0.925) between phosphatase and microbial population activity and
between dehydrogenase and microbial population activity (r = 0.934).
It is important to note how variations in population/enzyme profiles
provide some indication of the impact of land management practices. For
example, high levels of activity were noted in the 0 to 5 cm depth for pas-
ture compared with adjacent sugarcane fields. Sugarcane fields were sampled
before and after harvesting and burning of the leaf and stem bulk. A 50%
drop in microbial activity was noted as a result of this "slash burn" tech-
nique. Below the burn zone, microbial and enzyme activity was much higher in
48
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Table 5. Field Sites: Soil Profiles with Depth.
Site
Number
1
2
3
4
5
6
7
8
9
10
11
Location
Sugarcane
Sugarcane
Sugarcane
Pasture
Pasture
Pasture
Swamp
forest
Swamp
forest
Swamp forest
Natural
swamp
Natural
swamp
Depth
0-5 cm
10-20 cm
30-40 cm
0-5 cm
10-20 cm
30-40 cm
0-5 cm
10-20 cm
30-40 cm
0-5 cm
10-20 cm
Profile
fine textured soil, granular, very dry,
fire scarred; rapid water runoff
fine textured soil, granular, moist,
considerable root rhizosphere debris
more pronounced clay content, marbled, iron
oxides evident in rusty soil color, moist
fine textured soil, highly organic, moist,
rapid water runoff
fine textured soil, more obvious clay content,
moderate root rhizosphere debris, moist
high clay content, water table at 50 cm
mark, high iron oxides, very marbled
high litter content, moist but not water-
logged; dark, rich soil having a very pro-
nounced musty odor; intermittent flooding
high clay content, moderate root rhizosphere
debris, marbled
heavy clay below water table
high litter, overlaying water level 5 cm,
fine suspended sediment; perennial flooding
high clay content, moderate root rhizosphere
debris, accumulated litter moderate,
12 Natural
swamp
30-40 cm
13 Impounded 0-5 cm
swamp
14 Impounded 10-20 cm
swamp
15 Impounded 30-40 cm
swamp
moderate clay content
heavy clay below water table and organic
level; very anaerobic
extensive litter deposits; finely separated
sediment material, overlaying water level,
9 cm-microcosm sediment source; perennial
flooding; static water flow
moderate litter deposits and root fragments,
moderate clay material; microcosm sediment
heavy, compact clay below water table and
organics level; very anaerobic
49
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Table 6. Fluctuation of Microbial Population with Depth.
Field
sites
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Filamentous
Fungi (103)»
35.0
72.0
21.0
140.0
22.0
1.0
88.0
5.0
1.0
47.0
3.0
<1.0
95.0
10.0
<1.0
Yeasts
(103)*
2.0
4.0
<1.0
3.0
<1.0
<1.0
11.0
1.0
<1.0
12.0
1.0
<1.0
13.0
1.0
<1.0
Bacteria
(105)*
25.0
335.0
35.0
226.0
74.0
12.0
74.0
34.0
10.0
91.0
37.0
7.0
145.0
55.0
15.0
Actinomycetes F,Y:B,A
(105)* 103 : 10s
10.0
20.0
14.0
42.0
17.0
2.0
18.0
18.0
2.0
17.0
14.0
6.0
20.0
13.0
5.0
1 . 05 : 1 . 0
.21:1.0
.43:1.0
.53:1.0
.24:1.0
.07:1.0
1.07:1.0
.12:1.0
.07:1.0
.55:1.0
.08:1.0
.08:1.0
.65:1.0
.16:1.0
.05:1.0
Colony Forming Units (CFU)/dry weight in grams.
See Table 1: Sites 1-3 Sugarcane; 4-6 Pasture; 7-9 Swamp Forest;
10-12 Natural Swamp; 13-15 Impounded Swamp.
50
-------
Table 7. Enzyme Activity at Various Sites with Soil Depth.
Site
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Dry
Weight
(in grams)
9
8
8
6
7
7
3
5
5
2
5
5
2
5
5
.38 g
.89
.80
.30
.12
.68
.25
.59
.99
.40
.40
.49
.22
.26
.34
Phosphatase Activity Dehydrogenase Activity
Absorbance mg/ml--" mg/g Absorbance mg/ml** mg/g
.170
.465
.263
.367
.457
.257
.287
.345
.203
.209
.231
.087
.254
.407
.130
.030
.075
.047
.073
.044
.013
.067
.037
.014
.048
.044
.010
.057
.022
.007
.032
.084
.053
.116
.062
.017
.206
.066
.023
.200
.081
.018
.257
.042
.013
.198
.674
.324
.584
.291
.104
.489
.244
.116
.329
.289
.094
.391
.159
.065
.06
.17
.09
.125
.165
.085
.100
.121
.075
.076
.080
.030
.084
.140
.045
.064
.191
.102
.198
.232
.111
.308
.216
.125
.317
.148
.055
.378
.266
.084
p-nitrophenol.
-V*
TTF.
the root rhizosphere area (10 to 20 cm). Differences in moisture content may
have played an important role in these variations.
Swamp forest profiles appeared to be comparable to those obtained in
pasture except that bacterial and fungal populations are more stratified with
depth. This is indicative of the high organic material accumulating on the
forest floor as a result of litter fall. Below the 20 cm depth, a signifi-
cant decrease in microbial and enzyme activity indicates changes in soil
organic content and increasing anaerobiosis.
Magnitude of microbial populations between the swamp forest and the
impounded swamp were comparable. Higher fungal concentrations were noted for
both compared with that present in the natural swamp. Bacterial populations
were more comparable in profile, with both the swamp forest and impounded
swamp having a higher bacterial biomass than the natural swamp. It is note-
worthy that phosphatase and dehydrogenase levels were in close agreement with
51
-------
microbial population levels, giving indications of the degree of activity of
bacteria and fungi present. Dehydrogenase levels were higher for the swamp
forest and impounded swamp, probably correlated with the preponderance of
high litter fall.
Initial Static Microcosm Studies for Total
Heterotroph/Chitinoclast Enuraeration--
Fig. 17 shows variations in total heterotrophs in natural and chitin-
amended soil to Guthion concentrations of 0, 10, 25, and 100 ppm. Statistical
analysis indicated significant variations in total bacterial concentrations
(P < .01) for both control and chitin-amended soils. Fig. 17 indicates high
bacteria concentrations after only two days of exposure to Guthion, espe-
cially for soil amended with 100 ppm of the pesticide. A high bacteria
concentration after only two days of exposure to the pesticide is seen.
'/^M Bacteria
i i o Phosphatase Activity
0-1
20-
40-
E
o
o.
UJ
Q
Fungi:
Bacteria (X105)
Enzyme Activity
mg/g :
Dehydrogenase Activity
SUGARCANE FIELD
20
100
.100
40
200
.200
60
300
.300
80
.400
.400
100
500
.500
120
600
.600
140
700
.700
?
Q.
UJ
Q
Fungi
\s
20-
Q , Q * ^sss Bacteria
^r~ ^^ 4-
j^^ ^^^ \ mi»O
^^* ,/' \ a
M \ *>
r /
V Cf
PASTURE LAND
Fungi
Phosphatase Activity
Dehydrogenase
: (X103) 20 40 60 80 100
Bacteria : (X loS) 100 200 300 400 500
120
600
Activity
140
700
Enzyme Activity
mg/g : .100 .200 .300 .400 .500
.600
.700
Figure 16. Variations in Microbial Populations and Enzyme Activities.
52
-------
u
?
£ 20-
Fungt (X
Bacteria (X
Enzyme Ac
mg/g:
^&^~~^* '
-+
i^
I ^ O
IMPOUNDED SWAMP D
103 ) 20 40 60 80
105 ) 100 200 300 400
tivity
.100 .200 .300 .400
100
500
.500
Bacteria
Fungi
Phosphatase Activity
Dehydrgenase Activity
EPTH
1
120
600
.600
1
140
700
.700
O-i
~ Bacteria
« + Fungi
i o Phosphatase Activity
lium Q Dehydrogenase Activity
SWAMP - FOREST
Fungi: (X'O3) 20 4Q
Bacteria (X"5) 10o 200
Enzyme Activity
(mg/g) : .100 .200
O-i
6Q
300
.300
80
400
.400
100
500
.500
I
120
600
.600
140
700
.700
E J**fT
O 20H4^*> D
I W /
IJI/
0
«v>o Bacteria
o Phosphatase Activity
1 " O Dehydrogenase Activity
NATURAL SWAMP
Fungi : (X'O3) 20
Bacteria : (X105) 100
Enzyme Activity
(mg/g): .100
40
200
60
300
80
400
100
500
120
600
140
700
200 .300 .400 .500 .600 .700
Figure 16 (cont.) Variations in Microbial Populations and Enzyme Activities
53
-------
STATIC MICROCOSM STUDY
HETEROTROPHS. CONTROL SOIL
*
STATIC MICROCOSM STUDY
HETEROTROPHS; CHITIN- AMENDED SOU
Guthion
control (0 ppm}
10 ppm
25 ppm
100 ppm
y
X
X
X \
EXPOSURE TIME (in doyi)
EXPOSURE TIME (in days|
Figure 17. Effect of Guthion on Heterotrophs and Chitinoclasts in Chitin-
Amended Soils.
The data indicates that Guthion has a pronounced positive effect on
microbial growth. Concentrations of the pesticide above 10 ppm tend to
increase microbial biomass. Treatment variations of total heterotrophs in
control and chitin-amended soils is shown in Fig. 18. Chitin-amended soil had
a higher least square means for each treatment than did control soil.
The static microcosm study indicated that pesticide levels significantly
affected microbial concentrations. Since chitin was the only definable
variable between soils, Guthion seemed to have a positive effect on chitin
turnover and, in all likelihood, on levels of chitinoclasts. However, due to
inherent limitations in the static microcosm design, a more definitive con-
clusion could not be reached. Mortality of invertebrates (nematodes and
larger organisms), as noted in data from field community tests, certainly may
have contributed to the number of degraders. However, this contribution was
not as highly significant in Guthion-amended controls as for chitin-amended
soils. Confirmation of this positive effect on chitin turnover was made in
subsequent continuous flow microcosm studies.
54
-------
100
Control Soil
Chitin - Amended Soil
25
100
AZINPHOSMETHYL CONCENTRATION (in ppm)
Figure 18. Static Microcosm Study. Treatment Variation of Total
Heterotrophs.
Continuous Flow Microcosm Studies for Total
Heterotroph/Chitinoclast Enumeration
Fluctuations in microbial biomass, expressed as colony forming units
(CFU), in control and Guthion-fortified (100 ppm) microcosms are shown in
Figs. 19 and 20. Data are given for chitinoclasts and heterotrophs under
aerobic and anaerobic conditions. Statistical analysis showed significant
variations in total heterotrophs and chitinoclasts (P < 0.01). A rapid
increase in microbial biomass occurred within the first three days of con-
tinuous application of the pesticide. After a 10-day application period,
microbial concentrations returned to initial levels, with no significant
change noted after the ten day period.
Under anaerobic conditions, differences in the heterotrophic and chitin-
oclastic populations in the control and Guthion-fortified microcosm were
evident. Significant increases, attributable to Guthion application, were
noted under both aerobic and anaerobic conditions. Statistical analysis
55
-------
CONTINUOUS FLOW AEROBIC MICROCOSM STUDY
CHITINOCIASTS
CONTINUOUS FLOW AEROBIC MICROCOSM STUDY
130-
131-
100
7}-
50
25-
ncicKuiKurna
70-
Microcoim A
control (0 ppm)
60-
/\ "' Microcosm B
/ X
/ \ |iOO ppm Gulhion) 50-
/ I 0
/ \ 2
/ > Microcosm C x
/ ' --4
' _.---\ \ I'00 PPm Gulhion) 5.
1 ' . 1 U
/ V »
// \ \ 30-
'/ \*
'/' \*
/ \
V \\
f \ ,
/ 1
/\" I
\' '
10-
l^r ..
),',.' °
It
1 I
1 1
1 I
1 I
1 |
' I
' 1 " Microcosm A control (0 ppm)
' / \ l . ._.. Microcosm C (100 ppm Gulhion)
' ' ' \
' ' M
i
' / \\
1 1 \>
' . .1
// 'i
' ' *
'/ i
/ \
' »
i i-
1 / i1.
/' ,-
'' i *
i/ \ « - ~
t i *~
^ x ^^^^ .
y/ ^^^
x^-.
CONTINUOUS APPLICATION TIME (in doys)
CONTINUOUS APPLICATION TIME (in doyi]
Figure 19. Effect of Guthioa on Heterotrophs and Chitinoclasts in Continuous Flow Microcosms
(Aerobic).
-------
CONTINOUS FLOW ANAEROBIC MICROCOSM STUDY
HETEROTROPHS
Microcosm A (0 ppm Guthion)
Microcosm B (100 ppm Guthion)
Microcosm C (100 ppm Guthion)
130.
173
100
73
30'
23
I
l> Microcc
/ 1
1 1 Microcc
/ I
V Microcc
/ \
i \
; V
/ A>
//\
/ /' » V
it ( '
/ / \ \
/ / i v
' / > v
/ ' ' \ \
' / '"-^ '
/ ^"~-
/
CONTINUOUS FLOW ANAEROBIC MICROCOSM STUDY
CHITINOCIASTS
Microcoim A |0 ppm Guthion)
Microcosm B (100 ppm Guthion)
Microcosm C (100 ppm Gulhion)
0 3 10 17
CONTINUOUS APPLICATION TIME (in dayi)
0 5 10 17
CONTINUOUS APPLICATION TIME (in days)
Figure 20. Effect of Guthion on Heterotrophs and Chitinoclasts in Continuous Flow Microcosms
(Anaerobic).
-------
shows significant variations in total heterotrophs (P < 0.01), however, no
significant variation in chitinoclasts occurred after five days of continuous
application of Guthion. Populations returned to initial levels, probably due
to containerization effect of the microcosm system, and remained invariant
after 17 da.
Chitinoclasts comprised a significant portion of the aerobic microbial
biomass prior to pesticide application and increased with continuous Guthion
application. A correlation between substrate turnover and enzyme activity
with increased pesticide application can also be projected. Fig. 21 shows
variations by treatment of heterotrophs under aerobic and anaerobic condi-
tions and represents a comparison of relative population concentrations
between aerobic (xlO6) and anaerobic (xlO5) conditions. Equipment limita-
tions prevented tests under aerobic and anaerobic conditions to be conducted
simultaneously. Significant increases attributable to Guthion application
were noted under both aerobic and anaerobic conditions. Numerical values of
total heterotrophs were, as expected, higher for aerobic than for anaerobic
100-1
80 -
O
x" 60-
U 40 -
Aerobic
Helerotrophs
u
80 -
60 -
40 -
20 -
0 -
Ar
H
aerob
Hi
Heterotrophs
100
100
AZINPHOSMETHYL CONCENTRATION (in ppm)
Figure 21. Treatment Mean Variations for Heterotrophs and Chitinoclasts
in Guthion Supplemented Continuous Flow Microcosms.
58
-------
conditions. Variations in chitinoclasts by treatment with the total popula-
tion were apparent. Declining CPU reflects, of course, the utilization of
the available food source, chitin and additional dead invertebrates, in
Guthion-amended microcosm units. These concentrations are important for two
reasons. In all likelihood, variations by a major physiological group within
the total population indicates selectivity of pesticide interaction within
the total population. A correlation between substrate turnover and enzyme
activity with increased pesticide application can also be projected. Chi-
tinoclasts constituted a significant percentage of the aerobic population
prior to pesticide application. This percentage of the total population
increased after continuous application of Guthion. Chitinoclasts are not a
significant physiological group within the anaerobic portion of the biomass
and remained invariant.
It can be postulated that variations in the chitinoclastic group, as
affected by Guthion, is indicative of the selectivity of pesticide interac-
tion within the total microbial population. These and other data support the
hypothesis that substrate turnover, whether from chitin itself or from
invertebrate mortality and specific enzyme activity, are directly affected by
Guthion application.
Changes in Sensitivity and
Utilization of Azinphosmethyl--
A test for changes in bacterial sensitivity to, and utilization of,
azinphosmethyl was conducted in conjunction with enumeration of total heter-
otrophs for the aerobic continuous flow microcosm study. Table 8 depicts
these changes by the microbial community at different pesticide levels and
type of growth media. Colonies were transferred from sample plates to
pesticide-amended plates using the Laderberg replica plating technique. The
values expressed are the percentage of transferable colonies enumerated after
incubation. Media amended with acetone served as a control to avoid possible
inhibitory effects attributable to acetone alone.
Slight variations in sensitivity to Guthion at concentrations of 10, 25,
and 100 ppm noted were not statistically significant. Minor variations in
growth of transferable colonies (Table 8) indicate a relatively low microbial
sensitivity to Guthion by sample treatment populations.
Slight variations in utilization of Guthion at levels of 10, 25, and
100 ppm were noted but were not statistically significant. The growth of
transferable colonies indicate a relatively small percentage of the total
population can utilize Guthion as a sole carbon source. These numbers
increased slightly for pesticide-treated microcosms, giving some indication
of adaptation by the microbial community to Guthion utilization.
Selected cultures obtained from the above tests for Guthion utilization
were grown in three types of media amended with the pesticide to a level of
225 ppm. Table 9 presents the relative growth of these selected axenic
cultures. A few isolates exhibited good growth rates using the pesticide as
a sole carbon source. Two actinomycete cultures exhibited good growth for
59
-------
Table 8. Changes in Sensitivity to and Utilization of Azinphosmethyl During Continuous
Flow Aerobic Microcosms.*
Application
Time
(Days)
B
> B '-v
o a c
0 p, 0
0 < -H
M 0 ,C
o c -u
H <-\ 3
S ^ 0
w E /~v
o a. c
o a. o
O pq -H
^ O J3
0 O 4J
3 ^ o
B
ui B /-^
0 O. C
o a o
0 U -rl
o o *>
is ^ o
0
3
10
0
3
10
0
3
10
N.A""
Acetone
90.1
99.0
98.3
89.2
99.0
99.0
98.3
99.0
99.2
N.A
10 ppm
90.1
96.0
95.1
89.2
98.7
98.3
92.5
98.2
99.2
N.A
25 ppm
90.1
96.0
95.1
79.6
98.2
96.7
87.5
98.2
99.2
MEDIA
N.A M.S.
100 ppm Acetone
90.1
91.0
88.5
86.0
97.4
95.9
87.5
97.3
99.2
24.3
22.2
30.3
45.1
42.2
48.6
23.4
42.3
48.0
M.S
10 ppm
29.7
19.8
30.3
40.3
50.5
51.8
28.1
52.3
50.3
M.S
25 ppm
29.7
22.6
18.1
41.3
49.5
55.6
26.6
46.6
52.6
M.S
100 ppm
27.0
18.8
18.1
36.5
49.5
54.1
15.6
45.5
52.6
ON
O
Expressed in Terms of % Transferable Colonies Enumerated After Incubation.
i*
Nutrient Agar (Difco, Inc.).
v
Minimum Salts & Basal Agar (Difco, Inc.).
-------
Table 9. Axenic Culture Study. Growth of Selected Axenic Cultures Isolated from Continuous Flow
Aerobic Microcosms.
MEDIA
Isolates
HI P2
1I2 P2
II p
11 p
IIG
AH P4
A2o P4
Nutrient Broth
and
225 ppm Guthion
XXX
XXX
XXX
XXX
XXX
XXX
XXX
Minimum Salts
and
225 ppm Guthion
XX
XX
0
XX
XXX
XX
XXX
0.1% Chi tin
Minimum Salts and
225 ppm GuthLon
0
0
0
0
XXX
XXX
XXX
C/16 XXX XX XXX
C/19 XXX 0 XXX
C/9 XX X XXX
Cu P2 XX 0 XX
C14 P2 XX X XXX
Time: 8 Days
0 = No Growth XX = Moderate H = Heterotroph
X = Adequate XXX = Good Growth A = Actinomycete
C = Chitinoclast
-------
all three types of media. This is noteworthy in view of the relative abun-
dance of chitinoclastic actinomycetes in both upland and flooded soil micro-
bial populations. Chitinoclasts and heterotrophs grew well in the presence
of the pesticide. No estimation of actual degradation rates was made of
those cultures growing solely on Guthion.
Pesticide Accumulation--
The flow pattern of the continuous flow microcosm involved removal of an
amount of fluid from the reaction vessel equivalent to amounts introduced.
Preliminary tests in pesticide loss from the system due to flushing showed a
depletion rate of 10%. At the prescribed rate of pesticide addition (0.105
ml/min) for 500 ml, complete replacement of water within the microcosm with
100 ppm pesticide amended water would occur in 3.31 da. The detectable
amount of pesticide after three days was considerably less. A significant
accumulation of pesticide by the flooded soil occurred as a result of the
continuous application, with high (1000 ppm) pesticide levels noted in sedi-
ment in both aerobic and anaerobic microcosms. Similar accumulation pheno-
mena was noted in physical/chemical studies. This indicates that the
sediment was an effective reservoir (sink) for the pesticide. It is note-
worthy that the total raicrobial population under both aerobic and anaerobic
conditions was not adversely affected by the extremely high concentrations of
Guthion.
Initial Enzymatic Studies--
Dehydrogenase Activity. Measurement of dehydrogenase activity provides
an insight into the metabolic activity of microorganisms in soil. Fig. 22
shows the mean treatment effect on dehydrogenase activity of two amended and
control soils, indicating no significant changes in dehydrogenase activity.
Starch was used as a second nutritive source to chitin to make ortho-
gonal comparisons on dehydrogenase activity between amended and control
flooded soils. Although not statistically significant, inferences can be
made on dehydrogenase activity being affected by amending with chitin and
starch. Dehydrogenase levels for chitin-amended soils were perceptibly
higher than those for control and starch-amended soils. Dehydrogenase levels
in chitin-amended soils increased with pesticide level, but not to signifi-
cant levels.
Phosphatase Activity. Fig. 23 shows the mean treatment effect on phos-
phatase activity of three soil types with addition of Guthion at levels of 0,
10, 25, and 100 ppm. Statistical analysis of treatments for each soil type
indicated no significant changes in phosphatase activity. Similar to dehy-
drogenase activity, inferences can be made on phosphatase activity being
affected by amending the natural soil with chitin and starch. Slight de-
creases in phosphatase activity are noted for pesticide-treated soils due to
the increased availability of phosphorus provided by the pesticide.
Determination of Pesticide Half-Life. Residual pesticide concentrations
in soil from dehydrogenase and phosphatase activity studies are shown in
Table 10. Half-life for the pesticide for each soil type at different levels
62
-------
O
CO
0.05
0.20 -
Control Soil ChitinAmended Soil StarchAmended Soil
0.15-
8,
0.10-J
Illlllllllll
0 10 25 100 0 10 25 100 '0 10 25 100
AZINPHOSMETHYL CONCENTRATION (in ppm)
* TRIPHENYLTETRAZOLIUM FORMAZIN
Figure 22. Effect of Chitin-Addition on Dehydrogenase Activity In Situ.
-------
0.80 -
Control Soil Chitin-Amended Soil Starch-Amended Soil
0.60-
Illlllllllll
0 10 25 100 10 25 100 0 10 25 100
AZINPHOSMETHYL CONCENTRATION (in ppm)
*P-NITROPHENOL
Figure 23. Effect of Guthioa on Phosphatase Activity in Three Soil Types (Control, Chitin-Amended,
Starch-Amended).
-------
Table 10. Residual Guthion Concentrations in Soil from Dehydrogenase and
Phosphatase Activity Trials
Exposure Time: 17 Days
Soil
Type
Natural
Soil
Chitin-
Amended
Soil
Starch-
Amended
Soil
Initial
Cone .
0
10
25
100
0
10
25
100
0
10
25
100
Final
Cone.
0
0
6
0
0
1
0
0
3
0
.1923
.5824
.083
0
.1873
.7405
.878
0
.1907
.7488
.759
Percent
Recovered
1
1
2
6
1
2
1
1
2
3
.923
.923
.330
.083
.873
.962
.878
.907
.995
.759
Half
Life
(Days)
2
3
4
2
3
2
2
3
3
.982
.134
.209
.962
.348
.964
.976
.359
.591
Mean
Half-Life
(Days)
3.44
3.09
3.31
of concentration compare closely. The half-life (71 = 3 days) for the pesti-
cide in our study was longer than half-life values noted in field application
studies (T, = 2 days). However, this value was shorter than values given by
Yaron (197^) of 5 days in upland, agricultural soils.
Initial Carbon Metabolism Studies--
The carbon metabolism microcosms was designed to test for the rate of
N-acetyl-D-glucosamine (NAG) utilization, as well as for relative respiration
rates, by using NAG (glucosamine-14C). The objective was to obtain a more
definitive picture of substrate utilization, i.e., NAG, as affected by pesti-
cide treatment, compared with earlier studies using chitin, and to determine
more precise fluctuations in the microbial biomass due to pesticide addition
based on 14C02 production.
As noted in Figs. 24 and 25, both 14C02 respiration and 14C-glucosamine
rates increased with Guthion addition. Variation in evolution of 14C02 with
time by treatment proved to be highly significant (P < 0.01). An immediate
response to the tagged NAG was detected for static microcosms containing
Guthion. A somewhat delayed response was noted for static microcosms without
the pesticide. Evolution of 14C02 for all microcosms leveled off after 48
hours and remained invariant for a period of 24 days. The 14C02 evolved
during these experiments accounted for between 27 to 42% of the added 14C.
65
-------
ON
U
-------
Microcosm A&B (0 ppm Guthion)
Microcosm C&D (100 ppm Guthion)
EXPOSURE TIME (in hours)
0 1
CPU
Figure 25. Effect of Guthion on 14C-Glucosamine Uptake in Carbon Metabolism Microcosm.
-------
Earlier, Bartha et al. (1967) reported organophosphates cause an initial
increase in total C02 production in upland soil, the magnitude of which is
directly related to concentration applied.
As data in Fig. 26 suggest, Guthion has a positive and rapid action on
NAG utilization, corresponding to increases in microbial activity concentra-
tions and 14C02 evolution. This increase in microbial activity correlates to
the response noted earlier in chitin-amended continuous flow-through micro-
cosms. Thus, a stimulatory effect by Guthion on chitin and its breakdown
products is postulated. This stimulatory effect by Guthion is augmented by
benthic invertebrate mortality resulting in additional chitin input into the
total substrate pool. Chitin, an energy-rich substrate readily colonized by
the extant microbial community, we feel provides an absorptive medium for the
pesticide. Faust (1977), in his extensive review of organic pollutants in
aquatic systems, noted the effect of organic matter content in controlling
pesticide adsorption. Presence of Guthion has a positive effect on chitin
utilization, which may trigger a more complete and rapid mineralization of
the xenobiotic.
Determination of Pesticide Half-Life--
The breakdown products of many organophosphates have been reported to
have an inhibitory effect on microbial respiration. However, this was not
apparent for Guthion for the 24-day study period. Table 11 lists the residual
pesticide concentrations in soil from the carbon metabolism microcosm study.
A half-life of 7-7^ days was determined. A longer T! , for these studies,
reflected in part the utilization of NAG rather than chilin as an energy rich
substrate. For a 24 day test period, over 90% of the initial pesticide added
was degraded to various breakdown products, possibly anthranilic acid and
Table 11. Residual Guthion Concentrations in Soil from Carbon
Metabolism Microcosm Study.
Microcosm
A
6
C
D
Exposure Time:
pH:
Temperature:
Initial
Cone .
(ppm)
0
0
100 ppra
100 ppm
24 days
7.0 - 7.2
30 C
Final
Cone.
(ppm)
0
0
9.467
10.960
Half
Life
Percent (T^)
Recovery Days
9.467 7.057
10.960 7.524
68
-------
O\
vo
TREATMENT MEAN
14CO2 RESPIRATION RATE
2.0-
o
-------
benzazimide (Liang 1972). Present data suggest that these and other possible
breakdown products are not toxic to microbial respiration. Nevertheless,
continuing studies, that amend flooded soils with these compounds are needed
to present a thorough picture of the total spectrum of effects of Guthion on
the composite soil environment.
Our field studies support observations of other investigators on effect
of organophosphate pesticides. Sethunathan (1973) noted a more rapid degra-
dation of parathion in soils under flooded conditions compared with those
under non-flooded conditions. Guthion is similar in action on the microbial
community in flooded soils as are other organophosphates previously tested in
upland, agricultural soils (Atlas et al. 1977). Rapid increases in bacterial
concentrations coincide with rapid degradation of the pesticide. Higher
pesticide concentrations correlate with higher bacterial concentrations. No
significant variations in phosphatase and dehydrogenase activity are noted as
a result of pesticide addition. These aspects, correlated with data from
microcosm studies, are of significance in the selection and use of pesticides
on upland, agricultural soils adjacent to highly productive estuarine
systems.
Studies with Guthion, Methyl Parathion and Kepone
Microbial/Enzymatic Correlations:
Guthion and Methyl Parathion
In field tests with Guthion and methyl parathion, enzyme activity,
phosphatase, and dehydrogenase correlated with increases in microbial activ-
ity. Results of field tests for methyl parathion are given in Fig. 27. Soil
phosphatase and dehydrogenase activities were examined in terms of bacterial
and fungal populations with depth and the particular soil environment. High
correlations were noted between phosphatase and microbial activity as well as
between dehydrogenase and microbial activity. These variations in population/
enzyme profiles provided indications of the impact of land management prac-
tices on impounded freshwater swamp-forest habitat compared with pristine
sites. Organic material accumulation from litter-fall on dry and submerged
swamp forest floors were reflected by highly stratified bacterial and fungal
populations. Xenobiotic addition reflected shifts in these stratifications,
indicating microbial stress and response. Addition of methyl parathion was
evidenced by increases in both bacterial and fungal populations, along with
enhanced enzyme activity. Subsequent microcosm studies showed these biomass
increases to be highly significant.
Continuous Flow Studies (Part 2)
Microbial and enzymatic responses in control and Guthion-fortified
microcosms over a 20-day period are shown in Fig. 28A. Fig. 28A represents
the control set while Fig. 28B shows the effect of pesticide addition.
Guthion was added to the test microcosms after 13 days. In general, enzyme
activity was correlated with increases in microbial activity, with an order
of magnitude change in fungal biomass. Similar responses in both fungal and
bacteria levels (Fig. 29) were noted in different tests using methyl para-
thion.
70
-------
0-5-
10-20-
QL
LU
Q
30-40-
,.0o 53
0 50
Colony Forming
Units (CPU)
SUGARCANE
Fungi (control) x 103) ^. 5ppm MP
Bacteria (control x 10s) o 5ppm MP
Phosphatase (control) ^ 5ppm MP
Dehydrogenase (control) Q 5ppm MP
100
150
mg/g PNP H
mg/g TTF |-
200
250
.5
1.0
.100 .200
Enzyme Activity
300
1.5
.300
E
o
0-5-
10-20-
a.
UJ 30-40-
O
0 50
Colony Forming
Units (CFU)
B W PASTURE
* Fungi (control x 103) $ 5ppm MP
Bacteria (control x 10s) o 5ppm MP
* Phosphatase (control) O- 5ppm MP
Dehydrogenase (control) D 5ppm MP
100
1
150
mg/g PNP
mg/g TTF
200
250
300
1.0
1.5
.100 .200
Enzyme Activity
.300
Figure 27. Effect of Methyl Parathion on la Situ Microbial and Enzymatic
Activity.
71
-------
0-5-
^
g
O
z 10-20-
Q.
UJ
^ 30-40-
. 0 .^ Mp » 0
^^'^^^'^ .v*'*' / 'X
^/^* '^^^^ £'' ' /
s^^**^^ ./ ' s
iff* ?? -^
U ^ «
I A M SWAMP FOREST
4 o n -o
* Fungi (control x 103) O-Sppm MP
Bacteria (control xlO5) o 5ppm MP
* Phosphatase (control) -O-Sppm MP
Dehydrogenase(control)D 5ppm MP
0 50 100 150 200 250 300
Colony Forming
I
0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1.0 1.1
0 .100 .200 .300
Enzyme Activity
0-5-
O
z 10-20-
|_
O.
UJ
° 30-40-
^gfSi*^-0 ,.<:%?*
^sif^^^^ .^^^
<(&w^' o"i*"i^-t>'
III/ :/ / /
III/ 'ii 1 /'
/* i//'
J j l^- IMPOUNDED SWAMP
* Fungi (control x 103) O 5ppm MP
Bacteria (control x 10s) o 5ppm MP
#. Phosphatase (control) . . . , , , .
0 .5 1.0 1.5
0 .100 .200 .300
Enzyme Activity
gure 27 (cont.) Effect of Methyl Parathion on In Situ Microbial and
Enzymatic Activity.
72
-------
VARIATION IN MICROBIAL / ENZYME ACTIVITY
FOR CONTINUOUS FLOW- THROUGH
MICROCOSM (CONTROL)
VARIATION IN MICROBIAL/ENZYME ACTIVITY
FOR CONTINUOUS FLOW - THROUGH MICROCOSM
(GUTHION TREATMENT 10 ppm)
175-
e
o
I
o
tc
2
8
125-
75~
50-
25-
0 3
_._._ .Bacteria (XIO>)
I Fungi (X103)
"" o Phosphatase Activity
> oDehydrogenase Activity
-.175 .700
A!
1%
T
T
-.150 .600
-.125 .500
-.100 .400
-.075 .300
-.050 200
-.025 .100
III!
at aft
-.200 1.000
-.175 .875
-.150 .750
-.125 .625
7 10 13 f7 20
TIME IN DAYS
.100 .500
.075 .375
050 .250
025 .125
7 10 13 t7 20
TIME IN DAYS
Figure 28. Variation in Microbial/Enzyme Activity in Continuous Flow Microcosm (Control and Guthion-
Treated) .
-------
MICROCOSM A (MP)
(control)
MICROCOSM D
(methyl-parethion treatment Sppm.)
200-
175-
150-
e
u
1 ,25-
a
!
£ 100-
c
o
o
O
75-
50-
25-
» Fungi (UK)*)
o Bacleiia («10*>
D Aclinomycetes (xlO'l
* Phosphalase
Dehydrogenase
.....
..*'
^ ~~ ' ~ " v '*
s \
' \ *:.
.^^^^^V. '-..
v
\s
.0 L\,^^\
o..,., °" " *-'~-^6r^ O
X m
O I
Jut 3 <
D ID O
35 3S
"2 g
S i
4 Fung. (MO1)
o Bacteria (>I06)
D Acl.nomyceltb (xlO1)
Phospnaiast
Dehyrjiogenase
^.
h\
j \\
1 ^ \
j \ \
i * * * ^^-1^^
/ * """^ ^^^^^***^
/ ^ v " \
// \ ,\
/ ^ V
a v \ * ''
'/ -^ \ '"
lil \ \ ""*
rf// ^ \
'/ x \
*/ v- *
/ .Au. *^vv----*
* ^^'"' V"VVS:'-»'''^N;»
.' "o*
/'
b
~l 1 1 1 11 T '
0 3 7 10 I 13 17
End ol appl'Colicn
Application Time In Days
-cc -coo
875 C6T-
- 750 C750
626 CC25
iCC C5CC
375 C27t
25C C25C
-.25 C',25
n n
T m
O X
? I T 3)
3 > 3 O
~> "g
m >
Figure 29. Variation in Microbial/Enzyme Activity in Continuous Flow Microcosm (Control and Methyl
Parathion-Treated).
-------
Bollag (1972), in his discussion on the biochemical transformation of
pesticides by soil fungi, noted that bacteria and fungi will react to xeno-
biotic compounds differently; bacteria will often attach and degrade a com-
pound to its mineral components whereas fungi will induce minor chemical
changes. Cognizant of the high fungal biomass found in both sugarcane and
impounded swamp soils, significant degradation and utilization of organo-
phosphates such as Guthion by fungi, may be a contributing factor in the
relatively short half-life, i.e., 1-1\ d, of Guthion in soils.
The mean treatment variations for the various test parameters are given
in Table 12. Analyses of data for the dependent variables, bacteria and
fungi showed significant variation between the control and test microcosms.
Using analyses of variance revealed significant (P < 0.01) variation in
biomass numbers. The Duncan's multiple range test of variations in treatment
means showed significant variations at an alpha level of 0.05. Significant
variation in phosphatase level by respective treatment was not noted, in
contrast to the significant variation in phosphatase activity observed over
time of application. Highly significant variations (P < 0.01) in dehydro-
genase activity were noted by treatment and time. Duncan's multiple range
tests for dehydrogenase indicated significant variation between the control
and experimental microcosms at an alpha level of 0.05.
Table 12. Mean Treatment Variations and Enzyme Response for Total
Heterotrophs in Continuous Flow Microcosms.
3
Filamentous Fungi (x 10
Yeasts (x 103)
Bacteria (x 10 )
Actinomycetes (x 10 )
Phosphatase (mg/ml PNP)"
Dehydrogenase (mg/ml TTF)"
Treatment I
(control)
24.5
4.3
34.5
4.1
0.416
0.036
Treatment II
(Guthion)
58.8
2.0
50.6
5.1
0.437
0.048
PNP - p nitrophenol.
tf
TTF - triphenyltetrazolium formazin.
75
-------
Continuous-Flow Studies (Part 3)
and Carbon Metabolism Studies (Part 2)
(R)
Microbial response to addition of methyl parathion (5 ppm) and Kepone
(0.5 ppm) in control and test static carbon metabolism microcosms is shown in
Figs. 30 and 31. Data are given for fungi and bacteria, expressed as the log
of colony forming units (CFU), for both compounds. Statistical analysis
indicated significant variations in both microbial groups with a single
application of methyl parathion. After incubation for 10 days, microbial
concentrations returned to initial levels, with no significant changes noted
subsequently. No significant ^otal treatment variation was seen in the
static carbon metabolism Kepone microcosms with either bacteria or fungi,
although bacterial CFU were higher in the control microcosm at day three and
fungal CFU were higher in control microcosms at day seven. Nevertheless, in
general, control microcosm chambers had higher CFU than did experimental
units.
Figure 30. Microbial Response to Methyl Parathion (5 ppm) in Static Carbon
Metabolism Microcosm.
76
-------
Figure 31. Microbial Response to Kepone (0.5 ppm) in Static Carbon
Metabolism Microcosm.
Fluctuations in bacterial and fungal biomass, expressed as log CFU/ml,
in control and xenobiotic-amended continuous flow microcosms are shown in
Figs. 32 and 33. Significant increases in both bacterial and fungal log CFU
occurred within the first three days of continuous application of methyl
parathion. Xenobiotic input was terminated after 7 days with no significant
variation in bacterial biomass after 10 days. However, fungal populations
declined more rapidly after 10 days, with the control microcosms having the
greatest decrease after day 17.
Microbial response to continuous application of Kepone (Fig. 33) was
similar to responses noted in static microcosm tests (Fig. 31). Significance
by treatment variations due to Kepone addition was not evident between con-
trol and test microcosm units. Fungal log CFU in the control unit were
higher at 7 days than in the kepone-amended microcosm. Bacterial log CFU for
both control and test microcosms were comparable, although the control-units
were somewhat higher. Controls for both methyl parathion and Kepone were
not significantly different in terms of bacterial and fungal log CFU
(Fig. 30), in contrast, fungal log CFU were higher for Kepone . It appears
that variations in microbial populations in the microcosm information provide
77
-------
CO
Figure 32. Microbial Response to Methyl Parathion (5 ppm) in Flow Through Microcosms.
-------
Figure 33. Microbial Response to Kepone (0.5 ppm) in Flow-Through Microcosm.
information on xenobiotic selectivity and interaction with the total micro-
environmental community, indications of possible mortality substrate contri-
butions from higher trophic levels and indications of compound recalcitrance
and fate.
ATP Response--
Estimates of viable biomass, as provided by adenosine triphosphate (ATP)
concentrations, for control and test static carbon metabolism and flow-
through microcosms are shown in Figs. 34 and 35, respectively. Log ATP
estimates for static microcosms (Fig. 34) provided indications of significant
increases in microbial biomass for methyl parathion, in contrast to Kepone
addition. These observations were further confirmed in flow-through micro-
cosms (Fig. 35), wherein a significant increase in microbial ATP was noted
following application of parathion. ATP levels remained consistently high
for the tegt microcosm, declining rapidly after day 13. ATP concentrations
for Kepone were statistically invariant with both control and test micro-
cosms having variable high and low data points throughout the growth period.
79
-------
2.5
2.0
I1'5
O)
3
oT 1.0
5
o>
o
0.5
MP - Control
oMP- Experimental \5ppml
A KP - Control
A KP - Experimental [o.5 ppmj
x ^-.
/' /x
0 3 17 10 13 17 21 24 27 30 days
Figure 34. Response of ATP to Addition of Methyl Parathion and Kepone in
Static Microcosms.
2.5
-------
ATP concentrations, in continuous flow through microcosms (Fig. 35),
showed comparable responses for methyl parathion. ATP levels were signifi-
cantly higher for methyl parathion microcosm units, with peaks occurred
within three days of continuous application. ATP levels dropped by day 10
except for a transitory peak in the control microcosms at this time. ATP
levels dropped rapidly Jor both control and test units after ten days.
Concentrations in Kepone units again were not significantly different from
those of the control. However, after day three, Kepone -amended microcosms
had somewhat lower ATP levels compared to those of the control. A moderate
increase in ATP concentrations on day 21 for both control and test microcosms
is difficult to explain. This may be due to a containerization effect of the
microcosms or to subsequent degradation^ of the lignocellulose component
present in the sediment. Probably, Kepone itself was not solely responsible
for this ATP increase, although a slight increase in 14C02 release in static
microcosms was noted during this time.
Further Enzyme Responses--
A notable decrease in phosphatase activity was seen between Kepone -
amended and control static microcosm units (Fig. 36). Phosphatase levels,
initially higher than that of controls for Kepone , steadily dropped after
day three. In contrast, phosphatase levels fox methyl parathion again were
significantly higher than controls for Kepone , and steadily dropped after
day
O>
E
a.
a.
a
"a
O)
o
5.0
4.0
3.0
2.0
1.0
PHOSPHATASE
. MP- CONTROL
oMP- EXPERIMENTALCSppm]
* KP- CONTROL
& KP- EXPERIMENTALfo.Sppm]
5.0
4.0
O)
3.0
a
in 2.0
. -°
0 3 7 10 13 17 21 0 days 0
OEHYDROGENASE
.MP-CONTROL
o MP- EXPERIMENTALCSppm]
* KP- CONTROL
- EXPERIMENTAL [O.Sppm]
3 7 10 13 17 21 30 days
Time
Figure 36. Response of Phosphatase and Dehydrogenase to Addition of Methyl
Parathion and Kepone in Static Microcosms.
81
-------
three. Levels of dehydrogenase for methyl parathion were higher at day three
but subsequently dropped to, or below, control levels. Hence, no statisti-
cally significant methyl parathion by treatment variation was discerned.
Dehydrogenase levels for Kepone , although somewhat lower than controls, were
not statistically significant.
Significant increases in both phosphatase and dehydrogenase activity in
flow-through microcosms were noted for continuous methyl parathion addition
(Fig. 37). These increases coincided with those for total microbial CEU, as
noted previously for methyl parathion. Dehydrogenase levels for Kepone were
somewhat below control values, but were not statistically significant.
5.0
4.O
O>
I 3.0
a.
£ '
a
o>2.0
1.0
5.0
PHOSPHATASE
. MP - CONTROL
oMP- EXPERIMENTALCSppm]
A KP- CONTROL
A KP- EXPERIMENTAL[0.5ppm]
u. 3.0
o
a
o
§>2.0
1.0
DEHYDROGENASE
MP-CONTROL
o MP- EXPERIMENTAL[Sppm]
» KP-CONTROL
a KP- EXPERIMENTAL [O.Sppm]
10
^
13 17 21
End MP Addition
^End KP Addition
30 days 0
10
^-
13 17 21 30 days
EndMP Addition
^ End KP Addition
Figure 37. Response of Phosphatase and Dehydrogenase to Addition of Methyl
Parathion and Kepone in Flow-Through Microcosms.
Radiotracer Analysis
Residual levels of 14C-methyl parathion and 14C-Kepone for replicate
microcosm series, based on 14C02 mineralization of the parent molecule, is
shown in Fig. 38. After 13 days under microcosm conditions outlined pre-
viously, a significant loss of methyl parathion was evident. Rapid bio-
transformation and degradation of the parent molecule occurred with the first
week of application. This initial degradation period correlated with
increasing CFU, phosphatase, dehydrogenase, and ATP levels, as mentioned
previously. Fig. 39 shows maximal 14C-assimilation of both parent molecules,
or their major metabolites, within the CFU, occurring by day seven for methyl
parathion. A sharp decrease is noted in 14C-assimilation by day 13, along
with similar decreases for 14C02 levels.
82
-------
Remaining Xenobiotic I Based on 14CO2 release, theoretical I
Figure 38. Residual levels of 14C-Methyl Parathion and 14C-Kepone in Static
Microcosms.
Extremely low levels of 14C02 were detected for Kepone during the 30
day incubation time period (Fig. 37). Over 98% of the original Kepone , or
possibly major metabolites, remained after this period. Although it is
possible that the 14C02 detected could be small ^quantities of Kepone ,
follow-up sterile microcosm tests with- 14C-Kepone resulted in no 14C02
detection. 14C-assimilation of Kepone gradually increased during this
period, raising the possibility of its being actively assimilated by the
microbial component, however, with minimal utilization.
In carbon metabolism studies with 14C-nL-methyl parathion and chitin
(Fig. 40) a more rapid evolution of 14C02 occurred in chitin-supplemented
microcosms compared with control microcosms. Although rates of 14C uptake
were approximately equal for CFUs for both control and chitin-supplemented
microcosms, the total (jCi of 14C assimilated was greater for chitin-
supplemented microcosms due to a significant increase in CFTJ. Methyl para-
thion degradation was further induced by the presence of this energy rich
substrate.
83
-------
I4C ASSIMILATION WITHIN CPU
Figure 39. 14C Assimilation Within Colony Forming Units (CFU) With Methyl
Parathion and Kepone in Static Microcosms.
Particular attention in our research has been given to the structural
polysaccharide chitin, probably the most abundant biodegradable polymer in
marine and estuarine environments. Turnover of this energy and nutrient-rich
material in estuarine environments is a critical basic transformation process
in the functioning of trophic food webs (Fig. 41). Analyses of microbial
ecology and chitinoclast populations requires careful study of chitinoclastic
processes and substrate mineralization as affected by a variety of xeno-
biotics.
In earlier sections of our report, data were presented on interactions
between a labelled breakdown product of chitin, N-acetyl-D-glucosamine (glu-
cosamine-14C), and the organophosphates, azinphosmethyl (Guthion) and methyl
parathion. In these studies, significant increases in chitinoclastic popu-
lations, were indicative of rapid substrate transformation and utilization,
coinciding with disappearance of the xenobiotic in question. The compound
N-acetyl-D-glucosamine (NAG) is a key pathway substrate in enzymatic
hydrolysis of chitin (Hood 1973) and appears to be an ecologically signif-
icant material in in situ chitin transformation (Hood and Meyers 1977a, b).
Interaction between NAG and a variety of xenobiotics as will be shown in
84
-------
b
X
80-
60-
00
(Jl
b 40H
a.
20-
TREATMENT MEAN
14CO2 RESPIRATION RATE
mwmmm
14
C METHYL
60-
CFU
PARATHION/CONTROL
14 C- METHYL
PARATHION/CHITIN
TREATMENT MEAN
14C- UPTAKE RATE
CPU
14 C METHYL
PARATHION /
CONTROL
14C- METHYL
PARATHION/
CHITIN
-80
O
60 «
o
40 O
E
u
-20
Figure 40. Effect of Chitin on 14C02 and 14C Uptake in Methyl Parathion Carbon Metabolism Microcosms.
-------
Energy-Rich Chitin
Constituents Available
to Food Chain Organisms
Chitin Production ,
(crustaceans, insects,
annelids, fungi, etc.)
i
Chitin Deposition
(anthropod exuviae, dead
animal exosketetons)
Abiotic Chitin- Heavy Metal
Complexing/Physical Breakup
of Chitinous Exoskeletons)
Chitin Degradatbn
(Heterotrophic Nutrition )
Microorganism hydrolysis ^
of chitin to glucose, ammonia,
and acetate
Biodegradable Substrate
(source of chitin
precursurs)
.Residual Chitin in Water
Column and Sediment
abiotic chitin-heavy
metal complex ing
chitin processed
by filter-feeding
organisms (oyster,
clam, zooplankton, etc. )
Figure 41. Chitin Turnover in an Estuary. .
subsequent research with methyl parathion, Kepone and selected phenols,
provides an extremely sensitive monitoring "tool" that can be effectively
utilized in microcosm systems.
(R)
In subsequent investigations, 14C-uL-methyl parathion and 14C-Kepone
were introduced into both continuous flow and static chitin-amended micro-
cosms at concentration levels noted in previous work (5.0 ppm and 0.5 ppm,
respectively). Figs. 42 and 43 present microbial population responses of
continuous flow studies for methyl parathion and Kepone , respectively. In
the methyl parathion run, significant increases in bacterial, fungal and
total chitinoclasts were noted in experimental microcosms for days-. 3 and 7
compared with controls. Microbial population levels for Kepone -amended
microcosms were lower than controls for days 3, 7, and 13, with fungal and
chitinoclastic groups showing significance (p < .05).
Similar responses, for phosphatase, dehydrogenase and microbial ATP were
noted for both Kepone and methyl parathion as evidenced in earlier investi-
gations with chitin, except that dehydrogenase and microbial ATP levels were
significantly higher in methyl parathion/chitin microcosms. Fig. 44 shows
residual levels of 14C-methyl parathion and 14C Kepone for comparable micro-
cosm series based on 14C02 ^release. Chitin produced no effect in compound
disappearance in the Kepone study, whereas in the methyl parathion study,
chitin contributed to both compound assimilation and utilization.
86
-------
Co
-J
METHYL PARATHION/CHITIN
[ Flow Through Studies 1
Figure 42. Microbial Response to Methyl Parathion in Chitin-Amended Flow Through Microcosms.
-------
oo
oo
a>
?,
6
KEPONE/CHITIN ADDITION
[Flow Through Studies]
Figure 43. Microbial Response to Kepone in Chitin-Amended Flow Through Microcosms.
-------
Remaining Xenobiotic I Based on 14CO2 release, theoretical)
Figure 44. Residual Levels of 14C-Methyl Parathion and 14C-Kepone in Carbon Metabolism Microcosms
-------
Understanding of the substrate and enzymatic events in chitin breakdown
is critical in terms of discerning effects on pathway flow and points of
maximal compound utilization. These involve particle size reduction, fol-
lowed by decalcification and deproteinization of the chitinous matrix.
Effects can be direct, i.e., on the process itself, or indrect, i.e., via
inhibition or reduction of activities of the chitinoclast biomass. Investi-
gators have noted that adsorption effects (surface phenomena) are especially
important in chitinoclastic processes and related colonization activities.
Analyses of chitinase elaboration as a major activity factor and its rela-
tionship to xenobiotics should provide relevant data on environmentally-
significant surface phenomena. Xenobiotics may physically block active sites
on the chitin substrate, thus inhibiting inception of breakdown processes.
The products N-acetylglucosamine and glucosamine resulting from activity of
chitinolytic enzymes can be monitored to study various physiological deter-
minants intrinsic to the composition of the microbial community and trans-
formation of ecologically significant substrates such as chitin.
Axenic Flask Studies--
Growth responses of representative microcosm isolates are shown in
Table 13. In all instances with methyl parathion, a moderate to high re-
sponse (> 30% change) was noted in overall ATP levels. This correlated with
rapid assimilation of the parent molecule in mixed enrichment media as desig-
nated and a notable total biotransformation. A low response was evident for
isolates No. 1, 2, and 3 for methyl parathion in minimal salts media, whereas
a negative response occurred for the bacterial isolate No. 4 along with a
moderate response in the chitin medium. The most appreciable loss of methyl
parathion occurred with fungal Isolate No. 1: 38% after seven days in
Martin's media. However, when methyl parathion was the sole carbon source,
only low responses were noted. These results are comparable to those of Rao
and Sethunathan (1974), using enrichment culture techniques, in which the
fungus Penicillium waksmani, tolerated high levels of parathion and actively
converted parathion to aminoparathion. Our observations also indicate the
important role that fungal species may play in sequential biotransforraation
of a xenobiotic. Munnecke and Hsieh (1976) alluded to similar findings using
specifically adapted mixed raicrobial cultures for detoxification of para-
thion.
All representative microcosm isolates grown in the presence of Kepone
showed minimal sensitivity to the compound at a concentration of 100 |Jg/l.
Relatively low levels of 14C were assimilated by each isolate during the
seven-day incubation period. Total 14C biotransformation was not noted for
any of the Kepone microcosm isolates. However, the actinomycete isolate
from the methyl parathion microcosm induced a noticeable total 14C biotrans-
formation, suggesting possible xenobiotic biotransformation by this
particular organism. Bourquin et al. (1977) mentioned the possible inter-
ference of methyl parathion degradation by Kepone . While species specific
responses to xenobiotic input are noted in axenic culture, extrapolation of
such data to provide information on the metabolic integrity of an aquatic
system may present an incomplete picture.
90
-------
Table 13. Microcosm-Isolated Species Level Response to Tagged Xenobiotic Addition.
Isolate No.
1 - Fungal
2 - Yeast
3 - Actino.
4 - Bact.
5 - Fungal
6 - Yeast
7 - Actino.
Microcosm C-MP
Source +
Enrichment
MP MS
MAR
CHT
MP MS
MAR
CHT
MP MS
JEN
CHT
MP MS
JEN
CHT
KP
KP
KP
14 14 14
C-KP ATP C Total C
+ Response Assimilation Biotransformation
Enrichment
+++ +++ +++
+-H- ++ ++
+++ +++ +++
+;; *:: *::
MS
MAR +++ +
CHT +++ +
MS - +
MAR +++ +
CHT +++ +
MS
JEN +++ +
CHT +++ +
-------
Table 13. (Continued)
VO
to
Isolate No.
8 - Bact.
1 - Fungal
3 - Actino.
Microcosm UC-MP 14C-KP ATP UC
Source + + Response Assimilation
Enrichment Enrichment
KP MS
JEN +++ +
CHT +++ +
MP MS
MAR +++ +
CHT +++
MP MS
JEN ++ +
CUT +++ +
14
Total C
Bio trans format ion
_
-
-
-
-
-
_
+
+
MP - methyl parathion (500 pg/£)
KP - Kepone (100 (jg/£)
MS - minimal salts
MAR - Martin's agar medium*
JEN - Jensen's agar medium*
CHT - Chitin agar*
- negative response
+ low response (<10%)
++ moderate response (<30%)
+++ high response (>30%)
incubation time - 7 days
incubation temperature - 30 C
*see p. 38
-------
Gas Chromatography Analysis--
Residual sediment samples analyzed for methyl parathion via GC tech-
niques showed close correlations between laboratory and in situ rates at
experimental plots. The latter ranged from 3.7 to 5.2 days for pesticide
half-lives. Microcosm half-life values for methyl parathion. were 3.3 days
(mean treatment determination). Field applications of Kepone were not con-
ducted. However, GC analysis of microcosm sediments showed appreciable,
accumulation levels of Kepone in flow-through systems and negligible loss of
the compound in static microcosm systems, i.e., 92.4% remaining.
CONCLUSIONS
Our studies, using a combination of microbial and enzymatic approaches,
and environmental correlations, support the value of the benchtop microcosm
as an analytical tool. Rates of utilization and breakdown product generation
can be effectively monitored. Furthermore, varying rates of utilization and
biotransformatioEL of two dissimilar chemical toxicants, i.e., methyl para-
thion and Kepone , can be discerned within the microcosm. The rapid disap-
pearance of methyl parathion coincides closely with ^n sjitu results. ATP and
other enzymatic tests provide sensitive indications of initial degradation of
the parent compound, and can be used as early indices for fate analysis.
Axenic flask studies have further confirmed these findings. Control micro-
cosm units have repeatedly reflected a comparable microbial, biomass found in
control field sites and also have exhibited comparable microbial diversity
and specific enzyme levels. Exposed microcosms, statistically identical to
control units, provided reproducible information on initial environmental
effects, as well as data on environmental fate of the target pesticides used
in this study. Variations in microbial biomass, microbial diversity and
specific enzyme level responses were more apparent and precise in exposed
microcosms than in exposed field sites. However, these variations on a
broader scale were indeed noted in field exposed plots. The relative half-
life, T! , of target compounds were of the same order of magnitude in both
microcosm and field studies. Multivariant statistical analysis of all
parameters analyzed in field plots provided a correlation matrix similar, if
not at times identical, to comparable multivariant studies of microcosm
parameters. The concept of the microcosm itself is validated and verified in
that it can provide a straightforward, reproducible, and accurate means of
simulating an ecosystem to generate analytical data on stress and impact of
abiotic and biotic factors.
The importance of monitoring chitinoclasts (chitin-decomposing micro-
organisms) and chitin substrate cycling in terms of energy flow in estuarine
food webs is clearly evident. Use of the microcosm in analyzing the effect
of xenobiotics on chitin transformation provides a valuable "tool" in dis-
cerning early trophic level responses as well as overall environmental impact
by target toxic substances.
It is equally important in validation of the microcosm approach to
establish whether recalcitrant compounds, such as Kepone , behave similarly
in the laboratory microenvironment. A rapid biodegradation rate for such a
recalcitrant would indicate a containerization effect attributable to either
93
-------
incorrect parameter assessment or to the microcosm design itself. As shown
in these studies, Kepone was recalcitrant, with minimal 14C02 expiration.
ATP and specific enzyme systems, i.e., phosphatase and dehydrogenase, sup-
ported this finding, together with lack of significant variations in both
bacterial and fungal components of the microbial biomass.
Certainly, statistical tests, including analysis of variance and multiple
regression techniques, are essential methods to be employed in evaluating
data generated in our studies. Types of data correlations evaluated are
summarized on the following page. Along with the above tests, Duncan's
multiple range test have been used to elucidate microbial/enzymatic responses
of treatment variations. In our conceptual approach of aquatic microcosm
techniques, a logical and economical subsequent step would be mathematical
model interpretation of microcosm results. The systems developed represent
prototype models that closely approximate important critical parameters of
the target environment.
The approaches and experimental data given here should allow development
of a quantitative baseline of major microbial groups and related enzyme
activities for interconnected ecosystems within an ecologically important
drainage basin. Development of information on enzyme/microorganism inter-
actions is relevant to understanding determinants of xenobiotic impact on
productivity in aquatic regions such as the Barataria Basin. Furthermore,
total microbial biomass and species diversity may serve as valid indices of
biodegradable substrate turnover and productivity, and should provide infor-
mation on microhabitat features as affected by the addition of a variety of
toxic substances.
Microcosms provide a means for replacement of possible sensitive micro-
organisms by tolerant species, as well as monitoring sequential biodegrada-
tion processes among important indigenous microbial populations. Fungal and
actinomycete groups appear to be particularly significant in this regard.
The approaches and experimental data given here should permit further devel-
opment of a quantitative "baseline" of major microbial groups together with
relevant enzyme activities for evaluation of ecosystems within larger aquatic
regions. With careful consideration given to those physicochemical and
microbial processes needed to simulate environmental conditions, microcosm
approaches reported here can generate valuable and relevant information on
xenobiotic activities in wetland and aquatic ecosystems.
94
-------
DATA CORRELATIONS
-OVERALL
Phosphatase
ATP
Dehydrogenase
14C02
14C Assimilation
(^Microbial Biomass)
Phosphatase
14C02
Dehydrogenase
I4C Assimilation
^SPECIFIC
ffiacteria
Phosphatase
14CO,
Dehydrogenase
14C Assimilation
Phosphatase
14C02
Dehydrogenase
14C NAG
Assimilation
Chitinoclasts
Phosphatase
ATP
Dehydrogenase
Phosphatase
ATP
Dehydrogenase
Assimilation
.9208
.9375
.9478
.8948
.9140
BAG
CHT
Fungi
.9153
.9371
.9965
.9072
.7571
.9564
.8778
.7434
.9535
.9010
.9061
.5203
.9043
.9146
.8798
.8741
.8462
.8814
.9649
.8372
.9148
.7214
.9967
.7185
.9182
.6556
.7264
.6367
Compound
MP
MP
K
MP
K
MP
K
Guthion
Guthion
MP
MP
MP
K
MP
MP
K
MP
K
95
-------
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the Committee to Review Methods for Ecotoxicology. National Academic
Press, Washington, B.C.
Atlas, R. M. , D. Pramer, and R. Bartha. 1977. Assessment of pesticide
effects on non-target soil microorganisms. Soil Biol. Biochem. 10:
231-239.
Bartha, R., R. P. Lanzilotta, and D. Pramer. 1967. Stability and effects of
some pesticides in soil. Appl. Microbiol. 15:67-75.
Bourquin, A. W. , P. H. Pritchard, and W. R. Mahaffey. 1977. Effects of
kepone on estuarine microorganisms. Develop. Ind. Microbiol. 19:489-497.
Bourquin, A. W., and P. H. Pritchard (eds.). 1979. Microbial degradation of
pollutants in marine environments. EPA 600/9-79-012. 552 pp.
Bollag, J. M. 1972. Biochemical transformation of pesticides by soil fungi.
CRC Critical Reviews in Microbiology 2:35-38.
Bollag, J. M. 1974. Microbial transformation of pesticides. Adv. Appl.
Microbiol. 13:75-130.
Casida, L. E., Jr. 1977. Microbial metabolic activity in soil as measured
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Chan, J. C. 1970. The occurrence, taxonomy and activity of chitinoclastic
bacteria from sediment, water and fauna of Puget Sound. Ph.D. Disser-
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Faust, S. D. 1977. Chemical mechanisms affecting the fate of organic pol-
lutants in natural aquatic environments. Pages 317-366 in I. H. Suffet,
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Giesy, J. P., Jr. (ed.). 1980. Microcosms in ecological research. Technical
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Gillett, J. W. 1980. Terrestrial microcosm technology in assessing fate,
transport and effects of toxic chemicals. Pages 231-249 in R. Hague
(ed.), Dynamics, Exposure, and Hazard Assessment of Toxic Chemicals.
Ann Arbor Science Publ. Inc., Ann Arbor, Mich.
Gillett, J. W. , and J. M. Witt (eds.). 1977. Terrestrial microcosms and
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Hague, R. 1980. Dynamics, exposure and hazard assessment of toxic chem-
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Heuer, B. 1976. Effects of phosphatases on the persistence of organophos-
phorus pesticides in soil and water. J. Agric. Food Chem. 24:611.
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Holm-Hansen, 0., and C. R. Booth. 1966. The measurement of adenosine tri-
phosphate in the ocean and its ecological significance. Limnol.
Oceanogr. 11:510-519.
Hood, M. A. 1973. Chitin degradation in the salt marsh environment. Ph.D.
Dissertation, Louisiana State University, Baton Rouge, La. 158 pp.
Hood, M. A., and S. P. Meyers. 1977a. Microbiological and chitinoclastic
activities associated with Penaeus setiferus. J. Oceanogr. Soc. Japan
33:235-241.
Hood, M. A., and S. P. Meyers. 1977b. Rates of chitin degradation in an
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Hsu, S. C., and J. L. Lockwood. 1975. Powdered chitin agar as a selective
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Jensen, H. L. 1930. Actinomycetes in Danish soils. Soil Sci. 30:59-77.
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Liang, T. T., and E. P. Lichtenstein. 1972. Effect of light, temperature,
and pH on the degradation of azinphosmethyl. J. Econ. Entomol. 65:
315-321.
Martin, J. P. 1950. The us.e of acid, rose bengal, and streptomycin in the
plate count method for estimating soil fungi. Soil Sci. 69:215-233.
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of parathion. Appl. Environ. Microbiol. 31:63-69.
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of Lateolabraux japonicus. Bull. Misaki Mar. Biol. Inst. 19:.l-47.
Orndorff, S. A., and R. R. Colwell. 1980. Distribution and characterization
of kepone-resistant bacteria in the aquatic environment. Appl. Environ.
Microbiol. 39:611-622.
Portier, R. J. 1979. Microcosm studies on the effect of azinphosmethyl
(Guthion) in agriculturally-affected wetlands. M.S. Thesis, Louisiana
State University, Baton Rouge, La. 105 pp.
Portier, R. J. , and S. P. Meyers. 1981a. Analysis of chitin substrate
transformation and pesticide interactions in a simulated aquatic micro-
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Portier, R. J. , and S. P. Meyers. 1981b. Use of microcosms for analyses of
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Pritchard, P. H. , A. W. Bourquin, H. L. Frederickson, and T. Maziarz. 1979.
System design factors affecting environmental fate studies in micro-
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Microbial Degradation of Pollutants in Marine Environments. EPA 600/
9-79-012.
Rao, A. V., and N. Sethunathan. 1974. Degradation of parathion by
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Reissig, J. L., J. L. Strominger, and L. F. LeLoir. 1955. A modified color-
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Seki, H. 1965. Microbiological studies on the decomposition of chitin in
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Sethunathan, N. 1973. Microbial degradation of insecticides in flooded
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adenosine triphosphate assay: conceptions and misconceptions. ASTM
Special Technical Publication 695:99-116.
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for assay of soil phosphatase activity. Soil Biol. Biochem. 1:301-307.
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98
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SECTION 4
IN SITU EFFECTS OF GUTHION ON BACKSWAMP AQUATIC
INVERTEBRATES AND SHALLOW WATER LEAF LITTER DECOMPOSITION
J. W. Day Jr. and F. H. Sklar
INTRODUCTION
Substitute chemicals, like Guthion, move throughout the environment
whereby their total impact cannot be evaluated by tests that deal with only
one species or a single component of the particular environment. The
application of Guthion in this portion of the overall study was in situ, and
experiments were designed to answer two major questions:
a. How are the aquatic invertebrate communities affected by Guthion?
The invertebrate biota was chosen for a number of reasons: 1) they are
important as fish food items and are considered to be ecologically
significant members of the swamp food web; 2) they occupy all trophic
levels; 3) their biomass and activities in general reflect the
environmental conditions. Furthermore, the limited motility of the benthos
makes it less difficult to observe real population changes, based on ease of
sampling.
b. What is the effect of Guthion on swamp litter decomposition rates?
Nutrient recycling, export of inorganic and organic materials, and
incorporation of the latter into sediments, are critical resultants of
wetland decomposition processes that could be adversely affected by
increasing use of pesticides on agricultural lands adjacent to swamps and
marshes. To our knowledge, the effects of pesticides on leaf litter
decomposition rates in wetland habitats have not been examined, although a
number of terrestrial studies have been conducted (Neary and Merriam 1978;
Gottschalk and Shure 1979) with variable results.
Insecticides accounted for nearly half of the 340 x 10 Kg of
pesticides used in the USA in 1964 (Thompson and Edwards 1974). Of this
volume, 31.8 x 10 Kg were organophosphates (Brown 1978). By 1974, the use
of organophosphates had increased to 84 x 10 Kg. Currently, between
1.61-4.27 (x 10 )Kg/yr of the organophosphate Guthion is applied to nearly
17,000 hecatres of sugarcane in the Barataria Basin hydrologic unit
(Hopkinson 1978; Dozier 1978). The majority of these farmlands are adjacent
to freshwater swamps and marshes, thus the need for the tests described here
to evaluate the impact of Guthion runoff on the biota of these latter
localities.
99
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Guthion is not as tightly bound to soil particles as organochlorine
insecticides such as endrin. When a mixture of Guthion and aldrin are added
to a 60 cm column of soil, Guthion readily percolates through the column but
aldrin does not (Lichtenstein et al. 1966). Because of their greater water
solubilities and higher mobility in soils, the organophosphates are not
readily volatilized into the atmosphere and are more likely to enter aquatic
environments via runoff (Westlake and Gunther 1966). Percent runoff can
vary from 8.6% for a 2.8 cm/hr rainfall, to 66% for a 11.2 cm/hr rainfall in
the upper Barataria Basin (Hopkinson 1978). Based on the Hopkinson runoff
model (Hopkinson 1978), maximal Guthion concentration in the runoff after a
1" rain (2.5 cm/hr) would be 328 ppb if Guthion were applied at the usual
dose of 0.85 Ibs/ acre (Dozier 1978). As rainfall increases, the
insecticide is diluted but the percent runoff increases. Assuming all of
the applied Guthion dissolves in rainwater, then the maximal concentration
in the runoff after a 4" rainfall, i.e., 10.2 cm/hr, would be 616 ppb.
When endrin was used on sugarcane, the runoff rate was 5 ppb in runoff
and 1 ppb in groundwater, and was considered to be safe to aquatic life
(Willis and Hamilton 1973; Pionke and Chesters 1973; Lauer et al. 1966). At
that time, the majority of the fish kills in the Barataria basin were
attributed to pesticides (U.S. Army Corps of Engineers 1974). Endrin was
postulated to be the major factor in the extensive fish kills in the lower
Mississippi River in 1960 and 1963, and its runoff is now considered to
represent an environmental hazard (Brown 1978).
The toxicity of Guthion for fish is less than that of endrin, but
greater than endrin for crustaceans, nematodes, and other invertebrates
(Brown 1978; Torgeson 1972). Guthion could, therefore, pose a more indirect
hazard to fish. This chemical, soluble in water at 24 ppm (at 25C) is a
non-systemic phosphor odithioa te or ganophospha te with high mammalian
toxicity, i.e., the acute oral 1,050 for rats is 15 mg/Kg (Eto 1974).
Laboratory measured toxicities to aquatic fauna range from a high of 0.20
ppb for the cladocerans (Daphnia) to a low of 3,300 ppb for channel catfish
(Table 14).
Destruction of any food web component can affect an entire ecosystem.
Attempts to restock insecticide-contaminated rivers with fish often are
unsuccessful because the fish food organisms do not return (Gray 1974).
Subtle biological changes can occur over long periods of time which could
induce permanent ecological changes. For example, methyl parathion affects
prey selection of gulf killifish (Farr 1978) and sediment turnover rates of
lugworms (Bourquin et al. 1979). Malathion produces spinal deformations in
juvenile bluegills at 10 ppb (Eaton 1970). DDT affects avian eggshell
thickness (Anderson and Hickey 1972; Bitman et al. 1970). Mosquito fish
accumulate parathion and produce lethal excreta (Mulla et al. 1966). These
types of changes affect all trophic levels.
100
-------
Table 14. Toxicity of Guthion (ppb) to aquatic fauna
Organism
Brown Shrimp
Amphipoda (Gammarus lacustus)
Cladoceran (Daphnia magna)
Oyster Eggs (Crassostrea virginica)
Clam eggs (Mercenaria mercenaria)
Stovefly (Pternarays California)
Cope
LD Exposure
(ppb) (in Hrs)
1
0.25
0.20
620
860
1.50
48
48
48
48
48
96
Reference
Brown 1978
Sanders 1969
Sanders and Cope
1966
Davis and Hidu
1969
Sanders and
Tadpole (Bufo woodhousii fowleri) 0.13 96
Crawfish (Procambarus clarkii) 100 96
Fish Rainbow trout 14 96
Coho salmon 17 96
Yellow perch 13 96
Bluegill sunfish 22 96
Largemouth bass 5 96
Channel catfish 3,300 96
Fathead minnow 240 96
Carp 700 96
Mosquito fish 78 96
Stickleback 4.80 96
Sheepshead minnow 3 72
Pinfish 10 24
Spot 20 24
1968
Sanders 1970
Baker 1974
Macek and
McAllister 1970
Carter and Graves
1973
Katz 1961
Coppage 1972
Coppage and
Mathews 1974
EXPERIMENTAL
Short term effects of a single exposure to Guthion. Test Series I;
In the fall of 1977, four 48 cm diameter plastic enclosures were placed
in the open area of an impounded swamp. Guthion, dissolved in acetone, was
used to inoculate three enclosures; an equivalent quantity of acetone alone
was added to the control enclosure. Concentrations of Guthion in the
treatment enclosures were 1 ppm, 10 ppm and 25 ppm. Samples of water, mud,
and floating vegetation were taken after 1, 5, and 8 days for analysis of
Guthion. Mud and water samples were stored in dry ice. The floating
vegetation (Lemna s pp. ) and associated invertebrates were sampled by
scooping water and vegetation with a 15 cm diameter corer (Paterson and
Fernando 1971) and slowly pouring the liquid through 4-6 layers of
cheesecloth. The animal/duckweed mixture retained in the cheesecloth was
stored in a 10% formalin rose bengal solution to aid sorting by coloring the
invertebrates a bright red (Masen and Yevich 1967). Material retained by a
500 u sieve was sorted under a dissecting microscope at 2.5 x magnification.
101
-------
A settling experiment was conducted to demonstrate that the fauna
living in the floating duckweed do not remain there after death. Thus, any
species retained in the cheesecloth will represent living specimens
unaffected by short-term exposure to Guthion. Four 1-gallon plastic jugs
were filled with swamp water, duckweed and invertebrates, and two of the
jugs treated with 5 mg of Guthion in 5 ml of acetone while the other two
were treated with 5 ml of acetone alone. Containers were kept at room
temperature (18-24 C) for 48 hr. Differences between number of animals in
the floating vegetation, and on the bottom and sides of the jugs were
recorded for contaminated and control containers. The additional interfaces
supplied by the walls of the containers decreased the number of animals
found in the floating vegetation by 9% (Fig. 45). The lethal dose of
Guthion decreased the number of animals in the floating vegetation by 100%,
all sank to the bottom soon after death. Animals appeared unaffected by the
1 ppt acetone solution.
FLOATING VEGETATATION
SEDIMENT
40 -
3O -
cc
LU
a
cc
HI
CD
20 -
10 -
81%
C
A
P
O
"tf
A
P
D
A B
:ONTROL
t
0%
A B
GUTHION
16
12
cc
LLJ
a.
cc
UJ
CD
5
3
Z
9%
A
CONTROL
100%
B
GUTHION
Figure 45. Effects of Guthion on Settling of Biota of Floating
Vegetation (F.V.).
The data from Test Series 1 begin with Figure 46. The amount of
dissolved Guthion in each of the swamp enclosures decreased very rapidly.
After 1 day, the 25 ppm Guthion concentration in the water, decreased to 1.5
ppia, a 94% reduction. The reduction rates after 8 days for the 25 ppm, 10
ppm and 1 ppm enclosures were 97.6%, 96% and 95%, respectively. As the
Guthion concentrations in the water decreased, concentrations in the
102
-------
7°PPm
25ppm
Guthion Concentrations
In The Water
10ppm
1ppm \ ^ Guthion Concentrations
In The Sediment I Top 10cm I
Figure 46. Changes in Guthion Concentrations in Sediment and Water.
103
-------
Effects of Guthion (1ppm)
on the F.V. Community
Figure 47.
Effects of Guthion (lOppm)
on the F.V. Community
Effects of Guthion (1 and 10 ppm) on the Biota in Floating
Vegetation (F.V.).
104
-------
sediment increased (Fig. 47). Guthion was probably rapidly sorbed to
sediments, unless added at concentrations above solubility. As much as 20%
of the initial concentration was found in the top 10 cm of sediment 24 hr
later. In this sediment Guthion disappeared slowly; seven days after the
first appearance of Guthion in the sediment, the concentration in the
sediment of the 25 ppm, 10 ppm and 1 ppm enclosures was 1 ppm, 0.56 ppm and
0.10 ppm, respectively. The effects of this sediment Guthion on benthic
populations was part of another experimental design (Test Series II). The
floating vegetation community was immediately affected by the presence of
Guthion (Figs. 47 and 48).
Effects of Guthion [25ppm 1
on the F.V. Community
Figure 48. Effects of Guthion (25 ppm) on the Biota in Floating
Vegetation (F.V.)
2
The control enclosure increased in total population, from 11,544/m to
18,584/m in 8 days (Fig. 49). The populations in 1 ppm, 10 ppm, and 25 ppm
enclosures decreased to 680/m , 737/m and 441/m , respectively, in 8 days.
The large population of amphipods (i.e., Hyallela azteca) was eradicated
after 8 days at all concentrations tested. Hydrozoans and zooplankton
tolerated 1 ppm Guthion for the first day; however, by day 8, hydrozoans or
zooplankton were absent. The diptera population (i.e. 1,400/m ) was
eradicated after 5 days at 25 ppm Guthion, but was still found after 8 days
105
-------
at 10 ppm and 1 ppm Guthion.
abundant animals after 8 days.
Gastropods and oligochaetes were the most
CONTROL
Effects of a plastic enclosure
on the F.V. Community (
Figure 49. Effects of Plastic Enclosure on the Biota in Floating
Vegetation (F.V.).
Long term effects of a single exposure to Guthion. Test Series II;
The benthic habitat was sampled with a 20-cm-diameter stovepipe corer,
manually forced into the sediment. All contained materials were removed
with a long handle ladle to a depth of 20-25 cm and poured into a 500 u
sieve. Samples were preserved in a 10% formalin-rose bengal solution.
According to Weber (1973), a corer, is the only quantitative device suitable
for sampling shallow-water benthic habitats. Estimates of standing stock
comparable to those obtained with an Ekman grab can be obtained with a
corer, but with less effort because of the smaller volume of sediment that
requires sorting (Flannagan 1970; Kajak 1971).
106
-------
The benthic population within the 48-cm-d iame ter polypropylene
enclosures was sampled with the stovepipe corer 16 days after inoculation
with Guthion during summer 1977. Concentrations of Guthion in the
enclosures were 0.01 ppm, 0.1 ppm, 1 ppm, and 10 ppm. An equivalent
quantity of acetone alone was added to the control enclosure. The long
period between inoculation and sampling allowed for decomposition of any
invertebrates killed by the Guthion. A dissecting microscope at 2.5
magnification was used for sorting.
In spring 1978, another long-term experiment was designed to study the
rate of recovery of the invertebrate community after a lethal dose (i.e., 25
ppm) of Guthion. A study to determine effects of a sublethal continuous
exposure of Guthion was conducted at the same time (outlined in Test Series
III). A series of 34, 1-m high, 20-cm diameter galvanized steel stovepipes
were used as enclosures. Twelve of the enclosures were treated with 25 ppra
Guthion and twelve with a equivalent quantity of acetone. Every 6-7 days
all the floating vegetation from the enclosures, from each treatment, was
removed with a ladle, stored on cheesecloth, and that retained by a 500 p
sieve was preserved in 10% formalin-rose bengal solution. The sampling
schedule is given in Table 15. Sorting was done with a dissecting
microscope at 2.5 magnification.
Another long-term experiment, May through September 1979, was designed
to study the effects of Guthion on leaf litter decomposition rates. Tupelo
and ash leaves were dried at 30 C for 7 days; 5 grams of leaves were placed
in 20 x 20 cm nylon bait bags with a 1.6 mm square mesh. Fifty-four of
these litter bags were soaked for 24 hrs. in each of the following
concentrations of Guthion-t rea t ed water, 100 ppb, 1 ppm and 100 ppm.
Fifty-four bags were soaked in an equivalent amount of acetone-treated
(i.e., 2 ppm) water. Treatments were spaced a minimum of 30 M apart in a
tupelo stand within the impounded swamp. Bags were hung from nylon cord and
spaced approximately 60 cm apart. Six samples of nine replicates were
collected from each treatment through the course of the experiment.
Amphipods, were particularly reduced; the control enclosure had 350/0.0314
m , the 0.01 ppm Guthion enclosure, only 5 animals/0.0314 m . Freshwater
clams were present in all treatments except for the 0.01 ppm enclosure.
Aquatic worms (Plesiopora) were highest in the 0.01 and 0.1 ppm enclosures,
but completely absent from the 1 and 10 ppm enclosures. Although nematodes
were not counted, their absence was noted from all treatments. The control
enclosure had one less taxa than the surrounding swamp and the density of
invertebrates was very similar.
107
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Table 15. Sampling Schedule for che Test Series II (April-June, 1978)
and III (June, 1978)
Treatment
Control
25 ppm
10 ppb
Natural
Swamp
1 2
Acetone
Inoculated S
Acetone &
Guthion- S
Inoculated
Acetone &
Guthion- S
Inoculated
S
Period of
(in
8 15
S S
S S
Exposure
Days)
22 30 36 43 50
S S
S S
R S&R R S&R R S&R R
S
S S
57
S
S
S
S
S= Sample the floating vegetation; R= re-inoculation with Guthion.
The invertebrate population from the pesticide treatments sampled 16
days after inoculation averaged only 9% as dense as the control or natural
swamp community. The results of this experiment (Table 16) indicate smaller
numbers of amphipods, dipterans, and nematodes in the Guthion enclosures.
Table 16: Effects of Guthion on Back-Swamp Invertebrates
(Sediment plus Floating Vegetation)
Number Per Sample (20-cm diameter corer)
TAXA
Amphipoda
Heterodonta
Odonata
Diptera
Plesiopora
Nematoda
Basoramatophora
Isopoda
Collembala
Hemiptera
Ephemeroptera
Total
Natural
Swamp
335
3
1
3
4
Present
1
347
Control
350
2
7
9
Present
1
369
.01 ppm
5
2
26
Absent
8
1
1
43
0.1 ppm
6
2
37
Absent
8
2
55
1 ppm 10 ppm
9 7
1 5
Absent Absent
1
10 13
The results of the spring 1978 experiment to measure recovery rates are
given in Tables 17 and 18. The population of invertebrates living in the
108
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duckweed from control and treatment enclosures decreased with time. The
experiment could not be continued beyond May 1978 due to death of the
enclosed floating vegetation. After one week, the invertebrate density was
reduced by 99% and 82% for the 25 ppm enclosures and 82% for the control
enclosure. After one month, the invertebrate density for all enclosures
averaged only 3% of the density recorded at the start of the experiment.
Table 17. Test Series II. Use of Stovepipe Enclosures
(25 ppm Guthion)
Taxa
Day-
Date-
0
4/19/78
6
4/26/78
13
5/3/78
28
5/18/78
B
Totals
677
Number per .03 m
Amphipoda
Oligochaeta
Diptera
Basommatophora
Coleoptera
Odonata
Hemiptera
Collembola
Lepidoptera
Tricladida
Nematoda
Zooplankton
9
520
19
126
3
P
P
1
420 4
16
42 2
1
1
2
P A
P A
6
1
1
1
A
A
6
10
11
4
A
A
2
13
11
1
A
A
1
1
14
3
2
A
A
1
4
4
8
3
A
A
483
9 31
27 21
20
Note P = Present; A = Absent
109
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Table 18. Test Series II - Use of Stovepipe Enclosures
(Control)
TAXA
Day-
Date-
0
4/19/78
6
4/26/78
13
5/3/78
28
5/18/78
B
Number per .03
Amphipada
Plesiopora
Diptera
Basommatophora
Rhynchobdellida
Ephemeroptera
Coleoptera
Neuroptera
Hemiptera
Odonata
Acari
Lepidoptera
Collembola
Araneida
Cyprindonti£ormes
Zooplankton
4
300
145
2
1
1
P
Totals 453
12
340
104
15
1
2
2
1
1
1
P
479
16
89
10
1
8
5
1
P
130
1
30
1
1
8
1
P
42
1
1
25
3
27
1
3
A
61
1
21
1
12
2
1
A
38
1
1
5
1
2
2
1
A
13
5
2
1
2
1
A
11
Note P = Present;
Absent
Leaf litter weight loss, due to decomposition during the summer of
1979, was not affected by Guthion (Fig. 50). The 1 ppm Guthion-treated
leaves appears to have a greater decomposition rate, however, the rate of
weight loss for all treatments, including the control, was not significantly
different throughout the course of the entire experiment. On the average,
43% of the leaf litter decomposed in 127 days. This represents a 16.9 mg
dry wt. loss/day during the summer months. No invertebrates were found in
any of the litter bags.
Effects of continuous exposure to Guthion (0.01 ppm). Test Series III;
Ten stovepipe enclosures were re-inoculated with Guthion each week over
a four week period, to a concentration of 10 ppb. The original design
called for an eight week experiment (Table 19) but death of the floating
vegetation invalidated further sampling. The sampling technique was the
same as that used in the previous recovery rate experiment of Test Series
II.
110
-------
.
0 01
10 ppm Guthion
100 ppb Guthion
Control
Figure 50.
20 40 60 80 100 120 140 Days
May- June July Aug.- Sept. Months
Effects of Guthion on Leaf, Little Weight Loss Due to
Decomposition.
Ill
-------
The results of the experiment are given in Appendix A.
The invertebrate population living in the floating vegetation and not
enclosed in a 20 cm diameter stovepipe, ranged during the 4 week experiment
from 517 individuals/0.03 m2 to 1765 /0.03 m (Table A-l). Concurrently the
population in the control enclosures steadily decreased from 466
individuals/0.03 m2 to 12 /0.03 m2 (Table A-2). The number of taxa
decreased from 11 to 8. The population in the 0.01 ppm enclosures (Table
A-3) decreased only slightly more than that in the control enclosures.
Density decreased from an original average of 631 individuals/0.03 m to a
final population of only 9 /0.03 m . The number of taxa decreased from 13
to 6.
Aquatic worm density was most affected; a population of 300-500
i ndi vidua Is / 0 . 03 m was reduced to zero in both control and treatment
enclosures.
A summary of all three treatments is given in Table 19. The average
density for the 0.01 ppm Guthion treatment was slightly higher than that for
the control, but was not significantly different. However, both were
significantly different from the natural swamp situation. The diversity was
statistically the same for all three situations, although the 0.01 ppm
Guthion treatment averaged one less taxon than the natural swamp. The
distribution of invertebrate density, i.e., evenness, was the same for all
treatments.
Short term comparison of Guthion with Furadan, Methyl Parathion and Azodrin.
Test Series IV:
In October, 1978, four insecticides were tested on floating vegetation
invertebrate communities contained within 20-cm diameter stovepipe
enclosures over a 48 hr period. The insecticides were: Guthion, Furadan (a
carbonate used on rice), methyl parathion (an organophosphate used on
soybeans) and Azodrin (a systemic organophosphate used on cotton.) Each
pesticide was tested at two concentrations; 1 ppb and 10 ppb. Three to five
replicate enclosures were used for each treatment. Control enclosures were
not treated with acetone. Stovepipe enclosures were placed randomly in the
impounded swamp in October, 1978, and the entire floating vegetation, plus
water column from each enclosure, was separated by a 500 ja sieve 72 hr
later. Samples were stored in 10% formalin-rose bengal and sorted at 2.5 x
under a dissecting microscope.
The results from this experiment are tabulated in Appendix B.
Replicates varied greatly, with coefficients of variation ranging from 4.6
to 78%. While the density of invertebrates in the floating vegetation in
the surrounding swamp did not change during the course of the experiment,
densities in the enclosures were greatly reduced. Those in the control
enclosures were less than the natural swamp, i.e., 68/0.016 m and 225
individuals/0.016 m , respectively. The average density of the insecticide
enclosures ranged frop a low of 17/0.016 m with 10 ppb methyl parathion to
a high of 34/0.016 m at 1 ppb Furadan. The dominant taxon in the control
112
-------
and natural swamp treatments were Amphipoda and Oligochaeta. Azodrin
treatments were completely devoid of oligochaets and very few (maximum
4/0.016m ) were found in the other three insecticide treatments. Amphipoda
populations ranged from 4 individuals/.016 m at 10 ppb Guthion to 27/0.016
m at 1 ppb methyl parathion. The average density of Amphipoda in the
control and natural swamp samples was 33/0.016 m and 56/0.016 m~,
respectively. The population of damselflies and dragonflies (i.e. Odonata)
appeared unaffected.
Table 19. Average density, diversity and evenness of animals found in
the floating vegetation after 30 days of continuous exposure
Natural Swamp
n = 6
Control
n = 8
.01 ppm
n = 6
Natural Swamp
n = 6
Control
n = 8
.01 ppm
n = 6
DENSITY
2
(Number per .03 m )
X S.E. MIN
992
153
225
260
70
130
386
11
8
DIVERSITY
( p. Inp )
X S.E. MIN
1.29 .09 .92
1.13 .11 .73
1.2 .13 .77
EVENNESS
Diversity
MAX
1850
479
700
MAX
1.5
1.7
1.6
Number of
Taxa
14
15
13
(Maximum Diversity)
X
Natural Swamp .61
n = 6
Control .61
n = 8
.01 ppm .67
n = 6
S.E.
.04
.07
.10
MIN
.47
.38
.33
MAX
.72
.89
.90
113
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The average density and evenness of each treatment are shown
graphically in Fig. 51. No differences were noted among the four
insecticides. Methyl parathion at 10 ppb, had the lowest density of
invertebrates and Furadan at 1 ppb had the highest. The evenness of the
invertebrate population distribution was highest in the insecticide
treatments and lowest in the natural swamp.
1.00
.90
.80
-------
CONCLUSIONS
Containment of a parcel of swamp water, by a 20 cm stovepipe for
periods greater than 2 weeks, has an adverse effect upon the community
contained within. The effect of small field enclosures can mask effects due
to the insecticides. Floating vegetation within the 20 cm diameter
stovepipe enclosures was dead after 30 days. Thus, the recovery rate of the
community to 25 ppm Guthion (Test Series II) could not be evaluated.
Similarly, conclusions cannot be drawn as to the effects of continuous
exposure since density of the control group and treatment group decreased at
approximately the same rate. Conclusions can only be made from those
experiments where the control replicates were found to be unaffected by
enclosure. They include the experiments that used the larger 48 cm
enclosures and all short-term experiments.
The complex community of swamp invertebrates is composed of crawfish
( Procambarus), amphipods (Hyallela azteca), aquatic worms (Haplotaxida),
snails (Basoramatophora), diptera larvae (Chironoraidae, Ceratopogonidae,
Culicidae), springtails (Collembola) , aquatic caterpillars (Synclita
accidentalis) , various beetles (Haliplidae, Dytiscidae, Noteridae,
Hydrophilidae, Helodidae, Curculionidae), predacous Odonata (Anisoptera,
Zygoptera), Hemiptera (Corinadae, Notenectidae, Naucoridae, Belastomatidae
Mesovelliidae), nematodes, zooplankton (Cladocera), flatworms (Tricladida),
leeches, and spiders. These contribute to the stability, integrity and
function of swamp ecosystems by transferring and regulating the flow of
energy and materials. Guthion can, at concentrations as low as 1 - 10 ppb,
alter the density and distribution of the community in the water column and
in the flooded soil. Crustaceans and insects were the most sensitive to
Guthion in the water, while molluscs and annelids were the most tolerant.
The aquatic worm Aulophorus spp. and the Basoramatophora snails tolerated 25
ppm Guthion for short periods and 0.1 ppm for long periods. This is in
agreement with the work of Harman (1974) who found snails generally
unaffected by insecticides. In fact, snail populations can increase after
insecticide treatment (Pimentel 1971). Annelids also exhibit natural
resistance to insecticides (Ferguson 1969; Davey 1963). Our data presented
here show the resistance of the annelids to Guthion over short periods with
decreasing tolerance over extended time. Brown (1978) reported that Guthion
was harmless to the terrestrial worm Eisenia spp. at 5.6 Kg/ha. The death
of the floating vegetation and annelids in the control enclosures of Test
Series III and IV suggests a sensitivity to material or conditions other
than Guthion. Brinkhurst and Cook (1974) noted that aquatic worms are
influenced more by the quality and quantity of organic matter than by the
chemical parameters of the water or sediment. According to Anderson et al.
(1965), when organic matter is high and there is a scarcity of fauna other
than worms, the existence of toxic substances is suggested.
The worms were not resistant to all of the organophosphates tested.
The 10 ppb methyl parathion treatment and all raonocrotophos treatments of
Test Series IV were completely devoid of oligochaetes. Parathion has been
reported to have no effect on earthworms at 8 Ib/acre in England, although
toxic at 8 Kg/ha in Germany (Brown 1978). The fact that monocrotophos is a
115
-------
systemic could contribute to its toxicity. Other organophosphorus systeraics
have been found to eliminate earthworms when applied at normal agricultural
rates (Edwards et al. 1967).
The population density of amphipods (H. azteca) was reduced more than
any other group of invertebrates by treatment with Guthion. In Test Series
I, a concentration of 1 ppm reduced the population from 9,000/m to zero in
24 hr. In Test Series II a concentration of 0.01 ppm reduced the population
from approximately 11,000/m to 160/m over a period of 16 days. The short
term results of test Series III and IV showed the same trends. The amphipod
population was reduced by approximately 50% in the presence of 1 ppb
Guthion, methyl parathion, Furadan, or Azodrin. Farlow (1976) found H_.
azteca to be very susceptible to field application of Dimilin and
recommended its use as an indicator organism to determine the impact of
mosquito control chemicals.
Swamp amphipods in floating mats of Lemma spp. can reach densities of
60,000/m (Sklar unpublished data). Densities of such magnitude imply that
amphipods play a dominate role in the cycling of materials, and their
reduction due to insecticide runoff could have significant ecological
consequences. For example, in a stream contaminated with BHC, an
organochloride used on sheep, a thick layer of benthic algae Cladophora and
Spirogyra, developed due to the destruction of the herbivorous Gammarus
amphipods (Hynes 1961). In terrestial systems, removal of the dominate pest
herbivores results in the elimination of most predators and parasites,
culminating in an even greater infestation of some other undesirable pest
species (Pimentel 1961; Brown 1978). Hulbert et al. (1972) found that the
organophosphorus chlorpyrifos applied to shallow freshwater ponds created an
ecological imbalance favorable to the very insects it was meant to control.
We need to know what ecological changes will result from low level
contamination of amphipod-rich wetlands.
Aquatic insect populations were very resistant to short-term exposure
to organophosphates. After 8 days at 10 ppm Guthion, a population of
diptera larvae still remained. In Test Series IV, the total number of
insects was as high in the 1 ppb treatment enclosures as in the unenclosed
swamp (see below).
Natural Swamp - 28 Methyl Parathion - 22
Control - 20 Carbofuran - 26
Guthion - 30 Monocrotophos - 34
The total number of insect orders was highest in the 10 ppb Azodrin
treatment and included one species of Ephemeroptera (i.e., mayflies). The
mayflies have generally been considered to be very sensitive to pollutants.
Their appearance in organophosphate-treated water in Test Series II and IV,
and in the data of Roback (1974), indicate that as a generality, this is not
true. Roback found that the majority of aquatic insects are not good
"indicator organisms" because of their ability to tolerate a broad range of
water chemistry and physical conditions. However, our data indicate that
116
-------
their ability to tolerate Guthion decreases with time. Not a single insect
species was found at 100 ppb after 16 days in Test Series II.
Toxicity of an insecticide depends on its concentration and its
distribution through the water column. The more insoluble the chemical the
more rapidly it is lost from the water (Thompson and Edwards 1974). Beynon
et al. (1971), however, reported that even the comparatively soluble
organophosphorus insecticide, chlorfenvinphos, was rapidly deposited on
bottom sediments. Guthion was found to decrease very rapidly from the
water, much of it appearing in the sediments within 24 hr. Edwards et al.
(1964) demonstrated that the susceptibility of diptera larvae to TDE was due
to the close association of these animals with the organic sediments on
which the insecticide was absorbed. The effect of Guthion on the benthos
was demonstrated in Test Series II. Nematodes were not found in any of the
Guthion treatments, even at concentrations as low as 10 ppb. The natural
neraatode density was not calculated, only the presence or absence of the
species retained by a 500 >i sieve. As a result, the effectiveness of
Guthion as a nematicide in cypress tupelo swamp sediments is unknown. The
sensitivity of nematodes to Guthion is not surprising since other
o r ganophospha t es (i.e., Parathion, Furadan, Dasonit) have been found to
decrease nematode populations and are sometimes used as soil nematicides
(Brown 1978; Torgeson 1972).
The other benthic invertebrate, the clam (Sphaerium spp.), was
unaffected by Guthion at the concentrations tested, i.e., 10 ppb to 10 ppm.
This too is not surprising since mortality caused by pesticides in mussels
and clams has not been demonstrated (Brown 1978; Fuller 1974) even though
these animals can concentrate .insecticides at levels greatly in excess of
natural concentrations in water (Miller et al. 1968). Mainly, the immature
stages are affected by organophosphates. The LD.-Q of Guthion for the
larvae of the hard clam, Mercenaria mercenaria, is 860 ppb, and 620 ppb for
the eggs of the american oyster, Crassostrea virginica (Davis and Hidu
1969). The ecology of swamp bivalves might be affected by Guthion as a
result of its ability to interfer with progeny and not its direct effect
upon adults.
Guthion did not affect the rate of ash-tupelo litter decomposition.
All treatment results were fit into a daily linear regression model with the
following results:
Untreated 0.1 ppm 1 ppm 10 ppm 100 ppm
Correlation 0.93 0.87 0.94 0.90 0.89
Coef. (r)
Daily wt. loss 14.1 12.4 15.0 14.1 13.3
(nig)
No trends were observed and significant differences were not obtained. It
has been noted that, in general, organophosphate insecticides do not affect
bacterial populations at practical application levels (Brown 1978).
117
-------
Parathion applied at 10 Lb/acre had no effect on the numbers of soil fungi
or bacteria (Eno 1958). In some instances, the microbial activity is
stimulated by applications of organophosphates (Tu 1970). There is a need
to expand this type of experiment to other wetland types, especially in
areas where microarthropods and annelids play an important role in
comminuting dead plant material, making it more available for bacterial
decomposition. When soil raicroarthropods were eliminated by naphthalene,
the leaf litter decomposition rate was reduced by as much as 25% (Brown
1978). In the current study, the sediment is relatively devoid of micro and
macrofauna that could aid in decomposition. Other swamp and marsh habitats
have significant numbers of arthropod decomposers (Sklar and Conner 1979).
Before it can be said conclusively that Guthion does not affect aquatic
decomposition processes, more litter decomposition experiments must be
conducted in other major wetland soil types.
The aforementioned can be summarized below:
Guthion added to backswamp aquatic environments is rapidly removed from
the water column, much of it appearing in the sediments within 24 hr.
Guthion concentrations, ranging from 0.1 ppm to 100 ppm has no effect upon
the raicrobial degradation rate of ash-tupelo litterfall in the aquatic
environments of a permanently flooded swamp. The density and distribution
of backswamp aquatic invertebrates from a permanently flooded cypress-tupelo
swamp is altered by contact with the insecticide Guthion at concentrations
ranging from 0.01 ppm to 25 ppm.
Annelids and molluscs are the most tolerant backswamp aquatic
organisms. They can survive high dosages of Guthion (i.e., 1-25 ppm) for
periods greater than one week when the insecticide is injected into habitat
enclosures and allowed to degrade "naturally". In situ survivability of
these animals to long term exposure is still unknown. The dominant
backswamp invertebrate, the amphipod, Hyallela azteca, is the most sensitive
aquatic organism. Large populations (e.g., 9,000/sq. m) can be reduced to
zero in 24 hr. at Guthion concentrations greater than 1 ppm.
Aquatic insect populations were able to tolerate a broad range of
insecticides (i.e., Guthion, methyl parathion, carbofuran and
monocrotophors) and dosages (i.e., 0.01-10 ppm) for short periods of time.
Their natural genetic variability coupled with their high rate of
reproduction, give aquatic insects, like their terrestrial counterparts, the
ability to develop resistant populations.
118
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translocation of diazinon-C and parathion-p off a model cranberry
bog and subsequent occurrence in fish and mussels. Trans. Amer. Fish.
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Mulla, M. S., J. 0. Keith, and F. A. Gunter. 1966. Persistence and
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121
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Patersoa, C. G. , and C. H. Fernando. 1971. A comparison of a simple corer
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123
-------
CONCLUSIONS
The procedural methodologies developed in this multidisciplinary tiered
approach to analysis of the environmental effect and fate of selected xeno-
biotics in agriculturally affected wetlands provides a useful "tool" for
further evaluation of other EPA priority-pollutant classes of compounds,
i.e., phenols, phthalate esters, chlorobenzenes. In addition, information is
contributed with regard to modifying current agricultural practices and to
furthering development of coastal zone management strategies. The continuing
formulation of valid predictive models will generate needed information on
the fate (and effects) of a variety of toxic chemicals in fresh, brackish,
and saline ecosystems. Furthermore, data should be obtained on microbial and
related physicochemical transformation processes in_ situ and under laboratory
conditions that affect the environmental fate of toxic chemicals.
In the projected evaluation of other EPA priority pollutants, the com-
bination of field and laboratory methodology, the latter employing a variety
of microbial, benthic, enzymatic, and physicochemical transformation activ-
ities, is the preferred approach. This allows for on-going formulation of a
valid computer data base program for statistical and numerical model analysis
to standardize laboratory methods and to establish a ranking system for other
classes of toxicants. In continuing these evaluations of xenobiotic impact
in economically and biologically important wetland systems, we feel addi-
tional information should be garnered in the following areas:
(1) The further identification of relevant abiotic processes, parti-
cularly Eh/pH, which complement biotic transformations of "target"
compounds.
(2) The role of ecologically-significant metabolizable substrates,
i.e., chitin, which provide an active microbial and enzymatic pool
for both acclimation to, and subsequent enzymatic adaptation of,
"target" xenobiotics.
(3) The ultimate bioavailability of selected xenobiotics to the afore-
mentioned microbial pool and estimation of minimal, or threshold
levels, at which biotransformation processes can proceed, and over
what duration.
Biotransformation test protocols must include an evaluation of sorption
reactions affecting rate(s) of microbial transformations. In addition, there
needs to be a monitoring of microbial/benthos at varying Eh, pH, and salinity
levels for comparative purposes.
124
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Particular attention should be given to the structural polysaccharide
chitin, among the most prevalent biodegradable polymer in marine and estua-
rine environments. It is recognized that turnover of chitin is a critical
key transformation process in the functioning of trophic food webs. The role
of chitinoclastic microorganisms in bioconversion of this carbon/nitrogen-
containing substrate needs careful examination. Analyses of microbial
ecology and the extant chitinoclast population requires careful study of
chitinolytic processes on fate of a variety of toxic substrates. Similarly,
correlations between the latter compounds and chitin mineralization warrants
thorough investigation.
Analyses of chitinase elaboration as a major activity factor and its
relationship to toxic substances should reveal noteworthy data on environ-
mentally-significant surface phenomena or adsorptive effects. Toxic
substances may physically block active sites on the chitin substrate, thus
inhibiting initiation of essential breakdown processes. Products resulting
from such activities, i.e., N-acetyl-glucosamine and glucosamine, all enter
into the various physiological determinants of the microbial community.
Correlations between these processes and toxic compound fate are germane to
elucidation of the effect of environmental and chemical perturbances on
transformation of such ecologically significant substrates as chitin.
The physicochemical experiments conducted during the course of this
investigation have provided particularly useful information on mobilization
or immobilization processes occurring in soil environments. Implications as
to the development, migration, and permanency of aerobic/anaerobic layers in
both wetland flooded soils and adjacent agricultural soils with regard to
pesticide environmental fate were presented. In recognization of the impor-
tance of oxidation-reduction conditions on the environmental chemistry of
pesticide compounds, agricultural practices near sensitive wetland systems
should be examined in terms of such physicochemical processes. Possible
modifications should be designed to consider the following in overall agri-
cultural practices:
(1) The implementation of draining vs. non-draining practices, parti-
cularly in rice growing regions, to take advantage of oxidation-
reduction variations to regulate pesticide residence time.
(2) The monitoring of pH effects based on individual crop needs as well
as pesticide environmental fate.
(3) The careful analysis of wetland soils adjacent to agricultural
areas, with regard to their various physical and chemical
properties to ascertain accumulation effects and residence time
phenomena of pesticides.
Additional research is needed on mobility and degradation of toxic compounds,
especially related to runoff activities. Incorporation of such data into
appropriate data information bases, in an effort to further refine EPA's
agricultural runoff model, is anticipated.
125
-------
The overall purpose of these investigations has been to incorporate
experimental data and overall experimental design, primarily developed for
federal and state monitoring and management strategies, into environmental
impact and hazard assessment of toxic chemicals in waterways and wetland
systems. More information is needed on aquatic soil/sediment processes that
are critical in productivity and food web dynamics. In continuing to pursue
these aforementioned objectives, the overall conceptual approaches involved,
and the ultimate use of information obtained, is illustrated below.
MONITORING IN SITU MICROBIAL
ENZYMATIC PROFILES
LABORATORY/
MICROCOSM
APPROACHES
HAZARD
ASSESSMENT
OTHER PERTINENT
INFORMATION
STATE AGENCIES
EVALUATIONS/RESPONSES
WETLAND MANAGEMENT
DECISIONS & PRODUCTIVITY
ASSESSMENT
AI MICROCOSM STUDIES ON FATE & TRANSPORT O F TOXIC
CHEMICALS.
81 MICROCOSM STUDIES ON EFFECT OF TRANSPORT BREAK-
DOWN PRODUCTS ON KEY SUBSTRATES: CHITIN,
CELLULOSE.
Cl MONITORING MICROBIAL INTERACTIONS IN WATER/SOIL
ISEDIMENTI/SUBSTRATE PHASES.
Dl EVALUATION OF POSSIBLE SALINITY EFFECTS.
The implementation of effective and optimal management practices for
maintaining agricultural productivity must include an economic analysis of
cost and energy use. Nevertheless, such practices should be deleterious
adjacent to highly productive wetland areas. Development of economically
efficient strategies for managing and monitoring agricultural pollution need
to be based on use of the best agricultural and wetland environmental science
and technology. Procedural wetland research efforts, such as presented here,
must continue to resolve both compatability as well as possible conflicts
between hydrologically-linked habitats within a major aquatic ecosystem.
126
-------
BIBLIOGRAPHY
Portier, R. J. 1979. Microcosm studies on the effect of azinphosmethyl
(Guthion) in agriculturally affected wetlands. M.S. thesis, Louisiana
State University, Baton Rouge. 105 pp.
Jones, R. D. , and M. A. Hood. 1980. Effects of temperature, pH, salinity,
and inorganic nitrogen on the rate of ammonium oxidation by nitrifiers
isolated from wetland environments. Microb. Ecol. 6:339-347.
Jones, R. D. , and M. A. Hood. 1980. Interaction between an ammoniura-
oxidizer, Nitrosomonas sp., and two heterotrophic bacteria, Nocardia
atlantica and Pseudomonas sp.: A note. Microb. Ecol. 6:271-275.
Portier, R. J., and S. P. Meyers. 1981. Chitin transformation and pesticide
interactions in a simulated aquatic microenvironmental system. Dev.
Indust. Microbiol. 22:543-555.
Portier, R. J. , and S. P. Meyers. 1982. Use of microcosms for analyses of
stress-related factors in estuarine ecosystems. In Wetlands Ecology and
Management (B. Gopal, R. E. Turner, R. G. Wetzel, and D. F. Whigham,
eds.). Intern. Scientific Publ. Jaipur, India. pp. 375-387.
Portier, R. J., and S. P. Meyers. 1982. Monitoring biotransformation and
biodegradation of xenobiotics in simulated aquatic raicroenvironmental
systems. Dev. Indust. Microbiol. 23: in press.
127
-------
APPENDIX
Table A-l. Test Series III: Various Taxa of
Animals in Natural Swamp
TAXA 4/19/78 4/26/78 5/3/78
A B A B A B
5/
A
18/78
B
Individuals/0.03 m2
Amphipoda 37
Oligochaeta 290
Diptera 69
Basommatophora
Tricladida
Ephemeroptera
Coleoptera 5
Hemiptera 3
Odonata 3
Collembala
Lepidoptera 3
Homoptera
Rhynochobdellida
Araneida ^A
Zooplankton P
Total 410
481
345
88
40
5
11
1
1
P
981
352
524
658
274
3
7
8
20
1
3
P
1850
258
600
561
223
3
1
14
15
3
1
P
1679
157
101
77
19
5
13
11
3
P
386
334
154
90
35
3
19
13
P
648
* **
Sampled with F.V. Scoop; P = Present
128
-------
Table A-2. Test Series III: Various Taxa of
Animals Present (Control)
TAXA 4/19/78
A B
Amphipoda 4
Oligochaeta 300
Diptera 145
Basommatophora 2
Rhynchobdellida
Ephemeroptera
Coleoptera
Neuroptera
Hemiptera 1
Odonata
Acari
Lepidoptera
Collembala
Araneida
Cyprindonti-
forrnes^ 1
Zooplankton P
Totals 453
12
340
104
15
1
2
2
1
1
1
P
479
4/26/78
A B
5/3/78
A B
2
Individuals/0.03 m
1 1
16
89
10
1
8
5
1
P
130
1
30
1
1
8
1
P
42
1
25
3
27
1
3
A
61
21
1
12
2
1
A
38
5/18/78
A B
1
1
5
1
2
2
1
A
13
5
2
1
2
1
A
11
P = Present; A = Absent
129
-------
Table A-3. Test Series III: Various Taxa of Animal
Present (0.01 ppm Guthion)
TAXA
4/19/78 4/26/78 5/3/78
A B A B A B
5/18/78
A B
Individuals/0.03 m2
Amphipoda
Oligochaeta
Diptera
Basoauna-
tophora
Tricladida
Ephemeroptera
Coleoptera
Hemiptera
Odonata
Collembala
Araneida
Homoptera
Lepidoptera .
Zooplankton
Totals
3
556
46
67
2
5
2
3
8
2
P
700
28
400
39
79
2
4
5
1
1
1
1
P
561
1
3
4
13
1
1
1
A
24
4
9
22
2
6
3
I
A
47
1
1
1
4
1
1
A
9
1
3
4
A
8
P = Present; A = Absent
130
-------
APPENDIX B
Results of Test Series IV Expressed
_ as density per unit area
(H = Average Diversity. V = Average evenness.
C.V. = Coefficient of Variation.)
Control
TAXA (order)
A
B
C
X
S.D.
Individuals/0.016 m2
Amphipoda
Oligochaeta
Diptera
Basommatophora
Odonata
Lepitoptera
Coleoptera
Hemiptera
Cyprinodentif ormes
Zooplankton
TOTAL
-£p. Inp
(Diversity)
H = 1.29
H/!} max
(Evenness;
25
3
1
7
2
1
1
1
41
1.3
.63
53
48
3
16
7
1
1
. Yes
129
1.3
.67
21
2
1
7
2
1
34
1.27
.71
33
18
2
10
4
1
68
C.V.
S.E.
17
26
1
5
3
0
53
= 78%
= 30.6
.67
131
-------
TAXA (order)
Natural Swamp - 10/16/78
ABC
S.D.
Individuals/0.0113 m2
Oligochaeta
Amphipoda
Basoramatophora
Odonata
Tricladida
Hemiptera
Hydroida
Diptera
Nematomorpha
Lepidoptera
Coleoptera
Zooplankton
TOTAL
-Ip Inp
H - 1.T5
S/H max
V = .69
TAXA (order)
128
59
32
5
44
1
269
1.33
.74
Natural
A
101
57
20
1
25
1
5
Yes
210
1.32
.68
Swamp -
B
98
51
24
1
22
6
1
1
1
205
1.4
.64
10/19/78
C
109
56
25
2
30
4
228
C.V.
S.E.
X
17
4
6
2
12
3
-~
36
= 16%
= 20.6
S.D.
Individuals/0.0113 m2
Amphipoda
Oligochaeta
Diptera
Tricladida
Basommatophora
Odonata
Hemiptera
Coleoptera
Ephemeroptera
Zooplankton
TOTAL
-Ip Inp
H i 1.1*
H/H mav
51
64
5
8
9
1
Yes
138
1.22
.68
64
203
7
7
13
3
2
1
Yes
300
1.0
.48
53
142
9
16
12
4
1
1
Yes
238
1.21
.58
56
136
7
10
11
3
1
225
C.V
S.E
7
70
2
5
2
2
1
~
82
. = 36%
. = 47.2
132
-------
TAXA (order)
Amphipoda
Oligochaeta
Diptera
Odonata
Basommatophora
Hemiptera
Cyprinodentif ormes
Zooplankton
TOTAL
"'Pi In ^
H = 1.32
i/H max
V = .80
TAXA (order)
Odonata
Amphipoda
Diptera
Basommatophora
Oligochaeta
Homoptera
Hemiptera
Collembola
Zooplankton
TOTAL
-'Pi lnPi
H = 1.43
-/H max
V = .87
24
10
1
9
9
2
Yes
31
1.37
.85
30
11
8
4
2
Yes
25
1.22
.88
Guthion (1 ppb)
Sample No.
18 4
Individuals/0
5 26
3
5 3
2 1
5 5
1
1
18 39
1.47 1.12
.91 .63
Guthion (10 ppb)
Sample No.
16 12
Individuals/0
4 8
19 4
2 2
9 3
4
1
1
Yes
38 19
1.32 1.53 1.
.82 .85
.016 m2
8
.016 m2
3
3
1
1
1
1
Yes
10
64
92
X
14
1.3
6
4
4
29
C.V.
S.E.
X
7
8
2
4
1
.5
.5
23
C.V.
S.E.
S.D.
11
1.5
3
4
2
11
= 36%
= 6.1
S.D.
4
8
2
3
2
.6
.6
12
= 51%
= 5.9
133
-------
Methyl Parathion (1 ppb)
TAXA (order)
Diptera
Oligochaeta
Odonata
Amphipoda
Hemiptera
Cyprinodentif ormes
Basommatophora
Coleoptera
Zooplankton
TOTAL
-rPiinPi
H = 1.34
H/H
_ IU&.X
V = .75
TAXA (order)
Odonata
Diptera
Amphipoda
Basommatophora
Hemiptera
Cyprinodentif ormes
Zooplankton
TOTAL
-Tpi lnpi
H = 1.10
H/H max
V = .83
13
3
3
3
3
1
1
Yes
14
1.7
.95
Methyl
32
6
2
Yes
8
.56
.81
Sample No.
5 1
Individuals/0.016 ra2
3 3
1
2 6
27 19
2 (Dead) 1
2 5
1
Yes Yes
36 36
.9 1.42
.56 .73
Parathion (10 ppb)
Sample No.
- 28 25 17
Individuals/0.016 m2
6 9 1
7 2
12 5 5
7 1 1
2
1 1
Yes Yes Yes
34 18 8
1.49 1.27 1.07
.93 .79 .77
X
3
1
4
16
1
2
~
29
C.V.
S.E.
X
6
3
6
2
.5
17
C.V.
S.E.
S.D.
0
1.5
2
12
.6
2.5
13
= 44%
= 7.3
S.D.
3
3
5
3
.6
12
= 72%
= 6.1
134
-------
Furadan (1 ppb)
TAXA (order)
15
Sample
9
No.
2
X
S.D.
Individuals/0.016 m2
Odonata
Amphipoda
Diptera
Lepidoptera
Oligochaeta
Basommatophora
Hemiptera
Zooplankton
TOTAL
-rPiinPi
H = 1.39
H/Hmax
V = .81
TAXA (order)
5
16
6
1
4
1
33
1.41
.79
33
2
7
5
3
7
Yes
24
1.51
.94
Furadan (
Sample
29
5
24
1
2
11
1
Yes
44
1.24
.69
10 ppb)
No.
26 21
4
16
4
3
6
34
C.V.
S.E.
X
2
9
3
1
5
10
= 30%
= 5.8
S.D.
Individuals/0.016 m2
Odonata
Hemiptera
Amphipoda
Diptera
Basommatophora
Oligochaeta
Zooplankton
TOTAL
-cpi Inpi
H = 1.25
H/H
__ max
V = .88
13
2
7
4
1
Yes
27
1.3
.81
5
10
14
Yes
29
1.02
.93
8 8
6 8
7 4
4 1
1
Yes
26 21
1.47 1.2
.91 .87
9
8
7
2
26
C.V.
S.E.
3
2
5
2
3.4
= 13%
= 1.8
135
-------
Azodrin (1 ppb)
Sample No.
TAXA (order)
Odonata
Diptera
Lepidoptera
Amphipoda
Basommatophora
Heraiptera
Homoptera
Zooplankton
TOTAL
-% Inpi
5 = 1.33
H/H max
V = .83
34
6
1
2
19
4
Yes
38
1.34
.83
31
5
6
1
-
1
1
10
Individuals/0.016 m2
4
3
6
1
1
X
5
4
1
9
4
.5
S.D.
1
4
1
10
3
.6
Yes Yes
14
1.3
.81
Azodri
20
1.35
.84
a (10 ppb)
24
C.V.
S.E.
12
= 52%
= 7.2
Sample No.
TAXA (order)
Odonata
Amphipoda
Diptera
Hemiptera
Lepidoptera
Basoramatophora
Coleoptera
Cyprinodentif ormes
Epheraeroptera
Zooplankton
TOTAL
-Ip. Inp.
H = 1.3*1
H/Hmax
v = 78 1
35
9
5
1
2
1
18
1.27
.79
23
5
16
6
1
1
Yes
29
1.19
.74
22 11
Individuals/0.016 m2
8 7
11 18
6 2
3
6 9
1
1 1
1
Yes
30 38
1.82 1.35
.94 .75
X
7
13
4
2
4
.5
29
C.V.
S.E.
S.D.
2
6
2
1
4
.6
8
= 29%
= 4.1
136
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