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 ~"
DESCRIPTORS

Release to public
b.lDENTIrlERS/OPEN ENDED TERMS

IS. SECURITY CLASS (TliitHeponi
Unclassified
20. SECURITY CLASS fTliispafC)
Unclassified
c. COSATI Held/Group

21. NO. OF PAGES
nfi
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.
                                                 r™m
 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.

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     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

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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

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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).

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     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

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     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

-------
 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

-------
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

-------
 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

-------
                              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


  JS


I'  20


  IS


  10


    I

  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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Bourquin,  A.  W.   1975.  Microbial-malathion interaction  in artificial salt-
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-------
Chemagro Division Research  Staff.   1974.   Guthion (azinphosmethyl):   organo-
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DeLaune, R. D. , C.  N.  Reddy, and W. H.  Patrick, Jr.   1981.  Effect of pH and
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Fries, G.  F.   1972.   Degradation of chlorinated hydrocarbons under anaerobic
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Garnas,  R.  L.   1977.    The  fate  of  methyl  parathion   in  a marine  benthic
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Harvey, J.  C. ,  G.   A.  King, and  J.  W.  Young.   1969.   The  decomposition of
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Heuer, B. , B.  Yaron,  and Y. Birk.   1974.   Guthion half-life in aqueous  solu-
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Jolly, A.  L.,  J. B.  Graves, J. W.  Avault,  and  K. L. Koonce.   1978.   Effects
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Kaufman, D. D.  1974.  Degradation of pesticides  by soil microorganisms.   In
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     of America,  Inc.,  Madison, Wisconsin,  pp.  133-202.

LaFleur, K.  S.,  W.  R.  McCaskill,  and  G.  T. Gale,  Jr.    1978.   Trifluralin
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Lambert, S. M.  1967.  Functional  relationships between  sorption in  soil  and
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                                      33

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Meyer,  F.  P.   1965.   The experimental  use of Guthion  as a  selective  fish
     eradicator.  Trans. Amer. Fish. Soc. 94:203.

Munnecke, D. M., and D. P. H. Hsieh.  1976.  Pathways of microbial metabolism
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Murphy,  S.  D. , and K.  P.  DuBois.  1957.   Enzymatic  conversion  of  the dime-
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Nakatsugawa,  T. ,  and  P. A.  Dahm.   1962.  Activation  of Guthion  by  tissue
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Parr, J. F. ,  and  S. Smith.   1973.   Degradation of  Trifluralin under labora-
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Patrick, W.  H. , Jr.,  and D. S. Mikkelsen.  1971.   Plant nutrient behavior in
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Patrick, W.  H. , Jr.,  B. G.  Williams,  and J. T. Moraghan.   1973.   A  simple
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Pionke,  H.   B. ,  and  G.  Chesters.   1973.   Pesticide-sediment-water interac-
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Probst,  G.  W. , T.  Golab,  R.  Herberg,  F.  Holzer,  S.  Parka,  C.  Schans,  and
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Rajaram, K.  P., and N.  Sethunathan.  1975.  Effect of organic sources  on the
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     296-300.

Rajaram, K.  P.,  and  N.  Sethunathan.   1976.   Factors  inhibiting  parathion
     hydrolysis  in  organic  matter-amended soil  under  flooded  conditions.
     Plant Soil 44:683-690.

Reddy,  K. R. ,  and W.  H. Patrick, Jr.  1975.  Effect of alternate aerobic and
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                                      34

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Sethunathan, N.   1973.   Organic matter and parathion  degradation in flooded
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     in the soil environment.   Agric.  Food Chem. 22:439-441.


                                      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

-------
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

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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

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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

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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

-------
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

-------
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

-------
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   Chitin—Amended Soil   Starch—Amended 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).

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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* ?? -^
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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 \ * ''••
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lil \ \ ""*
rf// ^ \
'/ x \
*/ v- *
/ 	 .Au. *^vv----*
* ^^••'"' V"VVS:'-»'''^N;»
.' "o*
/'
b 	

~l 	 1 	 1 	 1 	 1—1 	 — — 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

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                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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Atlas,  R.  M. ,  D.  Pramer,  and  R.  Bartha.   1977.   Assessment  of pesticide
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Bartha, R., R. P. Lanzilotta, and D. Pramer.   1967.   Stability and effects of
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Bourquin,  A.  W. , P.  H. Pritchard,  and  W. R.  Mahaffey.   1977.   Effects of
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Bollag,  J. M.   1974.   Microbial transformation  of pesticides.   Adv.  Appl.
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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
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Faust,  S.  D.   1977.   Chemical mechanisms affecting the  fate  of organic pol-
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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,
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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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                                      96

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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
     estuarine environment.  J. Oceanogr. Soc. Japan 33:328-334.

Hsu, S. C., and J.  L. Lockwood.   1975.   Powdered chitin agar as a selective
     medium  for enumeration  of   actinomycetes   in  water  and  soil.   Appl.
     Microbiol. 29:422-426.

Jensen, H.  L.  1930.   Actinomycetes  in  Danish  soils.    Soil  Sci.  30:59-77.

Karl,  D.  M.   1980.   Cellular nucleotide measurements   and  applications  in
     microbial ecology.  Microbiol. Reviews 44:739-796.

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.

Munnecke, D. M., and D. P. H. Hsieh.   1976.  Pathways of microbial metabolism
     of parathion.   Appl. Environ. Microbiol.  31:63-69.

Okutani, D.   1966.   Studies  of chitinolytic systems in  the digestive tracts
     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-
     environmental system.  Dev. Indust. Microbiol.  21.

Portier, R. J. ,  and S. P. Meyers.  1981b.  Use of microcosms for analyses of
     stress-related  factors   in  estuarine  ecosystems.   Int. Wetlands  Con-
     ference,  New Delhi, India (September 1980).


                                      97

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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-
     cosms.   Pages 251-272  in A. W. Bourquin  and P. H.  Pritchard,  (eds.),
     Microbial  Degradation of  Pollutants  in Marine  Environments.   EPA 600/
     9-79-012.

Rao,  A.  V.,   and  N.  Sethunathan.   1974.   Degradation  of  parathion  by
     Penicillium  waksmani  Zaleski isolated  from  flooded  acid sulphate soil.
     Arch. Microbiol. 97:203-208.

Reissig, J. L., J. L. Strominger, and L. F. LeLoir.  1955.  A modified color-
     imetric  method  for  the estimation  of n-acetylamino sugars.   J.  Biol.
     Chem. 217:959-966.

Seki, H.   1965.   Microbiological  studies on the  decomposition  of  chitin in
     marine  environments.   X:  Decomposition of  chitin  in marine sediments.
     J.  Ocenaogr. Soc. Japan 21:261-269.

Sethunathan,  N.   1973.   Microbial  degradation of  insecticides in  flooded
     soils and  in anaerobic cultures.  Residue Rev. 47:143-165.

Stevenson,  L.   H. ,  T. H.  Chrzanowski,  and  C.  W.  Erkenbrecher.  1979.   The
     adenosine  triphosphate  assay:   conceptions  and misconceptions.   ASTM
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Yaron, B., B.  Heuer,  and Y. Birk.   1974.   Kinetics  of azinphosmethyl losses
     in the soil environment.  Agric. Food Chem. 22:439-441.
                                      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

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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

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     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

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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

-------
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

-------
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

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             .
             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

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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

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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

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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

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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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Gray,  I.  E.   1974.  Systems  with pesticide,   ln_ H. T. Odum, B.  J. Copeland,
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    123-141.
                                    120

-------
Harman,  W.  N.   1974.   Snails  (Chapter 9).  In C. W. Hart and S.  L.  H.  Fuller
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Hopkinson,  C.  S.   1973.  The  relation of man and nature in Barataria  Basin,
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    Rouge.   235 pp.

Hulbert, S. H. ,  M. S. Mulla,  and H.  R.  Willison.    1972.   Effects   of  an
    o r ga nophos pho ru s  insecticide  on the phytoplankton, zooplankton and
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Hynes,  H. B.  N.   1961.   The effect of sheep-dip containing the insecticide
    BUG  on the  fauna  of  a  small  stream.    Ann.  Trop. Med.   Parasit.
    56:192-196.

Katz,  M.   1961.   Acute  toxicity  of some organic insecticides to three
    species of  salmonids  and to  the threespine stickleback.   Trans.  Amer.
    Fish.  Soc. 90:264.

Kajak,   Z.   1974.    Benthos  of standing water.   In W. T.  Edmondson,  G.  G.
    Winbert (eds.),  Secondary  Productivity  in Fresh  Waters.   IBP Handbook
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Lauer,  G.  J. , H.  P. Nicholson, W.  S.  Cox,  and J.  I. Teasley.   1966.
    Pesticide  contamination of  surface waters by sugar cane farming in
    Louisiana.  Trans. Amer.  Fish  Soc. 95:310-316.

Lichtenstein,  E. P.,  K.  R.  Schulz,  R.  F.  Shrenting and Y.  Tsukano.   1966.
    Toxicity  and fate  of  insecticide residues in water.  Arch. Environ.
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Macek,   K..  J. , and N. A.  McAllister.  1970.   Insecticide  susceptibility of
    some  common  fish  family representatives.  Trans. Amer. Fish. Soc.
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    Bengal stains  to  facilitate  sorting benthic samples.   Trans.  Amer.
    Microsc. Soc.  86:221-223.

Miller,  C.  W.,   B.  M.   Zuckerman,  and A.  J.  Charig.    1966.    Water
    translocation  of  diazinon-C    and  parathion-p   off   a  model  cranberry
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                                    121

-------
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                                    122

-------
Weary, G.  C.,  and H.  G.  Merriam.  1978.  Litter decomposition in a red  maple
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                                    123

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                                 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

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     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

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                                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

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                                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

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                  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

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              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

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                        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

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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

-------