PB-243 825
REVIEW AND EVALUATION OF AVAILABLE TECHNIQUES FOR
DETERMINING PERSISTENCE AND ROUTES OF DEGRADATION
OF CHEMICAL SUBSTANCES IN THE ENVIRONMENT
SYRACUSE UNIVERSITY RESEARCH CORPORATION
PREPARED FOR
ENVIRONMENTAL PROTECTION AGENCY
MAY 1975
DISTRIBUTED BY:
KTin
National Technical Information Service
U. S. DEPARTMENT OF COMMERCE
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing}
. REPORT NO.
EPA-560/5-75-006
2.
3. RE
4. TITLE AND SUBTITLE
Review and Evaluation of Available Techniques for
Determining Persistence and Routes of Degradation of
Chemical Substances in the Environment
REPORT DATE
May 1975
6. PERFORMING ORGANIZATION CODE'
'. AUTHOR(S)
P.H. Howard, J. Saxena, P.R. Durkin, L.-T. Ou
8. PERFORMING ORGANIZATION REPORT NO
SURC TR 74-577
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Life Sciences Division
Syracuse University Research Corporation
Merrill Lane - University Heights
Syracuse, New York 13210
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
EPA 68-01-2210
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final Technical Report.
14. SPONSORING AGENCY CODE
13. SUPPLEMENTARY NOTES
16. ABSTRACT
This report reviews and evaluates the present state of techniques which
have been used to determine the environmental persistence (biological, chemical and
photochemical degradation) and routes of degradation of chemicals released in the
environment by human activities. The information sources included relevant papers,
books, and review articles, abstracting services, and computer searches such as
National Technical Information Services, and the current investigators files of the
Smithsonian Science Information Exchange.
The techniques that were identified were reviewed and then evaluated for their ability
to simulate natural environmental conditions, convenience of procedure, time require-
ments, necessary equipment and reproducibility. The ultimate evaluation was based on
how well the methods have worked with well-known environmental contaminants. A cost
analysis of the test methods was undertaken to determine the feasibility of compre-
hensive screening of chemicals for environmental persistence.
A relationship between chemical structure and environmental persistence is presented
and some theoretical grounds for such correlations are discussed. An attempt has been
made to categorize chemicals for their suitability to various test methods based on
consideration of physiochemical properties, toxicity, environmental release factors
and commercial economic factors.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTlFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Degradation test methods, persistence, bio-
logical degradation, photochemical tech-
niques, chemical transformation, metal
transformation, polymer breakdown, routes
of degradation, chemical structure and
persistence, cost analysis
PRICES SUBJECT TO CHANGE
19. SECURITY CLASS (ThisReport)
Unclassified
18. DISTRIBUTION STATEMENT
Document is available to public through the
national Technical Information Service,
Springfield. Virginia 22151
21. NO. OF PAGES
20. SECURITY CLASS (This page)
EPA form 2220-1 (t-73)
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EPA-560/5-75-006
May 1975
REVIEW AND EVALUATION OF AVAILABLE TECHNIQUES
FOR DETERMINING PERSISTENCE AND ROUTES OF DEGRADATION
OF CHEMICAL SUBSTANCES IN THE ENVIRONMENT
by
P.H. Howard
J. Saxena
P.R. Durkin
L.-T. Ou
Contract No. 68-01-2210
Project No. L1210-05
Project Officer
Michael J. Prlval
Prepared for
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
Document is available to the public through the National Technical
Information Service, Springfield, Virginia 22151.
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NOTICE
This report has been reviewed by the Office of Toxic Substances,
EPA, and approved for publication. Approval does not signify that
the contents necessarily reflect the views and policies of the
Environmental Protection Agency, nor does mention of trade names or
commercial products constitute endorsements or recommendations for
use.
ii
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TABLE OF CONTENTS
Page
LIST OF TABLES xiv
LIST OF FIGURES xvi
SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS 1
I. INTRODUCTION 23
A. General 23
B. Methods and Approach 29
1. Literature Search 29
2. Scope and Organization of the Report 30
II. TYPES OF ENVIRONMENTAL DEGRADATION 33
A. Biological Degradation 33
1. Metabolic Pathways 35
2. Metabolic Activity 38
3. Availability of Synthetic Organics to Various Taxa 40
B. Photochemical Degradation 42
C. Degradation by Chemical Agents 45
III. BIODEGRADATl'ON OF CHEMICALS IN AQUATIC OR SEWAGE TREATMENT
CONDITIONS 49
A. Techniques for Determining Biodegradation of Chemical
Compounds in the Aquatic Environment 49
1. Biochemical Oxygen Demand 51
a. Dilution Method 51
(1) Standard 5-day BOD procedure 52
(ii) 10-day BOD procedure 55
(iii) Long-term BOD Technique 55
(iv) Two-bottle - Single Dilution Reaeration Method 56
b. Respirometry 58
(i) Warburg Method 61
(ii) Modification of Warburg Apparatus 63
(iii) Differential Manometer 64
(iv) Electrolytic respirometer 66
(v) Oxygen electrode respirometer 67
(a) Galvanic cell oxygen electrode respirometer 68
(b) Clark-type oxygen electrode 69
(c) Other Oxygen Electrode respirometers 70
(vi) Miscellaneous Techniques 71
iii
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TABLE OF CONTENTS
(Continued)
2. River Die-Away Test 72
a. Original River Die-Away Test 73
b. River Die-Away with Fortified and Inoculated Waters 76
c. River Die-Away Test with Polluted River Water 79
d. Anaerobic and Microaerophilic River Die-Away Test 81
e. Die-Away Test with Marine Water 82
3. Shake Culture Test 83
a. Shake Cultures Inoculated with Natural Communities
of Microorganisms 83
(i) Degradation tests using activated sludge
or sewage as source of microorganisms 83
(a) Original shake culture method for study
of surfactant biodegradation 83
(b) Shake culture test of the Soap and
Detergent Association (SDA) 85
(c) Bunch and Chambers Test 86
(d) Shake culture test utilizing preserved seed 87
(1) Air-dried activated sludge - The
Aeration Test 88
(2) Shake culture test using sludge
preserved by lyophilization ( 89
(e) Degradation methods utilizing composite
seed 92
(f) Slope (slant) culture technique 94
(g) Shake culture employing seed acclimated to
increasing concentration of the test
chemical 96
(h) Other modified tests 97
(ii) Degradation Test Using Lagoon Microorganisms 100
(iii) Shake culture test without initial inoculation 100
(iv) Shake cultures inoculated with lake sediments 101
b. Shake Culture Studies Using Pure Cultures of
Microorganisms ; 102
(i) Pure culture obtained from commercial sources
or from research laboratories 103
(ii) Pure Cultures Isolated from Natural Sources 104
(iii) Pure Cultures Obtained from Enrichment 105
(a) Enrichment with test chemicals 105
(b) Enrichment for Cometabolic Degradation 107
(c) Enrichment for Marine Microorganisms 110
(iv) Pure Cultures Isolated from Naturally Enriched
Environment 112
(v) Cell-free'Extract Studies 114
(vJ) Multiple diffusion chamber of study interaction
among pure cultures of microorganisms ' 115
iv
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TABLE OF CONTENTS
(Continued)
4. Continuous Culture Technique
5. Terrestrial-Aquatic Model Ecosystem
6. Model Aquatic Ecosystem
B. Techniques Simulating Sewage Treatment Conditions 126
1. Introduction 126
2. Activated Sludge Systems 129
a. Continuous-Flow Systems 129
(i) Official German Test Method 134
(ii) Miniature Continuous-Flow Units 136
b. Semicontinuous and Batch Systems 139
(i) Batch Systems 139
(ii) Semicontiauous Systems 140
3. Trickling Filter Systems 144
a. British WPRL Pilot-Scale Trickling Filters 145
b. Recirculation Filter Test 146
4. Anaerobic Systems 148
5. Field Tests 150
C. Analytical Procedures 151
1. Extraction and Clean-up 152
2. Analytical technique 153
a. Chromatographic Methods 153
b. Radiotracer Technique 153
c. Colorimetric Methods 154
d. U.V. and I.R. Spectrometry 156
e. Measurement of C02 Evolution 157
f. Oxygen Consumption 157
g. Microbial Growth 158
h. Bioassay 159
i. Determination of Total Carbon 159
j. Others 160
D. Evaluation of the Techniques used for Determining
Biodegradation of Chemicals in Natural Water Systems 161
1. Factors affecting biodegradation 162
a. Type of Inoculum 162
b. Mineral Salt Composition 165
c. Test Compound Concentration 166
d. Supplementary Nutrients 167
e. Oxygen Requirement 169
f. Temperature, pH, Light, etc. 170
2. Comparison of Methods 172
3. Correlation between laboratory and field results 175
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TABLE OF CONTENTS
(Continued)
" 4
J
4. General Discussion of Various Test Methods
a. Rapid Screening test for biodegradability
Biochemical Oxygen Demand
River Die-Away Test
Shake Culture Test Inoculated with Natural
Communities of Microorganisms 184
Model Ecosystems 186
b. Biodegradation Test Methods for Determination of
the Routes of Degradation J?90
E. Evaluation of Techniques Used to Determine Biodegradation
Under Biological Treatment Plant Conditions ₯99
1. Introduction 199
2. Factors Affecting Biodegradation Under Waste Water'
Treatment Conditions 200
a. Acclimation and Deacclimation of the Microorganisms 200
b. Temperature 202
c. Analytical Methods 203
3. Correlation Between Laboratory and Field Results 203
4. General Comparison of Laboratory Methods 205
a. Biodegradation Potential, Reproducibility, and
Direct Comparisons of Techniques 205
b. Advantages and Disadvantages of Individual Techniques 209
(i) Screening Tests ' 209
(ii) Continuous and Semicontinuous Techniques 211
F. Cost Analysis 215
1. Techniques for Studying Biodegradation of Chemicals
in Water 216
a. Preliminary test to Determine Biodegradability 216
b. Intensive Biodegradation Study to Identify
Metabolites and Elucidate Pathway of Degradation 219
2. Techniques Which Simulate Sewage Treatment Plant
Conditions 220
IV. BIODEGRADATION OF CHEMICALS IN THE SOIL ENVIRONMENT 223
A. Techniques Used for Determining Biodegradation 224
1. Laboratory Tests 224
a. Natural Communities from Soil 224
(i) Soils Incubated with Test Chemicals 225
(a) Aerobic Studies 225
(b) Flooded Conditions 227
(c) Anaerobic Conditions 228
(ii) Soils Suspended in Aqueous Solution 228
(iii) Soil Perfusion Technique 229
vi
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TABLE OF CONTENTS
(Continued)
b. Pure Culture Studies 231
(i) Pure Cultures Isolated from Soil Enrichments 231
(a) Enrichment: Cultures Obtained by Treatment
of Soil with Test Chemical 232
(b) Naturally Enriched Cultures 233
(c) Soil Perfusion Cultures 234
(ii) Other sources of Pure Cultures 235
c. Cell-free Extract Studies 237
d. Miscellaneous Methods 238
2. Greenhouse Studies 241
3. Field Studies 241
IJ. Analytical Procedures 243
1. Chemical Analyses 243
a. Extraction and Clean Up Procedures 243
, b. Chromatographic Methods 246
(i) Gas-Liquid Chromatography 246
(ii) Thin-layer Chromatography 246
(iii) Paper and Column Chromatography 247
c. Spectrophotometric Methods 248
(i) UV Absorption 248
(ii) Visible Spectrophotometry 249
(iii) Infrared Spectrophotometry 250
d. Radioassays 250
(i) Assay for the Loss of Radioactivity of
Test Chemicals 251
(ii) Identification of Metabolic Intermediates 252
(iii) 1I+C02 Evolution 253
e. GC-MS Techniques 254
f. Q£ Consumption 254
g. C02 Evolution 255
2. Bioassays 256
a. Plant bioassays for herbicides 256
b. Insect Bioassays; for Insecticides 258
C. Evaluation of Biological Techniques 259
1. Factors Affecting Degradation 259
a. Soil Type 259
b. Soil Depth 261
c. Test Chemical Concentration 262
d. Soil Microorganisms and Acclimation 263
e. Physical Environment - pH, Temperature, Oxygen
Availability, Redox Potential and Moisture Content
of the Soil 265
f. External Carbon Source 271
vii
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TABLE OF CONTENTS
(Continued)
2. Correlation Between Laboratory and Field Results
3. General Discussion of Various Test Methods
i
D. Cost Analysis for Testing Biodegradabillty of Chemicals
in Soil ; 279
V. PHOTOCHEMICAL AND CHEMICAL ALTERATIONS -2'83
A. Degradation of Chemicals in the Atmospheric Environment »283
1. Introduction "'218*3
2. Techniques Used for Determining Atmospheric Degradation 285
a. Long-Path Infrared Cells '285
b. Plastic containers '*28-9
c. Glass Flask Reactors +290
d. Smog Chambers 296
(i) Rose and Brandt Smog Chamber 296
(ii) Wayne and Romanovsky Smog Chamber 298
(iii) Korth, Rose and Stahman Smog Chamber 298
(iv) Bartlesville Petroleum Research Center
Smog Chamber 300
(v) Stainless Steel Chambers 302
(vi) Stanford Research Institute Smog Chamber 302
(vii) Battelle Memorial Institute Smog Chamber 304
. 3. Analytical Procedures 305
a. Long-Path Infrared Spectrometry 305
b. Gas Chromatographic Analysis 307
c. Colorimetric Analysis and Instrumental Methods 309
d. Mass Spectrometry 311
e. Bioassay 312
4. Evaluation of the Techniques 313
a. General 313
b. Factors Affecting Degradation 314
(i) Spectral Distribution and Intensity of Light 314
(ii) Concentration of Reactants 315
(a) Humidity 315
(b) NO-Hydrocarbon Concentrations 316
(c) Other Reactants 318
(iii) Temperature 318
(iv) Chamber Configuration, Construction Materials,
and Cleaning Techniques 319
c. Internal Consistency of Results 323
viii
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TABLE OF CONTENTS
(Continued)
d. Comparison of Laboratory Results to Behavior in
the Natural Environment 324
(i) Hydrocarbons 324
(ii) Fluorocarbons 327
e. General Discussion of the Advantages and
Disadvantages of the Various Methods 328
5. Cost Analysis 331
a. Loss of Test Compound 332
(i) Only One Compound Studied 332
(ii) More Than One Compound Per Year for 5 Years 332
b. Loss of Test Compound and Isolation and Identifi-
cation of Breakdown Products 333
(i) Only One Compound Studied 333
(ii) More Than One Compound Per Year for 5 Years 333
c. Summary 334
B. Photochemical and Chemical Alterations in the Aqueous
and Soil Environment 335
1. Photochemical Alterations 335
a. Introduction 335
b. Techniques Used to Determine Photoalterations 339
(i) Light Sources 339
(ii) Solution Photochemistry 344
(a) Photochemical Equipment 344
(b) Experimental Conditions 347
(iii) Adsorbed or Thin Film Photolysis 350
2. Techniques Used to Determine Chemical Alteration '352
a. Introduction 352
b. Techniques Used to Study Chemical Alterations 352
3. Analytical Procedures 354
. a. Isolation and Detection of Degradation Products 354
b. Identification of Degradation Products 356
4. Evaluation of the Techniques 357
a. General 357
b. Factors Affecting Chemical and Photochemical
Degradation 358
(i) Light Wavelength 358
(ii) Reaction Media 359
(iii) Sensitizers 361
(iv) Hydrogen Ion Concentration . 362
(v) Other Factors 364
ix
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TABLE OF CONTENTS
(Continued)
i Page
c. Extrapolation of Laboratory Results .to Field
Conditions 364
(i) Photolysis of Dieldrin to Photodieldrin 365
(ii) Photodegradation of the Sodium Salt of
Pentachlorophenol '368
(iii) Photolysis of Polychlorinated Biphenyls '-368
d. Summary, - '3.71
(i) Photochemical Studies
(ii) Chemical Studies
Cost Analysis
a. Photolysis Studies
b. Hydrolysis Studies
VI. THE INTERCONVERSION OF ALKYLATED AND INORGANIC FORMS OF CERTAIN
METALS AND METALLOIDS .*?37-9
A. Introduction "379
B. Chemical and Biochemical Transformation -of Metals and
Metalloids 380
1. Valance Changes 380
2. Methylation 382
Mercury ,382
Arsenic 384
Selenium and Tellurium 385
3. Chelation 385
C. Test Methods for Studying Transformation 386
1. Biological Transformation in Aquatic Environment 386
a. Methylation of metals 386
(i) i Mixed Culture Studies - 386
(ii)' Pure Culture Studies 389
(iii) Field Studies 391
b. Degradation of Organometallic Compounds 392
2. Biological Transformation in the Soil Environment 393
3. Model Ecpsystem and Aquarium Studies 394
4. Test Methods for Photochemical Studies 395
a. Broad-band (>290 nm) and monochromatic (313 nm) light
from a mercury lamp 396
b. Sunlight 396
5. Test Methods for Studying Chemical Transformation 396
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TABLE OF CONTENTS
(Continued)
p. Analytical Procedures 397
: 1. Isolation Steps 398
2, Analysis 398
a. Gas chromatography 398
b. Use of labelled compounds 399
c. Atomic Absorption 399
d. Spectrophotbmetric procedure 400
e. Neutron Activation 401
f. X-ray fluorescence 402
g. Polarographic methods 402
3. Identification 402
E. Evaluation of Techniques 404
1. Factors Affecting Transformation of Elemental Contaminants 404
a. Factors Affecting Methylation of Metals 404
. (i) Concentration of the Metal 404
(ii) Microbial activity 406
(iii) Adsorption and Chelation of Metals 407
(iv) Presence of other chemicals 407
(v) Physical parameters such as pH, temperature and
redox potential of. the test medium 409
b. Factors Affecting Degradation of Organometallic
Compounds 410
2. General Discussion of the Test Methods Used for Determining
. Environmental Transformation of Organometallic and
Elemental Contaminants 411
I 3. Correlation Between Laboratory and Field Results 415
F. Cost Analysis 417
VII; ENVIRONMENTAL DEGRADATION OF SYNTHETIC POLYMERS 419
A. Introduction 419
B, Techniques for Determining Degradation 420
1. Biological Test Methods 420
a. Screening Tests 422
(i) Pure Culture Test on Agar 422
(ii) Mixed Culture on Agar 423
(iii) Humidity Cabinet Test 424
b. End-Use Tests 425
(i) Soil Simulation Tests 425
(ii) Aquatic Submersion Test 426
c. Field Tests 426
(i) Above-Surface Exposure 426
(ii) Soil Burial Test 427
(iii) Aquatic Submersion 427
xi
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TABLE OF CONTENTS
(Continued)
2. Physiochemical Test Methods 428
a. Degradation by Liquids 428
b. Degradation by Gases 429
c. Degradation by Light 430
C. Analysis Procedures 431
1. Changes in Mechanical Properties 'V431'
a. Water Absorption or Transmission (ASTM, E96-66,t1971) ''/4'3£
b. Electrical Properties f£'33.
c. Elasticity/Embrittlement , -*'4'35^
d. Hardness #4'3i6.;
e. Tensile Strength 'V4>37
f. Weight Loss -*438
2. Response of Biological Systems J V438
3. Molecular Alteration » ^44L
D. Evaluation of Techniques -447'
\
1. Factors Affecting Degradation 447
a. Biological Degradation 447
(i) Selection of Polymer Formulation ' 447
(ii) Pretreatment of Test Specimen 448
(iii) Selection of Degrading Organism 449
(iv) Choice of Media . 450
(v) Conditions of Growth - 451
(vi) Duration of Exposure 452
b. Physiochemical Degradation .' 453
2. Internal Consistency of Results 455
3. Comparison of Laboratory Results to. .Behavior in *the
Natural Environment 456
E. Cost Analysis 457
VIII. RELATIONSHIP BETWEEN CHEMICAL STRUCTURE AND' ENVIRONMENTAL ;,
PERSISTENCE .r 461
A. Relationship of Chemical Structure andtBiodegradabillty 461
B. Atmospheric Stability of Organic Chemicals 467
C. Categorization of Elements 472
-i
D. Structure-Degradability Relationships of Synthetic
Organic Polymers , 474
';f.
1. Biological Degradation 474
2. Physiochemical Degradation 477
xii
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TABLE OF CONTENTS
(Continued)
Page
IX. CATEGORIZATION OF CHEMICALS IN.TERMS OF THE SUITABILITY OF
VARIOUS TEST METHODS 481
REFERENCES 487
xiii
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LIST OF TABLES
1. Figures From a Hypothetical Grasslands Soil
Community . . . | 40
2. Effect of Seed Type and Quality on-BOD-Results .... 53
3. Summary of Standard 5-day BOD Procedure Used by
Various Investigators 54
4. Warburg Respirometry Conditions < 63;
5. River Die-Away Test for Determining Biodegradability
of Organic Chemicals 74
6. Composition of the Basal Medium , '8.4
7. Composition o'f Medium §5
8. Pure Culture Obtained from Commercial Sources
or Research Laboratories 104
9. Pure Cultures Isolated from Natural Sources . . ..... 1.Q6
10. Degradation Studies Using Pure Cultures
Isolated from Enrichment 109
11. Degradation Studies Utilizing Pure Cultures Isolated
from Naturally Enriched Environment 113
12. Characteristics of Bench Scale Continuous
Activated Sludge Units ................. 131
13. Batch Sludge Die-Away ......... 141
14. Trickling Filter Conditions .............. . 145
15. Anaerobic Die-Away Procedures . 149
16. Comparison of Biodegradation of LAS and TBS
by Different Experimental Techniques T78
17. Summary of Principal Field Test Results 204
18. Comparison of Alcohol and Alkylphenol Ethoxylate
Biodegradability under Laboratory and Field Conditions* 205
19. Comparison of Biodegradation Test Methods ........ 207
20. Comparison oflBiodegradation Test Methods 208
21. Cost Analysis for Preliminary Biodegradability Test . . 217
22. Cost Estimates for Techniques Used to Simulate
Sewage Treatment Plant Conditions . .......... 221
23. Plant Bioassays for Herbicides 257
24. 1£*C02 Evolution from Five Soil Types Each
Receiving 2 ppm of 1LfC^Carbaryl 260
25. Characteristics of Sharpsburg Silt Clay,Loam
and Keith Sandy Loam at Various Depths . 262
26. Effects of pH '"on Ability of Lipomyces sp. to Degrade
10~**M Paraquat .in Three Media at 22* ......... 266
27. Effects of Temperature on Ability of Lipomyces sp. to
Degrade lO'^M^Paraquat in Mineral Salts Medium . . . . 267
28. The Rate of Degradation and Arrhenius Activation Energy
of Selected Triazine and Uracil Herbicides Applied to
the Soil at 8,'ppm '. 267
xiv
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List of Tables
(continued)
29. Liquid Chromatographic Analyses of Residual Oils
from the Aeration Experiment after 5 days of Growth
at 30°C 269
30. Radioactive Carbon Dioxide Collected from Culture of
Rhodotorula gracilis during a 10-day Incubation Period. . 272
31. Cost Analysis for Preliminary Biodegradability Test
in Soil Environment 281
32. Interfering Infrared Absorption Bands from Background
Contaminants or Common Products 306
33. Minimum Concentration for Sodium Bisulfide Collection
Technique . 310
34. Ranking of Reactivities of Hydrocarbon Consumption
When Photolyzed in Presence of NO under Static Conditions 324
35. Relative Rates of Percentage Loss of Hydrocarbons
Averaged over Four Hour Irradiation . . 325
36. Comparison of Acetylene, Ethylene, Propylene Ratios
of Two Ambient Air Samples 327
37. Advantages and Disadvantages of Static Vs. Dynamic
Procedures in Studying Atmospheric Reactions ...... 329
38. Experimental Conditions of Pesticide Photolysis 336
39. Approximate Wavelength Limits for Transmission of Various
Materials and Water at Room Temperature 343
40. Environmental Transport Processes of PCS's 370
41. Absolute Limits of Detection (in g) for
Atomic Spectrometric Methods 400
42. Instrumental Limits of Detection in Trace Metal Analysis. 403
43. Cost Estimates for Evaluation of Environmental Fate of
Elemental Contaminants and Organometallies . 417
44. Cost of Selected Procedures in Determining the Degradation
of Synthetic Organic Polymers 458
45. Relationship Between Chemical Structure and Biodegradation 465
46. Olefin Relative Reaction Rate Comparison 468
47. Classification of Elements from the Standpoint of
Environmental Pollution 472
48. The Biodegradability of Various Synthetic Organic Polymers 475
xv
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LISTOF FIGURES
1. Percent Total Metabolism by Different-Taxa
of a Hypothetical Soil Population
in a Meadow . V; 39
2. Spectral Energy' of Sunlight^Relative to Chemical Bond
Energies and Low-Pressure Mercury Arc Lamp . . 43
3. Modes of Formation and Deactivation of^Molecules
in the Excited'State . . -S44
4. Schematic Representation of Individual Biodegradation
Reaction Units i>57
5. Warburg Manometer . . . . . . ":'62
6l Diagram of Differential Manometer ............. 3"65
7. Schematic Diagram of one of the six units of the
Sapromat A6 Respirometer . .... . . ... . . V67
8. Completely Filled Electrode-Respirometer . . ... ... . ^<70
9. Experimental Apparatus used for Simultaneous
Microaerophilic and Aerobic Tests ............ 82
10. Oxygen'Uptake by Sludges Preserved by Four Methods ... 91
11. General Protocol for a 10 Un^.t Biodegradation; Test Unit . 93
12. Continuous Culture System . ''. . . ... . 117
13. Schematic Drawing of Model Ecosystem for Studying
Pesticide Biodegradability and Ecological-Magnification . 119
14. Aquatic Ecosystem Simulator. , . . . . . . . . . . . . . . 125
15. Flow Pattern of Domestic Activated Sludge-Waste
Disposal Plant '. 127
16. Schematic 'Diagram of a Serial Type Aerated, Chamber
Laboratory Model Activated"-Sludge: Unit .... 130
17. Various Completely Mixed-*Aerator Model. Activated
Sludge Unit . . . . . . .-."-. ... . . ..... . . . . . 133
18. Apparatus for Activated-sludge:Test .135
19. Continuous Activated; Sludge;:.Unit . . ... ... . . .... 138
20. Miniature Complete Mixing Continuous.Activated .Sludge
Unit . . . .'."'. ...................... 138
21. Soap and Detergents Association's Semicontinuous
Activated Sludge Aeration, Chamber . . 143
22. Recirculation Filter Apparatus ....... 147
23. Methylene Blue'Dye . . . . . . . . . ..... ... . ... 154
24. Soil Perfusion Apparatus . . . . . . 230
25. Soil Perfusion^Apparatus . . . . . . . . . . . . ... . 230
26. Cross Section of a Lysimeter . . . . . . 240
27. Effect of Soil1Type on Amitrole Degradation ....... 261
28. Time 'Course of .Breakdown of 250 ppm of Pyrazon in
Different Soils 264
29. Disappearance of CIPC in perfused soil, etc. ...... 264
xvi
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List of Figures
(continued)
30. Disappearance of CEPC in perfused soil, etc. 264
31. Disappearance of Trifluoralin from Soil Suspensions as
a Function of Redox Potential and Time 270
32. Schematic Diagram of Vapor-Phase Photoreactor 294
33'. Environment Irradiation Test Facility 297
34. Chamber Light Energy for Korth et al. Chamber ...... 299
35. Spectra of Teflon and Tedlar Films in the UV Region . . 301
36. SRI Smog Chamber 303
37. Emission Spectrum of the Low-Pressure, Medium Pressure,
and High Pressure Mercury Arcs 341
38. Spectral Distribution of a Low-Pressure Mercury Lamp;
Fluorescent Sunlamp; etc 342
39. Sunlight-Simulating, Laboratory Photoreactor . 344
40. Photoreactor Equipped with a Gas Lift for Continuous
Extraction . 345
41. Quartz Immersion Well Photochemical Reactor ...... 346
42. Effect of Soil Sterilization on Amitrole Degradation in
Hagerstown Silty Clay Loam . . . . _. 353
43. Basic and Acidic Photolysis of Trifluralin . , 362
44. Acid and Base Catalyzed Hydrolysis of
Organophosphorus Pesticides ... 364
45. Photolysis of Dieldrin 365
46. The Biological Cycle for Mercury 383
47. The Biological Cycle for Arsenic 384
48. Anaerobic Microbial Reactor System .... 388
49. Aerobic Microbial Reactor System 388
50. Concentration of Methylmercury in Botton Sediment After
Addition of Inorganic Mercury Followed by Incubation
for Seven Days 405
51. Design of Differential Manometer . 439
52. Products of Atmospheric Degradation of Olefins 470
53. Selective Reactivity of Benzylic Hydrocarbons 471
54. PhyBiochemical Degradation of Polyethylene 480
xvii
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SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS
The determination of persistence and degradation products of a chemical
contaminant in the environment is an important parameter in the overall eval-
[l uation of a compound's potential environmental hazard. This report reviews
i!
and evaluates techniques which have been used to study the persistence and
degradation metabolites of chemicals in the environment.
A literature search was conducted using papers, books, and review
i articles, abstracting services, and computer searches such as the National
Technical Information Service and the current investigator files of the
Smithsonian Science Information Exchange. Relevant articles were gathered
and examined, and approximately 800 references were used in the review and
evaluation report.
A major difficulty encountered in this review is the lack of precise
definitions or criteria for evaluating a chemical substance's stability in
the environment. Such terms as environmental persistence, alteration, and
biodegradability have rarely been assigned quantitative definitions. This is due
to the numerous environmental factors which affect the degradation rate and
: the lack of precision inherent in characterizing biological systems. Thus,
' even if one microenvironment is exactly simulated in the laboratory, appli-
> cation of the results to other environments can be at best only quantitative.
This is in contrast to the half-life of an isotope, for example, where the
rate is independent of environmental systems.
Several definitions have 'been suggested, especially for biodegradability:
Primary biodegradation - biodegradation to the minimum extent
necessary to change the identity of the compound (WPCF, 1967)
-------
Ultimate .biodegradation - biodegradation to (a) water, (b) carbon
. ;.- j
dioxide, (c) inorganic compounds (WPCF, 19617)
Acceptable biodegradation - biodegradation to the minimum extent
necessary to remove some undesirable property of the compound
* . . .
such as foaminess or toxicity (WPCF, 1967)
Very biodegradable - a compound that can be utilized as a carbon
£
and energy sources (Prochazka and Payne, 1965)
Biodegradable - a compound that can be attacked by the. enzyme
i .
apparatus acquired by microbes during the course of
evolution (Dagley, 1972b)
In addition, some researchers have defined biodegradability in terms
of specific analytical methods and test systems. For example, the presumptive
test of the Soap and Detergent Association (SDA, 1965) defines as biodegradable
any anionic surfactant that loses greater than 90% of its methylene blue
activated substances (MBAS) in the SDA shake culture test. These types of
.1 ' *
definitions are useful for comparison of different compounds in closely related
chemical groups. Furthermore, there is considerable dispute over whether a
compound that cannot be used for growth, may be cometabolized (concomitant
metabolism of a non-growth substrate) to a significant extent in the natural
.
environment. All of these definitions have some merit and drawbacks. We
».
would suggest that ai truely biodegradable compound is one which can be con-
verted in a relatively short period of time to low-molecular-weight metabolic
1 . *"
intermediates (e.g. glutamic acid, succinic acid, etc.).
.«
The qualitative nature of these definitions is apparent and it is probable
that these definitions will remain relatively inexact because of the variety
of chemical compounds being considered.
-------
A chemical that is released into the environment may be affected by
physical transport processes or altered chemically by exposure to various
biological and chemical agents in the environment. The physical processes
i
have a tendency to distribute and dilute or concentrate but not destroy
.the contaminant and these transport processes can have considerable impact
on the biological and chemical reactions that may take place. On the
other hand, chemical and biological reactions in the environment result
in alterations and frequently degradations of the contaminants to substances
which in many cases may be innocuous, thus ending the pollution potential
of the contaminant.
The alteration and degradation processes can be divided into three
categories: (1) biodegradation - effected by living organisms, (2) photochemical
degradation - nonmetabolic degradation requiring light energy, and (3)
degradation by chemical agents (chemical degradation) - nonmetabolic degradation
which does not require sunlight. Biodegradation of organic compounds appears
to be the most desirable because it results generally in completely mineral-
ized end-products. In contrast, photochemical and other nonmetabolic processes
usually result in only slight modifications in the parent compound. Microorgan-
isms appear to have major responsibility for determining persistence or break-
down of an environmental chemical and, therefore, techniques which use micro- ,
organisms have been focused upion in this review. Many compounds that enter
the environment (e.g. pesticides and hydrocarbons) have been shown to be
photochemically labile, but the importance of the process for soil and water
contaminants is unknown. Hydrolysis of environmental compounds by chemical
agents has been extensively studied and correlation between laboratory and
-------
.
field results is 'facilitated by the ease of measuring one of the more impor-
tant rate-determining factors, pH, bothi in.the laboratory and in the field.
However, other nonme'tabolic processes have received little attention.
i
A variety of techniques have beeiisused to determine blodegradatlon in
soil and water. They are briefly, tabulated below and advantages and dis-
advantages are outlined in the subsequent paragraphs. Following thp.se;
paragraphs are discussions of techniques.used to study photochemical', and.,
other nonmetabolic processes, metals, and polymeric materials. Thencon--
elusions and recommendations are presented.
Water
Biochemical Oxygen Demand (BOD)
Dilution Method
Respirometry
River Die-Away
Shake Culture Test
Mixed Cultures
Pure Cultures
Model Ecosystems
Activated Sludge
Official German Test
Miniature Continuous
Semicontinuous
Trickling Filter
Anaerobic Systems
Soil
Laboratory Techniques
Natural and Mixed Cultures
Soils Incubated with
Test Chemical
Soils Suspended in.
Aqueous Solution
Soil, Perfusion
Pure Cultures
Cell.Free Extracts
Greenhouse Studies
Field Studies,
Many of the above techniques are generally considered suitable for rapid
screening of biodegradability, whereas^ the others have been more commonly used
..-a.
for detailed investigation of the biodegradation process. With all the.
-------
techniques used, the analytical method provides the greatest spectrum of
cost and desirable information. Using radiolabelled compounds is undoubtedly
\ .
the best way to study a chemical's persistence and breakdown, but the cost of
synthesizing the compound is frequently high. At the other end of the spectrum
are techniques which require no analytical development work (e.g., BOD, CO.
evolution and total organic carbon). In between are techniques such as thin
layer and gas chromatography,
Rapid Screening Tests for Biodegradation in Water
Prior to an extensive investigation of the pathways of biodegradation of
chemical substances, a rapid screening test is usually run. Perhaps the most
frequently run test is the determination of biochemical oxygen demand either
by the dilution method or respirometry. Since the method requires no specific
analytical technique for estimation of the chemical compound, the test is
rapid and can be applied to a variety of chemical compounds. Further, since
the procedure measures oxygen consumption and not the compound, there can be
little confusion about disappearance of the compound due to physical adsorption
of the test chemical. The major difficulty with this technique is quite
often the Interpretation of the results. The method does not provide
any information about the nature of the degradation products. Also, since
oxygen is utilized for a number of complex metabolic reactions (including
synthesis of cell material) .and not simply for oxidation of the test chemical,
the correlation of oxygen utilization data to loss of test compound to
assess the extent of biodegradation is some times difficult unless extremes
of oxygen uptake are noted (0% or 100% of theoretical 0.). In addition,
the endogenous oxygen uptake rate is usually subtracted from the oxygen
uptake measured in the presence of the test chemical, ignoring the possibility
-------
of stimulation or inhibition of the endogenous rate sometimes caused by
addition of the test chemical. Simultaneous measurement of soluble carbon
during disappearance of the test compound may be used to confirm and supplement
the respirometric data. The respirometric method is generally considered
more precise as far as measurement of oxygen demand is concerned, and
since the microbial concentrations used in the respirometric method are
i
usually high, the technique simulates treatment plant conditions more
closely than does the dilution method. The polarographic method of respiration
measurement is generally less time consuming and allows continuous monitoring
of oxygen demand (BOD is normally only measured after 5 days) but since
the reaction period is so short little acclimation is allowed.
'''!
The river die-away test is relatively simple and requires a minimum of
equipment. The test chemical is placed in a natural water sample and the
disappearance of the compound is monitored. The reaction conditions closely
approximate the conditions encountered in nature. However, one of the more
serious shortcomings of the test is the variations in bacterial count and
i
composition between different rivers, between different points in the same
river, and even at the same point in a given river, which may cause considerable
fluctuation in the results.
Shake culture systems inoculated with natural communities of micro-
organisms are also used as a screening test, the most standardized being the
SDA (1965) method. The source of microbial inoculum has included such
sources as sewage (most frequent), lagoons, and lake sediments, but the
well defined medium composition which is used, provides somewhat better re-
producibility than the river die-away test. The technique allows for flexible
-------
operation including the use of acclimated and unacclimated seed, a defined
external carbon source, and even a preserved seed (better reproducibility,
but less like a natural population). Generally high concentrations of micro-
organisms are used. As a result the test period is relatively short but the
test conditions may be more favorable for degradation than generally encountered
in the natural environment.
To date, model ecosystems are oriented more towards answering questions
of bioaccumulation and metabolism of chemical substances in upper levels of
the food chain. Although metabolism in higher food chain organisms is
important in terms of toxicological effects and bioaccumulation, its
Importance in terms of total environmental persistence or degradation appears
to be relatively minor in comparison to the role of microorganisms. Thus
it appears to make little sense to use elaborate model ecosystems which are
time consuming and difficult to set up to test for environmental persistence
and biodegradability. However, it should be noted that many of the results
obtained from model ecosystems for highly degradable or very persistent
chemicals seem to be in good agreement with environmental monitoring data and
microbial test systems. However a striking exception is benzole acid which
is very biodegradable in microbial systems but in the system of Metcalf et _§!_.
(1971) the compound is accumulated and conjugated in the food chain organisms.
Test Methods for Determining Biodegradability in Biological Waste
Water Treatment Plants
Techniques used to simulate biological waste water treatment plants differ
in microbial population and concentration and frequently in the amount of
acclimation that is developed with the test compound. Since numerous chemicals
-------
which potentially could enter the environment first pass through biological
*
waste water treatment plants, it is important to determine their fate in
** .
these systems.
Activated sludge systems are most frequently modelled in the laboratory.
Both continuous and semicontinuous procedures are used. The semicontinuous
units are much more economical since they do not require the constant
attention of a continuous system and, they use much less feed and test
i.
material. Also, semicontinuous operation avoids the difficulty of maintaining
satisfactory circulation of sludge which is sometimes encountered using
continuous systems.
The official German activated sludge test has been required by German
law since 1964 for testing anionlc sutfactants. The technique has the
advantage that it can be run in any well equipped laboratory (no sewage
effluent or sludge needed as inoculum because the sludge is developed from
airborne microorganisms). Disadvantages include the fact that several
researchers have found it difficult to maintain a stable biochemical operation
and satisfactory circulation of sludge. The latter problem seems to be
remedied by the porous pot modification.
The miniature continuous-flow activated sludge systems provide economy
in preparation, storage and handling of feeds as well as savings in time and
f ' * .
labor. Although miniature systems are further removed from the characteristics
of full scale treatment plants, the large scale units are so far removed from
\ *
full scale that another factor of 10 probably has little affect. Both
natural and syntfietic feed have been used, the natural feed providing shorter
-------
acclimation time but requiring that radiolabelled material be used with
surfactants because of the background of surfactants in the natural feed.
Although the semicontinuous technique does not exactly simulate the
continuous operation of a full treatment plant, it is similar to plants
where the feed and recycled sludge are mixed at the entrance to the aerator.
The 24 hour cycle of the SDA procedure is convenient because it requires
no overnight attention.
Trickling filters are somewhat easier to operate than continuous
activated sludge systems, since no sludge needs to be recycled. Also,
scale-up factors from laboratory studies to commercial filters can be made
without great worry since the most important dimension is the depth of the
bed. However, disadvantages include: (1) a long acclimation period (14 weeks
for development of a mature film and 4-8 weeks acclimation),
(2) a fly nuisance, (3) lack of easy accommodation in a constant-temperature
room or bath, and (4) operational conditions can not be readily adjusted
(especially retention time). Recirculating filter tests, which are very
similar to soil perfusion tests (except that the supplemental nutrients
and carbon are higher in the recirculating filter), seem to be of less value
because their high biodegradability potential does not allow distinction
between relative degrees of biodegradability.
Soil Screening Tests
Unlike natural water, soils are generally rich in microorganisms and,
therefore, have been used extensively as microbial inoculum without amendment
or added microorganisms. Test methods utilizing natural communities of
microorganisms have generally been used as screening tests for biodegradability.
9
-------
Common techniques Include soil perfusion and incubating of 'the test chemical
with soil or with soil suspended in water. Using the soil as the test
medium closely simulates the conditions encountered in nature. However,., soil
from different geographic locations may give different results in degradation
studies. The presence of a complex medium such as soil may introduce addi-
tional steps in extraction and clean up and some metabolites may be unextract-
able. Also, the presence of soil may preclude the assay of biodegradatipn by,
/
many analytical procedures (e.g.,. oxygen consumption perhaps due to the high
endogenous rates of soil respiration). ^
In degradation test methods where soil is suspended in water, one
deals with a suspension of soil, rather than soil as it is encountered under
natural conditions. Since soil is used as an inoculum in the test procedure
and not as a medium for degradation, the quantity of soil suspended in the
aqueous medium is very small, and this may limit the availability of many
undefined nutrients present in soil and considerably affect the test results.
The advantage of the method is that in cases where a dilute soil suspension
is used, certain analytical measurements (e.g., disappearance of U.V. ab-
sorption) can be made directly on the sample or on the supernatant obtained
after centrifugation. The extraction and clean up steps In such cases will
'
be minimal. Thus if a large number of compounds are to be tested, this tech-
nique may be contemplated.
Soil perfusion systems consist of soil columns through which a con-
tinuous flow of water passes. The test compound may be either adsorbed on
the soil or dissolved in the water. Similar to aqueous suspensions, soil
perfusion systems deal with a solution rather than just soil and the constant
10
-------
exposure of microorganisms to air, water and the test chemical may poorly
simulate what occurs under natural conditions. The system is very potent
and provides an unusually high biodegradability potential. Thus it appears
that although the soil perfusion system Is an excellent tool for enriching
microorganisms which will degrade the test chemical, such a system is un-
suitable for use as a routine biodegradation test method due to its unusually
high biodegradation potential and difficulties in handling many units.
Enrichment and Pure Cultures
Mixed culture tests are more frequently used as screening tests because
mixed cultures of microorganisms are easier to obtain and the results may be
extrapolated to natural conditions easily. However, in some cases enriched
or stock cultures have been used to determine biodegradability. The procedure
with stock cultures usually involves screening a large number of different
organisms obtained either commercially or isolated from natural sources
(without enrichment) to determine the number that will grow on the test
chemical. The number of different organisms that grow on a chemical can be
considered somewhat indicative of the compound's biodegradability. Pure
culture studies, however, fail to account for breakdown of a compound by
synergistic processes and to consider the relative population density of
the particular organisms as it naturally occurs in the environment.
Enrichment culture techniques usually consist of isolation of cultures
which have first been enriched on the test chemical as the sole source of
carbon. The result is a pure culture that is able to use the test compound
as. a sole source of carbon and energy. This is similar to the result from
screening large numbers of pure cultures, except that the enrichment procedure
11
-------
is normally less tine consuming. Enrichment culture using an external carbon
!
source (chemical analogue of the test chemical - analogue enrichment; un-
related chemical - co-substrate enrichment) allows for the isolation of
3
organisms which may cometabolize the test compound. This procedure is
^-
t
particularly important for compounds which have been found to be recalcitrant
to being used as a carbon and energy source.
Techniques for Determining the Routes of Biodegradation
Elucidation of, the pathways of degradation involves identification of
the degradative intermediates and assignment of places in the scheme of deg-
radation. However,, from the point of view of understanding the environmental
i t- . .
behavior and hazards of a chemical, it may be sufficient to identify the degra-
dation intermediates only. . ..
/>'
The isolation and identification of metabolites using the complete in-
cubation mixtures from the above-mentioned screening methods is generally very
difficult. The researcher is dependent upon available extraction techniques
and on his knowledge of the type of breakdown products that might be expected.
Also, in a mixed culture laboratory system a metabolite may be degraded rapidly
and not accumulate in detectable levels. To overcome these problems, re-
searchers have frequently used pure cultures and cell-free extracts in metabolic
studies.
Major advantages of using pure cultures or cell-free extracts are (1)
complications originating from the complexities and variability of the soil
'.-, . . .
and water systems are eliminated, (2) the extraction and clean up procedures
will be simpler, (3) data will be more reproducible, and (4) it is possible
to study individual degradation steps because of the specificity of the
12
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enzyme systems contained in different species. The disadvantages Include
(1) failure to allow breakdown by synergistic processes and (2) the general
remoteness of the conditions from natural processes in soil or water.
Cell-free extracts are normally used where intact cells provide (1)
difficulty in manipulation of physical and chemical parameters, (2) rapid
reactions that do not allow examination of intermediate metabolites, and (3)
-difficulty in studying the separate enzymes involved in degradation. Major
disadvantages in the use of cell-free extracts are that (1) they are further
removed from natural conditions because the permeability barrier (cell
membrane) is absent and (2) the intermediates that are detected may never
appear outside the cell.
Catabolic pathways are generally determined by using radiolabelled
material. This is because of the higher sensitivity of radiotracer detection,
and the fact that radiotracer studies provide total balance sheets of the
fate of the compound. The techniques used for studying biodegradation are
generally similar to those which have been used by microbial physiologists
for studying metabolic pathways of a natural substance. Metabolic infor-
mation with intact cells has been derived using a number of techniques, in-
cluding (1) analysis of culture fluid for metabolites (2) removal of the
intermediates from the cell by extraction (3) accumulation of an intermediate
by addition of an enzyme inhibitor (4) sequential enzyme induction by the
substrate to enable cells to oxidize intermediate oxidation product. When
cell-free extracts are used, efforts are usually made to break the reaction
link at different points and thus study the reaction in small segments.
13
-------
This is generally accomplished by inactlvation or removal of a particular '
enzyme. Some information on metabolic pathways can also be obtained by
**" *
demonstration of the presence of appropriate enzymes in the cell-free
extracts. It is clear from the above that for elucidation of metabolic
pathways, there is'"no single method which will provide all the needed
. .- . 1,
information; the researcher generally has to try to put together a pathway
from the information which is derived from all the studies. For this
\
reason, detailed-studies are quite time consuming (and costly) and require
a well equipped laboratory.
Photochemical Techniques *;
Photochemical, techniques can be divided into two categories (1) simula-
tions of atmospheric phenomena, and (2) simulations of other environmental
photochemical processes. ,
- Atmospheric studies are usually conducted on relatively volatile
I . .
compounds and techniques that have been used include (1) long-path infrared
(LPIR) cell reactors, (2) plastic bag reactors, (3) glass'flask reactors, and
(4) smog chambers. The system has the. advantage of requiring minimum
. ^
analytical development since.most compounds have diagnostic infrared absorptions.
However, because of the nature of the analytical system, relatively high
concentrations and simple reaction systems must be used. Use of plastic-
containers and glass reactors provides inexpensive -and versatile methods
of studying atmospheric degradation. However, the development of analytical
.I ,
techniques may be quite time consuming, especially because of the small
sample size. Smog 'chambers have the advantage of a large size (lower
surface/volume and, therefore, less wall reactions; larger analytical
samples and thus lower concentrations of reactants) and the potential for
14
-------
dynamic operation (closer to natural conditions). In addition, it is easier
to control humidity and temperature in a smog chamber, but cleaning is more
difficult than with plastic or glass reactors. Good correleation between labor-
atory and field results has been demonstrated for all systems, perhaps because
of che better reproducibility of chemical systems. Few low volatile compounds
have been studied under atmospheric conditions, although this may be a major
reaction media for photolysis in the environment.
Photochemical processes in other parts (e.g., soil and water) of the
environment are poorly understood. A number of well known environmental
contaminants (e.g. pesticides) have been shown to be photochemically labile
under laboratory conditions. However, the relative importance of the process
to over-all environmental degradation is poorly demonstrated. Laboratory
techniques have included photolyzing aqueous and organic solvent solutions,
thin films, and absorbed films of the test compound both with and without
photosensitlzers. However, extrapolation of the laboratory results to
the field is extremely difficult because of the lack of understanding of the
effect that adsorption on soil or sediment or exposure to natural chemicals
in the environment may have on the photochemical process. Complicating the
problem is the fact that frequently the photooxidative product is the same
as the product obtained microbially. It is suggested that these studies.
have somewhat lower priority for routine testing than do biological or
>i
chemical processes because of the difficulty Inherent in interpreting the
results. When a compound is tested, light containing wavelengths no less
than 290 run and a variety of test media such as water (various pH's), soil,
silica gel, and thin films on glass should be used. Also, the possibility
of sensitized photochemical alterations should be investigated.
15
-------
3
Techniques for Studying Degradation Induced by Chemical Agents ':'
. ' '' «
Environmental processes effected by chemical agents are usually studied
^
* '*
by eliminating the,possibility of biological degradation through sterilization
or using distilled.water. The most frequently studied chemical process,
hydrolysis, is normally determined with distilled water at various pH's. The
(
number of compounds that should undergo this type of testing can be signi-^
% . >
ficantly reduced by considering the chemical structure of, the compound;.
t
Esters, amides, and carbamates are obvious candidates for testing. Compoujids
that have good leaving groups (e.g. halogens) located at positions that would
stabilize a carbonium ion (allylic, benzylic, etc.) also should be tested.
Correlation of results from the laboratory to the field is facilitated by
the ease of measuring in both places the pH, which has a major impact on the
'''.' * .
reaction rate.
Chemical processes in soil can be studied after sterilization of the
soil by autoclaving, chemical treatment or y-irradiation. The sterilization
processes often alter the soil to such an extent that any process observed
! . ' '-.''
could be artificial. Nevertheless, it should be noted that any biodegradation
technique where the test chemical is incubated with a natural medium (e.g.
.-*.'
soil or water) would allow transformations effected by chemical agents to
take place, and, therefore, these processes would be partially considered
during biodegradation testing.
i ' ' .
Techniques for Studying Metal Transformations
The study of metals and metal compounds transformations in the environ-
ment requires a variety of different considerations than for organic chemicals,
. i ; .
, ty. ' ' .
since the metal portion of the compound can not be converted to innocuous
16
-------
end products, as with the organics (C02> H-0). Also, some of the reactions
are reversible and thus the kinetics become very important to the cycling
of the metal through the environment. A variety of processes may take place
such as degradation of organometallic forms to inorganic forms and valence
changes, methylation, or chelation of the metal. As with organic compounds,
natural communities and pure cultures and model ecosystems have been used.
However, in most cases these techniques only answer the qualitative aspects
of metal transformations (e.g. will a metal be methylated). Qualitative
information is important for organometallic compounds and organic compounds,
since the processes are irreversible. However, with metals, methylation and
demethylation can both take place so that the reaction rates are extremely
important.; These reaction rates will vary from test method to test method
and within various environments. Thus, although laboratory techniques are
Important for determining organometallic degradation and the possibility of
metal methylation, the field studies should be used to determine the kinetics
of metal transformations.
Techniques for Studying Polymeric Materials
Polymeric materials, especially plastics, are major environmental
contaminants. However, the tests used with these materials have been oriented
at determining deterioration (loss of commercial function) rather than
degradation. This is reflected in the test methods (screening, end use,
field test) and the analytical methods used (e.g. hardness, embrittlement,
.electrical properties). Nevertheless, the screening, end use, and field
tests used all Indicate that most of the synthetic organic polymers are
extremely persistent, and this Is supported by monitoring data.
17
-------
The apparent recalcitrance to microbial attack of most commercially :
.' ' " '*'' ' ! ' ' '
important synthetic-polymers may well reflect physical nonavailability of
.' ft
the polymer to appropriate extracellular enzymes. This is to say that most
,... ; .... ^ . .. ..., , .. , ;. _
synthetic polymers cannot be assimilated by microorganisms without prior
chain cleavage either by exo-enzymes or physiochemical processes. While
standard tes.t methods do expose the polymers to exo-enzymes and selected
-*4. , '
physiochemical factors such as light and water, the inter-relationships
v " "
between biological and physiochemical degradation are only beginning to be
explored. Further,0the effects of a typical microenvironments is little
understood. Lastly,''the environmental degradation of some polymers may be
an extremely slow but nonetheless significant process and one which ,most
A ' * '''''.
exposure and analytical methods would fail to detect. Thus, more varied and
prolonged exposure conditions and perhaps the use of radlolabelled material
may be required to determine with confidence the environmental fate of polymers.
Conclusions and Recommendations . .,...-
V Review of the techniques available for studying environment persistence
'/''
and pathways of degradation provides no .single .technique that is quick,
reasonably.priced, and provides'information^meaningful to a large number of.
processes in nature." In fact,-none of the methods provide results that can be
more than qualitatively relied upon in terms of persistance of the test chemical
in nature because ofjthe varying conditions encountered in the environment.
A good method would be to radiolabel the compound .and then test for its
persistence and identify the breakdown products in a variety of simulated
* ' . '. ' i.f :
environments (not model ecosystems). This certainly should be undertaken with
chemicals that are toxic and/or are reaching the environment in large quantities.
18
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However, with chemicals that are produced in small quantities or have just
reached commercial production, the justification of such extensive testing
is questionable, unless the compounds exhibit a high level of toxicity. Thus,
it appears that the amount and type of testing to be recommended should be
decided on a case by case basis. :
A number of parameters can assist in the decision making process. The
quantity produced and released to the environment, the persistence, and the
toxicity of the material provide some indication of the environmental hazard
involved and thus the degree of testing needed. Routes into and residence in
the environment provide information on the type of medium that should be used
(sewage, natural water, soil, air). If the release is in only a few places
it might be worthwhile considering a test that closely simulates those places
(e.g. river die-away test). On the other hand, when the release is not well
understood or if the compound is expected to be widely dispersed, it might be
advisable to use more standardized and, therefore, somewhat more reproducible
i
techniques. Physical properties may be useful in deciding the type of
testing and the experimental procedure. For example, non-volatile compounds
should not be tested in atmospheric systems and water insoluble compounds
i
require special experimental precautions to prevent adsorptlve losses. Chemical
structure may also be helpful in setting priorities as well as in estimating
persistence and breakdown pathways. This has been reviewed in detail in
Section VIII., p. ',61.
During this review a number of areas were Identified where research is
needed. Some of these areas are listed below:
Biomass and Metabolic Rate Considerations - Although it would
appear that microorganisms determine the persistence or
degradation of a chemical contaminant, much better biomass
19
-------
and metabolic rate information is necessary to compare
microorganisms with higher organisms. '
Qualitative and Quantitative Comparisons Between Laboratory
; Techniques and the Field - The lack of quantitative comparisons
isv,distressing and is normally attributable to a lack of
!.->''' " :- '
planning for physical transport processes in field monttjor-ing
research. Also, considerably more qualitative compajrispjis
. i '. ' ' '
are necessary.
»
Field Tests to Evaluate Photochemical Processes - Althqugh, a
* J * * i
number of chemicals have been found to be labile to, sunlight,
(
only one compound has been shown to be affected by sunlight
i
attentuation in the field. More field tests are needed in
order to allow the interpretation pf la^pTatory results.
Test a Wider Variety of Chemicals - A considerable amount of
information i% available about pesticides, detergents, and
hydrocarbons but little information is available about other
.' \j .--.i . '» ?rf ; ,r . ' - .1 -.r V . . *'.'''.' «T
chemicals used in large commercial quantities.
Clarify Effect of Nutrient Recipes ^ A variety of nutrient recipes
? .
have been used either for convenience or for the ease of using
. f. -
a particular analytical method. The effect" of varying the
'- ,, - . ' . > '
nutrients is little understood.. :
Determine importance of Cpmetabolism as an Environmental Process -
i" '
A number of researchers have considered to be biodegradable
' /: - ''-... , '':-
those chemicals that can be used as carbon and energy sources.
i.
However,: this does not take into account cometablie processes
20
-------
which could be equally Important and could alter the test ap-
t* ...
proach considerably (add external carbon source).
Study Chemical Structural Relationships to Environmental Stability -
Only a few studies were uncovered which attempted to correlate
chemical structure to environmental persistence. More under-
j«' k
standing along these lines would be extremely helpful. Few
attempts have been made to correlate many physical properties
of chemicals to their biodegradability.
' '
Determine Kinetics of Various Metal Transformation Reactions in
the Environment - Prior to doing this though,'it might be
necessary to understandVthe actual role of different biological
systems in transformation. For example, at the present time
it is normally assumed but .not proven that microorganisms
methylate metals which are then,taken,up, by higher food chain
organisms. Also more efforts should be made to find ways
for predicting the ability of a metal to undergo methylation
' i " . . . '
' «' in the natural environment. t ":'..
21
-------
I. INTRODUCTION
A. General
Commercial development of chemicals has had a beneficial impact on
the way man works and lives (ACS, 1973). However, in many cases, these com-
mercial applications have resulted in an increased burden of chemical contam-
inants in the environment. The Council on Environmental Quality (1971) has
suggested that "selected metals, their compounds, and certain synthetic organic
/
chemicals are perhaps the best examples of toxic substances which can adversely
affect man and his environment."
A major factor for assessing the potential hazard of these environ-
mental contaminants is the consideration of the chemicals' environmental persis-
tence and degradation products. For example, it is quite possible that "highly
toxic, readily biodegradable substances may pose much less of an environmental
problem than a relatively harmless persistent chemical which may well damage
a critical wild species"' (Goodman, 1973). This report reviews and evaluates
techniques which have been used to determine the environmental persistence and
degradation pathways of various chemicals.
The importance of knowing the alterations and ultimate fate of a
chemical in the environment can vary depending upon the chemical. Under-
standing the environmental alterations of metals and organo&fttallic compounds
is^required because the toxicity and .mobility of the material may be greatly
affected by various changes in chemical form. In addition, the kinetics of
the alteration reactions in the environment may be extremely important since
some of the degradation routes (e.g. methylation-demethylation) are reversible
(Wood, 1974).
Preceding page blank
23
-------
With organic; compounds, environmental- reactions are usually irrever-
I ' ''
sible aridj furthermore, the organic compounds can potentially degrade to water
and carbon dioxide. For organic compounds, the term "persistence" has much
more relevance, and the kinetics of the reactions, although still quite impor-
tant, have received somewhat less laboratory study than the qualitative assess-
ments of whether a .degradation process takes place or not. This isiundoubtedly
due to the^ fact that modelling the dynamic environment in the laboratory i;s;
,i
most difficult in that the environment consists' of numerous microenv-ironments
which can vary considerably in temperature, pH, microbial population,-etc.
Thus, quantitative data-developed'under one set of laboratory conditions^ may
!
be somewhat misleading for another set of environmental conditions, while the
qualitative indications of environmental degradation may, be relied upon in
(
many different microenvironments.
Some definition of nomenclature is necessary. Such terms as environ-
i
mental persistence, alteration, biodegradability and degradabilityjare lacking
in precision and are often dependent upon, the: perspective of" the author. .Soil
i
scientists frequently refer to pesticides that are'not retained in the soil
for long periods of time as non-persistent:,: although, in many cases, the loss
may be due to physical removal processes, such as vaporization and leaching
(resulting in dilution but not destruction). Biodegradability is frequently
r
v
considered by detergent scientists as synonymous with treatability or remov-
ability in a sewage^ treatment plant. The interpretation of these terms used
i '' ' '
in this report Is briefly reviewed.
The terms persistent and degradable are applied in this report only
1 s
to the total environment rather than any individual medium (air, soil, water)
24
-------
and, therefore, any transport processes of a chemical are irrelevant to a
determination of persistence or degradability other than it may change the
possible reactions that may take place (e.g., vaporization into the atmos-
phere almost eliminates the possiblility of biodegradation). Degradation
means "to reduce the complexity of a chemical compound by splitting off one
or more groups or larger component parts" (WPCF, 1967). Biodegradation means
that the destruction process is accomplished by the action of living organisms
(e.g. see Swisher, 1970). Environmental alteration means that the
compound has undergone a chemical change that is not necessarily a reduction
in the complexity of the molecule.
Biodegradation has been further divided into the following three
categories (WPCF, 1967):
"1. Primary Biodegradation - Biodegradation to the minimum extent
necessary to change the identity of the compound.
2. Ultimate Biodegradation - Biodegradation to (a) water, (b) carbon
dioxide, and (c) inorganic compounds (if elements other than C, H and
0 are present).
3. Acceptable Biodegradation - Biodegradation to the minimum extent
necessary to remove some undesirable property of the compound such as
foaminess or toxicity."
Primary biodegradation is basically the loss of the parent compound, although
this is somewhat dependent upon the analytical methods used. Ultimate degra-
dation is the complete mineralization of the organic chemical, the third
category falls between the two extremes but results in perhaps the haziest
definition. In another approach, Dagley (1972b) has suggested that a man-
made compound is biodegradable if it is attacked by the enzymic apparatus
acquired by microbes during the course of evolution. Painter (1973a) has noted
that two general criteria have been used to assess the biodegr'adability:
25
-------
,
(1) the rate and extent to which natural or enriched mixed cultures degrade
the compound, (2) the proportion of tested species, strains or isolates whichi
are able to use the compound as a sole source of carbon and energy. Prochazka
and Payne (1965) feel that the ability to isolate microorganisms that use the
test compound as a sole source of energy and carbon is the best criter-i*a 'for '
biodegradability.
These definitions are obviously only of a qualitative nature. .'As
mentioned earlier, this is due to the numerous Chemical compound's to 'which
. 4
the definitions are applicable and the lack of precision Inherent in biological
'i
systems, this non-quantitative nature is in contrast to the half-li'fe 'of an
isotope, for example, where the rate is independent of environmental events
:. t
(Dagley, 1972b). thus, assessment of a chemical's persistence or degradation
rate in the environment requires that the compound be evaluated in some type
;
of test method or technique and the results are billy meaningful when stated with
*
the conditions of the technique, the components essential to such techniques.
are the test chemical, the analytical method, and the environmental component.
i
A good test method" should meet the following criteria: (1) be as simple as
*
possible, (2) be as economical as possible, (3) be reproducible and (4) report
results which can be correlated to field conditions (see SDA, 1965). These
* . . . '
components of various techniques have been reviewed and evaluated in detail
s . 1
I
in the following sections.
- '* ' .' . ' >'
Well over 9,000 synthetic organic chemicals are produced in commercial
X1-
. I .'*
quantities (Council on Environmental Quality* 1971) and annually total approxi-
mately 140 billion pounds in the United States (U. S. Tariff Commission)..
Iliff (1972) has suggested that world-wide up to 40 billion pounds of manufactured
26
-------
organic chemicals enter the environment annually. Included are organic com-
pounds used as pesticides (over 1 billion pounds, U.S. Tariff Commission),
which are Intentionally distributed in the environment, and detergents (Swisher,
1970; Anon., 1963), solvents (Levy, 1973; Laity e_t al. , 1973), plastics (25
billion pounds in 1972), chemical intermediates (34 billion pounds in 1972),
plasticizers (1.7 billion pounds in 1972) and other compounds, which may be
inadvertently or accidently released during manufacture, transport, use, and/or
disposal. Compounds, such as polychlorinated biphenyls (PCB1s), phthalate esters,
hexachlorobenzene, and other organic chemicals (e.g., Miller, 1973; Kleopfer
and Fairiess, 1972; Little A.D., Inc., 1970), are typical of the latter
9
category. In addition, it has been estimated that more than 2 billion pounds
of oil are lost at sea annually (Friede e£ al.., 1972) and about 9.5 billion
pounds of total plastic wastes accumulate each year (Potts ejt al. , 1972).
These different categories of organic chemicals enter and reside in
different parts of the environment. For example, detergents usually pass
through a biological treatment plant before entering streams, rivers, lakes,
etc. Pesticides are normally sprayed on crops or, if they are aquatic herbi-
cides, they may directly enter aqueous systems. Those solvents which are fairly
volatile vaporize into the atmosphere. From the initial entrance point, all
of the above materials may be biologically or chemically altered or physically
transported from one point to another. Understanding the mobility and physical
transport mechanisms of a chemical is important in terms of determining the
potential effect of the environmental contaminant (Oak Ridge National Laboratory
under contract to EPA is reviewing techniques used to study the transport of
27
-------
chemicals in the environment). However* as long, as the pollutant remains,in
i
existence, it can enter the food chain or affect man directly (Alexander,
: v"
1967). On the other hand, if the organic compound is completely mineralized
to inorganic material (CO., H-O), its potential for being a pollutant is
*
removed. The possible biological and, chemical degradation processes, which may
take place in the environment are reviewed in Section II (p. 33). In
addition, biodegradation of chemicals under biological treatment condltdpna-
is considered since a compound's fate in these systems can be the dete^ining
factor in whether the compound becomes, an environmental contaminant, oj, degrades
to innocuous material.
28
-------
B. Methods and Approach
1. Literature Search
Papers, books and review articles relevant to this review were
gathered by a variety of approaches. Extensive use was made of such abstracts
as the Air Pollution Abstracts, Biological Abstracts, Bioresearch Index, Chem-
ical Abstracts, Water Pollution Abstracts, and Pollution Abstracts. Since
these abstracting services all have some amount of delay between when the
article is published and when it appears in the abstract (approximately 3-6
months), a search of recent, highly relevant journals was also undertaken.
(October 1973 - March 1974). In addition, a computer search was performed of
the reports published by the National Technical Information Service (NTIS) and
the current investigator files of the Smithsonian Science Information Exchange
(SSIE).
. The abstracts, obtained in this way, were screened for relevant
articles and the papers deemed pertinent were collected and examined. The
bibliography of highly relevant articles was examined to provide a double
check on the abstract search. In some instances, a citation search of
extremely pertinent,articles was conducted using Science Citation Index. :
Individual researchers, who were either professional acquaintances.
of the authors of this report or were identified by the SSIE computer search,
were personally contacted and requested to supply any recent articles they
might have published or have in press. In addition, in a number of cases, per>-
sonal visits were made to these researchers' laboratories in order to discuss-';
experimental techniques used. Furthermore, a number of experts in the field ,
-,
have been asked to review drafts of the final report.
29
-------
".- " '' ' * \
i
Unfortunately, no articles have reviewed the cost of the various
techniques and, therefore, the prices that are quoted in this review are only
i
estimates. Information which was used to calculate these estimates included
>'
(1) time requirements cited in papers, obtained from researchers, or estimated '
' » '
by the authors of this report, (2) complexity of the procedure and nece's'sary
technical training of the personnel, and (3) capital investments inequipment.
The cost may fluctuate considerably depending upon the chemical being ;tested
and the laboratory doing the test and, therefore, the projected costs have
only an order of magnitude reliability.
2. Scope and Organization of the Report
i
This report reviews techniques that have been used to study the
persistence and breakdown of chemicals in the water, soil, and air medium of
the biosphere. With the exception of the model ecosystem technology, techni- .
ques used to study metabolism of chemicals in higher trophic levels (e.g., plants,
fish, mammals) have been excludedi It was felt that these processes, although
very important to considerations of bioaccumulation and toxicity, are less
relevant to environmental degradation and persistence. A full discussion of
this rationale is .presented in Section II A. (pi33 ).
This report is organized in a-variety of categorizations. In
some instances^ the type of chemical has determined the category (e.g., metals
ivij .
and polymers). In other cases, biodegradatibn ;is divided by different media
(e.g., soil, water, -sewage). In one instance, the type of degradation
:--\ ' . . ' '.
(chemical and photochemical) is the distinguishing factor. For the most part,
these categories were chosen as a matter of convenience in'reviewing the avail-
able information. ,
30
-------
Most sections are divided into (1) a review of the techniques
(2) analytical techniques (3) evaluation of the techniques, and (4) a cost
analysis.
The quality and quantity of the information has varied con-
siderably depending upon the chemical and the medium. By far, the most in-
tensively studied group of chemicals in their appropriate medium have been the
pesticides in soil and water, the detergents in sewage and water, and the hydr
carbons in the atmosphere. The approaches and techniques used with these
materials have been reviewed for their applicability to other chemical groups
of potential environmental contaminants.
Most of the techniques reviewed are applicable to a determinati
of degradability or persistence. Techniques for studying the routes of degra-
dation have not been reviewed in a great amount of detail since they are very
similar to procedures used to study metabolic pathways of natural substrates.
31
-------
II. TYPES OF DEGRADATION IN THE ENVIRONMENT
*,
Chemicals which are placed in the environment are subjected to chemical
alterations and physical processes by sunlight, water, soil, inorganic and
organic material, and biological agents and by the combined weathering
action of rain, wind, temperature and humidity (Van Middelem, 1966). The
physical processes, such as adsorption on colloidal substances, volatilization,
bioaccumulation or leaching, have a tendency to distribute and dilute or
concentrate but not destroy the contaminants. On the other hand, chemical
and biological reactions in the environment result in alterations and fre-
quently degradations of the material to innocuous substances. The alteration
and degradation processes can be divided into three general categories: (1)
biodegradation - effected by living organisms; (2) photochemical degradation -
nonmetabolic degradation requiring light energy; and (3) degradation effected
by chemical agents (chemical degradation) - nonmetabolic degradation which
does not require sunlight. Alexander (1967) has suggested that of these three,
biological degradation of organic compounds is the most desirable because it
results generally in end-products that have been completely mineralized to
inorganic compounds. In contrast, photochemical and other nonmetabolic processes
usually result in only slight modifications in the parent compound. However,
all of these processes are important from the standpoint of the environmental
fate of the chemicals, and will be discussed in detail In the following
sections.
A. Biological Degradation
In order to insure growth, maintainance and development, living
systems must obtain carbon, other essential elements, and energy from their
Preceding page blank
33
-------
environment. For non-photosynthetic organisms, this process involves the
:3
absorption of various naturally occurring organic compounds, the subsequent
metabolism of these compounds to yield energy and essential elements which can
be incorporated into the structure of the organism, and the elimination of
metabolic products. Although biological transformations of organic chemicals
generally occur via relatively specific processes, there is sufficient latitude
or non-specificity in many organisms to allow them to perform a significant role
in the degradation and/or removal of various synthetic chemicals which are
introduced into the environment. The factors governing this ability to degrade
a natural or synthetic compound are: (a) the compound must be able to reach
the organism or the enzyme site; (b) the compound must not be lethal; (c)
.the enzymes necessary^to alter the chemical must be present or able to be
induced; and (d) the environmental conditions must permit the operation of
i ' ' .
the enzyme(s) (Alexander, 1966). Thus, if absorbed by a given organism, a
synthetic chemical may be (a) degraded to serve as a source of carbon and/or
V
energy; (b) degraded by cometabolic processes (see Horvath, 1972a); (c) slightly
modified and then stored or excreted; (d) stored or excreted without chemical
alteration. Frequently there is a lag between the exposure .of the chemical
to the biological agent and the beginning of degradation or transformation.
In the case of microorganisms, the lag is attributed to the need for
f~
"acclimation." This term will be frequently referred to in this report and,
therefore, needs to be clarified. Acclimation is a broad term, usually
referring to a variety of processes (such as enzyme induction, selection of
a species of microorganism, etc.) which take place during the lag period.
Although biological degradation might conceivably be accomplished
by any living organism, available information indicates that by far the most
34
-------
significant biological systems involved in ultimate blodegradation -
degradation to CO., H.O, and other inorganic- compounds - are the bacteria and fungi.
This conclusion is based on(l) what is known about the metabolic pathways of
bacteria, fungi and higher organisms; (2) the projected metabolic activity
of various organisms, and (3) the availability of synthetic organics to the
bacteria and fungi. The following paragraphs are devoted to an elaboration
of the above factors.
1. Metabolic Pathways
Various forms of life Including wild life, fish, various marine
species, domestic animals and even many have been shown to contain some residues
of environmental chemicals (Smith and Isom, 1967; Davis and Hughes, 1965;
Rawls, 1966; Durham, 1969; Edwards, 1973; Holden, 1970). Whether the eventual
loss of chemicals from these organisms is due to metabolism or excretion ia
unclear. "toefl!ier~anld~VanOverbeek""(i9'7i) have stated that "organisms with an
effective excretory mechanism [which perhaps includes most animals] will, as a
rule, not degrade the compound that does not fit into the normal sequence of
metabolic events as completely ae do those organisms in which this means of
disposing of a compound is absent." This is based on the fact that a compound that
is sufficiently water soluble (allows excretion) requires less energy for excretion
than does the large number of reactions required to degrade a compound into
small molecular fragments or even gaseous products (Loeffler and VanOverbeek, 1971).
On the other hand,'-water insoluble compounds, e.g., chlorinated hydrocarbons,
insecticides, tend to accumulate, and storage of residues often occurs in the
adipose tissue of the organism (Edward, 1973; Kenaga, 1972).
Higher plants usually do not have an efficient excretion apparatus.
The metabolic tendency in such cases is to convert the chemical into some
neutral, water-soluble form in which it can be stored in cell vacuoles (Crosby,
35
-------
1973). Examples of these transformations prior to storage include (1) con-
M
' ' ;''
version of naphthaleneacetic acid to its glucose ester, followed by the forma- '
tion of the: more stable amino acid conjugate (Veen, 1966), (2) conversion of
the insecticide carbaryl into persistent glycosylated metabolites (Kuhr and
Casida, 1967); and, (3) binding of certain pesticides to plant proteins (Brian,
; *
I960) or incorporation into structural lignins;, pectic substances, etc. (Chin
, . f
ejt al., 1964; Meagher, 1966).
In contrast to the'metabolic processes of high organisms,, the,
.i
catabolic versatility of bacteria and; fungi suggests that these organisms are
i
likely to play a major role in. the ultimate degradation of synthetic chemicals
which enter the environment. Alexander (1973a) has stated that all evidence
points to the fact that microorganisms are responsible for converting to in-
organic products many complex natural and synthetic organic molecules which
cannot be significantly, altered by higher life forms. Enzymatically, the
degradative activity of bacteria and fungi may be based on their ability to
catalyze the initials-steps in degradation resulting in metabolites that can.
enter existing metabolic pathways (Dagley, 1'972'a). Furthermore, microbial
communities in the environment seem: to vary more in the number of different
microbial groups present. This high:species diversity in microbial communities
t
results in a multitude of possible biochemical pathways which may provide a
. *.
major advantage to microorganisms over other organisms in catalyzing degradation
i
of the many structurally different environmental chemicals.
Invertebrate forms including, the protozoans and lower metazoans
have not been as Intensively studied as the vertebrates, higher plants, bac-
\*
teria, and fungi. These invertebrates have not as yet been directly implicated
36
-------
in the ultimated degradation of synthetic chemicals although they are associ-
ated with the dispersion of such compounds (Wright, 1971) and thus make the
chemicals more accessible to the bacteria and fungi (Alexander, 1966).
A great number of studies can be cited in which a compound has
been found to be degraded by the action of microorganisms but not by higher
organisms. Funderburk (1969) has reported no metabolism of paraquat in higher
plants. Numerous microorganisms have, however, been found to use paraquat as
a sole source of carbon or nitrogen (Baldwin e_t al. , 1966; Anderson and Drew,
1972). DDT, a pesticide well known to be persistent in organisms of higher
trophic levels (Edwards, 1973), has recently been shown to undergo extensive
degradation by the combined action of two bacteria, as shown by in vitro studies
of Pfaender and Alexander (1972). Another good example is the herbicide endo-
thal. Sikka e* al. (1974) have shown that blue gills removed less than 1% of
the total amount of herbicide; the removed amount was present in the fish in
unchanged form. Shake culture and aquarium studies (Sikka and Saxena, 1973;
Slkka and Rice, 1973) have, however, shown that endothal is rapidly degraded
by microorganisms. When mixed function oxygenases from liver microsomes
(from animals) attack naphthalene, an epoxide is formed which may isomerize to
give naphthol, or undergo enzymic hydrolysis to the trans-dihydrodiol; the
product formed is usually excreted as conjugates (Gibson, 1972; Dagley,
1972b). By contrast, microorganisms oxidize naphthalene and break down the
rings to provide a source of carbon and energy for growth (Gibson, 1972;
Dagley, 1972b).
Degradation of several natural materials by microorganisms can
further substantiate the unique role of microorganisms in governing environmental
37
-------
persistence of chemicals. Pristane,.an isoprenoid alkane which is formed by .
*' -I
crustaceans of the genus Calanus, has been found to accumulate unchanged in the
.locations .where it is not susceptible to microbial attack (McKenna and Kallio,
1971; Dagley, 1972b). Under favorable conditions, however, pristane is readily
* -
i . .
metabolized by a variety of microorganisms {McKenna and Kallio, 1971; :Dagley,
1972b). Another natural product, atrppine, -which is probably even more ;toxic
than many pesticides, has been found to serve as a source of carbon'for .certain
microorganisms (Niemer e_t al., 1959; Niemer and Bucherer, 1961). In other
i
living forms, these compounds evoke mechanisms of detoxification and elimination
(Dagley, 1972b).
This relative metabolic advantage of microorganisms .over-high
organisms might be,attributable to the difference in their evolutionary pro-
cesses. Microorganisms, for the most part, reproduce more rapidly and have
£'...
a high rate of mutation, thus allowing them ito-develop enzyme systems over
the .years that will metabolize a ,greater variety of organic structures. How-
ever, the diversity in microbial communities'.which provides a multitude of
possible biochemical pathways is .probably even more important.
.1- .....'
2. Metabolic Activity
The amount of material consumed by any given group of organisms
.will depend largely on the product of their biomass and metabolic rate/unit
-; . .. / ' - - ':
mass. In general-, the rate of metabolism increases with increasing surface/
.'-«
volume ratio (Thimann, 1966).
Organism Surface/volume ratio
200-lb. man 0.3
,'r '
Hen's egg 1.5
Amoeba 400
Bacteria (0.5p or less) 120,000
38
-------
The bacteria and fungi may thus be expected to be immensely more metabolically
active per unit weight than higher life forms. For example, a lactose-fermenting
bacterium will break down from 1000-10,000 times its own weight in lactose in
one hour, whereas a man would require 250,000 hours to break down 1000 times
his own weight in sugar (Thimann, 1966).
An analysis of the metabolic activity of different organisms
in soil by Macfadyen (1957) has demonstrated the importance of bacteria in
a limited ecosystem (see Figure 1). The data indicate that bacteria alone
account for the major portions of the total metabolism in the soil.
Figure 1: Percent Total Metabolism , ers
by Different Taxa of a Spiders
Hypothetical Soil Community Fly Iorvae
in a Meadow (Macfadyen, 1957) Beetles
Reprinted from Animal Ecology. Springtails
Aims and Methods, Copyright o .
Pitman Publishing. Protozaa
Nemoiodesl 16
[Bacteria 65% |
This can be attributed to a much larger magnitude of biomass and higher meta-
bolic rates of soil microorganisms compared to soil animals (Table 1)
(MacFadyen, 1963). Dagley (1972b) has also estimated that microbial biomass
.(including algae) is much greater than that of the combined animal biomass.
This approach of analyzing ecosystems on the basis of metabolic
activity is particularly relevant to an assessment of the relative importance
of various taxa in that it illustrates the relative rates of energy consump-
tion, which is related to organic material consumption.
39
-------
- . i
Table 1: Figures From a Hypothetical Grasslands. Soil Community Showing
Order of Magnitude of Numbers, Biomass, and.;Metabo!ism for the
Main Biological Groups (Macfadyen, 1963)
Metabolism
Group of
Organisms
Bacteria
Fungi
Protozoa
Nematoda
Lumbricidae
1
Enchytracidae.
Mollusca
My ri apod a
Isopoda
Opiliones
Acari-
Parasitids
Oribatei
Araneae
Coleoptera
Diptera
Collembola
Weight of
Organism
(mg)
_9
10
i
0.05
0.001
5000
0.14
1500
25
22.7
18
0.2
0.25
10
250
610
V
: .0.46
Approximate estimates grassland
per sq m
Numbers
15
10
8
5 x 10
7
10
3
10
5
10
50
500,
500;
40 :
5 x ,10 J
2 x ID.1?
600,
100
200;,-
5X1P*
"
Mass (g)
1000
400
38
12
120
12
10
12.5
5
0.4 .
1.0
2.0
6,0
1.0.
1.0,
5.0
Calories, per
day.per^g
at* 16°G:
575^
16-1;
14,
i
144,
7
100
29
36
36
53
280
72
27
39
29
144
3. Availability of Synthetic Organics to. Various Taxa
Even given the high order of metabolic activity and versatility
shown by the bacteria and fungi, their role in biological degradation might
40
-------
be somewhat limited if significant amounts of synthetic organic chemicals
were stored by higher organisms, thus making the chemicals unavailable for
. ' ! ' ' V.
microbial degradation. However, such a situation does not seem likely in
.'.-'.,' i . i
the< environment. For example, Edwards (1973) has calculated the amount' of ',
orgaaochlorine insecticides which is locked up in the soil biota. The
calculations are based on the average figure of 1.0 ppm of organochlorine
residue in the living organisms. Considering that there Is an average of ;
: .j
25 tons of living organisms per ha (Stockll, 1950), the amount stored in the '
biota will be equivalent to 0.025 Kg/ha (0.022 Ib./acre) (Edwards, 1973), which
is in the range of 1-2% of total (of many herbicides) normally applied;per
acre (application rate: 1-2 Ibs. actual/acre, Thomson, 1967). DDT may be ,
typical of this group of Insecticides. Woodwell and coworkers (1971) have
calculated that the total biota in oceans, fresh water and soil probably con-
tains 3.3% or less of the amount of DDT produced in one year (during the mid-
1960 fs). Compatable with this projection, the Study of Critical Environmental
I : '.
Problems (SCEP, 1970) estimates that of the 500,000 tons of DDT existing in
the worlds oceans, only 0.12% is stored by fish. Although the data presently .
available is somewhat fragmentary, it does suggest that synthetic organic
chemicals which are not readily degradable by higher life forms are also not
stored in significant amounts in higher life forms and are thus available for
microbial degradation.
Thus, the bacteria and fungi seem to serve and will probably
continue to serve as the major factors in the ultimate biodegradatlon of
synthetic organic chemicals in the environment. Consequently, this review
has focused on techniques which utilize for the most part microorganisms ;
as the biological agents of degradation.
41
-------
"' B. Photochemical Degradation *
The photolysis of a chemical in the environment appears to be an
/ '"
important process which may have a considerable effect.on a chemical's per-
sistence and degradation. This is especially true of atmospheric contaminants
(Altshuller and Bufalini, 1971; Leighton, 1961) and many organic pesticides
(Crosby and Li, 1969; Crosby, 1969a,b; Plimmer, 1970).
. t
In order for a chemical to react photochemically, it must^benable?
to derive energy from the incident light available to it. In the environment,
sunlight is the incident light source. The ozone in the atmosphere.-affectively
absorbs all sunlight
-------
900
115
800
o
CM
600
400
200^
95
82
H - CH2 OH
250
Figure 2:
300
350
X,
72
63
400
450
Spectral Energy of Sunlight (B) Relative
to Chemical Bond Energies and Low-Pressure
Mercury Arc Lamp (A)
(modified from Crosby, 1969b and Crosby, 1972a)
A molecule may become excited by direct absorption of light or by
accepting energy from an excited donor molecule (sensitizer). The ultra-
violet absorption spectra provides an indication of the ability of the
molecule to absorb energy at various wavelengths (extinction coefficient),
and that is dependent upon whether the transition is "allowed" (determined
by selection rules, see Jaffe and Orchin, 1962). This absorption is very
dependent upon the matrix of the molecule (vapor phase, absorbed on a solid,
dissolved in various liquid). In fact, it is quite possible that a compound
43
-------
that does not absorb at">290 run in a solvent such as hexane, may absorb
light in the environment. For example, Plimmer (1972b) has noted a 60 ran
red shift for trifluralin (a nitroaromatic herbicide) when it was adsorbed
on silica gel. Sensitization occurs when an acceptor molecule has an excited
state of correct energy relative to a donor molecule and it requires some
interaction between the; donor and acceptor.
Once the molecule is excited, it can release its energy in -several
non-chemical ways (Figure 3) including fluorescence and phosphorescence
I
(irradiative decay) and radiationless decay (energy transferred to -the matrix).
On the other hand, the molecule may use the energy to chemically alter the
>
molecule. The efficiency of each of these processes (usually stated as
quantum yield) is,again somewhat dependent upon -the matrix.
Excited^ Electronic Stoles and
Transitions Between States
btorptlon
Figure 3. Modes of Formation and Deactivation of
Molecules in the Excited State (Owen, 1971).
Reprinted from Organic Chemicals 'in Aquatic
FnviroiCTentfl. by courtesy of Marcel Dekker, Inc.
44
-------
Photochemical alteration can also occur by the Interaction of the
compound of Interest with a photochemically excited molecule (e.g., singlet
oxygen, Pllmmer, 1972a, Crosby, 1972b; or diethylaniline with DDT, Miller and
Narang, 1970). Again the neighboring molecules are extremely important to
the process.
Because of these effects which are induced by the matrix, laboratory
simulation of environmental photolysis is very difficult. The conditions that
are used are sometimes very remote from natural conditions and the results
obtained should be extrapolated with a great deal of caution.
C. Degradation by Chemical Agents
Other nonmetabolic decompostion processes besides photolysis may
also be important in the environment (Rosen, 1972 a; Crosby, 1970; Crosby, 1969a)
Ubiquitous substances, such as air, water, soil, etc. may react with or
catalyze environmental degradation or alteration reactions. Unfortunately,
with the exception of hydrolysis processes, relatively few non-photolytic
chemical (as opposed to biological) reactions are known (Rosen, 1972a)-.
Crosby (1970) has suggested that this low number of reported chemical
reactions, at least in soil, is due to a lack of distinction between biological
and nonbiological reactions in experimental studies. Such differentiation
is extremely difficult because of the difficulty of sterilizing soil without
changing the soil structure and chemical composition. In contrast, this
distinction between the two processes (biological and chemical) is extremely
simple in reactions that take place in the atmosphere since biological
processes are minimal. Some of the major non-photolytic chemical processes
/
in the environment will be briefly reviewed.
45
-------
The free-radical character and solubility in water make oxygen a '
most effective environmental reactant (Crosby, 1970). Many compounds upon
. i
exposure to air will be readily oxidized. In many cases the presence of light
accelerates the oxidation process but a considerable number of reactions can
v
take place in the dark (Crosby, 1969a).
Pesticides such as phosphites, sulfpxides and dithiocarbamates are .
readily oxidized tdsphosphates, sulfones and thiuram disulfides, respectively.
Oxidation reactions are particularly facilitated in the atmosphere (Haagen-
Smit and Wayne, 1968) where the medium consists of 20% oxygen. Other oxidation
reactions initiated by oxidants or free radicals formed in photochemical smog
may also be important to the environmental fate of a chemical substance (Crosby,
^4'
1969a). "
^i
Water, besides being an important medium for chemical reactions, is
reactive itself through its ability to form either hydroxyl or hydrogen ions.
Consequently, pH becomes important in nonmetabolic decompostion of chemicals in
an aqueous medium. Normally drinking water pH is between ;5 and 8, although
values as high as.10.5 have been noted (Crosby, 1969a). In soil, the water
is seldom neutral and can vary from a ,pH of 3 to 10.5 (Crosby, 1970). Al-
though solvolysis .and elimination reactions have been noted (Rosen, 1972a).
Crosby, 1969a), the most widely recognized and studied reaction with water
is hydrolysis. The reaction is usually .pH dependent and can be catalyzed by
i; " ;. . .
various agents (e.g., hydrolysis of parathion is catalyzed by common amino
acids, hydroxylamine derivatives, metal ions, and metal ion chelates). With
brganophosphorus esters, the hydrolysis reaction mechanism is different under
acidic or neutral conditions (alkyl-oxygen bond is attacked) compared to under
'I.:
alkaline conditions (phosphorus-oxygen bond attacked).
46
-------
Many chemical substances that enter the environment become associated
with soil and dust particles. Some mineral constituents of soil, such as
clays, can act as catalysts. For example, they can cause endrin to isomerize
from an epoxide to a ketone. The organic constituents of soil can serve as
oxidizing and reducing agents since they contain a relatively high concentration
of stable organic free radicals.
All the above processes can be accelerated by an increase in
temperature and, therefore, temperature is an important degradation parameter.
In general, non-photolytic reactions effected by chemical agents
have received considerably less attention than other environmental processes.
However, it should be kept in mind that in many systems used to study bio-
logical or photochemical degradation, degradation by chemical agents cannot
be excluded.
47
-------
III. BIODEGRADATION OF CHEMICALS IN AQUATIC OR SEWAGE TREATMENT CONDITIONS
Organic chemicals entering the aquatic environment can be degraded by
chemical or biological agents. Degradation techniques which are used to study
processes effected by chemical agents are reviewed in Section V. p, 283. In thij.s
section, the more frequently used biodegradation techniques are reviewed. The
section has been divided into two major subdivisions; (1) static tests which
use low bacterial concentrations and simulate natural water systems, and (2)
dynamic systems which maintain high concentrations of bacteria and thus more
closely simulate conditions of a biological waste water treatment plant.
A. Techniques for Determining Biodegradation of Chemical Compounds
in the Aquatic Environment
Chemical compounds can enter water in a variety of ways. These include
runoff from land, discharge of industrial waste, home use and garbage disposals
(sewers), dumped through carelessness and accidents, and direct application to
water to control pests. Oceans are contaminated by oil at an estimated rate of
5 million tons per year (Blumer and Sass, 1969; Blumer ea al., 1973). Chemicals
can also enter the marine environment due to run-off from treated lands into
the rivers and finally into estuaries and oceans. Even when present at Iqw con-
centrations, these compounds may be hazardous since a large number of them can
bioaccumulate in the food chain.
The origin of microorganisms in the aquatic environment is not clearly
understood. It has been suggested that the variety of microorganisms present in
natural waters is of allochthonous origin; e.g. , they are Introduced from the
washings from soil or with sewage (Wuhrmann, 1964). Specialized types of micro-
organisms may be found in a stream due to the unusual chemical conditions, with
regard to its Inorganic and organic composition. The environmental conditions
Preceding page blank
49
-------
in the aquatic system differ in many other respects from those of soil and
' ' l
sewage. The differences, which include concentration and types of organic
- . I fl
I * !
.chemicals, microblal community, supplemental nutrients, light conditions, etc.,
can significantly affect both the extent and rates of biodegradation. Natural
-. j »
water represents an extremely diluted medium where continuous' replacement of
A » ' ,* »
the metabolic substrates and removal of end products takes place due to
-------
or less regarded as a !blodegradability test method, it has been considered as a
separate class.
1. Biochemical Oxygen Demand
Many organic compounds are degraded by natural microbial com-
munities in the presence of oxygenwhich usually results ultimately in the
conversion of the substrate acted upon to CCL, water, and other inorganic pro-
ducts. Consequently, measurement of oxygen uptake has proved to be a useful
method to estimate the extent of biodegradation. However, interpretation of
the oxygen measurement result is somewhat difficult since microorganisms oxidize
organic matter to carbon dioxide and water while simultaneously also synthesizing
new cell material, metabolites, etc., which have oxidation states different than
that represented by carbon dioxide and water. In most instances, therefore, the
measured biochemical oxygen demand is considerably less than the amount of oxygen
needed for complete oxidation (calculated theoretically).
Several methods which have been used for determination of biochemical
i
oxygen demand can be grouped into the following categories:
a. Dilution Method
The BOD test consists of measuring the depletion of dissolved
oxygen during or after a period of incubation. The dissolved oxygen level is
normally measured either by chemical methods, or by the use of an oxygen-sensi-
tive electrode. The oxygen available for degradation is limited to the amount
which is dissolved in the water at the start in the test (8-10 ppm, Swisher 1970).
This limits the initial concentration of the test compound as well as the size of
inoculum in the test system (to obtain the desired range of oxygen depletion in
a given period). The temperature is usually maintained at 20°C, which is similar
to the temperature of surface waters during summer.
51
-------
(1) Standard 5-day BOD procedure '
i* ' ' .
The procedure is described in detail in the standard manual
tf '
of the American Public Health Association (APHA, 1971, p 489).
-« , (
The test uses the incubation bottles of 300 ml capacity having
i
a special shape designed to hold a water seal so that oxygen
. ' . ii.
transfer from the atmosphere into the bottle will be minimal.
J '
,1
Dilutions are prepared for each test chemical so that oxygen
depletions fall in the range of 2 to 7 mg/S. (convenient range
for measurement);
\
In the standard BOD tests, the dilution water which con-
tains necessary trace nutrients for proper growth of micro-
4
organisms is prepared (APHA, 1971, p 489), and then the water
=jt ' ,
is seeded at a concentration of 1-2 ml/fc of dilution water
r '
with settled domestic sewage which has been stored at 20°C
for 24-36 hours. In certain instances specialized seed material
containing organisms adapted to the use of the organic compounds
may. also be used. Such adapted seed is often obtained from
the effluent of a biological treatment process receiving the
waste in question. Adapted seed may also be developed in the
r 3.>
laboratory by continuously aerating a large sample of water
'.> ':
and feeding it small dally increments of the test chemical
together with soil or domestic sewage.
The effect of various seed types on BOD results is shown
in table 2.
52
-------
Table 2. Effect of Seed Type and Quality on BOD Results
......_ JCAPHA,_li7D
5-day
Seed Mean Standard
Correction 5-day BOu Deviation
Type of Seed
Settled fresh sewage
Settled stale sewage
River water (4 sources)
Activated-sludge effluent
Trickling filter effluent
mg/A
>0.6
>0.6
0.05-0.22
0.07-0.68
0.2-0.4
mg/fc
218
207
224-242
221
225
mg/fc
±11
± 8
±7-13
±13
±8
* BOD In the absence of added carbon source
** For Standard Solution (150 mg each of glucose and glutamic
acid)
The bottles are incubated for 5 days in the dark at 20°C
and then the dissolved oxygen in the samples is determined
using an iodometric method or a membrane electrode. A summary
of variations employed in the test method as used by various
investigators is given in Table 3.
For this test the American Public Health Association has
recommended that the quality of the dilution water, the effec-
tiveness of the seed and the technique of the analyst should be
checked periodically using pure organic compounds on which the
BOD is known or determinable. For this purpose a solution con-
taining 150 mg/& each of reagent grade glucose and glutamic acid
53
-------
can be used. The5 day BOD value for the Standard Solution
' r
may vary slightly according to the type of seed used (Table 2).
If,,however, the results vary; appreciably from those given in
Table 2 after considering the .seed source, the technique is
; ' .'i
questionable.
Table 3. Summary of Standard 5?-day, BOD Procedure Used
by Various Investigators
Reference .
Began and Sawyer
(1955)
Sawyer et al.
(1956)""
Ryckman and lawyer
(1957)
Conway and Waggy
(1966)
Pfeil and Lee
(1968)
Compound tested Concn.
Synthetic
detergents
Surfactants.,
Surfactant
Nitrilotriacetic
acid (NTA)
3-10mg/A >
0-320mg/£
Type of Seed t..
Natural sewage,
Acclimated activated
Sludge
Acclimated activated
Sewage
Acclimated laboratory
activated sludge
Raw sewage aged for
24 hours. Activated
sludge, Acclimated
activated; sludge
Thompson and Duthie NTA
(1968)
Dias. and Alexander; Aliphatic acids
(1971") and alcohols1
Sturm (1973)
Nonionic
Surfactants
2 and 5 mg/A- Seed' acclimatized to
NTA in BOD- water for
14 days
Domestic sewage aged
for 24-48 hours
Sewage microorganisms
acclimatized to test
chemical and yeast
extract (2mg of carbon/A)
carbon/A
2 and 5 mg/&
temperature 25°C; each compound was also tested in combination with glucose
54
-------
(11) 10-Day BOD-Procedure
Mills and Stack (1954) have described a 10-day BOD test
and used It for studying biological oxidation of synthetic
organic chemicals. The reason for using a longer Incubation
period was due to the occurrence of a lag period of one to two
days before oxygen depletion was observed In control bottles
(without the chemical). These Investigators Inoculated the
dilution bottles with dispersed seed (Heukelekian, 1949) de-
veloped from settled sewage which had been acclimated. The
desirable characteristics of the seed Included Insignificant
adsorption of the chemical and ready availability of the seed
for batch experiments. The method has also been referred to
as the "dispersed seed aeration system" (Mills and Stack, 1954).
Another modification made In this test was that air containing
1% CO. was used for aeration whereas In the standard BOD test,
air Is used. This was done to control the pH of the mixture
being aerated by preventing the loss of CO .
(Ill) Long-term BOD Technique
This technique was developed by Elmore (1955) for de-
termining BOD when (a) the compound requires long acclimation
periods (b) the compound requires long periods for complete
degradation, or (c) higher concentrations of the test chemicals
are used. In this test a large bottle (several liters capacity)
is filled with the test solutions and aerated. From this a
series of several standard BOD bottles are filled and Incubated.
55
-------
The large bottle is also incubated. On subsequent days a
\ <
single bottle is removed and analyzed for dissolved oxygen.
When all the small bottles have been analyzed or when the
. ' i'
t
dissolved oxygen level is near exhaustion, the large bottle is
t
reaerated and from this another series of small bottles is pre-
pared for analysis on subsequent days. This procedure is re-
' i,
peated as many times as necessary until the degradation of the
chemical is complete.
(iv) Two-Bottle - Single Dilution Reaeration Method
This method was originally described by Orford _et ,al. (1953)
and-later used by Ryckman (1956), Ryckman and Sawyer (1957),
i
'Young et_-al., (1968) and Buzz ell e_t al. (1968). It permits
withdrawing many samples for analysis and has provision for re-
i
plenlshment of oxygen as it is depleted.
The biodegradation reaction unit consists of two 9-liter
pyrex bottles with connecting siphon as shown in Figure 4. -The
lower-^bottle is kept full and when the samples are withdrawn from
this bottle for dissolved oxygen analysis, the second bottle re-
plenishes the liquid through the siphon.
Similar to other BOD tests, the dilution water is inoculated
with raw sewage to provide seed for biodegradation. If necessary,
the contents of both bottles may be reaerated by directing air
through the sampling tube.
' Buzzell et_ al. (1968) expanded the Two Bottle - Single
Dilution Reaeration Method to make it more nearly quantitative
56
-------
and more general in nature. These researchers measured the
following parameters in the sample removed from the biodegradation
reaction unit:
Utilization of oxygen to determine BOD, chemical oxygen
demand, and total organic carbon to monitor removal and
transformation of carbon.
Changes in bacterial population numbers to assess the
deleterious effect of the chemical, if any, or utiliza-
tion of carbon from the organic compound.
Onset of nitrification to estimate noncarbonaceous oxygen
utilization.
The total information obtained above was considered in
assessing the behavior of organic chemicals.
RESERVOIR
BOTTLE
f
HOM
damp
MMPIWO
r
Figure 4, Schematic Representation of Individual
Biodegradation Reaction Units (Young e_t al. 1968) v
Reprinted with permission from Journal Water Pollution*
Control Federation, Vol. 40(8 Part 2), 354-368, Wash., D.'C.
57
-------
b. Respirometry n
Similar to the dilution method, respirometric technique deter-
mines biodegradability by measuring oxygen consumption linked to the oxidation
j.*, ,
of the test chemical. 'The methods described in this section, however, differ
\
from standard dilution methods in that no serial dilution of the test sample
is necessary. The oxygen consumption measurement .in the respirometric method is
generally made for a period much shorter than 5 days. The seed used in the
**
respirometric test has generally been acclimated to the test chemical
whereas, in the BOD dilution method, acclimation is assumed to occur during
the test period. Since the concentration of microorganisms used in respirometric
*;
methods is generally higher, treatment plant conditions may be simulated'
. , ,
more closely. Furthermore, unlike the standard dilution method, which
measures oxygen consumed at 5 day intervals, the respirometric technique
allows continuous measurement of oxygen uptake. The- measurement can,
therefore, be stopped as soon as the oxygen consumption-rate has leveled
off.
-ft
The respirometric technique used for studying biodegradation can
be divided into two categories depending on whether or not sewage (or external
carbon source) is oxidized along with, the organic compound of'interest.
Systems containing organic compound, external carbon
,i \ ...-,..-
':' ' '
source'and microorganisms. The concentration of the external
carbon source is normally high while the concentration of the test
organic compound is kept low. Three respirometric runs are made:
TO '.'.
one with microorganisms alone, one with microorganisms and external
carbon source, and one with microorganisms, external carbon
i
source*and the test organic compound. The advantage of this
technique is that sewage could supply organic matter to those
58
-------
microoganlsms that can oxidize the organic compound but not utilize
It for carbon and energy. A problem of interpretation of results may
arise in this method because the organic matter present may effect
the decomposition of the test compound. Therefore, the method is
not frequently used.
Fbr studies of the degradation of surfactants, the common pro-
cedure is to use concentrations of suspended solids and sewage
which approximate the concentrations found in activated sludge
systems. The surfactant concentration is kept as low as would be
found in sewage entering a treatment plant (usually about 100 mg/£
or somewhat higher) (Bogan and Sawyer, 1954; Barden and Isaac, 1957;
Hunter and Heukelekian, 1964).
System containing organic compound and microorganism.
In this technique only two respirometric runs are required: one
containing microorganisms only and one containing microorganisms
and the test organic compound. External carbon source or sewage is
not added and therefore the system is less complicated. For this
reason, the procedure is often preferred for the determination
of biodegradability (Vath, 1964; Bogan and Sawyer, 1955; Sawyer et al.,
1956; McKinney and Symons, 1959; Blackenship and Piccolini, 1963).
If sludge is used as the source of microorganisms, unassimilated
organic matter is removed prior to its use as seed in the respiro-
meter. This can be accomplished either by not feeding the sewage
several hours preceedlng its use, thus forcing the microorganisms to
burn unasSimulated carbon (Bogan and Sawyer, 1955), or by washing
the sludge with saline solution prior to its use in respirometry
(Blackenship and Piccolini, 1963).
59
-------
r The type of inoculum used in respirometric studies has varied
'.' *'
considerably. Researchers have used both mixed and pure cultures of micro-
.. 0 S
organisms. The use of]the mixed population of microorganisms has been more fre-
quent since they more,closely represent'natural environmental conditions. Sources
of mixed-population have included sewage, activated sludge, trickling filter
slime, river water, and river or lake mud (Hunter and Heukelekian, 1964).. "Pure
i
cultures for respirometric studies have generally been isolated by enrichment
culture technique (see section III A.3.b.iii. , p. 105) or obtained -from isitock
. i . .
cultures of microorganisms (Heyman and Molof, 1967; Ellis et^ al. , 1957:; -Walker
: ''.:
and Cooney, 1973a and 1b). !
The duration of the respirometric test is relatively %hort and
acclimation of the organisms to the chemical may not occur during this period.
'."'-'
Consequently, microorganisms acclimated beforehand are often used. Ryckman and
.Sawyer (1957) found that degradation of alkylbenzene sulfonates-was delayed for
* . < o
several days when organisms native to sewage were used. However, when acclimated
i : '
activated sludge was.used, degradation .was rapid. Activated sludge from a unit
acclimated to the surfactant has been used as the inoculum for studying degradation
of surfactants (Bogan and Sawyer, 1954, 1955;- Sawyer £t al., 1956; Barbaro and
Hunter, 1965; Nelson et al_. , 1961; Brink and Meyers, 1966). Okey and Bogan (1965)
used cultures which were derived from soil and sewage inoculum and grown on un-
1 -U' .. ' .
substituted homologs of the chlorinated test substrates. In other instances, they
used the activated sludge grown on a homologous substance. In order that the seed
- ' ' '''.' '-
used in one laboratory can be easily duplicated elsewhere, Buzzell e_t al. (1969)
used activated sludgei acclimated to the synthetic sewage which had been preserved
by lyophilization (freeze drying). The microorganisms were reactivated by mixing
dried sludge with synthetic sewage prior to use in the Warburg apparatus.
' ...-'' .. "'-:-
- ' - >'
' ' -:-V;" . 60, ' ' ' ' '
-------
A number of techniques have been devised for respirometric
studies. Some measure gas exchange by manometric means, e.g., Warburg respiro-
»
meter. Others measure oxygen by means of polarography, e.g., oxygen electrode.
Researchers have modified these systems according to their requirements. Various
respirometric techniques and their modifications are discussed below.
(i) Warburg Method
In this test, oxygen is supplied to the system in the form
of the gas phase which is continuously transported into the
liquid phase by agitation. The Warburg respirometer determines
oxygen uptake by measuring pressure changes while the gas and liquid
volume are held constant. The higher level of microbial activity
which is normally obtained in a respirometer flask simulates more
nearly a biological treatment unit rather than a lake or a river.
A Warburg respirometer unit is shown In Figure 5. It consists
of a U-shaped capillary tube of uniform cross section, both arms
of which are graduated in millimeters. Attached to one arm is
t
a sample vessel. In a typical respiration experiment, the flask
contains an oxygen containing gas phase, a liquid phase containing
an organism and its substrate, and a center well containing po-
tassium hydroxide solution to absorb produced CO . The flask is
Immersed in a water bath at constant temperature and shaken or
swirled to promote a rapid gas exchange between the liquid and
the gas phase. From the magnitude of the change, which is
61
-------
Indicated by the difference in the height of the fluid in one arm
of the manometer, the quantity of oxygen consumed can be calculated.
Controlled temperature
water bath
Manometer-^.
flask &
support in
place
Adjusting
I /screw
Manometer
&
support assembly
hater mechanism wit
attachments lor
manomer sunorts
Figure 5. Warburg Manometer
(F.ckenf elder et. al.. 1972)
Courtesy- of Springer-Verlag
Changes in the barometric pressure are compensated for by
'the use -.of a blank" flask containing water, which is known as the
thermobarometer. The extent of endogenous oxygen uptake is
/
estimated from a control run which is identical to the test run
except that the test compound is not added.
Summary of the Warburg conditions used by various investigators
is given in Table 4:
62
-------
Table 4. Warburg Respirometry Conditions
Reference
Bogan and Sawyer
(1955)
Sawyer e£ al.
(1956)
Stelnle e£ al.
(1964)
Okey and Bogan
(1965)
Brink and Meyers
(1966)
Heyman & Molof
(1967)
Test
Chemical
Synthetic
detergents
Synthetic
detergents
Surfactants
containing
ethylene oxide
monomers
Hydrocarbons
Anionic
surfactants
Surfactants
Concentration used
50-100 ppm
50-100 ppm
50 mg/fc
1 mg/fc (substrate
containing
chlorine)
Period of
Incubation
6, hours
6 hours
48 hours for poorly
degradable compounds
8-10 hours for
easily degraded
compounds
10-72 hours
12-24 hours
0.01-0.005 M 2-26 hours
Primary alkylbenzene
sulfonate
0.005M secondary
linear compounds
(ii) Modification of Warburp Apparatus
Caldwell and Langelier (1948) modified the Warburg apparatus
to accommodate large samples (10-75 ml instead of 3 ml). They
increased the volume of the reaction flask to 125 ml. Gellman
and Heukclekian (1951) used reaction flasks of 140 ml volume and
samples of 2 to 50 ml. Jaegers and Niemitz (1952) used the War-
burg apparatus with 125 ml flasks specially designed to have a
low hydrodynamic resistance, thereby reducing the strain on the
manometer tubing.
63
-------
f'
Nelson et al. (1961) used a Warburg apparatus with 125 ml
flask for studying biodegradation of alkylbenzene sulfonates. The
reaction volume was 20 ml, and included concentrated activated
sludge and alkylbenzene sulfonate (ABS) solutions. Oxygen uptake
was measured over a period of 28 hours. Buzzell e_£ al. (1969) used
similar flasks for studying the biodegradability of several families
t
of organic: chemicals. Oxygen consumption was measured up to a
*_,<<.,
period of'12 hours. At the end of the run, these researchers also
determined; the soluble carbon remaining in the Warburg flask to
v
support the oxygen consumption data and to confirm the completeness
of the degradation process. Further, to obtain a complete picture
i
of the behavior of the organic chemical, Buzzell et^ t&. (1969)
carried out parallel shake culture studies, (see Section III A.3,
* * -r
p. 83 ) in 'order to determine changes in total organic carbon, and
used a dehydrogenase enzyme assay to follow the changes in
the metabolic activity of the biological agent.
Barbaro and Hunter (1965) utilized the Warburg apparatus
equipped with flasks of 140 ml capacity to study the effect of
'.,
clay minerals on surfactant biodegradability. Use of a large
"*
flask permitted them to increase the sample volume to 50 ml and
therefore data on oxygen uptake could be collected for up to
i .
10 days.
(Hi) Differential Manometer
Dick (1964) has reported that the accuracy of the Warburg test
is low fo'r relatively small 0. uptake rates in waste waters. Schulze
j". * .
and Hoogerhyde (1967), therefore, tested the utility of the differ-
ential manometer for BOD measurements. A modification of the
64
-------
instrument developed by Schulze and Hoogerhyde (1967) is shown
in Figure 6. The unit uses a 300 ml flat bottom sample flask and
i
a 300 ml flat bottom reference flask connected by a ground-glass
joint to a capillary glass tube, which is connected to a manometer
by two ground ball-joints. The flasks are submerged in a constant
temperature water bath and paraffin oil is used as manometer fluid.
The sample is agitated by a 1-inch long Teflon-coated magnetic stirring
bar. The sample flask is also equipped with an 1/2-inch diameter
hanging glass cup to which is added 10% KOH solution to absorb CO .
The oxygen consumed is measured by a micrometer syringe. The
measuring and sampling technique is described in detail by Hoogerhyde
(1965).
Figure 6. Diagram of differential manometer (Schulze & Hoogerhyde, 1967)
a) sample flask, b) micrometric buret, c) manometer, d) reference
flask, e) equilibration valve, f) valve to atmosphere, g) ball joints,
h) KOH cup, i) magnetic., stirring bar.
Reprinted from Develop. Ind. Microbiol., JJ, 284-297, Society for
Industrial Microbiology. ,
65
-------
(Iv) Electrolytic respirometer
i
Liebammn and Offhaus (1966) have described a sapromat A6
reaplrome|ter, a patented electrolysis respirometer which is
available from J.M. Voith, GmbH, Heidenheim, West Germany.
Schematic diagram of the unit is shown in Figure 7. The
oxygen pressure in this type of respirometer is automatically
i
maintained at a constant value by an electrolysis cell, for
<:' . '
the duration of the experiment. The current passing through
the cell, which is proportional : to the oxygen consumed'by /.the
sample, may be integrated and/or continuously recorded. The
apparatus operates independently of the changes in barometric
rpressure. The whole apparatus is placed inside an incubator
to minimize the changes due to temperature variations. The sapromat
:i. . .
test is usually run for a period of 120 hours.
Pauli and Franke (1971) have used the sapromat apparatus to
evaluate the effect of preservatives and disinfectants on the bio-
1
logical degradation of a standardized sewage. The test was run for
120 hours with intermediate samples drawn at 24- and 48 hours. Th'e
.degradation obtained without the test chemical was expressed as 100%.
V '- ' ;.'. ..''.. ' .
A sapromat value higher than 100% was considered to be due to the
i '
degradation of the teat! chemicals.
-vj ' - . ' '
66
-------
Figure 7. Schematic diagram of one of the six units of the Sapromat A6
Reapirometer (Paul! and Franke, 1971).
1) magnetic stirrer; 2) sample; 3) C02 absorber; 4) manometer;
5) electrolytic cell; 6) measuring and control unit; 7) printer
(optional). Reprinted from Biodeterloration of Materials
published by Applied Science Publishers Eta". '
(v) Oxygen electrode respirometer
Oxygen electrode respirometers measure dissolved oxygen in
the liquid phase, unlike the manometric method in which pressure
changes in the gas phase are measured. This method allows an
Investigator to record continually the oxygen depletion which Is
related to the oxidation of the organic chemical. Although an
oxygen-sensitive electrode can be used as an analytical tool
to measure dissolved oxygen In 5-day dilution BOD test (APHA
1971,), the respirometric methods described in this section
are somewhat different than the dilution method. First,
67
-------
the concentration of microorganisms in oxygen electrode respir-
^
ometer test is generally higher and the test-,is run for a much
shorter period than the dilution method. Second, the oxygen
electrode respirometer has the .reaction vessel linked to the
...<
metering .device and this permits continuous measurement!: of oxygen
uptake.
(a) Galvanic cell oxygen electrode respirometer
»
' The assembly described by Gannon et^ al. (1965)' consists
of a standard two-liter sealed reaction flask wi-thva-specially
constructed .galvanic cell oxygen electrode (Gilmam>Instrument
Co.) as developed by Mancy eral. (1963). The oxygen electrode
is connected to a standard strip chart recorder to permit con-
f'
tinuous recording of residual oxygen concentration with time.
' .( ' '
j . ' t
When the oxygen concentration in-the 7flask drops to 2, mg/Jl,
the flask is aerated by means of compressed air. and the_
procedure is repeated.
The oxygen analyzer consists of a silver-lead Galvanic
couple separated from the tear.sample by a polyethylene
V
membrane with a 1 M KOH/solution between rht couple- mn\
the membrane. One modification of the analyzer IH tlio -^
' _i ' . ' . ,
substitution of 1% methylcellulose (Dow Chemical Co.) in
\- ' . '
place of the lens paper as a carrier for the 1 M KOH to
increase the sensitivity of the electrode.
68
-------
This procedure has the same advantages as the reaeration
technique In that no dilution is involved but has an added
advantage of keeping the original sample intact, rather than
splitting it up into several sealed BOD bottles.
(b) Clark-type oxygen electrode
This is a gold-silver electrode system which is protected
by a thin gas permeable Teflon membrane (Clark et al., 1953).
The membrane isolates the sensor element from its environment,
except that oxygen can diffuse into the sensor.
The sensor is 25 times more sensitive compared to conven-
tional gas phase manometric systems (Yellow Spring Instrument
Co., 1970).
Hammond and Alexander (1972) and Dias and Alexander (1971)
have used Clark-type polarographic electrodes for BOD determin-
ation; the electrode was mounted in a rubber stopper so as to
fit the neck of the BOD bottle. The advantages of using oxygen
sensor over chemical methods in BOD determinations have been
discussed by Reynolds (1969).
The sample volume in this assembly is restricted to
3 ml and, therefore, the amount of oxygen available is limited.
The problem can be overcome by using a Macro bath assembly which
allows an investigator to use sample sizes between 20-50 ml.
This assembly may be more suitable in degradation studies where
increased quantities of oxygen are desirable.
69
-------
(c) , ..Other Oxygen Electrode respirometers
:. ' Eye et_ al. (1961) used a solid oxygen electrode
connected to a pH meter for BOD measurements. Samples
were diluted as in the dilution test and dissolved oxygen
was continuously measured by the oxygen electrode..
The apparatus developed by Lamb e£ al^. (1964)fis .
shown in Figure-8. This is similar to other systems:
using oxygen electrodes except that a diffuser is provided
for aeration of the respirometer contents.
OXYGEN
ELECTRODE
LUCITE CAP
WATER
OUTLET
WATER
JACKET
Figure 8. Completely filled electrode respirometer (Lamb et^ al^, 1964)
Reprinted with permission from Journal Water Pollution Control
Federation, 3£, 1263, Wash., D.C.
70
-------
(vi) Miscellaneous techniques
A complete description of all the respirometric methods is
available in the review by Montgomery (1967). Some brief descrip-
tions of some of the other respirometry techniques will be discussed
in this section.
The apparatus described by Popel e_t al. (1958) consists of a
vertical reaction column connected to a manometer and a gas
burette; at the bottom of the column is a gas diffuser. Air is
continuously pumped in closed circuit through the column and through
an external gas-washing bottle containing alkali, by means of a
diaphragm pump. Excessive foaming was a problem with the apparatus
when samples containing detergents were examined.
An automated apparatus is described by Snaddon and Jenkins
.(1964) in which instead of noting the change in manometer reading,
a measured volume of oxygen is added to balance the manometer,
automatically by a system of pipettes and electromagnetic valves.
A modified automatic respirometer is described by Wilson (1967)
which has a provision for circulating the gas phase continuously
through an alkali scrubber.
Busch e_£ al. (1962) developed a technique to study biodegrad-
ability by determining the theoretical oxygen demand (BOD corresponding
to exhaustion of the substrate plus the oxygen equivalent of the
new cells formed). Sewage was allowed to settle for 24 hours,
filtered, and then served as the seed in this test. The BOD
measurements were carried out by standard methods. Cell yield
was determined by weighing the cells after filteration. Oxygen
71
-------
equivalents were determined,by assuming that the general formula
for the cell contents .was,C5H7N02., It has been suggested that
the assumed .formula might not always, be correct and therefore error
«. ' -
could be introduced (Grady and Busch, 1963).
2. River Die-Away Test
The -river die-away test is a .static type biological system..,
!
and has .been extensively used in bio.degradation studies. This^.,-
: '$
type of test system is an attempt to.simulatebiodegradation conditions
I
of a river, lake, lagoon, or marine type environment. In this test,
raw water is collected from a river and. allowed to stand for a day
and the water is decanted to remove*large particles and mud. The
chemical compound of interest is added to-the river water contained
in a glass, jar and the solution is analyzed:at various intervals to
determine degradation by one of a variety?of analytical techniques.
The microbial content of the?:river water:.-is normally low, although
:....;;
the number :could vary significantly depending, on the site chosen. Rivers^
receiving domestic and industrial waate^are likely to have a much greater
microbial population. The size of the water sample is largely dependent
on the analytical requirements. In studies reported, the sample size.
has varied anywhere between 1-20 liters. The type of containers used
include mason ^ars, glass bottles, etc. The period of incubation of
the test chemical with river water normally ranges from a few days to
up to 8 weeks,; depending on the observed rates of biodegradation. This
.'*'''
period Includes the time required for acclimation of the river bacteria,
o
which varies from compound to compound.
72
-------
a. Original River Die-Away Test
The river die-away test has been extensively used in
studying biodegradation of surfactants. In these studies,
various sources of river water and test chemical concentrations
have been used. A summary of a number of river die-away tests
is given in Table 5. Selected other studies are discussed below
in greater depth.
The river die-away test orginially described by Hammerton
(1955) was employed to study the degradation of sodium lauryl
sulfate at 4 ppm. The period of standing ranged from 18-36
hours. When the depleted solution was redosed with detergent,
its concentration was reduced even more rapidly than the first
time. Degradation was also examined when water was boiled or
phenylmercuric acetate was added to inhibit bacterial action,
or when detergent was dissolved in distilled water. In a
modified river die-away test, Borstlap and Kooijman (1963) used
distilled water supplemented with a small quantity of activated
sludge, instead of river water. Lashen j2t al. (1966) carried
out river die-away tests with nonionic-surfactant alkylphenol
ethoxylate in the water samples collected from several locations
on a number of different rivers.
73
-------
Table 5. River Die-Away Test for Determining '
Biodegradability of Organic Chemicals
Reference
Compound Tested Concn. Source of River Water
Sawyer et al.
(1956)
Blackenship and
Piccolini (1963)
Well and Stirton
(1963)
Setzkorn et al.
(1964)
Weaver and Gouglln
(1964)
Bacon (1966)
Osburn and Benedict
(1966)
Swisher (1967a)
Warren and Malec
(1972)
Swisher and Gledhill
(1973)
Detergents 5 ppm
Nonionic 20 ppm
detergents
30 anionic and -
6 nonionic
detergents
Detergents 10 ppm
Surfactants 20 ppm
Surfactants 7 ppm
Polyethoxylated
alkyl phenol
Surfactants 5 rags
Nitrilotriacetic 20 ppm
acid and related
imlno and amino
acids singly or
in combination.
o-benzyl-p-chloro- 0.1 mg/fc
phenol (S'antophen I)
Two sources in
Massachusetts
Delaware River
Schuykill, Susquehanna
River and Delaware
River
Mississippi, Missouri,
Ohio, Wabash, Kansas
and Arkansas Rivers
Ohio River
Detroit River at the
intake to City Water
Plant
200 ppm Ohio River
Detroit and Meramec
Rivers
Meramec River
74
-------
Sample sites included highly industrialized areas where microorganisms
may be expected to be acclimatized, rural suburban areas which
probably had some exposure to nonionics, and representative
water from non-industrialized area having little or no exposure
to nonionic surfactants.
Only a few reports are available on studies concerning biodegra-
datlon of pesticides in river water. Eichelberger and Lichtenberg
(1971) have studied the persistence of 28 common pesticides in
river water from the Little Miami River over a period of 8 weeks. The
concentration of pesticide in this study was initially 10 mg/&.
The concentration is somewhat higher than usually found in
surface waters, but was used to facilitate the identification
of decomposition products. The mixture was incubated in sealed
glass jars on a laboratory bench under simulated sunlight.
The contents were shaken periodically to redistribute the
pesticide and any suspended matter that might have settled out.
Identification of chemical degradation was made by running a
parallel experiment in which distilled water was used as a replace-
ment for river water. i
Blodegradation of urea in river waters under winter conditions
was studied by Evans.'et'al. (1973). Winter conditions were chosen
since urea^ among other things, is used as a deicing agent. Sam-
ples of Yorkshire river water (England) of varying composition
were obtained and bottom mud was added. The concentration of urea
75
-------
was kept low, between 1-15 mg/llter. Evans and David (1974)
in the biodegradability studies of glycols used river waters
of varying composition and topographic origin obtained from 4
different sources. Studies were carried out at temperatures
ranging from 4-20°C.
' . ./j
b. Rive? Die-Away with Fortified and Inoculated Waters
Since the composition of the river water chosen for the
river-die-away tests varies considerably in microbiological
and chemical terms, the reproducibillty of the biodegradation
results has been .poor (Swisher, 1970). Furthermore, in view
of the fact that the concentration of microorganisms and essential
nutrients required for their growth is generally low in river
.A
water, the time of incubation of the test chemical with the
i
river.water has been long. To overcome these difficulties,
attempts have been, made to devise fortified waters for use In
this test.
Garrison and Matson (1964) inoculated river water with
activated sludge from, a predominantly domestic source. Ther
sludge was filtered and added.to a concentration of 0.1%
(w/v). Conway and Waggy (1966) in their test have used half-
gallon bottles charged with one liter of river water containing
O.S.Z of settled sewage. Surfactant concentration was 10 tag/A.
The bottles were loosely capped during the test. Two bottles,
to which surfactants were not added, served as controls. Eden
j£t ali (1967) Incubated for periods up to three weeks, a dilute
. 75
V! .
-------
solution of the detergent material in river water seeded with
a small quantity of sewage effluents. The test was conducted
in open or closed bottles in a manner similar to the BOD test
(see Section III A.I,a.1, p.52). Since a limited amount of oxygen
is available in the closed bottle test, both the amount of in-
oculum and the initial concentration of surface active material
is limited.
In a new accelerated biodegradation test (Hitzman, 1964),
the procedure employed was the same as for the river die-away
test described by Proctor and Gamble (1964) except that the
natural water was fortified with microorganisms obtained by
continuous centrifugation of water from a municipal sewage plant
effluent or river water itself. In order to achieve reproduci-
bility between the results obtained by different laboratories,
the fortified water was diluted to a standard optical density so
that the number of cells in each test was somewhat similar.
The French IRChA tecnhique (Instltut National de Recherche
Chimique Appliqiue) (Brebion e_£ al., 1966) uses river water
medium fortified with additional microorganisms from a culture
developed from sewage-polluted river water. Nutrients are
added in the form of meat extract and peptone to sewage-
polluted river water and microorganisms are allowed to multiply.
For the test, unpolluted river water containing surfactant
(10 ppm) was inoculated with 10-20% of the above culture (bac-
terial count between 200 and 400 million/ml). The test mixture
was redosed with surfactant on the seventh day.
77
-------
t
, A technique to demonstrate acclimation of river microflora
to alkylphenol ethoxylate has been described by Lashen and Booznan
i
(1967) and is based on the presumption that,acclimated bacteria,
'. i
when diluted greatly in unacclimated river water solution, should
'initiate an immediate degradation when the test compound is bio-
t, .
. degradable. In this procedure, a fresh uninoculated Delaware
river water sample was Inoculated with 1% of the acclimated river
water. A portion of the Delaware river water was .left uninoculated
to serve as a control.
To study .the biodegradability of the organic pesticides,
>'.' .
Sey.in, 1-naphthol, baygon, pyrolan and dimetilan, Aly and El-Dib
(1972) have used Nile River water buffered with phosphate to
maintain a pH of 7.2 ±0.1. Insecticides were added each at a
concentration of 4 mg/liter and 10 liter portions of each solu-
tion placed in a 5 gallon container. The incubation was carried
' T ' '
.out at room temperature and:aerobic conditions were maintained
v<
by bubbling a gentle stream of air. The disappearance of the
.compound was also followed after redosing with insecticide, to
check for possible ^acclimation. If.an insecticide was found to
be degrading slowly, .river water was supplemented with 1% of
.settled sewage to increase the rate of degradation.
Biodegradation of sulfonated amide surfactants was studied by
' -v
Sheers et al. (1967) using the water from the North Branch of the -
.' "I-"** " '
Rarltan River at a point which had a minimum possibility of
contamination by detergents. The water, following filtration
^«
through cotton, was distributed in 16-liter portions in 20-liter
78
-------
bottles. Inorganic nutrients vere added to .furnish the
necessary F and N concentration based on the COD values of
each bottle. Test material (5 and 10 mg/fc) was added and
the bottles were aerated.
Horvath (1972b) modified the river die-away test to study
cometabolic degradation. A biodegradable analogue of the test
chemical was added along with the test chemical. In studies
on the degradation of trichlorobenzoate, the biodegradable
analogue used was sodium benzoate, which was added at twice
the concentration of the test chemical.
/
c. River Die-Away Test with Polluted River Water
In several biodegradation studies, polluted water collected
from rivers receiving domestic and industrial waste has been used
because such waters contain greater microbial populations and
often contain certain specialized microorganisms. The source of
bacteria utilized by Wayman and Robertson (1963) in the degradation
of anlonlc and nonanionlc surfactants was South Platte River
water sampled near the discharge pipe of the Denver sewage
treatment plant effluent. The chemicals discharged into
this water over a three-year period included ABS in the
range of 2-8 ppm. Biodegradation was studied in solutions
prepared by dilution of river water by as much as 100 times
with distilled water or by using the river water undiluted.
The surfactant concentration was 25 ppm. The stability of
detergents was studied under aerobic, as well as anaerobic
79
-------
'Conditions. Aerobic systems, were studied by continuously
bubbling filtered air through solutions. Anaerobic systems
<.]«
were studied by use of a Brewer anaerobic jar or without
*.; '" ' ,
bubbling air through the. surfactant solutions (the later
system need not. necessarily be anaerobic). Various tempera-
tures ranging from 10 to 35°C were employed in the study.
Ettinger and Ruchhoft (1950) have examined the- fatee of
.mbnochlorophenols in polluted river water obtained*from the
Great Miami River and the Little Miami River. In their, studies,
the compound was, attacked more readily when added to-river
water than when added to. diluted sewage. The more rapid
attack was attributed to. the presence of microorganisms in ,/
I''1'' ;
the river water capable of destroying prtho- or para-chloro-
,X;
phenol. Persistence of pyridine bases at a concentration of
I'ppm using polluted, river, water from several different
sources - Ohio River water sampled both, above and below
Cincinnati; Great Miami River water taken near the mouth -
was examined by Ettinger. et al. (1954). With the exception
of,Little Miami water, all the river waters were known to
have a history of pollution by waste containing pyridine
bases. In an initial persistence experiment, these investi-
gators also studied the degradation of pyridine in tap water
containing 1% dilutions of Cincinnati sewage.
Yasuno e_t al. (1966) studied the degradation of organo-
phosphorus insecticides in polluted water. As a source of
polluted water, these investigators used (1) sewage water
80
-------
obtained from a natural breeding place of the test insect
(Culve pipiens molestus) used for the biological assay, and
(2) artificially prepared polluted water (3 gm of unsterilized
laboratory animal food powder added to one liter of tap water
and kept for 7 days at 28°C). The concentration of insecti-
cide added to pure and polluted water started from 80 ppm,
and twofold dilutions were made such as 40 ppm, 20 ppm, 8 ppm,
4 ppm, 2 ppm, 0.8 ppm, up to the lowest of 0.0008 ppm for some
insecticides. The insecticldal activity of the medium was
tested at 1, 4, 8 and 16 days.
d. Anaerobic and Microaerophilic River Dle-Away Test
Degradation of several anionic detergents under anaerobic
and mlcroaerophillc conditions has been studied by Maurer
et al. (1971) using river water as the source of inoculum.
Detergent solution of 5 or 10 ppm prepared in Schuylkill
«
River water was transferred in 2 liter portions into vented
3 liter aspirator bottles. The bottles were equipped with
extrecourse spargers through which nitrogen gas or air was
passed (Figure 9). Bottles had been wrapped with foil to
minimize the growth of algae which may release oxygen into
the system.
81
-------
Figure 9. Experimental apparatus used for simultaneous
microaerophilic and aerobic tests (Maurer et al., 1971)
Courtesy of J. of the Amer. Oil Chemists' Society.
'" The anaerobic die-away test used by Conway and Waggy
(1966) is simply a moderately seeded river die-away test
conducted under anaerobic conditions. Surfactant was dis-
solved in oxygen-free river water which was seeded lightly
with sewage. The solution was purged with nitrogen to re-
move dissolved oxygen. The bottles were sealed, inverted
-'' '
and stored in dark. Aerobic die-away tests were concurrently
run for comparison.
' > '
e. Die-Away Test with Marine Water
Recently, efforts have been extended to the study of
compounds using sea water as inoculum. In the test described
by Atlas and Bartha (1972, 1973), degradation of petroleum
was studied in the sea water collected off the east shore of
. i
Sandy Hook, N.J. Sea water samples were supplemented with
KNOr and Na2HPO, as nitrogen and phosphorus sources, respec-
tively. One-hundred-mllliliter portions of the sea water
82
-------
were Introduced into the flask component of the gas train
assembly for CO. evolution measurements. Filter sterilized
crude oil was added to the sea water as 800 mg fresh or 560
mg "weathered oil" and the flasks were continuously aerated
with CO -free air. Since the temperature in the ocean is
lower than optimum for many microorganisms, these investigators
maintained the temperature between 5 and 20°C.
3. Shake Culture Test
In this technique a pure culture, or a mixture of microorgan-
isms from natural sources such as soil, water, activated sludge,
sewage, etc. or from active mixed laboratory cultures are grown
and/or adapted in a medium containing the organic compound to be
tested. The medium used for this type of test generally contains
balanced nutrients and essential mineral elements. The flasks
are aerated and at certain intervals aliquots are withdrawn to
test for biodegradation. Based on the type of inoculum used
and whether the microorganisms used for biodegradation have been
adapted, the shake flask culture test can be run in several ways.
a. Shake Cultures Inoculated with Natural Communities of
Microorganisms
(1) Degradation tests using activated sludge or sewage
as source of microorganisms
(a) Original shake culture method for study of
surfactant biodegradation.
Huddleston and Allred (1963) have described
a shake culture test in which they employed a mixed
83
-------
culture obtained from an activated-sludge-type waste-
fi
treatment plant as the source of inoculum. The culture
was adapted by three successive transfers in a medium
(composition shown in Table 6) containing 30 mg/liter
'( i
detergent. The adapted seed (10 ml) was finally^used
*
i
,to inoculate sterilized 1 liter medium containing^30^mg
of test detergent in a 2 liter Erlenmeyer flask. SiThe
culture was incubated at 25°C on a rotary shaker. -Samples
' !
* i
.were withdrawn at desired intervals for analysis. --The
-.,'
test has been:used extensively to study the biodegrada-
tility of surfactants (Allred et al., 1964;-Huddleston
and Allred, 1965).
Table- 6.
'.-^
Composition of the basal medium '(Huddleston and Allred, 1963)
............... ..... . . 3.00 g
K2-HP.Oif .................... 1.00 g
;MgSOu 7E20 ...... ..... ...... 0.25 g
KG1. . ............. ....... '0.25 g
. . . . ....... , . . . . . . . . . Trace
Yeast extract. . . . ... . . . . . ...... 0.30
Distilled water. .......... ..... 1.0 liter
pH after sterilization . . . .... ..... 7.1
Detergent. ...... ............ 0.030 g
84
-------
Setzkorn and Huddleston (1965) in a modified test
used reduced concentrations of inorganic salts and yeast
extract (Table 7). This was done to facilitate the use
of U.V. analysis (described in detail in Section III C.2.d.,
p.156) to follow the rupture of the benzene ring system.
The incubation was performed in 1/2 gallon fruit jars
which contained 5 ppm of the test compound and 5 ml of
raw .sewage in 1 liter of synthetic medium.
Table 7.
Composition of Medium (Setzkorn and Huddleston, 1965)
Compound Wt. g
NH^Cl 0.1500
K2HP02 0.0750
MgSOi*. 0.0125
KC1. 0.0125
Yeast extract. 0.0050
Deionized water. ............... 1.0 liter
(b) Shake culture test of the Soap and Detergent
Association (SDA)
The subcommittee on biodegradation test methods of
the Soap and Detergent Association has adopted, with
minor modifications, the shake flask method described by
Huddleston and Allred (1963) to be used as a screening or
presumptive test (SDA, 1965). If the degradation in the
presumptive test equals or exceeds 90Z, the compound is
85
-------
considered adequately biodegradable by SDA. 'if, however,
degradation falls between1 80 and 90%, the biodegradability
(
is confirmed by semicontinuous activated sludge procedure
(see -Section III B.2.b, p. 139). The medium used in the shake
\ ' .
culture test is the same as described by Huddleston -arid"1'-'
Alired (1963) (see Table 6). The microbial Inocul-um^for^
tKe test may be one of the following:
i '
1. Natural Sources (soil, water, sewage,
; activated sludge-, etc . )
.2. Laboratory culture (activated sludge,
river die-away, '.etc.)
: 3. Culture obtained from:
>*. '
Laber co Laboratories, Inc.
' 123 Hawthorne Street
Roselle Park, N.j. 07204;
The seed' is adapted by making two '72-hour transfers. For
the "final.- inoculation"! ml culture is- used for each 100 ml
of fresh mediumVand tbe test- is ruh-f or 8 days-.
(c) Bunch and Chambers Test
Bunch and Chambers (1967) described- a shake culture
test' in which they used BOD" dilution water supplemented
with' yeast- extract- as the basal medium. Their preference
for 'BOD water over tlie nutrient medium oi Huddles ton and
Alired (1963) was due to the fact that BOD dilution water
is already available in many laboratories that would be using
86
-------
a teat for biodegradation. In thla test 10 ml of settled
sewage is used to inoculate 90 ml of BOD dilution water
containing 5 mg of yeast extract and a suitable amount of
test compound (approximately 2 mg/100 ml). The test is
run in 250 ml Erlenmeyer flasks which are aerated on a
shaker at ambient temperature 25°C ± 5°. Weekly subcul-
tures are made in fresh medium for three consecutive weeks
(total time of the test is 28 days). The flasks are
examined the day following inoculation and if no turbidity
is found then the test is repeated with the same and three
/
lover concentrations of the test compound.
Leigh (1969) modified the Bunch and Chambers test in
order to study the degradation of compounds such as chlorinated
hydrocarbons, which have low solubility in water. Aqueous
solutions of the insecticides (saturated) were prepared in
distilled, deionized and charcoal-filtered water. Inorganic
salts and yeast extract were added to the insecticide solution
as in the preparation of BOD water. The mixture was seeded
prior to distribution into the Erlenmeyer flasks.
(d) Shake culture test utilizing preserved seed
; The variations in the type of inoculum used by different
laboratories for testing biodegradability has sometimes made
it difficult to make comparisons between the results. For
this reason attempts have been made to use a seed which could
87
-------
be readily duplicated in other laboratories or conveniently
stored. This includes means of preserving the seed in the
dormant form which could be rendered viable at the time of
use.
'(1) Air-dried activated sludge - The Aeration Test
I
. Truesdale e_t al. (1969) have described a test
referred to as the "aeration test" which involves in-
tubation of a dilute solution of the detergent in stan-
idard BOD dilution water under aerobic conditions with
air-dried activated sludge as the inoculum. This test
was adopted by British Standing Technical Committee on
Synthetic Detergents (British STCSD) for testing bio-
degradability of detergents. The test 'is also referred
t.to as the British STCSD Test (Swisher, 1970). Air-dried
ractivated sludge was .prepared by evaporating to dryness
a thin layer of sludge at room temperature. Dried
sludge retained its activity for many weeks without
special conditions for storage. These investigators
'discarded freeze drying as a method of preservation since
it required the .addition of a preservative which could
i
complicate the results.
;:i
The test was used for studying the biodegradability
of synthetic detergents (Eden ejt a_l. , 1967; Truesdale
et al., 1969). The detergent concentration was kept
at 10 mg/liter of BOD water. The mixture was inoculated
88
-------
with 30 mg/liter of dried sludge and aerated by gentle
stirring. Measurements were made for a period of 12-21
days.
Patterson e_t al. (1967, 1968) used the aeration test,
after introducing a few variations, for studying the bio-
degradation of alkyl phenol polyethoxylates. In order to
remove visible dried sludge particles from the solution,
which may adsorb the detergent, these investigators filtered
the mixture through a Whatman No. 1 filter paper, after
overnight aeration in BOD water. Furthermore, whenever the
detergent concentration was increased, the proportion of
dried activated sludge was also Increased to accelerate the
degradation.
In order to avoid lengthy acclimation periods for
compounds which degrade very slowly, Truesdale et^ al. (1969)
modified the aeration test by using an acclimated microbial
culture which was obtained from a recirculation filter. The
presence of active inoculum from this source increased the
rate of degradation considerably. However, the authors com-
mented that with this inoculum, the degradation observed was
considerably greater than achieved in normal treatment plant
practice.
(2) Shake culture test using sludge preserved by
lyophilization
Buzzell e£ al. (1969) have investigated .and evaluated
various methods for preserving standardized activated sludge.
89
-------
The sludge was prepared in a Bench Scale continuous-flow model
activated sludge plant (see Sec. Ill B.2a, p.129), which was
' 1'
inoculated with activated sludge obtained from a municipal
treatment plant receiving sewage- largely of domestic origin.
In order to provide a source of nitrogen and phosphorus^ a-
synthetic sewage feed consisting of glucose and peptone -arid-
inorganic salts dissolved in tap water was used with the' model
activated sludge plant (Wiener, 1966). Activated sludge-;
acclimated to the synthetic sewage was preserved using the <
following procedures:
1. Freezing slowly at -15°C (deep freezer unit).
2. Freezing quickly at -76°C (acetone and dry ice).
3. Freezing-quickly at -192?C (liquid nitrogen).
4. Lyophilization
The activity of the-sludge:-preserved by the
procedures described above was evaluated by measuring
oxygen uptake in a Warburg respirometer with glucose
!?'.'' . '
plus peptone as substrate. The-results indicated
(Figure 10) that the lyophilized sludge was the most
vigorous since it used oxygen'at significantly gruuter
'.')
rates than sludges preserved by other methods. Con-
sequently lyophilization was preferred.
90
-------
4 6
TIME - hours
10
Figure 10. Oxygen Uptake by Sludges Preserved by Four Methods.
Substrate: Glucose plus Peptone (Buzzell et '_al., 1969)
Courtesy of Manufacturing Chemists AssociaTTon.
In order to reactivate the dried sludge for use in
shake culture studies, it was mixed with synthetic sewage.
' The rejuvenation process was carried out in batch units which
were fed at 12-hour intervals. A rejuvenation period of
24 hours was found to be optimum since it gave rise to micro-
organisms with the highest respiration rates.
91
-------
Shake culture studies were carried out in 500 ml
wide-mouth flasks which contained 100 ml of reactivated
sludge and 100 ml of organic chemical solution (180 mg/
liter as carbon). The flasks were covered loosely with
aluminum foil and shaken at 120 oscillations per minute
during the 12-hour test period.
In the shake culture studies described above, the
adverse effect of the test material on the biological
agent was monitored by measuring dehydrogenase activity,
an enzyme system responsible for oxidizing or dehydro-
genating unspecified organic compounds in the cell.
Furthermore, the pattern of oxygen utilization was also
observed using a Warburg respirometer (see p. 61). The
results were combined with the shake culture studies and
all the derived information was considered for evaluating
the behavior of the organic chemical.
(e) Degradation methods utilizing composite seed
j . . ' ' .
Sturm Q973) suggested a biodegradability screen-
ing test which used a composite seed prepared by
j . -
mixing equal volumes of cultures, each of which had
i
been acclimated to a test compound. The advantages
of common composite seed are: (1) it allows using a
single blank for many test units, and (2) it provides
a bacterial population acclimated to a variety of
chemicals. Using this procedure these investigators
92
-------
studied the degradation of eight nonionic surfactants.
Individual acclimation cultures for each chemical and
a dextrose control were prepared in 2-liter capacity
wide-mouth flasks, which contained settled raw sewage
as a source of microorganisms, yeast extract as an
easily utilizable nutrient source, BOD water as a
diluent and source of inorganic nutrients, and a
test material; the mixture was incubated for 14 days
in the dark. Equal aliquots from each of these
cultures werie used to make a composite seed. A 10%
solution of the composite seed in BOD water was
prepared and aerated for 24 hours. Subsequently,
test material (20 mg/liter) was added and degradation
was studied (see Figure 11).
MCIMATION CUlTURf
USD * BOO WHO
ISO ml Senltd Inllutm Smt(<
SO «n/l TMII (Uriel
20 (/! It it MilCiitl
cotmtiTf sue
co. tist
tOO Hi CompolKt $»<
$400 ml BOO Willi
UO «) R*spici»t
Till MitiMiU
Figure 11. General Protocol for a 10 Unit
Biodegradation Test Unit (Sturm, 1973)
Courtesy of J. of the Amer. Oil Chemists' Society
93
-------
Microbial cultures used by Garrison and Matson
(1964) In their degradation studies were obtained from
the Soap and Detergent Association (SDA) which had adapted
"d
them to a medium containing 30 ppm of linear alkylbenzene
sulfonate (LAS) prepared from dodecene-1. The above
investigators modified the cultures by growing them in
a SDA medium containing mixed detergent feed. The authors
suggested that the adaptation procedures described above
will develope cultures which can be used in studies with
a wide variety of detergents.
For use as seed, Langley (1970) developed a hetero-
genous population by growing the microorganisms present ,.
in settled sewage supernatant on a mixture of primary
alcohols (ethanol, 1-propanol, 1-butanol, and isobutanol).
The cultures were maintained by daily removal of one-third
of the culture fluid and addition of fresh alcohol mix-
ture. The microorganisms adapted in this manner were used
to study the degradation of higher molecular weight al-
cohols. In case of insoluble substrate, e.g., 1-hexadecanol
and 1-octadecanol, a series of flasks, each containing i-
dentically treated medium, were used. For periodic
analysis, the whole flask was sacrificed.
(f) Slope (Slant) culture technique
In this test, acclimated seed is prepared by in-
oculating an agar slant containing approximately 10 mg/
liter of the test detergent with the mixed bacterial
94
-------
culture from sewage works effluent (Cook, 1968). Agar
slants are incubated for 7 days and the bacterial growth
is used to inoculate a detergent solution prepared in
standard dilution water. Organisms suspended from two
slants were used to inoculate one 500 ml of the detergent
solution. Samples were analyzed for degradation prior
to inoculation and for a further 15 days. In the initial
studies, Cook (1968) used four different effluents having
varying proportions of industrial waste, domestic sewage
and agricultural waste as the source of inocula for the
slant culture technique to see if the reproducibility of
the results was affected. It was concluded that any
effluent could be used as the source of inoculum without
significantly affecting the results. Degradation was also
studied using unacclimatized seed prepared by inoculating
agar slants without the test detergent. Since the bacteria
that grew on the slants not containing the detergents
degraded the test detergents Just as effectively as those
which had been exposed to the test detergent, the author
*
concluded that the bacteria had not been acclimated by
slant culture.
In several tests Cook (1968) also used suspensions .
of organisms obtained from recirculating filters (see
p. 146), as the source of inoculum for agar slopes. .The
microbial population developed in the form of visible
95
-------
slime (organisms grown on test detergent as the sole
source of organic carbon) in the recirculating filters
was transferred into Ringer solution in McCartney bottles
and shaken. The suspension thus prepared was used to
inoculate agar slants without added detergent.
Dobane 055, a synthetic anionic detergent, was not
degraded by acclimated or unacclimated cultures obtained
from the agar slants inoculated with sewage work effluent
(Cook, 1968). However, when the inoculum prepared on
agar slants from recirculating filter inoculum was used,
the detergent was degraded after an initial lag. It
was known that the bacteria were not acclimated by the
slope culture technique and, therefore, the author spec-
ulated that the acclimation must have occurred during
!
recirculation.
(g) Shake culture employing seed acclimated to
increasing concentration of the test chemical.
Schwartz (1967) studied the degradation of pesti-
cides utilizing microorganisms from an activated sludge
unit and the effluent from a primary sedimentation basin
of the Whittier Narrows Water Reclamation plant. In
addition, samples of the flow and slime from a channel
containing refinery waste effluents were used. The medium
contained mineral salts (Gray and Thornton, 1928), various
amounts of pesticides, supplemental organic carbon source
96
-------
and microorganisms. The breakdown was studied in a
series (one to six) of adaptation stages; once signifi-
cant or complete degradation became evident, transfer was
made using the microbial inoculum from the preceding
adaptation stage. During these stages the amount of pesti-
cide was increased and the supplemental nutrient concentra-
tion was decreased.
Hemmett (1972), using a similar approach, studied
the biodegradability of phenoxyacetic acid herbicides and
phenols. Activated sludge, which was used as a source of
aquatic microorganisms, was incubated in a mixture of
synthetic sewage and an appropriate concentration of the
herbicide. By addition on alternate days, the herbicide
concentration was increased and the synthetic sewage con-
centration was decreased. At the end of the adaptation
period (approximately 14 days) the cultures were used in
degradation studies.
(h) Other modified tests
Thompson and Duthie (1968) prepared acclimated seed
for studying breakdown of NTA by exposing microorganisms
present in settled raw municipal waste water to nitrilo-
triacetic acid (NTA) in Bunch and Chamber (1967) medium.
The adaptation was continued for 14 days. Degradation
97
-------
studies were performed in a 8-20 liter carboys which
contained diluted seed (1:10 with BOD water) and NTA.
The mixture was aerated with CO -free air.
< Cordon £t al. (1968) studied the biodegradability
of anionic tallow-based detergents in 1-gallon wide-
mouth jars which contained 3 liters of deionized water,
10 mg of activated sludge per liter (on dry weight
basis), nutrient salts (free of sulfate to permit the
assay of sulfate ion formation from anionic detergents)
and 40 mg of detergent per liter. Activated sludge was
obtained from a treatment plant which treats mostly
domestic sewage. The detergent already present in the
sludge was removed by deacclimation in a laboratory
model activated sludge plant (Ludzack, 1960). A similar
method was used (Cordon e_t al., 1972) to study the
degradation of sulfonated alkanol amides. The test was
.run under aerobic and microaerophilic conditions. The
microaerophilic test was carried out as described by
Maurer ejt _al. (1971) (see p. 81 ) except that instead of
using river water, an inorganic nutrient salt solution
was used as the medium.
Ferguson e_t al_. (1973) studied the environmental
fate of nitrilotriacetic acid (NTA) using four sources
of organisms: a small stream, a farm pond, a river below
'-*
Athens sewage effluent, and the pilot plant treatment
98
-------
plant at the Robert A. Taft Water Research Center,
Cincinnati, Ohio (with prior exposure to NTA). Shake
flasks contained 5 ml seed, basal salts (Payne and
Feisal, 1963) and 10 mg/liter NTA. NTA degradation was
also studied in the presence of an external carbon source;
the investigators used glucose (10-100 mg/fc) instead of
the more commonly used yeast extract. The flasks were
incubated for 22 days and samples were withdrawn for
analysis every 48 hours.
Pawlowski and Howell (1973a & b) used activated
sludge and soil as the source of mixed culture of micro-
organisms. To maintain a constant inoculum for experiments,
the inoculum was first acclimated at 28°C in a chemostat
with a feed medium containing 100 mg/liter phenol. Two
separate residence times in the continuous culture appa-
ratus gave rise to two populations of microorganisms
which were used in the degradation studies.
In his shake culture studies, Gledhill (1974) used
a Bellco waffled Erlenmeyer flask which had been equipped
14
to trap C0_. Using this system he examined the break- -
down of trlchlorocarbanilide with raw sewage and activated
sludge seed (from laboratory semi-continuous activated
sludge unit) diluted with BOD water. For aeration, the
flasks were sparged periodically with 70/30 O./N mixture.
99
-------
The activated sludge obtained from a local treatment
plant was found to display an 8-10 weeks lag before
' 4
acclimation was gained.
(11) Degradation Test Using Lagoon Microorganisms
'j
A method has been suggested by Halvorson et_ _al. (1971)
for testing the biodegradability of insecticides using resting
cell suspensions of the bacteria from a sewage lagoon. The
use of lagoon microorganisms was preferred because, bacteria
indigenous to this environment have been shown to exhibit a
wide spectrum of physiological properties. The bacteria re-
covered from 40 liters of lagoon water were washed and resus-
pended in 200 ml of potassium phosphate buffer (pH 7.0)
8
(about 4 x 10 cells/ml). Organophosphate insecticides were
added at an initial concentration of 50 ppm. For studying
biodegradation of chlorinated hydrocarbons lower cell popu-
lations and lower concentrations of the test chemical were
used. Small aliquots of the reaction mixture were incubated
in small vials. The entire contents of the vial were analyzed
at regular intervals.
(ill) Shake culture test without Initial inoculation
Considering the possibility that the cultures ordinarily '
used in biodegradation studies may be overspecialized and,
>»
t ' '
hence, may give unrealistically good results, Swisher (1966)
in"his studies used the microorganisms which develop in an
. -.Mf . .''-..
100
-------
uninoculated test medium. This development of organisms
from ambient sources is also used in the detergent continuous
activated sludge test required by German law (German Govern-
ment, 1962) (see Sec. Ill B.2.a, p.134). Swisher (1966)
used the medium developed by Allred e_t al. (1964) which was
prepared and handled without aseptic technique. Surfactant
was -added to the medium contained in 1 liter Erlenmeyer flasks
and then shaken on a rotary shaker. After one week, 1% of
this mixture was inoculated into fresh medium plus surfactant
and successive weekly transfers (for 4-5 weeks) were continued
.in this manner.
Swisher (1968) in his later studies on linear alkylbenzene-
sulfonate (LAS) degradation used the cultures which had de-
veloped in the uninoculated medium containing C LAS, and
maintained through 61 weekly transfers on SDA medium. In
much of the degradation work, Bunch and Chambers (1967)
medium was used since the interference with the analytical
test (U.V. analysis) was minimal. The transfers were made
at biweekly intervals Instead of weekly.
(iv) Shake cultures Inoculated with lake sediments
Graetz e_t al. (1970) have used the water obtained by
centrlfugation of Lake Tomahawk sediment as a source of
oicrobial species. Sterilized 2% peptone solution was in-
oculated with the water obtained as described above; after
36 hours, when cloudiness had developed, the culture was
101
-------
diluted twofold with a 5 rag/liter parathion solution and ,.
degradation was studied. Degradation was also followed in.
"a sediment system consisting of 90 mg Lake Tomahawk sediment
and 200 ml of a 5 mg/liter parathion solution fortified with
5 ml of inoculated and incubated 2% peptone solution.
b. Shake Culture Studies Using Pure Cultures of Microorganisms
i
Shake culture studies using pure cultures of microorganisms have
i
been extensively used in evaluating biodegradation. Biodegradability
tests utilizing pure cultures will be expected to be more reproducible
than if undefined mixed cultures are used as inoculum. A variety of
sources ,of microorganisms have been used to obtain the pure cultures.
These include pure cultures obtained from commercial sources or from
a laboratory, pure cultures isolated, from natural sources (without
exposure to the test chemical), and pure cultures enriched from the
natural population. In this method, sterile mineral salts culture
medium (or salts medium supplemented with easily utilizable carbon
source) is prepared and the organic compound of interest is included
as the major potential nutrient. The medium is inoculated with an
appropriate culture of microorganisms and aliquots are removed at
various intervals for analysis.
Pure cultures are generally not considered as satisfactory inocula
i
for preliminary screening. It is unlikely that a single species
could be found which will be capable of assimilating all the chemicals,
since the number of necessary constitutive and inducible enzyme systems
capable of degrading organic compounds is limited in any single culture
102
-------
test system. By using a large number of pure cultures this problem
can be partially overcome. When a large number is employed, the
relative degree of biodegradability of a compound can be assessed
from the proportion of the tested species which can utilize the com-
pound as sole carbon and energy source (McKenna and Kallio, 1964;
Painter, 1973a). In addition, pure culture studies should be of
value in the detailed study of specific metabolic reactions.
On the basis of the sources used for procuring pure cultures of
microorganisms for biodegradation studies, these studies could be
grouped as follows:
(i) Pure culture obtained from commercial sources or from
research laboratories
A number of pure cultures are generally tested for their
ability to degrade the test compound. The organisms selected may
be those which have been reported to metabolize certain synthetic
organic compounds. Payne & coworkers (1970) and Painter (1973a)
have suggested the use of species of genera such as Klebsiella,
Escherichia. Serratia, Candida, Pseudomonas, Flavobacterium.
Achromobacter, and non-parasitic species of Mycobacterium and
Nocardia. Filamentous fungi such as members of the genus
Aspergillus could also be added to the list since these organisms
have been found to grow on a variety of substances (Murray et al.,
1970).
A summary of the studies in which investigators have used
pure cultures of microorganisms obtained from commercial sources
or research laboratories is given in Table 8.
103
-------
Goodnow
&
Harrison
(1972)
VanAlfen
ft Kosiige
(1974)
Table 8 ture Culture Obtained from
CQjqmetcial Sources on Research Laboratories
Reference
Huddles ton
& ''Allred
(1963)
Klug &
Markovetz
(1967)
t
Organism Source
Escherichia
colif" Serra'tia
raarcensens3.
Proteus vulgaris"1,
Pseudomonas
f lucres censa
110 species of 1. North Regional
Candida Research Lab . ,
Peoria, 111.
Test. Chemical
Sulf onated
alkylbenzenes
N-alkanes,
even-numbered
1-alkenes
External
Time of Carbon
Cone. Incubation Source
30 mg/Jl 3 days None
,
0.2ml/ 2 weeks yeast
20 ml nitrb'gen
base
19 Genera,,34
species and 45
strains of
Bacteria, e.g.
Acetobacter,
Azotobacter'.
Bacillus,, j
Chrdinbbactertum,
Escherichia, etc.
2. Dept. of Micro-
biology, Univ.
of Iowa
3. Amer. Type Cul-
ture Collection
(ATCC)
4. Dr. Phaff, Univ.
of Calif., Davis
5. Dr. Azoulay,
France
ATCC
Dr. Engley
Univ. of Missouri
Medical School
Surfactants
0.004-
0.5 g/fc
3 days
Trypticase
soy broth
without
glucose
E. coli
Pseudomohas
cepacia
Dr. Clark, Univ.
of California,
Berkeley
Dr. Lorbeer,
Cornell Univ.
2,6-Dichloro- 2 ug/ml 2 days
4-nitrbaniline
None
Adapted to straight''chain ABS by successive transfer
:'v; ' .
(ii) Pure Cultures Isolated from Natural Sources
' ' '-. In order to investigate the fate of organic chemicals in
*
certain specific natural environments, many investigators have
used the approach of isolating microorganisms from a particular
''.'&
environment and using those species in degradation work. Samples
c-,
obtained from natural water systems are diluted with synthetic
medium or sterilized natural water and placed on the agar plates.
104
-------
Isolated colonies are picked up and propagated on an appropriate
medium and used in studying degradation of organic chemicals.
The procedure differs from the enrichment culture technique
(see the following section) since the medium used for initial
isolation is formulated in such a way that it permits many of the
organisms to proliferate. Similar to other test methods utilizing
pure cultures, these studies individually fail to simulate the
natural environment or to provide information on degradative cap-
abilities of microorganisms under conditions which permit inter-
action with other species. However, if large enough numbers of
pure cultures are examined, the studies may provide some indi-
cation of biodegradability.
Salient features of the degradation studies performed using
microorganisms isolated from natural environments are given in
Table 9. '
(iii) Pure Cultures Obtained from Enrichment
(a) Enrichment with test chemicals
This test, which is also referred to as the Elective
Culture Method, is based on the principle that from any
natural population the organisms capable of utilizing a test
compound as a nutrient source should increase in number during
the enrichment period. The term enrichment refers to the
opportunity for outgrowth of certain types of organisms over
the others.
105
-------
Table'9. Pure Cultures Isolated from Natural Sources
Reference
Matsumura
et al.
TT97T)
Strzelczyk
et al. (1972)
Gonealez ec al.
(1972)
Alexander
(19730)
Natural Source
lake Michigan
and its
tributaries
Eutrophlc Lake
Jeziorak ,
Poland
'
Solar brine pond.
Great Salt, Lake,
Utah
Sea water and
- sediment material
fron Connecticut
coast
Organism
Identified As
approx. 450
isolates
(unidentified) -
Corynebacterium sp.
Nocardia sp.
Bacillus sp.
unidentified
Bacterium T-52
(unidentified)
100 marine
bacteria
Test Chemical Cone, of
Studied Test Chemical
DDT 10" 5H
pounds e.g.
p-Hydroxy benzole
acid, phthallc
acid, salicylic
acid, etc.
Ethylene glycol 10 ml/1
DDT 0.5 gg/ml
External
Period of Carbon
Incubation Sourcei"
30 days yeast
.extract
6 days '.None
3-5 days * glucosi
7-18 days yeast
extract
Soli (1973)
Walker &
Colwell (1973)
Walker et al.
(1973)
'Not given
Chesapeake Bay
water & sediments
Chesapeake Bay
5 Bacterial
strains .
(unidentified)
Cladosporiua sp.
Penicillium sp.
Alternaria sp.
Trichoderma'sp.
unidentified
bacteria and
actinomycetes
Cladosporiuin .
resinae
Synthetic oil
containing normal
paraffins,
isnparaffins,
cycloparaffins and
aromatics
Motor oil
(non-detergent)
aliphatic and
aromati'c hydro-
carbons , organo
phosphorus &
chlorinated' hydro-
carbon pesticides
10 days None
10 gm oil 21-28 days None
powder/i
Solids 17, w/v 30 days yeast
Liquid 102 w/v extract
Reproduced from
best available 'copy.
106
-------
In this method a source of natural population (e.g.,
river or lake water, sewage effluent, etc.) is enriched by
the addition of low, non-toxic concentrations of the test
compound. Transfers are made into fresh medium and enrichment
is continued. After several such transfers, the enriched
organisms are isolated by streaking on agar medium containing
the test chemical, and single colonies are picked. Although
the isolation of an organism which uses the test chemical
as the sole source of carbon and energy is in itself an in-
dicator of extensive biodegradability of the organic compound
(Prochozka and Payne, 1965), most researchers, as an added
proof, have further studied the biodegradation of the test com-
pound employing the isolated species in the shake culture test
or in a respirometer. Organisms isolated by enrichment culture
technique have been utilized extensively by investigators in
establishing the pathways of degradation of synthetic organic
chemicals. A voluminous literature is available on the use of
enrichment culture technique in isolation' of an organism for
studying the breakdown of chemicals. Some of these studies
are summarized in Table 10.
(b) Enrichment for Cometabollc Degradation
Cometabolism is defined as the metabolism of a substrate
by a microorganism which cannot use that substrate as a
nutrient. This term was first provided by Jensen (1963) and -;
later emphasized by Alexander (1967b) (also see Horvath, 1972a).
107
-------
Cometabolism may account for the degradation of many synthetic**
chemicals which do not sustain microbial growth and evidence
for, the ecological significance of cometabolism is rapidly
increasing. The elective culture method as it is normally L
-4 | ' -
used 'completely^ ignores cometabolism as a factor in the decom^
position of organic chemicals. Several investigators; have-
recently attempted to measure the contribution of cometabolic
'- I
degradation. Organisms can be enriched by application-r of.; bi'b-^
degradable analogues of the pollutant (analogue enrichment)
o.r by adding a co-substrate in. addition to the test chemical
(co-substrate enrichment) to a natural mixed population of
microorganisms.
Focht and Alexander (1971) employed the elective culture
technique to obtain microorganisms capable of- growing on di-
pheriylmethane, a npn-chlorinated analogue of DDT. The ability
of the isolated species (a strain of Hydrogenomonas) to grow
in a media containing DDT as the sole carbon source or comet-
abplize it was/investigated:(Focht and Alexander, 1970, 1971).
Horvath and Alexander (1970)obtained twenty isolates repre-
.--.).
senting nine/bacterial genera^by,, enrichment culture, and these
isolates metabolized substituted benzoates which failed to
serve as the sole' carbon sources for growth.
V*'i
Horvath (1973) later investigated the ability of a
structurally unrelated substance to serve as an enrichment
:'& '''. '
agent when added in the presence of the test compound. The
108
-------
Table 10. Degradation Studies Using Pure Culture Isolated from Enrichment
Reference
Payne &
Felsal
(1963)
Payne (1963)
Prochaeka &
Payne (1965)
Bernards
at al. (1965)
Horvath &
Kofi (1972)
Forsberg (,
Undquiet
(1967)
Cook (1968)
Rogers &
Kaplan (1970)
Teat Chemical
Dodecyl sulfata
Dodecyl benzene
sulfonat*
Alkyl bensene
aulfonate
Nitrilotri-
acetic acid"
JNX
LAS
Concn.
During
Enrichment
IX solution of
9SZ sodium
dodacyl sulfate
or dodecyl
benzene aul-
fonate
M
0.1X
10 mg/t
30 mg/i
Source of
Mixed
Inoculum
Soil near the
outfall of sewage
disposal plant.
ii
ii
."
River water
Agar slopes
inoculated with
sawage work
effluent
Activated sludge
Organisms
Isolated
Unidentified,
designated C12,
and C12B
(Pseudononaa sp.)
bacterium C12B
ii
HK-1
(Pseudomonss sp.)
Unidentified
Degradation Test
Cone, of
Tent Tint: of
Chemical Incubation
0.015-0.025 M 30 houru
SLS
0.1 M DBS
0.1X (w/v) 72 hours
straight chain
saturated
alcohol, carbon
atom 6-18
various concna . 24-48 hours
of sodium dodecyl
sulfate, dodecanol.
Mixture of CIQ-CJO
secondary alcohol
sulfate
0.1X 24 hours
as sole carbon 225 hours
source, 1 g/t;
ae sole nitrogen
source, 0.5 g/t
4 unidentified 12 mg/t 16 weeks
gran negative rods
2 Klebsiella sp.
20 bacterial Isolates Same as in shake
belonging to geners culture test of Soap
Peeudomonas. Achromo- (, Determent Aaaocia-
bactet. Paracolo- tion
. . bacterium
Rip in at el.
(1970)
Focht &
Williams
(1970)
Enzlngor
(1970)
Focht &
Joseph
(1971)
. Mined 6
Focht (197J)
Baggie si. ii-
(1972)
*
Sikka d
Saxena (1973)
Kaiser 4
Hong (1974)
LAS ,
p-Toluene-
Bulfonate
Alpha
trinitrotoluene
Nitrilotriacetic
acid
Blphenyl
p-Chlorobiph«nyl
Fhenylalkanes
Endothal
Polychlorinatad
blphenyls
(Aroclor 1242)
1 gm/i
1 g/l
Subcultured In
Increasing TNT
concn. up to
100 ppm
0.01 M
0.1*
1 g/l
0.1*
Soil, sewage,
river waters,
scrapings from
laundry drains,
woodland soil
Sewage effluent
Nixed liquor
suspended solids
from the TNT test
unit
Sewage effluent
Sewage effluent
Lake water and
hydrosoil
Hamilton Harbor,
Ontario
Mlxed culture
containing
various organisms
Pseudomones sp.
Pseudomonas sp.
Pseudomonaa sp.
Acromobacter
BP
Accoinobacter
pCB
Peeudomonas
acldovorans
Two Nocardla
strains
Arthrobacter
Unidentified
*As aole source of carbon and sulfur
30 ug/ml 72 hours
0.2-1 g/l 120 hours
100 ppn 5 days
O.U1 M 10 days for
growth; 4 hours
Cor degradation
by resting cells
0.1Z 66 hours
few drops
(exact concn.
not given)
250-1000 ppm 18 hours
0.1% 2 months
Reproduced from JPfi|
best available copy. >^!§r
109
-------
co-substrate enrichment: technique has been utilized in
w
studying degradation of chlorobenzoates. The enrichment
medium containing 25 mg/fc chlorinated hydrocarbon substance
also ^received 500 mg of glucose/ S, which served as the co-
substrate.
(c) Enrichment for Marine Microorganisms
. In an attempt to; investigate the breakdown of organic
t
chemicals in the marine environment, researchers have fre-
quently isolated pure cultures of microorganisms from 'marine
environments .by enrichment culture technique. Marine ;inud and
i "
water samples collected from- coastal areas are generally used
as a source of microbial population. The basal medium for
JjJ .
isolation is usually' sea water supplemented with inorganic
;
salts, e. g. , phosphate and; nit rate, in which sea water is
deficient. The test compound is added to serve as the sole
source of carbon and -energy. Organisms capable of utilizing the
test material are isolated as described before. Although a.
large number of chemical contaminants have been detected in
r '
'oceans, most of the breakdown studies have been restricted to
oil 'pollutants, a major marine contaminant.
Bartha (1970) 'isolated 40; strains of oil-degrading
marine microorganisms from oil-polluted as well as non-pol-
?
luted sea water and marine sediment using a basal medium
(sea water or Bushnell-Haas broth supplemented with 3% NaCl)
supplemented with individual hydrocarbons or crude oils as the
110
-------
sole added carbon source. The ability of these microorganisms
to degrade crude oil (light and heavy), and aliphatic, alicyclic
and aromatic hydrocarbons was evaluated. The crude oils as
well as other hydrocarbons were filter-sterilized prior to
addition to the shake flasks. Atlas and Bartha (1972) se-
lected two bacteria for further study from those isolated by
Bartha (1970) on the basis of their rapid growth and wide
range of hydrocarbon utilization. The aim was to establish
the order in which individual components are degraded, and
to quantitate the overall rate and extent of degradation
and mineralization by marine isolates. The degradation
studies were carried out using a gas train arrangement
mounted on a rotary shaker (described in detail in Section
III C.2.e, p. 157). In view of the fact that sea water
contains very low concentrations of nitrogen and phosphorus,
these researcher studied the degradation of crude oil after
the addition of olephilic N and P sources. The method
is recommended to promote oil biodegradatlon on the
high seas and may not be suitable as a degradation test
method.
Cerniglia.et al, (1971) investigated the degrac...
dation of petroleum by microorganisms maintained in
stock culture and found that fungi were superior to
bacteria. Perry and Cerniglia (1973a, 1973b) subse-
quently isolated filamentous fungi Cunninghamella "','.
Ill
-------
elegans and Penicillium zonatum from coastal water by en-
richment with crude oil. The basal medium used in these
studies was sea.water supplemented with a source of N and P.
NH.C1 was reported to be better for growth than NaNO..
*'A 3
Reisfeld et_ al. (1972) obtained a mixed popula-
tion of microorganisms by enrichment culture technique
using crude oil as the source of carbon. The medium
was supplemented with yeast extract. The enrichment
culture was streaked into supplemented oil agar
plates to obtain pure cultures of microorganisms.
Degradation was studied .with mixed as well as pure
cultures. Kator (1973) has enriched mixed cultures
t>
of/petroleum utilizing marine enriched sea water
medium (Miget _et al., 1969) supplemented with crude
oil. An oil-free .suspension of enriched cells pre-
pared from the vapor-grown cells (substrate is provided
in the vapor phase instead of being .added to the growth
medium) was used in degradation experiments.
(iv) Pure Cultures Isolated from Naturally Enriched Environment
When a synthetic chemical enters the "environment, one or
> '
a group of indigenous -populations possessing requisite-enzymes
or which can synthesize nccescary induciblc enzymes frequently
.\
multiply and make use of the introduced substrate. If a particular
'chemical is continuously dumped into the environment, the species
-------
Subsequently there is a much greater possibility of isolating
an organism capable of degrading a particular synthetic chemical
(if the chemical supports microbial growth) from an environment
which has previously been exposed to the chemical of interest.
In these studies, organisms have been isolated from such con-
tinuously exposed sources by plating appropriate samples either
on nutrient medium containing a readily available carbon source
or a medium in which the test chemical serves as the carbon source.
Isolated colonies are picked and pure cultures of microorganisms
are used for degradation work. The isolation procedure is different
from the enrichment culture technique since no transfers into
fresh medium containing the test chemical are necessary. Studies
which have made use of this technique are summarized in Table 11.
Table 11. Degradation Studies Utilizing Pure Cultures
Isolated from Naturally Enriched Environment
. Source of
>efer«nce Microorganism
Buhl 1
PaMon
(195 j)
Urock i
Oppenheloar
(WTO)
taaagi *
.Ontfhl (1971)
Cooey .
MlUur (1973)
Degraded
plastic
filiBB
011-aoslc«d
oil
Coametlc
product*
Contaminated
Jet Fuel
Byatena
Orgenien
Identified M
Aap«r»lllua
yaraicolor .
Peauooaonae
aarualnoaa '
Unidentified
23 etralna of
bacteria
25 actalna of '
y«eet
17 etrala* of
fungi
(sooe Identi-
fied)
C. raalnaa
Taat Cone, of Kktemal
Chemical Teat . Period of Carbon
Studied Chemical Incubation Source
Plaaticliera 3Z for eetara 7 day! yeoat
21 for alcohoie extract
Mineral oil - 5 d«y. Nona
karoaene mixture
Ingredient* 11 20 daya None
uaad In formulation
of coaoaelc producta
e.g., hydrocaxbooa,
elllconea, alcohola.
eatera» fatty aclda
o-alkanaa - 20-36 daye Nona
113
-------
(v) Cell-free Extract .Studies
. u Degradation studies using extracts of microorganisms have
,1
generally been carried'out for two purposes: 1. To invest!-
i
gate the pathways of degradation; 2. To study the enzymes
involved in degradation. Cell-free extracts are suited^ for
this type of investigation because physical and chemical .para^
meters affecting degradation can be controlled easily; and'.
the problem of cell permeability has, been bypassed.'-,
, I
' ; Heyman and Molof (1968) used cell-free extracts obtained'
from Pseudomonas C12B. an organism isolated by enrichment on
sodium lauryl sulfate (Payne and Feisal, 1963). The extracts
were prepared by grinding the cells with alumina in a chilled
mortar. Lijmbach and Brinkhus (1973) investigated the mech-
anism of biodegradation"of secondary n-alkyl sulfates and
secondary alkanols, using the cell-free extract prepared from
the bacteria isolated by enrichment on 2-butanol. The organ-i
isms were tentatively identified as a Pseudomonas species.
These researchers used an ultrasonic disintegrator for dis- '
rupting the cells.
Pfaender and Alexander (1972) studied the microbial
degradation of DDT _in vitro in the presence of enzyme-systems
from two species of bacteria. The selected bacteria included
a strain of Hydrogenomonas grown on a biodegradable DDT
114
-------
analogue, diphenylmethane, and an Arthrobacter sp. isolated from
sewage by enrichment using p-chlorophenylacetic acid (a
metabolite formed from DDT by Hydrogenomonas sp.) as the source
of carbon.
(vi) Multiple diffusion chamber of study ineraction among pure
cultures of microorganisms
An instrument has recently been developed (EcoloGen
Model E-40, New Brunswick Scientific Co., New Brunswick, New
Jersey) which can be used to study the combined action of
several pure cultures of microorganisms in the degradation of
organic chemicals. It consists of a central diffusion reser-
voir surrounded by four peripheral growth chambers, each sealed
with a membrane filter. The pore size in the membrane filter
allows only the metabolic product of the interacting pop-
ulation to diffuse to other chambers. A metabolite formed
as a result of degradation of a chemical compound by a
single microbial species will thus become available for
attack by different species in another growth chamber.
4. Continuous Culture Technique
In continuous culture, bacterial population density is regulated
by automatic addition of the fresh medium to the bacterial culture.
The technique allows an investigator to maintain a steady state
growth of microorganisms at a desired rate between zero and near max-
imal. The continuous culture technique has recently been
applied to the study of various degradative processes. One advantage
115
-------
in the continuous culture system is that toxic products and metabolic
i
wastes will not accumulate and their effect on the degradation process
will not increase with time. The system is more like the natural
&
aquatic environment in this respect where a continual removal or dilu-
tion of the toxic products is expected.
Pritchard and Starr (1973) have utilized continuous culture Ce'fchni-
que to study oil degradation. In this method, the water or nutrient
solution is continuously passed underneath the hydrocarbon layer while
maintaining a stable two-phase system in which hydrocarbon floats on
the surface of the water column. Aliquots of the culture are continu-
ally cycled out of the growth vessel, aerated and recycled back
(Figure 12). Studies were performed"using mixed bacterial population
present in Lake Ontario water as well as with pure cultures of bacteria
which became predominant in the continuous culture due to their rapid
rate of growth. Several researchers have also utilized the continuous
culture technique for enrichment of aquatic'microorganisms for use in
breakdown studies (Jannasch, 1967).
116
-------
RUBBER TUBING
COTTON
MEDIA
RUBBER STOPPER
CULTURE
VACUUM
AERATION UNIT
GLASS TUBING
SAMPLE PORT
HYDROCARBON
OR OIL LAYER
GLASS
STIRRING BAR
Figure 12. Continuous Culture System
(Pritchard and Starr, 1973)
Reprinted from Mlcroblal Degradation of
Oil Pollutants. Louisiana State Univ.
117
-------
i I
5. Terrestrial-Aquatic Model Ecosystem
i '
A model ecosystem is a laboratory simulation of a dynamic biological
environment characteristic of the natural ecosystem. A large number
( -
of contaminants enter the aquatic environment indirectly due to leaching
or runoff from the soil. A well-designed terrestrial-aquatic ecosystem
could simulate the application of the chemical to a crop in a terrestrial
situation and provide for transport from land to a typical lake water
situation involving a food web of both herbivorous and carnivorous
i
organisms. By varying the number of the food chain elements, ^both-very
i
i
complex and simple ecosystems have been developed. The system has been
used to examine both the biodegradability and ecological magnification
of environmental contaminants.
The laboratory model ecosystem described by. Metcalf et^ al., (1971)
utilizes a 10 x 12 x 18" aquarium and contains sloping soil/air/water
interfaces, plants and food chains of at least 7 elements (Figure 13).
The reference water (Freeman, 1953) contains inorganic salts to provide
satisfactory mineral nutrition: for plants and algae. The model
ecosystem contains snails (Physa),.algae (Oedogonium cardiacus),
Daphnia and a few milliliters of old aquarium water to provide the
plankton. The terrestrial portion of the ecosystem consists of washed
white quartz sand molded into a sloping surface into which sorghum is
planted. All the food chain organisms are raised in the laboratory.
118
-------
Figure 13,' Schematic Drawing of Model Ecosystem for
Studying Pesticide Biodegradability and
Ecological Magnification (Metcalf, et_ al., 1971)
Reprinted with permission from Environ. Sci. Technol.,
.5(8), 709-13. Copyright by the Amer. Chem. Society
Radiolabeled test compound is added (at approximately the same rate
as used in field application) after about 20 days equilibration. Culex
quinquefasciatus mosquito larvae are added to the ecosystem after
26 days and Gambusia affinis fish after 30 days. The aquarium is
housed in a plant growth chamber with a 12-hour daylight exposure
(5000 ft. candles) and the water is aerated continuously. The experi-
ment is. terminated, after 33 days and the fate of the test chemical
in food chain organisms and in the water is determined.
The food-chain pathways for the pesticide in the model ecosystem are:
(1) Sorghum -» Estigmene (larva)
(2) Estigmene (excreta)" * Oedogonium (algae)
(3) Oedogonium > Physa (snail)
(4) Estigmene (excreta) -* Diatoms
(5) Diatoms -» Plankton
(6) Plankton -t Culex (larva)
(7) Culex-» Gambusia (fish) -
119
-------
The authors claim that the model ecosystem techniques provide a con-
<,
eiderable possibility for the test contaminant to be degraded by simulated
,f"
sunlight, air, water, and during.;passage through the food chain.
Since its first reported use-, the model ecosystem of Metcalf e£ al.
(1971) has been used in a great: number of other studies to examine bio-
degradability and ecological magnification of environmental contaminants.
The compounds studied have included polychlorinated biphenyls, organo-
chlorine pesticides including DDT and its analogues, phthalate':plasti-
cizers, phosphate and carbamate ester insecticides (Metcalf & Lu,, 1973;
i
Metcalf, 1974; Kapoor e£ .-.al., 1972; Hirwe et^ al., 1972; Kapoor;;et'ai.,
1970; Metcalf et^ al_., 1973a; Metcalf e£ al., 1972). The ecosystem has
i
also been used by Metcalf et. al.. (1973b) to study the interaction among
pesticide chemicals.
Sanborn and Yu (1973> have used the.model ecosystem described by
Metcalf et ia. (1971) but modified the aquatic food chain slightly.
In addition to the standard components of the Metcalf ecosystem, these
researchers also added one crab (Uca.minax), two small fingernail clams.
v
(Corbicula manilensis) and a water-plant :Elodea. Yu et^ al^ (1974.) used
<,>
the model ecosystem,described by Sanborn and Yu (1973) but in addition
included frogs in the food chain. Most of .the food chain organisms were
obtained from stock cultures in the laboratory except clams, crabs, frogs
and Elodea which were .obtained from-the local pet shop. They investigated
the fate" of carbofuran :in the model ecosystem. Due to-the toxic nature of
carbofuran, most of the organisms were killed shortly after the intro-
duction of Insecticide and, therefore, tanks had to be restocked every
1*1
5-7 days. ~The exposure period for most aquatic organisms was hence reduced.
120
-------
6. Model Aquatic Ecosystem
Several ecosystems which simulate the aquatic environment alone
have been developed to study the environmental fate of those compounds
which are either applied or supposedly formed in water. These systems
generally contain elements of an aquatic food chain in an aquarium which
is filled with natural or reference water (see p. 118) containing inorganic
nutrients. The complexity of the ecosystem is generally determined by
the food chain pathways simulated in the ecosystem.
One of the simple ecosystems has been described by Sharman (1964)
for studying biodegradation of synthetic detergents. The system used
a 14 gallon aquarium which contained 44 & of river water. Eleven
small fish (guppies, white clouds and cat fish) were introduced and
the system was allowed to approach steady state for 7 weeks. In order
to stimulate acclimation of the living components, small amounts of
the detergent (0.5 mg/£) were initially added. Formal test was
started 4 weeks later by the addition of branched and straight chain
ABS at the concentration of 1 mg/&.
In the study of metabolism of DDT by natural microbial communities,
Pfaender and Alexander (1972) used a water-sediment ecosystem. The
system uses a 4-£ glass bottle to which two-liter portions of sewage
and fresh water containing sediment collected from a rural stream were
added. Sterilized air was passed over the surface of the liquid.
Autoclaved samples of sewage and fresh water plus sediment were incu-
bated in a similar fashion to show nonbiological change. Sikka and
Rice (1973), in their studies on endothall degradation, used 10-gal.
121
-------
capacity aquaria to which 7-gal. of pond water and 1^' layer of pond
hydrosoil was added. An aquarium containing endothall in autoclaved
pond water served as control.
->'}
The ecosystem devised by Metcalf and Lu (1973) is specially
suited for compounds which are volatile. Metcalf (197A)
has claimed that the system provides a rapid 3-day evaluation of,eco-
logical magnification, biodegradability and degradative pathways as
against more complex terrestrial-aquatic model ecosystems (see p.. 118).
In this system a 3-neck flask (3Jt capacity) fitted with reflux con-
14
denser and two traps to collect volatile products and C0? is used.
The aquatic fauna and flora (reared in the laboratory) mixed with
standard reference water are transferred to the flask. The radio-
labelled test compound is added to the ecosystem and after an exposure
period of 3 days, analysis is performed.
The aquatic ecosystem has been used by these investigators to study
the degradative fate of several industrial .organic chemicals such as
benzene derivatives, chlorinated biphenyls, and such pesticides as
DDT, aldrin, etc.
, ,i.
Isensee and coworkers (Isensee et al.., 197-3; Isensee and Jones, 1974)
modified the terrestrial-aquatic, model ecosystem of Metcalf at a_l. (1971)
to make it suitable as an aquatic ecosystem to study the environmental
\ . .
fate of aquatic contaminants. These researchers eliminated the terres-
'3 .
1 - i
trial phase from the ecoystem and modified the standard reference water
by increasing the NH^IK^ and K2HPOit concentration five-fold to obtain
122
-------
satisfactory algal growth. Since the major node of entry of certain
pesticides into water systems is from erosion of pesticides adsorbed
14
on soil, the researchers adsorbed the C-labelled compounds on soil
and then placed the soil in the aquarium tank and added water. The
temperature in these studies was maintained at about 21°C (since 21°C
is the optimum temperature for one of the food chain organisms) unlike
the Metcalf system, where the temperature was 26°C. In the modified
ecosystem, the food chain pathway was as follows: Water -» Algae -»
Snails, and water -» diatoms, protozoa, and rotifers -» Daphnia -» fish.
Johnson (1974) has developed a food chain model representing three
aquatic trophic levels to study bioaccumulation and biodegradation of
xenobiotics. These simulate warm water (>16C) or cold water (<16C)
food chains and have the following food chain pathways:
1. Microorganisms -» filter feeder (Daphnia) -» fish (blue gill) or
2. Detritus (leaves) + scavenger (scud) -» fish (rainbow trout),
respectively.
Cochrane et_ al. (1967) used plastic pools (30" high and 10 ft. in
diameter) to study persistence of Silvex. About 8 cubic feet of soil
(3 major soil types characteristic of the southeast) was added to the
pool within an area 6.5 feet in diameter. The pool was then filled
with approximately 80 cubic feed of water. Alligator weed was planted
in the pool and, when the growth became established, the herbicide was
applied by spraying.
123
-------
The Southeast Environment Research Laboratory (EPA, Athens)
t , .
(Sanders & Falco, 1973; Lassiter & Kearns, 1973; Falco & Sanders, 1973)
has recently developed an aquatic ecosystem simulator to study eco-
logical processes which involve interactions between chemical and
biological systems (Figure 14). The system consists of an environ-
mental chamber (22 meter x 3.66 meter) which houses a water-.channel
(19.5 meter long x 0.46 meter wide x 0.6 meter deep). Thev:level of
turbulent mixing in the channel is regulated by rotating paddle-wheels.
.<_
The channel is equipped with a radiant energy system (consisting .of red,
green and blue fluorescent light, and Incandescent light) and an air
circulator system. Water is supplied to the channel after passing
through a deionizer, still and heat exchanger.
No reports are available at this time concerning the use of
aquatic ecosystem simulator for determining environmental persistence
of chemicals. E.P.A. has recently Announced, however, (EPA, 1974) that
studies will soon begin with the-simulator to determine net degradation
rates under conditions when various modes of degradation are competing
under simulated natural conditions. It is anticipated that the studies
will enable: researchers'to predict what happens to a chemical when it
is discharged into.natural waters.
124
-------
\
ANALYTICAL
CHEMISTRY
LABORATORY
CONTROL
PANEL
COMPUTER
FACILITIES
AREA
CHAMBER
A. Floor plan - schematic of chamber facilities.
COILED HEAT
EXCHANGERS
COOLANT
WATER
ENTRY
AND
RETURN
AIR
ClRCULATION
FAN
GREEN
RED
BLUE
AlR VENT
NCANDESCENT
LIGHTS
B. Schematic of ecosimulation chamber light source.
Figure 14. Aquatic Ecosystem Simulator
(Sanders and Falco, 1973)
Courtesy of Pergamon Press Ltd.
125
-------
B. Techniques Simulating Sewage Treatment Conditions
1. Introduction
The techniques discussed previously simulate to varying degrees
conditions in nature. However, many researchers have found it very difficult
to extrapolate results of those techniques to estimates of removal during
waste water treatment because of the very great differences in such:parameters
as temperature, microbial concentration and types of microorganisms, 'food 'source
and acclimation. This lack of correlation has led to development -of 'techniques
which better simulate actual water treatment conditions.
Water treatment techniques which are currently modelled
in the laboratory include activated sludge processes, trickling filter
processes and septic tank treatment. A basic understanding of these treat-
ment processes is necessary in order to evaluate the modelling techniques
which are used. *>
Although some physical-chemical waste water treatment
techniques are currently being considered (e.g. Lewieke, 1972), biological
treatment is by far the most prevalent. Since many chemical substances will
pass through such treatment processes before being released to the environment,
it is Important to understand the degradation which may take place. However,
a careful distinction should be made between the "treatability" versus the
biodegradability ofva chemical substance under treatment conditions. Often
a substance is removed during treatment by physical processes such as adsorption
on sludge. Although the quality of the water receiving the effluent is improved
the overall environmental quality may be reduced since the aludge is often
126
-------
dumped somewhere else (e.g. landfilled or dumped in the ocean) and the chemical
may persist in that environment. On the other hand, one method of sludge dis-
posal is incineration and that technique may totally degrade the chemical sub-
stance removed by treatment.
Activated sludge systems are one of the most commonly used
treatment processes, especially in municipal sewage treatment. The system
basically consists of primary removal of suspended solids followed by secondary
treatment with aerated activated sludge (see Figure 15 ). Chemical substances
in the effluent stream can be removed (a) by oxidation in the sewer, (b) by
absorption on activated sludge followed by oxidation, (c) by adsorption on activated
sludge which is then "wasted" as excess sludge (Stennett and Eden, 1971) or
the substance may be removed with settled^material in the primary clarifier.
i
SLUDGE USTINO
BCD
Figure 15. Flow Pattern of Domestic Activated
Sludge Waste Disposal Plant
(Huddleston and Allred, 1964)
Courtesy of J. of the Amer. Oil Chemists' Society.
In terms of biodegradation, oxidation by activated sludge
is most important. Activated sludge is a bacterial floe in which are embedded
living and dead cells, cell wall fragments, and inert particle material, both
organic and inorganic. The concept of activated sludge treatment involves
four general steps (1) aeration of the sludge and wastewater for some signifi-
cant period of time, (2) solid-liquid separation at the end of the aeration
period, (3) discharge of the liquid fraction as process effluent, and (4)
127
-------
return of some or all of the separated/solids to the aeration stage of the
process [concentration of mixed liquor, solids (MLSS) is maintained at about
1200 to 3000 mg/1] (Eckenfelder et al., 1972). The time frame for each of
Ithese steps can vary considerably. The aerator can be long and baffled and
* $ .
designed for only a single pass from inlet to outlet or it can be a completely
mixed system (Swisher, 1970). In some cases the sludge is allowed to,'absorb
contaminants for short periods of time (15-30. minutes), and then separated
and aerated for a period of time (.commonly 2-6 hours) sufficient for ;solubili-
zation and metabolism of the sorbed organic matter (contact stabilization).
Other designs include extended retention times of 1-2 days which, has the
advantage of decreasing the amount of sludge disposal (economically attractive
for small plants). Another long retention time design is the oxidation ditch
which has been used extensively in Europe. The design consists of a
circular ditch in which the activated sludge liquor is repeatedly recirculated
(Swisher, 1970). ' ".:
The flocculation ability of the sludge is extremely important
to the treatment efficiency, since the separation of the sludge from
.the effluent and thus the efficiency of removal requires agglomeration and
settling of the sludge. Occasionally, when a plant is not properly operated,
filamentous microorganisms are formed producing a "bulking sludge" condition
which retards settling.
;- In contrast, trickling filters (percolating filters, bacterial
bed) are not dependent upon flocculation. The effluent to be treated
is passed over a biological film which has grown on packing material (usually
a few centimeters to a number of inches). Although exposure to the film is
128
-------
only a few minutes, soluble material is absorbed on the film and may be degraded
over a long period of time (Swisher, 1970).
In both activated sludge and trickling filter processes,
the bacteria are the principal agents of biodegradation (Buzzell e£ al., 1969).
Extracellular enzymes solubilize the compounds in order to allow suitable
entry into the cell wherein the cell can use the soluble chemical either for
energy or to build new cell substances. Higher microbial and invertebrate
forms, such as protozoa and worms, feed on the bacteria and maintain a population
balance (see Pike and Curds, 1971, for a detailed description of the micro-
biology of the activated sludge process). WltB. trickling filters, this feeding
process appears to he important in prolonging the operation of the filter.
A commonly used sewage treatment process in rural areas
is a septic tank system. The system basically consists of a tank which is
maintained under anaerobic conditions followed by an underground drainage
field or tile field. The anaerobic conditions and short retention time provide
little biodegradation and the principal action in the tank is settling of
insolubles which are mechanically removed periodically.
2. Activated Sludge Systems
Laboratory activated sludge units are generally of two types
(1) continuous and (2) batch or semi-continuous. Because of the large number
';
of procedures reported, only the techniques that have been widely used will
be described in detail.
a,. Continuous-Flow Systems
The size and dimensions of the activated sludge units have
129
-------
..varied depending upon the investigator'and the treatment process that is being
'modelled. Ludzack apd Ettinger (1963) have compared the capacity (size of the
aerator and settler), the surface to volume ratio, and the dimension of the
aerator and settler of a number of units. A recent review of continuous-flow
; systems is provided by Swisher (1970) in his book on the biodegradation,;of
surfactants. The characteristics of the procedures reviewed by Swisher,-as
well*as some newly reported procedures are summarized in Table 12.
' . *
Continuous-flow activated sludge systems can be categorized
into two general types (1) the serial type aerator which consists of an;,aerator
""" "".' ~ " " V
divided into compartments in series and (2) a completely mixed aerator. ,The
i,
serial type is used in simulation of what Swisher (1970) calls the classical''
treatment plant; a single pass from inlet to outlet. The.configuration, used
by Eldib (1963) (See Figure 16) is typical of this type of aerator. As can
be seen in Table 12 , the serial configuration was popular in ;the late 1950?s
and early I9601s, but is infrequently used now.
SEV/AGE-
Figure 16.
Schematic Diagram of a Serial Type Aerated Chamber
Laboratory Model Activated Sludge Unit (Eldib, 1963),
Courtesy of Soap/Cosmetics Chemical Specialties,
Hac Nair-Dorland Co., Inc.
-------
Table 12. Characteristics of Bench
Capacity
Aarator/ Dloaolvad
aettlar Aarator Battling Oi Laval
Bo f trance (lltara) Moaaaiou Diaonglono (ppeO
Truaedalo at al. (1939)
Orbaneial. (1963)
Xuaaw at .1. (1961)
J«Bdrayko and
SittCbeaberg (1963)
Dagane at .1. (1955)
Uuddlaeton ad
Allred (1964)
KeCaubey and
Uein (1959)
McXinnay and
Donovan (1959
Ludaeck (1960)
lann jj aj. (1964)
Cldlb (1963)
Fitter and
Tucko (1964)
Caiman Covamaent
(1962)
Svaaney and Foota
(1964)
Svleher at el. (1964)
Svleber at al. (1967*)
Svleher at aj. (1967k)
Buiiall at al. (1949)
SMnnatt and
Eden (1971)
Choi at al. (1974)
6.3/0. 3 4 section. 2-3
vertical
bafflaa
4.7/ 3 compart- conical 1-2
attnta In vith rotary
aarla* acraper
200/400 4-30 t c 3 mj/l
«al. It" dl*r 13" daap
matar
3/ Poroua-pot
udmlfua
2.W/ ' Coaplata 4
2.43 alxlai
Scale
M1.SS
(PP»)
3000
3000
2400-
1000
2000-
3000
2000-
6000
1000-
2000
(4 I/day)
3000-6000
(6 I/day)
1300
2000-1000
1000
500-2000
2000-6000
2300
2300
Continuous Activated Sludge Units
Tait katantioo Analytical
had Chamlcil Tlma AcolUatlon Hathod
Natural Surfactanta
aavaga up to 13 pps
Bynthatic Surfactant
aa«a|* (300 ppa 13 ppa
nutrlanta)
Natural aavaga Surfactant
(4800 t/day) 20 ppa
natural aavaga Surfactant
(3 t/hr) up to
(BOB 323 ppa) 30 ppa.
paptona 20 ppm
10X natural
aflwaga
Synthatlc. and ns
Natural 3 to 10 ppa
23S ranovad
in aynth.
301 raaovad
in natural
Natural and
aynthatlc
(200 ppa
nutrlant broth)
aavaga
4 I /day
Surfactant
20 ppa
240 ppm bacto- Surfactant
paptona + 44 ppa 30 ppa '
KHjPOj
SynChatle ' Surfactant
aawaga 20 ppa
250 ppa nutrianta
1 t/oay
Natural aavaga Surfactant
2 al/aln. 3 ppa
Synthatlc LAS, ABS
aavaga up to
(130 ppa 200 ppa
paptona)
or Natural
Synthatlc Organic
aavaga chaalcala
glucoaa 160 ag/1 180 ag/t
paptona 160 ng/1 aa C
uraa 2«.6 ag/1
Synthatlc Surfactanta
aavag* >~10 t/1
Natural .FCB'a
aawaga 1.63 ppa
(clarlflad)
6 - 8 hr. 6 vaaka Hathylana
(for T1S or Blua Actlva
LAS) Subatancaa
(NBAS)
11 hr. 4-12 vaaka KBAS
1 hr. KBAS
1 hr. 1-2 vaaka NBAS
(1.3 br.
in aattlar)
1 vaak
MB A3
7.3 hr. NBAS
3-« hr. KBAS
S hr. NBAS
. Sludga HBAS
dava loped
In praaanca
of tha aur-
f act ant
6 hr. 8-9 day 33« radlo-
oparation laballad
aurfactant
1-7 hr. KBAS
Ultraviolet
abaorption
Parric iron
chalatlon (NTA)
Acclimated to TOC. COD
aavaga but Dahydroganaaa
not teat chea- anayaa
leal (uaa activity
lyophllltad
eludga) alug
loading
} br 1-4 vaaka KBAS
5-10 hra. Slug loading Extraction
folloved by
gaa chroaat.
131
-------
Figure 17 depicts some of the apparatus I used in completely
mixed systems. The most automated system is the Huddleston-Allred (1964)
I,
system. The level is controlled automatically, the foam is repressed auto-
matically, and even the analysis for surfactant is made with an AutoAnalyzer.
However, of the completely mixed systems, the best known are the official
German test method and the miniature continuous flow units.
132
-------
, TO gECOND UNIT
IUPCU.*:H SHAFT \
\ / ANT.-rOAM CON 1 HOI. rt«
LCVEI. CONTROl. PROBE --. \ / fOCTEflttNT «.f.O POUT
AN".:'«"V'»»: \\ ii / / »r....... .
IlltHMOMEftH WULL-
MtOIUM ftiO PORT
II //
n//??:: - ""'«"«"UM(
IMPlLU.Uri "
Oxidation Vessels
(Huddleston and Allred, 1964)
Waulewuter
Kct-U
Efflutnt
Sedlmtncatlon
Tank
Sludge Out
(Choi e_t ai, 1974)
----- [III. Mil!
ron
uniinfl i sntin
CQUPAnTUftlTS
TOP »!(
1 Oltl
4"
nRlt
Clo
ate
r nirta
ailKlai
Hn.Mti.
iKln. h
U bf n
n IMt 1
ion l 10" tec
intii cut In
On It on inri c
t of the tov
0" PlotlRl.i
r tio coinpar
connncteil h| a H i 4" op
Inn
ill
.it
end
went I
ninp.
ler proof "ruhhir to net,i|"
nint uted to join Plsiljijjj ta
..
dratn to a cone.
Coining Clan torn
Spl. App. Oiv. Refirinit
Ho. Onnoil TI40
-Reproduced from
best available copy.
(Ludzack., 1960)
Figure 17. Various Completely Mixed Aerator Model
Activated Sludge Unit.
Courtesy of J. of the American Oil
Chendsts' Society
133
-------
(i) Official German Test Method
In ,1962, the German Government, because of high detergent
»'
concentrations in rivers (see Houston, 1963), passed a law
requiring that detergents had to be at least 80% biodegradable
as demonstrated by the 'Official German test method. The-details
of this standard test are described below:
The apparatus used, depicted in Figure 18(a), consists 'o'f
a complete mixing aerator (3£) and settler (2.2£) with an air
pump sludge return. A synthetic sewage containing 250 ppm
of nutrients and 20 ppm of surfactants is fed at a rate
of Ifc/hr. The air flow into the aerator provides agitation'
and is adjusted to maintain a dissolved oxygen level at above
2 ppnr (Swisher, 1970). The sludge is developed as a result of
chance inoculation from the air and is allowed to build up to
a concentration of about 2500 mg/Jl. It is presumed that the
sludge is acclimated since it is developed in the presence of
the test chemical.
The time between development of the sludge and the start
of the test period is left to the discretion of the investigator
What is required is a resonable steady removal of detergent
over a '21 day period, the average removal representing the
biodegradability. The effluent is collected in 24 hour
composite samples and analyzed for MBAS content. The percent
degradation is calculated by comparing the influent content
to the effluent content and the 21 daily values are averaged.
The minimum time required for the test Is about seven weeks
134
-------
OFFICIAL GERMAN METHOD
(a)
Ch)
Figure 18. Apparatus for activated-sludge test: A, storage
vessel; B, metering pump; C, aeration vessel;
Cp, porous aeration vessel; D, sedimentation vessel;
E, air-lift pump; F, effluent collection vessel;
G, sintered-glass diffuser; H, air-flow meter;
I, outer impermeable vessel. (Stennet and Eden, 1971)
Courtesy of Pergamon Press Ltd.
135
-------
if everything goes well - about four weeks for sludge gener-
ation and three consecutive weeks for the test (Houston, 1963).
.1,
This procedure has been adopted by the Organization for Economic
Cooperation & Development (OECD) and the Council of European
Communities (Council Directive, 1973).
Stennet and Eden (1971) have suggested a "porousr-pot"
technique modification of the German method addressedrat over-
coming difficulties with recirculation of the sludge. !. The
apparatus is depicted in Figure 18(b). The basic'difference
is that the sludge is separated from the effluent by retention
in a porous polythene vessel (average pore size" 50 ym) rather
than by flocculation and settling in a clarifier. All other
procedures are Identical to the official German test with the
exception that the porous vessel needs to be cleaned (1:6
diluted hypochlorite solution) once a week. .The mixed liquor
i
is transferred to a spare porous vessel to allow continuous
operation. This porous pot modification has been used by a
number of researchers from the .Water Pollution.Research Labor-
atory, Stevenage^ England (Eden jet a_l., 1972; Stiff and Rootham,
1973; Stiff ._et'al., 1973) to study tne effect of temperature
on the removal of surfactants during sewage treatment.
. i ' .
(ii) Miniature Continuous-Flow Units
Two miniature activated sludge units have been reported:
(1) the 600 ml aerator size used by Sweeney and Foote (1964)
and (2) the 300 ml aerator size used extensively by Swisher
(1964, 1967a, b).
136
-------
The configuration of the two apparatus is quite different
as can be seen by comparison of Figure 19 and 20. However, both
systems add the influent to the top of the aeration chamber and
provide agitation and circulation by bubbling air from the
bottom of the aeration chamber. However, Sweeney and Foote (1964)
felt that it was also desirable to provide gentle stirring (150 rpm)
In order to maintain agitation without having to keep the air
flow rate high.
Sweeney and Foote (1964) elected to use a natural sewage
feed with an activated sludge from a commercial activated sludge
treatment plant (same source as the sewage). The sewage was a
settled sewage obtained from a commercial plant and sterilized
at 120°C for 0.5 hr. The activated sludge was obtained immediately
before every run. This use of natural sewage and sludge shortens
i
the acclimation time required (two days elapsed before samples
are taken). The residence time Is 6 hr. and the test can be
run for as little as 8 or 9 days if "(a) fresh commercial sludge
is used, (fc) fairly steady values for surfactant removal are
obtained and (c) the results are normalized by using a control"
(standard surfactant for normalization) (Sweeney and Foote,
1964). The test surfactant is Introduced at 3 ppm so that the
total surfactant level (test surfactant and surfactant in the
natural sewage) would not be abnormally high (6-11 mg/1 typical
in U.S.). Because of the background surfactant In the natural
35
sewage, radlotracer techniques ( S) are used for analysis
(ether extract of acidified sample).
137
-------
'""'" TRUBORE
BEARING
COARSE
FRITTED
GLASS
FILTER
Figure 20.
Swisher (1964, 196?a, b) -
Miniature Complete Mixing""
Continuous Activated
Sludge Unit
Reprinted from Surfactanc-
Biodegradation, p. 169, by
courtesy of Marcel Dekke.
Inc.
Figure 19. Sweeney and Foote (1964)
Continuous Activated Sludge Unit
Reprinted with permission from
Journal Water Pollution Control
Federation, 36. 14-37, Wash., D.C.
138
-------
The miniature activated sludge unit used by Swisher
(1964, 1967a, b) is about half the size of the Sweeney and
Foote (1964) unit. Swisher has studied the biodegradation of
linear alkylate sulfonates (LAS) and nitrilotriacetic acid (NTA)
with these units and Gledhill (1974) used the apparatus to
study the biodegradation of 3,4,4'-trichlorocarbanilide. The
activated sludge cultures, originally from a municipal treat-
ment plant seed, are developed on and acclimated to a synthetic
sewage and the test chemical for several months. Swisher (1970)
also notes that natural sewage can be used with this unit.
The analytical method used is dependent upon the chemical
studied and study objectives. Swisher used a combination of the
MBAS method and ultraviolet absorption to study the breakdown
of the LAS benzene ring (1967b) and a chelometric technique
for NTA (1967a). Gledhill (1974) determined the biodegradation
14
by CO- evolution.
b. Semlcontinuous and Batch Systems
(i) Batch Systems
Continuous activated sludge systems require a great deal
of effort to i'et up end k««p running and therefere o&ny
investigators have elected to use batch and semi-continuous
systems. Swisher (1970) has noted that the batch unit is much
more economical, although somewhat more remote from simulation
of full scale practice.
The simplest test to run is the one batch activated sludge
die-away. This consists of aerating a sample containing activated
139
-------
sludge, sewage (natural or synthetic), and the test compound and*
(
analyzing for the loss of the chemical over a few days or weeks.
Actually, with exception of the feed (activated sludge and
sewage), this technique is little different than a river die-away
test,(see Sec. Ill A.2 p 72) Swisher (1970) has reviewed the
V > *
conditions of a number of batch sludge die-away systems and these
are presented in Table 13. One interesting variation of this
technique is reported by Gledhill (1974). He studied the bio-
degradation of radiolabelled 3,4,4f-trichlorocarbanilide
(200 Mg/1) using a shake flask apparatus, raw sewage, and
activated sludge (MLSS - 1000 mg/1) from a semicontinuous
activated sludge unit (only natural sewage feed). The shake
flask was a closed system which allowed the monitoring of
14
CO- evolution by KOH absorption.
(ii) Semicontinuous Systems
By operating a batch system in series (running batch after
batch},.)-a semicontinuous system evolves. This is often termed
a fill-and-draw process. The process basically consists of
(1) aeration (2) settling of sludge (3) drawing off the
supernatant liquor (treated effluent) (4) filling with fresh
feed and (5) aerating the new cycle. Analysis is normally
done at the end of the cycle. Thus, the fundamental differ-
ences between a batch and a semicontinuous process is that
the sludge has an opportunity to acclimatize to the test compound
since it is retained from cycle to cycle. Interestingly enough,
140
-------
Table 13. Batch Sludge Die-Away
(Swisher, 1970)
Reference
Test Compound
Aeration
Feed
Removed
>ierp and Thiele,
1954
Alkyl sulfate
Alkylaryl sufonate
Bubbled air - 3 his.
Bubbled air - 3 hrs.
No aeration
Bubbled air - 3 hrs.
it. ti
No aeration
Activated sludge
& sewage
So sludge
Activated sludge
& sewage
Activated sludge
& sewage
No sludge
Activated sludge
& sewage
40-60%
4%
80-90%
70-80%
9%
House and Fries,
1956
TBS
Bubbled air - 8 hrs.
Activated sludge
& sewage
25-85%, depending
on sludge adapta-
tion
Vaicum and Ilisescu
1967
(Romanian ISCH
Test)
Alkyl sulfate
& ABS
Bubbled air - 3 days
Unacclimated
sludge (MLSS-
1000-1500 ppm)
Official German
synthetic sewage
"Closely paral-
leled results
from 6 tests
(river water, 3
continuous acti-
vated sludge, BOD,
and Warburg"
(Swisher, 1970)
-------
the semicontinuous process had Its beginnings in the acclimation
of sludge seed for Warburg respirometry (Swisher, 1970).
By far the most commonly used semicontinuous process is
the standard procedure developed for ABS-type surfactants by
the Soap and Detergents Association's Subcommittee on Biodegra-
dation (SDA, 1965). The full standard procedure consists of a
two-step approach (1) a simple shake flask screening or presumptiv<
step (two 72 hr. adaptive transfers) (see p. 85), and (2) a semi-
continuous 'activated sludge step. If the surfactant is only
80-90% removed in the screening step the semicontinuous con-
firming test must be run. If the surfactant is >90% removed,
it is considered to be adequately biodegradable; if <80%, it is
considered not adequately biodegradable for a surfactant.
The semicontinuous step consists of a 24 hr. cycle (23 hr.
for aeration; 1 hr. for setting, drawing off the effluent, and
filling with fresh feed). The aeration chamber consists of a
i
cylinder with a cone shaped bottom, the dimensions of which
are illustrated in Figure 21. The activated sludge, initially
obtained from a sewage treatment plant, is maintained at a
mixed liquor suspended solids (MLSS) level of 2500 ± 500 mg/K,
by discarding solids as necessary. The surfactant is added at
a concentration of 20 mg/X. and if the sludge is not accli-
mated to the surfactant, the final concentration is built
up to in increments over four days. The minimum operation time
142
-------
D
opproi
ttmm
»-» 4 mm ( fi OR GREATER
hoUfordroini fLU5" wl
d..,c« 01 ihij INSIOE W4J-L
500ml l«vel i- \-
RUBBER STOPPER
-^
254 mm (I") OIA HOLE
CENTERED IN CHAMDCR
Figure 21. Soap and Detergents Association*a Semlcontinuous
Activated Sludge Aeration Chamber (SDA, 1965).
Courtesy of J. of the Amer. Oil Chemists' Society.
is 15 days - 5 for surfactant build-up, 3 for equilibration
to 20 mg/Jl, and 7 for level operation. A control standard,
pure C.«LAS, is run in parallel and results are only valid
when the standard gives a removal over 97.5%.
The SDA Subcommittee on Biodegradability, with the assistance
of twelve laboratories, conducted a statistical evaluation of
the semicontinuous procedure with seven ABS-type surfactants.
Analysis was determined by the methylene blue procedure.
Reproducibility for LAS surfactants was good, but the more
143
-------
highly branched ABS surfactants gave much more varying results.
i
The SDA Subcommittee also evaluated other nonionic surfactants,
but because of the ambiguities of the analytical methods no
clear-cut performance standards were established (SDA, 1969).
Swisher has also used the SDA semicontinuous procedure to study
the ultimate biodegradability of LAS (Swisher, 1967b), quaternary
ABS compounds (Swisher, 1969) and the biodegradability of NTA
(Swisher ^t ail., 1967).
3. Trickling Filter Systems
Trickling filter systems have been used extensively by many researchers
(e.g. the British Water Pollution Research Laboratory (WPRL) at Stevenage) to
study the degradability of surfactants. The start-up period is usually quite
lengthy since the biological film which has to be developed on the packing
material often requires weeks or months to reach a steady state. On the other
hand, difficulties with scale-up are much less since the most important dimension
is the depth of the bed (full scale - 6 ft. or 180 cm.) (Swisher, 1970).
Truesdale and coworkers have shown that trickling filters agree well with sewage
treatment works (Truesdale et al., 1959) and have even used the results as a
standard of reference for comparing other laboratory techniques (Truesdale
et ail., 1969). Swisher (1970) has reviewed the conditions used with trickling
filters and these are tabulated in Table 14. The rotating tube noted in Table 14
. simply consists of an empty hollow tube mounted slightly off horizontal and
rotated slowly along its axis. A more elaborate description of the WPRL
trickling filter and the recirculating filter reported by Jenkins et^ al., (1967),
follows.
144
-------
Table 14. Trickling Filter Conditions
(Swisher, 1970)
Reference
Dlmcloaa of MounioH of feed lit*
the Filter the pcokUf, vol. feed/
TO!.filter/
day
FMd
Acclimation
«( lloloflcal to Surfactant
Film Surfactant Concentration
Trickling Filters
Trueedale at al. 15 x 180 en
(1959) (WPRI.)
Busman et_ al. (1963) 15.5 x 55 cm
Schonborn (1962a) 10 x 110 cm
2.5 cm average 0.6
diurnal
variacions
. li cm 1.0
1.0
Detergent free 14 weeks 4-8 weeks
natural sewage
Natural sewage
or peptone,
glucose, salts
synthetic
sewage
Develop film 4 weeks
on natural- Test
on synthetic
13 ppm
10-200 ppm
15 ppm
RotatlnR Tube
Gloyna£C.£l. (1952) 6. 5x60 cm
Weaver U*62)
Renn (1965)
3 A/day
5 gal/day
Synthetic
sewage
Synthetic
sewage
10 ppm
10 ppo
Recycle TricklloR Filter
Burnop & Bunker (1960) 5 x 90 cm
(glass)
Edellne & Lambert U x 124 cm
(1965) ,
Jenkins eC al. (1967) 3.75 x 75 cm
Alexandra (196?) 10 x 200 on
unspecif .
vol. recycled
7 times per
hour
1-3 cm stone >j - 1 1/hr.
(up Co 110 OB)
5 X 8 m washed recycled
gravel 7-18 times/
day
1 cm ponolana
granules
(up to 150 cm)
Synthetic
(malto-
peptone)
Official previously None
German developed
BOD water
salta
10 ppm
NH3-N
24 hours
10-50 ppm
20 ppm
3-20 ppm
sole carbon
source
> a. British WPRL Pilot-Scale Trickling Filters
The trickling filter extensively used by the British Water
Pollution Research Laboratory consists of a cylinder, 15 cm in diameter and .;
180 cm deep, filled with 2.5 cm clinker (Truesdale, et al., 1959). A detergent-
free sewage (^8 gallons/day) (prepared from excreta and other normal constituents
3 3
of sewage) is applied to the filters (average 0.6 m /m of filter per day) in such a ,.
manner as to simulate the diurnal variations in flow at a sewage works ("square
145
-------
wave dosing"). The common pattern is 1.5 times the average flow during the day
and 0.5 times the average flow at night (1.5/0.5). This "square wave" dosing
was used because uniform loading gave unusually high biodegradability values
in the laboratory in comparison to actual practice. However, Klein and McGaughey
i
(1965) using a much less drastic "square wave" loading (1.33/0.67) found ,no:,.
difference between uniform and "square wave" loading after sufficient
acclimatization was allowed.
The test: is usually continued for several weeks until steady
state conditions are reached. In some cases considerable acclimation
time must be allowed - usually 5 to 8 weeks, but sometimes 12-13 weeks - and
about 1.4 weeks is required to develop a mature film (.S wisher, 1970). The
effluent.Is analyzed for surfactant [usually MBAS although Klein and Mc&uighey
' 35 '
(1965) have uaed s radtolabelled material] and the percent removed Is
calculated. Truesdale e£ £1 (1969)1 have suggested tnat "results obtained in
this test agree well with those obtained at an efficient sewage treatment works."
b. Recirculation Filter Test
Even small trickling filters require substantial amounts of feed
and test chemical during the many weeks or months necessary for attaining steady
state and acclimation. To overcome this difficulty many researchers have switched
from once through operation to recirculating the effluent in a batchwise procedure.
The procedure used by Jenkins and coworkers (1967) is somewhat typical of this approacl
146
-------
The apparatus used by Jenkins et_ al. (1967) Is depicted In >
Figure 22. It consists of a black PVC tube (length - 75 cm, diameter - 3.75 cm),
containing a column of 5-8 mm washed gravel. The feed is a solution of the test
l-iltcrod air
(.apply
Recirculaled
effluent
r
s
Gravel filter
Detergent solution
rocirculatod
Figure 22. Reclrculation Filter Apparatua
'' (Jenkins et al. 1967)
Courtesy of Pergamon Press Ltd.
chemical (10-20 mg/1 surfactant) made up initially in a 1% dilution of
sewage effluent or soil-extract in BOD water and 10. ppm NH, nitrogen
'(heeded,for smooth Biological oxidation of detergent?). The solution is
circulated through the column at a rate varying from 7 to 18 cycles/day and
when the test chemical concentration reaches a low level, it is replenished.
After several replenishments, the gravel was sufficiently inoculated with
microorganisms acclimated to the test chemical that reasonably consistent results
147
-------
could be obtained. It is at this point that the inoculate (1% sevage or soil)
is omitted and the test chemical becomes the sole source of carbon during
the test cycles.
4. Anaerobic Systems
Two anaerobic systems which are modelled in the laboratory and,are
important to sewage treatment are (1) septic tanks and (2) anaerobic digesters.
Septic tanks are used in most rural areas and in many suburban areas. Chemical
substances passing through these systems potentially could affect the quality of
ground and surface water. Anaerobic digesters are used to reduce the volume
of the sludge from an activated sludge treatment plant before it is dried.
The simplest procedure is the anaerobic die-away system. Briefly,
this consists of batches of feed and test chemical kept in an anaerobic jar
for periods of up to a month of two. The conditions used are outlined in
Table 15. When anaerobic digester conditions are simulated, high concentrations
of activated sludge or digester sludge are used. Hill and McCarty (1967) have
used this procedure with digester^sludge to study the anaerobic degradation of
organochlorine pesticides, although they note that this type of degradation
would probably occur only slowly in less favorable natural conditions.
Swlsher (1970) has briefly reviewed semlcontlnuous operation of
laboratory simulated septic tanks and anaerobic digesters with surfactants.
These semicontlnuous conditions are much closer to actual field conditions.
Only the two simulated septic tanks and drainage fields used by Straus (1963)
and Lashen e£ al. (1967) will be described.
148
-------
Table IS. Anaerobic Die-Away Procedures
(Swlsher, 1970)
1
References
Wayman & Robertson
(1963)
Vath (1964)
Klein (1965)
Manganelli et al.
(1960)
Meinck & Bringmann
(1961)
Fitter (1964b, c)
Apparatus
Brewer anaerobic
Jar
Amber bottles
Graduated
cylinder
Container with
provisions for
gas evolution
Food
Primary sewage
effluent
ti it
2 £- "
Activated
sludge &
digester sludge
(30,000 ppm
susp. solids)
Surfactant
10-25 ppm
20-100 ppm
25 ppm
100-750 ppm
Length of
Test
l«-2 months
2 weeks
40 days
40 days
; The septic tank used by Straus (1963) consisted of two cylindrical
compartments (first - 2 £, second 1 I). This was followed by a drain field
which consisted of a "series of columns packed with soil to the inlet, then
gravel past the inlet, topped off with more soil." The septic tank was seeded
with sludge from a full-scale septic tank and feed sterilized raw whole sewage.
. ' 05
The surfactant, an alkylbenzene sulfonate tagged with S, was added at a
"i ' '."-'' . '
V concentration of 10 ppm. The feed was added 4 minutes each hour for 16
consecutive hours each day. The average residence time was 5 days.
The Lashen jjt al. (1967) model septic tank-percolation field consisted
of a 1 gallon jar septic tank and a 3 in. diameter column of sand (0.5% peat
moss added) 2 ft. in height above a water table. The average retention time
was 67 hr. and the system was fed 3 times a day at a rate of 2.5 gallons per
day per square foot of surface area In the percolation field. The septic tank
149
-------
was Initially seeded with mixed liquid from an activated sewage plant and
fed a synthetic sewage (official German recipe).
5. Field Tests
The ultimate test of a chemical's biodegradability under water treat-
ment conditions can only be provided by a field trial. However, the expense
is so great that field trials are rarely attempted except in the case of
detergents. Swisher (1970) has identified at least 30 field tests undertaken
with surfactants. These will not be reviewed here. However, since one of ,
the best evaluations of a laboratory technique is provided by a comparison
of results with a field test, some of the procedures and difficulties
involved in field testing will be discussed.
Swisher (1970) has identified two major parameters which contribute
to the uncertainties of field testing surfactants: CD the inherent variability
'i
of the operating parameters of the treatment plant, and C2) analytical inter-
ference by surfactants already present in the incoming sewage. The first
parameter may result in widely varying treatment efficiencies. Some control
over these variations may be gained by comparing the removal of surfactant
to the removal of more natural components of sewage Ce.g. BOD). The analytical
difficulty results from the fact that the commonly used methylene blue procedure
(MBAS) is not a specific method (see p. 154). Other analytical methods are avail-
able (e.g. IR, desulfonation-GC, radiotracer techniques) but have been infre-
quently used because of the speed and sensitivity of the MBAS procedure. Two
techniques have been used in order to adjust for the deficiencies of the MBAS
procedure (1) substitution technique and (2) spiking technique (Swisher, 1970).
150
-------
In the substitution technique, all the detergents in a service area are re-
placed by the test detergent. Thus, in theory the MBAS measured should only
be the test detergent. However, this replacement is usually quite difficult
to accomplish and it may take years before all the detergent supplies are re-
placed and all the absorbed or trapped previous detergent is removed from the
sever lines.
The spiking technique is much simpler. It requires an addition of
the test surfactant just before treatment operation with analysis before addition,
after addition, and after treatment. With this technique, blank runs must be
run to determine the removal of the already present surfactant and this may be
a large source of error.
C* Analytical Procedures
A number of direct (e.g., estimation of parent compound or its
metabolite) and Indirect (e.g., measurement of growth or oxygen consumption)
analytical methods have been used to study biodegradation in the aqueous
environment. Although any analytical method can provide data relevant to .
biodegradability, there are important differences in the utility of the
results. For example, some methods reveal nothing about the nature of the
intermediate metabolites and, therefore, have somewhat limited application.
The analytical procedures involved in the assessment of biodegradability
in the aquatic environment are somewhat similar to those used in the soil
studies. The major difference is that the extraction and clean-up procedures
are relatively less complex in the natural water samples. Biodegradation
studies utilizing pure-cultures of microorganisms and cell-free extracts have
generally used the same standard methods regardless of the environment the
organism was isolated from.
151
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1. Extraction and Clean-up: Aqueous medium is generally much
,. (
t
less complex than soil and therefore extraction and clean-up is; not always
necessary. The requirement for extraction is more dependent on the type
of analytical method which the researcher is planning to use for analysis. '
Sometimes extraction is essential because of certain unusual binding char-
acteristics of a particular compound. The system used for extraction of a
chemical from water is largely dependent on the characteristics of the
chemical and less dependent on the water sample. The most frequently used
technique is partition of the chemical from the water into a water-immiscible
organic solvent. For example, hexane has been used for extraction of chlori-
nated hydrocarbons (Leigh, 1969), chloroform for polyethoxylated alkylphenols
(Osburn and Benedict, 1966); 15% ethyl ether in hexane for organochlorine
compounds; 20% benzene in hexane for organophosorus compounds; chloroform for
carbamate compounds (Eichelberger and Lichtenberg, 1971); a mixture of benzene
and ethyl ether (2:1, v/v) for crude oils (Kator, 1973). Desorption of ABS
and several other surfactants from microorganisms has generally been accomplished
by extraction with hot water (Hartman, 1963; Kelly e£ al., 1965; Tomiyama et al.,
1968), ethanol (Assoc. Amer. Soap and Glycerine Producers, 1961; Maurer et^ al^., 1965)
or methanol (Roberts and Lawson, 1958; Fischer, 1962; Bruce ^t .al_., 1966).
Allred _et al. (1964) has suggested boiling of samples with HC1 in order to
liberate surfactant from bacterial cells. Several researchers have used
alkaline conditions - e.g., alkaline aqueous acetone (Gould, 1962), methanolic
sodium hydroxide (Huber, 1962, 1968) for desorbing anionics. In certain cases
ionic strengths of the test solution is increased by addition of Nad prior to
extraction with organic solvent (Osburn and Benedict, 1966).
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2. Analytical technique;
a. Chromatographic Methods; Chromatographic techniques have been used
to isolate and sometimes, by co-chromatography, to identify degradation products
and the starting test compound. Paper, TLC or column chromatography is generally
used. The parent compound and/or the metabolites are extracted from the bio-
degradation test medium, the extracts are concentrated by evaporation of the
solvent, and the residue is chromatographed. Gas chromatography can be
successfully used, but its utility is limited to chemicals which can be
volatilized and are thermally stable. Extraction and clean-up of the sample
is generally required before GC analysis can be performed. In several cases,
however, aqueous samples have been directly injected into the GC for analysis
(Baird-.et al., 1974; Baker, 1966). Anionic surfactants are salts and, therefore,
are not volatile enough for GC analysis. Several researchers have developed
procedures for desulfonation of ABS giving rise to alkylbenzene which can be
used with GC analysis (Setzkorn and Carel, 1963; Swisher, 1963). . The desulfon-
ation-GC technique was found to be particularly suitable for measurina relative
rates of disappearance of the Individual components in a complex ABS mixture
(Huddlestori and Allred, 1963; Swisher, 1963; Allred et al., 1964).
b. Radiotracer Technique; Radioactive isotopes have been
extensively us.ed in studies on metabolism and degradation of environment?!
contaminants. The major advantage of this technique is that metabolites that
are formed are also tagged and, therefore, a mass balance of the parent
compound and breakdown products is possible. A large number of recognized
environmental contaminants are available from commercial sources in radio-
labelled form. In certain cases researchers have synthesized the desired
153
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chemicals in their laboratory (Metcalf & Lu, 1973). In a majority of degradation
studies, compounds tagged with C have been used (Metcalf, 1973; Focht and
Williams, 1970; Gledhill, 1974; Vath, 1964; Lashen e£ al., 1966). 3H-ring-labelled
compounds synthesized by a relatively simple technique (Hilton and O'Brien, 1964)
have also been used in several studies (Kapoor £t _al., 1970, 1972). Isotope
t
effects are possible with this technique, but would only be encountered if
o
breakage of the H-C bond is the rate determining step. Labelling the sulfonate
group with S has been frequently used in the case of detergents (Robeck e_t jil. ,
1963; Straus, 1963;'Sweeney and Foote, 1964). Blodegradation is measured by
determining the amount of inorganic sulfate formed. The major drawback of using
sulfonate-labelled material is that no information can be derived about the fate
of the ring.
c. Colorimetrie Methods; The methylene blue method for following the
biodegradation of anionic surfactants has been widely used because of its sim-
plicity and accuracy, >and because the loss of methylene blue activity closely
parallels the loss of foaming ability, one of the major water quality problems
from detergents. Also, the fact that about 75% of the U.S. surfactant production
consists of anionic materials (SDA, 1969) has contributed to the extensive use
of the technique.
Methylene blue (Figure 23) is a cationic dye which in the form
r ^'V^N^X^ ~i*
Figure 23. Methylene Blue Dye
154
-------
of an inorganic salt (e.g. Cl or SOi* ) is very water soluble. However, when
combined with a surfactant anion it becomes less water soluble and can be
extracted by organic solvents. The intense color of the dye allows for
detection of low concentration of anionic surfactants (e.g., 10 pg of ABS -0.1
ppm in a 100 ml sample - can be readily detected).
The procedure, as outlined in Standard Methods (APHA, 1971),
consists of additions of the dye solution to the sample followed by
chloroform extraction of the surfactant-dye salt. The concentration is deter-
mined by measurement of the absorbance of the chloroform solution at 652 my
(for LAS). The precision and accuracy of the method is acceptable. Standard
Methods reports a relative standard deviation of 9.1% and relative error of
1.4% in 110 laboratories with a river water sample which contained 2.94 mg/1
of LAS.
The major drawbacks to the procedure are (1) the method only
determines the test surfactant and not the metabolites and, therefore, only
provides an assessment of primary biodegradation, and (2) the method is not
specific for anionic surfactants and can be interfered with by organic sulfonates,
carboxylates, phosphates, phenols, inorganic cyanates, chlorides, nitrates, and
thiocyanates (complex or form ion pairs with methylene blue - gives high results)
and by organic materials especially amines (compete for the methylene blue - low
results) (APHA, 1971). Interferences from chloride ions (forces some .
methylene blue into the chloroform) precludes application of the method in
seawater without precautionary measures.
The partition of the surfactant - methylene-blue salt
into the chloroform phase is dependent upon the hydrophobic group in the
155
-------
surfactant. Above octylbenzenesulfonates the partition into the chloroform
is close to stoichiometric and therefore the method is extremely sensitive
for the parent alkylbenzenesulfonates. However, as the side chain is
1
metabolized during biodegradation (Swisher, 1970), the hydrophobia character
V
is reduced and this is why the method is only good for the parent surfactant.
The methylene-blue dye is not specific for anionic sur-
factant and thus the results are often reported as methylene-blue activated
substances (MBAS). In biodegradation tests, this problem is minimized to the
greatest extent possible by running a blank that does not contain surfactant.
d. U.V. and I.R. Spectrometryt Measurement of the infrared
absorption spectrum can provide information regarding structure, but its
i
relative insensitivity limits any quantitative application. However, an
I.R. method based on the measurement of the absorption of the sulfonate
group has been used for determination of ABS (SDA, 1965; Ogden et al., 1961;
Frazee and Crisler, 1964). I.R. spectrometry has also been used in biodegra-
dation studies of nonionics (Frazee et^ al., 1964; Osburn and Benedict, 1966).
The U.V. method of analysis for following the cleavage of the
benzene ring has been frequently used in degradation studies in the -aquatic
environment. The disappearance of U.V. absorption during degradation indi-
i
cates rupture of the benzene ring system. The U.V. analysis can usually be
made directly without concentration or extraction steps. However, in several
cases the technique cannot be used if some component of the medium or an
impurity absorbs strongly in the region of interest. The concentration of
the test material and medium components is kept low if U.V. analysis is used
(Setzkorn and Huddleston, 1965). U.V. measurement has been used for studying
156
-------
biodegradation of surfactants in continuous and semicontlnuous activated sludge
studies and in river die-away tests (Swisher, 1967b; Setzkorn and Huddleston,
1965). U.V. analysis usually falls to reveal minor modifications of the test
compound since the U.V. absorption will often remain unchanged unless ring cleavage
occurs.
e. Measurement of C0? Evolution! In the process of biodegradation,
VBVBHMHMMMVM^BMWMMMMHMMH. _
an organic molecule may be broken down to the ultimate end product, CO., and
therefore, measurement of COx evolution has frequently been used to follow bio-
degradation. A gas train assembly consisting of the reaction flask and CO traps
is generally used to measure GO. production (Atlas and Bartha, 1972, 1973; Sturm,
1973; Thompson and Duthie, 1968). The whole gas train arrangement may be mounted
on a rotary shaker and agitated, but often it is not shaken. During the test
CO -free air is bubbled through the test unit and the effluent gas is passed
through the CO. absorbers (e.g., solution of barium hydroxide or potassium hydrox-
* ,.
ide). Absorbed CO. is determined by titration, or by radioassay if a radio-
labelled test compound is used. Non-biological evolution of the gas is evalu-
ated by aerating a sterile control with COfree air. The quantity of CO
evolved may be compared with the theoretical maximum of GQ« production determined
/
by wet ashing of the test compound (Atlas & Bartha, 1972). In the interpretation
of CO2 production data, it must be remembered that, while oxidizing the test
organic compound to carbon dioxide and water, microorganisms are also synthe-
sizing new cell material from the compound. Measurement of CO- evolution is
especially useful to measure ultimate biodegradability.
f. Oxygen Consumption! Oxygen consumption linked to the oxidation
of the test chemicals can also be used to determine biodegradability of chemical
compounds in the aquatic environment. Methods for measurement of oxygen include
157
-------
manometry, polarography and chemical methods, and these have been discussed in
f
greater depth in Sec. Ill A.I, p. 51.
i
Oxygen consumption studies have been done both with mixed'
i
cultures (Nelson £t a_l., 1961; Hunter and Heukelekian, 1964; Blakenship and!
Piccolini, 1963) and with pure cultures of microorganisms (Heyman and Molbf,
1967; Ellis e_t al., 1957; Walker and Cooney, 1973a). Measurements of?oxygen
consumption linked to the microbial oxidation of the test compound is an>indirect
method for assessing biodegradability. The technique fails to reveal information
about the nature of the intermediate compounds formed during degradation. The
I
advantage of the technique, like CO evolution, is that it can be used without
developing an analytical method for the test compound. The extent of biodegrad-
ability is usually derived by comparison with theoretical oxygen demand for the
test compound. In this comparison one must take into account .the fact that, in
most instances, biochemical oxygen demand is considerably lower than theoretically
possible for complete oxidation since often 5-40% of the test* carbon is used
by microorganisms for synthesizing new cell material. When the test is run in
the presence of external carbon source or when the endogenous respiration rates
are higher, the interpretation of the results may be difficult because of the
possible influence of the test compound on the oxidation of external carbon
f. '
source or on endogenous respiration.
L '
g. Microbial Growth; A simple test for determining the bio-
degradability of a chemical compound is to show microbial growth on that com-
pound. A basal salt medium supplemented with the test compound is Inoculated
with the potential organism (commonly stocked organism or isolated by enrichment
158
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culture technique) and growth is measured by one of several methods; e.g., tur-
bidity, cell count, dry weight determination (Prochazka and Payne, 1965; Payne
and Feisal, 1963; Forsberg and Lindquist, 1967). Prochazka and Paype (1965) have
shown a direct correlation between culture turbidity (a growth indicator) and
degradation using mixtures of C..-C2() secondary alcohol sulfates. The use of
bacterial growth as an indicator of biodegradability can be applicable only for
those compounds wihch are susceptible to extensive degradation and can support
growth; i.e., serve as a source of carbon and energy for a bacterium. Therefore,
this assay method has somewhat limited utility.
h. Bioassay; Bloassay methods involve exposing some type of living
organisms to the test solution to measure the concentration of the test compound.
One such method, but just one of very many, is described by Yasuno e_t al. (1966)
for determination of organophosphorus insecticides. The test insect used for
the biological assay was young 4th instar larvae of Culex pipiens. Some 30-50
larvae were added to 250 ml of the blodegradation test medium containing the
insecticide and the results were read after 24 hours of exposure. Advantages
and disadvantages of the bioassay technique are discussed in detail in Section
IV B.2., p. 256.
1. Determination of Total Carbon; Measurement of total carbon
concentration can be used to monitor the course of biodegradation. The methods
for total carbon determination include chemical oxygen demand (COD) by dichromate
method (APHA, 1971) and dry and wet combustion methods (Pickhardt c£ a.1, 1955,
Weber and Morris, 1964). An alternate approach (combustion method) involves com-
plete oxidation of the sample in a stream of air or oxygen which passes over
a catalyst; the carbon dioxide produced may be measured by I.R. absorption (Van-
Hall and Stenger, 1964) or by one of several other procedures.
159
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If degradation of a mixture of chemical compounds, or the
test organic compound in the presence of exogenous carbon is investigated, the
' i
method fails to provide a direct demonstration as to the extent of degradation
or which materials in a sample are degraded. No information can be obtained
by this method concerning the nature of the intermediates formed. The differences
in the susceptibility of chemical compounds to combustion may sometimes introduce
i
error in the results (especially with COD).
j. Others;
j
Surface Tension; This test has generally been used for
surfactants and is based on their ability to reduce the surface tension of water.
The measurement is made with an interfacial tensiometer (Allred £t _al;, 1964;
Huddleston and Allred/ 1965; SDA, 1969). Blankenship and Piccolini (1963) have
pointed out that surface tension can only be used as a qualitative measure in
a biodegradlng system because different surfactants (and perhaps their inter-
. i |-.
mediate degradation-products) may differ in their degree of lowering the surface
tension.
Foam Threshold; Determination of the foaming potential is
another method which has been used inbiodegradability studies with surfactants.
Foamability is determined by shaking a sample of the test medium in a glass
stoppered graduated cylinder and reading the,amount of the foam present (Huddleston
'(_'''-'
and Allred, 1965; Lashen .et .al., 1966). The foam present can also be read by
'the foam test machine (Bacon, 1966). The method is not suitable for quantita-
tive estimations and has only been used by workers in cases where chemical analy-
sis is difficult.
:*. '
Oil Dispersion; Oil dispersion has been used to assay for the
microbial degradation of crude oil. Oil dispersion is measured by determining
. .
160
-------
the turbidity of the reaction medium after vigorous shaking (Reisfeld et al.,
1972). A sterile medium containing the crude oil is used as a blank.
D. Evaluation of the Techniques used for Determining Blodegradation
of Chemicals in Natural Water Systems:
In evaluating the test methods which have been used for determining
persistence of chemicals in the aquatic environment, a number of criteria
can be successfully used. These include comparing the results obtained
from a particular test method with those obtained by other test methods,
wherever possible with the results of the field experiment and with the
known environmental persistence of the compound as assessed from the monitor-
ing data. Although any method can be evaluated for its ability to yield
accurate qualitative as well as quantitative information, the data available .-
in the literature thus far have permitted only qualitative comparison,
e.g., whether a compound is shown to be biodegradable. A comparison
based on the quantitative results, e.g., rates of biodegradation, obtained
from different test methods, or a comparison of the degradation rates with
known environmental persistence of a compound has generally not been
attempted. This is perhaps because the quantitative results from different
degradation test methods and from the environment have varied considerably,
making it difficult to draw any conclusion about the actual rates of break-
down. The factors which affect biodegradation are numerous and have varied
from test to test and from laboratory test to natural conditions. Prior to
attempting to evaluate the test methods, it will perhaps be useful to discuss
these factors to understand how and to what extent they are responsible
for discrepancies in the degradation results.
161
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1. Factors affecting blodegradation;
In the elimination of polluting substances from waters, the organ-
isms primarily responsible for degradation are microorganisms.' In most cases,
s-*
the factors which influence the activity of these, microorganisms will affect the
biodegradation results. Some of the important variables pertinent to biodegra-
dation are: type of microorganism, mineral salt composition, test chemical^con-
centration, supplementary organic nutrients, 02, temperature, pH, light, etc.
a. Type of inoculum:
Perhaps one of the most important factors that affects'-:the .results
of a biodegradation test method is the nature and quantitiy of the microorganisms
used to metabolize the test organic compound.
In biodegradation tests, the pure strain or a mixture of micro-
organisms has been used. Ordinarily natural microbial communities have been pre-
ferred, since populations may complement each other in their biochemical capa-
bilities. Furthermore, it is unlikely that a single species of microorganisms
would be able to catalyze biodegradation of so many structurally different mole-
cules since the number of enzyme systems is limited in any one population.
The work of Cook (1968) and many others has demonstrated that pure
strains of microorganisms quite often are not as effective as a mixed
'
-------
test chemical was used as blodegradablllty criteria. The results obtained from
these large numbers of pure cultures may be considered somewhat similar to
mixed culture.
The number of microorganisms initially present in the biodegra-
datlon test solution Is generally not so important in terms of degradation, be-
cause of the short generation time for most microorganisms. Only the Initial rate of
degradation may be affected by the number of microorganisms. This point is
evident from various studies (Eden j£ jijL , 1967; Garrison and Mat son, 1964;
Conway and Waggy, 1966) in which researchers have used river water fortified with
added microorganisms to Initiate rapid biodegradation.
The source of microorganisms can also be very important- bqth in '
terms of the type of microorganisms that may be present as well aa the number
'"-.'
of each type. Swisher (1970) has found that the degradation of TBS (alkylbenzene
sulfonate derived from tetrapropylene) was higher in the river die-away test than
Y
when examined by shake culture and batch or continuous activated sludge test.
It should be emphasized here that this is not generally the case and may be :
true only for TBS. Since river water contains relatively few bacteria, the ['
. ' '" i f
high degradation activity was attributed by Swisher (1970) to the natural species ...
distribution in the river water. The source of microorganisms for testing bio- . ',
degradation in the aquatic environment has generally been from such sources as
sewage, activated sludge, river water, river or lake mud, and trickling filter
slime. Many of these sources have been examined for their bacterial composition
and have been found to be considerably different from each other. For example, .
activated sludge from varied sources was found to contain 72 different species
classified in 14 genera (McKinney and Wetchlein, 1955). Hoadley and McCoy (1965)
163
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'have reported Isolation of 11 species of bacteria from the lakes and streams.
i i
Many researchers have not added any inoculum to their biodegradation test medium,
and have relied on the organisms already present in the sample or those intro-
duced from the atmosphere (Swisher, 1966).
Another very important parameter which should be considered-while
studying biodegradation of a chemical compound is the effect of microbialadapta-
tion to the test chemical. From the biodegradability point of view, .orgjanic
compounds can be 1. readily utilizable, 2. utilizable after acclimation,.3.
slowly utilizable under all circumstances, or 4. not utilizable. For synthetic
organic compounds which are not likely to be found in the environment, acclima-
tion of organisms prior to studying degradation can be extremely important. ^
The enrichment culture technique is a means of isolating or developing bacterial
strains capable of degrading a particular organic compound. Importance of
acclimation is indicated from the work of Huyser (1960), who reported that 5-6
1 t
days were needed for disappearance of 8-phenylpentadecane LAS in unacclimated
river water but only 1-2 days in acclimated river water. Pfeil and Lee (1968)
observed that no obvious degradation of NTA was catalyzed by nonacclimated seed
organisms, whereas acclimated.seed organisms were able to degrade NTA fol-
lowing a lag period of three days. Las hen et_ al^ (1966) observed rapid and
v ' ' .
extensive degradation of octylphenol ethoxylate in river water (from below
heavily industrial urban area) bearing an acclimated microflora whereas very
'A '''.
slow degradation was noticed in other river waters. Acclimation is sometimes
<' ..'
unpredictable, and even after baceria have become acclimated to a chemical .
:-''.. . .
. ; - _
under one set of conditions, there is not guarantee of immediate action under
164
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other conditions. However, studies by Cook (1968) have indicated that accli-
mation may not always be required for biodegradation. She found that control
I
cultures grown without surfactant in agar were Juet as effective as! the "accli-
mated" ones. Others have made similar observations with different compounds.
b. Mineral Salt Composition; ,
For proper growth and function of microorganisms in the biode-
i
gradability test, elements such as nitrogen, sulfur, phosphorus, and magnesium
j
at relatively high concentrations, and various other elements, such, as iron,
copper, manganese, and zinc, in trace amounts are required. Certain bacterial
i
species may also require some preformed organic co-factors, e.g., certain
vitamins. Hattingh (1963) has pointed out that the optimum feed should contain
at least 5.3 parts of nitrogen and 1.2 parts of phosphorus for each 100 parts/of
BOD. Requirement for nitrogen and phosphorus supplement for biodegradation is
also suggested from the studies of Atlas and Bartha (1973). These authors found
' \
that the sea water contains very low concentrations of nitrogen and; phosphorus
and, therefore, degradation of petroleum in the sea water is slow. {When sea
i
water was supplemented with these essential nutrients, degradation was stimu-
lated several fold. '
Biodegradation tests have generally been performed using the micro-
organisms in one of the following two phases: (1) Growing cells, and (2) Resting
cells. In studies with growing cells, degradation of organic compound is linked
to the growth of the organisms while in the case of resting cells, no increase
in cell population usually occurs, and, therefore, degradation is the result
of the preformed enzymes in the microorganisms. The composition of the medium
used in the two types of studies has been considerably different. For example,
resting cells are generally suspended in buffer alone (Focht and Joseph, 1971;
165
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Focht and Williams, 1970), whereas with growing cells complete media have gen-
erally been used (Huddleston and Allred, 1963; Bunch and Chamber, 1967).
Several modifications in the mineral salt compositions have been
j
made by researchers to facilitate the use of a particular analytical method.
For example, Cordon .et ol. (1968) have used nutrient salt medium free of sulfate
to permit following sulfate ion formation from anionic detergents.
In the literature, there are almost as many different 'mineral
salt media reported'as there are biodegradability studies, but there is no
self-evident reason and probably no basis for this practice. This is mainly
because each researcher has preferred his own recipe. No attempts have been
made to determine if these different variations in the compositions of the
mineral salt medium affect the biodegradability of a chemical compound, but the
effect is probably small.
c. Test compound concentration;
The concentration of the test chemical initially added in the
biodegradability test medium can affect the rate and/or the extent of biological
degradation. The concentration of organic chemicals found in surface water is
normally on the order of a few milligrams per liter or lower. Investigators
have generally used much higher concentrations in biodegradation test methods for
many reasons, including a limited sensitivity of the analytical method. For
i
closer simulation .of field concentrations, it may be desirable to use lower con-
centrations. An important phenomenon which is very much concentration dependent
is acclimation of the bacterial cells. Unusually low substrate concentrations
either fail to allow acclimation or require prolonged incubation periods. !
166
-------
Ffell and Lee (1968) have investigated the biodegradation of NTA in!aerobic
systems at various concentrations. These Investigators found no measurable
degradation after 20 days incubation at NTA concentration of 5 mg/£. However,
in cultures where NTA concentration was 10 mg/4, extensive degradation was
initiated after 10 days of lag, possibly due to the development of some acclimated
organisms. The upper limit for the concentration of the test chemical may be
i
imposed by the toxic or Inhibitory action of the chemical on the microorganisms
or by a low solubility in water. Fuhrmann e£ _al_. (1964) observed a Significant
i
lengthening of the induction period and a lower reaction rate with an increase
in concentration of the surfactant. These authors concluded that the inhibitory
effect may be due either to bactericidal or bacteriostatic effects of the surfactant
at higher concentration, and/or decrease in oxygen in water with increasing
surfactant concentration. An increase in the lag period with increase in
concentrations was also observed in the studies of Aly and El-Dib (1972) with the
pesticide Baygon. The inhibitory effect of high chemical concentration is
also clear from the studies of Balrd et al. (1974) with phenols. Using
Warburg respirometry, these researchers found that even at relatively low
concentrations certain phenolic material produced deleterious effects on
respiration. Although all the seven phenols tested were degraded 100% at
1 mg/Jl concentrations, at higher concentration the degradation was severely
inhibited.
d. Supplementary Nutrients:
Several researchers have studied biodegradation of organic
compounds in the presence of a readily utilizable external carbon source.
167
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Addition of a carbon source to the biodegradation test medium may be expected
to enhance degradation in one of three ways:
1. The external carbon source may stimulate the growth
of microorganisms responsible for breakdown of the
5
test chemical so that the test vessel has a higher
cell density.
2. It may induce enzyme systems which, in addition to
catalyzing a reaction involving the natural sub-
strates, may also attack the test compound.
3. It may provide energy for a reaction sequence in-
volving an initial endergonic step.
The presence of a supplementary organic nutrient may permit
cometabolic degradation for those chemicals which fail to serve as a sole
source of carbon and energy for microorganisms. Cometabolism is a process
where a non-growth substrate is metabolized by a microorganism needing a dif-
ferent compound as a carbon and energy source occurs. Horvath (19.72a) has
criticized test methods which have been used for demonstration of biodegrada-
bility but do not allow for possible cometabolic degradation. The effect of
the presence of external carbon source on biodegradation of environmental
pollutants has been investigated by many workers. Horvath and Koft (1972) have
reported that although a branched chain ABS, tetrapropylene benzene sulfonate
(TBS), failed to support growth of Pseudomonas, when glucose was added growth
occurred and TBS was degraded. Decomposition of the herbicide 2,3,6-trichloro-
benzoic acid by a Brevibacterium was reported to be accelerated by the addition
of benzoic acid to the culture (Horvath, 1972b).
168
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Addition of an external carbon source In many cases has been
found to slow down or even stop degradation of the test chemicals. This has
generally been attributed to the preferred metabolism of the external carbon
source, or it is possible that the external carbon source may have a toxic
effect on the biodegradation of the test chemical. Also, it is possible that
populations using the added compound deplete the supply of some essential
nutrient. Fuhrmann Q al_. (1964) observed that biodegradation of ajstraight
l
chain ABS was delayed if an easily utilizable carbon source (Lactose broth)
]
was added during the course of biodegradation. Clattoni & Scardignp (1968)
i
have reported the inhibitory action of glucose on biodegradation. In
i
river water, 50 ppm of glucose delayed the onset of degradation of 10 ppm
,1
of LAS as long as glucose was replenished and maintained above 30 ppm. The
>
inhibitory effect may be attributed to a reduction in the rate of synthesis
i
of certain enzymes of LAS degradation by readily metabolizable carbon source
such as glucose (the phenomenon is referred as catabolite repression, for
details see Paigen and Williams, 1970).
Sikka and Saxena (1973) have compared the influence of two
different types of external carbon sources on the degradation of herbicides
endothall (1) glucose, which is a good source of carbon and energy, and (2)
i
yeast extract, which serves predominantly as an exogenous source of carbon
but also contains vitamins and other nutrients. Their findings indicated that
while the presence of yeast extract stimulated the degradation of endothall,
glucose caused a slight inhibition of degradation.
e. Oxygen Requirement;
For the oxidation of organic chemicals by microorganisms, a
terminal electron acceptor is necessary. Dissolved molecular oxygen, under
169
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aerobic conditions, and nitrate, sulfate or carbon dioxide under anaerobic
conditions can serve as1"acceptors. Extensive degradation of chemical compounds
in the environment is, for the most part, an aerobic process (Maurer et al.,
1971; Wayman and Robertson, 1963). However, replacement of an air stream
with a stream of pure oxygen results in decreased degradation (Fuhrmann £t^al>,
1964). This has been postulated to be due to the unavailability of CO. to micro-
organisms, but it could also be the result of 0 toxicity.
Under anaerobic conditions, biodegradation is generally slowei
However, some compounds are more susceptible to attack under anaerobic/condi-
tions. For example. Hill and McCarty (1967) have investigated aerobic and
anaerobic degradation of chlorinated hydrocarbon pesticides. These investi-
gators concluded that initial stages in the degradation were more rapid under
anaerobic than under aerobic conditions for the compounds studied.
f. Temperature, pH, light, etc.;
The majority of the known biochemical reactions increase in
rate.with an increase in temperature up to a certain optimum temperature.
Best growth of most bacteria is usually observed between 20 and 37°C. Proper
temperature is also important for acclimation of microorganisms to a new sub-
strate. It has been reported that at 5°C acclimation to a .new chemical may
> ' . .
take about five times as long as at 30°C (Water Pollution Research Laboratory,
1972). The effect of temperature on biodegradation by mixed populations such
as those in rivers and activated sludge is further complicated since a change
-------
Fuhrmann et al. (1964) have studied the degradation of straight
chain ABS at various temperatures and found that essentially no degradation
took place between 0 and 5°C. Such temperatures may be expected for 3-6
months of the year in many industrialized areas of the world. The above re-
searchers also found that with an increase in temperature the acclimation
period was shortened and the rate of breakdown increased. Similar observations
concerning the effect of temperature were made by Evans et^ _al. (1973), Evans
and David (1974), and Atlas and Bartha (1972) for biodegradation of urea,
ethylene glycols, and petroleum, respectively. The effect of temperature on
biodegradation of chemical compounds in waste treatment system (Hawkes, 1963)
and in respirometric test conditions (Montgomery, 1967) has been reviewed.
The pH of the biodegradation test medium is an important factor
which significantly affects the biodegradation rates. Strongly acidic and
basic pH can inhibit the development of microorganisms; most microorganisms
thrive best not too far from neutrality (pH 7). Heukelekian and Gelman
(1951) Investigated the effect of initial pH on the oxidation of industrial
wastes in respirometers and found that oxidation was most rapid in the pH
range 6 to 8. Studies of Aly and El-Dib (1972) have indicated another role
for pH of the test medium in biodegradation. These investigators found that
carbamate insecticides, pyrolam and dime tilam resisted biological oxidation;
however, the hydrolysis products formed under strong alkaline conditons could
readily undergo biological degradation in natural waters. These findings
suggest that the rate of chemical hydrolysis (which is pH dependent) may be
a limiting factor in biodegradation of certain chemicals.
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Light has rarely been shown to have a direct effect, on
microbial degradation of organic chemicals. It is possible, however,
that in certain cases, the photoproducts (see Section V, p. 283) rather
than the original compound may be attacked more easily by microorgan-
isms. , For such compounds"biological degradation in the presence of
light may vary considerably.
Investigators have generally preferred to incubate
biodegradation reaction medium in room light (Sweeney and Foote, 1964;
Weaver and Coughlin, 1964), or in dark to prevent the growth of algae
(Warren and Malec, 1972; Barstlap and Kortland, 1967). Under anaerobic
degradation conditions, incubation in dark is-particularly desirable to
minimize the growth of algae since they can release oxygen into the
system (Maurer et^ al. , 1971).
2. Comparison of Methods;
The physical and chemical parameters employed in testing
biodegradability have varied from one test method to another. The importance
of these parameters in affecting biodegradation results has been discussed
earlier. These conditions may be more favorable for biodegradation in some
test methods than in others. These variations between the test methods may
introduce qualitative as well as quantitative differences in the biodegrad-
ability (rate and/or extent) of a test compound. In order to evaluate the
internal consistency of the results obtained by various biodegradation test
methods, some investigators have examined the fate of single chemical com-
pounds by different test methods. Their studies are summarized in this
section.
172
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Swisher (1970) has compared various methods based on the results
of primary degradation of tetrapropylene derived ABS (henceforth referred
to as TBS). He found that the closed (dilution) bottle test and shake culture
test hardly accomplished any degradation of TBS, whereas In the river die-
away test It was often degraded to the extent of 65-75% (based upon loss
of MBAS). The author concluded that although the bacterial concentration
in river water is low, the higher biodegradability potential of this test
must be due to the natural species distribution and the natural medium.
Garrison and Matson (1964) determined the relative biodegradability of several
classes of nonionic surfactants by shake flask, die-away and Warburg respirometry.
Straight chain alkylphenol was found to be more biodegradable in either the
shake flask or die-away tests than was the corresponding branched chain product.
With branched chain alkylphenol, a somewhat greater amount of biodegradation
was indicated in the die-away test than in shake flask test. In the respirometry
test, none of the phenols or alkylphenol-straight or branched chain ethoxylates
were significantly degraded. Direct comparison of bacteriological slant culture
technqiue, river die-away test and shake culture test on various representatives
of the ABS group of detergents was attempted by Cook (1968). Relatively slow
degradation was indicated for the slant culture technique in comparison to
the other techniques. River die-away and shake culture test in this study
gave more or less comparable results.
There are no reports In which attempts have been made to compare
the results from model ecosystems to those obtained with other biodegradation
test methods. Certain chemicals which have been tested in Metcalf's ecosystem
have also been Investigated by certain other test methods by some Investigators
173
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and a comparison is possible in those instances. The extraordinary environmental
i '
stability of organochlorine pesticides dieldrin and DDT is shown in the Metcalf
system by very low overall biodegradability index values* (0.00094 for dieldrin
and 0.04031 for DDT). Eichelberger and Lichtenberg (1971) studied the persistence
of the above organochlorine pesticides in river water and, similar to Metcalf's
findings, reported no measurable degradation or chemical change. Comparison
of the results from Metcalf's ecosystem to those obtained with other tesf
methods for certain water-soluble compounds is somewhat more difficult.
For example, benzole acid is stored in the tissues of various organisms in
substantial quantities and is perhaps not available for microbial attack,
while aniline is not stored in appreciable quantities (Metcalf and Lu, 1973)
and can be degraded by microorganisms. Distribution of benzole acid and degrad-
ation products in the ecosystem (Metcalf and Lu, 1973) indicated that approx--
14
imately 75% of the total C in benzole acid was present in the nonpolar forms
which suggested (according to Metcalf's definition of biodegradability) a
i
persistent nature of benzole acid (overall biodegradability index in Metcalfs
system = 0.335). Aniline, another water-soluble compound, which is not stored
in significant quantities in food chains, was extensively degraded in the
14
ecosystem (only 20% of the total C in the nonpolar form, overall biodegradability
index = 3.8). Both these compounds have been shown to be extremely biodegradable
by other test methods (Buzzell jet a^. , 1968, 1969). From this comparison,
it appears that ecosystems, which have a tendency to store some compounds
(see page 35) and thereby make them unavailable for microbial attack, may
give misleading results concerning environmental persistence in the case of
certain compounds.
* Metcalf and Lu (1973) defined the biodegradability index as the polar
' products in the organism/non-polar products. For comparison purposes,
we have calculated the overall biodegradability index which is the total
polar products/total non-polar products.
-------
3. Correlation between laboratory and field results;
The laboratory techniques used for studying biodegradation
in the aquatic environment have generally attempted to retain as many important
factors of the normal aquatic environment as possible. These factors are,
however, so numerous and their influences so interwoven that any laboratory
test method may be unable to account for all these parameters. In spite of
these shortcomings, suitable test methods have provided information which
can be extrapolated to the natural conditions. The ultimate evaluation for
a test method is a comparison of its results to natural environmental results.
There has generally been good agreement between laboratory
biodegradation test results and environmental persistence for compounds which
are known to be extremely resistant to biodegradation. For example, DDT,
which is known to persist in the environment as shown by its widespread
occurrence (Edwards, 1973) has been found to be persistent when tested
by any of the several biodegradation test methods (Model ecosystem,
Metcalf £t al., 1973b; River water test, Eichelberger and Lichtenberg, 1971;
Modified Bunch and Chambers test, Leigh, 1969; Shake culture test using
microorganisms from sewage lagoon, Halvorson et al., 1971). A substantial
amount of monitoring data is now available on various isomers of PCB and,
therefore, this group of chemicals can also be used as an example for com-
parison. Veith (1972) has found that the proportion of higher chlorinated
isomers increases downstream in some Wisconsin rivers, whereas the lower
chlorinated isomers disappear. Nisbet and Sarofim (1972) proposed that,
since it is unlikely that the lower isomers are differentially retained in the
sediments (Veith, 1972; Nisbet and Sarofim, 1972), their disappearance probably
is due to their rapid microbial decomposition in the river environment. The
only laboratory study which has investigated the relative biodegradability
of various isomers of PCB was done by Metcalf and Lu (1973) in the model
i
175
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ecosystem. A comparison of environmental behavior of trichloro-,
tetrachloro- and pentachlorobiphenyi in the model ecosystem (Metcalf and
' f
Lu, 1973) showed a decrease in the overall biodegradability index with
increasing chlorination (BI = 0.1093 for trichloro-, 0.0606 for tetra- {
chlord-, and 0.02366 for pentachlorobiphenyi). These results are in agreement
with the suggested environmental behavior of PCB isomers discussed above.
Kaiser and Wong (1974) have investigated the degradation of Aroclor 1242
(mixture of PCB's containing mainly monochloro-, dichloro-, trichloro-, and
tetrachlorobiphenyl) in shake cultures inoculated with pure cultures of
bacteria enriched on Aroclor 1242. These researchers have also found that
less chlorinated biphenyls are degraded preferentially.
Some other compounds have been studied in enough depth that
they can be used to evaluate some of the test methods. Frank (1966) has
investigated the fate of monouron in simulated ponds; their results indicate
that the pesticide applied at the rate of 40 kg/ha persisted in excess of
2 years. In the river die-away test, however, monouron did not persist in
significant concentrations after 4 weeks (Eichelberger and Lichtenberg, 1971).
Sikka and Rice (1973) studied the persistence of the herbicide
endothall (7-oxybicyclo[2.2.1]heptane-2,3-dicarboxylic acid) in a farm pond.
They reported that endothall could not be detected in the farm pond) top one
inch of hydrosoil or pond water) after 30 days. Since endothall is water
soluble and its loss due to volatilization is negligible, it was assumed that
loss of endothall was due to biodegradation. In parallel aquarium studies,
these researchers observed a several-fold higher rate of degradation of
endothall. The aquarium studies were conducted using water and hydrosoil
from a pond which had been previously treated with the test chemical and,
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therefore, the microorganisms probably had been acclimated. That endothall
is rapidly degraded by microorganisms was also shown in a shake culture test .
inoculated with a species of Arthrobacter isolated by enrichment culture
technique (Sikka and Saxena, 1973). Oxygen uptake studies with the Warburg
method indicated that resting microbial cells oxidized endothall vigorously
and at a linear rate (Jensen, 1964). The final oxygen uptake value, however,
corresponded to only 37-43% of the theoretical oxygen demand of endothall.
These results are difficult to interpret since Jensen (1964) in his experi-
ments observed only 9-14% of the theoretical oxygen demand of glucose.
Urban e_t_ ji^. (1965) have studied the persistence of LAS and
TBS in a 6.6 mile canal with a flow-through time of 6 hours. The results
indicated 35% degradation (as revealed by the loss of methylene blue activity)
during passage of LAS compared to 15% for TBS. When the retention time
was increased to 10 days, LAS was degraded about 100% compared to 22-35%
for TBS. That LAS was more biodegradable than TBS is also supported from
the monitoring data of Merrell ejt _al. (1967) as discussed below. In 1961,
at Santee, Calif., the influent of the treatment averaged about 16.5 ppm
in MBAS, the effluent 8.5 ppm. It was down to 6.7 ppm after the oxidation
pond. Upon change from TBS to LAS by the U.S. detergent industry, the
oxidation pond effluent dropped to about 1 ppm, suggesting rapid degradation
of LAS.
The biodegradation of TBS (near C . alkyl group) and LAS
(mixed chain lengths) has also been tested by various laboratory test methods
by a number of investigators and the results are summarized in Table 16.
177
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Table 16. Comparison of Biodegradation of LAS and TBS
by Different Experimental Techniques
(Taken from Swisher, 1970)
Substrate: LAS
Method
River die-away test*
n n ' n
n n n
Shake flask*
ii n
n n
BOD
Method
River die-away test *
'n n n
n n ii
Shake flask*
M n
BOD
n
Extent of
Degradation %
97
100
100
90-92
91-96
100
19**
Substrate:
Extent of
Degradation %
27
25
30
17-20
18
2**
Time of
Incubation
(Days)
30
7
9
4
-
8
7
TBS
Time of
Incubation
(Days)
30
32
30
4
5
5
22
Reference
Knaggs et al. , 1965
Lang et al. , 1965
Wayman and Robertson
, 1963
Tomiyama et al. , 1969
Oba et al. , 1968
Lang et al. , 1965
Krone and Schneider,
Reference
Berger, 1964
Renn, 1964
Knaggs et_ al. , 1965
Renn, 1964
Allred et al . , 1964
Ryckman, 1956,
Ryckman and Sawyer,
Ryckman, 1956
1968
1957
* Analytical Method: Loss of methylene blue activity
** Percent of that calculated for complete oxidation of the substrate
178
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Since LAS and TBS are crude mixtures and their composition can vary somewhat,
the results should only be used to derive qualitative conclusions. The
laboratory shake culture and river die-away test results appear to be in
agreement with the field results and monitoring data. These tests have also
revealed approximately 18-30% biodegradability of TBS and 90-100% of IAS.
Since the degradation criteria in the river die-away and the shake flask
method is the loss of the parent compound, the data from the above tests can
not be compared with BOD data.
Although it is extremely valuable to know the rates at which
a chemical will degrade in the natural environment, extrapolating the labora-
tory biodegradation rates to the rate of breakdown in the natural aquatic
system has not been very successful.
4. General Discussion of Various Test Methods;
The formulation of a meaningful method for testing bio-
degradation of organic chemicals involves the selection of (1) an appropriate
test environment (or environments) with control of the experimental variables,
and (2) a suitable means of following the biodegradation. This literature review
has revealed a number of test methods for assessing biodegradability in the
aquatic environment. These have varied from quite complicated techniques such
as the "model ecosystem" of Metcalf et al. (1971) or the aquatic ecosystem at
EPA Athens laboratory, to relatively simple techniques, such as BOD.
In many cases, researchers have attempted to simulate to a
degree the natural environment. The simulation has involved adjustment of
the parameters such as microblal concentration, diversity of the mlcrobial
community, external carbon and energy source, oxygen, nutrient concentra-
tion, etc. A particular test method may be subject to other problems besides
179
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I i
failing to simluate the natural environmental conditions, such as inconven-
ience of procedure, time requirements, equipment or lack of reproducibility.
i
The test methods are compared in this section from the point of view of
these parameters. The methods described in the literature can be suitable
for: (1) preliminary screening for biodegradability, and (2) determination
of intermediate metabolites and the routes of breakdown. These two categories
of biodegradation test methods are evaluated separately.
a. Rapid Screening Test for Biodegradability;
In evaluation of the environmental persistence of a
chemical compound, it appears logical to first run a rapid test to screen for
biodegradability. The preliminary screening test may reveal if it is necessary
to detect the metabolites formed during degradation. For example, if
the BOD for a compound is close to the theoretical oxygen demand calculated for com-
plete oxidation, the question of accumulation of any undesirable metabolites
is less likely to arise. A number of screening tests have been used by
researchers to obtain information about overall biodegradability. Most
researchers have preferred using natural mixed cultures of biological material
in these tests because they are readily available and the results can be
extrapolated to natural conditions with less difficulty. Biodegradability
tests such as biological oxygen demand, river die-away test, shake culture test
using mixed inoculum, and perhaps more complex model ecosystems could qualify
for this category.
Biochemical Oxygen Demand;
Biochemical oxygen demand, whether determined by dilution
method or by respirometry, is an indirect test for assessing biodegradability
and does not provide any information about the nature of the degradation
products. However, the method requires no specific analytical method for
180
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estimation of the chemical compound and, therefore, the test is relatively
simple. Since the procedure measures oxygen consumption, there can be
little confusion due to physical absorption of the test chemical.
Since oxygen is utilized by microorganisms for a
multitude of complex metabolic reactions and not simply for oxidation of
the test chemical, interpretation of oxygen consumption data is some-
times difficult. A portion of the test chemical may be used for the
synthesis of cell materials which is much less oxidized than ultimate
degradation products CO.. and H_0. Because of uncertainties, oxygen
uptake methods would, at best, give only qualitative results. The problem
of interpreting the oxygen uptake data is further magnified due to
possible changes in the endogenous oxygen uptake rate (oxygen uptake in
the absence of test chemical) of microorganisms, which is generally
subtracted from the oxygen uptake rates obtained in the presence of the
test chemical. It is possible that the presence of test chemical may
cause a considerable change in the endogenous respiration rate and,
therefore, subtraction of endogenous rate will not be representative of
the oxygen used for biodegradation of the test compound. The problem
can be overcome by adjusting the inoculum to give very little or no
endogenous oxygen uptake.
If oxygen uptake is measured in the presence of an
exogenous carbon source (other than the test chemical), the exogenous
source could be subject to oxidation, confusing the test. In extreme cases,
the test compound may stimulate the oxidation of exogenously added carbon
source without being degraded itself. Alternatively, the test compound may
inhibit the oxidation of exogenously added carbon and give misleading results.
Error can also be introduced in oxygen uptake data if oxygen is also being
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consumed for nitrification processes. This will lead to erroneously high oxygen
consumption rates. This problem can be easily overcome by determination of
NO. formed in the medium (see Buzzell et al., 1968).
j . ~~*~
If the investigator desires, samples can be removed
during the course of oxidation to correlate the oxygen utilization curve
with other analytical data. Realizing that the oxygen utilization data
alone may be inadequate to help in understanding the behavior of organic
chemicals in the aquatic environment, Buzzell et al. (1968) followed the
carbon removal, changes in bacterial population and nitrification in the
samples removed for BOD assay. The carbon removal data normally support the
oxygen data and indicates the completeness of biodegradation; bacterial
enumeration measurements are of value in determining both biodegradation and
toxicity of the organic chemical, and measurement of nitrification gives
data concerning noncarbonaceous oxygen utilization which is corrected from
the total oxygen consumed. According to these researchers, the BOD test
method can thus be made more truly quantitative and allow wider applications.
Oxygen uptake measurements have been performed by
dilution method or by respirometric methods. The respirometric method is
generally considered more precise as far as measurement of oxygen demand is
concerned. Both methods, however, have the same interpretation problem
as discussed above. The microbial concentration used in respirometric
method is higher and thus it simulates treatment plant conditions more
closely than does the dilution method. The respirometric tests are normally
less time consuming than the 5-day dilution test. Among the respirometric
methods, polarographic methods, e.g., oxygen sensing electrodes, are generally
preferred over manometric methods for measurement of oxygen since the latter
method is more time consuming and less sensitive.
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River Die-away Teat;
This is one of the earliest tests used to measure bio-
degradability of chemical compounds. The reaction conditions in this test
more closely approach the conditions encountered in nature. The test is
simple to run and requires a minimum of equipment. However, one of the more
serious shortcomings of the test is the variation in bacterial count and
composition between different rivers, between different points in the same
river, and even at the same point at different times in a given river. Con-
siderable variations in the bacterial count in river water will also be
introduced due to seasonal variations. The river water from different sources
may also vary in the concentration of the nutrients and toxins present. These
fluctuations may cause variations in the results between laboratories studying
the biodegradability of the same material. In addition, the low concentrations
of microorganisms in river water have resulted, in some cases, in long periods
of incubation before noticeable degradation.
Rivers in many urban industrial areas may contain organisms
which are already acclimated to several synthetic chemicals. River water from
such sources may exhibit rapid degradation rates while river water from those
rivers which receive very little or no organic chemicals may require a long
acclimation period. Lashen e£ _al_. (1966) have investigated the role of natural
acclimation of river microorganisms in biodegradation. They reported that
samples from the Schuylkill, Delaware and Ohio Rivers below heavily industrial
urban areas show a rapid and extensive degradation of octylphenol ethoxylate.
River water from the Schuylkill, Delaware and Ohio rivers taken from areas
where usage of octyl- and nonlyphenol ethoxylates is estimated to be very low,
on the other hand, gave slower rates of degradation.
183
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The low organic nutrient levels and low solids content
of the river water system, may facilitate isolation and analysis of the test
chemical. On the other hand, the variations in the composition of the river
water may introduce variability in the extraction procedure and/or quantitative
measurement.
The various modifications made in the river die-away test
include the use of fortified and inoculated water, or polluted water.; Although
these modifications have eliminated the need for prolonged incubations, the
variations in the river water would still be a problem. Furthermore, with
the introduction of exogenous material in the natural river water, the
extrapolation of the results to natural conditions will be more difficult.
The anaerobic and microaerophilic river die-away test is a useful test for
assessing biodegradability in systems containing no oxygen or low concen-
trations of it. Such conditions are likely to occur in places such as the
deeper sediments of rivers and lakes.
Shake Cultures Inoculated with Natural Communities of
Microorganisms;
Shake culture test is a rapid test designed primarily
to assess biodegradability of organic compounds under aerobic conditions.
The test has been accepted as a screening or presumptive test for bio-
degradability by the Soap and Detergent Association (1965) Subcommittee on
Biodegradation Test Methods. The test generally uses high concentrations of.
microorganisms; subsequently, the duration of the test is relatively
short. The medium used for the test is of defined composition resulting
in good reproducibility. The test can be run in the presence or absence of;
an external carbon source, the latter conditions permitting the occurrence
of cometabolic degradation. The medium used in the test is most often
184
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specially formulated and contains all the essential nutrients required for
proper growth and function of many microorganisms and provides opportunity
for biodegradation. The conditions in this method may be much more favorable
for degradation than generally encountered in the natural environment. Sub-
sequently, extrapolation of the results to natural conditions may be difficult.
Kost researchers have preferred to use acclimated seed to obtain rapid
degradation. Under natural conditions, however, unless a particular compound
is continuously released into the environment in sufficient quantities, such
an acclimation may be quite slow. However, the use of acclimated seed is
desirable in that the rate of biodegradation under Idealized conditions is
obtained. In actual practice, more complete information about biodegradation
is obtained if the test is run with both acclimated and unacclimated seed.
Acclimation of the seed is generally carried out by frequent subculturing of
the seed in fresh medium containing the test compound (Huddleston and Allred,
1963; Bunch and Chambers, 1967). When acclimating a seed to a chemical which
is toxic to the biodegrading populations, the preferred technique is to
acclimate the seed organism in slowly increasing concentrations of the test
chemical (Schwartz, 1967; Hemmet, 1972).
The source of microblal Inoculum in the shake culture test has
varied considerably. Researchers have used microorganisms obtained from
sources such as sewage, lagoons, lake sediments, etc. Sewage microorganisms
have been used most frequently. The use of unidentified and nonspecific mixed
cultures in a test may always be criticized as an unknown variable and may
never give desired confidence to the test. Swisher (1966) used as inoculum
the microorganisms which had developed in an uninoculated test medium.
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Inoculum developed in this manner may vary from one laboratory to another.
In an attempt to minimize the variations in the type of inoculum used by
different laboratories, several researchers have attempted to preserve the
seed so that the same seed can be made available to other laboratories for
testing biodegradability. Some researchers,"*however, feel that it is unnecessary
because the biological composition of the natural seed (e.g., sewage) obtained
from various sources does not vary significantly (Swisher, Alexander, personal
communication). Furthermore, subjecting seed to preservation (e.g.,
lyophilization, air drying) may cause loss of activity. The slant culture
technique was evaluated by Cook (1968) and found not suitable for determining
biodegradability. The technique gave variable results which were not
reproducible and the degradation achieved was very low in comparison with
that achieved by other methods.
Model Ecosystems:
Model ecosystems used by researchers to date are oriented more
towards answering questions concerning bioaccumulation of the chemical com-
pounds than environmental persistence or biodegradation. Most of these
systems are not well defined in terms of their microbial composition and,
therefore, their reproducibility and subsequently their use in environmental
persistence studies is uncertain. However, the metabolic fate in the food chain
is quite reproducible in these systems. In Metcalf's ecosystem, the source for
microorganisms is old aquarium water; the microbial composition of aquarium water
can vary considerably from one laboratory to another and from day to day in the same
laboratory. In other ecosystem studies the source of microorganisms is not specified,
186
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although some microorganisms, e.g., those associated with the food chain
organisms or present in the aqueous medium are undoubtedly present in
the ecosystems.
Biodegradability in these studies generally refers to the
breakdown occurring in the food chain organisms rather than in the environ-
ment. For instance, the biodegradability index (polar products in organisms/
nonpolar products) in Metcalf's studies is calculated for fish and for
snails. Since Metcalf's system measures biodegradability in terms of the
ratio of polar and nonpolar metabolites, it assumes that conversion of a
compound to a polar metabolite is an indication of biodegradability. Although
this may be quite often true (see Section VIII, p. 461) there are examples
where a compound is water soluble and persistent. For example, a portion of
the soluble organic carbon of the oceans is not attacked by microorganisms
(Alexander, 1973a).
As pointed out earlier in Section II,(p.33 ) the role played by
unicellular microorganisms in the overall environmental persistence of the
chemical compound seems far more important than that exhibited by organisms
at higher tropic levels. Alexander (personal communication) feels that for
testing biodegradability alone, it is unnecessary to use a complex ecosystem
which contains the total aquatic food chain. Food chain organisms at
higher trophic levels (henceforth referred to as higher organisms) and
microorganisms are generally considered widely different in their catabolic
capabilities. Chemical compounds can be categorized into the following
187
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classes, depending upon whether they are degraded by microorganisms
i
and/or higher organisms:
1. Compounds which are degraded by higher organisms
as well as by microorganisms.
2. Compounds degraded predominantly by microbial action.
3. Compounds degraded predominantly by higher organisms.
There are apparently no known examples in the class.
4. Compounds not degraded at all.
Model ecosystem studies have generally provided excellent
information concerning environmental fate of compounds falling in Class 1
and Class 4. This is predominantly because the response of higher
organisms and microorganisms is generally similar if a compound is
highly biodegradable (e.g. glucose) or if the compound is highly persist-
ent (e.g., chlorinated-hydrocarbon pesticides). Sufficient numbers of
compounds falling in Group 2 have not been evaluated by model ecosystem
studies and, therefore, universality of the application of model ecosystem
technology in determination of environmental fate of chemical compounds
is hard to assess. Benzoic acid provides an example of a compound which
is not known to be environmentally persistent as shown by several test
methods (Buzzell e£ al., 1968, 69) but is persistent in the higher
organisms (Metcalf and Lu, 1973) due to its ability to be stored in the
higher organisms or conjugated.
188
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The terrestrial phase in the Metcalf ecosystem is prepared
from washed white quartz sand which is molded into a sloping surface.
The problems associated with sand include (1) microorganisms associated
with soil are absent, (2) failure to account for the effect of highly
important parameters such as adsorption or binding of the chemical to
the soil and reactions with soil colloids. These drawbacks have been
addressed in the ecosystem studies of Isensee and Jones (1974) who have
added soil to their ecosystem. In an effort to simulate erosion of
pesticides on soil, they adsorbed the compound on soil first and then
placed it in an aquarium and added water.
Metcalfs ecosystem also fails to permit sufficient rates of
photodecomposition, contrary to what has been claimed. Their aquarium
units are housed in environmental plant-growth chambers; the light
source used in these chambers (combination of fluorescent and incandescent
lamps) provides only a small fraction of the 290-350 nm light present in
the normal sunlight (see Fig. 2,p. A3). The intensity of this effective
component of U.V. light is further reduced in their system when the
chamber is covered with plexiglas. As shown in Table 39 (p.343), plexi-
glass is capable of absorbing significant portions of ultraviolet of
wavelengths less than 350 nm. The importance of simulation of sunlight
both in terms of wavelength and intensity can be quite important with
certain compounds. Isensee (1974) has found in his model ecosystem
studies with dlnitroaniline that exposure of the aquariums to various
light conditions (e.g., white fluorescent light, subdued sunlight and
189
-------
direct sunlight) can result in a direct relationship between sunlight
and dinltroaniline bioaccumulation. The bioaccumulation index follows
closely the order of increasing ultraviolet content and increased
intensity; i.e., white fluorescent light > subdued sunlight > less
j
subdued sunlight > direct sunlight.
In summary, model ecosystems are more suitable for evalua-
tion of metabolism by higher organisms and bioaccumulation through food
chain organisms than biodegradation. If the radiolabelled test chemical
is not available from a commercial source, it will have to be custom
synthesized by a commercial firm or prepared in the laboratory which
could be both expensive and time consuming. Model ecosystem studies of
Metcalf, Isensee and others have provided excellent information regarding
distribution and metabolic fate of a large number of environmental pollutants
in higher organisms.
b. Biodegradation Test Methods for Determination of the Routes
of Degradation
The screening tests discussed in the earlier section can
only provide results concerning biodegradability of a test chemical.
The value of the test will also be very much dependent on the analytical
method which has been used for assessing biodegradation. For example,
if the disappearance of the parent compound is the only parameter
monitored, nothing will be revealed about the identities of the Inter-
mediate products of biodegradation. On the other hand, if a compound
can be shown to provide total theoretical amounts of C02 to obtain
biochemical oxygen demand approaching theoretical, it can be fairly
190
-------
safely assumed that the compound will be biodegraded to innocuous material,
unless small quantities of highly potent toxins accumulate (Ayanaba and
Alexander, 1974J Alexander, 1974). In reality, however, such clear cut results
are not encountered too regularly. Therefore, it becomes essential to elucidate
the identities of intermediate metabolites since they may be equally or even
more toxic than the parent compound. Moreover, certain metabolites may persist
for long periods of time under one set of conditions whereas they may be short
lived intermediates of a metabolic pathway under others.
A detailed study of specific metabolic reactions is generally
quite time consuming and requires a well equipped laboratory and, there-
fore, such detailed studies should be attempted only if preliminary
evidence concerning transformation of the chemical has been obtained.
The use of unidentified and nonspecific mixed culture in determination
of pathways of degradation may always be criticized as an unknown variable
which may not give reproducible results. However, if a researcher is
simply interested in identifying persistent and toxic metabolites which
may be formed during degradation, purification of a particular micro-
organism is often unwise. The disadvantage in using natural sources of
mixed culture, e.g., sewage sludge, lake sediment, etc., is frequently
that the metabolite may get bound to the particulate matter and may
escape detection. Extraction of the adsorbed metabolite by drastic
means may sometimes alter the nature of the metabolite (Chestera et al.,
1974). Still, this is the model of the natural ecosystem, and the
problems of extraction are often easily overcome. Although identification
191
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of persistent metabolites is also possible using Metcalf's ecosystem,
the metabolites formed in=this system refer to the breakdown in the food
4
chain organisms rather than in the microorganisms. For this reason
Metcalf's approach will not be elaborated in this section.
The information concerning the routes of degradation of
chemical compounds has been derived largely from pure culture studies.
Although very reproducible, the problem associated with pure culture
studies sometimes is that they fail to account for breakdown of a com-
pound by the combined action of two or more microorganisms or for one
species providing growth factors needed by the degrading population. The.
i '
importance of synergistic relationships in the degradation of chemical
compounds has been demonstrated by Gunner and Zuckerman (1968). It is
evident, therefore, that in certain cases pure culture studies may show
the accumulation of a toxic metabolite, which in the mixed culture
studies may have been decomposed by another microorganism. Alternatively,
Intermediates may accumulate in nature but not be found in a given pure
culture, as is known for certain metabolic sequences (e.g. see Alexander
1972). The origin of pure cultures of microorganisms for degradation
work has varied considerably. Pure cultures have been obtained from
t*
commercial sources, or isolated or enriched from natural sources.
Enrichment of pure cultures using the test chemical as the sole source
'''»" ' ' ' '
of carbon is far more commonly used. This procedure is normally less
time consuming than screening a large number of microorganisms for their
ability to degrade a particular test chemical.
192
-------
Determination of catabolic pathways has been aided by the
use of radiotracers. This is because the detection sensitivity of
radiotracer techniques far exceeds the chemical and physical methods and
because radiotracer studies provide a total accounting or balance sheet
of the fate of the compound. Furthermore, the use of radiolabelled
compounds allows the researcher to distinguish between the carbon
provided by the degrading compound as opposed to cellular carbon from
other sources. Also the technique affords the opportunity of following
the fate of the test molecule within the cell. The use of radiotracers
in studies concerning biological fate of chemicals has been discussed in
a number of reviews (Casida, 1969; Kamen, 1957).
Elucidation of the pathways of degradation involves (i)
identification of the degradative intermediates and (ii) assigning
places to the intermediates in the scheme of degradation. The former
is, however, more important from the environmental standpoint. The
study of metabolic pathways begins with the intact cell and continues
with fractionating of the cell into smaller units. Some limited in-
formation can be obtained by incubating the chosen microorganisms with
the test chemical and then analyzing the culture fluids for the products
of degradation. This approach was used by Alexander (1972) in determining
an intermediate in the degradation pathway of 2,4-D. The appearance of
2,4-dichlorophenol in the culture fluid during the growth of Arthrobacter
on 2,4-D, suggested the phenol as an intermediate metabolite. Chemical
analysis of the culture medium of bacteria growing on a-naphthol
193
-------
has revealed information about the pathway of 1-naphthol degradation
(Bollag et_ al., 1975). Frenne, et al. (1973) have used this procedure
to elucidate metabolic pathways of pyrazon degradation.
The compound which accumulates in the culture fluid, how-
ever, heed not necessarily be a degradative intermediate. Dagley and
Chapman (1971) have stated that the easier it is to isolate a compound
from metabolic fluids, the greater the caution to be exercised before it
can be assigned the status of a degradative intermediate. A metabolite
which accumulates in copious amounts may be suspected to be formed from
a side pathway. Alternatively, it may be the end product of metabolism.
Quite commonly, intermediates remain within the cell, and
thus have to be extracted out from the cell prior to their character-
ization. Considering this possibility, a number of researchers have
extracted the cells as well as the culture fluid. Sikka and Saxena
(1973) subjected the cell suspensions to hot methanol treatment in order
to release the intermediates of endothall metabolism. Miyazaki, et al.
(1970) in their metabolism studies deprbteinized the cells by trichloro-
acetic acid to facilitate leakage; this was followed by extraction of
the cells with ether.
Chemical intermediates generally are only present in the
cell in minute concentrations which makestheir extraction and identifica-
tion sometimes difficult. An increase in concentration of the chemical
intermediate will take place until the time that the rate of degradation
194
-.1
-------
equals or exceeds the rate of formation of the intermediate. A meta-
bolite can be made to accumulate if the reaction sequence can be broken
at a specific point. This can be accomplished either by inhibiting an
enzyme or by adding a trapping agent that combines with the reaction
intermediate.
Tiedje, e£ al. (1973) have used malonate (inhibitor of
enzyme succinic dehydrogenase), arsenite (inhibitor of dehydrogenases
containing dithiols, particularly ct-ketoglutanic dehydrogenases, see
Dawson, et al., 1959), and 2,4-dinitrophenol (uncoupler of oxidative
phosphorylation), in an attempt to accumulate intermediates of NTA
degradation. Fluoroacetic acid, an inhibitor of the tricarboxylic acid
cycle, was used by Sikka and Saxena (1973) to promote the accumulation
of an intermediate of the degradation pathway of the herbicide endothall.
When inhibitors are used in intact cell studies, caution must be exercised
in interpreting the results because the permeability barrier of the cell
may sometimes prevent the inhibitor from gaining access within the cell.
Further evidence for the status of a compound as an inter-
mediate in the pathway can be obtained by the technique of sequential
induction. The concept is based on Stanier's theory (Stanier, 1947)
which states that cells growing on a specific compound are sequentially
induced to oxidize the Intermediate oxidation products. For example, when
a cell grows at the expense of compound A, it will contain enzymes that
catalyze the breakdown of reaction intermediates B, C and D in the sequence.
For example, Alexander (1972) and MacRae & Alexander (1963) observed that
195
-------
growth of a Flavobacterium on dichlorophenpxybutyrate resulted in the induction
of enzymes capable of oxidizing not only .the growth substrate but also 2,4-
dichlorophenol, which suggested that 2,4-dlchlorophenol was an intermediate
in the pathway. The 2,4-dichlorophenoxyacetic acid (2,4-D) molecule (which
would have been formed ifiphenoxybutyrate underwent beta-oxidation) was elim-
inated as an intermediate because dichlorophenoxybutyrate-grown cells failed to
oxidize 2,4-D. This same approach has also been used to elucidate the
pathways of breakdown of 2,4-D (Loos e± al., 1967), 2-alkylalkanoic
acids (Lijmbach and Brihkhuis, 1973), and primary alkylbenzene sulfonate
and linear alkylated sulfonate (Cohn, £t al., 1953). A limitation of
the technique is that certain enzymes may be nonspecific and may catalyze
the oxidation of compounds which may not even be part of the pathway.
c '. .
Moreover, sometimes a true intermediate may not be oxidized because it
is unable to enter the cell. These and other limitations of this approach
are discussed by Stanier (1947) and by Dagley and Chapman (1971).
Elucidation of biodegradation pathways with intact cells has
sometimes been handicapped due to (1) difficulty in manipulating physical
and chemical parameters, (for example the permeability barrier of the
cell prevents the investigator from removing any coenzymes, in an .attempt
to promote accumulation of an intermediate), (2) rapid reactions that do
not allow examination of intermediate metabolites, (3) the difficulty in
studying enzymes involved in degradation separately. Due to these
difficulties, many researchers have used cell-free extracts prepared
from1the microorganisms for advanced degradation work. The major dis-
advantage in the use of cell-free extracts is the difficulty in extrapolation
of the results to the natural environment. Disruption could result in
196
-------
interference of the degradation of the parent compound or of some of its
metabolites due to inactivation of some essential enzyme (s), or in some
other way change or introduce artifacts in the metabolic process in the
intact cell.
Information on the metabolic pathways has been derived from
cell-free extracts by breaking the reaction links at different points. This
permits the Investigator to examine the reaction sequence in small segments.
Reaction links can be broken by inactivation of a particular enzyme by inhibi-
tors, heat treatment, removal of coenzyme by dialysis, or fractionation of crude
extracts by a method which removes one of the enzymes and leaves the others in a
functional state. Some of these techniques have been used by Alexander and his
associates (Bolleg et_ al_., 1968; Alexander, 1972) in elucidating the pathways of
breakdown of 2,4-D. For example, in order to identify the products formed from
phenol (an intermediate metabolite of 2,4-D metabolism) by the cell-free extracts
of Arthrobacter, these researchers separated the phenol metabolizing enzyme from
the next enzyme in the sequence by passing the cell-extracts through a Sephadex
G-200 column.
Another approach which has been used for studying metabolic
pathways is to determine if the cell-free extracts metabolize the postu-
lated intermediates at a significant rate. In order for a compound to
be a catabolite in the proposed sequence, it must be metabolized at
rates that are compatible with the overall rate of degradation. In
studies of 2,4-D metabolism, Loos, et al. (1967) found that the enzyme
preparations converting the phenoxy compound to phenol, failed to meta-
bolize 2,4-dichloroanisole at a significant rate. These results suggested
that 2,4-dichloroanisole was not an intermediate in the 2,4-D metabolism.
197
-------
The demonstration of appropriate enzymes In the cell-free
«
extracts and/or their purification is also a step in the direction of
establishing metabolic pathways. Ornston and Stanier (1966), by purl-
T
fying the enzymes concerned with the degradation of benzole acid and
p-hydroxybenzoic acid, have shown that the metabolic routes for the two
compounds are entirely, separate and distinct. In view of the non-specific
nature of a number of enzymes heavy reliance is generally not placed on
this approach. Furthermore, the inability to demonstrate the presence
of a particular enzyme is of little importance In eliminating a pathway,
since enzymes can be inactivated during preparation of the extracts.
From the studies reported so far, it appears that once a
pure culture of microorganisms that metabolizes the test organic com-
pound has been obtained and cell-free extracts prepared, the 'techniques
which are used for elucidating metabolic pathways are similar to those
which have been used by microbial physiologists for studying metabolic
pathways of a natural substrate (Dagley and Chapman, 1971). The appli-
\ .
cation of these basic techniques in studying metabolism of environmental
contaminants is well illustrated by studies oh 2,4-D, which is one of
the few synthetic organic chemicals of environmental significance for
which degradation pathways have been reasonably well worked out (Alexander,
1972). Although the pathways established in pure cultures of microorganisms
and/or in cell-free extracts may not be easily extrapolated to natural con-
ditions, Alexander (1972) has stated that such studies could serve as a
useful guide to what happens under natural conditions.
198
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E. Evaluation of Techniques Used to Determine Biodegradation Under
Biological Treatment Plant Conditions
1. Introduction
Numerous chemicals which potentially could enter the environ-
ment first pass through biological waste water treatment plants. Their
fate in these systems can be the determining factor in whether the
chemicals become environmental pollutants or degrade to innocuous materials.
This section will evaluate the techniques used to assess the biodegrad-
ability of compounds under sewage treatment conditions.
In general these techniques can be divided into two categories
(1) static or screening techniques such as respirometry, river die-away,
shake culture, etc. and (2) continuous or semicontinuous techniques.
/
The former techniques have been reviewed in Section III A, p. 49 while
the latter were considered in Section III B. p. 126. Static techniques,
with the exception of the Warburg method with acclimated seed and sometimes
shake culture methods using acclimated seed (e.g. SDA, 1965), provide
only limited insight into a chemical's behavior in a waste water treatment
plant. Thus this section will concentrate on the continuous and semi-
continuous techniques although still considering static techniques when
comparisons are made with biodegradability in treatment plants.
For the most part, the compounds that have been studied for
their biodegradability in waste water treatment plants have been surfactants
(e.g., Swisher, 1970) and the treatment process modelled the most is
activated sludge treatment.
199
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: As pointed out'earlier (p. 126), in evaluating the fate of a
chemical, a distinction should be made between "treatability" (removal
l
by physical processes) and "biodegradability". With water-soluble
materials the distinction is not much of a problem, since the adsorption
sites on the activated sludge will soon become saturated and thereafter
the amount of material removed will represent the amount degraded. With
i
water-insoluble materials physical separations may quite often account for
all of the removal due to the small amount of sludge that is usually
wasted (e.g. Choi et jil., 1974).
Important variables which affect the results of the test
systems include (1) the chemical structure of the test material, (2) the
nature of the biological system, (3) concentrations of the test material,
nutrients, and microorganisms, (4) temperature and oxygen and (5) other
physical, chemical,,pr biological factors such as agitation, pH, growth
promoters, growth inhibitors, etc. (WPCF, 1967). The impact of some of
these factors will be discussed in detail in the following section. The 1
affect of chemical structure is discussed in Section VIII, p. 461 .
2. Factors Affecting Biodegradation Under Waste Water Treatment
Conditions
a. Acclimation and Deacclimation of the Microorganisms
The assimilation of an organic compounds by microorganisms
may be dependent upon acclimation of the cells to the test compound.
Acclimation usually requires varying amounts of time and may involve
synthesis of appropriate enzymes, selection of a .species, etc. The
200
-------
process is ordinarily a reversible one and, therefore, if the test compound
is removed from the feed and then added again, a reacclimation period, usually
somewhat shorter, is often necessary. Furthermore, the addition of a readily
utilized substance to the feed may result in a repression of the induced enzymes
and consequently reduce bio degradation of the test compound (Swisher, 1970;
Gaudy e_t al., 1964).
Organic compounds being fed into sewage-treatment plants can
be categorized into four not very sharply divided categories (1) readily
utilized, (2) utilizable after acclimation, (3) always utilized slowly, and
(4) not utilizable. In many cases, surfactants frequently fall into the second
category and because they usually are released into sewage in relatively con-
stant concentrations, a major attempt is made to allow maximum acclimation in
the test systems used. Other organic chemicals may be released into sewage in
varying quantities (shock loading) resulting in great fluctuations in concen-
tration and in acclimation of mlcrobial population. Buzzell et al (1969),
aware of the importance of acclimation, purposefully used systems that were
not acclimated in order that their results would be representative of slug
(shock) loading in activated sludge treatment plants.
Acclimation may be an unpredictable parameter and may change
considerably under new conditions. Sludge developed under semicontinuous
flow conditions may do poorly for a week or so when transferred to a continuous-
flow system. Acclimation may also be dependent on the temperature (see p.202)
and source of the seed.
Several approaches have been used in attempts to standardize
the seed. The Official German test developes its sludge spontaneously from
201
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the-growth of bacteria entering from'-'.the general environment. Both Buzzell
££ ad. (1969) and Eden et_ al. (1967) have resorted to the preservation of
'i
activated sludge seed. Buzzell £t al. (1969) found that lyophilized (freeze-
dried) sludge retained the greatest degradation potential. Eden et_ al. (1967)
reported that freeze-drying required the addition of sugar and proteins which
might interfere with the biodegradation test so they resorted to using air-
dried sludge.
b. Temperature
Although the community of bacteria in activated sludge or
trickling filter films may be affected by many parameters such as pH, feed,
etc., temperature has been shown in several cases to have a particularly dra-
matic effect (Ludzack et jl., 1961). Using the porous pot technique, Eden
e£ al. (1972) found that while NTA is almost completely removed at 20°C (98
i
percent), at lower temperatures indicative of winter conditions, removal is
considerably less (66-82 percent). Stiff et al. (1973) also studied temperature
effects on removal of alcohol ethoxylates and alkyl phenol ethoxylates using
the porous pot technique. At 15°, 11°, and 8°C the two alcohol ethoxylate
surfactants tested were well degraded. The alkyl phenol ethoxylate was well
degraded at 15°C, but at lower temperatures removal depended upon concentration -
at 5 mg/1 90% was removed, but at 20 mg/1 removal flucturated between 40 to
..-.-(' ' '' .
95% at 11°C and between 20 and 80% at 8°C. Similar poor degradation results
were found for alkyl phenol ethoxylates in a field trial during the winter.
Stiff and Rootham (1973) studied both linear and branched-chain alkylphenol
ethoxylates and concluded that the temperature effect appears to be a feature
i
of these particular materials during treatment, since both types of alkylphenol
ehtoxylates exhibited the same temperature effect.
202
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c. Analytical Methods
Variations in analytical methods can provide quite different
results for a biodegradation test. An analytical method that monitors dis-
appearance of the parent compound might indicate a high degree of biodegrada-
bility, while methods that consider metabolites can give the opposite result
if one or more of the metabolites is persistent. For example, Janicke (1971)
compared MBAS analysis (see Section III C, 2c, p.ISA) and total organic carbon
(TOG) analysis of LAS in a laboratory activated sludge unit. He found that the
TOC method gave a lower rate of degradation thus suggesting that some of the
metabolites are not destroyed rapidly.
3. Correlation Between Laboratory and Field Results
Laboratory techniques allow more controlled conditions to be main-
tained but extrapolation of the results to actual field conditions is not always
clear. For this reason several researchers have gone to large scale experiments
using full-size sewage-treatment facilities. The approaches used and the
inherent difficulties which exist with field tests have been discussed in
Section III B.5, p.150.
Detergents have been intensively studied under full-scale sewage-
treatment plant conditions. Alkyl benzene sulfonate (ABS) and linear alkylate
sulfonate (LAS) have received most of the attention due to the switch over
from ABS to the more biodegradable LAS in mid-1965. Brenner (1968) and
Weaver (1965) have reviewed a number of the principal field tests. In general,
the field results confirm the laboratory conclusions that LAS is more bio-
degradable than ABS, although the quantitative results vary greatly and seem
to be quite dependent upon BOD removal (see Table 17).
203
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Table 17. Summary of Principal Field Test Results
(Brenner, 1968)
Location Process
Manassas, Va. Conventional
Activated Sludge
H it
Uoodbridge, Va. Extended Aeration
ii it ti
Kettle Moraine,
Wis. ,
i
it it H it
it ii iiv-i it
New Lisbon, N. J. Trickling Filter
it it H , it
Material
Used
ABS
LAS
ABS
LAS
ABS
LAS
LAS
ABS
LAS
LAS
Detention
Time in
Hours
6-16
6-16
47
*7
34.5
28.6
34.0
39.0
% Removal '
ABS or LAS
54
85
58-61
97.7
90.7
96.5
51.3
68.3
75.5
80.0
I Removal
BOD
89
91
85-91
94.6
96.0
96.0
46.0
84.0
83.0
Mann and Reid (1971a, b) have evaluated alcohol and alkylphenol
ethoxylates by field trials with a trickling filter sewage treatment plant
which served a small community. These compounds are part of a major class of
surface active compounds - the nonionlc surfactants. The ethoxylates based
on primary alcohols have been found to be readily degradable in laboratory
test systems and the .-field trials confirmed their extensive biodegradability
(see Table 18 ).. However, evidence concerning the biodegradability of branched
chain alkylphenol ethoxylates is conflicting (Mann and Reid, 1971a). Swisher
(1970, p. 248) has attributed this conflict in data to: (1) a failure to make
sufficient provisions for bacterial acclimation and (2) a failure of the
cobalt thlocyanate (CTAS) analytical method to respond to biodegradatlon
intermediates which still show substantial foaming and other surface activity.
204
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The results of the field trials showed that the alkylphenol ethoxylates tested
were biodegradable under bummer conditions (15°C - approximately 80% degraded),
but were fairly persistent (only 20% degraded) under winter conditions (5°C).
The laboratory and field test results of Mann and Reid (1971a) are summarized
in Table 18. A TLC analytical technique was used.
Table 18. Comparison of Alcohol and Alkylphenol Ethoxylate
Biodegradability under Laboratory and Field Conditions
(Mann and Reid. 1971a)
Percent Biodegraded
Product
Dobanol
Dobanol
Nonidet
Nonidet
Nonidet
25-9*
91-9*
P40b
b
- Summer
- Winterb
Clear Sewage
Effluent Inoculum
Die- Away Test
99
10
Official German
Activated
Sludge Test
95-98 (3 tests)
97-98 (2 tests)
20
Trickling Filter
Community
Trials
89
83
ca. 80
ca. 20
a* Alcohol ethoxylate Alkylphenol ethoxylate
Stiff and coworkers (1973) have been able to duplicate this climatic affect
on alkylphenol ethoxylate degradation in the laboratory using the porous pot
activated sludge technique.
A. General Comparison of Laboratory Methods
a. Biodegradation Potential, Reproducibility, and Directed
Comparisons of Techniques
Because of the coat involved and poor reproducibility of the
conditions, field test evaluations of chemicals have been only infrequently
undertaken. Only thirty such tests have been noted by Swisher (1970, p 190)
205
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and for the most part these tests have been almost totally restricted to
surfactants. Thus biodegradability information on chemicals under sewage
treatment conditions is provided mostly by laboratory and bench scale techniques.
These laboratory scale techniques can vary from extremely
i
simple screening tests, such as the river die-away test, BOD, or Warburg
t
technique, to tests simulating the dynamic process of biological treatment of-
sewage in a sewage works, such as the official German test, the SDA (1965)
semi-continuous activated sludge test, or the miniature activated sludge
technique used by Swisher (1970). In general, "the simple screening tests,
though giving a rapid Indication of the ease with which unacclimatized bacteria
will degrade the detergent [or test chemical], will in most cases under-estimate
the degree of removal of the detergent during protracted sewage treatment"
(Stennett and Eden, 1971) because the screening tests fall to consider adsorp-
tion on the activated sludge followed by oxidation or "wasting" of excess sludge.
. i
The laboratory techniques may also vary in the degree of
what Swisher (1970) has termed "biodegradation potential." Methods that
possess a low potential are generally the ones which use a low concentration
of bacteria (e.g., closed bottle test and shake culture test - TBS degradation
5 to 10%). On the other hand, although the river die-away test has a low
bacterial concentration, its biodegradation potential, at least for anionic
surfactants, is quite high (TBS degraded to the extent of 65-75%) presumably
due to the natural species distribution and natural medium (Swisher,
1970). A number of researchers have reported direct comparisons of
biodegradation methods for a variety of surfactants that serve to illustrate
the different results obtained with different techniques. Truesdale and
206
-------
coworkers (1969) have compared five different test methods with a variety
of different surfactants. The results are tabulated in Table 19.
Table 19. Comparison of Biodegradation Test Methods
(Truesdale et al., 1969)
rt
Percent Removed
Pilot Scale Standard Official SDA Semi- Recycling
Detergent Percolating Aeration German Continuous Trickling
Filters Test Test Sludge Filter
Dobane JNX LAS 92 89-91 (9) 93-94 (2) 96-97 (4) 90-93 (6)
Dobane JNQ LAS 96 95-96 (4) 97-98 (2) 98-99 (4) 96-99 (6)
Dobane 055 LAS 96 96-97 (2) 95-97 (2) 98 (4) 94-98 (10)
ABS-4b 90 28-42 (9) 66-68 (3) 98-99 (4) 96-98 (6)
Dobane PT TBS 72 15-22 (3) 34-36 (2) 75-76 (2) 86-91 (5)
Empilan KM9C 98 (2) 99 (2) 98-99 (6)
Empilan KM20
-------
Table 20. Comparison of Biodegradation Test Methods
(Cook, 1968)
Percent
i
t
Continuous Sludge
(Official German)
Slope Culture
River Water
Shake Culture
(SDA standard method)
Semicontinuous Sludge
(SDA, 1965)
Recycle Trickling
Filter
Dobane
JNX
61 ±
74 ±
88 ±
88
89 ±
92 ±
5.
8.
0.
o.
1.
2
8
9
4
6
Removal,
Dobane
JNQ
66
89
93
96
96
96
± 2.
± 1.
± 0.
± 0.
± 0.
9
6
6
3
7
MBAS
Dobane
055
75 ±
0 -
96 ±
91
98 ±
97 ±
5.0
66
0.3
0.3
0.4
Difficult
to Degrade
ABS
34 ±
20 ±
29 ±
34
70 ±
83 ±
5.
7.
1.
4.
1.
5
3
9
0
5
scale percolating filter are the "true" value. For other compounds besides the
surfactants studied, this similarity in results may not hold true. Cook (1968)
concluded that the slant culture technique "gave variable results which were
not reproducible and the, degradation achieved was very low in comparison with
that achieved by other methods."
These two comparison studies also demonstrated the fact that
the reproducibility of all the methods decreases for compounds which are rela-
tively non-biodegradable. In both the studies, the range of values is the widest
for TBS compounds or difflcult-to-degrade ABS materials. Similar low reproduci-
bility is noted for a tetrapropylene derived ABS in a cooperative study organized
by the Soap and Detergents Association (SDA, 1965). In contrast, the confidence
limits for six LAS samples (very biodegradable) in both the shake flask and
208
-------
semicontinuous test were very close. The reproducibility is also quite de-
pendent upon the analytical technique as has been demonstrated with non-ionic
surfactants (SDA, 1969).
b. Advantages and Disadvantages of Individual Techniques
(1) Screening Tests
As noted earlier, screening tests, such as the BOD, river
die-away, Warburg, and shake culture tests, provide important
blodegradability information on compounds which require little
bacterial acclimation. Some acclimation, however, is possible
with the Warburg and shake culture technique. The SDA shake
culture technique provides for acclimation by two transfers on
the medium containing the test chemical and the Warburg seed
is often acclimated to the test compound in a semi-continuous
activated sludge apparatus (which does not mean the seed is
necessarily acclimated under Warburg conditions). Also, the
information from these screening tests is quite pertinent to
chemicals which periodically enter sewage treatment plants (slug
loading). Buzzell e£ al. (1969) have found that a combination
of Warburg respirometer and shake culture technique with a
standardized seed can provide good indications of slug loading
biodegradability.
The BOD test provides little insight into the biodegrada-
bility of compounds under treatment plant conditions. The
bacterial concentration is not representative of activated
209
-------
sludge conditions and the biodegradable potential is low.
Under usual conditions, the test takes 5 days and only the ''
5-day biodegradation rate is measured. Finally, the oxygen '
uptake measurement, unless close to 0 or 100% of theoretical,
is difficult to relate to test chemical loss or metabolite for-
mation.
The Warburg technique provides incubation conditions more
similar ,to activated sludge conditions and allows for the study
i . "
of biodegradation rate. However, the short experimental ex-
posure time, although it makes the method faster, allows only
minimal acclimation. The oxygen uptake measurement has the same
difficulty as mentioned for the BOD test.
\ ' ' '. -
, The bacterial concentration in the river die-away test is
low compared to activated sludge systems, although biodegradation
potential, at least for some detergents, is high. The reproduc-
i
ibility from laboratory to laboratory has often been cited as being
relatively poor, attributable perhaps to the different biological
make-up of various river waters. In order to measure the loss
of test chemical some analytical development effort may be
necessary. Metabolites can be but are rarely identified.
The shake culture technique is somewhat similar to the
river die-away test, except that the conditions are more stand-
ardized (e.g., Buzzell e^ al., 1969; Truesdale je£ al. , 1969).
Acclimation steps are possible and relatively high concentra-
tions of bacteria can be used. Depending upon the analytical
210
-------
procedure used, both loss of the parent compound and formation
of tmetabolites can be determined and the rate of degradation
can be assessed.
(11) Continuous and Semlcontinuous Techniques
Modeling of biological sewage treatment systems is more
closely approximated in terms of acclimation, agitation, and
removal of metabolites with continuous and semicontinuous
techniques. Both the bacterial concentration and biodegradation
potential are similar and the process of adsorption on the
activated sludge can take place in the systems.
The difference between semicontinuous and continuous
systems is quite considerable in terms of space, time, and
money for installation, operation, and maintenance (Swisher,
1970). The batch unit is much more economical since it
does not require the constant attention of a continuous system
and uses much less feed and test material (which may be an impor-
tant factor if the chemical is still in the development stage
where only small amounts of the test compound are available).
Also, in some continuous techniques, maintaining stable biochemical
operation and satisfactory circulation of sludge may be difficult.
Because the official German activated sludge test has been
required by German law since 1964, a number of laboratories are
equipped to perform the test and a great amount of experience
with anionic detergents has been gained. The technique can be
211
-------
run lii any well equipped laboratory (no sewage effluent or
i
sludge needed as Inoculum) and has been adopted by OECD
(Organization of Economic Cooperation and Development) and
the Council of European Communities (Council Directive, 1973).
Disadvantages include the fact that several researchers have
found it difficult to maintain a stable biochemical operation
and satisfactory circulation of sludge (e.g., Stennett and Eden,
1971; Truesdale e£ ail., 1969). In some cases, the Germans have
added ferric hydroxide to weight the sludge floes to facilitate
rapid settling, which takes the test even further from conditions
' -7
of commercial operations. In addition, Truesdale et al. (1969)
have termed the operation of the system as "laborious." As
noted earlier, the reproducibility is less for poorly biodegraded
chemicals. Fischer (1965) reports a spread of about ±5%, ±2%,
)
and.. ±1% for substances with degradation of 75-85%, 85-90%, and
:'." i .
90-95%, respectively. Considerable time may be necessary for
acclimation of the system (4 weeks-Houston, 1963) and the.
test period is 3 weeks. The porous pot modification by
Stennet and Eden (1971) seems to remedy the difficult sludge
circulation problem.
.. Miniaturized continuous-flow activated sludge systems
provide economy in preparation, storage and handling of feeds
as.well as savings in time and labor. However, they introduce
the possible difficulties of "(1) further departure from
characteristics of full scale treatment plants, (2) unsteady
biological performance, and (3) limitations on sizes of samples
which may be withdrawn for analysis"(Swisher, 1970, p 168).
212
-------
Swisher (1970) has concluded that these are not serious since
(1) the large scale units are so far removed from full scale
that another factor of 10 probably has little effect, (2)
biological performance Is not any more unstable than large
scale units and (3) an ample analytical sample Is available
for surfactant work (MBAS). The last factor might be Important
for other chemical groups where a less sensitive (than MBAS)
analytical technique must be used. The technique of Sweeney
and Foote (1964) used natural sewage and fresh seed to shorten
the acclimation period. However, as with the river die-away
test, the variability of natural feed and seed may result in
low reproducibility and perhaps analytical interference. Also,
gathering the sewage, sterilizing it, and obtaining the inoculum
is time consuming. The turbine agitator may be advantageous
when foaming compounds such as detergents are being studied.
The miniature unit used by Swisher (1967a, b) is half the size
of the unit used by Sweeney and Foote (1964), thus reducing the
feed requirements. Both systems are small enough that radio-
tracer techniques can be readily used (Sweeney and Foote,
1964-35S; Gledhill, 1974-14C02 evolution).
The semlcontlnuous operation is advantageous because of
the reduced amount of feed and operating attention required.
The 24 hour cycle of the SDA (1965) is convenient because it
requires no overnight attention. Problems with sludge recycling
is also eliminated since a one-half hour settling period is
213
-------
allowed. In addition, the cycling that occurs In a semi-
continuous system Is similar to many activated sludge processes
where the feed and recycled sludge are mixed at the entrance
to the aerator.
Trickling filters are somewhat easier to operate than the . >
continuous activated sludge systems, since no sludge needs to ;
be recycled. Also, scale-up factors from laboratory studies -to
commercial filters can be made without great worry since the
most Important dimension is the depth of the bed; this is around
1
6 feet and, therefore, can be easily accommodated in the labor-
atory (Swisher, 1970, p 156). Furthermore, different degrees
of treatment can be studied in a single experiment by taking
samples at various depths (Stennett and Eden, 1971). Disadvan-
tages include: (1) a long acclimation period (14 weeks for
development of a mature film and 4-8 weeks acclimation - Swisher,
1970), (2) a fly nuisance, (3) lack of easy accommodation in a
constant-temperature room or bath, and (4) operational conditions
can not be readily adjusted (especially retention time)
(Stennett and Eden, 1971). Recirculating filter tests seem
to be useless because of their high blodegradability potential.
\ Anaerobic systems have received little study. However,,
the general considerations with relationship to die-away and
semi-continuous are still applicable. Operation on a semi-
continuous basis closely simulates the field conditions in .septic
2J4
-------
tanks and anaerobic digesters and allows acclimation to occur.
For compounds that will be treated by septic tanks, an anaerobic
biodegradation study may be essential.
F. Cost Analysis
For cost purposes, the techniques used to study the environmental
persistence and degradation of chemicals in water systems have been divided
into three sections. The first section (la) consists of preliminary or screening
tests which provide information on the relative degradability of materials. The
second section (Ib) provides costs for intensive studies of metabolic pathways;
and breakdown product identification. The third section (2) discusses the
dynamic techniques used for fairly detailed studies of biodegradation under
sewage treatment conditions.
, A major cost factor in biodegradation studies is that of the analytical
equipment. In prediction of the cost, it has been assumed that an average
life of small equipment (e.g., shaker, spectrophotometer, oxygen meter, etc.)
is approximately five years (220 working days/year), whereas that of other
capital equipment (e.g., scintillation counter, gas chromatograph, etc.) is
ten years.
The equipment costs for a test have been estimated on the basis of the
number of days required to perform the test, assuming that the instrument will
be occupied all that time. The cost for each test is calculated for one compound
and for twenty compounds. The labor cost for the test is calculated at the rate
of $60/day (MS level worker, including benefits). The overhead is assumed to
be 125% of the professional services.
215
-------
1. Techniques for Studying Biodegradation of Chemicals in Water
a. Preliminary Test to Determine Biodegradability
The cost estimates given in Table 21 are more appropriate for
a new laboratory, which is interested in undertaking biodegradation studies.
These estimates do hot represent the price of the test quoted by a commercial
laboratory which is engaged in testing biodegradability on a routine basis.
The results of the preliminary screening test will be helpful
in deciding the necessity and approach to be used in more intensive studies.
For example, if the preliminary test indicates low biodegradability, the
Intensive study might use an enrichment culture technique to determine if any
breakdown can be detected. If moderate degradation is noted in the preliminary
test, the intensive study may concentrate on metabolite identification.
V.i:
216
-------
Table 21, Cost Analysis for Preliminary Biodegradability Test
Equipment
Blodegredetlon Analytical Analytical Cost for
Test Method Method Equipment needed the test
and their coat J.
S cr 10 day Chemical
BCD (i) Analysis
5 or 10 day "
BOD (20)
5 or 10 day
BOD (1) Oxygen Mater
5 or 10 dey
BOD (20 Oxygen meter
Two bottle-single Chemical
dllution-reeera- analyals
tloa method (1)
Two bottle-single Chemlcel
dilution- reaara- Ana,lyele
tlon method (20)
Two bottle-single Cerboa anelyels
dilution reeere- Chemical
tlon method (1) Analysis
of Btuxell et al.
(1968)
_ __
Oxygen mater Negligible
$400.00
Oxygen meter . "
$400.00
«_
-
__
Hltrlte deter- 45
mlnatlon apparatus
(colorimeter) $430
Dlgb speed centri-
fuge $1200
Carbon analyaer
$8000
Two bottle-single Chemical Same aa above 323
dilution reaeratlon analyels
Method of Buisell carbon
et el. (19«8) (20 > analysis
Warburg
reapirometry (1). Werburg
Manometer
Warburg Warburg
tesplrometry (20) Manometer
Oxygen electrode Polarography
resplromster .(1)
Oxygen electrode
respirometry (20 Polarogrephy
River Dle-Away Chemical
test (1) analysis
River Dle-Avay
test (20)
River Dle-Awsy Gas chrometo-
tsst (1) graphy
River Die- Away "
test (20)
River Dle-Away O.V.
test (1) Absorption
River Die-Away "
test (20)
Warburg
Manometer 4
$930
Centrifuge
$1200
" 20
Oxygen polero- 7
graphy $900
Recorder $1000
Temperature
control unit $400
centrifuge $1200
Same as above 33
colorimeter negligible
$430
it ii
Gas chromato- 8
graph $8000
43
Spectrophoto- 4
meter $3000
Centrifuge $1200
28
vare end misc. supplies
50
MO
40
240
130
(Includes 4 blodegrada-
tlon units)
2000
(Includes 40 bio-
degradation unite)
130
2000
30
300
30
330
30
500
SO
700
40
XO
8 MO/day 12SZ of Total Cost/
Professional cost compound
services
60 7S 183 183
XO 373 . 973 48
40 30 IX IX
UO 150 610 X
120 130 320 520
($90, If
oxygen meter
is used)
720 900 3620 181
($3(0, if
oxygen meter
is used)
600 750 1545 1545
4200. 3230 11773 588
UO ISO 324 324
600 730 1870 93
«0 75 183 185
3<0 430 1160 30
90 110.25 230.25 250
600 750 1850 92
120 150 338 336
720 900 2363 118
60 75 189 179
300 373 973 48
Remarks
Following para-
meters are studied
in this tost:
Biochemical
oxygen demand,
Total carbon,
Bactarial pop-
ulation
Nitrification
.
Vf4' "
Labor coot could
vary fraa com-
pound to compound
depending on tha
complexity of
the chemical
method of
analysis
217
-------
Table 21. Cost Analysis for Preliminary Biodegradability Teat
(continued)
Sheke culture
teet (1)
Sheke culture
teit (20)
Shake culture
teat (1)
Sheke culture
teet (20)
Shake culture
teet (1)
Sheke culture
teet (20)
Sheke culture
teet (1)
Sheke culture
teet (20)
Shake culture
teet (1)
Sheke culture
teat (20)
Sheke culture
Colorlaatiry
"
Gee chroma-
tography
"
Radioactive
aeterlel
U0>2 Matured
or perent
coapound
"
COj evolution
"
Growth aaaeure-
Mnt
M
Cerbon analyela
Sheker $300 l*gll«lala
Colorimeter $430
Sheker $1300 20
Colorimeter $450
Shaker $300
Gee chromato- 8
graph $8000
Sheker $1300 to
Gee ehrceutto-
grepK $8000
Sclntllletlon 10
counter, $10,000
113
- '
- 1
Spactrophoto- negligible
Mter 1500
' " "
rreete drying 35
50
TOO
50
700
800
(Include* libelled
eterlel)
12000
(Include* labelled
eterlel)
75
700
90
450
100
90
600
120
720
120
1500
60
480
60
420
100
110.29
730
150
900
190
1873
73
600
75
515
375
50
2D70
126
2 '80
13490
210
1180
185
1395
810
190
103
328
119
1090
779
210
89
183
«7
810
teet combined enoaatry; apperetua $2500
with oxygen eucclnle dehydro- Verburg reaping
utlllaetlon and genaee by colorl- actor. $950;
eucclnlc dehydro- aatrlc aathod 'Centrifuge, $1200;
genaee activity .Carbon analyser
aeaey (1) <$«WO; colorl-
(tuttell « *1. aatar, 1430
1969)
San aa above for
20 compounda n .** '
Model ecoeyetaa
(1)
Following pere-
aetere ere
studied ID this
teet:
cerbon renovel
oxygen ut111ca-
tion, succlnlc
dehydrogeneee
activity
Redloactlve
aaterlel
Sclatlllation
coeiter, $10,000
Model ocoeyete*
(20)
373
.700
700
(Includes radto-
. ecttve aataclel end
aeterlel needed for
thin layer chroaeto-
grathy, euto radlo-
«repby)
3000
3600 4300 »220 461
1816 1816
4920 6130 16*45 R22
(The nxnnber in parentheses indicates the number of compounds tested)
218
-------
b. Intensive Biodegradatlon Study to Identify Metabolites and
Elucidate Pathway of Degradation
Once the biodegradability of a compound has been established,
a detailed study can be undertaken to elucidate biochemical pathways and
characterize the nature of intermediate metabolites formed. For this purpose,
pure cultures of microorganisms have generally been considered more suitable.
Some researchers have utilized cell-free preparations in these studies; however,
the results thus obtained are of very little ecological or environmental relevance,
This phase of biodegradation test is time consuming and expensive and, therefore,
should be undertaken only with those compounds for which preliminary evidence
for accumulation of an intermediate metabolite has been obtained. For example,
the CO- evolution or BOD is substantially lower than theoretically possible.
In the intensive study, radiolabelled material is generally used, which may
sometimes have to be custom synthesized. Organic synthesis of a number of
suspected metabolites may be needed In order to identify the unknown metabolites
of the test chemical. The cost estimate for intensive study of biodegradation is
as follows:
One Compound
t
Labor - Ph.D. level (18K) Six months 9,000
Overhead 11,250
Equipment - GC-Mass Spec, (have analysis performed by 2,750
1 a commercial lab) Scintillation counter
(cost for six months - $ 500)
Chemicals, glassware
Miscellaneous supplies - material for thin-layer 750
and paper chromatography,
autoradlography, etc.
Total Cost $23,500
219
-------
Twenty Compounds
Labor - Ph.D. level (18K) Three years 54,000
Overhead 67,500
Equipment - GC-Mass Spec, (cost $100,000, depreciate 33,000
over 10 years; cost for 3 years, $50,000)
Scintillation counter, cost for 3 years
$3,000.
Chemicals, glassware and misc. supplies 5,000
Total Cost $159,500
Cost/Compound $7,975
2. Techniques Which Simulate Sewage Treatment Plant Conditions
.: the costs for the major continuous and semi-continuous techniques
are detailed in Table 22. ' The costs of the screening tests have already
been reviewed in Table 21. The cost of the combined shake culture-Warburg
respirometry screening technique used by Buzzell et al. (1969) to study shock
loading should be somewhat less than the sum of the individual techniques.
The cost estimates in Table 22 used the same analytical technique
so that the difference in the total cost per compound is only a reflection
of the procedure. The analytical procedure can drastically affect the total
cost, the magnitude of which is indicated previously in Table.21. The costs
also demonstrate that continuous techniques are, for the most part, more time
consuming and, therefore, expensive.
220
-------
Table 22. Cost Estimates for Techniques Used to Simulate
Sewage Treatment Plant Conditions
to
Biodegradation .
Test Method *
Official German
Activated Sludge
Test (1)
Official German
Activated Sludge
Test (20)
Miniaturized con-
tinuous activated
sludge
Sweeney & Foote,
1964 (1)
(20)
Swisher, 1967a,b
(1)
(20)
Trickling filter
(1)
Trickling filter
(20)
SDA, 1965
semi- con it inuous
activated sludge
(1)
(20)
Analytical Analytical Test
Method Equipment Apparatus
Needed Costs
and Cost
Colorimeter
$450 500
MBAS
" " 2500
500
" " 2000
11 n 200
" " 800
300
" " 1800
11 100
1000
Chemicals,
Glassware
and Misc.
Supplies
500
5000
100
2000
250
2000
500
5000
100
1000
Labor Cost
@ $60/day
(7 weeks)
2100
8400
(9 days)
540
3780
(30 days)
1800
7200
(9 weeks)
2700
.
10800
(20 days)
1200
4800
Overhead
125% of
Profess.
Services
2625
10500
675
4725
2250
9000
3375
13500
1500
6000
Total
Cost
6175
26850
2265
12955
4950
19450
7325
31550
3350
13250
Cost/
Compound
6175
1342
2265
647
4950
972
7325
1577
3350
662
* The number in parentheses indicates the number of compounds tested.
-------
IV. BIODEGRADATION OF CHEMICALS IN THE SOIL ENVIRONMENT
Industrialization and growth of the human population have resulted in
more intensive use of the land for food production and waste disposal. In
order to increase food production to meet the need, fertilizers, insecticides,
fungicides, nematocldes, herbicides and other synthetic chemicals are applied
to the land, and the amount of these chemicals used is increasing from year to
year. Furthermore, many synthetic organic chemicals such as insecticides are
used for public health purposes and they also reach the soil environment. Mun-
incipal sewage sludge and waste discharged from industry is ever increasingly
deposited on the land either directly or indirectly. Chemicals used as food
additives, food preservatives and other household materials are also deposited
on the land due to land-filling of municipal wastes.
The chemicals found in soil result either from direct application or in-
direct application. For the most part, pesticides provide the largest source
of direct application. Indirect application results from spray fall-out, from
rainfall and dust, from industrial discharge into air and subsequent settling
down to soil, from translocation by animal and plant, and from flooding of
polluted water on soil. Indirect application can be a major factor in con-
tributing to widespread soil contamination by synthetic chemicals. Only a
portion of the chemicals found in soil results from direct application. For
example, many soils that have never been treated with DDT contain small amounts
of DDT; presumably the chemical comes from spray drift or atmospheric fall-out.
Soil can be viewed as a complex system that consists of chemical reactants
and chemical and biochemical catalysts. The biochemical catalysts are composed
Preceding page blank
223
-------
of free-istate and absorbed enzymes as well as living cells. Microorganisms
t
seen to be the major contributor to the degradation of these chemicals in soil
(see Section II A, p. 33). Chemical reactions and photoreactions (only to a
\ . . . '
very shallow depth) may also contribute to degradation. This section will evaluate
the existing methods used in determining the biodegradation of the synthetic
chemicals in soil. ,(
A. Techniques Used for Determining Biodegradation
i -..
The existing methods for determiningbiodegradation in soil can
-------
the use of microorganisms occurring in natural soil. Soils collected from the
field are treated with test chemicals in the laboratory. The treated soils may
be incubated under (a) aerobic conditions [open air or forced aeration with air]
or (b) anaerobic conditions [flushing with nitrogen or other inert gases].
The treated soils may be mixed with water or aqueous solutions containing
mineral salts and/or organic substrates and incubated under stationary or per-
fused conditions, or if an aqueous suspension is used the suspension may be
shaken. Methods that involve transferring a portion of the incubated soil to
liquid medium containing the test material as well as further transferring
are discussed elsewhere (see p. 233).
(1) Soils Incubated with Test Chemicals
This testing method has been widely used. Soil samples *
are well mixed with the test chemicals and incubated under
stationary conditions with or without flushing with air, 0.
(for aerobic conditions), or N2> N2 + C0_ (for anaerobic con-
ditions). In this regard, this method is more closely related
to the natural systems than the other laboratory testing methods.
Soils collected from the field are generally dried in air
and then are passed through a sieve. These soils, in many
studies, are subjected to a physicochemical analysis including
pH, particle size distribution, percentage of organic matter,
ion exchange capacity, total nitrogen content, total carbon
content, water-holding capacity, etc.
(a) Aerobic Studies
The majority of the degradation studies are carried
225
-------
out under aerobic conditions. The methods for maintaining
aerobic conditions are listed below:
(1) Soil samples after mixing with test material
are placed in containers which are open to air. The con-
tainers used include test tubes (Castro and Yoshida, 1971),
plastic pots (Harris, 1969; Messersmith et al., 1971), large
jars (Lichtenstein and Schulz, 1959), small rectangular, flats
(HcClure, 1970), and styrofoam cups (Altom and Stritzke, 1973).
, (2) Soil samples are placed in containers that are
covered but left with air space. The experiments have been
performed using differential respirometers (Bartha, 1968, 1969),
biometer flasks (Bartha jat &L., 1967; Kazano, e_t al. , 1972),
r< ' .
buckets and beakers covered with plastic sheets (Bro-Rasmussen
ejt al., 1970; Bartha, 1971) sealed or stoppered containers in-
cluding glass tubes (Hance, 1969; DeRose and Newman, 1947),
flasks (Walker and Stojanovic, 1973), jars (MacRae and Alex-
ander, 1965), Warburg flasks (Gilmour e_t al., 1958), covered
containers including beakers (Bartha et al., 1968), jars with
lids or with loose-fitting lids (Zimdahl ejt al., 1970; Mont-
!-' ' . ....'".
gomery £t al., 1972).
(3) Treated soils are placed in the containers that
are flushed continuously with a low flow rate of C0_-free air.
The containers include wide glass tubes fitted with two-hole
stoppers permitting inflow of CO.-free air and outflow of evolved
226
-------
C0_ (Kaufman et al., 1968), pint jars that are connected into
i ' ~~"""
an aeration system in an incubator to provide CO.-free air
(McCormick and Hiltbold, 1966), etc.
(b) Flooded Conditions
In order to observe the degradation in flooded soils
such as in rice fields or in organic soils below the water table,
some degradation experiments are carried out under anaerobic
conditions or in submerged soil. For simulating flooded soils,
water is generally added to air-dried soils that have been
treated with the test chemical previously. Alternatively,
aqueous solutions containing the test material are added to the
air-dried soil samples. The various ways in which the experi-
ments are set up include:
(1) Air-dried soils are screened to pass through
a small sieve, placed in large glass test tubes, and then
flooded with water containing test chemical to provide a water
level of 5 cm above the soil surface (Castro and Yoshida, 1971;
Sethunathan and MacRae, 1969).
(2) Soil samples are treated with the test chemical
and are then flooded with distilled water to give a water level
of 4.5 cm above the soil surface (Parr e^t al. , 1970).
(3) Soil samples in glass test tubes or glass bottles
are flooded with distilled water or with aqueous solution con-
taining test chemical, and the ratio of soil sample and water
227
-------
(weight basis) la 1:2 (Goswami and Green, 1971), 2:1 (Iwata
eral., 1973), 1:1 (Sethunathan and Yoshida, 1969).
(c) Anaerobic Conditions
Strictly anaerobic conditions can be achieved by
flushing the soil systems with inert gases such as argon and
nitrogen. In most cases, air-dried soils that are screened
to pass a small sieve are placed in bottles, and water is added
to provide the desired moisture conditions, for example, at
60% field capacity. Incubation is conducted using a multi-
purpose manifold assembly of the appropriate soil systems with
different inert gases or gas mixtures including argon, nitrogen,
and N. + CO. (80:20) (Parr and Smith, 1973; Parr et al., 1970;
i L i
Guenzi and Beard, 1967).
(ii) Soils Suspended in Aqueous Solution
This test method generally uses small amounts of soil
samples that are suspended in water containing the test
chemicals which may be supplemented with nutrients. The
mixture is incubated with or without shaking. There are a
few studies in which the soil suspensions containing test
chemical are incubated in aerobic conditions. The aqueous
V ? .
solutions contain only mineral salts, and the pH of the solutions
is adjusted to neutrality prior to addition of soil samples.
Some researchers have used these conditions with sta- '
tionary incubation. A small amount of soils serving as micro-
cr
bial inoculum is added to aqueous solution containing the test
228
-------
chemical. The aqueous solutions may contain only mineral
salts and test chemical (Miles e_t al., 1971; Alexander and
Lustigman, 1966) or may be supplemented with small amounts of
yeast extract, peptone, and glucose to provide an external
carbon source (Naik et^ al., 1972). The aqueous solutions before
adding test chemicals are adjusted to a neutral pH. The con-
tainers used for the experiments are either flasks or bottles.
(iii) Soil Perfusion Technique
The soil perfusion apparatus has been used for studying
detoxification of pesticides and nitrification in soil as well
as to study microbial systems utilizing water-insoluble sub-
stances such as sulfur. Although each soil perfusion apparatus
developed by different people might be different somewhat, the
principle they are based upon is very similar. In most cases,
the soil percolation apparatus consists of a liquid medium res-
ervoir, a soil column, a delivery tube that transfers solution
and air into the soil, an air inlet, and an air outlet (see
.Figure 24 and 25). The whole unit can be sterile and can be
continuously operated under sterile conditions for a long period
of time. Most soil perfusion units are operated under negative
pressure, although some are operated under positive pressure.
229
-------
Fig. 24. Soil perfusion apparatus (Collins and Sims, 1956)
Courtesy of Nature.
SOIL
GLASS WOOL-
AIR OUTLET
DELIVERY TUBE
SAMPLING
PORT
AIR INLET
-RESERVOIR
Fig. 25. Soil perfusion apparatus (Kaufman, 1966).
Courtesy of Weed Science Society of America, publication
- of Weed Science.
230
-------
Soils used for perfusion experiments are dried In air
and screened through a sieve. Soil samples may be treated
with test chemical in the perfusion unit in two ways: (1)
soil samples are treated with test chemical before starting
perfusions (Kaufman and Kearney, 1965; Kaufman, 1966), or (2)
soils are perfused with aqueous solutions containing the test
chemical (Audus, 1949; Audus, 1951). The amount of soil employed
in a perfusion experiment may vary from 10 to 50 g (air-dried
basis), and 200 to 250 ml of distilled water or dilute solution
containing a test chemical is often used for perfusion. In most
cases, the concentration of test chemical under study is in the
range of 5 to 100 ppm. Samples of perfusate can be taken from
the reservoir at assigned Intervals of time, or daily, for
analysis of the chemical residue and the degradation products.
The rate of perfusion varies according to the investigator.
The method requires either that the parent compound or a pro-
duct of interest appears in the perfusate.
b. Pure Culture Studies
(i) Pure Cultures Isolated from Soil Enrichments
In order to show soil microorganisms are involved in the
degradation of natural and synthetic chemicals in soils, and
to establish routes of breakdown it is helpful to isolate micro-
organisms in pure culture from soils. This demonstrates that
a particular microorganism or some combination of microorganisms
231
-------
are indeed degrading a particular chemical or structurally-
related chemicals. The most effective and the most commonly
employed method for isolating the effective microorganisms is
. /
by means of soil enrichment culture techniques. The basic
principle behind the techniques consists-of increasing the pop-
ulation of the effective microorganisms in the soils or in soil
suspensions by treatment with the test chemical. There are
three ways of carrying out soil enrichments. These methods are
described below:
j ,
(a) Enrichment Cultures Obtained by Treatment of Soil
with Test Chemical
The technique involves the treatment of soil samples
with test chemical solutions. Soil samples may be treated with
the test chemical added either to an appropriate mineral salt
solution (Burger ejt al. , 1962; Jensen, 1957; Hammond and Alex-
ander, 1972; Tiedje e_t al., 1973) or to water (Cavett and Wood-
row, 1968). In some studies the solution of the test chemical
has been supplemented with yeast extract (Belser and Castro,
v 19,71; Ohmori e_t al., 1973; Jensen, 1957). The treated soil
samples are incubated under stationary conditions or on a
rotary shaker. After incubation for a period of time, an
aliquot of the mixtures is transferred for incubation in a
fresh liquid medium containing the test compound. The incu-
bation temperature is generally controlled at room temperature
i
or at 30CC.
232
-------
The growth of microorganisms can be judged from the increase
of turbidity of the enrichment culture fluids, or by microscopic
observations. That microorganisms are active in the enrichment
cultures can be shown by measuring the decrease in the concentra-
tion of the test chemical. For example, this can be easily
determined by release of chlorine ion from chlorinated hydrocarbons
(Belser and Castro, 1971) or by a decrease in UV absorption of
aromatic compounds (Burger £t al., 1962). For the isolation of an
effective microorganism(s) from the enrichment cultures, agar plates
of the mineral salt medium containing the same test chemical are
streaked with the enrichment culture fluids. The isolates are
purified by repeated streaking on the same medium or on a rich
medium such as nutrient agar. Each pure culture is then tested
for its ability to degrade the appropriate chemical in an
appropriate medium. The active microorganism is generally main-
tained on agar containing the appropriate chemical or occasionally
in rich medium such as nutrient agar.
(b) Naturally Enriched Cultures
' This soil enrichment technique is similar to the
method described above except that natural soil samples which
had been treated with the test chemical are used for the source
of isolating effective microorganisms without further subcultures.
A Streptomycea that can degrade herbicide diazlnon was isolated
from submerged soils treated with the chemical (Sethunathan and
233
-------
*
MacRae, 1969). Microorganisms utilizing crude oil have been
s
isolated from soils which had been contaminated with crude oil
. 4
over a period of time.
(c) Soil Perfusion Cultures
Another soil enrichment'technique uses soil perfusion
to enrich the effective microorganisms. The technique of soil
perfusion has been described previously. Kaufman and Kearney
?
(1965) isolated effective microorganisms on isopropyl-N-3-chloro-
phenylcarbamate (ClPC) and 2-chloroethyl-N-3-chlorophenylcar-
bamate (CEPC) from perfused soils. Isolation of pure cultures
of effective microorganisms from the perfused soils is accom-
plished via a soil dilution plate method. Serial dilutions
are prepared with the enriched soil from the perfusion units
using a mineral salts plating medium. CIPC or CEPC are supplied
as a sole source of carbon for organisms obtained from perfused
soils treated with these chemicals. The formation of clear
zones surrounding certain of the colonies is considered indi-
cative of CIPC or CEPC degradation and utilization. Stock
cultures of organisms utilizing either CIPC or CEPC are isolated,
purified, and maintained on the mineral salt medium with CIPC
or' CEPC as the sole source of carbon. Soil perfusion technique
has also been used to isolate bacteria that degrade N-methyl-
isonicotinate (Orpin ejt al., 1972) and 4-chloro-2-methylphenoxy-
acetate (Gaunt and Evans, 1971).
234
-------
(11) Other sources of Pure Cultures
Other pure cultures used to study the biodegradatlon of
a compound may be obtained from unenrlched soils (Ohmorl et
al., 1973), from sources other than soils (Ohmorl e£ al., 1973),
and from cultures usually stocked In the laboratory (Miyazaki
_et .al..» 1970). A method of Isolating microorganisms from un-
enrlched soils Is carried out by direct sprinkling of soil samples
onto an agar medium containing a test chemical (Ohmorl et^ al.,
1973).
The degradation of synthetic chemicals by pure cultures
isolated by enrichment is generally studied in two ways:
(1) by growing microbial cells in liquid medium containing the
test chemical, and (2) by resting cell techniques. For
measuring degradation by cells growing in liquid medium con-
taining the test chemical, generally a small volume of cell
suspension is added to a sterile liquid medium containing the
test chemical with or without supplemental growth factors such
as yeast extract or vitamin B complex. If aerobic conditions
are being considered, the mixture may be aerated on a shaker
or by bubbling sterile air or sterile CO-free air or the mix-
ture may be incubated under stationary conditions. Generally
Erlenmeyer flasks are used for this kind of experiment. At
assigned intervals of time, a portion of culture fluid is with-
drawn for assays of the chemical residue and/or the metabolic
235
-------
products. The incubation time varies considerably depending
a
upon the species of active microorganism, test chemical, type
of medium, incubation temperature, aeration, pH of the medium,
i
etc. A species of Arthrobacter grown in mineral salt medium
containing 0.2% of MCPA as the sole carbon source completely
degrades 4-chloro-2-methylphenoxyacetic acid (MCPA) in 100
hours at 25 °C under aerobic conditions (Bollag e_t al., 1967),
while a species of Pseudomonas, grown in mineral salt medium
containing 0.04 M monochloroacetate plus 0.08% peptone completely
degrades monochloroacetate in 16 days (Jensen, 1957).
The other way of observing degradation by pure culture is
by means of resting cell techniques. Cells grown in the medium
containing the test chemical may be harvested in the logarithmic
phase of growth (Tiedje et al., 1973), at 18 hours of growth at
30° under shaking condition (Cavett and Woodrow, 1968), after
3-5 days of growth with aeration (Burger jet .al., 1962; MacRae
and Alexander, 1963; Belser and Castro, 1971). They are usually
washed with buffer (Cavett and Woodrow, 1968; Belser and Castro,
1971; MacRae and Alexander, 1963). The concentration of phos-
..i1 -
phate buffer used by MacRae and Alexander (1963) is generally
in the range of 0.01M to 0.067M and the pH value between 7.1 to
7.5. Washed cells are then generally suspended In the buffer
used .for washing the cells, and an appropriate amount of the
'*C ' '
test chemical is added to the suspension. The mixture in the
flask may be stirred slowly at 25°C with a magnetic stirrer
236
-------
(Belser and Castro, 1971), or the mixture may be incubated in
a shaking water bath at 30°C (Tledje et_ al. , 1973; Sethunathan
e_t al., 1969). Samples are removed periodically for assays
of the test chemical residue and intermediate products. If
oxygen uptake is to be studied, the mixture is generally placed
in Warburg flasks and standard Warburg manometric techniques
are followed (Tiedje £t al., 1973; MacRae and Alexander, 1963;
Cavett and Woodrow, 1968). The dry weight of cells suspended
in buffer solution can be obtained by drying similar volumes
of the cell suspension and phosphate buffer and the weight
difference is the dry weight of cells.
c. Cell-free Extract Studies
For proof that the degradation of the compounds was due to
enzymatic reactions, cell-free extracts have been used. In addition, cell-
free extracts can also provide information for establishing the pathway of
intracellular degradation. The techniques used to promote product accumulation
and establish metabolic pathways are similar to those described before (Section
III,D,4,b, p. 190).
For preparation of cell-free extract, the cells are often cul-
tured with aeration in a medium containing the test chemical as a source of
carbon. The cells are harvested at the late exponential phase of growth
(Bollag ejt al., 1967), or after 2-3 days of growth at 25°C (Orpin et al. , 1972),
or after 7 days of growth at 27°C (Clark and Wright, 1970) by centrifugation.
Before being subjected to disruption, the cells are washed with cold buffer
237
-------
(Bollag et al., 1967), or buffer supplemented with mercaptoethanol (Orpin
££.*! 1972). Washed cells are suspended in the same buffer solution and ,
sonicated (Bollag et al., 1967) or disintegrated by high pressure (Clark and
Wright, 1970), or by other methods (Heyman and Molof, 1968). If high pressure
is used, the cell suspension may be passed through a cooled French press at
s ' . .
a pressure of 1,500 psi (Bollag e_t al., 1967), a Biox X-press or a Hughes'
press (Orpin tilt al. J 1972) .
Enzymatic activities of the cell-free extract are assayed by
incubation of the cell extract in the buffer containing the test chemical.
The reaction may be terminated by the inactivation of the enzymes, and the
products then are determined. If oxygen uptake and CO 'evolution are to be
determined, conventional respirometric techniques (Warburg apparatus) are em-
ployed. Co-factors such as NADH, NADPH or others may be added to the reaction
mixture so that enzymatic reaction is enhanced (Buswell and Mahmood, 1972;
Orpin «st al., 1972).
d. Miscellaneous Methods
Chambers and Kabler (1964) used the Warburg respirometric
technique to determine oxygen uptake as an indication of degradation of
\ '
phenols and phenol-related compounds. The mixed cultures of microorganisms
obtained from soil and sediment from a waste lagoon of a catalytic cracking
^ "
plant were used as the source of inoculum. Pseudomonas species were active
in about 80% of "the cultures while, in the remainder, Archromobacter, Xantho-
4 . . J
monas, and Flavobacterium species were active. Hammond and Alexander (1972)
have studied the biodegradability of methyl-substituted aliphatic acids by
238
-------
measuring oxygen utilization by dilution method (for details refer to Section
III.A.I.a, p.51 ) using soil as the source of microbial inoculum.
McClure (1972) reported that a mixed suspension of microorganisms
containing at least 7 species of bacteria, fungi and actinomycetes were obtained
from an IPC (isopropyl N-phenylcarbamate)-enriched soil sample. The mixed
suspension was applied to IPC-treated soil sample to determine whether the
suspension can accelerate the degradation of the herbicides in soil as de-
termined by a plant bioassay. Rectangular wooden flats (19 by 23 by 10 cm
deep) are filled with composted Gloucester sandy loam soil. Rate of appli-
cation of IPC equivalent to 4 and 12 kg/ha is homogenized in a nutrient solution.
Sixty ml of the mixed suspension (0.5 mg dry weight per ml) is sprayed onto
the soil surface of the flat. The flats are placed in the greenhouse and the
foliage, if any, is harvested, dried, and weighed after 2 weeks.
Studies of the degradation of alkylbenzene sulfonate (ABS) and
herbicides 2,4-dichlorophenoxyacetic (2,4-D), and 2,4,5-trlchlorophenoxyacetic
acid (2,4,5-T) in lysimeters have been reported (Robeck e_t al., 1963). Figure
26 shows the cross section of a lysimeter. The lysimeter contains sandy soil
and graded gravel is placed at the bottom of the tank. The experiment is con-
ducted in order to see if the ABS in sewage could be broken down while trickling
through an unsaturated soil. To do this, the first 260 days the lysimeter
is fed 19 liters of sewage daily at 0.75 liter per minute rate for 25 minutes
and then allowed to rest the remainder of the day. During this 260-day period
the concentration of ABS in the influent averaged 11.5 mg/4. This concentration
of apparent ABS is reduced to 0.5 mg/£ in the effluent of the column.
239
-------
II; El.)
TANK
GRADUATED
IJONL)|N(J INDICATOR
0"
'I '
0"
'A* 4" .?:>';?' ^'^'"'.vu^r-;
: ;;... ]>" GRAVEL;
I
STAINLESS STEEL')
WIRE SCREEN ,;,}
NEWTOWN SAND
GRADED GRAVEL -
/
VENT PIPE
OUTLET
V^/V^/I/-ii'j'J?'?8^ Jf WATER TADLE
'-..- - ** - r-f
~ "" - )_
3-0' OIA
Fig. 26. cross section of a lysimeter (Robeck et al.
.Reproduced from
best available copy.
240
-------
2. Greenhouse Studies
Only a limited number of researchers have studied biodegradation
in soil under greenhouse condition (De Rose and Newman, 1947; Sheets et al.,
1968; Burger et al., 1962; Ahmed and Morrison, 1972); In this test, soils
obtained from the field are treated with various concentrations of test chemi-
cals before being placed in containers, or conversely the soils are placed
in containers then test chemical is applied to the soils. Treated soils are
kept in the greenhouse in order to observe the degradation. In most cases
the chemicals which have been tested are herbicides including phenoxyalkyl
carboxylie acids, benzole and phenylacetic acids. Determining the toxicity
of the treated soils to plants (i.e., bioassay) makes it a simple and con-
venient way for determining the herbicide residue in soil. Soil treated with
herbicide is watered to maintain adequate moisture.
3. Field Studies
Most field studies have been focused on the degradation of
organophosphate insecticides, chlorlnation hydrocarbon insecticides, and
chlorinated phenoxyalkyl carboxylic acid herbicides. Some of these compounds,
such as DDT and aldrln, can persist in soil for a number of years.
In most studies the field is divided into plots. Each plot
receives the test chemical with concentrations generally similar to actual
field application. Two kinds of plots in terms of test surface area are em-
ployed. One is so-called microplots and the other is regular plots. The sur-
face area of some plots may be smaller than one square foot (Read, 1969;
Wolf et al., 1973; Ahmed and Morrison, 1972).
241
-------
Each microplot is enclosed by a wooden, metal or concrete '
frame, and may be furnished with a 30 x 30 mesh plastic net bottom. The
surface area of regular plots Is generally from a few hundred square feet to
a few thousand feet (Burnside e_t al., 1971; Llchtensteln and Schulz, 1959;
Lichtenstein e_t al., 1970; Schulz and Lichtenstein, 1971; Stewart e_t al., 1971;
Stewart and Chlsholm, 1971; DeRose and Newman, 1947). Plots are separated
individually by ridges of soil or by cultivated strips that are built up
around each plot to prevent lateral surface movement of the test chemicals.
The treatment of test chemicals in soil plots is generally
carried out by applying the test chemicals evenly over the soil. The chemicals
may .then be incorporated thoroughly into the soil to a desired depth. In the
case of microplots, test chemicals are uniformly mixed by hand with a rake.
In most cases, test chemicals are mixed with soil to a depth up to 6 inches,
but chemicals applied to a 15-inch depth have been reported (Nash and Woolson,
1967). The amount of test chemical applied to the soils is generally in
the*range of 2 to 20 Ibs. per acre, which is not far from the actual dosage
of many pesticides applied to crop fields. Higher concentrations are also
used in some studies (Wolfe e£ al_., 1973). In many cases, several different
dosages may be applied to the soils to compare the persistence of the test
chemical in various concentrations. Such field experiments require adequate
replication. Furthermore, untreated control plots are often essential. The
chemicals can be applied to the soils only once or they may be applied an-
i(
nually for a few consecutive years (Lichtenstein e_t al. , 1970; Stewart et al. ,
1971; -Stewart and Chisholm, 1971). Soil samples are collected by means of
242
-------
a soil sampler (Ahmed and Morrison, 1972), or be a soil auger (Lichtenstein
and Schulz, 1959; Schultz and Llchtenstein, 1971). If the vertical distri-
bution of the chemical residues In the soil plots Is to be studied, samples
are collected from different depths. Soil samples are then subjected to
chemical analyses and/or biological assay.
B. Analytical Procedures
The accuracy of the experimental results from soil degradation
. i ' . '
studies depends to a great extent upon the analytical methods employed.
Good experimental setup without supporting, suitable analytical procedures
can make the results meaningless. This is especially true in the case of
degradation experiments of synthetic chemicals in soil where the concentration
of the test chemicals applied to soils is generally very low and the soil
is very complex.
The analytical procedures used for determination of the chemical
residues and their metabolic intermediates in soils can be generally divided
into two categories: (1) chemical analyses and (2) bioassays. Chemical
analyses cover chromatographlc methods, spectrophotometrie techniques, radio-
assays, CO. evolution, 0. consumption, and GC-mass spectroscopic techniques.
Bioassays include plant assays for herbicides, Insect assays for insecticides,
etc. -. ;/''.' .
1. Chemical Analyses
a. Extraction and Clean Up Procedures
Prior to analyses, chemical residues and their metabolites
in soils have to be extracted from the soil samples and then concentrated.
243
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Soil contains a variety of inanimate and colloidal mater-
ials which can react with both the parent and intermediary compound and there-
by make their extraction extremely difficult. Divising a technique for extrac-
tion of an intermediate may be particularly difficult because the sorption and
solubility characteristics of the intermediate are unknown.
t
Most chemicals under study as well as most of their meta-
bolites are organic in nature, and, therefore, organic solvents are commonly
used for extraction. The choice of organic solvent depends on the chemical
nature of the test compounds and their metabolites. Very often the solvent
may consist of a mixture of two or more solvents and the system chosen is
»
the one that gives the highest recovery yield. For example, acetone is used
for removing anilide (Bartha, 1968), organophosphorous insecticides (Bro-
Rasmussen, e_t al., 1970), and dichlobenil (Montgomery, e_t al_., 1972) residues
from soils. Hexane and acetone mixtures (1:1) have been used to extract
trifluralin (Parr and,Smith, 1973), and Dyfonate (Schulz and Lichtenstein,
19.71). Sethunathan and Yoshida (1969) used hexane, .then acetone and then
hexane fordiazinon. Extraction can take place by hand shaking, in a mechanical
shaker (Iwata, £t al., 1973), in a Waring blender (Bartha, 1968), by sonication
(Parr and.Smith, 1973), or in a Soxhlet extractor (Bro-Rasmussen, e_t al_., 1970;
Montgomery, e_t al.', 1972; Stewart and Chisholm, 1971; and Zimdahl, et al. ,
1970). The extract-may be concentrated to an appropriate volume and then
directly used for chromatographic analyses (Bartha, 1968; Stewart and Chisholm,
19.71).. In other cases, the extract needs further purification and/or clean-
up* in order to avoid unnecessary interference for chromatographic analyses.
244
-------
One method of clean-up requires that the concentrated extracts be purified
by passing though an alumina column (Montgomery, e_t aJL., 1972; Bro-Rasmussen,
ejt al., 1970; Llchtenstein and Schulz, 1959). Another method is to add acti-
vated carbon to the extract (Bro-Rasmussen, e_t al., 1970).
The problems involved in separation of the chemical resi-
dues and their intermediates in the culture solutions of the pure culture
studies or mixed culture studies are generally less complicated and simpler.
Unlike soil degradation studies, the chemical residues in the liquid medium
of the pure culture studies are generally not associated with soil particles
and are, therefore, easier to extract. By and large, the main techniques for
separation of the chemical residues and their intermediates from the culture
fluids are about the same as the spearation procedures used with soil samples.
In that case, the culture solution is extracted with solvent, and the extracts
are dried with anhydrous sodium sulfate and then concentrated (BoHag ejt al. ,
1
1967; Miles, ejt al., 1969). Clean-up may be required for some extracts (Schulz,
£t ajL., 1970; Miyaza&i, e_t al., 1970). Furthermore, the culture solutions
before being subjected to solvent extraction may have the hydrogen ion con-
centration adjusted to the appropriate pH by adding NaOH or HC1 so that
extraction efficiency is enhanced (Bollag, e_t a_l., 1967; MacRae and Alexander,
1963).
In most cases, the intermediates in both the culture
solutions or soil samples are by no means completely separated by solvent
extractions. This is particularly true of many water soluble Intermediates.
245
-------
b. Chromatographic Methods
' Chromatographic methods have been widely used for separa-
tion and determination of chemical residues and their metabolic intermediates
in soil. Among them, gas-liquid chromatography and thin-layer chromatography
are most extensively used.
l -
(i) Gas-Liquid Chromatography
. When the chemical under study and some of its metabolic
intermediates are volatile and thermally stable, gas-liquid
chromatography (GLC) provides a good tool for quantitative
determination. In most cases, a small volume (a few microliters)
of the extract is injected into the instrument. Electron capture
and flame ionization detectors are the most commonly employed.
Some of the chemicals or their metabolites (e.g. organic acids)
that are relatively non-volatile can be converted to volatile
derivatives by simple procedures such as esterification. For
example, fatty acids (MacRae and Alexander, 1963), MCPA
(4-chloro-2-methyl-tfhenoxyacetic acid)(Bollag, &t_ ad., 1967),
chloramben (3-amino-2,5-dichlorobenzoic acid) (Wildung, e_t_ al.,
1968), fenac (Harris, .et. _al_., 1969), are methylated prior to
gas Chromatographic analysis.
(ii) Thin-layer Chromatography
Many organic chemicals and their metabolites can be detected
and separated by means of thin-layer Chromatographic methods, if
the right solid absorbent and the right liquid solvent systems
246
-------
are chosen. By combination with radioisotopic techniques
(Sethunathan and Yoshida, 1969; Sethvmathan, e* al., 1969;
Jones and Hodges, 1974; Mlyazaki, et al., 1969; Baude, et al.,
1974), with GLC (Sethunathan, et al., 1969), or with spectro-
photometric methods, quantitative determinations can be achieved.
In essence, a small volume of the concentrated extract is spotted
to a TLC plate and is developed along with standards with a suit-
able solvent system in a TLC tank. Sometimes, two or more solvent
systems are used for developing the same sample along with standards
to ensure the identification of unknowns (Guenzi and Beard, 1967).
4 . .
The TLC'plates may contain fluorescent indicators that help to
locate the spots under a short wavelength UV light. In most cases,
one dimensional TLC is used, but two dimensional TLC may be
employed (Schulz, ejt ^1. , 1970).
(ill) Paper and Column Chromatography
Because of time involved and some limitations in quantitative
determinations, paper and column chromatography are not frequently
used for separation and determination of the chemical residues
and their metabolites. However, column chromatography has
been used for fractionating metabolized oil samples for sub-
sequent GLC analyses (Jobson, et al., 1972). Also, a modified
ion-exchange resin column technique has been used for paraquat
(a herbicide) residues in culture broth (Anderson and Drew,
1972). In addition, a descending paper chromatographic
technique has been used for determining metabolic products of
247
-------
MCPA (4-chloro-2-methylphenoxyacetate) by a soil
Paeudomonas sp. (Gaunt and Evans, 1971).
c. Spectrophotometric, Methods
Spectrophotometric determination of synthetic chemical
residues and their degradation products in soils usually does not have the
sensitivity of GLC and TLC analyses. It may not be able' to distinguish be-
r
tween the parent chemical, metabolites and hydrolysis products. Nevertheless,
it can be used for determining some chemical residues or their metabolites in
soils or can be used as a confirmatory technique with TLC or GLC. There are
three types of spectrophotometry often used in soil studies: UV (200 - 400 nm),
visible (400 - 700) and infrared (2 - 15 y).
(i) UV Absorption
UV methods for quantitative determinations usually
require a rigorous clean-up of the extract to ensure the
" final solution is free of any interference material in
the spectrum region to be measured. However, UV absorp-
tions have been used as an indicator of the decomposition
of aromatic compounds including phenoxyalkyl carboxylic
. acid herbicides, substituted phenols and mono- and dis-
substituted benzenes by soil microbial communities or by
pure cultures in aqueous solutions (Alexander and Aleem,
1961; Alexander and Lustigman, 1966; MacRae and Alexander,
1963; Burger, et al., 1962). Since the analytical pro-
cedure involves no extraction and clean-up, soils used
248
-------
for this type of study should release a minimum amount
of material that would interfere with UV measurements.
The problem can be partially overcome by using very
dilute suspension of soil. Alexander and Lustigman
(1966) for example, used 45 ml of mineral salts media
inoculated with 1.0 ml of a 1% suspension of Niagara
silt loam. The authors note that a major shortcoming,
Imposed by the UV analysis, is the possible unsuita-
bility of the test conditions.
(ii) Visible Spectrophotometry
Since most synthetic chemicals applied to soils and
their metabolites are not colored, a colored complex must
form before the chemical can be quantitatively determined
by a spectrophotometer in the visible region. Colorimetric
methods can be used with other analytical methods for
confirming degradation. For example, degradation of
chlorinated phenoxyalkylcarboxylic acids, such as 2,4-D
(2,4-dichlorophenoxyacetatic acid) and MCPA (4-chloro-
* "
2-methylphenoxyacetic acid) are shown by a combination
of the release of chloride ion (the colorimetric deter-
mination) , with the loss of UV absorption, loss of radio-
activity of parent compound, GC determination, and plant
bioassays (Bollag, £t ' al. , 1967; Burger, ejt al., 1962).
Kaufman and Kearney (1965) determined degradation of
herbicides CIPC (isopropyl-N-3-chlorophenyl carbamate)
249
-------
,by two colorimetric methods; one determines the release
of chloride and the other, the formation of aniline.
(iii) Infrared. Spectrophotometry
Infrared Spectrophotometry is mainly used for qual-
itative determination of degradation products (Miyazaki,
t
£t ad., 1970; Bartha, 1969; Buswell and Mahmood, 1972).
This requires the isolation of a relatively large amount
(yg-mg) of pure compound.
d. . Radioassays
Radioactive tracer techniques have been a most useful tool
for determining intermediary metabolism and detoxication mechanisms (Casida,
1969). The studies of the breakdown pathways of the chemicals (mainly pesti-
cides) in soils are no'exception. Radioassay usually can be achieved at nanor
gram leyels ,of; the labelled compound,.and the specificity of analysis depends
largely on the degree of separation and clean-up before scintillation counting
or radloautography. Chemicals, in soil .studies .are generally labelled with
li» t. .
C because.of the required long test period and because the detection of
lit ,
C02 evolution is a good.,indication of mineralization. The selection of the
site for:labelling is very important and depends upon what information one
wants,: to obtain. .For example,, if one is interested in the cleavage of the
acetjate moiety from the:aromatic ring of 2,4-D (2,4-dichlorophenoxyacetate)
ands:in the .fate of acetate.moiety in soil, the acetate moiety should be labelled
m .
with-.:;C. On the other hand, if one is interested in the degradation pathway
lit
of the aromatic rings^ the rings will be labelled with C (Tiedje et al.,
l
-------
into three sections: (i) Assay for the loss of radioactivity of the test
lit
chemical; (ii) Identification of metabolic Intermediates; (iii) C02 evo-
lution.
(i) Assay for the Loss of Radioactivity of Test Chemicals
The accuracy for the determination of the loss of
radioactivities of test chemicals from soil samples largely
depends on the extraction and clean-up. These procedures
have been previously discussed (see p. 243). To separate
1<*
the parent C test chemical from its metabolites, TLC is
usually employed. Then the spots are scraped and placed in
a scintillation fluid for counting radioactivity (Jones and
Hodges, 1974; Bollag and Liu, 1971) or the radioactivity
may be determined by a radioscanner (Baude, e_t al. , 1974).
The determination of total radioactivity remaining in the
lit
soil samples (residual C-labelled parent compound plus
1»»
any nonvolatile C-labelled degradation products) can
be done by combustion techniques (Baude, e£ al., 1974;
Kazano, e_t al. , 1972). In the case of pure culture studies,
the total radioactivity in culture fluid is determined
by directly transferring a portion of culture fluid to
a scintillation fluid and counting (Bollag, et_ al. , 1967;
Bollag and Liu, 1971). Radloassay techniques are also
applied in studies of the leaching of chemicals in soils
and binding to soil components (Jones and Hodges, 1974;
Kazano et al., 1972).
251
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(11), Identification of Metabolic Intermediates
Again the accuracy of results relies on the extraction
14 14
and clean-up. The C-labelled products and the Olabelled
parent compound are generally separated by TLC. Standards
of the probable metabolites of the test chemical (when
they are known) are cochromatographed with the unknown
(Goswami and Green, 1971; Sethunathan and Yoshlda, 1969;
Miyazaki, e_t al. , 1970; Schulz, e_t al. , 1970; Orpin, et al. ,
1972; Matsumura, e_t al., 1971). The radioactive spots in
plates are located by means of radioautographic technique
or by a radioscanner. By comparison of the R. values of
the unknown metabolic products with the standards, the
possible identity of the unknown metabolites may be ob-
tained. With the assistance of radioautography or radio-
14
scanner, the portions of unknown C-labelled metabolites
are detected, each portion is collected, extracted and
crystallized. When large enough amounts of the unknown
are available, the unknown can be identified with analysis
by IR, NMR, mass spectroscopy, etc. Besides TLC, GC has
14
.been used for separating C-propanol (a degradation
product of l,2-dibromo-3-chloropropane) from soil samples
and the n-propanol is trapped from GC by bubbling through
a dioxane scintillation .solution (Castro and Besler, 1968).
252
-------
(ill) 14C02 Evolution
Although the disappearance of the radioactivity of
14
C-labelled test chemicals in soil samples is a good
14
indication of degradation, the detection of ,C02 evolution
provides a definite indication of the mineralization of the
chemical, since water arid CO- are the final biodegradation
14
products. Soil samples treated with a C-labelled test
chemical (may be also treated with the same non-labelled
chemical) are generally placed in a glass container and
incubated under aerobic conditions (Kaufman, £t al., 1968;
Jagnow and Haider, 1972; Kazano, e_t al., 1972; Wildung,
e_t aJL , 1968; MacRae and Alexander, 1965). For the studies
14 14
of CCL evolution under submerged soils, evolved CO. is
periodically withdrawn by passing C02~free humidified air
(Goswami and Green, 1971). Any evolved C02 is adsorbed
in a gas trapping solution. The CO -trapping solution is
either a mixture of alcohol and amine (for examples, ethano-
lamlne and 2-methoxyethanol, 1:2; ethanol and ethanolamine,
2:1, etc); or KOH or NaOH solution (0.1 N or IN). The
14
radioactivity of CO, in the trapping solution is deter-
mined by transferring a known volume of the trapping
.solution to a scintillation solution and counting in a
scintillation counter (Miyazaki, e_t al., 1969; Skipper and
Volk, 1972; Kazano, e_t al., 1972). If a KOH or NaOH
253
-------
solution is used for trapping C02, the radioactivity of
14
COj can also be determined by converting it to BaCO
and its radioactivity is then counted (MacRae and Alex-
ander, 1965).
e. GC-MS Techniques
Although the gas chromatograph is an effective instrument
for the separation and quantification of degradation products, it provides
little insight into the identity of the chemical structure of the residue.
For that reason, several researchers have resorted to using gas chromatrography
combined with mass spectrometry. Both Hammond and Alexander (1972) and Tiedje
et al. (1973) have used the technique with extracts from pure culture studies.
The extraction procedures and derivatization techniques are the same as used
with gas chrpmatography alone. Detection and identification of amounts as low
as 10 grains are possible (Karasek and Laub, 1974).
f. 0» Consumption
Although respirometrlc techniques have not been applied as
frequently in recent years in studies of biodegradation in soil as they have in
water, they sometimes are used. In a study of the biodegradation of various
aliphatic acids, Hammond and Alexander (1972) used standard BOD dilution
bottles containing a nutrient salts media and enough organic acid to utilize
8.3 mg/1 of dissolved oxygen (inoculum 30 mg of soil). The oxygen uptake was
i
measured with an oxygen electrode. Oxygen utilization resulting from nitri-
fication and oxidation of soil organic matter was corrected for by subtraction
of an untreated (no organic acid) blank.
254
-------
A number of researchers have Isolated pure cultures from
soil and then measured CL consumption of the cells suspended in nutrient medium
with the test compound (Orpin e£ jal., 1972; Burger e_t ,al^., 1962; Fincher and
Payne, 1962; Tiedje et al., 1973; Clark and Wright, 1970). Warburg respiro-
metry is most commonly used. Similar procedures have been used with cell-free
extracts (Neujahr and Varga, 1970; Buswell and Mahmood, 1972).
j
Many studies have used respirometry techniques with
natural soil samples. For example, Gilmour and coworkers (1958) used 125 ml
Warburg flasks to study the oxidation of glucose and wheat straw in soil samples.
In a study of the biodegradation of anilide herbicides in soil, Bartha (1968)
used a constant-pressure differential soil respirometer constructed in his
laboratory to measure 0? uptake. The apparatus consisted of a 250 ml filter
flask connected to individual manometers. Glass syringes, connected to the
i .
apparatus by a syringe needle, are adjusted to the uppermost mark initially.
The 0. uptake is measured by returning the manometer fluid to its original
position by adjustment of the syringe. The results were reported as the
difference between treated and control soil samples and in all cases negative
values (representing inhibition) were noted at the chemical concentrations
employed.
g. CO. Evolution
As was mentioned under Section d., Radioassays, p. 250, the
14 14
measurement of CQ- evolution from soil treated with a C-radiolabelled material
is a good indication of the mineralization of the test chemical. However,
several researchers have used unlabelled C00 evolution as an indication of the
2
255
-------
rate of respiration of the added test compound (Parr and Smith, 1973; Messer-
smith £t jil.., 1971; Engvlld and Jensen, 1969). Messersmith and coworkers (1971)
measured the CO. evolved from a positive pressure soil perfvision system des-
' ' v ^ _
crlbed by Kaufman (1966) (see Section on soil perfusion technique, p.229 ). DeFrenne
et al., (1973) measured the CO. evolved from cell-free extracts used to metabolize
~ ~~ 2. .: '
2-hydroxy-muconic acid. The technique is basically the same as described for
14
CO. evolution (trap CO. with base), except that a control must be run to
subtract the endogenous respiration.
2. Bioassays
».--
The determination of the soil persistence of pesticides, in-
cluding insecticides and herbicides, often uses bioassays. This is especially
true with field tests. The method can- show the persistence of a pesticide
residue toxic to sensitive animals or plants in soil. Furthermore, bioassays
are desirable because they do not rely on extraction techniques and are rela-
tively inexpensive because they do not require the development of an analytical
procedure. A disadvantage of the method is that pesticides may form complexes
with soil, or firmly bind to soil and become non-toxic to sensitive animals
or plants, even though the same concentration in aqueous solution would be
very toxic.
a. Plant bioassays for herbicides '
The bioassays for the determination of the persistence of
herbicides in soils are based on the growth inhibition of sensitive plants.
The degree of growth 'inhibition is generally correlated with the concentration
of
-------
are total dry weight of the plant (McClure, 1972), total dry weight of the
aerial portion (Lavy, e_t al_., 1973; Messerstnith e_t al., 1971), dry weight
of seed yield per acre (Burnside £t al., 1971), fresh weight of shoots (Sheets,
et. al., 1968), lengths of roots (MacRae and Alexander, 1965), and percentage
of emergency and normal development (MacRae and Alexander, 1965). Table 23
lists a number of plant bioassays used to study herbicide persistence.
For field bioassays, soil plots receive various concentra-
tions of herbicide. Seeds are then planted at various intervals. For labora-
tory and greenhouse bioassays, soil samples to be tested are generally placed
in pots, treated with herbicide, and then the seeds are planted.
Table 23. Plant Bioassays for Herbicides
Reference
DeRose and Newman
(1947)
Burns Ide et al. ,
(1971)
Messersmlth et al. ,
(1971)
Sheets et al. ,
(1968)
Lavy et al. ,
(1973)
Audus
(1949, 1951)
Compounds
Studied
dicamba
pi dor am
2,"4-D ;2,4,5-T
dicamba
picloram
2,3,6-TBA
trlfluralln
2,3,6-TBA
dicamba
tricamba
2,4-D
2.4TD
MCPA
2,4,5-T
Plants
Soybeans and
oats
Field beans
Amsoy beans
Corn
Snap beans
Soybean
Cress roots
Time of
Planting
1st day of herbicide
treatment and
subsequent plantings
Two years after
treatment, every
year thereafter
Right after herbicide
treatment
Right after herbicide
treatment
Right after herbicide
treatment
Remarks
Field bloassay
Field bioassay
Greenhouse
bioassay
Greenhouse
bioassay
Greenhouse
bioassay
Perfusate from
soil perfusion
column
257
-------
b. Insect Bioassays for Insecticides
The persistence of insecticides, such as organophosphates and
DDT, can be determined by bioassays using sensitive insects. The technique
has, been used by Ahmed and Morrison (1972), Harris (1969), and Read (1969).
In most cases,, insect larvae are.placed in the soil sample and the analysis
is determined..by the number that survive.
258
-------
C. Evaluation of Biological Techniques
1. Factors Affecting Degradation
The key factors that affect the degradation of synthetic chem-
icals in soil appear to be soil type, depth of soil, test chemical concentra-
tion, soil microorganisms and acclimation, physical environment (including pH,
temperature, 0? availablility, redox potential and moisture content of the
soil), and external carbon source.
Studies which deal with the influence of these factors on
degradation of chemical compounds in soil are described below. However, a large
number of studies in the literature have determined persistence of a chemical as
a function of changes in the environmental conditions, but have not distinguished
between degradation and loss due to movement, adsorption, volatilization, and
minor alteration in the molecule. Keeping in view the scope of this report,
such studies have not been cited as examples in this section.
a. Soil Type
A very wide spectrum of soil types has been used in studies
of the degradation of synthetic chemicals in the soil environment. The soil
types vary from sand (low in organic matter content, usually less than 1%) to
muck soil or peaty soil (high in organic matter content, usually higher than
50%). Although it is known that soil type does influence the degradation of
synthetic chemicals in soil, it is less certain whether the rate of degradation
correlates with organic matter content of the soil. Considering the fact that
microbial activity is associated with organic matter content, the soil types
that have higher organic matter content should give higher degradation rates.
259
-------
However, the presence of organic matter in soil also affects the movement,
adsorption and vaporization of the chemical (e.g., see Broadbent, 1967) and
results in immobilization of free-state enzymes. Such effects may reduce the
chances of cells or enzymes finding their substrates and thus perhaps reduce
the degradation rates. In fact, Marshall (1971) has shown a decrease in the
level>of metabolic activity due to adsorption of cells and enzymes on soil
organic matter. -.
Kazano et al. (1972) found the rate of degradation of
carbaryl (1-naphthyl methylcarbamate) was influenced by soil type; 14CO_
evolution in six soil types varied from 3 to 35.2% of initial radioactivity
during 32 days of incubation (Table 24).
Soil Type
Table 24. ll|C02 Evolution from Five Soil Types
Each Receiving 2 ppm of 1^C-Carbaryl
(Incubation period: 32 days) (Kazano et^ al., 1972)
Organic Matter
pH
14C02 Evolved
Clay
Sandy loam
Clay loam
Loamy sand
Loam
-------
Kaufman e_£ al_. (1968) studied the degradation of amitrole
(3-amino-l,2,A-triazole) in many soil types and found that the most rapid and
extensive degradation of amitrole occurred in the silty clay loam soil (Figure
27).
% C14 evolv«d
as C'«02
80
Hogerstovm Billy clay loom
.Lohelond sandy loom
..Celuyville muck
Figure 27. Effect of Soil Type on Amitrole Degradation
(Kaufman et aL., 1968)
Courtesy of Weed Science Society of America,
publisher of Weed Science.
b. Soil Depth
With an increase of soil depth, there is generally a
decrease in oxygen tension, volatilization, and loss of the test chemical.
A change in the types and abundance of microorganisms also accompanies a
change In soil depth. Some characteristics of two soil types (Sharpsburg
silty clay loam and Keith sandy loam) at three depths are shown in Table
25. Lavy ejt al. (1973) studied the degradation of 2,4-D and atrazine in
261
-------
Sharpsburg silty clay loam and Keith sandy loan at various depths. They found
that whereas the degradation of 2,4-D was rapid in soil from all the depths
tested, the rates of degradation of atrazine decreased with an increase in soil
depth of the two soil types.
Table 25, Characteristics of Sharpsburg Silt Clay Loam
and Keith Sandy Loam at Various Depths (Lavy et_ al., 1973)
Count of
Percentage
Soil Type
Soil
Depth;
Microorganisms
PH x 10?/B
Organic
Matter
Clay
Sand
(centimeters)
Sharpsburg
Silty Clay
Loam
Keith Sandy
0 -
36 --
91 -
0 -
36 -
91 *
23
61
122
23
61
122
6.2
6.1
6.7
7.5
8.1
8.2
26
16
3
18
3
3
4.8
1-3
0.8
0.9
0.6
0.1
39
42
38
18
21
17
15
25
25
77
39
35
c. Test Chemical Concentration
The persistence of synthetic chemicals in soil is often
affected by the dose applied to soil. At higher concentration, the chemical
often persists longer than at low concentration. Ahmed and Morrison (1972)
found' that'the longevity of four organophosphate insecticides was always
longer at a higher rate of application to soils. Similar observations were
262
-------
made by Wolfe et_ ai. (1973) with the organophosphate insecticide, parathion.
Messersmith _et al. (1971) studied the rates of breakdown of the herbicide
trifluralin in two soil types treated at the concentration of 140 and 1 ppm.
14 14
The rate of CO- evolution from C-trifluralin was slower at 140 ppm of the
herbicide than at 1 ppm.
d. Soil Microorganisms and Acclimation
Organic chemicals in soil are broken down largely by micro-
organisms. Soils differ in the types and abundance of microorganisms they
contain and this affects the persistence of chemicals in soil. Degradation of
organic chemicals by different soil types has been discussed previously (p.259 ).
Although degradation rates vary considerably from one soil type to another, the
variations cannot be attributed exclusively to the microbiological composi-
tion of the soil, since in complex environments such as soil, other soil and
climatic factors such as temperature, moisture content, acidity, organic
matter, etc. are also critical factors which affect degradation.
Acclimation also affects the capacity of soil microbes to
inactivate chemical compounds. Evidence for acclimation of soil microorganisms
to a test chemical has been obtained by a number of researchers. Engvild
and Jensen (1969) found that in the previously untreated garden soil, the break-
down of herbicide pyrazon was not complete even after 3 months. Addition of
10% pyrazon-incubated soil initiated a rapid breakdown of the herbicide and
degradation was complete within 45 days (see Figure 28). Kaufman and Kearney
(1965) reported that phenylcarbamate herbicides (isopropyl-N-3-chlorophenyl-
carbamate [CEPC]) were degraded more rapidly when reapplied to the enriched
soil and perfused for the second time, as is demonstrated in Figure 29 and 30.
263
-------
§ ,. ..
' ' "" 1
A Garden soil
Figure 28. Time Course of Breakdown of
250 ppm of Pyrazon in Different
Soils
A. Untreated garden soil (No. 3)
B. Addition of 10% pyrazon de-
composing soil (No. 1) to
untreated garden soil (No. 3)
causes accelerated breakdown.
C. The garden soil has become
enriched with pyrazon decompos-
ers and further additions of
pyrazon are rapidly decomposed.
(Engvild and Jensen, 1969)
Courtesy of Pergamon Pces.3 Ltd.
B. Garden soil*
activated soil
C. Pyraion re-.
added to B
60 Days
100
16
100
20
Figure 29. Disappearance of CIPC in (A)
perfused soil treated with
CIPC, and (B) same perfused
soil after a second treat-
ment with CIPC.
(Kaufman & Kearney, 1965)
Courtesy of American Society
for Microbiology
Figure 30. Disappearance of CEPC in
(A) perfused soil treated
with CEPC, and (B) same
perfused soil after a second
treatment with CEPC.
(Kaufman & Kearney, 1965)
Courtesy of American Society
for Microbiology
26A
-------
e. Physical Environment - pH, Temperature, Oxygen Avail-
ability, Redox Potential and Moisture Content of the
Soil
The hydrogen ion concentration of the soil or of the
degradation medium can affect the rate at which both chemical and microbio-
logical decomposition 'occur. For biodegradation, the pH effect is generally
very specific, depending on the particular chemical and the particular microbe
involved. The herbicide dalapon was detoxified in soil much more rapidly at
pH 7 than at pH 4, whereas 2,4-D was detoxified more rapidly at pH 5 than at
pH 7.0 (Corbin & Upchurch, 1967). Organophosphate insecticides have been
reported to persist longer in acid soil (Griffiths, 1966). However, Konrad
e_t al. (1967) and Getzin (1968) found that the organophosphate insecticide
diazinon decomposed much faster in acid soils. With chlorinated hydrocarbons,
researchers have generally found no correlation between their persistence and
the pH of the soil (Swanson et^ al^., 1954; Fleming and Maines, 1953; Bollon et al. ,
1958). Corbin and Upchurch (1967) have reported that the pH optima under
laboratory conditions for degradation of the herbicides dicamba, 2,4-D, dalpon,
amitrole and vernolate were 5.3, 5.3, 6.5, 6.5, and 7.5, respectively. The
authors assumed these responses were due to the influence of pH on microbial
activity (but it could be due to pH effect on sorption). The effect of pH
on the ability of Lipomyces sp. (a soil yeast Isolated from paraquat-treated
soil) to degrade paraquat in three media was examined by Anderson and Drew
(1972). These researchers found that in malt and mineral-salt medium paraquat
was degraded at most pH values but the rates were decreased at extreme pH
265
-------
values (Table 26). In soil extract, the degradation was reduced at extremely
acid pH values and was nondetectable at values of pH 8.4 or above.
\
Table 26. Effects of pH on Ability of Lipomyces sp. to Degrade 10~4M
Paraquat
PH
i
3.6
4.2
4.8
5.4
6.0
6.6
7.2
7.8
8.4
9.0
9.6
in Three Media at
% Paraquat
(
Malt
Extract
0.0
12.3
70.5
100.0
93.4
95.4
94.7
90.5
94.1
92.6
49.3
22 °C (Anderson
Degraded After
Mineral
Salts
0.0
77.1
84.6
87.9
58.3
51.8
35.5
46.9
99.1
68.0
50.8
and Drew, 1972).
3 Days in
i
Soil
Extract
70.0
80.0
100.0
100.0
100.0
100.0
100.0
50.0
0.0
0.0
0.0
The influence of temperature on the breakdown of chemicals
in soil is a complex relationship. Increase .in temperature generally results
in increased rate of volatilization, desorption and leaching. Microbial
activity in the soil is also directly influenced by temperature. At low
.temperatures, the overall transformation of a chemical is slower than at
higher temperature (Edwards, 1964; Lichtenstein and Schulz, 1959; Patterson,
1962). DeRose and Newman (1947) tested the persistence of three growth regu-
lators in soil at temperatures ranging from 10 to 30'C and found that persistence
.was inversely proportional to the incubation temperature. Anderson and Drew
(1972) studied the effect of temperature on the degradation of paraquat with
a pure culture of a soil yeast (Lipomyces sp.) and found that degradation
was decreased at temperatures below 20°C and above 35°C (Table 27). The rates
266
-------
Table 27. Effects of Temperature on Ability of Lipomyces sp. to Degrade ICT^M
Paraquat in Mineral Salts Medium, pH 7.2 In Static Culture (Anderson
and Drew, 1972)
Paraquat Paraquat
Degraded Degraded
After 3 Days After 3 Days
(%) Temp. (°C) (%)
6
12
16
20
22
55.5
59.6
62.6
72.3
96.1
26
30
34
39
100.0
100.0
100.0
0.0
of degradation of three triazines and two uracil herbicides were determined by
Zimdahl et al. (1970) at temperatures of 13.2 and 31.2°C. Evaluation of the
rate constants at the two temperatures revealed always lower degradation rates
at the lower temperature (Table 28). Montgomery e£ al_. (1972) studied the
Table 28. The Rate of Degradation and Arrhenius Activation Energy of Selected
Triazine and Uracil Herbicides Applied to the Soil at 8 ppm
(Zimdahl et al., 1970)
Rate of Degradation in Arrhenius
Reciprocal Months at activation
Storage Temperature (°C) energy
Herbicide
Atrazine
Simazine
Ametryne
Bromacil
Terbacil
13.2
0.19
0.21
0.14
0.14
0.37
31.2
0.60
0.55
0.26
0.19
0.59
(kcals/mole)
10.8
9.2
6.1
3.0
6.1
267
-------
degradation of herbicide dichlobenll in soil at temperatures 6.7 and 26.7°C;
the half-life of the herbicide at 6.7°C was found to be 28 weeks (+10 weeks
lag), and 19 weeks at 26.7°C.
Most degradative reactions catalyzed by soil microbial com-
munities require the presence of oxygen. Anderson and Drew (1972) found no
degradation of paraquat under anaerobic conditions in the presence or absence
of alternate electron acceptors (e.g., NC>3 or S0^~), whereas under aerobic
conditions, degradation was complete within 3 days. The effect of different
levels of aeration (obtained-by varying the amount of medium in the flask
while keeping the agitation constant) on microbial utilization of crude oil
has been .studied by Jobson e_t al^. (1972). Their results indicated differen-
. tial utilization of crude oil components at different levels of aeration;
there-was more rapid utilization of the saturated fraction under conditions
of maximum aeration (Table 29). On the other hand, a number of chemicals
have been found to be degraded more rapidly under anaerobic soil conditions.
Guenzi and Beard (1968) have found fortyfold higher rates of degradation of
DDT under anaerobic conditions than under aerobic conditions, although the
extent of change in DDT was small. Sethunathan and MacRae (1969) have re-
ported that disappearance of diazinon under submerged conditions was more
rapid than that reported in studies under aerobic conditions. Jagnow and
Haider (1972) have Compared the rates of disappearance of dieldrin in sta-
tionary -and aerated cultures of several soil microorganisms and found that
there was greater release of C02 from C-dieldrin in stationary cultures
than from aerated cultures.
268
-------
Table 29. Liquid Chromatographic Analyses of Residual Oils from the Aeration
Experiment After 5 Days of Growth at 30°C (Jobson ejt al., 1972)
Weight (%) of "topped"
oil volume
Oil fraction
Benzene-soluble
asphaltenes
Benzene-insoluble
asphaltenes
Saturates
Soluble NSOb
Insoluble NSO
Con- 250
trol mla
2.5 7.6
6.8 6.1
51.0 35.9
8.6 14.4
0.3 1.0
500
mla
4.8
6.8
46.8
10.7
1.0
750
mla
3.3
5.0
45.6
10.2
3.2
1,000
mla
3.3
5.5
47.2
10.1
1.0
Per 2-liter flask
NSO component: fraction recovered by elution with a 1:1 benzene-methanol
mixture. This fraction should contain more polar compounds than those eluted
with benzene which yields the aromatic fraction of crude oil.
Willis e* al. (1974) have pointed out recently that
.. factors other than mere exclusion of oxygen (e.g., redox potential) may
determine the rate of pesticide degradation in anaerobic environments.
These researchers investigated the relationship between the oxidation
reduction potential (Eh) and the rate of degradation of herbicide trifluralin.
It was found that the rate of degradation of trifluralin was much more rapid
under more reducing conditions (Figure 31).
269
-------
Y = 96.4 - 17.6 X + 0.7 X1 R7 = 0.91
Figure 31. Disappearance of Trifluralin from Soil Suspensions as a Function
of Redox Potential and Time. The curve Y = 99.0 - 1.95X was
developed from soil suspensions at redox potentials at +450, +250,
and +150 mV. The curve Y - 96.4 - 17.6X + 0.7X2 was developed from
soil suspensions at redox potentials at +50, 0, and -50 mV.
(Willis et al., 1974) Reprinted from Journal of Environmental
Duality, 2» 2f2.,'American °«cl«t"T nf A»r«?».'wnr.
Soil moisture content can influence persistence of a
chemical directly by affecting soil microbial activity, or indirectly by in-
fluencing the initial adsorption of the chemical, the rate at which the chemical
diffuses into the soil or the availability of the adsorbed toxicant (Gerolt,
1961). DeRose and Newman (1947) reported that plant growth regulators 2,4-D,
2,4,5-T and 2-methyl-4-chlorophenoxyacetic acid disappeared rapidly as the
moisture content in the soil was increased. Yaron e£ al. (1974) reported that
)
TV . - ' .
the presence of water in soil caused an increase in the rate of loss of the
pesticide, azinphosmethyl; the authors explained it on the basis of the fact
that biological activity is reduced in a dry environment. The effect of soil
water content on the rate of degradation has been summarized by Hamaker (1972).
270
-------
The calculated rates indicated that the degradation in dry soil may be many
fold slower than in moist soil and that the rate tends to level of£ with
higher moisture content with a possible change at saturation values (Hamaker,
1972). .''.'
. £. External Carbon Source
The role of soil organic matter in degradation of organic
chemicals has been discussed in an earlier section. Several researchers in
their degradation studies have modified soils with the addition of external
carbon sources, which differ in type and quantity from the organic matter
naturally occurring in soils. This section is devoted to a discussion con-
cerning the influence .of external carbon source in the biodegradation test
medium on the persistence of environmental contaminants. Similar to other
factors which favor soil microbial activity, addition of a readily utilizable
carbon source generally enhances the degradation of chemical compounds in soil.
vThe addition of larger amounts of supplemental carbon source, however, fre- ,
quently results in lowering of the degradation rates, presumably as a result
of greater ease with which the organisms are able to utilize the added carbon
(Kaufman £t al_., 1968). McClure (1970) has reported increased degradation of
the herbicides diuron, monuron, dlphenamld, dicamba, chloropropham and atrazine
in soils to which nutrient broth had been added. Glucose has been shown to
accelerate the degradation of atrazine, diuron, and disodium methanearsonate
(McCormick and Hiltbold, 1966; Dickens and Hiltbold, 1967) and parathion
(Lichtenstein and Schulz, 1959). Miyazaki e_t al. (1969) have studied the
metabolism of the acaricides chlorobenzilate and chloropropylate by pure cul-
tures of Rhodotorula gracllis. These researchers found that degradation of
271
-------
chlorobenzilate was stimulated by the addition of sucrose. In contrast, the
breakdown of chloropropylate was reduced in the presence of sucrose although
the microbial population had increased considerably by the addition of sucrose
(Table 30). Kaufman e_t al. (1968) have also reported that addition of organic
amendments (e.g., starch, hay, or sucrose) to amitrole-treated soil stimulated
microbial activity but reduced amitrole degradation.
Table 30. Radioactive Carbon Dioxide Collected from Culture of Rhodotorula
gracilis during a 10-day Incubation Period (Miyazaki et^ al, 1969)
^G-chlorobenzilate ^C-chlorop ropy late
supplement co
basal medium (0.5%)
None
.Sucrose
Citrate
.'bi-Ketoglutarate
Succinate
Fumarate
ODa
0.022
0.395
0.050
0.060
0.060
0.159
14co2b
0.64
1.93
1.58
1.00
0.90
0.45
ODa
0.020
0.355
0.027
0.067
0.056
0.130
Iltco2b
0.10
0.01
0.29
0.26
0.08
0.19
a
.Optical density in 10-fold diluted samples was measured at 420 nm at the end
of incubation period.
Percent collected of the originally incorporated ll*C-Chlorobenzilate or
1 **C-Chloropropy late.
2. Correlation Between Laboratory and Field Results
Field studies concerning degradation of organic chemicals in the
soil .environment have generally been limited to pesticidal chemicals. In most
cases, the residue of the applied pesticide has been determined at various
intervals by bioassay or other analytical procedure (Burnside e_t al > 1971;
*
Schulz and Llchtenstein, 1971; Lavy e£ ad., 1973). In these studies researchers
272
-------
have not attempted to distinguish between the loss of the compound by phenom-
ena such as leaching, volatilization, percolation to subsoil, etc. and due to
degradation. Furthermore, parameters such as temperature, moisture, and other
environmental conditions are continuously varying in any field experiment,.
which makes a meaningful comparison between a field study and a controlled
laboratory study extremely difficult.
Bro-Rassmussen et al. (1970) studied degradation of eight phos-
phate insecticides in the laboratory by incubating soil with the test chemical.
The disappearance curves of the insecticides were defined by a first order rate
constant. When degradation was studied under field conditions, the data failed
to fit the first-order rate model since the degradation rates decreased much
more rapidly than would be expected for first order kinetics. These authors
attributed these differences to the lack of strict control of several para-
meters (e.g., water content of the soil and temperature) during the duration
of the field experiment. However, they found a reasonably good agreement in
the laboratory and field results in terms of relative order of persistence of
the organophosphorous insecticides tested. Only in the case of two herbicides,
bromophos and mercarbam, was the decomposition rate found to be more rapid
in the outdoor experiments than in the laboratory experiments. However, the
validity of these comparisons Is in question, since the field tests had no
controls for physical loss.
A number of other comparisons between laboratory and field
results are possible. However, the field studies only measure loss of parent
compound or soil persistence and, therefore, they do not allow comparisons
of degradation.
273
-------
3. General Discussion of Various Test Methods
Unlike the natural waters, soils are generally richer in micro-'
blal content and have, therefore, been used extensively as microbial inoculum
without amendment or added microorganisms, in degradation studies. A test
method utilizing microorganisms occurring in natural soil approaches more
closely the conditions encountered in nature. The use of soil eliminates the
need for adjustment of various biodegradation parameters such as microbial pop-
ulation and diversity, nutrient and oxygen concentration, in an attempt to sim-
ulate the natural soil environment. The use of soil in a biodegradation test
in the laboratory permits the Investigator to perform degradation studies
under controlled conditions; e.g., where losses due to volatilization, leaching,
etc. can be controlled. However, soil, when used in the laboratory, is somewhat
removed from many naturally occurring environmental conditions. For example,
the moisture content in the laboratory degradation study is usually maintained
relatively constant under natural conditions, the moisture content will
change substantially. The soil from different geographic locations will
vary considerably in terms of physicochemical properties and microbial compo-
sition which may introduce serious variations in the results of degradation
studies of different laboratories. Furthermore, the presence of a complex
medium such as soil will often introduce additional steps in extraction and
clean up. Also, the presence of soil may preclude the assay of biodegradation
by'many analytical procedures. For example, measurement of oxygen consumption
with natural soil as the biological material has generally not been used for
studying degradation; the reason being, perhaps, the high endogenous rates of
soil respiration which may make the interpretation of oxygen uptake data more
difficult.
274
-------
Soils Incubated with a test chemical under stationary condi-
tions with or without aeration more closely simulates natural systems than
soil suspended in aqueous solution and shaken or incubated under perfused
conditions. The former approach allows the investigator to study degradation
under aerobic or anaerobic soil conditions. These conditions are known to
exist in the natural soil environments and determination of persistence of
a compound under all these conditions is essential.
In a number of degradation test methods soil is suspended in
water, and therefore, one deals with an aqueous suspension of soil rather
than soil at moisture levels generally encountered under natural conditions.
The moisture content of the soil can affect degradation of a chemical in many
different ways (see Section IV.C.I.e., p.265). When sizable amounts of soil
are used with the water, the system simulates flooded soil or sediment condi-
tions. However, in many cases, soil is used as an inoculum in the test pro-
cedure and not as a medium for degradation and, therefore, the quantity of
soil suspended in the aqueous medium is very small. This might limit the
availability of many undefined nutrients present in soil; such nutrients may
be essential for certain soil microorganisms to proliferate under the test
conditions and degrade the test chemical. The advantage of the method is that
in case a dilute soil suspension is used, certain analytical measurements,
e.g., disappearance of U.V. absorption, can be made directly on the sample or
on the supernatant obtained after centrifugatlon. The extraction and clean
up steps in such cases may be minimal. Furthermore, when the soil suspended
in aqueous solution is used in degradation studies, a more uniform distribution
275
-------
of the test chemical may be obtained. This may permit a uniform exposure of
the test chemical to the entire microbial population, as well as removal of
relatively homogeneous samples for degradation analysis.
Soil perfusion systems are rapid and easy to use but are some-
V I
what difficult to set up and maintain. The test requires that substrate or
product appear quantitatively in the perfusate and, therefore, can not be used
with the compounds which adsorb to the soil. Furthermore, in soil perfusion
systems, one also deals with an aqueous'suspension rather than soil and the
system is not a good simulation o'f the soil environment. Soil perfusion systems
permit a constant exposure of microorganisms to air, water and the test chemical;
subsequently the degradation environment is perhaps more potent than occurring
under natural conditions. Kearney (personal communication) has stated that a
soil perfusion system is an excellent tool for enriching microorganisms which
will degrade the test chemical; however, 'such a system is unsuitable for use
as a routine biodegradation test method due to its unusually higher biodegradation
Sl... . i .
potential and difficulty in handling many units. 'Recycle trickling filters
which operate on the same principle as the soil perfusion system have also
' $.
been' reported to possess higher biodegradation potential compared to other
test methods (Swisher, 1970; Cook,'1968). A soil perfusion system has gener-
ally been used to' study aerobic degradation;'the simulation of flooded and
anaerobic soil conditions has not been attempted with-this test method.
Pure culture and cell-free extract studies are generally con-
sidered ''suitable for elucidation of biochemical pathways and mechanisms of
degradation, but are unsuitable for preliminary screening for biodegradability.
276
-------
According £0 Wright (1971), organising) which can metabolize a compound in pure
.1
culture are not necessarily those responsible for its degradation in the soil.
Thus, results from pure culture studies may be difficult to extrapolate to the
natural conditions. Degradation by pure culture of microorganisms will also
fill to allow interaction between natural environmental conditions as well as
interaction with other microorganisms. Gunner and Zuckerman (1968) have un-
covered a synergistic relationship between two organisms in the degradation
of a chemical compound. These researchers found that when the Arthrobacter
sp. and Streptomyces sp. were incubated separately, no change was evident in
the 'diazinon' molecule. However, when the two organisms were incubated to-
gether, extensive degradation of the pesticide occurred. Bollag and Liu (1971)
have reported that mixed cultures of investigated microbes were more effective
in transforming the herbicide Sevin than pure cultures. Since certain bio-
logical reactions are catalyzed by extracellular enzymes, these enzymes may
degrade organic compounds in soil. Recently, evidence suggesting that soil
enzymes contribute to the breakdown of some organophosphorus insecticides has
been presented by Getzin and Rosefield (1968, 1971). If pure cultures of
microorganisms are used in degradation studies, the extracellular soil enzymes
produced by other microorganisms will be absent.
The greatest advantage in using pure cultures in degradation
studies is that complications originating from the complexities and variability
of the soil system are eliminated and the extraction and clean up procedure
will be simpler. The data obtained from such test methods will be more re-
producible than when soil is used as the source of biological material. Other
277
-------
advantages and disadvantages in using pure cultures of microorganisms for
;
studying biodegradation will be similar to those discussed in Section III.
D.I. , p. 162).
Degradation studies utilizing cell-free extracts have been
used by researchers to study the enzymatic mechanisms of degradation. Since
the membrane permeability barrier is absent when a compound is exposed to
the cell-free extract of a microorganism, one can always question if the com-
pound will be degraded by the intact cell and, if so, will the rates be com-
parable to those observed with the cell-free extracts.
}
Among the techniques used to~ isolate pure cultures of micro-
organisms for studying degradation of organic chemicals, the enrichment culture
technique is the most widely used. This technique provides the investigator
with effective microorganisms without having to screen a large number of micro-
bial stock cultures for their degradation ability. In this technique, re-
searchers have generally attempted to enrich microorganisms which can use the
tfest organic chemical as the sole source of carbon and energy. However,
sometimes the organisms may be able to metabolize a compound to some extent
but not necessarily use it as a sole carbon source for growth (referred to as
cometabolic degradation; see Horvath, 1972a). This may account for some
failures in isolating organisms by elective culture technique. Beam and Ferry
»
(1973) showed, degradation of cyclohexahe in fertile soil but were unsuccessful
irr isolating microorganism(s) which could utilize the test chemical as the sole
source of carbon and energy. Based on their findings, these researchers pro-
. posed that their inability to isolate organisms from soil which can utilize
278
-------
cycloparaffinic hydrocarbons as the sole substrate source cannot be taken as
proof that such organisms are not present in the environment. However, the
relative importance of cometabolism, as an environmental process, is unknown.
In order to enrich for microorganisms capable of cometabolizing
an organic compound, techniques such as analogue enrichment (Horvath, 1972b),
and co-substrate enrichment (Horvath, 1973) have been suggested (see Section
III.A.3.b ., p.107). Using these techniques, investigators have succeeded in
isolating pure cultures of microorganisms capable of cometabolizing compounds
such as DDT (Wedemeyer, 1967a, 1967b; Pocht and Alexander, 1970b), 2,3,6-
trichlorobenzoic acid (Horvath, 1971), and 2,4,5-trichlorophenoxyacetic acid
(Horvath, 1970). Although researchers have succeeded in isolating microorganisms
which cometabolize DDT and trichlorophenoxyacetic acid, it should be emphasized
that these compounds are well known to be persistent, thus again questioning
the significance of cometabolic degradation from the standpoint of environ-
mental persistence.
Because of the complexities and variability of soil systems,
the pathways of degradation have generally been studied with pure cultures or
cell-free extracts. The techniques used have been reviewed previously in Section
III.D.4.b., p.190).
D. Cost Analysis for Testing Biodegradabillty of Chemicals in Soil
Degradation studies can be carried out in two phases; (i) to test
the overall biodegradability of a compound, and (ii) to identify metabolites
and establish pathways of degradation. The analytical method for preliminary
testing could be a direct measurement of the parent compound and/or the
279
-------
metabolite, or an indirect method, such as measurement of microbial growth
and, oxygen consumption, C0_ evolution, etc. The costs associated with the
direct analytical procedure applicable to degradation studies in soil are not
different than those described for the aquatic environment. However, the time
and effort (or the cost) involved in the extraction and clean up of the sample
if soil is used as the degradation-medium is generally higher. A detailed
biodegradation study, is sometimes performed using pure cultures of micro-
organisms. The cost Involved in the intensive study is the same regardless
of the environment the pure cultures of microorganisms are isolated from.
The cost estimate for detailed biodegradation study in the aquatic environment
»..
has been described in Section 111., p. 21$ and these estimates will also be
applicable for detailed study in the soil environment. The cost estimates for
preliminary, soil biodegradability tests are given in Table 31.
280
-------
table 31. Cost Analysis for Preliminary Blodegradabillty Test in
Soil Environment
4)lud«»r..d
.'hjiealcal
alinn Oiit Method Analytical Method
under tftatlnnary
i in
(20)
.-'ojll Incubated with ceat Radioactive material
'cunditlona '(1) parent material)
Soil [ncu
' cheraU.il
condit Ion
(20)
bated with teat Radioactive material
a (1) of lt*C02i e.g. , in a
biometer flask).
(20)
Equipment Naadad Equipment Coat
and Their Colt for the Ta.sc
Caa chromacograph 23
$8000; Centrifuge
$1200
1JO
Scintillation 30
counter, llO.OMi
213
Scintillation 15
100
Chamlcala,
dataware and Labor Coat
Nlac. Supplies « »60/d«y
7} 300
800 1800
900
(Includes labelled 300
material)
12000 2100
(Includes labelled
material)
950 180
(includes labelled
material)
13000 1200
(Includes labelled
material, biometer
flaaka)
70 1BO
Omrhead
12St of
Professional
Servtcae
' 375
2250
375
2625
300
1500
225
Total Coat
775
5000
1605
16950
1510
15800
475
Cnsi/Cnn
7)5
250
1605
650
1510
790
475
chemical under stationary
conditions (1)
(20) "
Soil suspended In Gaa chromatography
aqueous solution (1)
(20)
.'Soil suspended In Radioacctve material
'Squeous solution (1) (meaeure jloea of
. : parent coapound)
'20) "
Soil suspended in ' ladloactive material
of "COj)
(20)
Soi 1 suspended In U.V. Absorption
aqueous solution (1)
(20)
Soil Auspcnded in Colorlmetrlc
aqueoua solution (1) (e.g., chloride
ion release)
(20)
Soli porfuaion test (1) Colorlmetrlc
(e.g. , chloride
release)
(20)
.v./ll perfuslon teat (1) Gas chrooatography
(20) "
Reproduced from |Pj|
best available copy. TJjjjfj?
-
Caa chroaacograph, 25
$8000; Centrifuge,
$1200.
11 127
Scintillation 30
counter, $10,000;
Centrifuge, $1200.
18J
Scintillation 20
i 100
Speetrophotooeter, 0
$3,000; Centrifuge,
$1200.
' " «0
Spectrophotomatar, 12
$3,000; Centrifuge,
$1200.
" 60
Spactrophotometer 28
93,000; Centrifuge,
91200.
11 200
Gaa chronaeograph, SO
98000; Centrifuge,
$1200
" 305
281
500 1200
75 300
800 1500
750 300
(includes labelled
material)
12000 1800
(Includes labelled
materiel)
800 240
(Includes labelled
material, biometer
eeaambly)
13000 1200
(Includes labelled
material, biooater
flaaka or gaa train
aaeembly)
50 120
300 600
75 180
400 900
100 420
800 3000
100 600
1200 3600
1500
375
1S75
375
2250
100
1500
ISO
750
225
1125
525
3750
750
4500
3200
775
4 inn
1455
16235
1360
15800
328
1690
495
2485
1073
7750
1500
9605
160
775
7,5
1455
810
1360
790
328
85
495
125
1073
388
1500
480
-------
V. PHOTOCHEMICAL AND CHEMICAL ALTERATIONS
A. Degradation of Chemicals in the Atmospheric Environment
1. Introduction
Atmospheric -transport is a major route for the distribution of
chemical contaminants of the environment, (Risebrough e_t ail., 1968; Risebrough,
1969; Stanley e£ al., 1971; Compton e_t al, 1972; Wood, 1974). In addition,
for many of the prominent air pollutants (e.g., SO , NO , CO, hydrocarbons,
X X
particulate matter, photochemical smog, etc.), the atmospheric route provides
the predominant route of exposure; to man, and other biota. Thus, an
understanding of the atmospheric chemistry of these contaminants is extremely
important to considerations of their persistence and fate in the environment.
Unlike the alterations discussed in other sections of this report, atmospheric
alterations of chemical contaminants are entirely non-biological.
Substances emitted to the atmosphere, as with other medium, may remain
as the parent compound or be converted to more or less objectionable products.
The variety of physical and chemical Influences on a chemical contaminant is
extremely complex and reviews of the reactions and mechanisms of the assimila-
tion process are presented elsewhere (Haagen-Smit and Wayne, 1968; Altshuller
and Bufalini, 1971; Leighton, 1961). Presented here will be only a brief dis-
cussion of the unique conditions to which a contaminant is exposed in the
atmospheric environment and the experimental procedures that are used to study
a chemical's reactions in the atmosphere.
The atmospheric media provides an excellent matrix for photochemical
or oxidative alterations of chemical contaminants. The intensity and wavelength
(>290 nm in the lower atmosphere) of sunlight are capable of initiating
Preceding page blank
283
-------
a variety of reactions. For example, the energy provided by sunlight is able
to break carbon-carbon and carbon-hydrogen bonds, cause the photodissociation of
.(nitrogen dioxide to nitric oxide and atomic oxygen, and photolytically
excite a relatively large number of oxygen molecules due to their high
concentration. This relatively high concentration of oxygen (20.9% V/V)
makes it one of the most; important participants in various reactions with
air pollutants since the^ates of reactions are concentration dependent (Haagen-
Smit and Wayne, 1968). Similar reasoning can be used for reactions with
water vapor (0.1 - 5% y/V) and carbon dioxide (.03 - 0.1% V/V).
Conditions in the upper atmosphere are even more vigorous. Above
heights of 50 miles, oxygen exists almost exclusively in the atomic form* .
Between 10 and 20 miles above the earth a region of high ozone concentration
exists. Above this level the energy of the incident sunlight is much higher
(<290 nm) because the ozone level is not available to absorb the high energy
iigtitS It is generally believed that, through turbulence and diffusion,
contaminants from the lower atmosphere will be exposed to the upper atmos-
phere" and this may be a major sink for environmental contaminants.
Physical processes may also be important to both fate and the
i
residences time of chemicals in the atmosphere. Adsorption of substances
on ^psrticulate matter and subsequent gravitational settling appears to provide
a major'source of pesticide (and presumably other chemicals) removal from
the air (Risebrough £t ajL. , 1968). A "washout" mechanism caused by falling
drops of rain may also be important, especially for water soluble materials.
In addition, the adsorption of chemicals on participate matter may drastically
effect the photochemical reaction rate (Klein and Korte, 1971).
284
-------
Thus, the atmospheric conditions that need to he simulated in the
laboratory for the evaluation of a chemical's persistence and fate are
extremely complex. A variety of approaches have been utilized.
2. Techniques Used for Determining Atmospheric Degradation
Most studies of atmospheric chemistry ha"ve concentrated on
processes which have physiological or toxicological significance. These
have included processes important to smog formation, eye irritation, oxidant
levels, etc. For example, the reactivity of hydrocarbons are often studied
in relation to the rate of oxidation of nitric oxide to nitrogen dioxide,
an important first step that results in photochemical smog formation. Rarely
is a chemical species studied just to determine its chemical reactions in
the atmosphere. The interpretation of results varies considerably, depending
upon whether one is evaluating long-term (e.g., trace environmental contam-
ination) or short-term phenomena (e.g., smog formation). For example,
reactive hydrocarbons in the atmosphere are considered extremely undesirable
because of their contribution to the formation of smog. This is analagous
to dissolved oxygen depletion in water by high BOD compounds. The techniques
which are discussed in this report are considered mostly for their ability
to assess a chemical's persistence and fate rather than to predict air
pollution consequences.
a. Long-Path Infrared Cells
Long-path infrared cells have frequently been used to study the
atmospheric photooxidation of hydrocarbons and nitric oxide. Stephens and
coworkers (Hanst et_ _al., 1956; Stephens, 1958) were among the first to use
285
-------
long-^path infrared spectroscopy (LPIR) in the study of air pollution chemistry.
The apparatus they used consisted of a 3 meter, 18 inch diameter stainless
steel tube which contained a three mirror multiple-reflection system. The
total path-length was 216 meters (72 passes). The cell was connected to a
single beam infrared spectrometer.
i
Tuesday and Glasson (Tuesday, 1963; Glasson and Tuesday, 1970a,
1970b, 1970c, 1971) used a similar 3 meter stainless steel long-path cell,
but with only 40 passes (total path-length 120 meters). Irradiation was
supplied by a number of eight-foot black light fluorescent bulbs (F96 - 8/BL)
*.
mounted inside the long-path cell. The cell was attached to a modified
Perkin-Elmer Model 21 Infrared Spectrometer. A full description of the
apparatus is given by Tuesday (1961).
The experimental procedure consisted of first determining the
actinic light intensity of the lamps by measuring the rate of nitrogen dioxide
phbtodissoclation at very low concentrations in nitrogen gas. Because the
quantum yield is close to unity over the spectral region of interest, this is a
ecaambri method of determining the light intensity in order to compare it to
the intensity of sunlight (Tuesday, 1961). In all of Tuesday and Classen's
work the light intensity was 0.27 min.
/"''' (N02) init. 1
Kd'XN02) = In (N02> final = rate min"
The long-path cell was evacuated to a pressure of less than lOy Hg,
and known pressures of nitric oxide and nitrogen dioxide were expanded into the
cell from an attached glass vacuum system. Hydrocarbons with high
286
-------
vapor pressures at room temperature were also expanded into the cell from the
glass vacuum system while hydrocarbons with low vapor pressures were added
by a I0-pl syringe to an electrically heated stainless steel manifold con-
taining several high vacuum values and a silicone rubber septum. The hydro-
carbon was expanded from the manifold into the cell and the manifold was
flushed with nitrogen. Nitrogen was then added to the long-path cell to about
600mm of Hg. After the addition of oxygen (155 mm Hg), the final pressure
was brought to 760 mm. of Hg with a small amount of nitrogen. The concen-
trations of hydrocarbons in most cases varied from 0.25 to 5.0 ppm, wl.th
/>.'.',
initial concentrations of nitric oxide and nitrogen dioxide of 0.38 ppm and.
0.02 ppm, respectively. Quite often these concentrations were varied when.tne
investigators were studying the effect of nitric oxide concentration on hydro-
carbon photooxidation or vise versa. These concentrations are well within
those existing in polluted atmospheres [3 to 5 ppm hydrocarbon expressed as
ppm carbon and 0.2 to 0.6 ppm nitrogen oxides (Korth et al., 1964)]. The
amount of thermal oxidation of nitric oxide in the cell prior to irradiation
was less than 1% at the concentrations used (Glasson and Tuesday, 1970a). The
concentrations of nitrogen oxides, hydrocarbons and photochemical products
were measured by scanning the infrared spectra.
A more recent study used LPIR for an assessment of the
effects of fluorocarbons on the phenomena of photochemical smog (Japar
Q al., 1974). These researchers used two (reference and sample) standard
long pathlength cells which were internally coated with FEP (fluorinated
ethylene-propylene) Teflon in order to decrease any effects due to
wall reactions. The cell optics were adjusted to obtain a total
pathlength of 40 meters. Irradiation was supplied by an external
287
-------
17.5"'arc-length, 1200 watt Hanovia medium,pressure mercury arc which was
surrounded by a double-walled quartz jacket cooled with distilled water.
The light passed through six 3" x 3" irradiation ports placed along the
length of the cell. The port consisted of I/A" thick glass which passed
only wavelengths longer than 310 nm. The actinic intensity of the lamp
measured as nitrogen dioxide dissociation in nitrogen was 0.131 min.
This-intensity was noted to be comparable to a Los Angeles noontime
measurement of 0.37 min.
The reaction mixtures were expanded into the LPIR:cells from
an attached glass vacuum system. Concentrations of fluorocarbons, nitrogen
dioxide and olefin varied from 5 to 8 ppm, 10.5 to 12.5 ppm and 15 to 34 ppm,
i
respectively. The-LPIR cells were pressured to slightly less than 1 atmosphere
with compressed air which was passed through a Matheson #450 Gas Purifier
and a 3" diameter; 2.5.' Dierite column to remove any oil and most* of the
water present In the air. Concentrations of the fluorocarbon, olefin, and
nitrogen dioxide were measured by scanning the infrared spectra before,
.T
during, and after irradiation through sodium chloride windows in the LPIR
cells.
The Environmental Protection Agency at Research Triangle Park,
N.C. (Bufalini, personal communication) is presently using a 30 ft. long (six
sections) LPIR made of pyrex glass with Teflon joints. Fourier transform
infrared spectrometry is used which allows computer comparisons of results
oM instantaneous determination of the spectra. Concentrations as low as ppb
levels can be used. The fluorescent lights are mounted on the outside of the
rauction chamber.
288
-------
b. Plastic Containers
A popular method for studying the atmospheric chemistry and
photochemistry of both artificial and natural atmospheric samples is the use
of plastic containers. The. common bag material is either FEP (Fluorinated
ethylene-propylene) Teflon, Tedlar or Mylar plastic.
Saltzman and coworkers (1966) used this techniques to
evaluate the stability of sulfur hexafluoride, bromotrifluoromethane, and
octafluorocyclobutane for use as meteorological tracers. The gas being
evaluated (SF6 - 3 to 8 ppb; BrCF3 - 35 to 100 ppb; C4Fe - 100 to 500 ppb)
was put into the bag with air from a cylinder and small amounts of some of
the common reactive pollutants of the atmosphere (H20, 03 - 5.9 ppm,
S02 - 5 ppm, automobile exhaust 2.8% and H2S - 5 ppm)were added. Sufficient
water to provide a saturated mixture was injected into the bag before addition
of the gas mixture. Irradiation studies were carried out with fluorescent
black-lights (GE F42T6BL) with 100-liter FEP bags (good transparency in the
ultraviolet region). For studies where ozone was incorporated, Mylar bags
were used. A control run without the pollutants showed that the gases were
slowly lost and this was attributed to diffusion through the 0.002 thick
plastic film. No decomposition within experimental error was reported for
any of the tracer gases. The possibility of washout by rain fall was cheeked
by placing a bag saturated with water vapor into a 3°C refrigerator. The
cold air sample was then drawn into another bag and analyzed. Analysis in
all cases was provided by gas chromatograph (no preconcentration) with an
electron capture detector.
289
-------
Altshuller et al. (1967) used FEP plastic containers to study
the phbtooxidation of hydrocarbons in the presence of aliphatic aldehydes. The
reaction mixtures of olefin and aldehyde diluted with, air (150 liters) were
irradiated with sunlight-fluorescent lamps (maximum intensity at 310 nm) and
I .'.."
with natural sunlight. Rates of decomposition of the olefin were determined
by gas chromatography using a flame ionization detector.
Kopczynski and coworkers (Altshuller, ej^ al_. t 197Qb; Kopcznyski
et al., 1972) have used plastic containers to measure photochemical reactions
of actual atmospheric samples. In the more recent study, they used atmospheric
,«
samples collected in a 300 liter Tedlar bag in the early morning (particulates
not removed). Irradiation with sunlight was begun as soon as the sample was
collected. Duplicate samples were run and a comparison was made between full
sunlight and 58% attenuated sunlight. An important step in the procedure
was"the conditioning of the bag by irradiating an air sample the day before
an experimental run.. Analysis of the hydrocarbons was provided by low
temperature gas chromatography using a preconcentrated sample and flame
ionization detector(GG-FI).
Gay,and Bufalini (1971) also used plastic containers in their
study of nitrogen balance of irradiated hydrocarbons in the presence of oxides
of nitrogen'.- They used a 100 liter FEP Teflon bag irradiated with GE-F42-T6
black lamps with an energy maximum at 366 nm.
c. Glass Flask Reactors
A number of researchers have used glass flask reactors to study
atmosptieric chemical reactions. The size of the reaction vessel has varied
n? 100 ml spherical vessels to 72 liter borosilicate flasks.
290
-------
Wei and Cvetanovic (1963) In a study of the vapor phase reaction
of ozone with olefins used two matched 100 ml spherical reaction vessels made
of pyrex glass. These were incorporated into a conventional high vacuum
line. The reaction vessels were connected to a gas chromatograph in order to
facilitate analysis. A somewhat similar apparatus was used by Wiebe et al.
(1973) to study the photolysis of methyl nitrite in nitric oxide, nitrogen
dioxide, and oxygen. They used a 500 ml spherical pyrex flask connected in
a vacuum line. Irradiation was supplied by a mercury arc lamp (pyrex filters
out light below approximately 300 nm). The reaction mixture was analyzed
by a mass spectrometer which was connected by a capillary tube to the
reaction vessel. Relatively high concentrations of reactants were used
(03-2 mole%; olefins - 20 mole%). The same apparatus was used by Shortridge
and Heicklen (1973) to study the chain mechanisms important to the photo-
chemical conversion of NO to N0£. These authors used a quartz vessel and
a variety of light sources including a low pressure mercury lamp (mostly
253.7 nm). The reactions were studied at low pressure (25 torr) and high
reactant concentration.
Gay and Bufalini (1971) used two different sized borosilicate
flasks as well as a 100 liter plastic bag to study the nitrogen balance of
irradiated hydrocarbon and oxides of nitrogen mixtures. A 22 liter boro-
silicate flask, having a silvered outer surface was used to determine if
molecular nitrogen is a product of photolysis. A mercury lamp (Hanovia
//679A36) fitted in a double-walled, water cooled, immersion well was used
as a light source. Initial concentrations of 60 ppm ethylene and 15 ppm
N02 in helium or oxygen were used. A similar apparatus, although equipped
291
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with a quartz immersion well (Corex filter-cut off at 260 nm), was used
by Bentley e^ al. (1972) to study the photolysis of dimethyl sulfide in
air (68 tng/1). These conditions were used to determine the applicability of
photochemical methods of controlling odor problems in the paper industry.
The larger borosilicate flask used by Gay and Bufalini (1971)
was a 72 liter flask. This reaction vessel was placed inside an irradiation
chamber described by Altshuller and Cohen (1963). Irradiation is provided
by GE-F42-T6 black lamps with an energy maximum at 366 nm. The light
intensity of the system measured in terms of nitrogen dioxide dissociation
was 0.4 min. Before each experimental run, the flask was washed with
aqueous cleaning solution, 300 ml of acetone, and large amounts of distilled
water. The flask was allowed to dry and then equipped with a large magnetic
Teflon-finned stirrer and a top with several glass and Teflon lines which
served as inlets and exhausts, some of which were connected to gas chromato-
graphs. The flask was then evacuated and refilled with air and the pollutants
to be studied. The relative humidity of the tank air was 5%.
Bufalini e_t'al. (1972) compared the 72 liter borosilicate flask
i .
to a 335 ft smog chamber (Rose and Brandt, 1960) in a study of reasons for
poor interlaboratory comparisons of various smog chambers. The 72 liter
-flask was irradiated in the chamber described above with different light sources
{GK-F-40-BLB blacklights, N02 K^O.50 min"1 and Westinghouse FS-40 sunlights,
d
SSO^Kj = 0.14 min .) The rate of tetramethylethylene photolysis and ozone
iaftaation from biacetyl was studied in "clean" and "dirty" flasks. The dirty
flasks were ones that were not cleaned with dilute sodium hydroxide solution
.-before"-each run.
292
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Laity and Maynard (1972) studied the reactivities of gasoline
vapors with three experimental apparatus: (1) 400-liter stainless steel
chamber, (2) a 235-liter glass vessel, and (3) a 23-liter glass flask. The
large glass vessel is a capped, boroailicate-glass, spherical vessel which is
placed in a metal box for irradiation. The source of irradiation in all
cases is blacklight fluorescent lights. The light intensity is 0.4 min~
(K, N02, 2 ppm in N ). Before each run, the reaction vessels are cleaned by
-2 -3
ozone treatment and evacuation (10 to 10 torr, 36 houra, ^ 60°C).
Stephens and coworkers (Stephens, 1973; Stephens and Burlenson,
1967) have used 20 and 50 liter borosilicate carboys to irradiate ambient air
samples to study hydrocarbon reactions in air. Irradiation was carried out
with both natural sunlight and artificial light (blacklight fluorescent lamps
N02 K, 1/2 life =1.3 min) using a static technique with the 20 liter carboy.
When studying aromatics, where the loss of reactant by absorption on the wall
is possible, Stephens (1973) used a stirred flow dilution technique. With
this technique the air sample in the 50 liter carboy is continuously diluted
with dry, hydrocarbon-free, make-up air during the irradiation. No
difficulty with adsorption of aromatics was noted. Analysis was provided by
gas chromatography combined with a trapping technique in order to make 0.1 ppb
detection possible.
A 72 liter borosilicate glass flask of special design has
been used by Crosby and Moilanen (1974) to study the vapor phase photolysis
of aldrin and dieldrin. The apparatus is depicted in Figure 32. The flask
is painted dull black on the outer surface . The light from the lamp ( E in
2
Figure 32) is passed through the inside of a 59 mm id (27 cm in area)
borosilicate glass tubing painted dull black on both the inner and outer
293
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surface. The light exits through an unpainted circular area (c) 15 cm in
diameter. The removable light trap (D) could be replaced with a 1 liter
spherical flask silvered on the interior. A 3 mm-thick borosilicate glass
disc (b) prevents escape of vapor and filters out UV wavelengths below
300 nm. Inlet and outlet tubes are used only as vents. The lamp is either
a 100W low-pressure mercury lamp (254 nm filtered out, 365, 410, and 440 line)
or a 275W RS-sunlamp. In the procedure, the compound to be studied was formed
into a thin solid film (hexane evaporation) on a watch glass which is then
placed into the bottom of the reaction chamber and warmed to 35°C. After
Figure 32. Schematic Diagram of Vapor-Phase Photoreactor
(Crosby and Moilenen, 1974)
Courtesy of Springer-Verlag
allowing the chemical vapor to evaporate into the chamber (several hours), irrad-
iation is begun. After irradiation for an arbitrary period (45 - 168 hours), the
reactor is dismantled and washed with hexane. By comparing results with and
294
-------
without the mirror (varying the light impinging on the inner surface of the
reaction chamber), the effect of wall reactions, if any, could be determined. '
A completely dark control is also run in order to provide evidence that
non-photochemical reactions are not taking place.
Laity (1971) also used a spherical 22 liter borosilicate glass
flask fitted with a glass cap and containing an all-glass magnetically driven
stirrer to compare results using blacklight fluorescent lamps with results
using natural sunlight.
Urone e£ al. (1968} used a 2-liter reaction flask in the study
of sulfur dioxide reactions in air. A mixture of sulfur dioxide tagged with
S35(>2 in clean, dry air was prepared in 20-liter Mylar bags. These were
then transferred to one or more calibrated, 2-liter quartz or borosilicate
flasks. Triple-distilled water was added to the flask with a 50 \ii syringe
to obtain the desired relative humidity and the flask was kept in the dark
overnight. Just before irradiation nitrogen dioxide was added to the flask.
Irradiation was provided by a bank of 30 x 1.5 cm intermediate pressure
ultraviolet lamps, coated to give a spectral distribution from 310 to 420 nm
with 90% of the intensity near 350 nm. The intensity was measured by potassium
ferrioxalate actinometry and found to be "about seven times the noonday
sunlight in the same wavelength region." The rate of sulfur dioxide reaction
was measured in the presence and absence nf hydrocarbons and inert solids,
such as sodium chloride.
295
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d. Smog Chambers
There are three major drawbacks to the previously mentioned
j
experimental apparatus for studying atmospheric reactions: (1) their small
size allows the possibility of heterogenous reactions, (2) ratio of volume
to surface allows significant radical termination at the walls, and (3) in
some cases the contaminant concentrations have to be unrealistically high
in order to provide analytical precision (Doyle, 1970). In order to reduce
these difficulties, many researchers have resorted to large photochemical
''smog chambers," so named because of their unique capability in studying the
.phenomena of photochemical smog. The large size of the chamber also allows
for simultaneous evaluation of physiological effects such as eye irritation
and plant damage.
Most of the smog chambers reported in the literature can be
operated in a dynamic or static mode. The dynamic procedure requires the
continuous introduction of reactants and is though! to more closely simulate
,the dynamic characteristics of the atmospheric environment. It is particularly
suited to studies which required large analytical samples. The static procedure
consists of batch introductions of reactants into the chamber and because of
its simplicity and precision, it is more predominently used (Dimitriades, 1967).
(i) Rose and.Brandt Smog Chamber
One of the earliest chamber facilities reported in
the literature and one still used by EPA (Bufalini et al.,
1972) is the enviromental irradiation test facility of Rose
and Brandt (1960). The facility consisted of five major
components: a dynamometer unit, a dilution air purification
unit, exhaust dilution assembly, irradiation
296
-------
chambers, and an exposure facility to evaluate air pollution
effects of the irradiated pollutants (See Figure 33). The
dynamometer unit ran an internal combustion engine in a way
representative of various driving patterns. The exhaust gas
was diluted with a clean air supply (cleaned with particulate
and charcoal filters) which was maintained at temperatures
between 60 and 100°F and at a relative humidity from 35% to.
90% by employing a refrigeration air conditioning system. The
hydrocarbon concentration was several ppm. The two irradiation
chambers, which could be operated in series or parallel, were
constructed of aluminum with Mylar windows and had a volume of
335 ft3 each.
AUTOMOTIVE VARIABLES:
1. ENGINE TYPE AND CONDITION
2. DRIVING CYCLE
1. FUEL COMPOSITION
41 FUEL AND OIL ADDITIVES
i. IRRADIATION WTENSmr
*. imUOIATWNTIME
4. CHAMBER TEMFMMTURE ANIMMMIDITY ,
6. DILUTION RATIO ''
EFFECTS: :; >
t. CHEMICAL COHKWT1W .
2. VEGETATION DAMAOI
3. EYE IRRITATION ,
4.'VISIBILITY REDUCTION
». BACTERIA EFFECT I
«. ANIMAL EXPOSURE
Figure 33. Environment Irradiation test Facility
(Rose and Brandt, 1960)
Courtesy of Air Pollution Control Assn.
297
-------
Circulation in each chamber was maintained by two tube-axial
fans. The chambers were designed to approach ideal dilution
performance (concentration of output gas would be representative
t
of the entire volume). The average leak loss from one chamber was
0.004 cubic feet/min (cfm). Irradiation was applied to each chamber
by two. banks of 70 fluorescent tubes (black, warm white, and blue)
in order to simulate solar irradiation from 290 to 450 nm. The
exposure time was varied by changing the flow rate (20 to 100 cfm).
When Bufalini and coworkers (1972) used the chamber, they
determined the light intensity to be 0.40 min in terms of
NO- dissociation. They studied the oxidation of nitric oxide
in the: chamber and noted an oxidation rate much faster than
theoretical which they attributed to chamber wall contamination.
(ii) Wayne and Romanovsky Smog Chamber
3
Wayne and Romanovsky (1961) used a 1000 ft chamber in a
study of the photboxidation of automobile exhausts using six
different fuels. A dynamometer apparatus similar to that of Rose
and Brandt (1960) was used to generate the exhaust gases. The irra-
diation chamber was constructed of glass and held together with an
aluminum frame. Forty-eight 400 watt mercury-arc lamps externally
mounted provide irradiation through pyrex windows. An internally
mounted fan provides rapid mixing of the entering materials.
(iii) Korth, Rose and Stahman Smog Chamber
Korth. and coworkers (1964) in studies conducted at the
Taft Sanitary Engineering Center again used a five component
»i
facility in a dynamic study of automobile exhaust gases.
298
-------
3
The Irradiation chamber is a 335 ft vessel, presumably the
same as used by Rose and Brandt (1960). However, the light
intensity was increased (present configuration, Figure 34)
to be more representative of sunlight in the Los Angeles area
at a zenith angle of 20 degrees (sunlight curve #2, Figure 34).
CHAMBER LIGHT ENERGY
1400
PRESENT
CONFIGURATION
PREVIOUS
CONFIGURATION
""^-SUNLI
CURVE**
2800 3000 3200 3400 3600 3800
WAVELENGTH (ANGSTROM UNITS)
4000
Figure 34. Chamber Light Energy for
Korth et^ al. (1964) Chamber
Courtesy of Air Pollution Control Assn.
299
-------
Altshuller and coworkers (Aitshuller &t_ al., 1969a,b; 1970a)
have made extensive use of the Korth e_t al. (1964) chamber.
Although a few experiments have been conducted under dynamic
conditions, most of their experiments have used a 6 hour,
static-irradiation period. In the 1970 study, a temperature
of 31 ±1° and relative humidity of 50% was maintained. These
authors studied the photochemical reactivities of paraffinic '",
and aromatic hydrocarbons with low concentrations of nitric
oxide. Kuntz et al. (1973) used the chamber to study the
photoreactivity of benzaldehyde-NO- and benzaldehyde-hydrocarbon-
NO mixtures. They cleaned their make-up air by passage through
activated charcoal and particulate filters.
(iv) Bartlesville Petroleum Research Center Smog Chamber
Two irradiation chambers with volume capacities of 64 and
3
100 ft have been used for air pollution research by the
Bartlesville Petroleum Research Center of the U.S. Bureau of
Mines (Dimitriades, 1967). The 64 ft chamber was constructed
so that the gaseous contents only contacted aluminum and a
t
5 mm Teflon film. The Teflon film was preferred over the
previously used Tedlar (polyvinyl fluoride) because it is less
permeable to water vapor and more transparent to ultraviolet
light (see .Figure 35). The chamber is equipped with nine
sampling ports and is usually run using the static procedure.
There are two fans mounted inside for internal circulation.
The 100 ft chamber was similar to the 64 ft3 chamber except that it
300
-------
100 MS IK) IA (10 US 210 Z» MO HO MO 1TOU02»OMO HO MO 3T0400
Figure 35. Spectra of Teflon and Tedlar Films
in the UV Region (Dimitriades, 1967)
Courtesy of Air Pollution Control Assn.
had pyrex windows. The ozone half-life time at 1 ppm in the
email and large chambers with the lights on was 3.6 hr. and
6 hr., respectively. The irradiation system consisted of a
number of fluorescent lamps designed to produce a light
intensity similar to natural sunlight. The Kd N02 was
0.38 min . In order to provide analytic samples, 0.7 liters/min
was withdrawn from the chamber and replaced with pure N^
(dilution rate 2.3%/hr). Using this apparatus, Dimitriades
(1967) studied the effects of background reactivity (Chamber
reactivity), temperature, and humidity on hydrocarbon-NO
. X
reactions.
e.
301
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(v) Stainless Steel Chambers
The General Motors smog chamber used by Heuss and Glasaon
(1968) and the smog chamber used by Laity et al. (1973; Laity &
Maynard, 1972) were both, constructed of stainless steel. The GM
chamber consisted of a cylinder 9 ft. in diameter and 5 ft. high with
3
an internal volume of 298 ft . Spaced symmetrically throughout
the chamber were 19-vertical borosilicate glass tubes which
contain the irradiation lampsj the K. NO_ was 0.4 min~ .
'The chamber used by Laity e_t al. (1973) consisted of
a polished stainless steel cylinder, 4 ft. long and 2 ft. in
3
diameter (397 liters-14 ft ) with blacklight fluorescent lamps
.mounted inside. Temperature was controlled by circulating
liquids .around .the chamber and between experiments the chamber
was cleaned by evacuation and heating (50 - 60°C) overnight.
The half-life of. ozone (1 ppm) under irradiation varied from
1.3 to 2.5 hours and the intensity of the light was 0.4 min
(Kj N02) . The results from the chamber were compared to
results using two glass vessels (235 I and 23 I ). The
following experimental conditions were used: 5 hrs. irradiation,
temperature 32°C, ^ 1 ppm test chemical, 0.6 ppm nitrogen oxides
(0'.57 ppm NO, 0.03 ± 0.02 N02) and 20% relative humidity.
(vi) Stanford Research Institute Smog Chamber
The SRI smog chamber reported by Doyle (1970) is an
elaborate facility. The photochemical reaction chamber is
mainly constructed of rolled sheets of 7/32 inch Pyrex
302
-------
(transparent down to 300 nm) with some cast aluminum sheet
panels. All Interior surfaces are coated with Teflon. Sonic
Jet-type pumps are used to stir the chamber contents (avoids
difficulties from Impellers). The chamber configuration depicted
3 3
In Figure 36 has an internal volume of 7.6 m (269 ft ).
Irradiation is supplied by fluorescent lamps uniformly dis-
tributed above the exterior of the chamber. The number and
types of lamps were chosen to simulate sunlight intensity from
300 to 600 nm. The longer wavelengths were simulated because
tOHIC PUHP5
MOUNTING
rnaotr
linrnr
COUCJION toil
CHAMBER VOLUME: 7.6 CUBIC METERS
SURFACE-TO-VOLUME RATIO. 4.4 i m'1
(SURFACE AREA. JJ.BitT1)
Figure 36 * SRI Smog Chamber
(Doyle, 1970)
Reprinted with permission from
Environ. Sci. Technol., 4_,_ 907.
Copyright by the Amer. Chem. Soc.
the chamber was designed to study compounds that might abvorb
in that region. The leakage rate from the chamber was 1.5%/hr.
and the ozone half-life in the Irradiated chamber was 6.7 hr.
i
The temperature can be controlled down to 27°C with the lights
on and the make-up air is humidified to the desired level
(usually 35% at 1 atm. and 25°C). Sample ports are built in
303
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the 'chamber for destructive analysis and the optics of a LPIR
(40 m path length) are inserted into the chamber through a
Teflon-film diaphragm, thus allowing non-destructive analysis.
A major effort was made to purify the make-up air. This
was, .accomplished using catalytic combustion over platinum at
I
elevated pressure (5 to 8 atm.) and temperatures (600°C). The
I
pure air supply had the following analysis: <0.5 ppm CO,
0.1-0.3 ppm NOx, and some organic material as analyzed by
GC-FI. An 8 hr. irradiation of the pure air yielded no
detectable oxidant concentrations and little change in the
nitrogen, oxide concentrations.
f
(vii) Battelle Memorial .Institute Smog .Chamber
, ' Wilson e£ al. (1972a,b) have reported the use of the
Battelle 610 ft smog chamber made of polished aluminum with
Teflon FEP window for the study of a variety of pure organic
compounds (ct-pinene, cyclohexene, 1-heptene, and toluene).
The initial conditions were the following: temp. 88-90°F,
relative,humidity .39-43%, hydrocarbon-- 10 ppm, NO - 0-1 ppm,
and NO2 -- 1-2 ppm.- . Analysis of reaction products was provided
by gas chromatography interfaced with mass spectrometry (GC-MS)
Both the SRI smog chamber and the Battelle smog chamber
have been used in a-comprehensive study of air pollution
i
problems resulting from organic solvent use (Levy, 1973).
304
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3. Analytical Procedures
Analytical procedures have considerable influence upon the reaction
conditions used and the results obtained in studies of chemical reactions in
the environment. If the sensitivity of the method is low, it may require
unrealistlcally high concentrations or large samples for analysis. Measure-
ment of the reactivity of hydrocarbons and solvents determined by NO loss,
N02 formation, oxidant formation, hydrocarbon loss, eye irritation, or plant
damage can give drastically different results, This section briefly discusses
and evaluates the analytical techniques that are used most commonly in
atmospheric studies.
a. Long-Path Infrared Spectrometry
When LPIR cells are used as reaction vessels, or when smog
chambers are equipped with LPIR windows (t)oyle, 1970), the. concentrations of
the reactants and products are followed by scanning the infrared spectrum.
Theoretically any chemical that absorbs between 2 and 15 microns can be
monitored. However, in practice a compound must have sharp, high extinction
coefficient, diagnostic peaks which do not overlap other absorption bands and
this somewhat limits the number of chemicals that can be studied in any one
experimental run. These peaks will vary depending upon the chemicals being
studied. However, interfering peaks will in most cases be the same. Some
of the interfering peaks that have been reported are listed in Table 32.
305
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Table 32. Interfering Infrared Absorption Bands
from Background Contaminants or
Common Products
Carbon Dioxide
Water j
, *
Nitrous oxide
***
Nitrogen dioxide
**
Ozone
Peroxyacetyl nitrate
Formaldehyde**
**
Acetone
**
Photooxidation Product Bands
(isobutylene)
****
Acetaldehyde
*****
Methyl nitrate
* Stephens, 1958
- ** Glasson and Tuesday, 1970a
*** Glasson and Tuesday, 1971
**** Japar et al., 1974
****** Tuesday, 1961
****
Microns
2.9, 4.3, >15
2.9, 5.8-7.0, 8-9 some interference,
4.5 (can only be seen if C0»
is removed
6.15
9.5
8.6
3.6
8.2
7-9
3.7, 8.9
6.25, 7.75, 9.8, 11.72
Fortunately, many compounds have more than one diagnostic peak. For example,
Glasson and Tuesday (1971) have used the 6.5 micron peak to follow the
.'formation of nitrogen dioxide. The interference caused by water vapor
:<<;,absorption was corrected for by monitoring the water peaks at 6.4 or 6.8
;jj'Biierons. -However, with the six fluorocarbons that Japar et_ al. (1974) studied,
- Oft.Iy three compounds had sufficient diagnostic peaks to allow long duration
irradiations. Interferences from relatively inert contaminants (e.g.,
carbon dioxide) can be removed by using a double-beam (reference and sample
cell) infrared spectrometer.
306
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The sensitivity of the method Is dependent upon the effective
pathlength of the LPIR cell^ the Intensity of the incident infrared light, and the
extinction coefficient of the diagnostic peak. The effective pathlength of
the cell is limited by the reflection efficiency of the mirrors. Stephens
(1958) has demonstrated that the optimum pathlength is reached when the
energy reduced by reflection losses is the initial value. Glasson and Tuesday
(1970a.b.c) used a 120 meter path-leneth to measure low concentrations of nitrogen
dioxide (0.02 ppm) and hydrocarbons (>1.0 ppm). Japar e_t aJL. (1974) reported
an experimental error of ±1% (2% concentration error at 50% transmittance)
for fluorocarbons at 5 to 8 ppm with a LPIR cell path-length of 40 meters.
Bufalini (personal communication) is able to use ppb levels of hydrocarbons
with Fourier transform IR.
b. Gas Chromatographic Analysis
Since many of the compounds being studied are volatile organic
chemicals (e.g., hydrocarbons), extensive use of gas chromatography has taken
place. The simplest application of this technique is the direct injection of
the dilute air sample. Bellar et al. (1962) described a continuous sampling
system used with a dynamic smog reaction chamber. The system injected
1-cc volumes of air into a gas chromatograph with a flame lonlzatlon detector
(GC-FI) and had a detection limit of 0.001 ppm for low molecular weight
hydrocarbons in complex atmospheric mixtures. Direct analysis of aromatic
hydrocarbons in photochemically irradiated mixtures using a l,2,3-tris(2-cyano-
ethoxy) propane stationary phase with GC-FI was reported by demons et aj..
(1963). The limits of detection were as low as 0.01 to 0.1 ppm.
307
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Several researchers have found that direct injection gas chromato-
graphic analysis does not provide the sensitivity necessary (e.g., Altshuller
ejt al., 1970b and Kopczynski et^ al., 1972) and have resorted to preconcent ration
of the sample before analysis. Freconcentration or trapping techniques have
both advantages and disadvantages. The advantage is that the sensitivity moves
into the ppb range. The disadvantages include the need for larger air samples
(need larger reaction vessels) and the fact that the procedure is considerably
more time consuming. The technique usually consists of trapping the compound
£
of interest on some packing maintained in a "U" shaped cold trap. For analysis
the trap is connected to a gas chromatograph and the trap is warmed to release
the compound. Bellar et al. (1963) and Stephens and Burieson (1967) reported
o
a sensitivity as low as 0.1 ppb for 100-cm samples using the trapping technique
; with GC-FI. Jaffe and Smith .(1974), using a sampling loop, reported a lower limit
of detection of 0.03 ppm for acetaldehyde, total hydrocarbons, and propylene.
A-relative standard deviation of 10% is likely with this technique (APHA,
V1972, p 132).
Atmospheric samples have also been analyzed by gas chromatography
with electron capture detection (GC-EC) when the compound of interest is more
sensitive or more specific to electron capture. Peroxyacetyl nitrate (PAN)
is commonly analyzed with this type of detector. Kopczynski and coworkers
'(1972) report a sensitivity of 0.1 ppm using a preconcentration procedure
'With GC-EC. The detector is normally calibrated using a sample from a
->long-path infrared spectrometer cell which has been analyzed using reported
absorptivities. The electron capture detector is also used in the study of
halogenated compounds for which the detector is extremely sensitive and
.«spficific (e.g., 0.3 ppb for BrCF-; Saltzman e£ al., 1966).
308
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c. Colorimetric Analysis and Instrumental Methods
Colorimetrie methods are commonly uaed to analyze for nitrogen
oxides, aldehydes, and oxidanta in atmospheric reactors. The following
sections will discuss the analysis techniques used for each of these pollutants.
T.iese analytical procedures, because of their sensitivity, require relatively
. : 3
high continuous sampling rates (0.7fc/min or 2.3%/hr of a 64 ft chamber,
Dimitriades, 1967) which requires a fairly substantial initial volume.
Nitrogen Oxides
Nitric oxide (NO) and nitrogen dioxide (NO-) are the two nitrogen
oxides most frequently monitored because of their importance to the photo-
chemical smog process. Quantitative analysis of nitrogen dioxide is provided
by absorption in a mixture of sulfanilic acid, N-(l-naphthyl)-ethylenediamine
dihydrochloride, and acetic acid which forms an azo dye absorbing at 550 nm
(Griess-Saltzman Reaction) (APHA, 1972, p 329). A precision of 1% of the
mean can be achieved in the 0.005 to 5 ppm range with careful procedure, when
sampling is conducted in fritted bubblers (500 ml/min for 4 min. was used
by Altshuller and Cohen, 1963). Some small interferences are caused by SO-
(ten-fold ratio-no effect) , 0_ (five-fold ratio-gjaa.ll interference) , and PAN
(15-35% response for equivalent molar concentration to NO-).
Nitric oxide is analyzed by oxidation to nitrogen dioxide and
then quantitation by the colorimetric method described above, In order to
use this method, the background N0« must be chemically removed before the
chromic oxide oxidation of NO. The range of detection is 0.005 to 5 ppm
and the sensitivity is 0.01 ug/10 ml of absorbed material (APHA, 1972, p 325).
Nitric oxide can also be measured by chemiluminescence with sensitivities
in the low ppb (Jaffe and Smith, 1974).
309
-------
Aldehydes
Aldehydes are common reaction products and reactants in photo-
oxidative 'atmospheric reactions, and therefore are routinely analyzed in
atmospheric reactors. Formaldehyde is determined by a chromotropic acid
colorimetric method (APHA, 1972, p 194). A concentration of 1 ppm of
formaldehyde in a 25 £ sample can be analyzed by this method and therefore
the technique is only applicable for sizable reaction vessels. Similar high
volume sampling rates (22,/min) are necessary with a sodium bisulfide
collection systems to determine the following minimum concentrations.
Table 33. Minimum Concentration for
Sodium Bisulfide Collection Technique
1 (APHA, 1972, p 190)
Compound Quantitation Method Minimum Concentration
CH-0 Chromotropic acid 0.02 ppm
CH-CHO Gas chromatography 0.02 ppm
CH3CH2CHO " " 0.03 ppm
(CH3)2CHCHp " " 0.03 ppm
* CH- = CHCHO mercuric chloride-
hexylresbrcinol 0.01 ppm
Oxidants
The term oxidants can include ozone, nitrogen oxides, various
fjeroxy^compounds, and free radicals. The more specific methods, such as
-.-.,* ?- . .
infrared' and ultraviolet are not nearly as sensitive as the less discriminating
colorimetry (Cohen et^ aL^., 1967), The rate at which the compounds
varies and allows for different methods of analysis. The most commonly
weed methods utilize the oxidation of KI to iodine followed by amperometric
Of colorimetric techniques. As with the other colorimetric methods discussed
above, a relatively large sample is needed for sub-ppm analysis (150 ml/min)
310
-------
(APHA, 1972, p. 348). However, If continuous monitoring is possible, very
good accuracy in the 0.01-10 ppm range is possible (+5% from the mean).
Interferences from N02 and PAN may be subtracted out and SO,, interference can
be removed by filtering the air stream through chromic acid paper absorber.
However, other oxidants will react to liberate iodine so the term oxidants is
normally used rather than ozone. An alternative ozone specific technique is
reported by Jaffe and Smith (1974). They have used an ozone meter that relies
upon the chemilumlnescent reaction of ozone and ethylene and is accurate in
the low ppb (Jaffe, personal communication).
d. Mass Spectrometry
Because mass spectrometers are able to qualitatively deter-
mine reactions species, they have been used to analyze atmospheric reaction
mixtures. Wiebe e* .al. (1973) used a mass spectrometer directly connected
to the reaction vessel by a capillary tube to study the importance of NO and
N02 in scavenging CH-0 radicals. However, this direct interfacing of mass
spectrometers has been infrequently used. More common is the separation of
the compound of interest followed by mass spectral analysis.
Wilson et al. (1972b) used gas chromatography combined with mass
'spectrometry (GC-MS) in order to identify specific components of aerosols
formed from pure compounds in the Battelle smog chamber. The isolated acid
fraction of the aerosol was esterified with diazomethane and then injected
into the GC-MS. Analysis of the mass spectrum provided convincing evidence
of compound identification. Although the quantities used for analysis is
not reported, the amount of material obtained is dependent upon the amount
h
of particulate matter that is filtered. In a dynamically operated smog
chamber, this should not be a limiting factor.
311
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e. Bioassay
In studying atmospheric reactions physiological analysis is
often substituted for chemical analysis because of the poor correlation
between chemical analysis and biological affects Ce.g., "eye irritation does
not give a high linear correlation with any chemical measurement," Laity
e_£ jali', 1973). The two most commonly used physiological effects are plant
i .
damage and eye irritation.
. l '
For plant damage evaluation, pinto beans, tobacco and
petunias have frequently .been used to assess the phytotoxicity of gases
i
from irradiation chambers. In the plant exposure chamber used by Korth
et_ jil. (1964), the plants were uniformly exposed to the irradiated gas for
4 hours and the leaf damage was estimated on the third day (scale of 0-4).
Several different responses noted included time of collapse, age of tissue
affected, patterns of final injury, and microscopic changes in the progress
of'tissue collapse. Parameters such as soil type, nutrient content and
light intensity need to be controlled with this technique (Heck e_t .al. , 1969).
, An eye irritation evaluation facility consists of an opening
into-the reaction chamber up to which panelists can place their eye to
determine the irritability of the gas. The facility .used by Korth et al.,
(1964) consisted of glass manifolds where five of the ten panelists were
asposed .simultaneously. Activated charcoal respirators were used to
^separate odor response from eye irritation response. The degree of eye
-trsritation was rated on a scale from 0-3. The eye irritation ports designed
for £he SRI smog chamber fit tightly enough that a respirator was not
., considered necessary (Doyle, 197Q). Doyle 0.970} has noted that the necessity
for. simultaneous and uniform exposure in assessing eye irritation requires
large chamber facilities.
312
-------
4. Evaluation of the Techniques . ' .
a. General
The techniques that have previously been reviewed have, for
the most part, been used to study processes important to the formation of
photochemical smog. However, this review is concerned with techniques that
. .*
can .be used to study the persistence and degradation of chemical substances
in the atmosphere, and, therefore, the evaluation will be directed at this
limited question. Evaluation of the techniques for predicting photochemical
smog phenomena will be left for others to consider (e.g., Jaffe and Smith,. 1974)
"The natural atmospheric environment is a complex, highly
reactive system and its simulation requires the duplication of the photo-
chemical and secondary dark reactions involving organic and inorganic
substances" (Rose and Brand, 1960). A number of researchers have suggested
that in order to reproduce such a complex system in the laboratory some of
the following parameters must be controlled: temperature, humidity, purity
of the air supply, concentration of the pollutants, chamber design and opera-
tion, spectral distribution and intensity of the light, rate of exchange
between chamber and ambient atmosphere and reaction time (Rose and Brandt,
1960; Levy, 1973; Doyle, 1970; and Bufalini et al., 1972). The effects of
these parameters will be discussed in the following section.'
In atmospheric chamber studies, measurements of pollutant
disappearance, product formation, visibility reduction, particulate formation,
eye Irritation, and plant damage are usually recorded. For the purpose of
this review, the first two measurements will be considered the most relevant.
313
-------
b. Factors Affecting Degradation
v
(i) Spectral Distribution and Intensity of Light
In a comprehensive evaluation of the factors affecting
reactions in smog chambers, Jaffe and Smith (1974) compared
the 'reaction rates using a "full" spectrum configuration
(>290 nm, xenon arc lamp) to the rates using a "cut-off"
spectrum (>350 nm, 3/16" Plexiglass filter with xenon arc
.lamp)". The light intensity was kept constant for both
configurations (N02 K, = 0.3 min"~ ). Of the three independent
variables studied (chamber material, spectrum, and surface/
volume ratios), the spectral change caused the largest
variation; the "cut-off" spectrum clearly slowed the reaction
rate relative to the "full" spectrum.
,.In contrast, Stephens (1973) compared the irradiation of
an ambient air sample in a borosilicate carboy with natural
sunlight and blacklight fluorescent lamps (simulate sunlight
fairly well in the 300-400 nm range, see Crosby, 1969b). The
sunlight sample showed more reaction, but the relative reactiv-
ities of the hydrocarbons were very similar. This difference
in rate may be explained by the difference in intensity
(sunlight-brightest day, N02 half-life - 1 min; artificial
light, NO- half-life - 1.3 min) of the difference light sources.
Furthermore, Laity (1971) concluded that "the differences between
blacklight lamps and natural sunlight do not dramatically
influence photochemical smog formation with the systems
investigated."
314
-------
Kopcznski et al. (1972) determined the effect of light
intensity by irradiating an ambient air sample in a plastic
(Tedlar) bag with full sunlight and sunlight attenuated by
58%. They found that the ratio of losses for various hydro-
carbons comparing full sunlight to 42% of full sunlight was
quite different and concluded that on days of greater intensity
the less reactive hydrocarbons would participate more in
photochemical reactions in the atmosphere.
(ii) Concentration of Reactants
(a) Humidity: The concentration of water vapor in the
atmosphere being studied appears to have a large affect on
the reaction rates. Dimitriades (1967) demonstrated a con-
siderable fluctuation in the ethylene reactivity (1.65 ppm as C
in 0.50 ppm NO), as measured by rate of NO., oxidant, or
formaldehyde formation, when the relative humidity was varied
from 2.0 - 49.6% (77°F). He concluded that "the need for
humidity control in chamber operations is imperative"
(Dimitriades, 1967). In a study of the effect of water vapor
on the photooxidation of .ja-pinene, Ripperton and Lillian (1971)
observed that increasing the humidity decreased the net mean/
time oxidant and 0~ production and net maximum condensation
nuclei production, but had an insignificant effect on the
average mean/time NO,, NO and c^-pinene concentrations.
315
-------
Wilson and Levy (1970) have reported that the interaction
between SO. and photochemical smog is dependent upon the
water vapor concentration. In contrast, Bufalini and Altshuller
(1969) found no change in the rate of oxidation of nitric oxide
(with various hydrocarbons) when the water vapor was increased
from 1.1 to 11 mm. of Hg. This result conflicts with the
water vapor effect noted by Dimitriades (1967). Bufalini and
Altshuller (1969) have postulated that the increase in hydro-
carbon reactivity caused by the increase in water vapor may be
due to a deactivation of active sites on the wall by the water
vapor, thus enabling free radicals to have a longer lifetime.
They felt'that their .glass chamber was either conditioned for
free-radicals (from prior irradiations) or the glass system
is less reactive than the smog chamber of Dimitriades (1967).
Hydrolysis reactions in the atmosphere effected by
water vapor may also be important in some instances. For
example; Collier=(1972) found that.although bis-chloromethyl
ether was stable for 18 hours at 10 and 100 ppm in 70% relative
humidity air, chloromethyl.methyl ether hydrolyzed rapidly.
, (b) NO-Hydrocarbon Concentrations: The atmospheric
photopxidation of nitric oxide in the presence of hydrocarbons
is the first and one of themost important reactions in the
complex series that results in photochemical smog. For this
reason, the reaction has received a great deal of study
316
-------
(Altshuller and Cohen, 1963; Altshuller e£ al., 1969a;
Altshuller e£ al., 1969b; Glasson and Tuesday, 1971; and
Glasson and Tuesday, 1970b). Many studies have varied the
hydrocarbon and NO concentrations in order to determine the
kinetics of the reaction add the differences between studies
with individual hydrocarbons in comparison to mixtures.
Glasson and Tuesday (1970a) in a study of the effects of
nitric oxide concentration on the photooxidation of propylene,
ethylene, trans-2-butene, isobutene, and _ra-xylene using LPIR,
found the following: (1) low concentrations of NO increase the
NO photooxidation rate, (2) the rates of hydrocarbon photo-
oxidation reach a maximum at a certain NO concentration, above
or below which the HC photooxidation decreases, (3) decreased
hydrocarbon concentrations decreased the rate of hydrocarbon
disappearance and product formation at all NO concentrations.
The initial hydrocarbon concentration has also been found to
affect the NO peak concentration, N0» dosages, oxidant
dosages, peroxyacetyl nitrate formation and eye irritation
(Altshuller et al., 1970a). Glasson and Tuesday (1971) have
demonstrated that (a) the nitric oxide photooxidation rates
observed with hydrocarbon mixtures are consistently less than
the rates calculated from independent experiments with the
single hydrocarbon system and (b) rates calculated from linear
extrapolations of data obtained with 1.0 ppm of the individual
hydrocarbons agree closely with the observed rates for the
317
-------
1 ppm total HC mixtures. This result is consistent with
increased NO inhibition with increase in the total hydrocarbon
concentration.
(c) Other Reactants: Besides water vapor, NO and
A
hydrocarbons, a number of other contaminants have been shown
to have an impact on atmospheric reactions. For example,
Gitchelli et al. (1974) have demonstrated the inhibition of
NO photobxidation (with hydrocarbons) by phenol, benzaldehyde,
and aniline. In a study of the photochemical reactivity of
benzaldehyde with NO-HC mixtures, Kuntz et^ al. (1973) reported
a similar decrease in the rate of oxidant formation, hydrocarbon
consumption, and NO conversion. Westberg and coworkers (1971)
reported that carbon monoxide accelerates the photochemical
reaction of isobutene and NO. Wilson e£ a±. (1972a) demonstrated
that the presence of SO- reduces the maximum NO concentration.
Also, sulfur dioxide was found to reduce the maximum oxidant
obtained from 1-butene, 1-heptene, and 2,2,4-trimethylpentane,
but it increased the oxidant obtained from toluene. Altshuller
et ^. (1967) have reported that hydrocarbons can be photo-
oxidized with aliphatic aldehydes even in the absence of NO,
although the rates are somewhat slower than those induced by NO.
(iii) Temperature
Bufalini and Altshuller (1963) studied the effect of
temperature on the photooxidatlon reaction of trans-2-butene
r
and 1,3,5-trimethylbenzene with nitric oxide in air. The
318
-------
results indicate about a two-fold decrease in conversion times
for nitrogen dioxide over the 20° interval from 20° to 40°C
and a corresponding increase in rates of reactions (hydro-
carbons disappear almost twice as fast). The authors suggest
that the results imply that the temperature changes may be
dependent upon the nature of the hydrocarbon. With the trans-
2-butene-nitric oxide system,the temperature change has been
equated in magnitude to an Increase caused by changing the
light intensity from K, = 0.20 to K, = 0.37 min'1. Doyle (1970)
a a
~/
has concluded that these results suggest that the temperature
in a chamber should be controlled to within only a few degrees
since the lack of precision of most analytical methods would
make more precise temperature control useless.
(iv) Chamber Configuration, Construction Materials, and
Cleaning Techniques
The importance of surface or wall reactions as they relate
to "adsorption on the chamber surface, chemical reaction or
decay of the gases, chemical reactions catalyzed by the chamber
surface, and the formation of new components from the original
precursors" has been recognized for a long time (Rose and
Brandt, 1960). These processes are probably quite important
when considering atmospheric reactions of low volatile
materials in reaction chambers. Unfortunately, in most cases,
these processes are little understood. However, in an attempt
to minimize these effects researchers have varied the
319
-------
construction materials (made as inert as possible), the surface/
volume ratio (S/V), cleaning techniques, and chamber configuration.
These parameters will be briefly discussed in the following
paragraphs.
The shape of the .chambers seems to be more dependent upon
convenience than upon any suspected effect due to various con-
figurations. The elongated shape of the SRI smog chamber is
dictated by construction costs, type of stirring pump (sonic
jet-type), and the ability to accommodate a flve^membered eye-
i
irritation panel. Jaffe and Smith (1974) used a hexagonal
prism shape; Laity and Maynard (1972), cylinder; and Stephens
(1973), carboys. A basic consideration is that the chamber
contents should be homogeneous, since inhomogeneous mixing
can have an effect on the reaction rate (Donaldson and Hilst,
1972). As a result, most chambers contain some type of mixing
;apparatus. But even the rate of mixing may have an effect
as Wilson et. .al. (1971) have demonstrated for aerosol formation.
It is not certain whether- aerosol formation is inhibited or
whether the aerosol is coated on the fan blades and chamber
surfaces (Wilson et^ al^., 1972a).
«j
Cleaning techniques can make substantial changes in the
results obtained. An important source of contamination has
^
been reported by Gay and Bufalini (1971) in their study of
the nitrogen balance of an irradiated mixture of hydrocarbons
and oxides of nitrogen. They found nitric acid as a principal
320
-------
surface product and believed it was formed by the hydrolysis
of a nitrogen pentoxide intermediate with water vapor on the
wall surfaces. Bufalini ejt _al. (1972) suggested that "dirty"
chambers may be responsible for the poor quantitative agreement
between various smog chambers. They noted drastic differences
(no ozone formed) in the irradiation of biacetyl in a clean
borosilicate flask (rinsed with base to remove HNO-) in
comparison to results with a flask that had previously been
used for biacetyl-NO photolysis and not cleaned. With tetra-
methylene (THE) irradiation, they found no reaction in the
clean flask, although THE was degraded in an unclean flask
(one biacetyl-NO -irradiation, evacuated, and then flushed
A
with tank air) . These results may also explain the differences
observed between irradiation of ambient air samples in con-
ditioned and non-conditioned plastic bags (Kopczynski et al.,
1972). Conditioning was achieved by irradiating an air sample
in a new bag for a full day prior to an experiment. Jaffe
and Smith (1974) compared two cleaning techniques (1) purging
(4.6 chamber volumes) at 110°F or (2) vacuum off-gassing
(2y pressure or less for 16 hours). The different cleaning
techniques had an appreciable effect when used with stainless
steel systems.
The ratio of a chamber's surface to volume (S/V) can vary
considerably (0.78 to 4.91, Jaffe and Smith, 1974). The
parameter is directly related to the probability of a molecule
321
-------
in the chamber coming in contact, and possibly reacting, with
the chamber walls. In a study that varied the S/V ratios,
Jaffe and Smith (1974) reported that most of the parameters
that were measured (e.g., NO- formation rate, NO. maximum,
ozone maximum etc.) were affected by the S/V ratio for the
four materials studied (Teflon, Pyrex, aluminum, stainless
steel).,
The chamber construction material also appears to be an
important parameter affecting atmospheric chamber results.
Altshuller and Cohen (1963) attributed the difference between
Mylar film and FEP film photooxidation to the difference in
light transmittance. However, the inertness of the material
may also be important. Laity and Maynard (1972) compared the
reactivities of gasoline vapors in a glass and stainless steel
chamber and found that the HC-NO mixtures always reacted
X
faster in the steel chamber. Jaffe and Smith (1974) compared
the effect of the use of Teflon, Pyrex, aluminum, and stainless
steel as construction materials. For such reactivity mani-
festations as NO- formation rate, time to NO- maximum, ozone
maximum, 50% propylene. destruction, and NCL dose, the following
order of reactivity is maintained: Teflon, Pyrex, aluminum,
stainless steel. Pyrex and aluminum are similar in behavior
for most parameters. Preliminary analysis of the data suggests
that stainless steel behaves differently than the other materials
(Jaffe and Smith, 1974).
322
-------
c. internal Consistency of Results
Interlaboratory comparisons of various chambers have shown
poor quantitative agreement in results of irradiation of simple hydrocarbon-
nitric oxide systems (Bufalini e£ jal., 1972; Jaffe and Smith, 1974). In some
cases even results in the same chamber have varied over the years, due to
different operation, new light source, etc. (Laity e_£ _al., 1973). These
differences could not be attributed to analytical variations (Jaffe and Smith,
1974). However, although the quantitative agreement has been rather poor, the
qualitative results (ranking of reactivities relative to a standard) can be
correlated fairly well as long as the experimental conditions are somewhat
-,-. .
similar. Laity et _al. (1973)have reported a relative standard deviation of
±10% for these relative rates, aven though their chamber performance has
varied so much that ten toluene standards were required in the past four years.
Hydrocarbon-NO systems have been studied the most, but only
a few studies have determined the relative reactivity measured as hydrocarbon
consumption. These have been summarized by Altshuller and Bufalini (1971)
and are presented in Table 34. The determination of hydrocarbon reactivity
in terms of nitric oxide oxidation is much more common because of the simple
analytical technique involved. A comparison of two such studies in a review
by Altshuller and Bufalini (1971) showed that the ranking was generally in
good agreement and followed the same -general order as the relative rates
determined by hydrocarbon consumption. The recent, comprehensive study by
Glasson and Tuesday (1970b) on hydrocarbon reactivity (measured by NO photo-
oxidation) in the presence of nitric oxide also correlates well with these
result's.
323
-------
Table 34. Ranking of Reactivities of Hydrocarbon Consumption
When Photolyzed in Presence of NO under Static Conditions
(see Altshuller and Bufalini, 1971 for references).
Ranking of Hydrocarbons
Schuck and Stephens and
Doyle Scott Tuesday
Hydrocarbon (1959) (1962) (1963)
Tetramethylethylene 10 10 10
trans-2-Butene 6 8
cls-3-Hexene 6
Isobutene 1.5 2 2
1,3-Butadiene 1
Propylene 1 21
m-Xylene 1
p-Xylene 0.5 0.5
Ethylene 0.1 0.3
Hexanes, octanes <0.1
Pentanes <0.1
d. Comparison of Laboratory Results to Behavior in the Natural
Environment . '
Techniques which model atmospheric reactions have little
utility if they are not comparable to reactions in the natural environment.
This section will briefly discuss the comparability of the results for two
chemical groups: (1) hydrocarbons and (2)- fluorocarbons. This covers the
spectrum of fairly reactive substances to almost totally inert materials.
(i) Hydrocarbons
The reactivity of hydrocarbons has been studied under a
variety of laboratory conditions and considerable monitoring
data is also available. Evaluating the techniques with a large
number of compounds has an advantage in that the relative
rates of reaction can be compared without having to resort
324
-------
to the very difficult task of comparing absolute rates. However,
if synergistic affects are present in the mixtures, the com-
parison to pure compound reactivities would be extremely diffi-
cult .
Kopczynski et_ al. (1972) have irradiated with sunlight
ambient air samples collected in plastic bags. They compared
their data to information obtained from irradiated air samples
in glass carboys (Stephens and Burleson, 1967) and relative
reactivities determined on pure HC-NO mixtures (Heuss and
Glasson, 1968). The relative reactivities, which are presented
in Table 35, are in substantial agreement. Altshuller et al.
Table 35. Relative Rates of Percentage Loss of Hydrocarbons
Averaged Over Four Hour Irradiation
(Kopczynski e* _al., 1972)
Kopczynski et al.
(1972)
Stephens and
Burleson (1967)
Heuss and
Glasson (1968)
Hydrocarbon
Rel rate Std, dev. (4-hr irradn.) (6-hr irradn.)
Ethane
Propane
in-Butane
Isobutane
o-Pentane
Isopentane
2 ,4-Dlmethylpentane
Acetylene
Ethylene
Propylene
1-Butene
2-Me thy 1-2-b utene
Toluene ;:
m-Xylene
o-Xylene
sec-Butylbenzene
0.06
0.16
0.27
0.24
0.37
0.43
0.99
0.14
1.00
2.33
1.62a
9 . 39b
0.56
1.14
0.70
1.25c
0.09
0.09
0.13
0.26
0.07
0.13
0.27
0.07
0.42
0.52
1.99
0.34
0.45
0.28
0.45
0.18
0.37
0.33
0.58
0.17
1.0
2.5
2.5
0.11
1.00
1.6
1.9
2.1
0.87
1.2
1.2
0.47
a Contains Isobutylene.
b Averaged over first hour of irradiation.
c Contains 1,2,4-trimethylbenzene.
325
-------
(1970b) also irradiated with sunlight ambient air samples in
plastic bags. They were unable to detect acetylene reaction
and.the butanes reacted very slowly. However, the relative
rates of the other paraffinic and olefinic hydrocarbons were
reasonably consistent with other studies.
1 The studies cited above are still, in a sense, laboratory
studies since static conditions were used. In comparing these
results, the more dynamic nature of the open atmosphere must
always be kept in mind (Kopczynski et al., 1972). However,
direct comparison of ambient air monitoring data to laboratory
results is extremely difficult because of varying imputs of
different hydrocarbons. Nevertheless, some qualitative
comparisons are possible* Stephens (1973) has noted that
. acetylene, ethylene, and propylene form a unique set of compounds
«.i»
since (1) they.are derived almost exclusively from auto exhaust, (2)
they are emitted in known ratios to each other, and (3) they
differ quite widely in reactivity. If one assumes that the only
source of the three compounds is from automobile exhaust,
dilution should have no effect on the ratio; only the difference
in reactivity should affect the relative concentrations.
Table 36 lists the compound ratios for an air sample taken in
the early morning (little photodegradation) and one taken in
the late afternoon. The order of reactivity suggested by the
monitoring data Cethylenopropylene) is consistent with
reactivities derived under static conditions in the laboratory.
326
-------
Table 36. Comparison of Acetylene, Ethylene, Propylene Ratios
of Two Ambient Air Samples
(derived from data in Stephens, 1973)
Acetylene
Ethylene
Propylene
Hydrocarbons in
Morning Air
(0735-0800 hrs.)
(ppb)
78
64
14
Hydrocarbons in
Polluted Air
Ratio
C2H2- 1.00
1.00
0.82
0.18
(1500 hours)
(ppb)
27
16
1
Ratio
1.00
0.59
0.04
% Change
in Ratio
-
27%
78%
(ii) Fluorocarbons
Fluorocarbons are released to the environment in large
quantities mainly due to their use as aerosol propellants.
Three studies under laboratory conditions of the atmospheric
stability of these compounds are available. Saltzman et al.
(1966) examined bromotrifluoromethane and octafluorocyclobutane
as potential meteorological tracers by exposing the compounds
(100-^250 ppb) in a plastic bag to light, ozone, water vapor,
SO,, and diluted automobile exhaust. They found no reactivity.
However, these compounds are not commercially important and
no ambient monitoring data are available. Japar e_t al. (1974)
examined the photostability of fluorocarbon 11 (CC13F),
12 (CC12F2), 22 (CHC1F2), 113 (C12FCCC1F2), 114 (C1F2CCC1F2)
and 115 (CIF^CCFJ in a LPIR instrument in the presence of
327
-------
nitric oxide and an olefin. For irradiations lasting as long
as eight hours, no decrease in fluorocarbon concentration was
noted. Hester eit al. C1973) irradiated CC12F2 and CC13F in
the absence'of hydrocarbons for periods of up to two months
and found no degradation.
Lovelock et al. (1973) have detected CC10F levels of
j
49.6 ppb (by volume) above the Atlantic Ocean, and Su and
Goldberg (1973) report concentrations of 97 ppb and 700 ppb
of CC1-F and CCl-F., respectively, in a desert area well
removed from industrial sources. Based upon the above
monitoring data and estimates of fluorocarbon released to the
atmosphere, the above authors calculated minimum residence
times of 10 (CCl-jF- Love lock et al., 1973) to 30 years
t
(CC^F- - Su and Goldberg, 1973). Obviously, the atmospheric
stability of fluorocarbons based both on the laboratory studies
and monitoring data is in good qualitative agreement.
e. General Discussion of the Advantages and Disadvantages of
the Various Methods
The choice of a technique to study the atmospheric reactivity
of a chemical substance has both technical and economic trade-offs. This
section will discuss only technical considerations.
.'.';
In general, the techniques that have been reviewed have been
designed to study relatively short term (hours) phenomena. For example,
although 94% of trichloToethylene reacts in 6 hours of irradiation, trichloro-
ethylene'has been characterized as "moderately reactive" (Altshuller and
Bufalini, 1971). The applicability of these techniques to studies of long
term'processes (days, weeks, months), which are important to a determination
328
-------
of the persistence and fate of a chemical substance, Is unknown. In addition,
with the exception of the study by Crosby and Moilanen (1974), only compounds
with high vapor pressures have been studied.
Chamber studies can be conducted under either dynamic or
static conditions. The advantages and drawbacks of these two procedures are
noted in Table 37. The size of the chamber will effect (1) the surface/
volume ratio arid (2) the analytical sample available and thus the concentration
of reactants that must be used. The construction material can effect both
the wavelength and intensity of the light source and the ability of the chamber
walls to terminate radical reactions. The methods that have been used for
studying atmospheric reactions will be reviewed in the following paragraphs.
Table 37. Advantages and Disadvantages of
Static Vs Dynamic Procedures in
Studying Atmospheric Reactions
(Dimitriades, 1967; Doyle, 1970)
Dynamic
Static
Advantage
Disadvantage
Advantage
Disadvantage
Better approximates
the atmospheric
situation
Lower effective
s ur f ace / vo lume
ratio (lower
chance of wall
reactions)
Larger analytical
samples-use lower,
more realistic
concentrations
Large samples for
eye irritation and
plant damage studies
Instability of flow
,and composition
introduces source
of experimental
error.
Need elaborate dilu-
tion, purification,
and air handling
system
Limited time of
irradiation
Simple and reliable Less like the
More tractable,
better precision
Simplifies chemi-
cal reaction
system.
dynamic atmos-
phere
Higher effective
surface/volume
ratio
Smaller analytical
sample-need higher
concentrations
329
-------
Long-path infrared (LPIR) systems require a minimum of
analytical development time since most compounds have a diagnostic infrared
absorption. However, because of the analytical technique, the systems studied
have been relatively simple (mostly HC-NO systems) and, unless one resorts to
Fourier Transform IR, the concentrations of reactants are somewhat higher than
what would normally be found under ambient conditions. The use of stainless
steel with fluorescent lights on the inside of the chamber (Glasson and Tuesday,
1970a, b,c) seems somewhat less desirable than using more inert substances,
such as Teflon (Japar et ail., 1974), with the light source on the outside.
The,use of plastic containers appears to be a versatile method
of irradiating both artificial and natural air samples with sunlight or arti-
ficial light sources. FEP Teflon seems to be the best material because of its
UV .transparency. Analytical development of methods of analysis for the low
i
concentrations that would be desirable could be quite time consuming.
Similar, considerations apply to the use of glass flask reactors
with the expectation that the glass reactors should be easier to rinse out for
either sampling or cleaning.
Smog chambers' have the advantage of larger size (lower surface/
volume ratio, larger analytical, sample-lower concentrations of reactants) and
\.
the potential for dynamic operation. In addition, it is easier to maintain
control of humidity and temperature. However, cleaning operations are much
more difficult than with plastic containers or glass reactors.
330
-------
5. Cost Analysis
The equipment costs of the techniques previously reviewed can
vary considerably. For example, a smog chamber may cost approximately $250,000
and a Fourier Transform LPIR, approximately $200,000, whereas a glass reactor
or plastic bag Irradiation unit could be assembled for well under $10,000.
We find it rather unlikely that the more expensive apparatus will be used to
test a compound's degradation unless the laboratory already has the equipment,
but for the sake of comparison, we have included the capital costs in the
per-compound calculations.
Some assumptions have been made in order to calculate the cost
estimates. The labor estimates are based upon the cost of a bachelor level
researcher ($12,000/year, including salary and benefits). Since most of the
techniques have been designed for short-term reactions, we have assumed that
the exposure time in the chambers will vary anywhere from 2 hours to 2 days.
Major time commitments (and therefore costs) are involved in analytical
development of techniques to determine the loss of test compound (difficult
at the low concentrations required) and formation of degradation products.
With LPIR, analytical development costs should be much lower. The study by
Wilson £t al. (1972b), which determined the chemical structure (using GC-MS)
of the aerosols formed from a number of olefins, is perhaps typical of what
would be necessary for a thorough study. In terms of cost, we have distinguished
between studying the loss of test compound and identification of the breakdown
products.
331
-------
a. Loss of Test Compound
(i) Only One Compound Studied
LPIR
Glass Flask or
Plastic Bag
Smog Chamber
Equipment
60,000
(not Fourier
Transform)
10,000
250,000
Labor
(cost)
($125/day)
3 days
($375)
6 days
($750)
6 days
($750)
Cost per
Compound
60,375
10,750
250,750
(ii) More Than One Compound Per Year for 5 Years
# Compounds
LPIR
Glass Flask or
Plastic Bag
Smog Chamber
Per Year
150
50
50
Equipment
60,000
10,000
250,000
Labor/year Cost per
(1 person-B.S.) Compound
28,000
28,000
28,000
250
600
1,500
332
-------
b. Loss of Test Compound and Isolation and Identification of
Breakdown Products
(i) Only One Compound Studied
LPIR
Glass Flask or
Plastic Bag
Smog Chamber
Equipment
Labor
(cost)
($125/day)
10,000 15 days
(buy time on GC-MS ($1900)
$l,000/compound)
250,000 15 days
(buy time on GC-MS ($1900)
$1,000/compound)
Cost per
Compound
12,900
252,900
(ii) More Than One Compound Per Year for 5 Years
LPIR
Glass Flask or
Plastic Bag
Smog Chamber
9 Compounds
Per Year
20
20
Total 5 yr.
Equipment
10,000 and
GC-MS $100,000
depreciated
over 10 years
total for
5 yrs a 60,000
250,000 and
GC-MS - 50,000
total for
5 yrs » 300,000
Labor/year
(1 person-B.S.)
28,000
28,000
Cost per
Compound
2,000
4,400
* We find it unlikely that an intensive study like this would
be done with LPIR because the breakdown products, in most
cases, could not be identified by IR.
333
-------
c. Summary
These cost calculations are very approximate. The possibility
of having a 5 year program to test the atmospheric fate of chemicals is
rather remote and, therefore, the costs are probably somewhat more than the
estimates provided for the more than one compound studies. The estimates
for the one compound studies are obviously inflated by the start-up costs.
In addition, great fluctuations can occur due to analytical difficulties.
In order to put this data into perspective, one researcher in the field has
suggested a price of approximately $3-5,000 per compound for a complete smog
'potential evaluation (measure NO oxidation, 0_ formation, etc. - not necessarily
compound degradation and breakdown products) in a smog chamber.
334
-------
B. Photochemical and Chemical Alterations in the Aqueous and Soil
Environment <
1. Photochemical Alterations
a. Introduction
Many chemicals that are released to the environment, especially
the less volatile ones, eventually reside in the soil and water. These media
do not provide as good a matrix as the atmosphere for photochemical alterations
due to the possible attenuation of the incident light. In soil, only compounds
that reside on the very top are susceptable to photolysis. Although water is
transparent to ultraviolet light (transmits light >180 nm), the intensity
falls off with increasing depth and below a few meters photolysis probably
proceeds at infinitesimally small rates (Crosby, 1972a). Nevertheless,
photolysis has been shown to be Important to the environmental fate of chemicals
which have low vapor pressures such as pentachlorophenol (Kuwakara, 1966a, b), '
pyrethrins (Chen and Casida, 1969), and dieldrln (Henderson and Crosby, 1968).
This section will discuss the experimental techniques used
to study the photolysis of chemicals in the non-atmospheric environment.
For the most part, the chemicals studied have been pesticides, and therefore
the conditions that are used are attempts to simulate field conditions (Crosby, 1969b).
However, when an attempt is being made to determine the photochemical reaction
pathways, the experimental conditions poorly simulate the environment, but
are frequently necessary in order to isolate products. Table 38: lists a , '
considerable number of the more recent photolysis studies along with the
chemicals studied and the conditions used.
335
-------
Table 38. Experimental Conditions of Pesticide Photolysis
Reference
Add Is on tit al.
: (1974)
And el in* n and Sucsn
Alley at al. (1971)
Alley et >J . (1974)
Aly and El-Dlb (1971)
Aly and El-Dlb (1972)
Archer e_t al- (1972)
Handul and Caslda
(1972)
Baur et al. (1973)
Benson et al. (1971)
Bull* and Edgerley
U96B)
Chen tmd Casldn
(1969)
Chung ej aj_. (1972)
Cronby and Loitin
(1973)
Crosbv and Wong.
(1973a, 1973b)
Crbtfby and Mollanen
(1973)
Crosby and Tang
( 1969a)
Crosby and Tang
: (1969b)
"* Ciooby aad Leitia
(1969)
Crulcknhank and
Jar row (1973)
Frost « al.. (1972)
t'«wr at al. (1973)
.Hhhbfln and Galbel
(1971)
Chemical Studied
2 carbanaten (Matacll
and Land r In)
1.4-Benzpyrent:
Mlrvit
Hlrex & Kepone
4 carbamates-Sevin,
Uaygon, Pyrolan. and
tUmetllan
Endosulfan
2-gec-Butyl-4,6-
dlnitrophenol and
the isopropyl
carbonate
Pi dor am, 2,4,5-T,
Dlcanba/free acid
and salt
ChLordane
Aldrin, dieldrln,
& cndrin
Pyrethrlno
Rotenone
Trlfluraltn
2,4.5-T, 2,4-D
p-chlorophenoxy-
acetlc acid
PCB's
3-(p-chloroph«nyl>-
1,1-dimethylurea
1-Hoph tha leneace tic
acid
Chlorophenylacetlc
acids
Ethylenethiourea
DDT
2,2-Dlbromo-3-
nlt r 1 lop r op ion amide
Plperonylbutoxide
Physical State
of Tent Chemtcal
Solul Lon - ethanol
and cyclohexane
Absorbed on CaCOj
In aqueous solution
(pH 9)
Solutlon-cyclohexene
and iaooctane (0.04M).
H; flushed
Aqueous solution
(pH 5.0. 7.0. 9.0)
Thin flLa on boro-
silicate glaas
(0.2g on 20 * 20 x
5 dish)
Thin film on glasa
and bean leaves
Thin film on glass
SolutloD-acetonf
(solvent and photo-
sensltlzer) Thin film
glass plate
petri dish-quartz cover
Solution-water
(20-25 ug/1)
Thin film-glass
Solution-oxygenated
BBthanol beneene
Thin film-plant leaves
glass surfaces,
and silica gel
chroma c opiates
Suspension In water
(tap or deionlzed,
50 mg/1) varied pH
Adsorption on soil from
hexane solution-suspended
In water
Aqueous methanol
Solution - 100 og/1
Also used acetone and
rlboflavin aa senal-
tliera
(1-10 mg/1)
Solution in vater
(200 mg/1)
Solution in wacar
Solutlon-auapenalon in
water (100 mg/1)
Adsorbed on silica
gel plates
Solution in water
Absorbed on silica gel
chromatoplate
Solution in water
(1.0 g/500 ml)
Thin fl 1m on glass
Light Source
Hanovla high-pressure
1000 watt Xenon- Hg
lamp with 2 Corning
filters
White fluorescent lamps
Hanovla 450 W. medium
press, Hg lanp
Two-l5W geralcldal tubes
Irradiated Into open
9- cm petrl dishes
GE germicide! lamps
Sunlight
f 40BL Fluorescent
tubes
Hanovla 450V, Corex filter
Klmax filter
Germicldal lamps
Sunlight
One 15W germlcidal lamp
275W sun lamp
Sunlight
Sun lamp
High press. , Hg lamp
(450V, Corex filter)
15W garmicldal laap
Sunlight and
F40BL fluorescent,
lamp through
borosilicate glass
Sunlight and
Six- 4 ft. F40BL
fluorescent UV lamps
mounted in a
cylindrical chamber
F40BL fluorescent lamp
through borosilicate
glass
Sunlight
Sunlight
' F40BL fluorescent lamp
through boroaillcate
glaas
Sunlight
F8T5BL fluorescent lamp
through borosilicate
glass
360V, high press
Hg lamp
Sunlight
275W sunlamp
Pair of UV fluorescent
lamps (one 300 nm and
one 350 nm) through
Pyrex
8-300 nm phosphor lamps
8-350 urn " "
Hg lamp
Fluorescent lamp
Sunlight
Sunlamp
Sunlamp 275 U
Hanovla 450W HK Jnmp
Wavelength
300 nm
>350 nm
quartz f 1 Uer
254 nm
254 nm
>290 no
Xmax 356 nm
?280 nm
254 nm
>290 nm
254 nm
>290 00
>260 nm
254 nn
>290 nm
'290 nm
300-450 nm
300-450 nm
290 nm
300-450 nm
>290 nm
254 nm
>290 nm
,260 nm
some 265-300
mostly >300 nm
254 nm
366 run
-290 nm
peak 297 nm
TLC and (1C-F1
.3
of C/iCOj and i'V
i)«it>rntniitti>n ut
Cl", prcp.iral i vc.
CC, IR, NHR, MS
TLC
4-amlnoantlpyrlne
oeihud Tor plienols
101* ergs /cm2 TLC
GC-EC
Ether extract
TLC of radlolabellt-d
material
7- 11 x I03ergs/ GC
sec/cm;:
GC-EC
TLC, IR, NMR
MS
GC
TLC of radlolabellud
material
TLC, radlolabelled
IR. MS
TLC
390-785 GC-EC
IR, MS
f-'C- EC
GC-HS
TLC
CC. TLC
IR
CC. TLC
IK
3300 uW/cm2 TLC-radlolahcllvd
300 i.U/cm* material
1900 wW/cm2
TLC
. 850 iiW/cmz TLC - *J
GC x A
wlt!> and vlthout filters varied
SunliRhc >290 nm
-------
Table 38 (continued)
Hi.luim «l «l. U»M)
Cray ot «l. (1912)
.
(1971)
Henderson and Crosby
(1968)
Henderson and Croaby
(1967)
Ivic tt ul. (1973)
[vie and Caslda
11971.1)
[vie and CaKtda
(197Jb)
Jordan tt al.
(1964)
Knovl'es and
Sen C.uota
(1969)
(1969) (1966a.b>
Langford *t al.
(1973)
Liang and
Llctitent.t«ln
(1972)
Lumbardu et al.
( 19'.!)
Matsuo and Caul do
(1970)
Miizzorchl And
Rao (1972)
MtCulre et al. '
(1972) " '
Mitchell (1961)
rtol lanen and
Crosby (1972)
Hoi lanen and
Croaby (1974)
Hosier et al.
(1969)
Hosier and Guenzl
(197)) .
- Newson and Woo da
(1973)
Nordbloo and
Miller (1974)
Pape and Zablk
(1972a)
Pape and Zablk
(1972b)
clmmlc-41 Studied
HajKuchlor Epodda
6-Methyl-2,3-
qulnoxalinedlthlol
Dleldrln
Oieldrin and Aldrin
Arochlor 1254 -
Hethazole
23 Pesticides
Chlorinated
cyclodlenes
Phenylureas
N-U-Chloro-o-totyl)-
N.N-dinethylforu-
mldlne
Cu-ITTA complex
Azlnphosnethyl
Dlvldrln
Two dinitrophenollc
pesticides
(Dlnobuton and
Dlnosub)
J-(p-Chlorophenyl)-
nnti 3-Plicnyl-l,l-
dlnethyluraa
Heptachlor
141 Pesticides
3' , 4'-Dichloropro-
plonanlllde
Brooacil ,
DOT
Plchloraa
'Jlnltramlne
4.4'Dlchloro-
biphenyl
Asynaetrlc
trU2ln-5(4H)-ones
SyniMtrical
triazlnes
.'tiyslc.ilStatc
of Teat Chemical
Solid in Ur dlak
(0.33» w/w)
Solution In benzene
(30 mg/32i ml)
bubbling N3
Solution-aqueous THF.
aqueoua ethanol
H; flushed
Solution-water
Solutlon-hexane,
cyclohexane. met Hanoi
(water, hexana,
beniene)
Solution-methanol water
Thin filn-glasa
Absorbed on silica gel
chroaatoplate
with senaltlxer
Thin film on bean
Aqueous chloroplast
auspennlon
Absorbed on filter
paper
Absorbed on ailica gel
chronatoplates
Solutlon-951 ethanol
Solution-water
Thin flln-glast; plate
Thin film-glass plate
Thin film-bean leaves
Solution-methanol
anaerobic (Nj
purged)
SolutLon-hexane,
cyclohexane and
acetono
Adsorbed on paper
Solution-water
Solution-water
Thin -film-on quart!
Solution-water
Solution-water and
mat Hanoi
Adsorbed on sand
SolutloD-2-propanal.
nethanol, ethyl ether.
cyclohexano ,
acatonitrile (degaased)
Solution-CClt,, benzene,
met Hanoi, water
Solution- ITU- thanol,
watci
Light Source
Sunlight
Hg lamp
Hanovla 450U medium
prase. Hg lamp-
pyrax filter
Ray one t photoreactor
Sunlight
Low press. Hg lamp
. boroslllcate filter
Garolcidal lamp
lamp
Sunlight
Cermlcidal lamp
Sunlight
Sunlight
Sunlight
UV lamp
H« UV lamp
Fluorescent UV lamp
Sunlight
Sunlight
Rayonet photoreacLor
Sunlight
Germlcidal lamp
Sunlight
Lov press. Kg Lamp
Low preae . Hg lamp
Fluorescent lamp
Carmlcldal lamp
Sunliglit
Fluorescent lamp
boro.lllcate filter
Sunlight
Fluorescent lamp
boroalllcate filter
Germlcidal , low press.
Hg lamp
Fluorescent lamp
Sunlight
Fluorescsnt lam)>
Fluorescent lamps
(310 nm)
Fluoreacent lamps .
(300 and 350 nm)
boroslllcate filter
Hg UV lamp
Fluorescent lamp
Unvelength
290 nm
254 nm
>2BO nm
254 nm
300 nm
350 nm
290 nm
'280 nm
254 nm
254 nm
>2»0 nm
>290 nm
254 nm
254 nm
320-400 nm
.290 nm
350 nm
254 nm
254 nm
>290 nm
254 nm
254 nm
)00 nm
254 nm
»290 nm
>300 nm
>290 nm
>300 nm
254 nm
300-380 nm
290 nm
>300 nm
234 nm
300 nm
IntenHlty Analytic f'roccdurcs
IR
CC, MS. KM*
TLC, MS, IR
GC
IR, MS
CC
CC, TLC
GC
TLC-radlolabclled
compoundu
TLC-redloUhclled
compounds
TLC-radlolabelled
compounds
UV absorpt Ion
260 uU/cm7 TLC-raululabelled
680 nWcm:' compound
TLC
TLC-radlolabellcd
compound
CC, IR
TLC-radlulabelled
compound
GC, TLC
:C, EC
try
CC, TLC
GC. TLC
CC-KS
TLC-radlolabelled
compound
TLC-rndlolnbelled
compound
GC
CC, TLC-
radlolabelled
compound
cc-r.c
TLC, CC,
IR. UV, KMR
GC-KS
TLC, f.C
IR, UV, NMR
CC-MS ,
-------
Table 38 (continued)
R|..l|.rt!l»'f
1
faroiliettl nnd
Hem (1971)
I'llmoer nnd Humar
(1969)
I'll met nnd
Pllttwr ct al.
(19«7)
Pllnmer ct al.
(1970) .... .
Porter (1971)
Redemunn and
Youngaon (196K)
Rosen (1967)
Rotten and Strunz
(1968)
Roaen et al.
(1969)
Roaen CL al.
(1970)
Rouen and Slewlerskl
(1971)
Rosen and Slewlernkl
(1972)
ROHU and Croaby
(I97J)
Kuxo ec el.
(1974)
Ruto el al.
' (1973)
SuCc and Huttlnger
(1971)
SUdo and Smith
(1967)
Smith and Grove
(1969)
Su and Zahlk
(I972a)
SB «nd ZaMk
(1972b)
: T-BJUl.
"°(»f*)~
Wright and Warren
(1965)
**"**'
Z*pp a I dl.
Reproduced from /§
i:i.i!m|.Ml Studied
2-yO-r-henyl-
N-Netliyl CarbaAatL>
Trtriuralln. beneflu,
nltrolln
dl chlorobentolc
arid
2,4-uichlorophenol
An It role
DDT nnd DDE
36 Dye coBpounds
6-Chloroplcollnic
acid
N,N-dimethyl-2,2-
dlphenylacataalde
2 urea herbicides
Phenyl urea
herbicides
Chloroanlllnes
Aglypt
Pyraion
Ethylenethlourea
PCB's
s-tria,ine.
PCfl'a
Diquat
Paraquat
Diquat
Arylamlde
derivatives
an'N-aethylearbsnate
hydrochloride :
0,0-Dlnethyl S-
phthallnldomethyl
phosphorodi t bloat*
Rlam«thrln and
other pyrethrolda
Trlfluralln
Endrln
Phenylmercury
compounds
I'hyeLcal State
uf Test Chemical
mt-thanol
Spruyud on soil
Solution-water methaoolt
water-aodlum bisulfite
Solution-water
rlbuflavln sensitlcod
Solution-water
Solutlon-methanol
(with or within 02)
Solution-water
Solution-water
Solution-water
Solution-water :
Solution-water
Solution-water
FHN aenaltized
Solution-water
Solution-water
Adsorbed on silica
gel platee
Solution-water
(with and without
So 1 ut ion-hexane
methanol
Solutlon-methanol
n-butanol, water
Solutlon-hexane,
methanol
Solution-water
Adsorbed on silica
Solution-water
Adsorbed on silica
Suspaneton-dlat. & river
water
(pH 3.1 + 7.1)
(250 ppm)
Solution-disc, and
river water (250 ppm)
Solutloa-diethyl ether
Adsorbed on silica gal
chronatoplate
Solution -water
Thio film-glass
soil
Sol u tlon-hexant ,
cyclohexane
Solution-water
acetonltrlle.
Light Sourct:
(254. 300. 360 run)
Fluorescent lampn
(FS-40T12)
Imax - 310 nm
Hg lamp
borofcl .(cute filter
lilt lamp
boroslltr.ate niter
High press. Hg lamp
various filters
450U Hg Idfflp
Corex filter
Pyrex filter
Carbon arc-Pyrex filter
Sunlight
Sunlight
Hg lamp
Low-prese . Hg lamp
Sunlight
Sunlight
Low press. Hg lamp
Sunlight
Sunlight
Sunlight
Hg lamp. Pyrex filter
.Sunlight
Hg lanp, Corex filter
Sunlight
Fluorescent lanp
Fluorescent lamps
borosillcate filter
Fluorescent lamps
borosillcate filter
Fluorescent lamp
Sunlight
Hg lamp, no filter
Borosillcate filter
Hg lamp with boro-
aillcate filter
Sunlight
4 SOU High preaa.
Hg lamp
(Pyrex filter)
450V High presa.
Hg lamp
(Pyrex filter)
Hg lamp with and
without Pyrex filter
Sunlight
275V Sun lamp
Sunlight
Hg fluorescent lamp
Hg lamp with filters
Vycor
Corex
Pyrex
4 SOW med. press
H* lamp
Pyrex filter
Sunlight
290-340 nm
>280 nm
>280 nm
220 nm
260 nm
>280 ran
>280 nm
>290 nm
254 nm
254 run
>290 nm
>290 nm
254 nm
>290 nm
>290 nm
>290 nm
280 mo
>290 nm
>260 nm
>290 nm
>285 nm
>285 nm
Xraax 310 nm
>290 nm
254 nm
>300 nm
>290 nm
>286 rm 2 x 105 erga/
cm2/8ec
>2B6 nm 2 K 105 ergs/
cm /aec
.,
360-380 tin
500-600 nil
>210 nm
>250 nm
>280 nm
IR, UV, NMK
Blo.,s»ay
CC. TLC
NMR
CC-MS
TLC
TLC-rndlcilabclli!d
. compounds
CC MS
UV
UV, IR
TLC
TLC. CC
TLC. GC
TLC
TLC
TLC, IR, MS
TLC
CC-MS
NMR
TLC
TLC, f.C
Radio label led
compound
TLC-radlolobelled
compound
TLC
TLC
TLC
TLC-radlolaballed
material
bioassay
Bloasaay
CC, MS,
NMR, TLC
_^
co lor tme trie (»
-------
b. Techniques Used to Determine Photoalterations
Ci) Light Sources .
Perhaps the most crucial factor effecting the photo-
products and their rates of formation is the wavelength and
intensity of the light source. Consideration must be given
to both the light source and any media that the light passes
through before irradiating the test chemical.
Using actual sunlight provides the best indication that
photolysis of a chemical will occur in the environment and
the use of sunlight is a common experimental technique. The
test chemical or the test chemical solution is often covered
by quartz, borosilicate glass, or in some cases clear, plastic
wrap to reduce evaporation losses and contamination. These
substances are transparent to light wavelengths greater than
180 nm, 300 nm, and 200 nm respectively (Calvert and Pitts,
1966; Crosby and Tang, 1969a). However, the inherent varia-
tion of sunlight in both wavelength distribution and intensity
results in poor reproducibility, inconvenience, and lack of
experimental control, and therefore many researchers have
resorted to artificial sources of ultraviolet light (Crosby,
1969b).
Arc and fluorescent lamps are by far the most popular
artificial light sources because they provide a rich source
of UV light. The mercury arc lamp in which an electric
discharge is generated in gaseous mercury vapor has become
the standard UV source for most photochemical research. The
339
-------
arc can he operated at high, medium, and low pressure and this
results in a series of sharp spectral lines (low pressure)
or a fairly evenly dispersed irradiation intensity over a
broad wavelength range (high pressure) as is depicted in
Figure 37(Calvert and Pitts, 1966).
The higher pressure mercury lamps radiate much of the
energy as heat (^ 80%, Crosby and Li, 1969) and therefore
precautions must be taken to prevent overheating of the chemical
i
being photolyzed and to protect against glass breakage. The
low pressure lamps, sometimes referred to as "germicidal lamps,"
are, cooler and far more efficient (over 90% emitted as light),
but most of their intensity is concentrated at 253.7 nm, which
is far below the wavelength energies found in sunlight.
The fluorescent lamp is a well-known variation of the
lowr-pressure mercury arc. The lamp is internally coated with
'- ' $
a thin layer of mineral or organic phosphor. The phosphor''. J ,
absorbs the 254 nm radiation and then readmits the radiation
by fluorescence producing a diffuse, even light at somewhat
lower energy than the original arc. By varying the phosphor
composition a wide spectral range can be provided. The spectral
distribution of two fluorescent light sources as well as a
low-pressure mercury lamp, sunlight, and the UV absorption
curve of ordinary borosilicate (Pyrex) glass is depicted in
Figure 38.
340
-------
Figure 37. Emission Spectrum of the Low-Pressure Q-Qy)» Medium Pressure
(35 cm), and High Pressure (100 atm) Mercury Arcs
(Calvert and Pitts, 1966)
Courtesy of John Wiley & Sons, Inc.
341
-------
2
J
BOO
600
400
200
250
300
350
X. n
400 450
Figure 38. Spectral Distribution of a Low-Pressure Mercury Lamp (A,G);
Fluorescent Suhlamp (B); Fluorescent Black-Light (D); Normal
Sunlight (E); Daylight Fluorescent Lamp (F); and Transmission
Spectra of Borosilicate Glass (C) (Crosby, 1969b).
Reprinted with permission from Residue Reviews, 25^(1) 1969.
Copyright by Springer-Verlag.
As noted previously, the medium that the light passes
through before striking the test chemical is also important
to the intensity and wavelength of the light reaching the
test chemical. Table 39 lists a variety of construction materials
used to make photochemical apparatus. The transmissions indi-
cated should be taken into account when reviewing the studies
listed in Table 38.
342
-------
Table 39.Approximate Wavelength. Limits for Transmission
of Various Materials and Water at Room Temperature
(Calvert and Pitts, 1966)
Approximate X (nm) for
% Transmission Indicated
Material
Window Glass (standard)
Optical (white crown) Glass
Pyrex (Corning 774)
Corex D
Co rex A
Vycor 790
Vycor 791
Quartz, crystal
Plexiglas (polymethylmeth-
acrylate)
Water (distilled
Thickness (mm)
1
3
10
1.8
1
2
4
1
2
4
.2.9
2
1
2
4
5
10
2.5
5.0
10.0
20
40
80
50%
316
330
352
327
306
317
330
278
288
304
248
215
223
236
185
193
322
338
350
188
192
202
30%
312
323
342
320
297
309
319
267
280
292
243
213
217
225
192
310
325
342
186
188
194
10%
307
314
330
309
280
297
310
250
267
281
240
> 254
212
2i3
217
186
297
311
326
185
186
188
343
-------
(ii) Solution Photochemistry
(a) Photochemical Equipment
A frequently used technique for studying the photo-
chemistry of a chemical is to dissolve the material in a
solvent before irradiation. Solvents such as water,
methanol, ethanol, hexane, cyclohexane, and benzene are
i
often used.
The photochemical equipment used by many pesticide
photochemists includes (1) Crosby's sunlight-simulating,
laboratory photoreactor (Crosby and Tang, 1969a), (2) the
quartz immersion well apparatus often used by Plimmer
et_ al. (1967, 1970) and Rosen and Siewierski (1971, 1972)
and (3) the Rayonet Photochemical Reactor sometimes used
by Zabik and coworkers (Pape and Zabik, 1972a, b; Pape
et_ _al. , 1970). Crosby's apparatus is depicted in Figure 39.
The reactor is constructed of borosilicate glass and,
therefore, the wavelength of light reaching the solution
is no longer than 290 nm (see Curve C, Figure 38). A
germicidal (low-pressure mercury) lamp may be used, but a
fluorescent lamp is preferred (Crosby and Li, 1969).
Ic
Figure 39. Sunlight-Simulating,
Laboratory. Photoreactori Condenser
joint (A), Lamp (B), Gas Inlet (C),
Reaction Chamber (D) and
Thermocouple Well (E)
(Crosby and Li, 1969).
Reprinted from Degradation of Herbicides.
by courtesy of Marcel Dekker, Inc.
344
-------
Crosby and Tang (1969b) have also used a similar
device equipped fpr continuous extraction, in their
study of 1-naphthaleneacetic acid (Figure 40). This
allowed the trapping of the photosensitive intermediate
photdproducts before they were degraded.
Figure 40. Photoreactor (B) Equipped
with a Gas Lift (A) for Continuous
Extraction (C) (Crosby and Tang, 1969b).
Reprinted with permission from J. Agr.
Food Chem., L7(6), 1291-1293. Copyright
by the American Chemical Society.
H
s
fl
^
\
C
I
B
T
The quartz immersion well apparatus depicted in
Figure 41 is more flexible in the light energy that can be
imparted to the solution. Normally a medium or high
pressure lamp is placed in the immersion well. The quartz
well will transmit light >180 nm but often the light is
attenuated with various filter sleeves (Vycor > 210 nm;
Corex, >250 nm; and Pyrex, >280 nm).
345
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Water
Solution
Irradiated'
n
Lamp
Quartz
glast well
Figure 41. Quartz Immersion Well
Photochemical Reactor (Kearney
et al., 1969)
Courtesy of Springer-Verlag
The Rayonet apparatus differs from the immersion well
apparatus in that the light is provided by a bank of low-
pressure mercury or fluorescent lamps which surround the
reactor (A max. of the available lamps is 254, 300, and
350 run). Sometimes the solution is placed in a number of
cylindrical tubes which are rotated (Merry-Go-Round Reactor)
and construction materials may vary from quartz to Pyrex
glass.
Much simpler containers such as petri dishes have
also been used 'for solution photochemistry. For example,
Crosby and Tang (1969a) used a borosilicate glass baking
dish covered with a perforated clear plastic wrap (transmits
light >200 nm), when irradiating an aqueous solution
346
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With sunlight Aly and El^Dia (1971) used open petri dishes
when they irradiated a number of carBamate insecticides in
aqueous solution with germicidal lamps C254 nm). Henderson
and Crosby Q.968) used an inverted, rounds-bottom, quartz
flask for sunlight irradiation of an aqueous solution of
dieldrih.
(b) Experimental Conditions
Before photolyzing a chemical in a solvent, the researcher
must choose the experimental conditions to be used. Some of
the parameters to be considered include (1) the light source
(2) the kind of solvent, (3) a choice between aerobic or
anaerobic conditions (bubble oxygen or nitrogen) and (4) a
choice between sensitized (and what sensitizer) and
unsensitized photolysis. The conditions that are chosen
are very dependent upon the objectives of the researcher.
If one is attempting to simulate the aquatic environment,
water will probably be used. If one is interested in the
degradation pathways, the reaction may be run in methanol
or under anaerobic conditions to facilitate isolation of
photolysis products.
Commonly used solvents include water, methyl or
ethyl alcohol, hexane or cyclohexane, and benzene. Less
frequently, solvents such as acetone (solvent and sensiti-
zer, Benson £jt .aJL., 1971), aqueous tetrahydrofuran
(Grunwell and Erickson, 1973), 2-propanol (Nordblom and
Miller, 1974), diethyl ether (Tanabe fit ^J., 1974), and
347
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acetonitrile (Zepp et al., 1973) have been used. Water
is used to simulate the aquatic environment while cyclohexane
is used to approximate the surface of a leaf or an oil slick
on water. Plimmer (1970) has used methanol as a solvent
in studying the photolysis of halogenated aromatic compounds.
In the compounds studied, hydrogen from the solvent
(H-CH OH ^ 90 kcal) was substituted in the same position
formerly occupied by the chlorine atom. In water, both
hydrogen and hydroxyl substitution occurred but at a slower
rate. Thus, because the work-up of the methanol solution
is easier, methanol has frequently been substituted for water.
The solubility of some-of the test chemicals in water
precludes solution photolysis. For example, Crosby and
Moilanen (1973) photolyzed a suspension of 1 or 10 mg/1
of chlorinated biphenyl in water. Andelman and Suess (1971)
instead of photolyzing a suspension, absorbed 3,4-benzpyrene
on calcium carbonate and irradiated an aqueous suspension
of the calcium carbonate.
The light used either does or does not attempt to
simulate sunlight. High energy light is used to see if
»
a compound reacts at all photochemically. In some cases,
it even provides photoproducts observed in the natural
environment (e.g., dieldrin forms the same photoproducts
in sunlight and upon irradiation with high energy UV light).
348
-------
Less energetic light is used in an attempt to more closely
consider environmental processes. Low energy artificial
light is frequently used with high concentrations (0.1 to
1.0 g/1) of the test chemical in order to isolate and
identify the photoproducts. In many cases, these products
and the rate of formation are compared with a sunlight
photolysis at lower concentration (e.g., Crosby and
Leitus, 1973).
The .intensity of the light used has a considerable
effect upon the photolysis reaction rate and, therefore,
its measurement is necessary in order to compare experi-
mental results. However, in many cases, only the light
source is reported. When the intensity is determined, it
usually is done with a physical or chemical actinometer
(See Calvert and Pitts, 1966).
In many cases, the compound under study does not
directly absorb ultraviolet light available from sunlight.
However, in the environment, the chemical may come in
contact with a compound that absorbs light efficiently
and could donate its energy to the chemical under study
(see Lykken, 1972). Therefore, many researchers have
added photosensitizers to the photolysis solution. Both
natural (e.g., riboflavin-51-phosphate sodium (FMN))
CRosen et al., 1970) and artificial (e.g., acetone,
Crosby and Wong, 1973b and benzophenone, Rosen and
349
-------
,Siewierski, 1970), sensitizers have been used. When using
benzophenone, a hydrogen donor solvent such as cyclohexane
should not be used because it leads to the rapid destruction
of the sensitizer CRosen and Siewierski, 1970).
Most places in the environment that receive irradiation
from the sun are aerobic and, therefore, photooxidation
is often an important environmental process (Crosby, 1972b).
In recognition of the importance of photooxidation, solvent
photolysis is usually carried out in the presence of oxygen.
However, the products formed in the presence of oxygen
may be extremely complex and as a result anaerobic conditions
are sometimes used to provide insight into the product
intermediates as well as the mechanism (e.g., Plimmer et al.,
,:
1970). In some cases, oxygen must be excluded in order
to isolate intermediates that are rapidly oxidized in the
presence of oxygen. For example, Crosby and Tutass (1966) .;
found that 1,2,4-benzenetriol, a major photoproduct of
2,4-D, could only be isolated by adding excess sodium
bisulfite to the aqueous photolysis solution before photo-
lysis in order to inhibit oxidation.
(iii) Adsorbed or Thin Film Photolysis
A considerable number of chemicals, mostly pesticides,
have also been photolyzed as thin films on borosilicate glass,
quartz, or bean leaves or in an adsorbed form on paper, silica
gel, and calcium carbonate. Perhaps the first use of this
350
-------
technique was the work of Mitchell (1961). He photolyzed
141 pesticides that were adsorbed on 8" x 8" filter papers
with a germicidal lamp (254 nm), The filter papers were then
developed in a chromatographic tank to determine if the
chemical had reacted photochemically. A modern variation of
the technique is the use of silica gel chromatoplates (Cheng
et^ al., 1972; Cruckshank and Jarrow, 1973; Ivie and Casida,
1971aj Knowles and SenGupta, 1969; Rosen and Siewierski, 1972;
Slade and Smith, 1967; Smith and Grove, 1969; and Ueda et al.,
1974). The silica gel technique has been used both with and
without photosensitizers (Ivie and Casida, 1971a). The
adsorption of the chemical on silica gel may have a drastic
effect on the absorption spectra of the compound and thus
effect the amount of direct irradiation adsorbed by the
compound and the photoproducts that result (Plimmer,.1972b).
Other surfaces that have been used include sand (Newsotn and
Woods, 1973), soil (Parochetti and Hein, 1973; Wright and
Warren, 1965), and calcium carbonate (Andelman and Suess, 1971).
Both sunlight and artificial ligh_t has been used in the photo-
lysis of adsorbed pesticides.
Another technique that has frequently been used with
pesticides is solid state photolysis of thin films of the
compound on glass (Archer e£ _§!., 1972; Bandal and Casida,
1972; Baur e£ ail., 1973; Benson e± _al., 19.71; Chen and Casida,
1969; Cheng e_t al., 1972; Fishbein and Geibel, 1971; Ivie
ejt al., 1973; Ivie and Casida, 1971b; Liang and Lichtenstein, 1972;
351
-------
Lombardo et al., 1972; Wright and Warren, 1965), quartz
(Hosier e£ ail., 1969) and bean leaves CBandal and Casida, 1972;
Cheng et al., 1972; Ivie and Casida, 1971b; and Matsuo and
Casida, 1970). Normally the chemical is dissolved in a solvent
for application to the surface and then the solvent is evaporated.
The possibility of intermolecular reactions under these con-
ditions is probably quite high.
. An interesting and novel way of photolyzing chemicals in
the solid state was reported by Graham et^ a±. (1973). They
studied the photolysis of heptachlor epoxide in a KBr disk
(0.5% w/w). Both artificial light (254 nm) and sunlight were
used (same results) and the reaction was followed by infrared
absorption spectra.
, 2. Techniques Used to Determine Chemical Alteration
1 !
t a. Introduction
A chemical placed in the environment may undergo other chemical
reactions besides photochemical alterations. The exposure of a chemical to
water, soil, air, and inorganic and organic matter may catalyse many chemical
reactions, oxidation and hydrolysis being perhaps the most common. The few
s.tudies of these processes that are available have been reviewed by Rosen (1972a)
and Crosby (1969a). This section will very briefly review some of the techniques
that have been used. ,
b. Techniques Used to Study Chemical Alterations
In almost all instances, the techniques used to study bio-
degradation of a compound automatically take into account chemical alterations.
352
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However, many researchers in atudying the degradation of a chemical in simulated
environmental conditions have distinguished between chemical and biological
degradation. This is usually done by removing the possibility of biodegradation.
For example, Eichelberger and Lichtenberg (1971), in a study of the degradation
of 21 pesticides in river water, distinguished the chemical alterations by using
distilled water. Studies of the degradation of pesticides in soil usually
include a control sample that has been sterilized (by autoclaving or treatment
with ethylene oxide or potassium azide, or by y irradiation). Kaufman et al. ,
1968) found that autoclave treatment was the most effective for retarding the
biodegradation of amitrole in silty clay loam (see Figure 42).
% C'« evolved
os CI402
80
60
Nonsterile
Ethylene oxide
.Autocloved
Figure 42, Effect of Soil Sterilization on
Amitrole Degradation in Hagerstown
Silty Clay Loam (Kaufman et al., 1968)
Courtesy of Weed Science Society of
America, publication of Weed Science.
353
-------
In a modification of the sterile soil technique, Hance (1967)
Incubated at 107°, 95°, and 85°C a number of herbicides in an aqueous slurry
with two soils (silty loam, 22.6% clay, 3.45% organic carbon, pH 6.2 in 1:5
aqueous suspension; calcareous silty loam, 33.6% clay, 3.09% organic carbon,
pH 7.5 in 1:5 aqueous suspension). The elevated temperatures were used to
eliminate the possibility of biological action and also to accelerate any
processes that were'occurring in order to reduce the length of the experiments.
Conventional studies of the hydrolysis rate of various pesti-
cides in water have been reported by Gomaa and Faust (1972) ; Faust and Gomaa
". '
(1972) ; Aly and El-Dib (1972) and Exner e£ al. (1973). The experimental pro-
cedure consists of exposing the pesticide (organophosphorus compounds, carba-
mates, etc.) to an aqueous solution buffered to various pH levels (both acidic
and basic) and then analyzing for hydrolysis products. Also, the effect of
different temperatures is sometimes determined.
Pliflimer et_ al. (1967) in a study of the degradation of amitrole
found similarities between the mode of breakdown in hydroxyl-radical systems
(Fenton's reagent) and soil. Fenton's reagent consists of a 0.2M solution
of ferrous sulfate in 0.1M sulfuric acid (60 ml) and 1% hydrogen peroxide
(100 ml). The system might be used to provide some insight into chemical
degradative mechanismsin soil.
3. Analytical Procedures
a. Isolation and Detection of Degradation Products
As Table 41 shows, a variety of analytical techniques have
been used with studies of the photochemical degradation of chemicals. These
detection, isolation, and identification techniques vary with, the particular
354
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compound and reaction conditions. Thin-layer chromatograph (TLC) appears to
be the most commonly used technique for isolation despite the .frequent diffi-
culty of overlapping bands (Crosby, 1969b). The method is an obvious choice
when the photolysis is carried out on silica gel chromatoplates. The combina-
tion of the two techniques (photolysis on chromatoplates and TLC work-up)
provides an extremely fast technique for determining photoreactivity. For
example, Ivie and Casida (1971a) screened the photosensitizing ability of
14
175 unlabelled pesticides against each of six C -labelled insecticide
chemicals using the combination of silica gel chromatoplate photolysis - TLC.
A similar technique, only using paper chromatograph, allowed Mitchell (1961)
to screen the photoreactivity of 141 pesticides. The technique used to detect
the spots on the chromatogram varies for each chemical but may include
(1) exposure to various light sources (254 or 366 run), (2) exposure to
chromogenic agents (Mitchell, 1961) and, or (3) use of radiotracer techniques
when radiolabelled matter is available for testing. When small amounts or
concentrations are being used, radioanalysis is most desirable because of
\
the .low sensitivity (nanogram levels) that can be achieved (Casida, 1969).
The combination of TLC with radiotracer assay is frequently used by pesticide
photpchemists (see Table 41) perhaps because of the availability of the
radiolabelled material.
Gas chromatography (GC) has also been found to be satisfactory
for the isolation and detection of volatile, thermally stable, compounds formed
in photochemical reactions (see Table 41). In some cases, GC is used to
quantitate compounds isolated by TLC. The sensitivity is dependent upon the
detector which is in turn dependent upon the chemical being studied [minimum
-7 -13
detectable quantity can vary from 10 g (thermal conductivity) to 10
(electron capture)(Karasek and Laub, 1974)].
355
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Other techniques that have been used for isolation and/or
quantification include (1) UV absorption (Andelman and Suess, 1971; Porter, 1973;
Redemann and Youngson, 1968), (2) colorimetric analysis (Aly and El-Dlb, 1971,
1972; Zepp, 1973); (3) infrared analysis (Graham et al., 1973); (4) polarographic
analysis (Exner ._et _al. , 1973) and (5) bioassay (Parochetti and Hein, 1973;
Ueda, 1974; Wright and Warren, 1965). These methods have the advantage of
being relatively fast and inexpensive to run, but are usually only good for
analysis of the parent compound and one or two degradation products.
b. Identification of Degradation Products
Although radiotracer analysis combined with chromatographic
separation provides extensive insight into the degradation rates and numbers
of products that, might occur, the technique provides little help with
structure identification (Crosby, 1969b). Gas chromatography has similar
problems.. In many cases, identification is only possible by cochromatography
of an authentic compound from synthesis (Casida, 1969). When the degradation
product is not an obvious one, physical tools, such as infrared spectroscopy,
mass.spectrometry, and nuclear magnetic resonance (NMR) spectrometry, are
used. These techniques require the isolation of the pure compound in fairly
large, amounts, (ug-mg), especially for NMR. However, with photochemical and
chemical degradation studies, it is often possible to run preparative reactions
in order to generate enough material for identification. For example, Crosby
and coworkers.(Crosby and Leitis, 1973; Crosby and Hammadmad, 1971) have used
solutions concentrations of 50 mg/1 to 1 g/1. In fact, many laboratory studies
are undertaken in model systems, that do not reflect actual environment
~it
conditions, in order to facilitate the isolation and identification of
products that might be formed in the environment (Rosen, 1972b).
356
-------
A. Evaluation of the Techniques
a. General
The laboratory techniques which, have heen previously described
are oriented at understanding chemical and photochemical alterations of chemicals
In the environment. Although the importance of these nonmetabolic processes to
degradation of contaminants in the atmosphere has been well documented, the
magnitude of these processes in soil and water media relative to biological
processes is for the most part unknown, (e.g., pesticide photolysis - Rabson
and Plimmer, 1973). This is undoubtedly due to the fact that both metabolic
and nonmetabolic processes usually only represent normal chemical transformation
(Crosby, 1969a) and, therefore, the assignment of a reaction in the soil and
aquatic environment to either a metabolic or nonmetabolic process is most
difficult. This appears to be particularly true with photooxidations where
the photodegradation products often prove to be structurally identical with
products of oxidative degradation in living organisms (Crosby, 1972b). Thus,
these nonmetabolic processes have been demonstrated to be important in only
a few cases where an unusual and environmentally stable alteration product
is formed or where attenuation of some parameter, such as light intensity
or pH, has altered the rate of degradation under natural conditions. This
is not to say that these processes are not important; just that their magnitude
of Importance Is difficult to demonstrate.
The chemical and photochemical alterations of a large
number of compounds, mostly pesticides, have heen studied in model systems
in order to identify products that might be formed in the environment and
to determine the rate of the degradation process. This section will
157
-------
discuss the factors affecting results obtained in model systems and consider
the extrapolation of these model results to processes in the actual environment.
b. Factors Affecting Chemical and Photochemical Degradation
(i) Light Wavelength
As noted earlier the wavelength of the light source can
i
have considerable affect upon the reaction that occurs. Many
researchers have used a variety of light sources including
low pressure mercury lamps (254 nm) , >290 nm artificial light,
and sunlight. Sometimes the results are the same (e.g.,
Cheng .et al.., 1972; Benson et ^L, 1971; Chen and Casida, 1969;
Henderson and Crosby, 1968), but in many cases drastic
differences are noted. For example, Henderson and Crosby (1967)
reported that the dechlorination of dieldrin was wavelength
dependent. Rosen (1967) detected five photoproducts of
diphenamid using 254 nm light, while in sunlight the compound
.V
was,stable. Moilanen and Crosby (1972) studied the photo-
decomposition of bromacil with sunlight. Although it had been
reported that a 1 ppm. aqueous solution of bromacil was completely
decomposed in 10 minutes with 254 nm light, Moilanen and Crosby
(1974) detected little photodecomposition using sunlight wave-
^t
lengths. Liang and Lichtenstein (1972) found rapid decomposition
of azinphosmethyl in water using 254 nm light, but no decom-
position using yellow (589 nm) or red (653 nm) light. Redemann
358
-------
and Youngaon Q-968) suggested that "a change of light sources
can result in a change in relative photolysis rates of parent
compound and intermediate products". They photolyzed
6-chloropicolinic acid with sunlight and a mercury arc lamp
(254 nm) and detected 6-hydroxypicolinic acid as an inter-
mediate with 254 nm light, but did not detect the compound
with sunlight. This result was attributed to the greater
absorptivity and thus possible photochemical reactivity of
6-hydroxypicolinic acid in the wavelength range available
from sunlight.
(ii) Reaction Media
The reaction media used in a photochemical experiment
can also affect the results. Solution photolysis will reduce
the possibility of intramolecular reaction, but many solvents,
especially H-donor solvents, may react with the chemical being
studied. Photolysis of halogenated aromatic compounds in a
H-donor solvent, such as methanol, hexane or cyclohexane,
results ,in hydrogen substitution at the halogen position. The
possibility that this represents what occurs in oil slicks
(Ca-C22 fatty acids, Crosby, 1972a) on the surface of water
or on the surface of a leaf (Crosby, 1969b) seems feasible
but has never been proven, in water, both hydrogen and
hydroxyl substitution may occur at slow rates (Plimmer,
1970; Crosby, 1972a). The mechanism involved is quite
important because solvents, such as methanol, are often
359
-------
used in place of water due to the solubility properties of the
. j
compound being photolyzed (e.g., glimmer eit^ ad., 1973). Both
chlorine dissociation of the excited molecule to a free radical
(Plimmer, 1970) and nucleophilic displacement of the chlorine
with a solvent or substrate molecule (Crosby et^ al_. , 1972;
Crosby, 1972a) have been postulated. The free radical mechanism
is theoretically possible in methanol (H-CH20H = 92 kcal;
H ~~\O/ = 104 fccal/mole), but in water (H-OH = 118 kcal)
the energetics make the reaction by a free radical mechanism
highly unlikely. Recently, Nordblom and Miller (1974)
have demonstrated that a free radical mechanism does not seem
feasible even with some H-donor solvents (cyclohexane, ethyl
ether). Thus, extrapolations of methanol results to reactions
in water should be done with some caution.
Adsorption of the chemical on silica gel or formation of
a thin film on glass or leaves may affect the adsorption spectra
of the chemical and thus affect the ability to directly derive
energy from the incident light. Plimmer (1972b) has noted
quite sizable Xmax shifts for compounds adsorbed on silica gel.
Rosen (1972b) reported completely different results for a thin
film on glass photolysis of a synthetic pyrethroid as compared
to photolysis on silica gel. Whether this can be explained
by a Amax shift is unknown. Rosen (1972b) has concluded that
"ifwe cannot, in some cases, extend our results from silica
gel to glass, extension to soil and leaf surfaces may be even
360
-------
more risky." Tor example, Parochetti and Hein (.1973) noted
that trifluralln vas reported to photodecompose on glass
surfaces, in organic solvents, in aqueous solutions (Crosby
and Leitis, 1973), and as a vapor, but their results provide
no positive evidence of photodecomposition on a soil surface.
(iii) Sensitizers
In cases where direct irradiation results in a relatively
slow reaction or no reaction at all, sensitizers are added to
determine if the compound can react photochemically by indirect
excitation. Since natural sensitizers, such as riboflavin
and chlorophyll, are available in the environment, this
process is possible in nature. The sensitized photodecomposi-
tion and the photosensitizer activity of a number of pesticides
has been reviewed by Ivie and Casida (1971a, b) and Lykken (1972).
The effectiveness of the sensitizer is dependent upon,
among other things, the wavelength at which it absorbs lightf
the efficiencies of the conversion from its singlet to triplet
state, the efficiency with which it transfers energy to
the acceptor molecular, and its concentration (see
Turro, 1967). Therefore, the choice of sensitizer can have
considerable impact on the rate of reaction. For example,
rotenone is 100 times more effective as a sensitizer of the
photoisomerization of dieldrin than is benzophenone (Lykken, 1972)
A typical study which employed the use of sensitizers, is
the photolysis of ethylenethiourea (ETU) (Ross and Crosby, 1973).
Photolysis of ETU (Xmax = 240 nm) in deionized water revealed
361
-------
no loss, while, in the presence of acetone or rihoflavin, 95%
of a .064 ppm aqueous solution was lost in 4 hours.
(iv) Hydrogen Ion Concentration
The pH of the reaction solution can have a considerable
effect on the photochemical or chemical process that takes
place. 'For example, Lanford e£ a^. (1973) found that the
quantum yield decreased linearly with increasing pH over the
range 2-12 in the photodecomposition of copper complexes of
NTA irradiated with 350 nm light (Rayonet unit). Crosby and
Leitis (1973) reported that the principal product from photo-
lysis of trifluralin was completely different under acidic vs
basic conditions (see Figure 43).
H7C3 C3H7
trifluralin
CF:
C2H5
Figure 43. Basic and Acidic Photolysis of
Trifluralin (Crosby and Leitis, 1973)
362
-------
Crosby and Wong C1973b) also noted that the rate of, 2,4,5-T
photolysis was somewhat more rapid at pHS than at pH3,
The hydrogen ion concentration has been sho^n to have a
i **
drastic effect on the rate of hydrolysis of carhamates,
organophosphorus compounds, amides, etc. Aly and EL-Dib 0-972)
reported that two carbamate insecticides CSevin, baygon) were
rapidly hydrolyzed under neutral to alkaline conditions.
Faust and Gomaa (1972) studied the chemical hydrolysis of
organic phosphorus pesticides. They noted that the basic
hydrolysis occurs in a S 2 fashion by nucleophilic substitution
in which OH attacks the P and substitutes for a R-0 group
(breaks P-0 bond). In contrast, acid conditions cause the
R-0 bond to break (see Figure 4.4). In Faust and Gomaa's (1972)
extensive review, they demonstrate that the hydrolytic rate
is pH dependent as well as dependent upon the nature of
substituents. Similar correlation between pH and hydrolysis
was found for azinphosmethyl by Liang and Lichtenstein (1972).
The rates of hydrolysis can fluctuate greatly. For example,
Zepp jet al. (1974) reported that the hydrolysis half-life (25°C)
of the butoxyethyl ester of 2,4-D was 9 hours at pH 8 compared
to one year at pH 5.
363
-------
(RD)2 r-
'?'Z Or
(S)
0
(R0)2 -P-OH + R'OH
CS)
.+ Q(S)
> (R0)2 - P - OH + R'OH
(S)
Primary ester
Figure 44. Acid and Base Catalyzed Hydrolysis
of Organophosphorus Pesticides
(Faust and Gomaa, 1972)
(v) Other Factors
Chen and Casida (1969) have suggested that, besides the
factors noted previously, the purity of the irradiated materials
'and1 the methods of determining the degradation products may
have a considerable affect on-the results that are reported.
Furthermore, Baur jst_-.al. (1973) have shown that concentration
may be important in their film photolysis.
c. Extrapolation of Laboratory Results to Field Conditions
The ultimate evaluation of a technique used to study chemical
; photochemical alterations in the environment is a comparison of results
364
-------
obtained in the laboratory with actual environmental results. The number of
cases where indisputable field results- are available is rather limited. Many
studies of pesticide residues in the environment have been carried out, but
provisions have not been made for volatilization nor have nonmetabolic
reactions been distinguished from metabolic reactions. However, there are
two examples involving photochemical reactions which are relatively
unambiguous - dieldrin and pentachlorophenol. In addition, information on
the photolysis of FCB's has been reviewed and compared to environmental
monitoring data.
(i) Photolysis of Dieldrin to Photodieldrin
Roburn (1963) reported the presence of an unknown
contaminant on samples of herbage sprayed with dieldrin. The
substance was later identified as photodieldrin Csee Figure 45
by Parsons and Moore (1966), Robinson et al, (1966), and
Rosen e_t ji]L. (1966). In a monitoring survey, Robinson and
Ci
» A
Photodieldrin
Dieldrin
o
Dechlorinated Product
Figure 45. Photolysis of Dieldrin
365
-------
coworkers (.1966) found that phctodieldrin occurred in very small
amounts (16;1 to 1000;! compared to dieldrin) in the environment
(e.g., cooked meals, human fat, shag eggs). Photodieldrin was
also detected by Henderson and Crosby C1967) on corn leaves
treated with dieldrin and exposed to sunlight.
The photoproduct has been produced by 254 nm light
irradiation of a thin film deposited on filter paper (Robinson,
et al.,' 1966) or glass plates (Rosen et al., 1966) and by
sunlight irradiation of an aqueous solution (Henderson and
Crosby, 1968), a thin film deposit on a glass plate (Rosen,
et al., 1966), or on various media with a sensitizer (Ivie
%
and Casida, 1971b). Rosen and Carey (1968) formed photo-
dieldrin in 75% yield by benzophenone-sensitized photolysis
of dieldrin in benzene with 268-356 nm light for 21 hours.
Irradiation of dieldrin in hexane with 254 nm resulted in the
formation of a dechlorination product that is not detected in
the field and is not formed at light wavelengths greater
than 260 nm.
That dieldrin is photoaltered in the environment is, of
\J
itself, somewhat unusual. In hexane solution, dieldrin has
an absorption band appearing at 260 nm and reaching a maximum
at 215 nm. Since the compound is transparent to wavelengths
greater than 260 nm, it should not be directly excited by
- i
sunlight because wavelengths below 285 nm do not reach the
366
-------
earth's surface (Crosby, 1969b). Nevertheless, dieldrin in
distilled water, adsorbed on paper, or in a thin film on glass
is converted to photodieldrin by sunlight. Two explanations
are possible, Rosen (1971) has cited a report which suggests
that the solar flux reaching the earth between 200-285 nm is
significant, although quite small. Rosen suggests that
this energy is concentrated in the 200-220 nm range since the
absorption coefficient of ozone drops off sharply at 220 nm
and therefore "it is not surprising that they [cyclodiene
insecticides] undergo appreciable photolysis in sunlight "
(Rosen, 1971).
Another possibility is that the wavelength of absorption
is shifted in different media making direct excitation possible
(for examples, see Plimmer, 1972h). Unfortunately, no one has
measured the ultraviolet absorption of dieldrin in water. This
i
possibility of wavelength shift is somewhat supported by the fact
that dieldrin in hexane does not form the dechlorinated
product, which is favored in hydrogen donor solvent at wave-
lengths less than 260 nm (Henderson and Crosby, 1967).
Another explanation is that dieldrin is photosensitized
in the natural environment (Rosen and Carey, 1968; Ivie and
Casida, 1971b), but this would not explain the photodecomposi-
tion of dieldrin in distilled water (Henderson and Crosby,
1968).
A complicating factor with photodieldrin is that it can
be formed microbially (Matsumura and Boush, 1967). One would
367
-------
expect that the photodieldrin formed on the surfaces of leaves
is probably the result of photolysis, but the rate determining
process in soil and water is unknown.
(ii) Photodegradation of the Sodium Salt of Pentachlorophenol
The sodium salt of pentachloropb-enol (PCP~Na) is used in
Japan as a control of barnyard grass in rice paddys. This
substance is highly toxic to fisn., hut the toxicological
activity in the water disappears several days after treatment.
However, the fish toxicity can be prolonged by covering the
surface of the field water with sheets (ffunakata and Kuwahara,
1969) which is good evidence of the photodegradation of
PCP-Na by sunlight. In order to isolate and identify the
degradation products, Munakata and Kuwahara (1969) irradiated
with sunlight 1 Kg of PCP-Na in 50fc of water. In ten days
the solution turned purple and the PCP-Na content decreased
by 50 percent, which is qualitatively in good agreement.with
field data. Unfortunately, a quantitative comparison between
field and laboratory results 'of PCP-Na loss and degradation
product formation is not available.
(iii) Photolysis of Polychlorinated Biphenyls
PCB's provide a good example of the problems that arise
in attempting to extrapolate laboratory photolysis results to
behavior in the environment. Safe and Hutzinger (1971) found
that 2,4,6,2',4',6'-hexachlorobiphenyl was readily dechlorinated
to lower chlorinated isomers when irradiated with Xmax 310 nm
light in hexane. Upon irradiating 2,2',5,5'-tetrachlorobiphenyl
' I
under similar conditions (the solution is degassed) only 34%
368
-------
of the starting material remained af tejr 41 hours (Hutzinger
eJLJii/» 1972). The hexa~ and octachlorobiphenyl were
extremely photochemically labile under those conditions, much
more so than the tetra isomer. Photolysis in hydroxylic
solvents at pH 9 yielded hydroxyl and carboxyl products
(Hutzinger e_t al., 1972). Ruzo et al. 0-972) reported the
stepwise reductive dechlorination of 3,4,3',4'-tetrachloro-
biphenyl and 4,4'-dichlorobiphenyl in hexane under 300 nm
irradiation. Herring e_t al. (1972) irradiated Arochlor 1254
in hexane, water and benzene (all the solutions contain 1%
acetone) with 254 nm light and sunlight. Gas chromatographic
analysis showed that some of the peaks increased (due to
reductive dechlorination of higher isomers) and the degrada-
tion was fastest in hexane, then water, and slowest in benzene.
Crosby and Moilanen (1973) irradiated a variety of chlorobi-
phenyls (di, tri, tetra) in aqueous suspension with the
sunlight-simulating, laboratory photoreactor. They isolated
both reduced and hydroxylated product. These authors
concluded that "photodecomposition of the lower chlorinated
isomers in both polar and nonpolar solvents reveals the
operation of environmental mechanisms which could effectively
degrade these widespread contaminants." They further suggest
that "the lack of lower chlorinated biphenyls from most
environmental samples is suggestive of an important role of
369
-------
photodegradation in the environmental fate of PCB's" (Crosby
and Mpilanen, 1973), Nordblom and Miller (1974) photolyzed
4,4'-dichlorobiphenyl in degassed 2-propanol and methanol
With 310 nm light and formed 4-chlorohiphenyl. In ethyl ether,
cyclohexane, or acetonitrile no photoreduction occurred and
the authors suggested that this data does not support a
homoly'tic free radical mechanism. Ruzo et^ a]U (1974) studied
the photolysis rates of six tetrachlorobiphenyl isomers in
hexane and methanol solution. The rate of reaction does not
follow the magnitude of the molar extinction coefficient. The
order in which the chloride atoms are cleaved is ortho>meta»
para. In summary, the chlorinated biphehyls seem to react
photochemically with >290 nm light and the higher isomers would
seem to be more labile than the lower ones, although Crosby
and Moilanen (1973) have argued that the lack of lower isomers
in .the environment would suggest a photochemical process.
Nisbet and Sarofim (Panel On Hazardous Trace,Substances,
1972) reviewed the transport and fate of PCB's in the environ-
ment. They note that the following processes should affect
the PCB isomer ratio in the environment.
Table 40«,i, Environmental Transport Processes of PCB's
(Panel on Hazardous Toxic Substances, 1972)
Evaporation, codistillation, etc. - reduce lower isomers near
point of release
increase lower isomers remote
from the point of release
Photolysis - reduce higher isomers
Metabolism & excretion - decrease lower isomers
370
-------
The ratto of higher isomers of PCB's increases downstream in
some Wisconsin rivers. Kaiser and Wong (1974) have demonstrated
that the less chlorinated isomers can be utllizated by
bacteria. Nisbet and Sarofim concluded that the lack of lower
isomers in samples from relatively contaminated areas is due
. to metabolism or greater mobility.
In conclusion, the importance of PCB's photolysis as an
environmental degradation process has not been well demonstrated.
d. Summary
The previous discussion has pointed out our lack of under-
standing of photochemical and chemical alterations as environmental processes
in soil and water. A large number of studies have demonstrated that
numerous chemicals will be altered by light of wavelengths similar to sunlight
(Plimmer, 1970; Crosby and Li, 1969). However, rarely has the relative
magnitude of photochemical processes compared to other environmental degrada-
tion processes been determined. With the exception of reactions taking place
in water, other non-photolytic chemical reactions catalyzed by air, heat,
soils or dusts have not received a great amount of attention (Crosby, 1969a).
The ubiquitous nature of water makes it an important medium for chemical
reaction and considerable study has been undertaken of hydrolysis processes
which take place in water. These studies have generally been limited to
compounds containing esters, amides, and carbamates, although classical
solvolysis reactions with heptachlor have also been noted (Rosen, 1972a).
Extrapolation of these results to the environment is facilitated by the fact
that the major parameter affecting the rate of hydrolysis is the pH, an
371
-------
easily determined parameter, both, in the laboratory and environment. In
contrast, in order to extrapolate photochemical results ±n the laboratory
to reactions in the environment one needs to know the sunlight intensity and
wavelength and the effect of the environmental media on light absorption and
"" V
quantum yield, presence of photosensltlzers, etc. With, the aforementioned
1 i
in mind, the following conclusions are reached.
(i) Photochemical Studies
Until the Importance of photochemical processes in nature
is somewhat better understood, studies of the photochemical
reactivity of a compound, in terms of a routine evaluation
the compounds environmental persistence and fate, should only
be performed following biological and chemical degradation studies.
This is recommended since we are largely unable to apply the
results that are generated. In most cases, when a photo-
degradation product is formed with the techniques used, the
best application of the results is a monitoring program to
i
see if the compound-is actually formed in the environment.
In addition, it seems reasonable that more studies should
be undertaken under conditions which are likely to be important
in the environment. For example, atmospheric photolysis Is
^
probably the most Important photochemical process in the environ-
,1
ment for compounds with measurable vapor pressures, and yet, so
far, only one study has appeared where photolysis of a pesticide
in the vapor phase has been examined (Crosby and Moilanen, 1974). In
372
-------
addition, air monitoring of potential photodegradation products
is not available. This lack of experimentation under atmos-
pheric conditions is probably due to the experimental difficulties
involved in the vapor phase photolysis of low volatile sub-
stances. However, before the solvent and thin film techniques
described in this section can be substituted for vapor phase
studies, some correlation between the results needs to be
established.
When a study of a chemical's photoreactivity is undertaken
the experimental conditions should be varied as much as is
practical in order to consider the various possible photochemical
reactions. Sunlight and/or artificial light containing no wave-
lengths less than 290 nm should definitely be used. Light in
the 300 to 400 nm range is particularly important. If the
compound does not react with the low energy light sources, it
might be desirable to use 254 nm light in order to determine
whether the compound is at all light sensitive. The intensity
of the light source should be measured and some type of rate
measurement (quantum yield or at least a product study with
time) should be determined. The reaction conditions should
Include dilute solutions in a variety of solvents (hexane,
methanol, water at acidic and basic pH) as well as thin films
on glass and absorption on silica gel, soil, or other solid
material. Photolysis in water is particularly important since
it is a major environmental medium, although isolation and
identification of the products, which are usually very polar,
373
-------
is often very difficult. The possibility of sensitized photo-
chemical alterations should also be investigated by running
a reaction in solution (or perhaps on silica gel) with a
triplet sensittzer.
The interpretation of the results obtained must be
cautiously applied. If a compound does not react under any
of the conditions described above, it is pretty safe to assume
that photolysis is not an important degradation process for that
compound. If the compound undergoes change when exposed to >290 run
light,"7 one may conclude that photolysis may be an important
degradation process. The degrees of importance should be
determined by monitoring, field, and kinetic data.
(11) Chemical Studies
Determination of the hydrolysis rates of compounds at a
variety of pH levels is extremely important, especially for
compounds that are likely to reach the aquatic environment.
However, the number of compounds that should undergo this type
of testing can be considerably reduced by considering the
chemical structure of the compound. Esters, amides, and
carbamates are obvious candidates for testing. Compounds that
f.
have good leaving groups, (e.g. halogens) located at positions
that would stabilize a carbonium ion (allylic, benzylic, etc.)
also should be tested.
The rates that are determined In distilled water may be
* Theserconditions are basically those recommended by a panel of a NAS-AEC
sponsored Workshop on Photoalteration of Pesticides, see Raason and Plimmer,
1973.;
374
-------
quite different from rates in natural xater. Such processes
as adsorption may retard the hydrolysis rates, while on the other
I i
hand, some contaminants such as Cu may catalyze the process
(Crosby, 1969a).
With the exception of atmospheric reactions, other straight
chemical processes in the environment have not received a great
deal of attention. However, many of these processes (especially
in soil) should be identified by the sterile blank that is run
with the biological test.
5. Cost Analysis
(a) Photolysis Studies
The apparatus necessary for the techniques described
previously is relatively inexpensive. A complete set of
photolysis equipment can be purchased for $2^3,000. Therefore,
most of the expense is due to labor and chemical analysis.
Because of the degree of difficulty and the analytical procedure
will vary with each compound tested, the following cost figures
are only approximations. The estimations have been divided
into (1) preliminary studies where a determination of photo-
chemical loss of the compound will be provided and (2) intensive
studies where major pathways are determined.
In the preliminary test, we have assumed that the compound
will be studied in water, methanol, and hexane solvents, on
silica gel chromatoplatea, and as a thin film on glass. The
375
-------
analytical method vjLll he gas chromatography-, TLC, or some
other technique specific for the parent molecule*
One Compound Study Only
Labor MS/level ($15K/yr=salary + benefits)«
1 man/month $1,250
Overhead (125% of Professional Services) 1,560
Photolysis Equipment 3,000
TLC 500
Miscellaneous Supplies 500
, per compound $6,710
20 Compounds/year for 5 years
Labor MS/level. 5 man/years $ 75,000
Overhead 94,000
Photolysis Equipment 2,500
Gas Chromatograph 8,000
Miscellaneous Supplies 2,500
$181,000
, per compound $ 1,810
In the intensive study, we have assumed that the breakdown
products were isolated by TLC or gas chromatography and were
identified by mass spectrometer or GC-MS. Radiolabelled material
will probably not be used unless it has been previously synthesized.
376
-------
One Compound Study
Labor MS/Level 4 man/month. $5,000
Overhead (125% of Professional Services) 6,250
Buy GC-MS analysis (or IR, MS, NMR) 2,000
Miscellaneous supplies 500
per compound $11,750
10 Compounds/year for 5 years
Labor MS/Level 5 man/years $75,000
Overhead 9A,000
GC-MS (or IR, MS, NMR) depreciated over 10 yrs. 50,000
Miscellaneous supplies 2,500
$271,500
per compound $ 5,500
(b) Hydrolysis Studies
The hydrolysis breakdown products are much more predictable
(e.g., ester -> acid and alcohol) and, therefore, a sophis-
ticated qualitative analysis laboratory should not be required.
Again the costs are divided into (1) a preliminary study and
(2) an intensive study.
For the preliminary study, we have assumed that the
hydrolysis rate of the compound will be studied at acid, neutral,
and basic pH and only the loss of the parent molecule will be
measured.
377
-------
One Compound
Labor MS level (15K/year) « 0.25 man/months $ 310
Overhead 390
Miscellaneous Supplies 200
per compound $ 900
150 Compounds/year for 5 years
Labor 5 man/year (.lab or /compound = .35 man/wks) $75,000
Overhead 94,000
Gas Chromatograph 8,000
Miscellaneous Supplies 5,000
$182,000
per compound $ 240
The intensive study would involve determining the hydrolysis
half-life at a variety of temperatures and pH's.
One Compound
Labor 0.5 man/month $ 620
Overhead 775
Miscellaneous Supplies 200
per compound $ 1595
50 Compounds/year for 5 years
Labor 5 man/year (0.25 man/month/compoundj $ 75,000
Overhead 94,000
Gas Chromatograph 8,000
Miscellaneous Supplies 5,000
$182,000
per compound $ 690
378
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VI. THE INTERCONVERSION OF ALKYLATED AND INORGANIC FORMS OF CERTAIN METALS
AND METALLOIDS
A. Introduction
Elemental contaminants are introduced into the environment from various
sources which Include liquid and solid waste from animal and man, mining and
industry, and agricultural chemicals. These elements also exist in one form or
the other in the environment as natural constituents of the earth's crust. Elements
which are of major concern today include: mercury, arsenic, lead, copper,
cadmium, chromium, nickel and vanadium. These exist in the environment in
many different forms as air pollutants, contaminants of water and soil, and
as residues in food.
Pollution of our environment by mercury and its conversion to poison-
ous methyl mercury by microorganisms has caused concern over the effect of
such trace metals. The toxic metals in the environment may present a more
insidious problem than pollution by organic chemicals because metals, unlike
organic chemicals, cannot be degraded to innocuous products such as carbon
dioxide and water. The degradation of organometallic compounds by microorganisms
generally leads to the liberation of toxic elements in addition to other end
products (Nelson ejt al., 1973; Von Endt et al., 1968). Metals can undergo changes
in valance state, be converted into organometallic form or be mobilized
from one environment to another. The interconversions are generally rever-
sible and result in steady state concentrations of various forms in the
environment.
Heavy metals have been known to be toxic to humans, animals and
plants. The form in which the metal occurs (e.g., pure metal, Inorganic
379
-------
compound, or organometallic compound) strongly influences its toxicity. A
small disturbance in the dynamic biological and chemical cycling of these
toxic elements can have considerable impact on their concentrations in the
environment.
In view of the toxic nature of certain elements, it becomes important
to know the fate of these chemicals in the environment. In the case of metals
and metalloids, the mechanisms involved in their transport and transformation
from one form to another are extremely important from the point of view of
environmental pollution. In reviewing the test methods which have been used
to study the transformation and fate of heavy metals and their compounds in the
environment, the organization used in previous sections has been slightly modi-
fied. Realizing the differences in the type and nature of the reactions involved
in heavy metal transformations, it is necessary to review first the possible
chemical and biochemical transformation of selected elements in the environment.
B. Chemical and Biochemical Transformation of Metals and Metalloids
Microorganisms, especially bacteria and fungi, play an important role
in catalyzing the modification, activation, or detoxification of compounds of
the toxic metals and metalloids. These interconversions could yield a product
which may be more or less toxic to higher organisms. The pathways
of elemental and organometallic forms of heavy metals are the following:
i
1. Valance Changes:
Trace metals can exist in the environment in several valance
forms and each form may differ in its toxicity. By means of oxidative and
380
-------
reductive reactions catalyzed by biological or chemical means, the relative
abundance of each species is controlled. For example, mercury can exist in
the following three forms (Wood, 1974):
Kg2* -p-*- Hg2+ + Hg°
The relative amounts of each form will depend on the solubility or the amount
of dissociation of the mercuric compound formed and the extent to which
metallic mercury enters or leaves the system. Vaporization of elemental
mercury will shift the reaction to the right, whereas an increase in the
concentration of Hg° either due to return from the atmosphere or due to
addition from external sources will shift the equilibrium to the left.
These transformations are also catalyzed by microorganisms (Wood, 1974).
Microorganisms are able to reduce the pentavalent form (arsenate)
of arsenic to the trivalent form (arsenite) (McBride and Wolf, 1971).
On the other hand, a number of microorganisms are able to oxidize arsenite
to less toxic arsenate (Mendal and Mayersak, 1962; Turner, 1954; Turner
and Legge, 1954).
OH
I 2.
HO - As - OH ( » As - OH
II II
0 0
381
-------
2. Methylation:
Biological methylation plays an important role,, in the transport of
toxic-metals and metalloids. Researchers have reported methylation of mercury,
arsenic, selenium and tellurium. Conversion of inorganic forms of metal or
metalloids to methylated forms is sometimes employed by microorganisms as a
detoxification mechanism.
Mercury: Jensen and Jernelov (1969), and Fagerstrom and
Jernelov (1971), have reported formation of both monomethyl and dimethyl
mercury in lake and river-sediments. Bisogni and Lawrence (1973) have
pointed 'out that biological wastewater treatment systems and anaerobic
digesters may provide excellent environment for methylation of mercury.
Wood and coworkers (1968) have investigated the biochemical pathways for
synthesis of methylmercury compounds; These investigators have also
reported that transfer of the methyl group from Co3+ of methylcobalamine
to Hg?+ could be catalyzed nonenzymatically under mild reducing conditions.
DimethyImercury resulting from microbiological transforma-
tion can be transported to the atmospheric environment due to its volatility,
where it may be subjected to photolysis. According to Corner and Noyes (1949)
the photolysis of dimethyImercury might proceed as follows:
Hg (CH3)2 -^ CH3 + .HgCH'3
The monomethyl mercuric radical can further undergo decomposition to give
rise to metallic mercury and methyl radical. The methyl radicals probably
>
abstract hydrogen or recombine to form methane or ethane, respectively
(Wood, 1974).
382
-------
Spangler and coworkers (1973a, 1973b) have reported evidence
for the microbial degradation of methylmercury in lake sediments. It was
suggested that organisms responsible for degradation of methylmercury may
serve a useful purpose in maintaining the environmental methylmercury concen-
trations at a minimum. The bacterial species isolated in pure culture caused
conversion of methylnercury into volatile elemental mercury (Hg ) and methane.
The biochemical, chemical and photochemical transformations
of mercury compounds set up a dynamic system in the environment. The bio-
logical cycle for mercury suggested by Wood (1974) is shown in Figure 46.
Figure 46. The Biological Cycle for Mercury
(Wood, 1974)
Courtesy of J.M. Wood, Science, 183C4129), 1049-52
Copyright 1974 by the Amer. Assoc. for the Advancement
of Science
383
-------
Arsenic; Arsenic, similar to mercury, has also been found to
undergo methylation. Microorganisms, particularly bacteria and fungi, have
been reported to play active roles in these conversions (McBride and Wolf,
1971; Challenger, 1945; Cox and Alexander, 197 ). The biochemical pathway
leading to the formation of methylated forms of arsenic have been investigated
by McBride and Wolf (1971). In the scheme proposed, arsenate (+5) is first
reduced to arsenite (+3), which is methylated to form methylarsenic acid.
The latter component' is reductively methylated, forming dimethyl and trimethyl
arsine. Because of7their volatility, alkylarsines may appear in the atmospheric
environment where they may be rapidly oxidized. Kearney and Woolson (1973)
have reported alteration of organoarsenic compounds via two pathways: (1) an
oxidative pathway leading to C-As bond cleavage and (2) a reductive pathway
leading to alkylarsine production. The biological cycle of arsenic is shown
in Figure 47.
Air
Water
CH]
I _ I _
CH, As' CH, * H As1 CH,
Trimethylarsine Dimcthylarsine
-Bacteria
Molds.
OH
\
As3*-OH-
HO As5* OH-
Nr. II f
Bacteria " Bacteria
0 0
CH,
HO As1* OH-
N
CH,
Bacteria
Arsenate
Sediment
Arsenite
Melhylarsenic
acid
-HO As*CH,
Dimelhylarsinic
acid
Figure 47. , The biological cycle for arsenic (Wood,. 1974)
Courtesy of J.M. Wood, Science, 1.83(4129) 1049-52
Copyright 1974 by the Amer. Assoc. for the Advancement
of Science
384
-------
Selenium and Tellurium: Selenium and tellurium have been
known to be acted on by microorganisms to produce methylated compounds.
Fleming and Alexander (1972) isolated a strain of Penicillium from raw sewage
»
which produced dimethylselenide from inorganic selenium compounds. The same
organism also catalyzed the conversion of several tellurium compounds to
dimethyltelluride. The methylation of selenium was later also shown to occur
in soil (Alexander, 1973c).
3. Chelation
Natural and man-made chelatea, both organic and inorganic,
occur everywhere in the environment at low concentrations. One example of
this is trisodium nitriloacetic acid, which is contemplated as a possible
detergent additive. This could lead to an increase in the concentration of
the chelant in water and may play an Important role in making metals more
soluble and, therefore, accessible, by forming chelates. The anion forms
of NTA can react with appropriate metal ions to produce the metal chelate
(Thorn, 1971):
NTA3~ + M2+ (* NTA - M~
Swisher et aj.. (1973) have indicated that NTA metal chelates are biodegradable
and, therefore will not be expected to accumulate in the environment. However,
the actual contribution of metal chelation to the biological cycle of toxic
elements in the environment is largely unknown.
385
-------
C. Test Methods, for Studying Transformation
1. Biological Transformation in Aquatic Environment
a. Methylation of metals
(i) Mixed Culture Studies: Biological methylation of mercury
compounds by aquatic organisms was first studied by Jensen and
Jernelov (1969). They treated 1 gm samples of bottom sediments
from fresh water aquaria with HgCl2 (100 ppm). Untreated
samples and suspensions sterilized by autoclaving served as
controls. Samples were incubated for 5 to 10 days and then
analyzed for CH3Hg . The formation of dimethyImercury from
mercury or from monomethyImercury was studied using homogenates
of dead (rotten) fish (Xiphophorus maculatus) as the biological
material. The conversion was studied under anaerobic condi-
i- .
tions,which were obtained by flushing the flasks with nitrogen.
Bishop and Kirsch (1972) studied the biological generation of
methyImercury under anaerobic conditions using a tertiary
sewage lagoon and a methanogenic enrichment culture as the
microbial source.
In order to study the mercury transformations in the
aquatic environment, Bisogni and Lawrence (1973) constructed
'*
microbial reactors which could be operated under anaerobic
» T
(low redox potential) and aerobic (high redox potential)
conditions. In both phases the units are operated on a
semicontinuous basis. Inorganic mercury in the form of mercuric
386
-------
chloride (concentrations varying from 0.1 to 100 mg/1 of
mercuric Ion) is introduced with the feed solution. The
feed solutions for the microblal reactors consisted of
carbonaceous-nitrogenous media (nutrient broth-glucose mixture),
nutrient salts, buffer salts and tap water. Since the nutri-
tional requirements for anaerobic and aerobic microbes are
somewhat different, two different inorganic salt solutions
were used. The nutrient salt solution of McCarty and Speece
(1963), supplemented with Coda, was used for anaerobic
cultures and the nutrient salt solution of Bisogni and
Lawrence (1973) was used for aerobic cultures. Another
difference between the two systems was that for anaerobic
units a bicarbonate buffer system was used, whereas the
aerobic units used a phosphate buffer system. In order to
verify that methylatlon reactions were mediated by micro-
organisms, sterile reactors were run parallel to microblal
reactors.
The anaerobic microblal reactor used by Bisogni and
Lawrence (shown in Figure 48) was constructed from a 1
or 2 liter Erlenmeyer flask, and was equipped with a
dimethylmercury trap, a mercury scrubber system, and a gas
measuring cylinder. The system was Inoculated with the seed
obtained from an anaerobic digester at a municipal sewage
treatment plant.
387
-------
eed air
gas
measuring
cylinder
relief
1
j
»
i
h~
pressure
indicating
tube
by-pass
valve
To atmosphere
Hg° absorbing
activated carbon
CxJ- gas sampling
port
collector
and
Hg° collector
gas .measuring
fluid reservoir
sampling
port
Magnetic
stirrer
Figure A8. Anaerobic Microbial Reactor System
(Bisogni and Lawrence, 1973)
vacuum
compressed air
overflow
trap
(CH )2Hg orllg
collector
acidic perraangnate
Hg° absorbing solution
sampling and
feed port
2 liter
reactor
Figure 49 Aerobic Microbial Reactor System
(Bisogni and Lawrence, 1973)
388
-------
Their aerobic microbial reactor was constructed from a
2 liter Florence flask (Figure 49). Compressed air as a source
of oxygen was supplied through a gas diffuser stone. A dimethyl-
mercury trap and a metallic mercury vapor trap were attached
to the flask as shown. The seed for the aerobic units was
obtained from the aeration chamber of a municipal sewage treat-
ment plant and maintained on a fill and draw basis for a period
of three months In the laboratory prior to the use In the
aerobic reactor.
(11) Pure Culture Studies - Intact Cells and Cell-Free Extracts:
Tonomura e£ al. (1972) have studied the formation of methyl-
mercury by an anaerobic bacterium (Clostrldium cbchleatium)
isolated from the soil. The organism was incubated anaero-
bially (gas phase, nitrogen) In a medium containing organic
nutrients, cysteine, mercuric chloride and vitamin B12» and
the methylmercury formed was assayed. McBride and Wolfe (1971)
studied the biological formation of alkylarsines utilizing
pure cultures of a methanogenic bacterium (Methariobactefium
strain M.O.H.). This bacterium was chosen since the extracts
of this bacterium were earlier shown to catalyze the formation
of methylmercury from Inorganic mercury (Wood et^ al., 1968). In
order to assay for the formation of alkylarsine, whole cells
of the bacterium were Incubated with sodium arsenate in a
gas atmosphere of H2-C02 (80:20). The temperature was kept
at A08C.
389
-------
Fleming and Alexander (1972) studied the formation of
dimethylselenide and dimethyltelluride by a,strain of
Penicillium isolated from raw sewage. The fungus was iso-
lated by plating on the medium containing inorganic salts
and an organic carbon source as well as Na2Se03 (1000 ng/ml). ,
The ability of the isolated fungus to form the volatile
products (dimethylselenium and dimethyltellurium) was investi-
i
gated by inoculating the medium (defined medium or autoclaved
municipal sewage) containing sodium selenite with the washed
spore suspension of the fungus. The vessels were capped with
a foam plug during the first 72 hours and later with serum
stoppers (since the synthesis of dimethylselenide was found
to start after 72 hours). Gas samples were withdrawn at
various intervals and the volatile products were assayed.
Vonk and Sijpesteijn (1973) studied the methylation of
mercury during aerobic growth of several bacterial and
fungal species which are commonly found in water and soil.
These researchers used a sublethal concentration of mercuric
ion in their experiments (range between 5-20 mg/£).
'Wood e_t al^. (1968) have studied the synthesis of methyl-
mercury compounds using the extracts of methanogenic bacterium
(Methanobacterium strain M.O.H.). The organism was grown on
i
hydrogen and carbon dioxide (80:20). The reaction mixture
for the assay of the formation of methylmercury and dimethyl-
mercury contained crude extracts, adenosine triphosphate (as
390
-------
energy source), Hg2+, methylcobalamlne and potassium phosphate
buffer (pH 7.0). The gas phase was hydrogen which served as
the source of electrons. Assay for methylated forms of mercury
was performed after an incubation period of 50 minutes.
McBride and Wolfe (1971) used the extracts of Methanobacteriura
strain M.O.H. to study the formation of dimethylarsine from sodium
arsenate. The reaction conditions were the same as those used
by Wood et al. (1968). A reaction flask which was heated in a
boiling water bath prior to incubation was run parallel to the
experimental flask to determine the extent of chemical
methylation of arsenic.
(Ill) Field Studies: Jacobs and Keeney (1974) studied methyl-
mercury formation and Hg loss from mercury-treated river sedi-
ments during in situ equilibration. Bulk sediments were
collected from river sites, treated with mercuric chloride or ,
phenylmercurie acetate (approx. 1-100 ppm Hg, oven-dry basis)
and returned to the river with untreated controls. After equili-
bration with the river environment for various intervals (2-12
weeks) samples were removed for analysis.
Kania ejt al. (1973) have investigated the fate of mercury
in artificial stream channels 300 ft. long, 2 feet wide and
1 ft. deep. The flow of water was set with manual control
valves to provide 25 gallons per minute into each channel.
The average retention time in the stream was 2 hours. Feeding
was allowed to occur naturally in the channels except that
391
-------
mosquito fish were introduced a month before mercury was
added. The levels of mercury were low (1 and 0.01 ppb). Fish
and the sediment samples were analyzed for the total mercury
at various intervals.
b. Degradation of Organometallic Compounds
Spangler e_t al. (1973a) have studied the bacterial degradation
of methylmercury in lake sediment. They incubated sediment water mixture to
which was added Hgd2 under aerobic conditions to permit the formation of
methylmercury; an aliquot from this flask was used to inoculate the medium con-
taining methylmercury in tryptic soy broth and the mixture was incubated for
170 hours. Uninoculated controls containing methylmercury were run simul-
taneously. These researchers later obtained pure cultures of aerobic and
anaerobic microorganisms capable of degrading methylmercury, by direct iso-
lation or isolation from enrichment cultures (Spangler e_t a!L., 1973b).
Swisher et, al. (1973) studied the biodegradation of NTA metal
chelates in river water collected from the Meramec River. NTA was added at a
concentration of 5 mg/fc. Selected metals were added separately as chlorides
at three levels (equivalent to 5, 1.5 and 0.5 mg/£ NTA). Another sample
was set up with NTA and no added metal.
A few reports are available in the literature in which
researchers have utilized the mercury-resistant bacteria in biodegradation
studies of organic mercurial compounds such as phenylmercuric acetate (Nelson
et al., 1973; Furukawa et_ al., 1969). Furukawa e^ al. (1969) studied the
decomposition of phenylmercuric acetate by a strain of bacteria named K62,
belonging to the genus Fseudomonas, which has been shown to be resistant to
392
-------
organic and inorganic mercury compounds (Tonomura et al., 1968). Cells grovm on a
medium containing organomercurial compounds were incubated with the test
mercury compound (10-100 ppm) in phosphate buffer and decomposed products
were analyzed. Nelson et al. (1973) isolated strains of mercury-resistant
bacteria, which could degrade phenylmercurie acetate, from estuarine water
and sediment samples by plating on a medium containing phenylmercuric acetate
and mercuric chloride.
2. Biological Transformation in the Soil Environment
The degradation work in the soil environment has mostly been
restricted to organo-arsenical pesticides. Researchers have used many dif-
ferent types of soil in these studies. The decomposition of methanearsonate
(MSMA) was studied by Dickens and Hiltbold (1967) in 4 different soils:
Norfolk loamy sand, Augusta silt loam, Decatur clay loam and Vaiden clay.
Methanearsonate was added to 20 gm samples of soil to give a concentration
of 110 ppm. Herbicide decomposition was also investigated in the presence
of added decomposable organic matter. The soils were moistened to field '' , ':.
capacity and degradation was studied up to 80 days. Von Endt ejt al_. (1968)
in their studies on the degradation of methanearsonates used the following
soils: Sharkey clay, Hagerstown silty clay loam, Cecil sandy loam and
Dundee silty clay loam. The concentration of MSMA ranged between 10 and
100 ppm, and Incubation with soil was continued for up to two months. Steam
sterilized soils were also incubated with methanearsonate to determine the
chemical breakdown.
For studying persistence of cacodylic acid, Woolson and Kearney
(1973) used three soils: Lakeland loamy sand, Hagerstown silty clay loam
393
-------
and Christiana clay loam. The soils in these studies were brought to 75%
field capacity and incubated for 32 weeks. The incubation was carried out
under aerobic as well as anaerobic conditions.
Von Endt e± _al. (1968), using soil enrichment technique (see sec-
tion IV.A.l.b., p. 231) isolated a fungus, several actinomycetes and several bacteria
capable of metabolizing methanearsenate. The concentration of methane-
arsonate (monosodium salt) during enrichment was 100 ppm. Experiments
involving degradation of arsenicals were conducted in Roux bottles. Solidi-
fied universal salt solution (Kearney £t al., 1964) containing 3% agar and
100 ppm methanearsonate was inoculated with the isolates of soil microorgan-
isms and degradation was studied.
3. Model Ecosystem and Aquarium Studies
Aquatic ecosystems which simulate the aquatic environment
have been used in studies concerning fate and transformation of heavy metals
and metalloids. Bahr and Ball (1969) in their studies on arsenic metabolism
used separate aquariums for groups of similar organisms as well as a
complete ecosystem. Aquariums containing sand, pond mud or gravel and
water with plants, fish or invertebrates each in separate aquariums were
used to study the fate of arsenicals in the absence of interference by other
factors. A complete ecosystem containing 12 plants each (Potamogeton,
Elodea and Isoetes sp.), 6 green sunfish, 6 fat head minnows, 6 bull-heads
and 6 of each invertebrates (crayfish, snails, and dragonfly naiads) was also
used by these researchers to study the metabolism of arsenic. The ecosystem,
described above, appears to be more oriented towards the higher organisms
since nothing is mentioned about the extent of any microbial transformation
in these studies.
394
-------
Fang (1973) has studied the biotransformations of phenylmercurie
acetate in aquatic organisms. Studies were performed in an aquarium contain-
ing gupples, snails (genus Helisoma), elodea and coontail. Again there is no
mention about any microbial transformation in their studies also. Isensee
et a_l. (1973) have used a modified version of Metcalf 's model ecosystem
(see section III.A.5., p. 118) to study the distribution and fate of alkyl-
arsenicals. The terrestrial phase of the ecosystem of Metcalf et al.
(1971) was omitted but 10 grams of soil was added to treated and
control (untreated) tanks. 1^C-cadocylic acid was added directly to the
tanks while 14C-dimethyl arsine was first absorbed to the soil and then added
to the aquarium. In view of the fact that cacodylic acid is used predominantly
in controlling pests of cotton, Schuth et al. (1974) reexamlned the fate of
cacodylic acid in micro-ecosystem which contained bottom feeding organisms
(catfish and cray fish -indigenous to cotton producing areas) and duckweed,
(Lemna minor L), daphlds and snails. The pesticide was mixed with soil
^"~*^~ ~"~^^ ; ,
prior to the addition to the tank.
The fate of cadmium in an experimental ecosystem was Investigated
by Gasklns et, al. (1971). These researchers used an ecosystem (also called
microcosm) which contained soil, water, plants, and fresh water fish and
snails (names of species not mentioned) from a plot of land near the labora-
tory. The radioactive cadmium was added to the terrestrial part of the
ecosystem as a component of a single, simulated rainfall.
4. Test Methods for Photochemical Studies
For studying photochemical transformations of organbmercurials,
Baughman e£ al. (1973) and Zepp e_t al. (1973) irradiated samples by:
395
-------
a. Broad-band (>290 rim) and monochromatic (313nm) light from a
mercury lamp. The light was filtered through a pyrex sleeve for the broad
V*
band studies and through a pyrex sleeve and a solution of potassium chromate
In aqueous potassium carbonate to isolate-the 313nm line.
' b. Sunlight. Aqueous solutions (ICT^M) of methylmercury compounds
were degassed in quartz tubes which were sealed and irradiated by September
sunlight for approximately 17 hours.
In order to study sunlight photolysis of phenylmercury compounds
(cpncn. range 4 x 10~5 to 10~5M in water) samples were exposed to approximately
20 hours of sunlight. Acetone-sensitized photodecomposition of phenylmercury
i .
compounds was Investigated at pH 2.3 and pH 10.2. The samples were irradiated
by pyrex-filtered light (>290nm).
5. Test Methods for Studying Chemical Transformation
i '
Baughman et al. (1973) have studied the acidolysis of dimethyl-
mercury utilizing two procedures: in one.method, dimethyImercury was added
to an aqueous solution of tetrahydrofuran (the GLC internal standard); a
standardized acid solution was added and the mixture was transferred to a
kinetic bomb. The bomb was immersed in thermostated oil bath and periodi-
cally aliquots were removed and analyzed by GLC. In another method, the
acid solution was added to dimethylmercury solution under nitrogen in a
glove.box. The. solution was transferred to a conductance cell immersed
in a thermostated water bath and conductance of the solution was recorded
at appropriate intervals.
In order to investigate the chemical methylation of mercury, Wood
jet al. ..(1968) incubated methylcobalamine and propylcobalamine with two
396
-------
Individual batches of Hg2+ under mild reducing conditions and the resulting
products were Identified.
D. Analytical Procedures
Transformation of metals and.metalloids, like other environmental con-
'taminants, has been studied either by assaying for the disappearance of the
parent compound or by following the formation of the metabolites and products.
The volatility of the several intermediate metal compounds and their tendency
to adsorb on particles and surface may sometimes present a problem In accuracy.
The analytical procedure employed for studying metal transformation usually
Involves following three steps:
1. Isolation Steps
The choice of extractant is, perhaps, one of the critical steps in
any analysis. Volatile metabolites of heavy metals are usually first trapped
In a suitable mixture and then recovered by extraction with organic solvents.
HgBt2-KBr solutions are generally employed as the trapping solution. This is
particularly suited for trapping mercury vapors or organomercurials (dimethyl-
mercury) (Spangler et_ £l., 1973a; Spangler e£ £l., 1973b). A Hg(N03)2-HN03
trapping solution is equally effective In trapping, but is not widely used
due to low extraction efficiency for organomercurials (Spangler et_ ai_., 1973b),
Volatile alkylarsines have commonly been trapped by oxidation to nonvolatile
acids by treatment with nitric acid which is kept in ethanol-ice baths
(Ralziss and Garron, 1923; McBride and Wolf, 1971). Other researchers have
used silver diethyldithiocarbamate-pyridine solution for trapping alkyl-
arsines (Powers jit al., 1959; Sachs, et^ al,., 1971). A second method based
on the property of alkylarsines to react with the red-rubber stopper used
397
-------
to seal the reaction vessel has also been used (McBride and Wolf , 1971).
For extraction of organic mercury, the benzene-cysteine extraction technique
developed by Westoo (1967) is frequently used (Fang, 1973; Bisogni and
Lawrence, 1973; Vonk and Sijpesteijn, 1973). A few researchers have preferred
to use toluene Instead. of benzene for extraction (Spangler et al. , 1973a,
.- r*-
1973b) . Inorganic mercury is not extracted into the organic solvent phase and,
therefore, provides no Interference. The recovery in benzene-cysteine
*»"
extraction technique is 98 ± 3 percent. The technique can also be used to
measure phenylmercury (Gage, 1961). For measurement of dlmethylmercury,
the gas samples containing dime thy Imercury are passed through a solution of
mercuric chloride in 2N HC1 prior to subjecting to benzene-cysteine extrac-
t.
tion. For extraction of mercury from fish, sulfuric acidtnitric acid
mixtures have been used. Sediment samples are autoclaved to release mercury
(Xfcnia et al. , 1973) .
2. Analysis
The following analytical procedures, have been, used for studying
trace metal transformations:
a. Gas chromatography! ,
The method is used widely for determining trace amounts of
volatile metabolites of heavy metals (Fleming and Alexander, 1972; Jensen
andstfernelBv, 1969; Spangler £t al. , 1973 a & b; Vonk and Sijpesteijn, 1973).
Methylated forms are. analyzed by gas' chromatographic detection of CHsMX or
CHaMCHa (where M = metal, X = halogen) using an electron capture detector.
Gfis* chromatography has also been used in trace metal analysis in which case a
volatile f luoroacetylacetonate derivative of the metal is prepared (Ross
T
398
-------
.et. ad.', 1965; Bayer et_ a±., 1971; Lisk, 1974). Dimethyl intermediates may
be first converted to monomethyl derivates prior to injection in the gas
chroraatograph in order to increase the sensitivity of the electron capture
detector. The technique is very sensitive and can be used to separate and
detect metabolites as well as the parent compound.
b. Use of labelled compounds:
One of the simpler methods to study the transformation of heavy
metals is by the use of radioactive elements or organometallic compounds
labelled in the element or in the carbon. Researchers have used ll*C-labelled
compounds for studying the fate of arsenicals in soil and in model ecosystems
(Von Eridt et al., 1968; Dickens and Hiltbold, 1967; Woblson and Kearney, 1973;
Isensee e£ al., 1973). ?lt As-labelled compounds have been utilized by McBride
and Wolf (1971), Bahr and Ball (1969). In studies concerning the degradation
of organomercurials 203Hg or llfC-labelled compounds have been used (Fang, 1973;
Furukawa e_t al., 1969; Nelson £t a!L. , 1973). Radioactive metabolites formed
are separated by thin-layer chromatography and may be identified by cochroma-
tography. Preparative chromatography can be used to obtain small quantities
of material for further identification of unknown metabolites. The radio-
labelled metabolites can be more easily detected and identified.
c. Atomic absorption;
In conventional flame atomic absorption spectrometry, the specific
element is burned in the flame and the energy absorbed at a specific wave-
length is measured. The conventional flame emission or absorption spectrom-
etry is not widely used primarily because of the availability of newer
techniques which afford superior sensitivity. A recently-developed technique
referred to as flameless or cold vapor technique, involves chemical reduction
399
-------
of test metal to the elemental form, its volatilization into a long-path-
length absorption tube and measurement of the absorption at appropriate
j
wavelengths. The method is both sensitive (detection limit 0.2 ppb for
1
mercury, 0.5^1.0 ppm fpt arsenic, Wallace e_t £l . , 1971; Braman and Foreback,
1973) and rapid, and has been used in heavy metal analysis by Iskandar e^
al., 1972; Jacobs and Keeney, 1974; Hatch and Ott, 1968; Bisogni and
Lawrence, 1973. Chemical agents such as benzene, toluene, xylene, chloride
interfere with the method and can produce positive absorbance peaks. Limits
of detection for various elements in this method are given in Table 41.
Table 41' Absolute Limits of Detection (in g) for
Atomic Spectrpmetric Methods
(Karasek and Laub, 1974)
Element
Ag,
As
Cd
Cu
Hg
Zn -.
Pb
Atomic Atomic
absorption fluorescence
10-10 10-ii
10"8 10'8
10-10 1Q-13
10"9 10"10
ID"7 1Q-10
io> iQ-ii
, 10"9 10'9
Atomic
emission
10-8
10~5
10"6
10-9
10"5
10"5
10-7
d. Spectrophotometric procedure;
The diphenylthiocarbazone (dithizone) method is probably the
most widely used colorimetric method for detection of trace levels of mercury.
400
-------
In this method, dithizone is reacted with Hg2.* or Hg2+ in acid solution to
form a colored complex which is extracted with chloroform or carbontetra-
chloride (Sandell. 1959: Snell and Snell, 1949). The absorption of the com-
plex is measured at 490nm or alternatively the decrease in dithizone absorbance
at 610nm is measured. The procedure can be used to determine 0.5 to 50 ppm
of mercury. Presence of copper, silver, gold, etc. interferes with the
determination. Preliminary treatment to insure decomposition of organic
material is required in order to determine organic-bound mercury by this
method.
The silver-diethyldithiocarbamate (AgDDC) colorimetric method
has been used for determination of arsenic (.Powers et^ al. , 1959; Dickens and
Hiltbold, 1967). In this method arsenate is first reduced to arsenite,
which is then reacted with hydrogen to form gaseous arsine. Arsine is
trapped in silver diethyldithiocarbamate-pyridine solution and the stable
red complex formed is measured at 540nm. The lower limit of detection for
this method is 2 ppb (Braman and Foreback, 1973). The method can be used
for determination of arsenic in organic arsenicals. However, the carbon-
arsenic bond should be first ruptured by digestion with nitric acid-sulfuric
acid oxidation procedure (Sachs e_t al., 1971).
e. Neutron activation;
This procedure can be used for determination of several elements
with great sensitivity. The technique involves exposing a sample to a source
of neutrons to produce a radioactive nuclide of the element (Lyons, 1964). For
mercury, for example, irradiation results in 2 radioactive nuclides, 197Hg
and 203Hg. Although the method is extremely sensitive, its use has so far
401
-------
been restricted to monitoring only for trace levels of metals in environmental
samples. The major disadvantage of this technique is that it requires special
irradiation facilities and data handling.
f. X-ray fluorescence;
In this method low-energy photons are used to excite the character-
istic x-ray energy of the element (Wallace et al^., 1971). The x-rays emitted
are then sorted and^measured using a solid-state Ge(Li) or Si detector coupled
to a -multichannel analyzer.
g. Polarographic Methods;
This technique is more suitable as a supplementary and reference
method, since its sensitivity is not high enough for use in analysis of
environmental samples (Wallace ejt al., 1971).
Limits of detection of. various methods;
The wellrknown instrumental methods which have been used in study-
' ing transformation of metals are summarized in Table 42.
3. Identification
-Thin-layer chromatography is one of the commonly used techniques
for separation and identification of mercury compounds. Most frequently
researchers have used silica gel and alumina thin-layer plates (Westoo, 1969;
Tatton and Wagstaffe, 1969). Johnson and Vlckers (1970) have used thin-layer
plates to separate various organic mercurials and inorganic mercury. In
several studies organomercurials have been reacted with dithizone to form
^ithizcmate complexes prior to separation by thin-layer chromatography.
vori Endt ej^ al. (1968) have used thin-layer plates coated with silica gel G
(calcium sulfate binder) to separate various inorganic and organic arsenicals.
402
-------
Table 42. Instrumental Limits of Detection in Trace Metal Analysis
(Karasek & Laub, 1974)
Method
Uses
Minimum
Detectable
Quantity, g Price Range, $
1."Gas -chromatography (GC)
A. electron capture
.B. helium ionization
2.'Mass ^spectrocietry (MS)
-A, .spark source
-B.. .electron.impactdirect
.probe
. :C. GC/MS/-computerized
D. ion-probe
3. .Neutron, activation analysis
'(NAA)
4.-Atomic .absorption spectroscopy
(AAS)
A. flame
B, flameless
5. Electron spectroscopy (ESCA)
6. X-ray fluorescence
7. Ion-scattering spectrometry
(ISS)
8. Polarography
electrophilic organics
e.g. CH3HgCl
all volatile compounds
all..ele3icmts, most compounds
all elements, most compounds
all elements
metallic elements
metallic elements
10
-13
10
-13
10
10
-13
-12
10
all- elements, -some inorganics 10
and -organics
-15
10
,-12
10
_LO
-9
-12
most elements, -some compounds 10
-7
elements with atomic number >12 10
-7
all elements, some inorganics 10
and organics
most metallic elements and 10
compounds, some inorganics
,-15
,-6
500 to 10,000
20,000 to 250,000
>250,000
500 to 20,000
60,000 to 250,000
50,000
40,000 to 65,000
500 to 1,500
-------
The compounds were Identified by cochromatography. A few researchers have
separated the arsenicals by electrophoresis on chromagram cellulose plates
(McBride and Wolf, 1971).
Combined gas chromatography-mass spectrometry has been extensively
used for detection-of metabolites (Fleming and Alexander, 1972; Jensen and
JernelBv, 1969). The technique has an almost universal ability to separate
components and provide a mass, spectrum for identification as well as detec-
i
tion. Overall fragmentation pattern of authentic methylated form of the
trace metal can be.compared with that of unknown compounds for identification.
In, certain cases, however, absorption of the chemical on the large surface
area in GC/MS systems and thermal decomposition may greatly reduce the appli-
cability of this technique.
E. Evaluation of Techniques
1. Factors Affecting Transformation of Elemental Contaminants .
a. Factors Affecting Methylation of Metals
Factors which influence the methylation of metals are generally
'the same 'as those which affect other microbiological transformations. These
include the concentration of«the metal, number and type of microorganisms
u
:present and their growth rates, adsorption and chelation of the metal,
presence of other chemicals including supplemental nutrients, physical
parameters such as pH, temperature, and redox potential of the test medium.
(i) Concentration of the metal: Jensen and Jernelbv (1969)
studied the formation of methylmercury under aerobic conditions
as a function of inorganic mercury (HgCl2) concentrations in
the lake sediment. Their findings indicated (Figure 50) that
v
:'. 404
-------
160
!,
.I-
0 0-1
t-O
10
100
1.000
Figure 50. Concentration of Methylmercury in Bottom Sediment After
Addition of Inorganic Mercury Followed by Incubation for
Seven Days (Jensen and Jernelov, 1969). Two parallel
experiment series were run, to the first series 0, 0.1,
1, 10, 100 and 1000 ppm HgCl2 and to the other 1, 5,
10, 50, 100 and 500 ppm HgCl2 were added.
Courtesy of Nature.
methylmercury production increased with increasing inorganic
mercury dosage up to 100 yg/g of sediment. A further increase
in Inorganic mercury caused a sharp decrease in the formation
of methylmercury, probably due to the inhibition of the
methylating microorganisms. An increase in methylmercury
formation with increasing inorganic mercury concentrations
i
has also been observed under anaerobic conditions (Bishop and
Kirsch, 1972; Bisogni and Lawrence, 1973). Fleming and
405
-------
Alexander (1972) studied the formation of dimethylselenide
from inorganic selenium. These investigators found that in
#
the medium containing 10 pig selenite/ml, as much as 13-24%
of the added selenite was converted to dimethylselenide,
whereas at 1000 yg selenite/ml, less than 2% of the selenium
was recovered in the dimethylated form.
(ii) Microbial activity: .Methylation of metals has been
demonstrated to be catalyzed by anaerobic as well as aerobic
bacteria (Tonomura et^ al., 1972; Vonk and Sijpesteijn, 1973;
McBride and Wolfe, 1971), and fungi (Fleming and Alexander,
1972; Cox and Alexander, 1973; Vonk and Sijpesteijn, 1973).
Since' all five organisms investigated by Vonk and Sijpesteijn
(1973) were able to methylate mercury, these researchers con-
cluded that a slight capacity to methylate murcury may be a rather
common property of aerobic bacteria. Since fungi generally do
not contribute significantly to the microbial activity in lake
sediments, their role in methylation may be more important in
soil.
In addition to the type and number of microorganisms,
microbial growth rate has also been found to influence
: >
methylation rates of mercury (Langley, 1971; Bisogni and
Lawrence, 1973). Bisogni and Lawrence (1973) have reported
that with doubling of net specific anaerobic growth rates
(from 1/24 per day to 1/12 per day) the net specific methyla-
tion. rate increased by a factor of approximately 3. A similar
406
-------
doubling of the aerobic growth rate caused approximately a
.two fold increase in the methylation rates.
(iii) Adsorption and Chelation of Metals: The adsorption of
elemental contaminants to inorganic and organic constituents
of water and soil systems is well documented (Ball Aglio,
1970; Hlnkel and Learned, 1969; Lagerwerff, 1972). Both
adsorption and complexation of the metal ion may Influence
the methylation process by making a particular metal ion
unavailable for methylation.
(iv) Presence of other chemicals: The presence of a number
of chemicals has been shown to influence the methylation of
inorganic metal ions. The effect is generally observed via
one of the following mechanisms: The chemical may influence
the metabolic activity of the test organisms; the chemical
may react with the metal ion thereby rendering it unavailable
for methylation; sometimes the chemical may be preferentially
methylated. Fagerstrom and Jernelbv (1971) were able to
show that methylmercury production is dependent on the bio-
chemical availability of inorganic mercury. These investi-
gators observed that sulfide was extremely inhibitory to the
methylation of mercury. Sulfide serves as a binding agent
for mercury whereby mercuric Ions are chemically converted
to Insoluble HgS. Sulfide is formed in anaerobic benthic
407
-------
regions of the environment from sulfate by sulfate-reducihg
bacteria. Tonomura e£ al. (1972) confirmed the inhibitory
effect of sulfide by studying the methylation of mercury by
a culture of £. cochlearium in the presence of sulfate-
reducing bacteria. These researchers observed very small
amounts of methylmercury formation in the combined culture
compared with that produced in the control.
Fleming and Alexander (1972), while studying the alkyla-
tion of selenite, observed that dimethylselenide formation
increased with Increasing sulfate concentrations. Cox and
Alexander (1973) have studied the effect of phosphate and
other, anions on the formation of alkylarsines. Their studies
4
with pure cultures of Candida humicola indicated that phos-
phate was able to inhibit the synthesis of trimethylarsine
from arsenite, arsenate and monomethylarsenate but not from
*
dlmethylarsinate. High antimonate concentrations also
depressed the rate of conversion of arsenate to trimethyl-
arsine, but nitrate was without effect.
The presence of biodegradable organic matter in the
reaction medium has generally been shown to enhance the
"rates of methylation. Bishop and Kirsch (1972) observed a.
stimulation of methylation rates in anaerobic systems when
organic nutrients, e.g., glucose-glutamic acid and acetate,
were added to the medium. Wood ejt _a_l. (1968) have suggested
that nutrient enrichment of methylation systems (for example
408
-------
by the addition of sewage) would Increase methylation rates
by increasing the numbers of bacteria capable of synthesizing
alkylcobalamlnes (a co-factor for methylation).
(v) Physical parameters such as pH, temperature and redox
potential of the test medium: The pH of the methylating
system could affect the rates of methylation of inorganic metal
either by affecting the mlcrobial enzyme system responsible
for this transformation, by affecting the responsible organisms
or by affecting the distribution and availability of the proper
species of metal ion for methylation. JernelBv et: al. (1972)
have reported that the pH optimum for methylation of mercury
either under laboratory or natural conditions is 4.5.
Several investigators (Langley, 1971; Bishop and Klrsch,
1972) have reported that the methylation process was depen-
dent on the temperature of the reaction medium. Blsognl
and Lawrence (1973) found that temperature (range 10-30°C)
significantly affects the aerobic or anaerobic methylation
reaction if a constant growth rate of microorganisms is main-
tained. These authors have suggested that the effect of
temperature on methylation observed by Langley (1971), and
Bishop and Kirsch (1972) may most likely be due to tempera-
ture related changes in microblal growth rate which also
affected the methylation process (see section on Microbial
Activity, p. 406.
409
-------
I
* A relationship between the conversion of mercury com-
pounds and redox potential has been studied by Tonomura et
al. (1972). They found that methylmercury is formed by
Clostridium cochlearium at about +50 mv. If the redox poten-
* ' ~
tial is lowered to -200 mv, sulfate reducing bacteria become
predominant, and produce large amounts of sulfide. Mercuric
ions chemically react with sulfide and are not available
for inethylation. These researchers failed to observe methylation
under aerobic conditions also (redox potential approximately
300 mv). Vonk and Sijpesteijn (1973), and Bisogni and Lawrence
(1973) were, however, able to obtain good methylation rates
under aerobic conditions. Whether anaerobic or aerobic
methylation plays a dominant role in methylation of mercury
under natural conditions is unclear.
b. Factors Affecting Degradation of Organometallic Compounds
Only a few reports are available concerning the effect of
various environmental and nutritional parameters on the degradation of organo-
metallic compounds. The biodegradation mechanisms of organometallic compounds
and' purely organic compounds are somewhat similar except that degradation of
organometallic compounds may give rise to an inorganic metal in addition
to other chemical metabolites and end products. In view of the similarity
between the biodegradation mechanisms of organometallic compounds and that
of other organic chemicals, it appears likely that important variables
pertinent to their biodegradation will be similar. These may include types
of microorganisms, mineral salt composition, test chemical concentrations,
410
-------
supplementary nutrients and physical parameters such as pH, temperature,
light, etc. The stimulation of the breakdown of monosodium methylarsonate
(MSMA) due to the presence of supplemental organic carbon source was reported
by Von Endt et, al. (1968). These researchers observed that soil isolates
released much greater amounts of llfC02 from ll*C-MSMA when yeast extract was
added to the culture medium, Woolson and Kearney (1973) have reported that
soil which had received cacodylic acid previously and presumably had an
adapted microbial population, metabolized cacodylic acid more readily than
fresh soil. These findings suggested that microbial adaptation could sig-
nificantly affect the biodegradation of chemical compounds in the environment.
2. General Discussion of the Test Methods Used for Determining
Environmental Transformation of Organometallic and Elemental
Contaminants
Toxic heavy metals can enter the environment either in'organometallic,
inorganic or elemental form. Organometallic compounds such as phenylmercuric
acetate, upon degradation, yield inorganic heavy metals which may be more or
less toxic than the organic compound forms. The resulting heavy metals may be
environmentally transformed via the routes previously shown in the biological
cycle for each metal. The fate of organometallic compounds has generally been
determined by similar methods as those employed for determination of environ-
mental persistence of an organic compound. The more commonly used test
methods have been shake culture test with mixed and pure cultures, and eco-
system studies. These methods have been evaluated in section III., p. 179. An
inherent problem in studying the fate of organometallic compounds by any
laboratory test method will be the accumulation of toxic heavy metal with
411
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degradation. The resulting heavy metal may inhibit further breakdown
of organometallic compounds. In contrast, in natural aquatic environ-
ments, the inorganic toxic metal formed is continuously removed by dilution.
The possibility of methylation-demethylation interconversion
of metallic compounds sets up a dynamic system of reversible reactions
which results in steady state concentrations of various metallic and
methylated forms in the environment. The disturbances produced in the
steady state concentrations caused by the introduction of metal into the
natural ecosystem as a result of man's activities will affect the natural
equilibrium which will, in turn, affect the concentrations of toxic inter-
mediates. In assessing the environmental fate of toxic heavy metals,
therefore, what needs to be answered is not only whether a particular metal
can be methylated but also if the kinetics of the process will allow signi-
ficant levels of the methylated form to build up.
The test methods used thus far have succeeded in answering only
part .of the kinetic question, e.g., can a particular metal be methylated.
The techniques used for these studies have included microbial reactors,
shake culture studies with pure and mixed cultures of microorganisms, and
microecosystems. The aerobic and anaerobic microbial reactions constructed
by Bisogni and Lawrence (1973) appear to be more suitable for investigational
use. They have used these reactors for studying the effect of various
i. .
environmental conditions on microbial methylation of mercury, where a careful
control of incubation conditions is desirable. More commonly used are
simpler tests in which investigators have incubated pure or mixed cultures
412
-------
of microorganisms with inorganic forms of a metal under aerobic and/or
anaerobic conditions and Have Identified the methylated product formed
(Vonk and Sljpesteijn, 1973; Jensen and Jernelttv, 1969; Fleming and
Alexander, 1972; McBride and Wolf, 1971).
Mixed cultures may sometimes give erroneous results since they
may be expected to contain microorganisms responsible for both methylation
and demethylation of the test heavy metal. Depending upon the conditions
chosen for the test, their activities could be affected and may influence
the results of the test. Natural communities, however, may be preferred
over pure culture of microorganisms, since it is unlikely that any single
microorganisms can methylate all the metals. Vonk and Sijpesteijn (1973)
observed that all the five aerobic bacteria investigated by them were able
to methylate mercury. From this they concluded that a slight capacity
to methylate mercury may be a rather common property of aerobic bacteria.
The use of aquatic microecosystems has become an effective tool for studying
biotransformation of metals (laensee ejt al., 1973; Schuth et_ al., 1974).
The ecosystem allows the investigator to examine the interconversions of
metallic compounds in a somewhat dynamic system. Since a large number of
metal transformations and transport reactions are expected to take place in
the model ecosystem due to the presence of a complete food chain, a kinetic
study of the metal transformation is also feasible. The information obtained
413
-------
couldfbe more easily'extrapolated to ths natural environment to assess if bio-
logical transformation of any metal would result in accumulation of toxic inter-
mediates in significant concentrations in the environment. There can, however,
always be doubt that the kinetic rate of each transformation reaction is the
same in the model ecosystem as that in the environment. Since the accumula-
tion of substantial quantities of any toxic intermediate is so much dependent
t
on the rate of each reaction, a change in the kinetic rate of one single
reaction in the model ecosystem could yield different results than what will
take place in the actual environment.
i
Numerous reports have become available from which it is clear
that mercury can be methylated either by enzymatic (microbial) or chemical
mechanisms (Jensen and Jernelbv, 1969; Wood e_t ad., 1968; Imura e_t al_., 1971;
Bertilisson and Neujahf, 1971). Whether these reactions occur to a signifi-
cant extent in the environment has yet to be determined. Spangler et^ al^ (I973b)
have"stated "the inability to find even traces of methylmercury in most sedi-
ments taken from areas highly polluted with inorganic mercury leads one to
question whether significant methylation occurs in sediment under environmental
conditions or whether the turnover rate of any methylmercury formed is such that
significant concentrations do not accumulate." These investigators (Spangler et al.,
1973b) have shown that a large number of microorganisms occurring in the lake
sediment possess the ability to aerbbically degrade methylmercury. These
species may be important in suppressing the methylmercury content of the
sediment. Similarly, species of microorganisms which degrade methylated forms
of arsenic have also been found to be present in the environment (Von Endt ejt
al., 1968).
414
-------
In spite of the fact that a few researchers have failed to detect
methylmercury in the lake sediment, it has generally been assumed that the
if
methylmercury found in fish is the result of formation before intake
beginning with methylation of inorganic mercury in sediments. Wood (1973)
has pointed out that measurement of steady state concentrations need not
reflect the rate of synthesis of methylmercury. He further states that the
rate of synthesis of methylmercury does not have to be very rapid in sediments
for fish to accumulate dangerous levels of it; when the rate at which methyl-
mercury is produced, then released into the water and taken up by fish exceeds
the rate of metabolism of methylmercury in fish, then methylmercury will
accumulate in fish (Wood, 1972). The kinetics of the process is more
important and, therefore, what should be measured according to Wood (1973)
is the concentration of total mercury in the sediment and the rate of methyl-
mercury uptake in fish. However, such measurements still would not distinguish
whether methylmercury was taken up by or formed in the fish. This literature
review has revealed no experimental studies in which measurement of kinetics
of methylation has been attempted.
3. Correlation Between Laboratory and Field Results
Very few field studies have been carried out to determine the
fate of heavy metals and the results are complex and difficult to interpret.
In one field study the fate of mercury was Investigated in artificial stream
systems (Kania £t al., 1973). However, the researchers analyzed only for total
mercury rather than determining the forms of mercury present.
415
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Braman and Foreback (1973) have analyzed the methylated forms of
arsenic In the environment. They have reported that dimethylarsinic acid was
the'major form of arsenic in the environment. Methylarsonic acid, although
found, was present in much smaller concentrations. Laboratory studies have
l.'
shown that microorganisms can methylate arsenic and that both dimethylarsinic
acid and methylarsonic acid may be intermediates in the arsenic methylation
sequence (McBride and Wolfe, 1971). in view of the fact that dimethylarsinic
i
acid may be resistant to oxidation, and if it is reduced by microorganisms
to dimethylarsine it will be readily oxidized back to dimethylarsinic acid,
this form of arsenic may predominate. The actual situation is more complex
than this since both dimethyarsinic acid and methylarsonic acid are added in
the environment in the form of pesticides and, therefore, one will expect to
find these forms in the environment. The estimated U.S. production for
arsenic pesticides indicates that methylarsonic acid is produced in much
larger quantity than dimethylarsinic acid [for example, in 1971, 70 million
pounds of disodium and monosodium methylarsonate was produced, whereas only
2 million pounds of sodium cacodylate (sodium salt of dimethylarsinic acid)
was produced, EPA Technical Report TS-00-72^04]. If no transformation of
these compounds occurred in the environment, one would expect to find methyl-
arsonic acid as the major form of arsenic in the environment. The fact that
dimethylarsinic acid was the predominant form as shown by the studies of
' Braman and Foreback (1973), can be interpreted to mean that methylation of
* .
methylarsonic acid was occurring in the environment and was responsible for
resulting in the accumulation of dimethylarsinic acid. McBride and Wolfe
(1971) were able to show transformation of methylarsonic acid to dimethylarsinic
acid in their laboratory studies with cell-free extracts of bacterium
Methanobacterium.
416
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F. Cost Analysis
The cost estimates given below are for evaluation of environmental
fate of at least 20 compounds (elemental contaminants or organometallics).
Since the analytical equipment cost for determining the fate of toxic metals
or their organic forms is extremely high, cost estimates for studying the
fate of one compound alone will be enormously high and perhaps misleading.
Table 43, Cost Estimates for Evaluation of Environmental Fate
of Elemental Contaminants and Organometallics. The
Total Cost Indicated is for Studying 20 Compounds.
Teat Method
Batch culture
studies vlth
mixed and pure
cultures of
microorganisms
Batch culture
tudleB with
Ixed and pure
cultures of
microorganisms
Aerobic and
en aerobic
alcroblal
reactors
Model eco-
system
Cell-free
extract
orudies
Cell-free
extract
studies
Analytical Method
Gas chromatography
Radioactivity
(It is desirable to use
label In the metal If
organometalllc compound
Is being Investigated.
Gas ehromaeography
Radioactivity
Gas chromatography
Radioactivity
Equipment needed
and its cost
Gas chromatograph -
Mass spectrometer
$100,000 (Depreciate
over 10 year period)
Scintillation counter
510,000
Gas chronatograph -
Mass Spectrometer
. 5100,000
Scintillation counter
$10,000
Gas . chromatograph -
Mass spectrometer
$100,000;
Ultras onlfler
9500
Scintillation counter
$10,000;
Ultrasonlfler $500
Equipment Chemicals
for the glassware end
test misc. supplies
1125 200
14,000
ISO (Includes custom
made labelled
material)
SOO
1350
205 ' 14,000
(Includes custom
made labelled
material)
900 200
115 14,000
(includes custom
made labelled
material)
Labor cost Overhead Total
9 $60/day 12JI cost Cost/Compound
2700 3375 7400 370
1800 2250 18200 910
3600 4900 9950 498
3600 4500 22305 1115
2700 3375 7175 360
1800 2250 18165 908
417
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VII. ENVIRONMENTAL DEGRADATION OF SYNTHETIC POLYMERS
A. Introduction
Stemming from crude attempts to mimic such natural products as wood,
i
rubber, and cotton; the synthetic plastics, elastomers, and fabrics have
characteristics which in many ways seem to improve rather than imitate
their natural counterparts. One of these characteristics and one of the
chief reasons for the versatility and commercial success of synthetic
polymers is their durability or, from an environmental point of view,
their persistence. The literature is rife with references to the recal-
citrance of most synthetic polymers (Alexander, 1973a), to the fact that most
do not degrade at "practical rates" (Potts et _al., 1972). However, it would
seem an error to substitute "inactive" for "recalcitrant". Degradation, if it
Is at all possible, is a function of time. What may be impractically slow
degradation in terms of present solid waste disposal may be practical over
decades of environmental exposure. On an evolutionary scale, the problem is
even more evident. It may have taken millions of years for a fungus to develop
that could metabolize lignin, but as the amount in the environment increase,
such fungi did evolve (Nickerson, 1971). This is not to say that an attempt
should or could be made to predict the fate of synthetic polymers over million
year periods, but rather that the assumption that a potentially hazardous
compound can be dismissed from consideration because it is polymerized may
not be entirely valid. References to the burgeoning production of synthetic
polymers are abundant (Alexander, 1973a; Titus, 1973; Scott, 1970). Although
such polymers are usually in bulk form and thus might be expected
Preceding page blank
419
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to be as environmentally immobile as they are stable, their potential ubiquity
is indicated by the plastic particles found floating on the surface of ,the
Saragasso Sea at an average concentration of 290g(3500 pieces)/sq. km.
(Carpenter and Smith, 1972). Further studies by Carpenter and co-workers
(1972) and Colten and co-workers (1974) demonstrate that aquatic pollution
with these polymers is becoming wide-spread.
The degradabllity of these polymers on prolonged exposure to the
various conditions likely to be encountered in the environment is difficult
to predict. Such predictions would best be made on a detailed understanding
of the mechanisms by which polymers degrade or persist. However, up until
the relatively recent interest in degradable plastics (Anon., 1972; Anon.,
1971 a and b; Rodriguez, 1971), techniques used to measure the degradability
rof synthetic polymers have concentrated on changes in physical characteristics
i
which might effect their utility rather than on an analysis of molecular
alteration (Wessel, 1964). This is evident by the predominence of such
techniques described below (see Analytical Techniques, p. 431). While some common
*
tests may well indicate whether a polymer will degrade in the test system,
they in no way illuminate the mechanism of degradation or recalcitrance.
Thus, an attempt will also be made to describe the various exposure or
analytical techniques that are available for determining molecular alteration,
.*
even though these are not frequently employed.
B. Techniques for Determining Degradation
1. Biological Test Methods
In designing an experimental procedure for determining
the'degradation of a polymeric substance, a variety of methods
i1
420
-------
are available and might be classified In a number of ways, e.g., by
media, duration,'type ot attack, etc. However, the most pragmatic
classification for the design of an experimental approach is a modification
of that proposed by Walchli (1968) based on the purpose of the test method.
Accordingly, techniques for determining degradability can be designated as
screening tests, end-use tests, or field tests. Screening tests are designed
to determine if a specific factor, such as a microorganism, will modify a
particular polymer. This type of test is conducted under carefully controlled
laboratory conditions with as few variables as possible. Because many of these
tests must be conducted in order to produce meaningful results, the test must
be simple, rapid, inexpensive, and yield reproducible results.
Opposite the screening test is the field, test. This test is
designed to determine if a specific polymer formulation will degrade under a
given set of field conditions for a prescribed period of time. In this case,
while various parameters may be carefully monitored, they cannot be controlled.
Consequently, interpretation may be difficult and results not readily repro-
ducible. The test will usually require a considerable period of time (i.e.,
the proposed or expected service time of the polymeric component), and
meaningful interpretation may require sophisticated equipment and highly
trained personnel.
Between these two methods of testing is the end-use test. This
type of test involves a varying amount of effort to simulate in the laboratory
the conditions to which a polymer might be exposed in normal use or dis-
posal. In such a test, certain factors affecting degradation may be
, 421
-------
experimentally altered in such a way as to make the test more simple, less
expensive, or more easily interpreted, while doing minimal damage to the
validity of the results. For example, shortening the duration of exposure
to microblal attack,may be off-set by elevating to a modest degree the ambient
temperature. As long as such balancing is kept within reason, end use tests
may yield accurate information. However, because of the many interactions
involved under actual field conditions, the design and interpretation of
end-use tests can be,precarious.
a. Screening Tests
(i) Pure Culture Test on Agar - The pure culture test on agar
may be described as a screening test designed to measure the
ability of a specific microorganism to degrade a specific polymer.
The test is popular not only to determine the degradability of
synthetic polymers but also to evaluate the efficacy of protective
agents on natural polymers (White and Siu, 1947). Although a
number of variations are possible, the technique outlined for
fungi by Rogers and Kaplan (1971) is exemplary:
(a) An inoculum is prepared of one or more strains of
a single organism by culturing the organism on a suitable nutrient
agar until a good spore crop is produced. The spores are washed
off and used as the Inoculum.
(b) Depending on the type of evaluation to be used,
samples of the polymeric material are prepared and sterilized
in ethylene oxide.
422
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(c) Sterilized samples are then placed in the previously
inoculated agar.
(d) The sample is then incubated for varying periods of
time, depending on the type of testing to be performed. Rogers
and Kaplan (1971), in testing for changes in physical properties,
incubated for 42 and 84 days. However, the method is equally
adaptable to 22-74 hour incubations if manometric techniques
are employed (Mandels and Siu, 1950).
Although this method is widely employed, it can be considered
only a crude measurement of the degradability of the test
compound in the environment.
(11) Mixed Culture on Afar - - As the name Implies, this
test Is quite similar to the previous test except that a
"representative" combination of test organisms is used (Awao,
et^ &±., 1971). As such, -this test attempts to approximate more
closely the actual conditions to which a material may be exposed
in the environment. The procedure for this test has been
described in some detail in ASTM D1924-70 (1970) but will,
because of its extensive use, be briefly summarized here.
The organisms generally used are:
ATCC No.* QM No.**
Aspergillus niger 9642 386
Penicilllum funiculosam 9644 391
Chaetomium globoaum 6205 459
Trlchoderma sp. 9645 365
Pullularia pullulans 9345 279c
* American Type Culture Collection
** Quartermaster Culture Collection
423
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Substitutions of fungi can be made when mandated by specific
test requirements. These cultures are maintained separately
under favorable conditions. For testing, separate spore
suspensions of each species are prepared to concentrations of
10 ± 10 spores/ml and verified by cell count. The separate
spore suspensions are then mixed in equal volumes and serve
as the inoculum. Inoculation is accomplished by spraying the
spore suspension onto the appropriate surface. The carbon
source may be either the test specimen or a piece of filter
paper, growth on the latter being used as a viability control.
The specimens are incubated for 21 days at 28-30°C and 85%
relative humidity (ASTM, D1924, 1970).
This technique has been used frequently without significant
modification (e.g., Darby and Kaplan, 1968;'potts .et al., 1972),
with and without attempts to sterilize the test sample (Berk
and Teitell, 1951; Hazeu, 1967; resp.). The only appreciable
modification of this procedure is the use of liquid rather than
solid media, and standardization of cell number by naphelometry
rather than by a counting chamber (Sharpe and Woodrow, 1971;
Pankhurst and Davies, 1968).
(ill) Humidity Cabinet Test - This test simply involves
inoculating the test specimen with the desired fungi and placing
424
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it In a cabinet at fixed humidity and temperature (Wessel, 1964).
The basic purpose of this test is to allow growth at optimum
temperature and humidity without introducing a complex soil
system. Cooney and coworkers (1973) used this test on irradiated
and non-irradiated samples to evaluate the effects of irradiation
on blodegradability.
b. End-Use Tests: It is often difficult to distinguish clearly
between screening tests and end-use tests. Prolonged exposure to mixed cultures
as discussed above might in some cases serve as end-use tests. However, such
fine distinctions may not be worth maintaining. For the purpose of this review,
end-use testing may be characterized by the presence of a laboratory system
of organisms and/or media so complex as to preclude complete and quantitative
description.
(i) Soil Simulation Tests - Soil simulation tests have been
used to assess the degradabllity of both natural and synthetic
polymers (Blake and Kitchin, 1949: Walchli, 1968, Potts £t al.,
1972). The samples to be tested are placed in a relatively small
amount of soil and given water and air. Usually, more than a
single type of soil is desirable and the pH may be adjusted.
After a given period of time, the samples are removed and tested.
Exposure to sterilized soil samples usually serves as the control.
Similarly, exposure of the test sample to a paste of garden soil,
mushroom compost, and diatomecous earth has been used to assess
the degradability of both natural and synthetic textiles (Lloyd,
1955; Pankhurst and Davies, 1968).
425
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(ii) Aquatic Submersion Test - This test is the aquatic
counterpart of the soil simulation test. Although the test as
designed by Muraoka (1966) specified sea water, it is equally
suitable, to fresh or brackish water. The samples are exposed
to sterile water, sterile water inoculated with microorganisms,
and sediment. The specimens may be exposed in both stressed
. and unstessed states at a constant 15°C for twenty-one months.
In all cases, measurements are made of the pH and oxidation-
reduction potentials of the water and sediment.
c. Field Tests: The technique of field exposure allows for
little experimental control, although many of the physical parameters can
be monitored. The emphasis is usually on the analytical procedures outlined
in-the latter part of this section (p. 431). In spite of the lack of experimental
\
control, these tests remain perhaps the most definitive source of the actual
environmental degradation of a polymer, although the mechanisms of such
>
.alterations can seldom be inferred. These tests fall into three basic categories
by media: above-surface exposure, soil burial, and aquatic submersion.
(i) Above-Surface Exposure - Above-surface exposure implies
i
little more than subjecting the polymer to weathering. Such
items as polymeric coatings and structural components might
commonly be tested in this fashion. Because any details of the
exposure procedure and experimental duration are defined by the
actual use conditions, no general procedure can be outlined
(Walchi, 1968).
426
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(11) Soil Burial Test - This test is the most common and well
established of the terrestrial field tests. It has been used both
on natural and synthetic polymers for many years and still enjoys
wide popularity (White and siu, 1947; Potts et al., 1972; Titus,
1973). Depending on the purpose of the test, the polymer is
buried in varying depths and type of soil. If non-microbial
attack is expected to Interfere with the results, appropriate
decoys can be employed such as wood cases for termites (Farkas-
Imrik, 1967). Commonly, such parameters as temperature and
water content are monitored. The physical dimensions of the
exposed sample are usually dependent on the type of analytical
procedure to be employed (Hueck and von der Toorn, 1965). In
most cases, the only control is an unburied sample, although
controls to test soil activity - e.g., rate of cellulose
decomposition - have been recommended (Jones, 1968).
(ill) Aquatic Submersion - Aquatic submersion techniques may
be considered simply as the aquatic counterpart of the previous
I
techniques in that aquatic testing most often involved both
exposure to water and sediment (Snoke, 1957). However, aquatic
systems do present somewhat unique problems in terms of exposure
periods, proper control, and retrieval. The most extensive
methodology is that described by Muraoka (1969). A large
number of separate specimens can be attached to a single sub-
mersible unit constructed so that the material is exposed both
above and below the sediment. In order to facilitate comparisons
427
-------
between different test sites, several parameters must be measured
including depth, temperature, oxygen concentration, salinity, pH,
hydrostatic pressure, current, and nature of sediment. When
possible, visual observation'is desirable, either directly or by
remote camera. If boring or fouling organisms must be considered,
various protective or bait devices can be attached to the test
samples (Connolly, 1963). Because a considerable time lag may be
anticipated between retrieval and laboratory analysis, care must
be taken to maintain as closely as possible the exposure environ-
ment during ^transport. This is especially critical when testing
, i
for water absorption. Although Muraoka's (1969) procedure is
specifically designed for pelagic testing, littoral exposure
requires about the same approach (Snoke, 1957).
2. Physiochemical Test Methods
Polymers may be degraded by a variety of non-biological agents.
In terms of exposure methodology, these agents may be classified as
liquids, gases, and light. In theory, all of the methods employing these
agents are less complex than biological exposures In that the experimental
conditions are usually well defined. In practice, however, many of the non-
.«.
biological exposure techniques require somewhat more extensive experimental
apparatus than their biological counterparts.
a. Degradation by Liquids: Water is by far the most important
liquid in the degradation of polymeric substances (Titus, 1973). It may
potentially affect a number of polymers having hydrolysable groups causing
-------
chain cleavage, strength loss, and changes In dielectric properties. As a rule,
exposure techniques consist simply of placing the polymer specimen in water
at a given temperature for a specific duration and then analyzing for
degradative changes (Anon., 1971; Brown and Reinhart, 1971). In addition,
this type of exposure is often used as a control in determining the effect
of aquatic microbial degradation as discussed in the preceeding section
(Muraoka, 1966).
b. Degradation by Gases: Exposure to gaseous agents such as
oxygen, ozone, and nitrogen is most often accomplished in a flow system at
a constant temperature and concentration for a given period. Ozone, for
instance, has been tested at 2% cone., 9g/hr., 50°C for 6 hours (Potts et al.,
1972) and 5.5% cone., 0.51g/min., 40°C, for 5 hours (Gutfreund, 1971).
This .method has also been used to test the effects of ^O^and Cl2 (Gutfreund,
1971). A modification of this method is the use of corona discharge. This
involves placing the polymer sample in an evacuated tube, flooding the system
with the desired gas flowing at a constant rate, and exposing the gas-polymer
preparation to a given electrical change for a specific period of time.
The reaction chamber, called a corona cell, consists of two non-conducting
concentric tubes separated by a small (0.5cm) gap. A ground electrode is
attached to the outer tube, while the inside tube is connected to a high
frequency generator (Carlssbn and Wiles, 1970). Although direct analogies to
environmental degradation requires caution, exposure to pure gases, either
with or without coronal acceleration, can be used to assess the potential
atmospheric degradabillty.
429
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c. Degradation by Light: Exposure to various wave lengths of
light is perhaps the .most critical area of physiochemical polymer degradation,
> and is comnonly conducted both in vacuo and with various gases (Day and
Wiles, 1972b). Depending on the nature of the polymer, experimental specimens
may consists of films, powder, or solutions. Films are by far the most
common type of specimen used and are prepared by dissolving the polymer in a
suitable solvent, pouring the mixture into a cast (e.g., glass plate), and
allowing the solvent to evaporate (Hummel, 1966). The key phase, however, is
"suitable solvent!" The possibility of solvation in some way effecting the
original polymeric structure must be considered. With polystyrene, for
instance, the quantum yield for hydrogen formation can be radically affected
by the type of solvent in which the sample is dissolved (Fox, 1967). This
potential solvent,.problem can be avoided by preparing the film from powder.
A known amount of polymer powder is placed evenly on a quartz plate and
then melted. The polymer surface is then covered with another quartz
plate and compressed. The samples are stripped from the quartz and vacuum
dried. This method of preparation does, however, involve alternate cooling
in dry ice and heating to 225°C. Thus, the thermal stability of the specific
polymer must be given careful consideration before this treatment is adopted
(Carlsson and Wiles, 1969).
The light used to determine photodegradation can be obtained
from a variety of sources (e.g., mercury vapor, xenon arc, carbon arc).
o o
Mercury vapor lamps of low pressure (2537A), medium pressure (2200-4000A), or
o
high.pressure (higher intensity in the 2000-4000A region) are common with
430
-------
filters being used on medium and high lamps to isolate the desired wave
lengths (Fox, 1967). The importance of fully characterizing the emissions
from the selected light source is evident from the work of Day and Wiles
(1972a). Comparing the emission curves of xenon arc with Pyrex/clear glass
o
filters and carbon arc with clear Pyrex globe, the actual output at 3400A
is more than double with the xenon arc. After 1000 hours of exposure of a
17,500 Mn polymer, the xenon arc reduced the Mn to 13,800, whereas the
carbon arc reduced it only to 16,900.
As with the biological techniques, physiochemical exposure
may take the form of a field test. Although the details of exposure condi-
tions may vary, it usually involves placing the polymer sample above ground
(minimizing biological affects) allowing attack by all three of the above
discussed degrading agents (Potts et al., 1972).
C. Analysis Procedures
A variety of methods have been used to assess the degradability of
polymeric substances. In general, they can be divided into three distinct
groups: those measuring changes in the mechanical properties of the polymer,
those measuring the response of biological systems, and those measuring
molecular changes. As with the methods of exposure, the type of analysis used
is usually based on the type, of test desired and the proposed use of the compound.
1. Changes in Mechanical Properties
The degradation of a polymeric substance might be reflected in
any of a host of physical or chemical changes. Certain factors, however,
can be discarded either because of difficulties in quantification or a lack
431
-------
of reliability. Consequently, such alterations as cracking, warpage, and
mechanical erosion, which often may indicate a form of degradation, may be of
»
little use because of the inherent difficulties in accurately describing such
processes (Carpenter and Smith, 1972). Similarly, changes in size may reflect
degradation but more direct factors such as absorption, leaching, or embrittle
ment are more readily measured and directly related (Muraoka, 1969). Even
very'obvious physical changes as discoloration must often be discarded in
analysis because they are not necessarily closely associated with actual
deterioration (Rogers and Kaplan, 1971).
Even excluding the above-mentioned factors from a quantitative
determination of degradation, several physiochemical parameters .for which
specific techniques:have been developed can be considered as analytical
candidates. These Include;
~. a. Water Absorption or Transmission
b. Electrical Properties
c. Elasticity/Embrittlement
d. Hardness
e. Tensile Strength
f. Weight Loss
a. Water Absorption or Transmission (ASTM, E96-66, 1971): The
amount of water in liquid or vapor phase that a polymeric substance will
transmit or-retain is related to macromolecular arrangement. Thus, changes
in these parameters can be used as an indicator of degradation if proper
consideration is given to other possible factors influencing absorption
(e.g., leaching of plasticizers). As a rule, water absorption is used for
relatively thick specimens while water vapor transmission is used for thin
sheets or films which might be expected to exhibit membranous properties.
.iV
432
-------
Water absorption has been used to determine the response of
plastics to marine exposure, although it could be adapted to other types of
exposure (Muraoka, 1969). Basically, water absorption is determined by
changes of weight in the specimen before and after submersion in water.
Because the primary concern is with changes in the percent water absorbance
by weight, two identical samples should be used. The sample should be
weighed prior to exposure (W..) , Immediately after submersion termination
(W2), and after conditioning at 23°C and 20% relative humidity until weight
stabilizes (W,). In marine exposure where borer attack may lead to
significant weight loss, VL must be discarded. Percent water absorption
is then wo~W3/W3 x 100. In cases where W is not obviously invalidated,
discrepancies between W--W- and W_ - W_ may indicate non-degradative weight
changes such as additive leaching or evaporation.
Water vapor transmission involves sealing the test specimen
over the mouth of a dish containing either water or a water absorbant substance.
The entire system is weighed, then placed in various temperature/humidity
systems at constant pressure. Weight gain [using desicant] or loss [using
water] can then be used to measure total vapor transmission over the experi-
mental period per unit of surface area, the accepted unit of measurement
2
being g/24h / m at specified experimental conditions (ASTM, E96-66, 197J).
b. Electrical Properties: Many factors, both, physical and
biological, can affect the electrical properties of polymeric materials.
In field testing it is often difficult to distinguish between the effects
of moisture and fungi, especially in that even subvisual growth of fungi
433
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can produce marked effects (Snoke, 1957; Greathouse &t_ ai^., 1951). However,
with proper experimental controls, the distinction can often be made and
the change in various electrical properties closely correlated to polymer
degradation (Muraoka, 1966).
For solid polymeric electrical insulating material, the
high-voltage, low-current dry arc resistance test can serve as a useful
screening test for degradation (ASTM, D495-70, 1971). This test involves
connecting either a tungsten rod electrode system or stainless steel strip
electrodes a given distance apart to the surface of a plastic sheet, applying
a specified voltage, and observing the arc formed. As the voltage is
V v
increased, failure of the insulating material will occur in a relatively
short period of time by the formation of a line of conductance and the
consequent disappearance of the arc into the material. Variations in
failure time may then be used in assessing degradation. Certain
types of material, however, may fail in different ways, such as combustion
;
or carbon formation. For these types of material, the test has little
relevance (ASTM, D495-70, 1971).
! An electrical property test more closely related to end-use
rather than screening is a measurement of changes in dielectric breakdown
voltage (ASTM, Dl49-6.4, 1971). This test is conducted at commercial power
.1
frequencies and can be .used to measure changes in insulating ability after
exposure to various degradative processes. The test basically consists of
applying a given voltage or voltages to the test material over a given period
of time under specified temperature and humidity. The specific conditions
of the test should simulate the actual use of the material to be tested.
434
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Dielectric breakdown is preferably determined by the physical decay or dis-
tortion of the material rather than an increase in current. The test iteeIf
can be conducted in any of these ways: a rapid voltage rise over a short
period from zero voltage to breakdown voltage, a slow continuous rise in
voltage for 50% breakdown to breakdown, and an even increment rise from 50%
of breakdown to breakdown.
While both the arc resistance test and the breakdown voltage
test measure changes in decomposition points from electrical stimulation,
more subtle characteristics such as the dielectric constant can also be
measured (ASTM, D150-70, 1971). This type of testing involves rather sophis-
ticated apparatus and careful interpretation which, in essence, involves
direct electrical measurement of capacitance by constructing an experimental
capacitor from the test material. Changes observed in dielectric constant
can be caused by physical or chemical changes, moisture absorption, and
surface ionization. A more manageable and readily measured parameter may
be the change in DC resistance caused by degradation. This can be determined
simply by direct measurement of voltage or current under specified circum-
stances (ASTM, D257-66, 1971).
c. Elasticity/Embrittlement: Changes in elasticity may indicate
actual alterations in the polymer. Photooxidation, for instance, of poly-
ethylene or nylon may lead to cross-linkings which result in embrittlement
(Lightbody .§££.!., 1954). However, the simple loss of plasticizer may also
lead to elasticity loss without necessarily Indicating any actual degradation
at the molecular level (Carpenter and Smith, 1972). Two quantitative methods
435
-------
can be used for the. measurement of elasticity, the cantilever beam technique
t
or the vibrating reed method.
The vibrating reed method involves calculation of the elasti-
city modulus through experimental measurement of the resonance frequency of
the sample (Hazeu and Waterman, 1965). The results obtained by this method
are highly dependent on the thickness of the sample and the sensitivity is
greatly diminished by samples approaching 1.0 mm.
The cantilever beam method-measures the elasticity of a
material by experimental determination of the force and angle of bend. Thus,
the deflection angle caused by a known force can be used to indicate stiffness.
i
The physical distortion of the test material, however, indicates both plasti-
city and elasticity; thus, derivation of the elasticity modulus is not possible.
However, this method is suitable for measuring relatively thick specimens
so long as the length-to-thickness ratio exceeds 15 to 1but loses accuracy
as the thickness decreases to 0.5 mm (ASTM, 747-70, 1971).
d. .Hardness: For thicker test specimens, hardness rather than
\elasticity may be' a more dependable indicator of degradation. The basic
procedure for testing the hardness of plastics (ASTM, D785~65, 1971) recommends
a thickness of at least 0.6 cm, although the method can be adapted for
testing tape samples (Pankhurst and Davies, 1968).
The basic premise in hardness testing is the hardness can be
correlated to the depth of penetration of a steel sphere, pyramid, or other
device under a fixed load. In practice, the index of hardness is determined
436
-------
by the Increase in penetration from a smaller initial indenter load to a larger
load. A variety of devices are available for measuring hardness by this method;
e.g., Rockwell Hardness Tester (ASTM, D785-65, 1971); Durometer (Muraoka, 1969);
Wallace Microhardness Tester (Pankhurst and Davies, 1968). While the scale
readings with each may vary, they can be converted to indentation depth, e.g.,
one scale division in Rockwell Tester may equal 0.002 mm. More Important in
comparing results of different tests is the size of the indenting sphere and
the interval of time between the minor and major strokes, both of which have
a marked effect on the depth of penetration. However, in measuring deterioration,
the main concern is in the difference between exposed and control samples, making
conversions often unnecessary. The prime factors in experimental testing are
that the samples be uniformly clean and conditioned (Pankhurst and Davies,
1968). When possible, such hardness tests should be compared to other relevant
parameters such as moisture absorption (Muraoka, 1969).
e. Tensile Strength: Changes in tensile strength has long been
a popular method for measuring the degradation of polymers, both natural
and synthetic (White and Slu, 1947). The basic method has altered little.
After exposure, the material to be tested is cleaned, dried, and conditioned.
It is then placed in an appropriate testing device. This tester usually
consists of two vise-like jaws, one of which separates from the other at a
fixed speed with a gauge for measuring load. Commonly, changes in breaking
strength are measured (Berk and Teltell, 1951). This basic method can be
modified to accommodate a wide variety of samples ranging from extremely
small samples of plastic (ASTM, D638-68, 1971) to nylon and polypropylene
ropes with tensile strength in the thousands of pounds (Muraoka, 1969).
437
-------
Detailed methods for conducting this type of test are outlined in ASTM, D638-68.
As a rule, this tensile strength is often a reliable indicator of degrad-
tion (Rogers and Kaplan, 1971; Pankhurst and Davies, 1968) and correlates
well with other parameters (Mandels and Siu, 1950, Potts et_ al., 1972).
f. Weight Loss: When dealing with a pure polymer or when the
effects of various additives can be precisely determined, weight loss can
be a very reliable indicator of degradation (Hazeu, 1967; Potts ejt al., 1972).
The procedure involves simply the weighing of the specimen both before and
after exposure using proper conditioning techniques to eliminate the effects
of moisture absorption (Hitz and Zinkernagal, 1967; Mandels and Siu, 1950).
t
When measuring biodegradability, an aseptic control may be introduced
(Sharpe and Woodrow, 1971).
2. Response of Biological Systems
The various tests for correlating biological response of selected
organisms to the degradation of polymeric substances all attempt to, in some
wayf monitor metabolic activity. This is most often done by either respira-
metric measurements or estimates of cell multiplication.
Perhaps the most common method is the determination of BOD.
This has been used by Snoke (1957) to screen for biological degradation of a
number of synthetic polymers. Water containing a known amount of oxygen
i
and the test polymer is inoculated with liquid from an enrichment culture.
Controls consist of polymers submerged in water. Incubation is carried put in
darkness in a water bath at a constant temperature. At various intervals
438
-------
(0,1,2,4,8 weeks) dissolved oxygen is measured. Although this test can be
used as a qualitative determination of the ability of the polymer to be used
as a carbon source, quantitative determinations are highly dependent on the
surface area of the test sample.
A direct determination of oxygen consumed over a relatively
short (48 hr.) period can be obtained using differential manometry (Mandels
and Siu, 1950), In this method, two flasks, experimental and control, are
connected to a manometer as diagrammed below.
Experimental Flask Control Flask
Figure 5"f. Design of Differential Manometer
[Mandel and Slu, 1950]
Courtesy of American Society for Microbiology
The experimental flask contains nutrient agar, the test substrate, and the
inoculum. The control contains only the agar. The CO. released Is absorbed
by KOH(10%) and the oxygen consumed is calculated by displacement of the
manometer fluid. Although rapid and technically uncomplicated, this test is
based on the premise that initial growth rate is directly related to degrad-
ability. While this assumption may be valid for measuring the relative rate of
439
-------
metabolism of readily degradable compounds, its applicability to the long-
range environmental stability of recalcitrant polymers is dubious.
A procedurally more complex but probably more precise direct
measurement of oxygen uptake is available using the Warburg apparatus. A
microbial inoculum is prepared by centrifugation. Standard Warburg flasks
containing aqueous NaOH in the-central chamber and the inoculum in the main
chamber are used. The side arms contain either the test substance or
distiller water. Thermobarometric control flasks contain only distilled
water (Pankhurst and Davies, 1968).
' The evolution of metabolic by-products have also been used as
indices of degradation. Given that certain bacteria metabolize endogenous
protein in the absence of external nutrients, the degradation of a test
material may be inversely related to NH, evolution. This test is conducted
in a non-nutritive broth containing the inoculum and test material. Test
samples are omitted in the control. Ammonia determinations are made from a
given volume of the broth after incubation. Protein is removed by precipi-
tation with trichloroacetic acid and centrifugation. Nessler's reagent is
added to the supernatant and optical densities determined spectrophoto-
metrically (Sharpe and Woodrow, 1971). A similar approach has been used to
determine anaerobic degradation by sulfate-reducing bacteria in sea water with
measurement of hydrogen sulfide (Snoke, 1957).
An alternative to the above methods is the visual determination
of bacterial growth. Using an agar plate, this type of observation can be
made over a wide if somewhat inexact range of quantitative tolerances
(Muraoka, 1969; Brown, 1946; Jones, 1968; Potts et^ ad., 1972; Walchli, 1968).
440
-------
Using liquid culture, however, comparatively exact estimates of
microbiological growth can be obtained by either direct or indirect measure-
ments. A viable cell count can be obtained by the surface drop method. This
Involves inoculating an agar plate with a fixed volume of liquid culture
after exposure to the polymer. Before inoculation, the sample is diluted
so that clear clones can be recognized. The number of clones established
allows for direct measurement of total number of viable cells in the polymer
culture sample (Pankhurst and Davies, 1968; Sharpe and Woodrow, 1971). An
indirect measurement can be obtained by nephelometric determination of
optical density in Inoculated cultures. With this method, samples may have
to be diluted as turbidity Increases with growth (Hltz and Zinkernagel,
1967). Using either direct or indirect liquid culture methods, the samples
must be shaken constantly to insure homogeneity of culture conditions.
3. Molecular Alteration
While changes in mechanical properties and response of biological
systems may indicate that degradation Is or is not occurring, these changes
only provide Indirect evidence for degradation. However, providing direct
evidence of degradation of polymers requires a radically different approach
from that of non-polymeric substances. Common analytical techniques, such
as gas chromatography, TLC, and mass spectrometry, which are frequently used
with lower molecular weight compounds, are not readily applied to most
polymers. Instead, changes in molecular weight and/or indications of bond
rearrangement or substitution are needed to satisfactorily describe polymer
degradation.
441
-------
Perhaps the only true indicator of polymer degradation is "backbone"
cleavage and the most reliable indicator of"backbone" cleavage is a decrease
in molecular weight. With most synthetic polymers, however, molecular weight
is more often an average than an absolute. Two common ways of expressing
this average are "w'eight average molecular weight" (MJ) and "number average
molecular weight" (M ). Values of M are based on techniques such as light
n w
scattering, sedimentation rate, and some viscosity readings, which determine
the weights of the various molecular fractions in the polymer sample. The
M is dependent on the number of molecules in a given mass and is usually
calculated from osmotic pressure measurements. As a rule, techniques used
to determine M are more influenced by the larger molecules in a given sample,
whereas M determinations measure large and small molecules with equal
sensitivity. Thus, in a given polymer sample, M will be greater than M
unless the sample is molecularly homogeneous. As the heterogenicity increases,
so will the ratio of M to M . This ratio (M /M ) can be used as an index
w n w n
of polymolecularity or changes in chain length distribution (Thomas and
Kendrick, 1969).
Sedimentation rates- from which M can be calculated are obtained by
ultracentrifugation of a polymer in a solvent of lower density than the
polymer. Change in refractive index of the solvent are used to monitor
polymer sedimentation. However, the rate of movement is dependent not only
.'on molecular weight but also on the shape of the molecules, with spherical
molecules settling more rapidly than cyclindrical ones. While this method
. '.» .
ia frequently used to characterize protein macromolecules, it is not
widely employed.on synthetic polymers.
442
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Light scattering measurements Involve comparisons of the amount
of light transmitted by a pure solvent compared to that transmitted by a
solvent-polymer mixture. The amount of-light scattered is related not only to
.>
the number of molecules present but also to their size, with large molecules
scattering proportionately more light than smaller molecules. Thus, only
an M can be derived from this method. While somewhat more popular than
w
ultracentrifugation (Fox, 1967), this method is rarely used for molecular
weight determination. However, light scattering measurement can also be
used with polymer films as an index of degradation without actually deter-
mining molecular weight (ASTM, D 1003-61, 1971).
Osmotic pressure measurements are conducted by dissolving the
polymer in a solvent and placing this mixture on one side of a membrane
selectively permeable to the solvent but not the polymer, with pure solvent
on the other side of the membrane. The osmotic pressure can be determined
either by static or dynamic methods. The static method measures the fluid
flux in a calibrated capillary tube connected to the polymer-solvent side of
the membrane, indicating solvent influx. This method is probably the more
accurate and requires less sophisticated apparatus, however, a considerable
period of time is usually required for the system to reach equilibrium. The
dynamic method utilizes an osmometer in which the flow of solvent is prevented
by the external application of a known pressure, the primary advantage being
the rapidity with which the test may be made (Golding, 1959).
Viscosity measurements to determine molecular weight is the most
popular method in dealing with synthetic polymers (Carlsson and Wiles, 1969a;
Day and Wiles, 1972b; Potts _et _al.,.1972). Basically, viscosity values are
443
-------
obtained by passing, a dilute polymer sample under a given pressure through,
a capillary tube,and measuring the rate ,of flow. In practice this is, done
. . ' -.-,* '1
by any number of, commercially available dilution viscome.ters. The intrinB.ic
viscosity [n] of^ a-homogeneous polymer is, related to molecular weight as:
. . -, ,.
[n] = KM . The constant "a" depends on .the rigidity of the molecule, varying
from 0.5 to 2.0 as.rigidity increases. The constant "R"is specific for. a
polymer-solvent mixture. For npn-hompgenepus polymers, the molecular weight
obtained is equa^l. to M if "a" = 1 (Golding, 1959). If "a" ^ 1, ,theimolecular
' , W ^ ' ' - ' ' i ' ':>..
weight.is often given simply a M , viscpsity average molecular weight
.'.."' 'V ' ''.' ! ' - !..,., ^ ._-£
(Potts ejt _al., 1972). The value is usually based on a standard [n]/$f curve
of analogous polymers of known molecular weight. Further, if, both "a" and
"K" are-known, it is possible to obtain M from viscosity measurements as done
".'-. ' ' ' n . :.' :-f-:-'
by Day and Wiles (1972a) for polyethylene terephthala.te:
» " ' ' ' !
[n] ="l.7 x 10-^M0-83 j
n
c f
Although changes, in. molecular weight are vital in monitoring
polymer degradation, more, precise information on types and degrees o.f changes
in bonding patterns can be obtained from standard infrared spectrpphptometry
(e.g., Delman £t al.., 1969; Gutfreund, 1971). As with non-polymer; compounds,
absorption characteristics in the infrared regions are highly specific and
changes in absorption patterns indicate molecular structure alterations.
_ - ' ' n
Samples used in spectrophotometric measurements may be prepared as films.cast
from solutions, pressed or melted films, microtomed samples, paraffin oil
mulls, or KBr disks. The thickness of the sample depends on the absorption
intensity of the polymer .and may vary from 0.005 mm to several millimeters
(Hummel, 1966). Polymers, of course, present highly complex spectra. In
444
-------
some cases, curve resolvers (e.g. DuPont 310) are necessary to resolve complex
infrared envelopes (Carlsson and Wiles, 1969b). When spectra are recorded
between sodium chloride discs, tetrachloroethylene has been used to preclude
interference bands (Day and Wiles, 1972a).
In that degradation of polymeric substances is often restricted to
the surface, an infrared technique has been developed which can determine
the penetration of the attack by identifying polar groups between 0.1 and
2 urn in depth (Cooney et al., 1973). This technique is referred to as
'attenuated total reflection spectroscopy." Basically, the technique involves
focusing an IR beam on a polymeric film with a suitable reflective backing.
The beam undergoes total internal reflection and various frequencies are
diminished depending on the absorption characteristics of the polymer (Carlsson
and Wiles, 1969). The absorption characteristics at varying depths are obtained
by varying the reflective element and the angle of incidence of the IR beam
(Carlsson and Wiles, 1971b).
Various types of thermal testing are also useful in determining
degradation. Perhaps the most elementary measurement is heat of combustion
(cal./g) using a bomb calorimeter. This procedure measures essentially the
amount of energy in a polymer. Degradative changes such as backbone cleavage
would thus be reflected in a decreased heat evolved during thermal decomposition.
More detailed information may be obtained from differential thermal analysis.
In this method, the polymer sample is slowly exposed to a wide temperature
range and changes in heat energy are compared with that of an Inactive high
thermal stability control. Decomposition is indicated by the polymer's heat
445
-------
absorption while condensation or crystallization is indicated by heat release.
' '
Changes in the temperature at which these thermal reactions occur indicate,
molecular rearrangement (Gutfreund, 1971).
Depending on the nature of the. polymer and the degrading agent,
various other types of analytical procedures ,may be employed. Simple light-
microscopy has been .used on microtomed samples of methane rubber to note
-( *
mycelial penetration (Awao, &t_ a^., 1971). Similarly, electron scanning-
can be. used-to characterize either surface changes in the polymer sample
(Po;tts et--al., 197.2) or growth of microorganisms (Cooney e£ al., 1973)i,
The degree ,of crystal linity can be measured by X-ray diffraction., although
this, technique, is not widely used to detect degradation. Chemical reactivity
' ' ' . c
can be used, to indicate, alternatives in end groups or side groups. Qnje.
method, is exposing, the. polymer to HNO. and then determining the amount of
residual nitrogen in the sample, by the Kj.eldahl test. This involves degradation
of the, polymer nitrogen with concentrated sulfuric acid and converting- it
to .ammonium sulfate which can then be measured photometrically (Gutfreund,
1971). Lastly, volatile products from the degradation of polymers may-,also
be measured. This can be done by standard mass-spectrometric and gas-^
chroma tog raphic methods (Day and Wiles, 1972c). When only one volatil'e .
decomposition product is evolved, manometric techniques may also be employed
(Fox, 1967).
446
-------
D. Evaluation of Techniques
1. Factors Affecting Degradation
a. Biological Degradation
The design of an experimental procedure for determining the
biological degradation of polymers can be divided into seven distinct phases
of evaluation.
(i) Selection of Polymer Formulation
(li) Pretreatment of Test Specimen
(Hi) Selection of Degrading Organism
(iv) Choice of Media
(v) Conditions of Incubation
(vi) Duration of Exposure
(i) Selection of Polymer Formulation: Polymers are seldom
used or disposed of in a pure form. Most contain a variety
of non-polymeric substances - such as plasticizers, stabilizers,
fillers, pigments, and lubricants - any or all of which may
undergo physical, chemical and/or biological deterioration or
dissipation. Such additives may lead the researcher to the
erroneous conclusion that the polymer is being degraded,
especially when surface growth due to the presence of a
degradable plasticizer or embrittlement due to the leaching
of plasticizers are used as degradation criteria. Thus, a
valid attempt is often made to determine the degradation of
the pure polymer (e.g., Wessel, 1964; Potts ejt al., 1972).
While admittedly a necessary approach, especially for
447
-------
+
I I'
screening tests, the possibility that these resultS'-may, have
little application to degradation of normal > polymer fo^rmUl&tlbns
(
must be evaluated. Consequently, in addition to testing. the
pure/ polymer, various formulations may; also require "testing.
One ^approach is to use various additives of known degradability
in the 'same polymer. For example, Hazeu: (1967) tested PVC films
witty three plasticizers, two of which readily degrade {'(di?-iso-
octyl sebacate and adipate) and one which does'. not i-(diriso-octyl
phthalate). By comparing results of these three formulation,
the1 /.investigator can distinguish between degradation attributable
to the plast-icizers rather than the polymer. While Hazeu *s"
.results cannot be refuted, the method may lead to misleading
conclusions if it is assumed that a material (Plasticizer)
degradable in pure form is -necessarily degradable in formula-
tion . Thus, Pankhurst and Davies (1968) demonstrated -.that
components of' a PVC type- may -degrade. separately vbut^ show; no
signs of doing , so r when compounded.
(ii) Pretreatment of Test Specimen: When exposing: a pplymer, to
degradation by a well-defined v'microbial colony, it' is ; of ten
desirable to avoid contamination with unidentified- organisms
by sterilizing' the test specimen. However, this sterilization,
itself, may cause some form of degradation' (Hazeu,, 1967;.
Pankhurst and Davies, 1968). Nevertheless, ethylene oxide
sterilization in addition- to phenyl mercuryborate -treatment
has been successfully employed (Hitz and Zinkernagal, .1967).
448
-------
Sterilization with ultraviolet irradiation is an alternate
approach, but possible effects that this might have on the
polymer must first be ascertained (Awao et al., 1971).
(iii) Selection of Degrading Organism: In attempting to deter-
mine biological degradation of a polymer, the choise of the
degrading organism can be based on any number of factors.
In general screening tests, an organism might be selected for
its common occurrence in nature or rapid growth rate (Berk and
Teitell, 1951)* In attempting to find organisms that degrade
a polymer, the organisms might be isolated by soil enrichment
techniques (Sharp and Eggins, 1970; Sharp and Woodrow, 1971).
In still other cases, the organism may be Isolated from the
material to be tested (Pankhurst and Davies, 1968). Whatever
the criteria, the existence of controls exposed to similar
physio-chemical factors is mandatory to assess the relative
importance of biological and non-biological factors. With
respect to pure vs. mixed culture exposures, the pure culture
technique is generally regarded as appropriate only for
screening tests, even though a single bacterial species may
predominate in nature (Walchi, 1968). In mixed culture
techniques using fungi, the problems of microblal antagonism
and the general failure to properly consider the role of
bacterial attack are common difficulties (Jones, 1968).
449
-------
(iv) Choice of Media: Under controlled laboratory. conditions j.
theiv-nutrient-salts agar ^recommended by ASTM D2676 andi,Dl924 (1970)
seems to have all but universal acceptance, for thev.cultivation
. > . '
of both bacteria and fungi: -
Potassium dihydrogen orthophosphate (KHvjPO^) 0.7g
Potassium ;monohydrogen orthophosphate' (^HPOi^) . : 0.7g ,
Magnesium, sulf ate (MgSO^ 7H20) . 0.7g^
Ammonium nitrate, (NH^NQs)- .l«P8;,'s;
^ -... '"" i '
Sodium chloride (NaCl) 0.005g.v,
Fer]rousv.sulfate (FeXOij 7H20) 0.002g
Zinc sulfate (ZnSQit 7H20) 0.002g ,
Manganese sulf ate (MnSOjt 7H20) O.OOlg ,
~i *
Agar 10. Og
Watcer IOOO,^J0 ml,.
Hitz and Zinfcernagel (1967') haye,,intr9ducedv.ra variation
on.; this method with the omission of .the agar. Theoresultingr
liquid medium .allows for nephelometric assessment -.of .micro-
biological growth, thus making quantitative estimates, more,
valid.
While the medium described .above may satisfy .the, mineral
requirements of many microorganisms, it hardiy.;apprpximatfes'
normal environmental . conditions . When .such ;an. approximation-
is desired, a number of media modifications are -possible; An
actual soil sample with a variety of added carbon sources.,:
may approximate environmental conditions in the laboratory .
(Lloyd, 1955; Hueck and. van der Toorn, 1965). Alternatively,
450
-------
a medium with a well defined carbon source (e.g., filter
paper) may be used (Sharp and Egglns, 1970; Walchi, 1968).
The screening tests which supply no external carbon source
to the organisms may be used to conclude whether a polymer
is readily used as a carbon source but may not indicate the
potential of a growing culture to alter a specific polymer
(Walchi, 1968).
(v) Conditions for Growth: For the most part, screening
tests are conducted under conditions as close as possible
to ideal for the test specimen. This Includes factors such
as optimum light, oxygen, temperature, and pH. Admittedly,
such conditions may seldom be encountered in the environment
and there is a potential for such tests yielding false
positives in terms of environmental degradability (Wessel,
1964). In addition, erros may be made in assuming polymer
biodegradation from field studies, using polymer-additive
formulations in which additive decomposition rather than
polymer degradation is observed (Berk and Teitell, 1951;
Rogers and Kaplan, 1971).
A potential source of false negatives in terms of degrada-
tion is that all screening and end-use tests encountered in
the degradation of polymers conducted in a relatively closed
system. While gases may diffuse in and out, the possibility
exists that toxic products of initial polymer degradation may
451
-------
accumulate and inhibit further degradation. Although this
has been cited as a possible source of error even in field
testing (Muraoka, 1969), there is no indication that such
inhibition actually has occurred in any laboratory te.sts.
(vi) . Duration of Exposure: If any major criticism.is. to 'be
leveled against exposure techniques for determining bio-
logical degradation, it might justly be made on the failure
of ,many tests to allow for sufficient time for degradation
to .occur. Many tests, especially those using respira.tory
techniques for analysis, are conducted over, extremely
short time periods with many using no external carbon source.
Thus, a compound that is degraded at a very low rate might
appear to be non-biodegradable in these short periods of
exposure. However., all investigators who have utilized
such methods (Mandels and Siu, 1950; Sharp and Eggins., 1970;
Sharpe and Woodrow, 1971; ZoBell and Grant, 1942) recognize
this factor. These tests are designed to determine if a
compound is readily degradable, not if a compound has the
potential to degrade or even will degrade under environ-
mental conditions.
452
-------
End-use tests and field tests, however, have somewhat
higher goals with consequent longer exposure periods of months
or years (Cooney e_t al^., 1973; Muraoka, 1966 and 1969; Hueck and
van der Toorn, 1965). However, the conclusions that can be
drawn from such exposure periods are still somewhat limited
to stating that a specific polymer does not degrade rather
than that it cannot be degraded. Given that the concentration
of polymeric waste is constantly rising and that it may already
be quite high in certain mini-environments (e.g., land fills),
the possibility of microorganisms evolving necessary enzyme
systems to degrade and metabolize synthetic polymers cannot
be dismissed (Nlckerson, 1971).
b. Physiochemical Degradation
In describing the various techniques for the exposure of
polymers to physiochemlcal degradation, a distinction was made between attack
by liquids, gases, and light. While a useful division for discussing procedural
details, this classification is rather artificial. Seldom will the environ-
mental degradation of a polymer be caused by any single element. As in the case
of polyethylene, non-biological degradation will usually involve the combined
effects of light, oxygen, and water (Scott, 1970). Thus, many of the exposure
techniques do not attempt to distinguish between these effects and, except in
cases where basic research is being conducted into the mechanism of degradation
453
-------
(Day and Wiles, 1972c)",'such distinctions .hardly seem indicated. What is of
more importance is the relevance of the exposure .to conditions which are .lik
to be met in the environment. ,
For experiments designed to .determine ,,the .effect-of 4water on
i
polymeric material, the critical factors are-pH, .temperature ,and .duration. Most
of the available information on hydrolysis is primarily concerned with those
polymers ,that are known to degrade rap.idly, such as hydroxyp.ropyl cellulose and
polyethylene oxide (Titus, 1973; .Brown .and Reinhart, 1971; Anon., If71b). How-
ever, a variety of ^polymeric substances whose hydrolysis. character^stics..haye
not been'well .defined are known to contaminate aquatic :envirpnmentsr(Carpen^er
and ..Smith, 1972). Although the work.of Muraoka (19.66 and 1969) would .tend to
indicate that most .common polymers do not undergo appreciable,^y.drolysis,
systematic studies would seem to be indicated.
Standard-exposure ..methods of /polymers to atmospheric .gases
and/or irradiation ;are often characterized as ''accelerated,tlweathering" tests
(Delman e£ .ail., 1969) . By ,and .lar.ge, .this: assiimption seems j.ustifjled. Photo-
chemical .degradation requires an energy .level of .around jBQ-lOO.kcal^mole ; if
polymeric, bonds :are to be cleaved (Gutfreund, 1971). In terms ofv^ave length,
this.energy can. be .calculated by the formula: .energy in kcal/mole '«
* O
2.86 x,105/X in A (Cooney et^ al. , 1973). Based on this formula, and,,the, above
information omenergy .levels, wave .lengths of 287-358 nm are. the. most appro-
priate for determining the photodegradability of synthetic^polymers.
A54
-------
2. Internal Consistency of Results
Two interlaboratory experiments have been conducted in an attempt
to standardize results and test methodologies which apply to synthetic organic
polymers. Hueck and van der Toorn (1965) report the results of nine labora-
tories using the soil burial test with residual tensile strength as an index
of degradation. Although these tests were conducted on Cu-naphthenate impreg-
nated cotton rather than a synthetic polymer, the results are indicative of
problems in quantitative comparisons of degradation testing. In general, the
results for the untreated cotton controls were consistent between laboratories
but the results with the impregnated specimens varied widely. Here a comparison
of impregnated cotton to synthetic polymers with additives may be valid. Dif-
ferences in leaching characteristics of additives or impregnates may markedly
affect the rates of actual or apparent degradation, making quantitative com-
parisons between laboratories difficult in cases where pure polymer specimens
are not used. Hazeu (1967) outlines similar results from an interlaboratory
experiment using mixed culture agar plate tests on plasticized polyvinyl
chloride. In this Instance, laboratories were in substantial agreement when
weight loss was used as an index of degradation but visual evaluation of the
test specimens proved misleading. By using three different plastlcizers,
only two of which are biodegradable, changes in weight appeared to be due
entirely to plasticizer metabolism rather than polymer degradation.
While quantitative comparisons of synthetic polymer degradation
may be difficult to make because of the effects of various additives, the
qualitative results are in relative agreement. In no case encountered in the
455
-------
literature do investigators disagree on the degradability of any.pure polymer
tested-by the same or different test methods. Details of these results in
terms of the effect of structure on degradability are discussed in
section VIII. ,: p. -461. ,
3. Comparison of Laboratory Results to Behavior in the Natural Environment
Because of the lack of systematic monitoring data, a satisfactory
*
correlation be'tween the environmental fate of synthetic-organic polymers and the
resultS'iof >the^ various test methods previously discussed cannot be^made. ..How-
ever, '-working -with the-scant .data that is .available, the results of tests for
biological arid physiochemical degradation are not in conflict with current
monitoring data.
Screening, .end-use, and field tests all indicate that^most of the
-.' . ' ' : f ''
synthetic-organic polymers which'have enjoyed commercial success .of the past
:two. decades are-rextremely persistent (see;.Section VIIL , p. 461). .Monitqr.ing data of
** ~ . i - 3 ' "'-'.
sea surface .contamination (e.g., Carpenter and Smith,:.19 72; Carpenter et :al.,
.'"'". -H:
1972;;;and Colt-en-et_
-------
leaching are common, actual polymer degradation is most often attributed to
physioehemical rather than biological factors (Eggins and Mills, 1971). This
is also in substantial agreement with the results of various test procedures
as discussed in Section VIII D. , p. 474.
E. Cost Analysis
The cost of determining the environmental degradability of synthetic
organic polymers may vary widely depending upon the exposure protocols that
are established and the types of analytical techniques that are employed.
Because the possible approaches which could be employed are numerous, the
cost analysis will be restricted to a representative series of screening,
end-use, and field tests using both mechanical and molecular indices of
degradation. In that the cost of exposure testing and analytical techniques
are independent of each other, cost breakdowns of various procedures are
given separately in Table 44. A daily labor cost including overhead for
a B.S. chemist or biologist is assumed at $120/day. Because the simultaneous
testing of more than one compound would most often be considerably more
economical, estimates are given for both one and ten compounds.
The following outlined costs are at best only crude approximations. A
variation of + 25% could easily be expected, depending on the volume of business,
local labor costs, variations in overhead, the specific compound to be tested,
etc. It should be noted in Table 44, that the equipment costs do not include
initial capital expenditures. It is assumed that only fully equipped labora-
tories would do this type of testing and that the per unit cost of such
equipment would be reflected in the overhead rate. Also, the cost of running
appropriate controls is included in the total estimate.
457
-------
Table 44- Cost of Selected Procedures in Determining the
Degradation, of Synthetic, Organic. ?olyine.r8^
' Number of Length, Labor EquIpMnt Total Cost/
Procedure 1 Compounds of Test Man-days.Cost Cost]. Cost Compound
EXPOSURE. TESTS
Screening Test
(Mixed Age*. Plate).
End-use Test
(Soil Simulation. Test)
Fiel.d
(Sot! Burial, Teat)
ANALYTICAL TECHNIQUES
Hardness or Tensile
Strength;
Weight Loss Test
Scanning,. Electron ,
Micrograph
Molecular Weight
Determination by.
Sedimentation
I.R. Spectra
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
(days)
21
21
6.0.
60
120
120
0.25
2.5
0.1
0.5
0.25
2.5
0.5
4.0
0.2
0.5
0.5
3.0
1.0
4.0
5
8
0.25
1.5
0.1
0.5
0.25
2.5
0.5,
4.0,
0.2
0.5
$ 60.
360,,
120
48CL
700
960
30 .;
180
12
60
30
180
60
480
24
60
"*
$ 10i.
80
20
100
50.
150V
*
* ^
*
*
200
1600
15
60
*
" " ' -
$ 70 r
440,
140.
58Ov
750
Hip,
30:,
18Q>;
12
60^
23Q;
1780:
75
5 20^
, ' i"
24
60V
$ 70,
44.
140.
58
750
30.
. iff,
12
6
230
178
75
52
24
6
Negligible
458
-------
For a complete evaluation of the environmental persistence of polymers,
all levels of testingi.e., screening, end-use, and field testswould probably
be necessary. Screening tests such as the mixed agar plate method are useful
in determining whether a polymer is readily degradable under ideal conditions.
However, polymers shown to be degradable under such conditions may degrade
much more slowly under environmental conditions. Conversely, the lack of
degradation in a screening test does not rule out the possibility of degrada-
tion over prolonged periods under natural conditions. In the same way, if
positive degradation is found in a field teste.g., soil burialit may be
desirable to determine the rates of degradation under controlled conditions.
Analytical techniques that are commonly used and quite beneficial in
determining product utility--!.e., tensile strength and hardness testsare
only of tangential use in assessing environmental degradation. Other types
of tests which do show molecular degradationsuch as spectral analysis and scanning
electron micrographyalthough different in price, yield much more useful and
definitive information on potential environmental fate. As estimated in Table 47,
the total cost of a complete series of tests - screening tests, end-use test,
field test, electron scanning, and I.R. spectra - would be about $775 per
compound if ten compounds were tested or $1722 per compound if one compound
were tested. This testing of many commercial polymers may be justifiable on
the basis of high sales volume, high unit cost, and/or cost spent in basic product
research and development.
459
-------
VIII. RELATIONSHIP BETWEEN CHEMICAL STRUCTURE AND ENVIRONMENTAL PERSISTENCE
Although a fair amount of information is available "about the influence
of molecular structure on the toxicity to human beings of drugs and certain
other chemicals, much less is known about the influence of molecular structure
on the environmental persistence" (Goodman, 1973). This lack of understanding
is indeed unfortunate since understanding the correlation between chemical
structure and environmental persistence could be extremely useful. For
example, such understanding would allow synthetic chemists to consider the
environmental stability of a chemical in the early stages of commercialization.
It would also allow inclusion of environmental persistence considerations when
setting environmental hazard priorities for new chemicals or chemicals which
have not been thoroughly tested. Similar reasoning applies to inclusion of
bioaccumulation considerations in priority setting since the potential for
"ecological magnification" (Kapoor et al., 1973) is closely related to the per-
sistence both in the environment and the food chain. In addition, understanding
relationships between chemical structure and environmental stability provides
insight into breakdown mechanisms and pathways in the environment and provides
a considerable advantage to researchers attempting to identify metabolites.
The following sections will discuss what is known about the relationship
between chemical structure and persistence in the environment. The sections are
categorized either by chemical group, reaction media, or mechanism of
degradation.
A. Relationship of Chemical Structure and Biodegradability
Part of the reason that only a few generalizations can be drawn
between chemical structure and environmental persistence is the lack of
Preceding page blank
461
-------
precise criteria 'for determining persistence. Two general criteria have'
frequently been used: the rate and extent to which a natural or'enriched
mixed 'culture degrades the compound, or the proportion of-tested speciesj -strains,
or isolates which is able to use the compound for carbon and energy source-(Painter,
1973a). Payne and :coworkers (Payne et_ ,al.., 1970; Prochazka and Payne1, 1965) have
concluded'that "no .more convincing evidence of degradability can be offered
than demonstration-;!.that an organic compound can be utilized-as a: source of
both carbon and 'energy by'microorganisms.11 Horvath (1972a), however ;r dis-
agrees with: the growth criterion and feels-that many compounds are.:terme'd
"recalcitrant" (Alexander, 1965) not due to microbial infallibility'but
instead.-due' to fallible biodegradability test'methods which do not: take;into
account co-metabolism (concomitant oxidation of a non-growth-substrate)'. The
importance of the co-metabolic process to environmental persistence is relatively
unknown. Many studies-have not examined the possibility of co-metabolic degra-
dation and, therefore, this lack of co^metabblic evaluation should^-*bevkept,In
mind in the following review of chemical strueture-biodegra\labil±tfy correlations'.
The only physical property that has been found to be relatedVto. bio-
degradation is the water solubility of the chemical. Water-insoluble4 mat'e'riais
are frequently expected to endure longer than water-soluble materials (Alexander,
1965, 1973a). Kapobr et^ al. (1973) found-good agreement between water -solubility and
their "biodegradability index" (BI=non-polar products/polar products), although' the
correlation between BI and the octanol-IUO partition coefficient was very
poor. Along this same line, Swisher (1970) has concluded that, with
surfactants, the "chemical'nature of the hydrophilic group is of-only minbr
importance in affe'cting biodegradability". The reason for increased* persistence
462
-------
with decreased water-solubility is not well understood but it is possible that
the hydrophobicity can contribute to: (1) failure of the chemical to penetrate to
the reaction site within the cell, (2) reduced rate of attack when biodegradation
is regulated by the rate of solubilization, and (3) inaccessibility of the chem-
ical due to adsorption or entrapment in inert material (see Alexander, 1973a).
Other parameters have also been suggested for correlation with biodegrad-
ability. For example, Torgeson (1971) suggested that "the rate at which a
molecule is degraded is dependent on a number of factors such as: (1) the ease
with which the molecule can penetrate the cell and reach the appropriate
enzyme site, (2) the extent to which steric effects interfere with enzyme
bonding, and (3) the extent to which electronic effects of molecular substituents
either interfere with enzyme bonding or alter the energy required to break the
critical bonds in the molecule." In order to quantify these factors, Torgeson
(1971) suggested the approach used by Hansch (Gould, 1972) for correlating
structure to biological activity which includes consideration of lipid solubility
(octanol-water partition coefficient), electronic effects (Hammet functions),
and steric hindrance (Taft steric parameter). Unfortunately, a comprehensive
comparison of biodegradability to these combined factors has not been undertaken.
However, Omori and Yamada (1973) have demonstrated a relationship between
electronic structure and position of hydroxylation on aromatic compounds by micro-
organisms and, therefore, it appears that pursuit of this approach might be
very fruitful.
463
-------
Most comparisons of chemical structure and biodegradability ;are much
less .aiuilyticalL than the approach suggested above, and are usually presented
as generalizations fpf functional groups. Painter (1973a) has suggested the
following parameters which affect resistence :tOjbiodegr-adatipn:./molecular size,
tertiary branching,, the nature, position and number,ofsubstituents -in .the
molecule, and :the presence of oxygen,.nitrogen, .chlorine >,and other atoms.
Table 4 5 summarizes a number of studies which have -examined ;the-:biodegr:,adability
" t
''of various, chemicals. From these .studies, some. generaliza>tlo.ns:;arev:apparent.
Highly, branched compounds are frequently, resistant, to biodegradatipn. i'YThis
is-attributed to the fact that increased substitution hinders, Pr-oxidation, the
process'.by? which alky! chains are usually broken down (Hammond -and^ Alexajider,
1972). 'SThis effect first became apparent .when studying Ithe,ibio4egradability
of'surfactants .(LAS compared to TBS) (Swisher,: 1970). .As ^.general, rule,
alcohols, aldehydes, acids, esters,: amides,, and^aminovacids seem to...-bev-.more
susceptible to,microbial attack than the corresponding, alkane,; olefins,-iketones,
dicarbpxylic'. acids, nitriles, amines,, and chloroalkanes :(Painter,: 19,73a) . Ether
functions are sometimes particularly.resistant to microbial. attackJiCRyckman
e_t_ .al. ,' !>196'6) . -Substitution on aromatic rings can have varying leffects. Groups,
such as carboxyl or hydroxyl, have a tendency.to increase .biodegradabilityAwhile
halogens and nitro groups reduce microbial attack (Alexander^ and-.Lustigman, 19.66).
Meta-disubstituted phenols and phenoxy compounds are usually more shdu'ld be
emphasized that these results are dependent upon the test methods used .and the
criteria of Wodegradation.
464
-------
Table 45. Relationship Between Chemical Structure and Biodegradation
Reference More Biodegradable
(leas persistent)
.Leas Biodegradable
(more persistent)
Alexander (1965, 1973a) Water soluble
Svlsher (1970)
Painter (1973u)
Water Insoluble
ALIPHAT1CS
Mohanrao and McKinney
(1962)
Van Der Linden and
Thljsse (1965)
[lift (1972)
McKenna and Kalllo
(1964)
Aliphatic
O:H,) ,
2 n
0 - C,. >C,
0
Perry and
Cernlglia (197}n) Normal alkanes. Straight chain, Gases, Alkanes, Branched Alkenes, Branched AromatlcB,
C10~C19 alkanes C2~C4 C5~C9 Alkanas. C3-Cn Alkenes,
C12"C19 " C12 Cycloalkoneo
Ryckman et_ al. (1966) R - CHjOH
RCHO R - CR
II
0
R - 0 - R
Painter (1973a)
Ludzack and
Erclnger (1960)
RCH.OH. RCHO, RCO.H
' 0 *
RCO,R. RCNR, R-CHCO.H
2 I 2
H, RCN. RNH.,
RCHR
I
Cl
Kaufman (1963)
CHCl
COjH
CHjCHjCCljCOjH
Ettlnger (1936)
CM, CH.OH
1 3 3
CH3(CH2)3(ra2 CHj-CH-CHjOH
0
II
CH COC H
J0 '
CHjCHjCHOH
OH
0
(HOCH2)2NH r^
0 0
CH3CCH2CCH3
HO(CHCH-
2-0)jH
Uatfleld (1937)
CHjOH, CHjCHjCHjOH,
HO(CH2-CH20)j-H HO(CH2CH20).,H
aldehydes, ketonee
Mills and Stack (1954) esters, sugars, straight HOCH CH OH. CH-CH-CH-OH ((CH,),CH),0, (HOCH.CH.)NH,
chain alcohols, acids, C1C11 CH OH (CH CH )C-0
----- ' (.1U12LH2UH, (CHjCH2;i.-0, (HOCH,CH,),N, HO(CH.CH.-O) H,
9 } 2 Nx
:,CM,JCH, (CHjCHjjjO, J^
Evans and David
HO(CH2CH20)jK
HO(CH2CH20)3H
Dlas and Alexander
(1971)
Hamnond and Alexander . X-CH,-(CH.) -CH.OH
(1972) Z 2 " .
X - Br, Cl. CH,
R - C(X) - CO-H
n £
R - C(X) = CH.OH
n.. L
465
Reproduced from
best available copy.
-------
Table 45 (continued)
Reference
More Biodegradable
(lens peruist^nt)
. MQHATICS
.? Alexander and
LuatigmAn (1966)
JC2T
Lesa Biodegradable
'(nbre petalatanl)
/NO,
.Alexander and Aleem
'.(1961)
acrae and
Alexander '1965)
Cl
Cl
-/oV
OH Br -< O V OH
/Cl
Cl-/oV
OH / O VOH
Cl Br
Cl
Cl Cl
«-( 6-
ri cl
Cljgj
C^~~^l
Cl
C/
o VLU?'
UO-U
(1971
R 4-Cl > 2,4-Cl - 2.4.5-C1 > 3-C1 > 3,4-Cl
£> ^tfams
Jkey and Boga- Culture
(1965)
OH
OH
cl
ci
Cl
.CHO
Cl
ci ci
,-.U_/O\- OCHjCOjH CJr-^OV^
Cl -( G V CHO
Reproduced from
best available copy.
466
-------
B. Atmospheric Stability of Organic Chemicals
Since the beginning of research on photochemical smog, there has been
strong evidence of a correlation between hydrocarbon reactivity and chemical
structure. Enough hydrocarbons have now been studied that some general
trends are apparent. Most reactivities of hydrocarbons have been measured
in terms of their ability to participate in the photooxidation of nitric
oxide to nitrogen dioxide (e.g., Glasson and Tuesday, 1970b). However, the
ranking of the hydrocarbons in terms of NO photooxidation follows the same
general order as that found for hydrocarbon consumption (Altshuller and
Bufalini, 1971).
For the most part, atmospheric reactivity seems to be directly
proportional to the nucleophilic character of the molecule. Glasson and
Tuesday (1970b) have reported the following order of reactivity: highly
reactive - disubstituted internal olefins > cyclopentenes > monosubstituted
internal olefins > unsubstituted internal olefins - cyclohexenes a tri- and
tetra- alkylbenzenes = diolefins > dialkylbenzene - terminal olefins > more
than 4C paraffins = monoalkylbenzenes > propane _>_ 2,2-dimethylpropane -
benzene > ethane > methane > little reaction. Thus, the reactivity increases
as the electron release into the double bond or benzene ring increases. As
further evidence that the species reacting with the hydrocarbon is electrophilic
in character, Glasson and Tuesday (1970b) compared the relative rates of NO
oxidation of several olefins to rates for oxygen atom-olefin and ozone-olefin
(in N-) reactions. This comparison is reproduced in Table 46.
467
-------
Tab;le ,46. Olefin Relative.Reaction Rate Comparison
J, (Glasson and Tuesday, 1970b)
Olef in
Ethylene
Propylene
1-rButene
3-,Meth35l-l4butene
Isobutene
2-Methyl-l-butene
2*Ethy;-l-butene
trans-2-Butene
trans-2rPentene
trans^Hexene
2tMethyl^ 2-but ene
2-rMethy 1- 2-pentene
2 , 3^Dimethyl-2-butene
2 , 3r-Dimethyl-2-:pente.ne
a
. Relative to isobutene rate
Cvetanovic (1960).
Wei and Cvetanovic (1963) .
Relative reaction
phptoox. O^atpm
0..48 0,0.4
1.0 0..23
0.83 0.23
0.77 ....
1.0 1.0
0.9,7, ...
0.66 ...
3.2 1.1
2.2
.1.7 '
5,4 3,2
4,6
17 4.1
15 ...
for indicated reaction.
.rates t "
0^ in Nj0
0,21
q.,95'
0.85
0.75
1.0
1.3
2.2
2.8
...
3.2
3.2
5.5
....
468
-------
Reactivity trends in monosubstituted benzenes (toluene > ethylbenzene >
iso-propylbenzene > tert-butylbenzene) is consistent with hyperconjugative
electron release. Not all the different reactivities can be explained by
electronic effects. The greater reactivity of cyclopentenes in comparison
to cyclohexene can be ascribed to the greater strain energy of the five-
membered ring. The greater reactivity of 1,3,5-trimethylbenzene in comparison
to 1,2,4-trimethylbenzene suggests some type of steric hindrance.
The reactivity of nucleophilic compounds can also be applied to other
compounds beside hydrocarbons. For example, trichloroethylene is relatively
reactive (95% loss in 6 hours irradiation) whereas tetrachloroethylene is
fairly inert (Altshuller and Bufalini, 1971). This can be explained by the
electron withdrawing effect of the additional chlorine atom.
When hydrocarbons are photolyzed in the presence of nitric oxide; ozone,
aldehydes and ketones, carbon monoxide, organic nitrates, and small amounts
of epoxides, alcohols, esters, and peroxides have been observed (Altshuller
and Bufalini, 1965). The products formed from reaction at olefinic double
bonds are perhaps best understood. Some examples are given in'Figure 52.
Recently, the reaction of some benzilic hydrocarbons to form
peroxybenzoyl nitrate (PBzN), a powerful lachrymator, has been noted (Heuss
and Glasson, 1968). The ability to form PBzN, rather than rupture the
benzene ring forming peroxyacetyl nitrate (PAN), is dependent upon the
presence of a benzylic carbon (see Figure 53).
469
-------
.Reference
Alleshuiler and Bufalihi
Reaction
GI
CH2 = CH
C * CH,
b
i II
- CH = CH
HCH
o
it
HCH
0 0
II II
HCH ^ CH2 = CH - CH
Wil'son .et 'al.
0
COOH
C00H
de-cm j
COOH
0
, COOH
Crosby and
Moirarien '(1974)
:Figifte 52. Products of Atinospheric 'Degradation of Olefins
'470
-------
R
-CH2CH2CH3
O ) - CH0-R >- \ ~ / ^-vn. QUO
0
CH3COON02
Figure 53 . Selective Reactivity of Benzylic Hydrocarbons
(Stephens, 1973)
Thus, from the above discussion, it can be seen that a great deal
more is known about degradability of organic chemicals in the atmosphere.
This probably is due to the more predictable nature of chemical systems
as opposed to biological systems.
471
-------
C. Categorization of Elements }
By examining the current knowledge of the, physical and chemical
.properties of;a toxic element, it is possible to jnake certain predictions
as to>* how, \some of these materials may behave: in.,the environment. .Such
.information could be helpful in deciding which of the toxic metal si/should
.be monitored in the .environment. Wood (1974) has classified elements on the
basis of itheirf.tpxicity and their relative availability for environmental
transformation, as .determined by their solubility characteristics and
concentrations at which ;they occur in.the environment; naturally. According
i ' .
j to,;this classification, .toxic, elements can be considered (i)-.non-critical,
; (ii),'.toxic and relatively accessible, or (ill) toxic but very insoluble
and-iyery-rare. -,The elements fit in these categories as .follows:
Table 47... Classification of Elements from the Standpoint
ol Environmental Pollution .(Wood, 1974)
:Noncritical Very toxic -Toxic but
.and relatively very Insoluble
accessible or very rare
- -- -
: Na
K
Mg
Ca
. H
0
N
C
P
Fe
. S
Cl
Br
*
F
Li
Rb
Sr
Al
Si
Be
Co
Ni
Cu
Zn
Sn
As
Se
Te
Pd
Ag
Cd
Pt
Au
Hg
Te
. Pb
Sb
Bi
Ti
Hf
Zr
W
Nb
Ta
Re
Ga
Lat
Os
Rh
Ir
Ru
Ba
iSome may argue with this designation, but fluoride
is added to drinking water. tAil the lanthanides are
very Insoluble and some are very rare.
472
-------
The elements classified as very toxic and relatively accessible should
be of major concern since they have the highest potential for environmental
hazard. The relative mobility of these elements in the environment, as well as
their toxicity, is somewhat dependent on their ability to undergo methylation.
Wood (1974) has predicted that tin, palladium, platinum, gold and thallium
will be methylated in the environment, but that lead, cadmium and zinc will
not be methylated. This prediction is based on the fact that the alkyl
metals of lead, cadmium, and zinc are not stable in aqueous systems and that
methyl B.^ does not transfer methyl groups to these elements.
Even if it can be predicted which metals can be methylated, it still
needs to be determined whether methylation will occur in the natural
environment, and if so will it result in accumulation of substantial quantities
of the methylated form in the environment. The methylation process in the
environment is considerably more complex than in laboratory studies, and
prediction of environmental methylation rates is extremely difficult. For
example, the kinetics of degradation of an alkylmetal [as shown by Spangler
.et. al. (lS>73a,b) for methylmercury] will be important in determining the net
quantity of the methylated form in the environment. No attempts have so
far been made to formulate a methylation model which will take into account
the kinetics of all the processes occurring in the environment.
In summary, some environment hazard priorities for elements can be
determined by considering availability, toxicity, and solubility of the
elemental forms and stability of the methylated form.
473
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D. Structure-Degradability Relationships of Synthetic Organic/Polymers
1. Biological Degradation
i
Very, little is presently known about the effect of structure on the
biological degradation of synthetic polymers. This question hasreceived'bniy
cursory attention ^n the published literature with exceptions such as'-the work
of Darby; and^r Kaplan (1968) and the exemplary-study by Potts and-:rcow6rkers
(1972). A listing of synthetic polymers which are subject or^resistant to
biological attack is presented in Table-48. ,
Although no absolute structure-degradability relatiohshipi can^-be
! .'
deduced-from this information, three distinct parameters affecting degradation are
apparent: chain,length or configuration, side group substitutiony and" chairi' bonding.
The-effect of chain length on configuratibn^is seen quite^ clearly in
the polyethylenes. Structurally, the polyethylenes are- closely allie'd' to the
natural, paraffinsj having the molecular formula CH3~(CH2) -CHji It has 'loiigV
been> recognized : that the lower molecular weight (smaller- chain lengtW)Ifp'oly-
ethylenearare susceptible to biological-degradation- while-the*higher :morecular
weight compounds*.are quite resistant (Lightbody jst jal., 1954)"J Recehtf tests
indicate;rthat the straight chain polyethylenes'below-500 MW can>be utilized
by microorganisms -but that comparable -sized molecules with methyl^branches1 on
every fourth carbon are resistant to attack (Potts ^t al., 1972)/suggesting w
a side-chain ..effect. Raines and Alexander (1974) have shown'that'normal alkanes
containing up. to 44 carbon atoms were metabolized by microorganisms1;'Vlf
The effect of the-side group is also evident-in both tfhe fcellulbse
esters and ethers. The cellulose derivatives have the general-formula:
OR CH2OR'
0
-------
Table 48 . The Biodegradability of Various Synthetic Organic Polymers
(Alexander, 1973a; Brown, 1946; Greathouse et_ jal., 1951;
Lightbody .et al., 1954; Titus, 1973; and Wessel, 1964).
Polymer Degradable
Acrylics
Polymethyl methacrylate No
Polyacrylonitrile No
Acrylonitrile-vinyl chloride
copolymer No
Cellulose
Cellulose acetate (low
acetylation) Yes
Cellulose acetate (high
acetylation) No
Cellulose acetate-butyrate No
Cellulose acetate-proplonate No
Cellulose nitrate Yes
Ethyl cellulose No
Hydroxypropyl cellulose Yes
Rayons
Acetate rayon No
Cuprammonium rayon Yes
Viscous rayon Yes
Polyethylenes
Polyethylene (low-^500-M.W.) Yes
Polyethylene (high M.W.) No
Polytetrafluoroethylene No
Polymonochlorotrifluoroethylene No
Polypropylene No
Polyisobutylene No
Polymer Degradable
Vinyls and Vlnylidenes
Polyvinyl chloride No
Polyvinyl alcohol No
Polyvinyl butyral No
Polyvinyl acetate Yes
Polyvinyl chloride acetate No
Polyvinylidene chloride No
Polyvinylidene fluoride No
Phenol-formaldehydes
Phenol formaldehyde No
Phenol-analine formaldehyde Yes
Resorcinol-formaldehyde No
Urea-formaldehyde No
Melamlne-formaldehyde Yes
Polyamides
Nylon No
Polyurethanes
Polyether linked Slight
Polyester linked Yes
'
Aliphatic Polyesters
Epsilone-caprolactine Yes
Polytetramethylene succinate Yes
Polyethylene adipate Yes
Aromatic Polyesters
Ethylene glycol terephthalate No
Chlorinated Polyethers No
Polycarbonates No
Epoxides No
Siloxanes No
Alkyd resins ; Yes
475
-------
In the natural, polymer cellulose, the R-groups are hydrogens. In cellulose
I.
acetate; - a cellulose ester, R = -C-CH3 -,- microbial resistance is imparted
only by, a high .degree of acetyl, substitution (Lightbody et'al;-,.- 1954', Alexander,
1973a). The nitrate group, however, does not;apparently protect the .molecule
from biological attack regardless of the degree of substitution.
Thet.polyurethanes exemplify the effect of chain bonding *on the
degradability of polymers. Darby and Kaplan (1968) tested a variety of
polyether.and polyester linked polyurethanes. The polyether polyurethanes
sho,wed, varying.^degrees of bipdegradability depending on the number of; side
chains in the^diol moiety and the degree of methyl substitution in'the polymer
chain. All of .the polyester polyurethanes, however, were highly degradable.
Although the general influences of chain length, chain linkage, and
side group substitution are apparent in the above examples, absolute structure
degradability relationships .cannot be drawn. As Darby and Kaplan-(1968)
indicate, each polymer or series of polymers must be evaluated on ;tne basis
t
of experimental evidence. For example, while the polyester linkages in
conjunction with aliphatic groups seem conducive to biological deterioration,
degradation is.blocked by extensive methylation of the polymer chain as in
polymethylmethacrylate. Thus, while specific aspects of polymer structure
can be correlatedito degradability, the predictive value of these correlations
is not very quantitative.
476
-------
2. Physiochemical Degradation
The roles of various physical and chemical factors which affect the
degradation of synthetic organic polymers have received far more extensive
'""' "'- "">.
characterization on the molecular level than the biological factors. Although
a given physiochemical agent seldom acts alone in polymer degradation, five
basic factors may be isolated for initial consideration: temperature, water,
oxygen, ozone, and sunlight. -^ .
The effect of temperature in the environmental range has received the
least attention of the above items because it cannot be divorced^ from a -t .$#&
consideration of at least one of the other factors. It $eems obvious that .£;'
as the temperature increases the effects of water, oxygen, and/or ozone
will be augmented. For instance, the hydrolysis of polyethylene terephthalate
proceeds rapidly in water at 100°C but negligibly at 70°C (Brown and Reinhart,
1971). Gutfreund (1971) has studied the thermal degradation of polyethylene
but under pyrolytic conditions relating to waste management rather than
environmental degradation. Studies on the potential effects of cryogenic
exposure have not been encountered.
The effects of water on polymer degradation are often difficult to
distinguish from the effects of fungi (Brown, 1946; Leutritz and Hermann,
1946). However, a variety of structural groups including acetals, amides,
esters, nitriles, and some ketones are subject to hydrolysis. Polymers
containing such groups as part of the chain backbone - e.g. polyamides, poly-
esters, and the regenerated cellulose fibers - will undergo chain cleavage
on exposure to water (Lightbody .et jil., 1954). While most often referred to
as an adverse reaction, hydrolysis characteristics have recently been used in
477
-------
the design of polymers intended to degrade in exposure to water, thus
facilitating solid-waste management (Anon., 197ib; Anon.* 1974). i
Oxygen is a major cause of polymer degradation under normal environ-
mental exposure. Lightbody and coworkers (1954) have cited oxygen as:the
degrading agent of greatest economic importance causing embrittlemejrit,
cracking, and granulation- in commercially important polymers such as p&iy-
ethylenei nylon, and: the cellulose esters; Similarly, oxygen may ^ead; to cracking,
embrittlement, or tackiness in some synthetic rubbers. On the molecular level,
oxygen-attack'may result in crosslinking, chain cleavage, oxygen addition,- or
hydropetoxide formation, and often involves autoaccelerating free radical
reactions (Gutfreuhd, 1971; Scott, 1970).
Ozone'-is involved in similar types of polymer degradation reactions
initially involving chemisorption (Gutfreund, 1971). Particularly affected by
ozone are.polymers containing aj. carbon-carbon-double bond. Thus-, most of the"
commercially important synthetic polymers are not especially attacked;' Even
! CZ ' ',..'.
neoprene rubber | is not extremely sensitive to
(l-CHi-C:- CH-CH2-]x[-SS-]y)
ozone because the double bond is deactivated by the adjacent chlorine
(Lightbody _et al., 1954).
Perhaps the key factor in the non-biological degradation' of synthetic
polymers is light-. In terms of the engineering of degradable polymers, photo-
degradation seems to be the most productive approach (Titus,- 1973). A variety
of photosensitizers, such as benzoin, cobalt nitrate, manganese nitrate, and
cobalt chromate, may be added^ to polymer- formulations to increase the"-rate of
photodegradation "(Gutfreund, 1971). However, many polymers are inhere'ntly
478
-------
photodegradable. A photodegradable 1,2- polybutadlene resin has been developed
in which the rate of degradation is directly proportional to the crystalinity
(Anon., 1971a). Similarly, a polybutene-1 film, reportedly degrades into
inert hydrocarbon powder after exposure to light for varying periods
(Anon., 1972). In addition to polybutene and polybutadiene, many
commercially important polymers - such as polyethylene, polystyrene, poly-
propylene, acrylonitrile-butadiene-styrene, and polyvinyl chloride - undergo
photolytic reactions (Titus, 1973).
The importance of structural impurities in the physiochemical
polymers has been repeatedly emphasized (e.g. Searle, 1971). For Instance,
CH3
polypropylene, | , if pure, would be transparent, would not
absorb light, and therefore would not undergo photodegradation. However,
polypropylene contains ketone groups which absorb ultraviolet irradiation and
result in Norrish Type I chain scission or Norrish Type II hydrogen extraction
(Cooney e£ _al. , 1973). Similarly, ketone groups have been purposely added
to polystyrene to accelerate photodegradation (Titus, 1973). Impurities are
conducive to other types of physiochemical attacks besides photolysis. Poly-
ethylene CH3~(CH2-CH2) -CHs, for instance, contains unsaturated carbon-carbon
bonds which can be attacked by oxygen or ozone and result in either direct
chain cleavage or hydroperoxide formation (Gutfreund, 1971).
As indicated previously, the physiochemical agents which can
degrade polymers seldom act in isolation from one another. This is illustrated
in Figure 54 with the mechanism for the degradation of polyethylene in which
the effects of impurities are discounted (Gutfreund, 1971).
479
-------
CH, CH,
/vwvkCH,-C - CH, vv\ _Ate-wv\CH, -C -CH./VVV* '-,
' i * ' *
H
?*' H,0 ?*' J (3)
i/wv^CH,-C -CHjVvv+bH^-LCH,-C -CHT
OOH
CH, CH,
OH +-CH, -C -CH, /w« --CH, -C » -CH,
Q Of
Figure, 54. Physiochemieal Degradation of Polyethylene
(Gutfreund, 1971) S
Although considerable progress is currently being.made in development
of degradable polymers and the elucidation of the structural Components
which are 'conducive to degradation, structure-degradability relationships,
other'than'the rather general characteristics outlined above, cannot be
drawn. HoWever, two points deserve particular emphasis. First, ,the physio-
chemical degradation of polymers does not usually result in monomeric units
but rather ends in a wide range of intermediate size molecules. 'This has been
illustrated in polyethylene (Gutfreund, 1971) and also seems to apply to
polybutene-1 (Anon.,. 19.72). Secondly, physiochemical degradation does .not
necessarily lead to biological degradation. This has been shown in:the
photodegradation of polypropylene (Gooney, et. al., 1973). Thus,.,while an
ideal polymer might be one that physiochemically degrades into units .which
can be biodegraded to carbon dioxide and water, this pattern has not-been
demonstr'ated in polymers which have been termed "degradable".
480
-------
IX. CATEGORIZATION OF CHEMICALS IN TEBMS OF THE SUITABILITY OF VARIOUS TEST
METHODS
After the possible persistence and degradation processes have been theo-
retically considered (see Section VIII, p 461), -an appropriate experimental
procedure needs to be developed to test the chemical's environmental behavior.
Deciding on the appropriate procedure requires the consideration of the physio-
chemical properties, quantities and sources of release to the environment
(release potential), commercial-economic factors of the compound of interest,
and the toxicity.
/
There are well over 2 million chemical compounds registered with the
American Chemical Society's Chemical Abstract Service (Council on Environ-
mental Quality, 1971). Fortunately, only 9 - 10,000 synthetic organic com-
pounds are in commercial use. These commercial (or potentially commercial)
compounds (and appropriate model compounds, to establish meaningful theoretical
models and generalizations) are the ones that should be tested since materials
produced in large quantities have a high potential for becoming environmental
contaminants. However, compounds likely to pose hazards because of their
potential toxicity should be tested too, even if their production is small.
Setting priorities for research on these thousands of commercial compounds
is an extremely complex process requiring the consideration of environmental
release potential, environmental stability, and toxicity (Howard, 1974). Some
of these same factors can be used for deciding which test method should be used.
For example, the quantity produced, the frequency of release, the environments
through which the chemical passes, and toxicity should have a considerable
effect on the technique chosen. The quantity, toxicity and potential for release
to the environment of the chemical provides an approximate indication of the
degree and complexity of the testing required. Thus, for compounds in the
developmental stage of commercialization, probably only a screening test is
481
-------
justified: As commercialization continues, more detailed studies should be
undertaken. When a compound is found to be produced and released into the.
environment in large quantities or in concentrations that might be(toxic or subject
to biomagnificatibn, it should receive an indepth (and extensive) study. Of the
three parts of a test system (chemical, analytical; methods, test media), the
analytical methods provide the widest spectrum of desirable information and costs.
The Indirect methods, such as oxygen uptake, carbon dioxide evolution, and ultra-
violet spectrbsco'py., are relatively Inexpensive but provide no insight into the
degradation pathways. In contrast, using radioassay techniques, although expen-
sive, allows a mass balance of the breakdown products and'considerable insight
\ . - * . i , i
into the degradation that may take place in the environment.
Although chemicals in our environment often move from one medium to another,
they frequently are first released into one specific medium. For'example, .
volatile materials usually vaporize into the atmosphere, (vapor pressure
it *
important), detergents usually pass through sewage treatment plants or a septic
tank, and plastics usually end up buried in landfills. Therefore, although a
chemical that enters the environment should be tested for persistence in a
variety'of media, some priorities can be provided by the routes into-the
environment 'and 'likely points of residence in the environment. Thus-volatile
materials ought to be tested in atmospheric chambers (e.g. smog chambers, LP1R,
plastic or glass containers). Hobbs (1974) has concluded that chemical species
with molecular Weights substantially higher than 200 to 300.will .notPreside
in the atmosphere as molecular species (vapors). Non-volatile,water^soluble
materials should be tested in aqueous media and the media in which the material
enters theenvironment,'but not in atmospheric systems. A contaminant found
482
-------
In water effluents should be tested under sewage treatment and natural water
conditions. The time of the year that the chemicals are used can also be
Important. For example, Evans et_ al. (1973) studied urea under winter condi-
tions because of Its heavy use during that season (de-icing agent).
The frequency of environmental discharge may sometimes be an important factor
in determining the degree of acclimation to be allowed. Buzzell je£ al. (1969)
found that degradation of compounds which were slug loaded into activated sludge
treatment plants was best modeled by using unacclimated sludge. Swlsher (1970)
concluded that with surfactants which are usually fairly evenly loaded into
sewage treatment plants, acclimation should be allowed to develop to its
fullest. Knowing the degradation rate under both acclimated and unacclimated
conditions can sometimes provide considerable insight into biodegradability.
Physical and chemical properties of the compound, besides being indicative
of routes into the environment and mobility from one medium to another, may
also have a considerable Impact on the technique to be used. For example,
volatility (vapor pressure) may be an important consideration in design of the
experimental apparatus. In atmospheric studies, some low vapor pressure
materials have a tendency to enter into unrecognized wall reactions and are
difficult to inject into the reaction vessel. Glasson and Tuesday (1971) used
a heated manifold In order to expand low vapor pressure compounds into their
long-path cell. Stephens (1973) used a stirred flow procedure with glass
carboys to study aromatic compounds with minimum loss of reactants due to
adsorption on the walls. Crosby and Moilanen (1974) used a special apparatus
which compensated for wall reactions for studying the vapor phase photolysis of
pesticides.
483
-------
In contrast, highly volatile compounds require special precautions in
s,pil,and^ater? systems. For closed, systems,-.such, as :BOD, volatile
i "
compounds .cause little difficulty. .However,,where;the reaction vessel is open
to the .a.tmpsphere, .physical loss can. result. ,..SeYeral,;aRp,r;paches. tq,,.compensate
or control such ^losses have, been used. Metcalf.and .Lu.(1973) used a system
With a^yapor trap instead of an open aquarium when working with,relatively
volatile materials., When working with laboratory pilot plant activated sludge
i : '
treatment plants, Buzzell et al. (1969) .ran,stripping studies to determine
i
volatilization losses.
i . >
Another physical property that, determinesv the suitability of .^the .experi-
mental, procedure and, method is the water solubility of a-.cfaemical-;^most,.water
insoluble;(hydrophobic) compounds .tend to adsorb on particulate,master, and
other.; solid .surfaces. .This effect is .especially prominent,.when,,test:ing;,for
persistence in an aqueous ,system. If;the comppund is^water soluble, aliquots
of,,the,,aqueous solution can be sampled to determinevdegradat:ion. ,v:Hpweyerfi when
.\t:he..-cpmppund is- very water- insoluble,.. (e.g., -prganochlorine. pesticides) a number
of frident ical^s^amples are prepared, so that .the. whole, sample .-can be ^extracted. For
example,;when using the river dier-away , test with a number of pestipides j ^Eichel-
berger and Lichtenberg (1971) prepared fifteen dosed samples, and a>complete
sample, was extracted at varying times up to eight weeks. A similar procedure
was reported by Halvprson et^ al^ (1971) .using the shake culture ..test with a
number of insecticides, and by Leigh (1969) using a modified.$unctv and Chambers
(1967).test with chlorinated hydrocarbon insecticides. Water solubility,may also
be important In- soil systems .where ;adsorption on soil particles.,may reduce .the
degradation rate, and complicate the analytical extraction procedure... On the
othershand, water soluble ionic species (e.g. diquat and paraquat^, Funderburk,
;:1969), may also be tightly bound to soil particles. .Water insoluble materialslSmay
--.. .:484
-------
also give misleading results in activated sludge treatment plant systems.
For example, Choi et al. (1974) reported that an activated sludge system re-
moved a high percentage of PCB's contained in waste water. However, PCB's
were adsorbed on the sludge but not degraded and, therefore, the PCB would be
deposited wherever the sludge was dumped (landfill, land spread, or the ocean).
In summary, commercial-economic, environmental release, biological effects,
and physical-chemical properties of a chemical can be used to determine the
extent and type of testing for environmental persistence and fate as well as
determine the experimental procedure. Some important parameters include (1)
quantity produced and released into the environment, (2) route and frequency
of release into the environment, (3) volatility, (4) toxicity, and (5) water
solubility.
485
-------
REFERENCES
ACS Committee on Chemistry of Public Affairs (1973), "Chemistry In The
Economy", American Chemical Society, Washington, D.C. [p. 23]
Addison, J.B.; Silk, P.J.; and Unger, I. (1974), "Photochemical Reactions of
Carbamates. II. Solution Photochemistry of Mtacil (4-dimethylamino-m-tolyl
N-methylcarbamate) and Landrin (3,4,5-trimethylphenyl N-methylcarbamate)",
Bull. Environ. Contam. Toxicol. 11(3), 250-5. [p. 336]
Ahmed, M. and Focht, D.D. (1973), "Degradation of Polychlorinated Biphenyls
by Two Species of Achromobacter". Can. J. Microb., 19, 47 (1973). [p. 109]
Ahmed, N. and Morrison, F.O. (1972), "Longevity of Residues of Four Organo-
phosphate Insecticides in Soil", Phytoprotection 1972, 13(2-3), 71-4.>
[p. 241, 243, 258, 262] ^ V ' .-;
Alexander, M. (1963), "Microbiology of Pesticides and Related Hydrocarbons",;
in Principles and Applications in Aquatic Microbiology, H. Heukelekian
and N.C. Dondero, ed., John Wiley and Sons, Inc.
Alexander, M. (1965), "Biodegradation: Problems of Molecular Recalcitrance
and Microbial Fallibility", Advance Appl. Microbiol., 7, 35-90. [p.
465] : ~ .,;'."
Alexander, M. (1966), "Biodegradation of Pesticides", in Pesticides and
Their Effects on Soils and Water. American Society of Agronomy
Publication, No. 8, 85. [p. 34, 37] ..-...,.- ..!.;'.,...;/r.-:'
Alexander, M. (1967a), "Pollutants thatf::Resist the Microbes"; Hew Scientist, ; .
35, 439-440. [p. 28, 33] % ^ ;. " . _ *'"
Alexander, M. (1967b), "The Breakdown of Pesticides in Soils", in Agriculture
and the Quality of our Environment, pp. 331-342 N.C. Brady, ed., Amer.
Assoc. Adv. Sci., Publication 85, Washington, D.C. [p. 107]
Alexander, M. (1971), Microbial Ecology, John Wiley & Sons, Inc., NY.
Alexander, M. (1972), "Microbial Degradation of Pesticides", in Environmental
Toxicology of Pesticides, F. Matsumura, G.M. Boush and T. Misato, ed.,
Academic Press, NY. [p. 192, 193, 195, 197, 198]
Alexander, M. (1973a), "Nonbiodegradable and Other Recalcitrant Molecules",
Biotechnol. Bioeng. 15(4), 611-47. [p. 36, 187, 419, 462, 463, 465,
475, 476]
Alexander, M. (1973b), "Microbial Degradation of DDT", U.S. Nat. Tech.
Inform. Serv.. AD Rep. No. 762469, 26 pp. [p. 106]
Alexander, M. (1973c), "Microbial Formation and Degradation of Pollutants in
Soil", from Jord-och Skogsbrukets Ansvar vid Avfallets Utnyttjande och
Cirkulation. Nordiske Jordbruksforskeses Forening, 1973, pp. 20-48.
[p- 385]
Preceding page blank
487
-------
Alexander, M. (1974), "Microbial Formation of Environmental Pollutants",
Adv. Appl. Microbiology, 18, 1-64. [p. 191]
Alexander, M. and Aleem, M.I.H. (1961), "Effect of Chemical Structure on
Microbial Decomposition of Aromatic Herbicides", J. Agr. Food Chem.
! 44-7. [p.] 248, 464, 466]
Alexander, M. and ,Lustigman, B.K. (1966), "Effect of Chemical Structure on
Microbial Degradation of Substituted Benzenes", J. Agr. Food- C^iem.
14(4), 410-13. [p. 229, 248, 249, 464, 466]
Alexandra, D. (1967), "Development of a Fast Test for the Biodegradation of
Detergents", Chem. Ind. (Paris), 98_, 1443-1448. [p. 145]
Alley, E.G.; Dollar, D.A.; Layton, B.R.; and Minyard, J.P., Jr. (1973),
"Photochemistry of Mirex", J. Agr. Food Chem., 21, 138i [.p.1 336]
Alley, E.G.; Layton, B.R.; and Minyard, J.R., Jr. (1974), "Identification
of the Phbtoproducts of the Insecticides Mirex and Kepone", J. Agr.
Food Chem., 22, 442. [p. 336]
Allred, R.C.; Setzkorn, E.A.; and Huddleston, R.L. (1964), "Detergent Bio-
degradability as Shown by Various Analytical Techniques", J. Am. Oil
Chemists' Soc., 41(1), 13-17. [p. 84, 101, 152, 153, 160, 178]
Allsopp, D.; Eggins, H.O.W.; and Hollingsworth,, B.S. (1973), "Microorganisms,
the environment, and materials", J. Oil Colour Chem. Ass., 56(6), 237-40.
Altom, J.D. and Stritzke, J.F. (1973), "Degradation of Dicamba, Picloram,
and Four Phenoxy Herbicides in Soils", Weed Sci., 21(6), 556-60. [p. 226]
Altshuller, A.P. and Bufalini, J.J. (1965), "Photochemical Aspects-of Air
Pollution", Photochem. and Photobiol., 4^, 97. [p. 469, 470]
Altshuller, A.P. and Bufalini, J.J. (1971), "Photochemical Aspects of Air
Pollution: A Review", Environ. Sci. Technol., 5_, 39. [p. 42, 283, -323,
324, 467, 469]
Altshuller, A.P. and Cohen, I.R. (1963), "Structural Effects on the Rate of
. Nitrogen Dioxide Formation in the Photooxidation of Organic Compound-
Nitric Oxide Mixtures in Air", Air Water Pollut., 7_(8), 787^5. [p. 292,
309, 317, 322]
Altshuller, A.P.; I.R. Cohen; and T.C. Purcell (1967), "Photooxidation
of Hydrocarbons in the Presence of Aliphatic Aldehydes",. Science
15J>(3777), 937-9. [p. 290, 318]
Altshuller, A.P.; Kopczynski, S.L.; Wilson, D.; Lonneman, W.; and
Sutterfield, F.D. (l969a), "Photochemical Reactivities of tv-Butane
and Other Paraffinic Hydrocarbons", J. Air Pollut. Contr. Assoc.,
1£, 787. [p. 300, 317]
488
-------
Altshuller, A.P.; Kopczynskl, S.L.; Wilson, D.; Lonneman, W.; and
Sutterfield, F.D. (1969b), "Photochemical Reactivities of Paraffinic
Hydrocarbon-Nitrogen Oxide Mixtures Upon Addition of Propylene or
Toluene", J. Air Pbllut. Contr. Assoc., 19(10): 791-797. [p. 300, 317]
Altshuller, A.P.; Kopczynski, S.L.; Lonneman, W.A.; Sutterfield, F.D.; and
Wilson, D.L. (1970a), "Photochemical Reactivities of Aromatic
Hydrocarbon-Nitrogen Oxide and Related Systems", Environ. Sci. Technol.
4(1): 44-49. [p. 300, 317]
Altshuller, A.P.; Aubrey, P.; Kopczynski, S.L.; Lonneman, W.A.; and
Sutterfield, F.D. (1970b), "Technique for Measuring Photochemical
Reactions in Atmospheric Samples", Environ. Sci. Technol.,
4.(6), 503-6. [p. 290, 308, 325]
Aly, O.M. and El-Dib, M.A. (1969), "Photodecomposition of Some Carhamate
Insecticides in Aquatic Environments", Org. Compounds Aquatic Environ.,
Rudolfs Res. Conf., 5th (Pub. 1971), 469-93. Edited by Faust, Samuel J.
Dekker: New York, N.Y.
Aly, O.M. and El-Dib, M.A. (1971), "Persistence of Some Carbamate Insecticides
in the Aquatic Environment. I. Hydrolysis of Sevin, Baygon, Pyrolan,
and Dimetilan in Waters", Water Res., 1(12), 1191-205. [p. 336, 347,
,356]
Aly, O.M. and El-Dib, M.A. (1972), "Persistence of Some Carbamate Insecticides
in the Aquatic Environment", Advan. Chem. Ser. Ill (Fate of Org. Pestic.
in the Aquatic Environ.), 210-43. [p. 78, 167, 171, 336, 354, 363]
Andelman, J.B. and Suess, J. (1971), "The Photodecomposition of 3,4-
Benzpyrene Sorbed on Calcium Carbonate", in Organic Compounds in
Aquatic Environment, p. 439 Samuel D. Faust and J.V. Hunter, ed.,
Marcel Dekker Inc. [p. 336, 348, 351, 356]
Anderson, J.R. and Drew, E.A. (1972), "Growth Characteristics of a Species of
Lipomyces and its Degradation of Paraquat", J. Gen. Microbiol, 70(1),
43-58. [P- 37, 247, 265, 266, 267, 268]
Anderson, J.P.E.; Lichtenstein, E.P.; and Whittingham, W.F. (1970), "Effect of
Mucor alternans on the Persistence of DDT and Dieldrin in Culture and in
Soil", J. Econ. Entomol., 63_(5), 1595-9.
Anon. (1963), "Biodegradability of DetergentsA Story about Surfactants",
Chem. Eng. News, 41(11), 102-14, 126. [p. 27]
Anon. (1971a), "Technology Concentrates-Degradable Polybutadiene", Chem. Engr.
News, Nov. 29, p. 29. [p. 420, 429, 479]
Anon. (1971b), "Three-layer Bottles Degrade in Water", Chem. Engr. News,
Nov. 29, p. 34. [p. 420, 429, 454, 478]
489
-------
Anon. ,(1972), "This Mulch Will Self-Destruct", Chem;vWeek, Feb; 16, p. 44.'
[p. :420, 479,, 480] .'
Anon. (1974), "Think Ecology When You Drink", Environ. Sci. Technott; 8_(2), 115.
[p. 478]
APHA (1971), Standard Methods for Examination of Water and WastewaterV
13th Edit., APHA, New York (American Public Health Association, 'American
Water Works Association and Water Pollution Control Federation), [p. 52,
53, 67, 155V159]
APHA (1972), Methods of Air Sampling and Analysis. APHA. Washington, D.C.
American Public Health Association, [p. 309, 310, 311] [
Archer, I.E.; Nazer, I.K.; and Crosby, D.G. (1972), "Photodecomposltion of
Endosulfan and Related Products in Thin Films by Ultraviolet Radiation",
J. Agr. Food'Chem., 20, 954. [p. 336, 351]
Asai, R.E:; Gunfher, F.A.; and Westlake, W.E. (1974), "Influence of Some?r-"
Soil Characteristics on the Dissipation Rate of Landrin Insecticide,"
Bullet. Environ. Contamin. Toxicol., 11, 352.
Assoc. Amer. Soap and Glycerine Producer (1961), "Determination of ABS in
Sewage", J. Water Pollut. Control Fed., 33_, 85-91. [p. 152]'
ASTM, D149-64 (1971), "Dielectric Breakdown Voltage and Dielectric Strength of
Electrical'Insulating Materials at Commercial Power Frequencies'.''Annual
Book of ASTM Standards, 30, 17-23. [p. 434]
ASTM, D150-70 (1971), "A-C Loss Characteristics and Dielectric Constant
(Permittivity) of Solid Electrical Insulating Materials." Annual 'Boofeof....:
ASTM. Standards. 30, 24-46. [p. 435] /
. " . , 3
ASTM, D257-66 (1971), "D-C Resistance; of Conductance of Insulating'Materials,"
Annual Book of ASTM Standards. 30. 84-104. [p. 435]
ASTM, D495-70 (1971), "High-Voltage, Low-Current, Dry Arc Resistance' of Solid'
Electrical Insulation", Annual Book of ASTM Standards,. 30, 125-^l38v^
[p. 434]
ASTM, D638-68 (1971), "Tensile Properties of Plastics", Annual Book :of ASTM-....,..
Standards.,30. 178-191. [p. 437, 438]
ASTM, D747-70 (1971), "Stiffness of Plastics by Means of a Cantilevet Beam",
Annual.Book of ASTM Standards. 30, 255-265. [p. 436]
ASTM, D785-65 (1971), "Rockwell Hardness of Plastics and Electrical Insulating
Materials"i Annual Book of ASTM Standards. 30, 274-279. [p. 437]
ASTM, D1003-61 (1971); "Haze and Luminous Transmittance of Transparent Plastics",
Annual^Book of ASTM Standards. 30. 377-383. [p. 443]
490
-------
ASTM, D-1924-70 (1970), "Determining Resistance of Polymeric Materials to
Fungi", Annual Book of ASTM Standards, 30, 29-33. [p. 423, 424, 450]
ASTM, D2676-67T (1970), "Determining the Resistance of Plastics to Bacteria",
Annual Book of ASTM Standards. _30, 42-5. [p. 450]
ASTM, E96-66 (1971), "Water Vapor Transmission of Materials in Sheet Form",
Annual Book of ASTM Standards. 30, 757-764. [p. 432, 433]
Atlas, R.M. and Bartha, R. (1972), "Degradation and Mineralization of
Petroleum by Two Bacteria Isolated from Coastal Waters", Biotechnol.
Bioeng., 14(3), 297-308. [p. 82, 111, 157, 171]
Atlas, R.M. and Bartha, R. (1973), "Fate and Effects of Polluting Petroleum
in the Marine Environment", Residue Rev., 49, 49-85. [p. 82, 157, 165]
Audus, L.J. (1949), "Biological Detoxication of 2,4-D in Soil", Plant and
Soil, 2, 31-6. [p. 231, 257]
Audus, L.J. (1951), "The Biological Detoxication of Hormone Herbicides in
Soil", Plant and Soil, 3., '170-92. [p. 231, 257]
Audus, L.J. (1964), "Herbicide Behavior in Soil. II. Interaction with Soil
Microorganisms", in The Physiology and Biochemistry of Herbicides,
L.J. Audus, ed., p. 163, Academic Press, N.Y.
Awao, T.; Komagata, K.; Yoshimura, I.; Mitsugi, K. (1971), "Deterioration of
Synthetic Resins by Fungi", J. Ferment. Techno1., 49_(3), 188-94. [p. 423,
446, 449]
Ayanaba, A. and Alexander, M. (1974), "Transformations of Methylamines and
Formation of a Hazardous Product, Dimethylnitrosamlne, in Samples of
Treated Sewage and Lake Water", J. Environ. Quality, 3_, 83-89. [p. 191]
Bacon, L.R. (1966), "Foam Generation Method for Evaluating Biodegradability",
J. Amer. Oil Chem. Soc., 43(1), 18-25. [p. 74, 160]
Baggie, G.; Catelani, D.; Galli, E.; and Treccani, V. (1972), "The Microbial
Degradation of Phenylalkanes: 2-phenylbutane, 3-phenylpentane,
3-phenyldodecane and 4-phenylheptane", Biochem. J., 126, 1091-1097.
(p. 109]
Bahr, T.G. and Ball, R.C. (1969), "Arsenic-74 Metabolism in an Aquatic Eco-
system", Michigan Academician, Vol. 1, No. 3 and 4. [p. 394, 399]
Baird, R.B.; Kuo, C.L.; Shapiro, J.S.; and Yanko, W.A. (1974), "The Fate of
Phenolics in Wastewater Determination of Direct-Injection GLC and
Warburg Respirometry", Arch. Environ. Contamin. Toxicol., 2_, 165.
[p. 153, 167]
Baker, R.A. (1966), "Phenolic Analysis by Direct Aqueous Injection Gas
Chromatography", J. Amer. Water Works Assoc., 58, 751. [p. 153]
491
-------
Baldwin, B.C.; Bray, M.F. ; and Geoghegan, M.J. (1966), "The Mlcrobial,
Decomposition of Paraquat", Blochem. J., 101, 15. [p* 37] '
Bandal, S.K. and Caslda, J.E. (1972), "Metabolism and PhotoaUeratlon of
2-8£C-rbutyl-4,6-dinitrophenol (DNBP Herbicide) .and Its laoprppyl,
Carbonate Derivative (Dinobuton Acaricide)", J. Agr. Food, Chem. ,
20, 1235. [p. 336, 351, 352]
Barbaro, R.D. and Hunter, J.V. (1965), "The Effect, of Clay Minerals on
Surfactant Bipdegradability", Purdue Univ. Eng. Bull. , Ext. Ser. No.
118. p. 189-196. [p. 60, 64]
Barden, L. and Issac, P.C.G. (1957), "The Effect of Synthetic. Detergents on
the Biological Stabilization of Sewage", Proc. Inst. Civil Engrs.
(London), £,371-405. [p. 59] s
Barstlap,- C. and Kort land, C. (1967), "Effect of Substituents in the- Aromatic
Nucleus on the Biodegradation Behavior of Alkylaryl Sulfonates", J. Amer.
Oil. Chem. Soc., 44, 295.. [p. 172]
Bartha,; R. (1968), "Biochemical Transformation of Anllide .Herbicides;, in
Soil", J. Agr. Food Chem. , 16(4), 602-604. [p. 226, 244, 255]
Bartha; R. (1969), "Transformation of Solan in Soil", Weed Sci., 17, 471-2.
[p; 226, 250]
Bar tha.'R. (1970), "Biodegradation of Oil Slicks in the Marine Environment "
U.S. Nat. Tech. Inform. Serv.. AD Rep. No. 715801, . 10 pp.. [p. ,110, 111]
Bartha, R. (1971), "Altered Propanil Biodegradation in Temporarily Air^-Dried
Soil", J. Agr. Food Chem., 19(2), 394-395. [p. 226] \ ' '
Bartha, R.; Lanzilotta, R.P. ; and Pramer, P- (1967), "Stability a
Effects of, Some Pesticides in Soil", Appl. Microbiol. , 15(1) ,. 67-75.
[p. 226]
Bartha, R. ; Linke, H.A.B.; and Pramer, D. (1968), "Pesticide Transformation:
Production of Chlotoazobenzene from Chloroanilines" , Science, 161, 582.
[p. 226]
Baude, F.J.; Pease, H.C.; and Holt, R.F. (1974), "Fate of Benomyl on Field,
Soil and Turf", J. Agr. Pood Chem., 22, 413. [p. 247, 251]
Baughman, G.L., Gordon, J.A. , Wolfe, N.L. and Zepp, R.G. (1973), "Chemistry
of Organomercurials in Aquatic Systems", Ecological Research Series,
EPA - 660/3-73-012, G.P.O., Washington, D.C. [p. 395, 396] ;
Baur, J.R. ; Bovey, R.W. ; and McCall, H.G. (1973), "Thermal and Ultraviolet .
Loss of Herbicides", Arch. Environ. Contam. Toxicol., 1^, 289. [p. 336,
351, 364].
492
-------
Bayer, E., Mullen, H.P., and Sievers, R. (1971), "Hexafluoromonothio-
acetylacetone A New Ligand for Gas Chromatographic Separation of
d8 Metals", Anal. Chem., 43, 2012. [p. 399]
Beam, H.W. and Perry, J.J. (1973), "Co-metabolism as a Factor in the
Microbial Degradation of Cycloparaffins", Arch. Mikrobiol., 91., 87-90.
fo. 278]
Bejuki, W.M. (1961), "Pseudomonas Aeruginosa as a Materials Biodeterioration
Test Organism", Devs. Ind. Microbiol., _2, 263-270.
Bellar, T.Sigsby, J.E.; demons, C.A.; and Altshuller, A.P. (1962), "Direct
Application of Gas Chromatography to Atmospheric Pollutants", Anal.
Chem., 34, 763. [p. 307]
Bellar, T.A.; Brown, M.F.; and Sigsby, J.E., Jr. (1963), "Determination of
Atmospheric Pollutants in the Part-Per-Billion Range by Gas Chromato-
graph", Anal. Chem., 35, 1924. [p. 308]
Belser, N.O. and Castro, C.E. (1971), "Biodehalogenation - The Metabolism
of the Nematocides cis- and trans-3-chloroallyl Alcohol by a Bacterium
Isolated from Soil", J. Agr. Food Chem., 19_(1), 23-26. [p. 232, 233,
236, 237]
Benson, W.R.; Lombardo, P.; Egry, I.J.; Ross, R.D., Jr.; Barron, R.P.;
Mustbrook, D.W.;.and Hansen, F.A. (1971), "Chlordane Photoalteration
Products: Their Preparation and Identification", J. Agr. Food Chem.,
19, 857. [p. 336, 347, 351, 358]
Bentley, M.D.; Douglas, I.B.; Lacadie, J.A.; and Whittier, D.R. (1972),
"The Photolysis of Dimethyl Sulfide in Air", J. Air Poll. Contr.
Assoc., 22, 359. [p. 292]
Berger, B. (1964), "Biodegradability of Animic Detergents Influence of
the Structure of the Hydrophobic Chains in Degradability", Ind. Chim.
(Paris), _51, 421-431. [p. 178]
Berk, L. and Teitell, L. (1951), "Effect of Fungus Growth on the Tensile
Strength of Pressure-Sensitive Electrical Insulating Tapes", Bull. Am.
Soc. Test. Mater., 174. 67-71. [p. 424, 437, 449, 451]
Bernarde, M.A.; Koft, B.W.; Horvath, R.; and Shaulis, L. (1965), "Microbial
Degradation of the Sulfonate of Dodecyl Benzene Sulfonate", Appl.
Micorbiol., 13, 103. [p. 109]
Bertilisson, L. and Neujahr, H.Y. (1971), "Methylation of Mercury Compounds
by Methylcobalamin", Biochemistry, 10, 2805-2808. [p. 414]
Bishop, P.L. and Kirsch, E.J. (1972), "Biological Generation of Methylmercury
in Anaerobic Pond Sediment", paper presented at the 27th Purdue Industrial
Waste Conference. Purdue University, May 2-4, Lafayette, Indiana.
[p. 386, 405, 408, 4091
493
-------
Bisogni, J.j. and Lawrence, A.W. (1973), "Kinetics of Microbially Mediated
Methylation of Mercuty in Aerobic and Anaerobic Aquatic Environments",
Technical Report 63, Cornell U. Water Resources and Marine Sciences
Center, Ithaca, New York. [p. 382, 386, 387, 388, 398, 400,\405, 406,
409, 410, 412]
J
Blake, J.T. and Kitchin, D.W. (1949), "Effect of Soil Microorganisms on
Rubber Insulation", Ind. Eng. Chem., 41, 1633-41. [p. 425]
Blankenship, F.A. and Piccolini, V.M. (1963), "Biodegradation of Nonionics",
Soap Chem. Specialities, 3£(12), 75-78, 181. [p. 59, 74, 158, 160]
Blumer, M. and Sass, J. (1969), "Oil Pollution: Persistence and Degradation
of Spilled Fuel Oil", Science, 176(4039), 1120-1122. [p. 49]
Blumer, M.; Ehrhardt, M.; and Jones, J.H. (1973), "Environmental Fate of
Stranded Crude Oil", Deep-Sea Res. Oceanogr. Abstr., 20(3), 239-59.
[p. 49]
Bogan, R.H. and Sawyer, C.N. (1954), "Biochemical Degradation, of Synthetic
Detergents", Sewage and Ind. Wastes, 26. 1069-80. [p. 59, 60]
Bogan,.R.H. and Sawyer, C.N. (1955), "Biochemical Degradation, of Synthetic
Detergents. II. Relation Between Chemical Structure and Biochemical
Oxidation", Sewage and Ind. Wastes, 2T_, 917-28. [p. 54, 59,. 6,0,.-63]
Bollag, J.M., .E.J. Czaplicki and R.D. Minard. (1975), "Bacterial.Metabolism
Of 1-Naphthol", J. Agr. Food Chem., 23(1), 85-90. [p. 194]
Bollag, J.M.,.Helling, C.S., and Alexander, M. (1967), "Metabolism, of
. 4.-chloro-2-methylphenoxyacetic Acid by Soil Bacteria", App. Microbibl.,
15, 1393-8. [p. 236, 237, 238, 245, 246, 249, 251]
Bollag, J.M. and Liu, S.Y. (1971), "Degradation of Sevin by Soil Micro-
organisms", Soil Bipl. Biochem., 3_(4), 337-345. [p. 251, 277]
Bollag, J.M., Helling, C.S., and Alexander, M. (1968), "2,4-D Metabolism.
. Enzymatic Hydroxylation of Chlorinated Phenols", J. Agr. Food'Chem.,
16, 826-828. [p. 197]
Bollpn, W.B.; ^Roberts, J.E.; and Morrison, H.E. (1958), "Soil Properties
and Factors Influencing Aldrln-Dieldrin Recovery and Transformation",
J. Econ. .;Entomol., 51, 214. [p. 265]
Borstlap, C. and Kooijam, P.L. (1963), "A Study of the Biodegradation of
Anionic Synthetic Detergents: A New Laboratory Test", J. Am. Oil Chem.
Soc., 40, 78-80. [p. 7-3]
Braman, R.S. and Foreback, C.C. (1973), "Methylated Forms of Arsenic in the
Environment", Science, 182(4118), 1247-1249. [p. 400, 416]
Brebion, G., Cabridenc, R. and Jullig, T. (1966), "Evaluation of the Bio-
degradability of Detergents by a Static Method", Tribune CEBEDEAU,
18. 13. .[p. 77]
494
-------
Brenner, I.E. (1968), "The Impact of Biodegradable Surfactants on Water
Quality", J. Am. Oil Chem. Soc., 45, 433-436. [p. 203, 204]
Brian, R.C. (1960), "Action of Plant Growth Regulators. III. Adsorption
of Aromatic Acids to Oat Monolayers", Plant Physiol., 35, 773.
Brink, R.H., Jr. and Meyers, J.A., III (1966), "Anionic Surfactant Bio-
degradability Studies by Warburg Respirometry" J. Amer. Oil Chem. Soc.,
43, 449-451. [p. 60, 63]
Broadbent, F.E. (1967), "Nature of Soil Organic Matter", Proc. West. Weed
Contr. Conf., 21, 31-5. [p. 260]
Bro-Rasmussen, F.; Noeddegaard, E.; and Voidurn-Clausen, K. (1970),
"Comparison of the Disappearance of Eight Organophosphorus Insecticides
From Soil in Laboratory and in Outdoor Experiments", Pestic. Sci.,
l.(5), 179-82. [p. 226, 244, 245, 273]
Brown, A.E. (1946), "The Problem of Fungal Growth on Synthetic Resins, Plastics
and Plasticizers", Mod. Plast., 23, 189-95. [p. 440, 456, 475, 477]
Brown, A.E. and Reinhardt, K.A. (1971), "Polyester Fiber: From its Invention
to its Present Position", Science, 173. 287-293. [p. 429, 454, 477]
Bruce, A.M., Swanwick, J.D., and Ownsworth, R.A. (1966), "Synthetic Detergents
and Sludge Digestion: Some Plant Observations", Inst. Sewage Purification.
J. and Proc.. 427-447. [p. 152]
Bryan, E.A. (1966), "Application of the Chlorate BOD Procedure to Routine
Measurement of Wastewater Strength", J. Water Pollut. Control Fed.,
38, 1350-1362.
Bufalini, J.J. (1974), personal communication, June 25.
Bufalini, J.J. and Altshuller, A.P. (1963), "Effect of Temperature on
Photochemical Smog Reactors", Air Water Pollut., 7^(8), 769-71. [p. 318]
Bufalini, J.J. and Altsuller, A.P. (1969), "Oxidation of Nitric Oxide in the
Presence of Ultravioltet Light and Hydrocarbons", Environ. Sci. Techno1.,
1, 469. [p. 316]
Bufalini, J.J.; Kopczynski, S.L.; and Dodge, M.C. (1972), "Contaminated
Smog Chambers in Air Pollution Research", Environ. Lett., 3_(2), 101-9.
[p. 292, 296, 298, 313, 321, 323]
Bulla, C.D. and Edgerley, E. (1968), "Photochemical Degradation of Refractory
Organic Compounds", J. Water Pollut. Contr. Fed., 40(4), 546-56. [p. 336]
Bunch, R.L. and Chambers, C.W. (1967), "A Biodegradability Test For Organic
Compounds", J. Water Pollut. Cont. Fed., 39_(2), 181-7. [p. 87, 97, 101,
166, 185, 484]
495
-------
1 . I
Burger, K.; MacBae, I.C.; and Alexander, M. (1962), "Decomposition of
Phenoxylalkyl Carboxylic Acids", Soil Sci. Soc. Am. Proc.,
26(3), 243-6.' [p. 232, 233, 236, 241, 248, 249; 255] j
Burnop, V.C.E. and Bunker, H.J. (1960), "Synthetic Detergents with Facile
Blodegradation", Centre Beige Etude Doc. Eaux., Bull. Trimestr.,
CEBEDEAU,.4_, 262. [p. 145]
> i . .
Burnside,, 0.;C.; Wicks, G.A. and Fenster, C.R. (1971), "Dissipation of
Dicamba, Picloram, and 2,3,6-TBA Across Nebraska1', Weed Sci., J9, 323-5.
[p. 242, 257, 272]
Busch* A.W., Grady, L., Rao, T.S. and Swilley, E.L. (1962), "Short-Term
Total Oxygen Demand Test", J. Water Pollut. Cont. Fed., 34, v354-362.
[P- 71]
Buswell, J.A. and Mahmood, A. (1972), "Bacterial Degradation-of ;£rMethoxy-
benzoic Acid", Arch Mikrobiol. 84(4), 275-286. [p. 238-,-250; 255]
Buzzell, J.C., Young, R.H.F. and Ryckman, D.W. (1968), "Behavior of^Organic
Chemicals in the Aquatic Environment, Part II. Behavior in Dilute
Systems", Research Report, Manufacturing Chemists Assoc;
Washington, D.C. [p. 56, 174, 182, 188, 217]
Buzzell, J.C., Jr.; Thompson, C.H.; and Ryckman, D.W. (1969), "Behavior of
Organic Chemicals in the Aquatic Environment, Part III. Behavior in
Aerobic Treatment Systems (Activated Sludge)", Manufacturing Chemists
Assoc., Washington, D.C. [p. 60, 64, 89., 91, 129, 131, 174,' 188!, 201,
202,. 209, 210, 218, 220, 483, 484]
Caldwell, D.H.. and Langelier, W.F. (1948), "Manometric Measurement of the
Biological Oxygen Demand of Sewage", Sewage Works J., 20, 202-18i [p. 63]
Calvert J.G. and Pitts, J.N., Jr. (1966), "Photochemistry", Wiley & Sons,
N.Y. [p/42, 339, 340, 341, 343, 349]
Carlsson, D.J. and Wiles, D.M. (1969a), "The Photodegradation of Polyprppylene'
Films.. II. Photolysis of Ketonic Oxidation Products", Macromolecules,
1,. 587-97. [p. 430, 443, 445] /
CarIsson, D.J. and Wiles, D.M. (1969b), "The Photodegradation of Polypropylene
Films. III. Photolysis of Polypropylene Hydroperoxide", Macromolecules,
2, 597-606. [p. 430, 445]
Carlsson, D.J. and Wiles, D.M. (1970), "Surface Studies by.Attenuated Total
Reflection Spectroscopy. I. Corona Treatment of Polypropylene",
Canadian J. Chem., 48(15), 2397-2406. [p. 429]
Carlsson, D.J. and Wiles, D.M. (1971a), "Photooxidation of Polypropylene Films.
IV. Surface Changes Studied by Attenuated Total Reflection Spectroscopy",
Macromolecules, _4, 174-179. .
496
-------
Carlsson, D.J. and Wiles, D.M. (1971b), "Photopxidation of Polypropylene Films.
V. Origin of Preferential Surface Oxidation", Macromolecules, 4^ 179-84.
[p. 445]
Carpenter, E.J. and Smith, K.L. (1972), "Plastics .in the Sargasso Sea Surface",
Science, 175. 1240-1. [p. 420, 432, 435, 454, 456]
Carpenter, E.J., Anderson, S.J., Harvey, G.R., Miklas, H.P., and Peck, B.P.
(1972), "Polystyrene Spherules in Coastal Waters", Science, 178, 749-750.
[p. 420, 456]
Casida, J.E. (1969), "Radiotracer Studies on Metabolism, Degradation, and
Mode of Action of Insecticide Chemicals", Residue Rev., 25, 149-59.
[p. 193, 250, 355, 356]
Castro, C.E. and Belser, N.O. (1968), "Biodehalogenation. Reductive
Dehalogenation of the Biocides Ethylene Dibromide, l,2-dibromo-3-chloro-
propane, and 2,3-dibromobutane in Soil", Environ. Sci. Techno1.,
1(10), 779-783. [p. 252]
Castro, T.F. and Yoshida, T. (1971), "Degradation of Organochlorine Insecticides
in Flooded Soils in the Philippines", J. Agr. Food Chem., 19(6), 1168-70.
[p. 226, 227]
Cavett, J.J. and Woodrow, M.N. (1968), "A Rapid Method for Determining
Degradation of Plasticized PVC by Microorganisms", Biodeterioration of
Materials; Microbiological and Allied Aspects, (A.H. Walters and
J.J. Elphick, editors), Elsevier, N.Y., pp. 162-169. [p. 232, 236, 237]
Cerniglia, C.E.; Hughes, T.J.; and Perry, J.J. (1971), "Microbial Degrada-
tion of Hydrocarbons in Marine Environments", Bacteriol. Proc., p. 56.
[P. HI]
Challenger, F. (1945), "Biological Methylation", Chem. Rev., 36, 315-61. [p. 384]
Chambers, C.W. and Kabler, P.W. (1964), "Biodegradability of Phenols as
Related to Chemical Structure", Dev. Indust. Microbiol., 5_, 85-93.
[p. 238]
Chen, Y.L. and Casida, J.E. (1969), "Photodecomposition of Pyrethrin I, Allethrin,
Phthalthrin, and Dimethrin. Modifications in the Acid Moiety", J. Agr.
Food Chem., 17_(2), 208-15. [p. 335, 336, 351, 358, 364]
Cheng, H.M.; Yomamoto, I.; and Casida, J.E. (1972), "Retonone Photo-
decomposition", J. Agr. Food Chem., 2£, 850. [p. 336, 351, 352, 358]
Chesters, C., Pionke, H.B., and Daniel, T.C. (1974), "Extraction and Analytical
Techniques for Pesticides in Soil, Sediment, and Water", in Pesticides in
Soil and Water, Chpt. 16, preprint, [p. 191]
Chin, W.T.; Stanovick, R.P.; Cullen, I.E.; and Holsing, G.C. (1964),
"Metabolism of Swep by Rice", Weeds, 12_, 201. [p. 36]
497
-------
Choi, P.S.K.; Nack, H.; and Flinn, J.E. (1974), "Distribution of Poly-
chlorinated Biphenyls in an Aerated Biological Oxidation Wastewater
Treatment System", Bull. Environ; Contam. Toxicol., 11, 12. (j
[p. 131, 133,. 200, 485]
Clattoril, P. and Scardigho; S. (1968); "New Contribution to the Knowledge of
Blbdegradability11, Riv. Ital. Sostange Grasse, 45(1), 15-28., [p. 169}
Clarkv C.G. and Wright, S.J.L. (1970), "Degradation of the Herbicide Isopropyl
N-phehyleafbamate by Arthrobacter and Achromobacter Species from Soil",
Soil Biol. Biochem., 2^4), 217-26. [p. 237, 238, 2551 ;
Clark, L.C., Jr., Wold, R., Granger, D. and Taylor, F. (1953)-, "Continuous
Recording of Blood Oxygen Tension by Polarography", J. Appl. Physio1.,
6, 189. [p. 69] : .
demons, C.A.; Leach, P.W.; and Altshuller, A.P. (1963), "lt2t-3-:Tritf(-2-
cyanoethoxy)propane as a Stationary Phase in the Gas Chromatographic
Analysis of Aromatic Hydrocarbons", Anal. Chem., 35, 1546v J[p. 307]
Cochrane^ D.R., Pope, J.D., Nicholson, H.P., and Bailey, G.W. (1967), "The
Persistence of Silvex in Water and Hydrosoil", Water Resources .Research,
.3(2), 517-523. [p. 123]
Cohen, I.R.; Purcell, T.C.; and Altshuller, A.P. (1967), "Analysis of the
Oxidant in Photooxidation Reactions", Environ. Sci. Techno!., 1^, 247.
[p. 310]
Cohn, M., Monod, J. and Pollock, M.R., Spiegelman, S., and Stanier, R.Y. (1953),
"Terminology of Enzyme Formation", Nature, 172, 1096. [? 196]
Collier, L. (1972), "Determination of Bis-chloromethyl Ether at the1Ppb Level
in Air Samples by High-Resolution Mass Spectroscopy", Environ. Sci.
Technol., ^, 930. [p. 316]
i
Collins, P.M. and Sims, C.M. (1956), "A Compact Soil Perfusion Apparatus",
Nature, 178, 1073-4. [p. 230] . ,
Colton, J.B., Knapp, F.D., and Burns, B.R. (1974), "Plastic Particles in
Surface Waters of the Northwestern Atlantic". Science, 185,: 491.
[p. 420, 456]
Compton, B.; Bazydlo, P.P. and Zweig, G. (1972), "Field Evaluation .of Methods
of Collection and Analysis of Airborne Pesticides", EPA Contract CPA
70-145, Syracuse University Research Corporation, Syracuse; N.Y. [p. 283]
Connolly, R.D. (1963), "Effect of Seven-Year Marine Exposure-on Organic Material!1,
Mater. Res. Std., _3, 193-201. [p. 428]
Conway, R.A. and Waggy, G.T. (1966), "Biodegradation Testing of Typical
Surfactants in Industrial Usage", Am. Dyestuff Rept. ^5(16)v 607-614.
[p.. 54, 76, 82]
498
-------
Cook, R. (1968), "Bacterial Degradation of Synthetic Anionic Detergents",
Water Res., 2.(12), 849-76. [p. 95, 96, 109, 162, 165, 173, 186, 207,
208, 276]
Cooney, J.J. and Walker, J.D. (1973), "Hydrocarbon Utilization by Cladosporium
Resinae". The Microbial Degradation of Oil Pollutants, D.G. Ahearn and
S.P. Meyers (eds.), Center for Wetland Res., LSU, Pubn# LSU-SG-73-01,
pp. 25-32. [p. 113]
Cooney, J.D.; Colin, G.; and Wiles, D.M. (1973), "Sunlight, and the Microbial
Susceptibility of Thermoplastics", Amer. Soc. Test. Mater., Spec. Tech.
Publ. No. 533, 17-27. [p. 425, 445, 446, 453, 454, 479, 480]
Corbin, F.T. and Upchurch, R.P. (1967), "Influence of pH on Detoxication of
Herbicides in Soil", Weeds, 15, 370. [p. 265]
Cordon, T.C.; Maurer, E.W.; Panasiuk, 0.; and Stirton, A.J. (1968),
"Analysis for Sulfate Ion in the Biodegradation of Anionic Detergents",
J. Amer. Oil Chem. Soc., 45(8), 560-562. [p. 98, 166]
Cordon, T.C.; Maurer, E.W.; and Stirton, A.J. (1972), "The Biodegradation
of Some Sulfated Alkanolamides", J. Am. Oil Chem. Soc., 49_(3), 174-177.
[P. 98]
Council Directive of November 22, (1973), "On the Approximation of the Laws
of the Member States Relating to Methods of Testing Biodegradability of
Anionic Surfactants", Official J. Europ. Commun., 16, (L347) 53.
[p. 136, 212]
Council on Environment Quality (1971), "Toxic Substances", Government
Printing Office, Washington, D.C. [p. 23, 26, 481]
Cox, D.P. and Alexander, M. (1973), "Effect of Phosphate and Other Anlons on
Trimethylarsine Formation by Candida Humicola", Appl. Microbiol.,
25(3), 408-13. [p. 384, 406, 408]
Crosby, D.G. (1969a), "Nonmetabolic Decomposition of Pesticides", Ann. N.Y.
Acad. Sci., 160(1), 82-96. [p. 42, 45, 46, 352, 357, 371, 375]
Crosby, D.G. (1969b), "Experimental Approaches to Pesticide Photodecompostion",
Residue Reviews, 25, 1. [p. 42, 43, 314, 335, 339, 342, 355, 356,
359, 367]
Crosby, D.G. (1970), "Nonbiological Degradation of Pesticides in Soils",
Pestic. Soil: Ecol., Degradation Movement, Int. Symp. (Pub. 1971),
86-94. [p. 45, 46]
Crosby, D.G. (1972a), "Photodecomposition of Pesticides in Water", Advan.
Chem. Ser., Ill (Fate of Org. Pestic. in the Aquatic Environ.), 173-88.
[p. 43, 335, 359, 360]
Crosby, D.G. (1972b), "Environmental Photooxidation of Pesticides", in
Degradation of Synthetic Organic Molecules in the Biosphere, Nat. Acad.
Sciences, Washington, D.C. [p. 45, 350, 357]
499
-------
Cr.os.by* D.G. (1973), "Fate of Pesticides in the Environment", Annu. Rev,*
Plant,,Physigl., 24, 467-92. [p. 35],
Crosby, D.G., and .Hammadmad, N. (1971), "The Phot or eduction of Pentachloro-
benzenes", J. Agr. Food Chem., 19, 1171. [p. 356]
Crosby^ D.G. and-Leitus, E. (1969), "Photolysis of ChlorophenylacetiCiAcid",
J.,.Agr, Food Chem., 17, 1036. [p. 336] ' ,
Crosby, D.G. andjLeitis, E. (1973), "Photodecomposition of Trifluralin .in
Water", Bull. Environ. Contam. Toxicol., 10(4), 237-41. [p. 336y 349,
356, 361, 362],
Crosby,'D.G. and,Li, M.Y. (1969), "Herbicide Photodecomposition", in
Degradation of Herbicides, ed. by P.C. Kearney and D.D,. Kaufman,, Marcel ^
Dekker; -Inc>'., N.Y. [p. 42, 340 344, 371]
Crosby, D.G. and-,Mpilanen, K.W. (1973), "Photodecomposition of Chlorinated
Biphenyls and Dibenzofurans", Bull. Environ. Contam. Toxicol.,, 9.(6),
372-7. [p. 293, 294, 329, 348, 369, 370]
Crosbyj D.G. and5Moilanen, K.W..(1974), "Vapor-phase Photodecomposition of
Aldrin and.Dieldrin11, Arch. Environ. Contam. Toxicol., 2_(.i), -62-74.
[p. .372, 470]
Crosby, D.G. and-.Tang, C.S. (1969a), "Photodecomposition of 3-(p,-chlQrophenyl)-
1,1-dimethylurea (Monuron)", J. Agr. Food Chem., 17(5), 1041--104y4. [p. 336,
339, 344, 346]
Crosby, D.G. and. Tang, C.S. (1969b), "Photodecomposition of 1-Naphthaleneacetic
AcidV, J. Agr. Food Chem., 17.(6), 1291-1293. [p. 336,.. 345]
Crosby, D.G. and^Tutass, H.O. (1966), "Photodecpmpositipn of 2,4-Dichloro-
phenoxyacetic Acid", J. Agr. Food, Chem., 14(6), 596-598. [p. 350]:
Crosby,-D.G. and. Wong, A. S. (1973a) , "Photodecomposition of pj-Chlprophenpxyr-
acetic Acid",' J. Agr. Food, Chem.., 21,, 1049. [p. 336]
Crosby, D.G. and Wong, A.S. (1973b), "Photodecomposition of 2,4,5^Trichloro-
phenoxyacetjic Acid (2,4,5-T) in Water", J. Agr. Food Chem., 21,^1052.
[p., 336, 349, 363]
Crosby, D.G.; Moilanen, K.W.; Nakagawa, M.; and Wong, A.S. (1972),.
"Photpnucleophilic Reactions of Pesticides", in Environ. Toxicol.
Pestic.. Proc. U.S. - Jap. 1971. 423-33. [p. 336, 360] -
Cruickshank, P.A. and Jarrow, B.C. (1973), "Ethylenethiourea Degradation",
J. Agr. Food Chem..,. _21(3), 333-335. [p. 336, 351]
Cvetanovic, R.J. (1960), "Electrophilic Character of Oxygen Atoms",
Can. J. Chem.,, j[8, 1678. [p. 468]
500
-------
Dagley, S. (1972a), "Microbial Degradation of Stable Chemical Structures:
General Features of Metabolic Pathways", in Degradation of Synthetic
Organic Molecules in the Biosphere, National Academy of Sciences Conference,
San Francisco, Calif., June 12-13, 1971.
Dagley, S. (1972b), "Summary", in, Degradation of Synthetic Organic Molecules
in the Biosphere. National Academy of Sciences Conference, San Francisco,
Calif., June 12-13, 1971. [p. 2, 25, 26, 36, 37, 38, 39]
Dall, Aglio, M. (1970), "Comparison Between Hydrogeochemistry and Stream
Sediment Methods in Prospecting for Mercury", Proc. 3rd International
Geochem. Symp., Toronto, April. [p. 407]
Darby, R.T. and Kaplan, A.M. (1968), "Fungal Susceptibility of Polyurethanes",
App. Microbiol., 1£, 900-905. [p. 424, 474, 476]
Davis, E.M. and Gloyna, E.F. (1969), "Anionlc and Nonionic Surfactant Sorption
and Degradation by Algae Cultures", J. Amer. Oil Chem. Soc., 46(11),
604-608.
Davis, J.T. and Hughes, J.S. (1965), "Annual Report", Louisiana Wildlife
and Fisheries Commission, 21-28. [p. 35]
Dawson, R.M., Elliott, D.C., Elliott, W.H. and Jones, K.M. (1959), Data
for Biochemical Research, p. 148, Oxford University Press, London.
Day, M. and Wiles, D.M. (1972a), "Photochemical Degradation of Poly (ethylene
terephthalate). I. Irradiation Experiments with Xenon and Carbon Arc",
J. Applied Polymer Sci., 16(1), 175-189. [p. 431, 444, 445]
Day, M. and Wiles, D.M. (1972b), "Photochemical Degradation of Poly (ethylene
terephthalate). II. Effect of Wavelength and Environment in the
Decomposition Process", J. Applied Polymer Sci., ^6(1), 191-202.
[p. 430, 443]
Day, M. and Wiles, D.M. (1972c), "Photochemical Degradation of Poly (ethylene
terephthalate). III. Determination of Decomposition Products and
Reaction Mechanism", J. Applied Polymer Sci., 1.6(1), 203-215.
[p. 446, 454]
De Frenne, £.; Eberspaecher, J.; and Lingens, F. (1973), "The Bacterial
Degradation of 5-Amino-4-chloro-2-phenyl-3(2H)-pridazinone",
Eur. J. Biochem., 3,3(2), 357-363. [p. 256]
Degens, P.N., Jr.; Van Der Zee, H.; and Kommer, J.D. (1955), "Influence
of Anionic Detergents on the Diffused Air Activated Sludge Process",
Sewage Ind. Wastes, 27. 10. [p. 131]
Delman, A.D., Landy, M. and Simms, B.B. (1969), "Photodecomposition of
Polymethylsiloxane", J. Polymer Science, Part A-l, 7_» 3375-86.
[p. 444, 454]
501
-------
DeRose; H.R. and Newman, A.S. (1947), "The Comparison of the Persistence of
Certain"Plant Growth-Regulators When Applied to Soil", Soil Sci. Soc.
Amer. Proc*. 12, 222-226. [p. 226, 241, 242, 257, 266, 270];
Dias, F. F. and Alexander,, M. (1971), "Effect of ChemicalV Structure on.the;
Biddegradability of Aliphatic Acids and Alcohols", Appl. Microbiol.
22(6), 1114-1118. [p. 54, 89, 187, 465]
-J . Ii
Dick, J.N. (1964), "The Evaluation of Bacteria Growth-Rate Constants^for
Municipal'Waste", Thesis, Michigan State Univ., East Lansing,, Michigan.
[P- 64] s
Dickens, R. and*Hiltbbld, A.E. (1967), "Movement and Persistence-of Methane-r
arsonates in Soil", Weeds J. Weed Soc. Amer. 15(4), 299-304. [p. 271,, 393,
399, 401] , ;
Dimitriadesi B.J.^' (1967), "Methodology in Air Pollution Studies Using:
Irradiation,Chambers", J. Air Pollut. Contr. Assoc., r7_, 460-466.
[p.-296, 300, 301, 309, 315, 316, 329]
Dole, M. (1973), "Polymers That Primarily Degrade on Irradiation!.1, Radiat.
Chein. Macromol., 2, 97-119. D. Malcolm, edit., Academic,
New York, N.Y. ' ' *
Donaldson, C.D.P. and Hilst, G.R. (1972), "Effect of Inhomdgeneous Mixing On
Atmospheric Photochemical Reactions", Environ. Sciv Techno!., 6^(9), 812T-816.
[p. 320] :
Doyle, G.J. (1970), "Design of a Facility (Smog Chamber) for Studying Photo^-
chemical Reactions Under Simulated Tropospheric Conditions"., Environ.,
Sci. Technol., 4., 907. [p. 296, 302, 303, 305, 312, 313, 319,, 329]
Durham; W.F. (1969), "Body Burden of Pesticides in ManV, Ann. N.Y. Acad.
Sci., 160, 183. [p. 35]
Eckenfelder, W.W., Jr.; Goodman, B.L.; and Englande, A.J. (1972), "Scale-up
of Biological Waste Water Treatment Reactors", Advan. Biochem.i Eng.,
2^, 145-80'. [p. 62, 128]
**'
Edelirie, F. and Lambert, G. (1965), "A Rapid Method for Measuring' >
Biodegradfebility", Tribune CEBEDEAU (Centre Beige Etude Doc. Eaux)
18, 120-6. [p. 145]
Eden, G.E., Truesdale, G.A. and Stennett, G.V. (1967), "Assessment-of
Biodegradability", Annual Conference: Institute of Water Pollution
Control, Conference Paper No. 2F. [p. 76, 88, 163, 202]
Eden, G.E., Culley, G.E. and Rootham, R.C. (1972), "Effect of Temperature on :
the Removal of NTA (Nitrilotriacetic Acid) During Sewage Treatment",
Water Res., £, 877-883. [p. 136, 202]
Edwards, C.A. (1964), "Factors Affecting the Persistence of Insecticides
in Soil", Soils and Fert., 27_, 451. [p. 266]
502
-------
Edwards, C.A. (1966), "Insecticide Residues in Soils", Residue Rev., 13,
83-132.
Edwards, C.A. (1973), Persistent Pesticides in the Environment, 2nd
edition, CRC Press, Cleveland, Ohio. [p. 35, 37, 41, 175]
Eggins, H.O.M. and Mills, J. (1971), "Biodeterioration and Biodegradation
of Synthetic Polymers", in, Microbial Aspects of Pollution, ed. Sykes,
G. and Skinner, F.A., Academic Press (London).
Eichelberger, J.W. and Lichtenberg, J.J. (1971), "Persistence of Pesticides
in River Water", Environ. Sci. Technol., 5.(6), 541-4. [p. 75, 152, 174,
175, 176, 353, 484]
Eldib, A. (1963), "Testing Biodegradability of Detergents", Soap, Chem.
Specialties, _39(6), 59-63, 109-12. [p. 130, 131]
Ellis, L.F.; Samuel-Maharajah, R.; Mendelow, L.M.; Ruth, L.; and
Pivnick, H. (1957), "Oxidation of Components of Soluble Oils", Appl.
Microbiol., _5, 345-8. [p. 60, 158]
Elmore, H.L. (1955), "Determination of BOD by a Reaeration Techniques",
Sewage Ind. Waste, 27. 993. [p. 55]
Engvild, K.C. and Jensen, H.L. (1969), "Microbiological Decomposition
of the Herbicide Pyrazon", Soil Biol. Biochem., 1_(4), 295-3CO.
[p. 256, 263, 264]
Enzinger, R.M. (1970), "Special Study of the Effect of Alpha TNT on
Microbiological Systems and the Determination of the Biodegradability
of Alpha TNT", U.S. Nat. Tech. Inform. Serv.. AD Rep. No. 728497, 24 pp.
[p. 109]
E.P.A. (June 1972), Technical report: TS-00-72-04. "The pollution
potential in pesticide manufacturing", Pesticide Study Series 5,
Washington, D.C. [p. 416]
E.P.A. (1974), Southeast Environment Research Laboratory, Freshwater Ecosystem
Pollution Research, March 22 Newsletter, [p. 124]
Ernst, W. (1972), "Degradation of [1I+C] DDT on Silica Gel G Chromatograms
Under Laboratory Conditions", J. Chromatogr, 67_(1), 179-181. [p. 336]
Ettinger, M.B. (1956), "Biochemical Oxidation Characteristics of Stream
Pollutant Organic Chemicals", Ind. Eng. Chem., 48, 256-9. [p. 465]
Ettinger, M.B. and Ruchhoft, C.C. (1950), "Persistence of Mono-chlorophenols
in Polluted River Water and Sewage Pollutions", U.S. Nat. Tech. Inform.
Serv.. PB Rept. 215489, June 10. [p. 80]
503
-------
Ettinger, M.B.; Lishka, R.J.; and Kroner, R.C. (1954), "Persistence of
Pyridine Bases in Polluted Water", Ind. Erig; Chert., 46, 791-3.
tP- 80]
Evans, W.H. and David, E.J. (1974), "Biodegradation of Mono-, Di-, arid
Triethylene Glycols in River Waters Under Controlled Laboratory
Conditions", Water Res., a(2), 97-100. [p. 76, 171, 465]
Evans, W.H.; David, E.J.; and Patterson, S.J. (1973), "Biodegradation of
Urea in River Waters Under Controlled Lab Conditions", Water Res.,
I, 975-85. [p. 75, 171, 483]
i . . f
Exner, J.H.; Burk, G.A.; and Kyriacou, D. (1973), "Rates arid Products of
Decomposition of 2,2-dibromo-3-nitrilopropionamide", J. Ajgr. Food
Chem., 21(5), 838. [p. 336, 354, 356] ,
Eye, J.D., Reuter, L.H. and Keshavan, K. (1961), "Plantiniini Electrpde ,
System Measures Dissolved Oxygen and Biological Oxygen Demand", Water
Sewage Works, 108. 231. [p. 70]
Fagerstrom, T. and A. Jernelov (1971), "Formation of Methylmercury from
Pure Mercuric Sulfide in Aerobic Sediment", Water Research,
5(3), 121. [p. 382, 407]
Falco, J.W. and W.M. Sanders III (1973), "A Physical Model for Simulation
of Aquatic Ecosystems", in Proceedings of Modeling the Eutrophl-
cation Process Workshop. PRW 136-1. pp 159-170. [p. 124] '
Fang, S'.C. (1973), "Uptake and Biotransformation of Phenylmercurie Acetate
by Aquatic Organisms", Archiv. Environ. Contain. Toxicol. _!(!),' 18'.
[p. 395, 398, 399]
Farkas-Imrik, M. (1967), "Resistance of Plastics to Tropical Biological
Damaging Agents", Muanyag Gumi, 4^(3), 77-80. [p. 427]
Faust, S.D. and Gomaa, H.M. (1972), "Chemical Hydrolysis "of Some Organic
Phosphorus and Carbamate Pesticides in Aquatic Environments",
Environmental Letters, 3_(3), 171-201. [p. 354, 363, 364]
Ferguson, B.C.; Todd, R.L.; Holm, H.W.; Pope, J.D., Jr.; and Kariia, H.J.
(1973), "Environmental Fate of Nitrilotriacetic Acid", Eng. Bull.
Purdue Univ., Eng. Ext. Ser., No. 140(Pt. 1), 271-83. [p. 98]'
Fincher, E.L. and Payne, W.J. (1962), "Bacterial Utilization of Etner
Glycols", Appl. Microbiol. 10, 542-7. [p. 255]
Fischer, W.K. (1962), "Detection Methods for Detergent", Miinchner Beitrage
zur Abwasser-,Fischerei-, und Flussbiologie £, 168-183. [p:. 152]
Fischer, W.K. (1965), "Practical Operations and Equipment' in the (Official
German) Detergent Test", Vom Wasser, 3£, 168-192. [p. 212]
504
-------
Fishbeln, L. and Gaibel, Z.L.F. (1971), "Photolysis of PestlcAdal Synergiets.
I. Peperonyl But oxide," Bull. Environ. Cont am. Toxicpl;, 5_, 546.
[p. 336, 351] :;; ' .' ',"
Fisher, M.W. (1954), "The Growth of Tubercle^ Bacilli in Silicone Coated
Glass Tubes", J. Bacteriology, 67_, 613r614.
Fleming, R.W. and Alexander, M. (1972), "Dliiethylselenide and Dimethyltellu-,,
ride formation by a Strain of PenicillJum", Applied Microbiol., Vol. £4)^ ^
No. 3, p. 424-429. [p. 385, 390, 398, 404, 405, 406, 408] ^|...,,.
Fleming, W.E. and Maines, W.W. (1953), "Persistence pf DDT in Soil of the ,,
Area Infested by the Japanese Beetle",-J. Econ. Entbmol., 46, 445.
[p. 265] ; V
i
Focht, D.D. and Alexander, M. (1970a), "Bacterial Degradation of Diphenyl-
methane, a DDT Model Substrate", Appl. Microbiol., 20(4), 608-611.
[p. 108]
Focht, D.D. and Alexander, M. (1970b), "DDT Metabolites and Analogs: Ring
Fission by Hydrogenomonas", Science, 170, 91-92. [p. 108]
Focht, D.D. and Alexander, M. (1971), "Aerobic Cometabolism of DDT Analogues
by Hydrogenomonas sp.", J. Agr. Food Chem. 19(1), 20-22. [p. 108]
Focht, D.D. and Joseph, H.A. (1971), "Bacterial Degradation of Nitrilo- .;
triacetic acid (NTA)", Can. J. Microbiol., 171(12), 1553-1556. [p. 19, :
165]
' ' -'';&i5
Focht, D.D. and Williams, F.D. (1970), "The Degradation of pj-Toluenesulfonate: ~'
by a Pseudomonas". Can. J. Microbiol., 16(5), 309-316. [p. 109, 154, 166]
Forsberg, C. and Lindquist, G. (1967), "On Biological Degradation of
Nitrilotriacetate (NTA)", Life Sci. i(18 Pt. 2), 1961-1962. [p. 109, 159]:;
Fox, R.B. (1967), "Photodegrad^tlbn of High Polymers" i Progress in Polymer
Science, ]., 45-89. [p. ^O^^l, 443, 446] I ;
Frank, P. A. (1966), "Persistence and Distribution of Monouron and Neburon
in an Aquatic Environment", Weeds 14, 219. [p. 176]
Frazee, C.D. and Crisler, R.O. (1964) , "Infrared Determination of Alkyl
Branching in Detergent ABS", J. Amer. Oil Chem: Assoc., 41,; 334-335.
[p. 156]
Frazee, C.D.; Osburn, Q.W. ; and Crisler, R.O. (1964), "Application of
Infrared Spectroscopy to Surfactant Degradation Studies", J. Amer.
Oil Chem. Soc., 41, 808-12. [p. 156]
Freeman, L. (1953), "A Standardized Method for Determining Toxicity of Pure
Compounds to Fish", Sewage Ind. Wastes, 25, 845-848. [p. 118]
505
-------
Freene, E.D., Ebexspiicher, J. and Lingena, F. (1973), "The Bacterial
Degradation of 5-tmino-A-dhiefO-2-pheflyl-3(2H)-pyridaBinonen,i Jtur. J.
Biochem., 33,' 337-363. [p. 194] *,
* - .
Frlede, J.; Gulre, P.} and Qheleen, R.£, (1972), "Aieeeament of Bio-
degradation Potential fot Controlling Oil Spille on the High Stti",
Ui£i|tot. Task- Iflfotffl. fiiivAfift, AB .Report 739-848. [p. 27 J
Puhtmann, R.J.; Van Peppen, J,) Fofd, W. (1964), "Role of External, Varieblte
in Degradation 01 Straight-Chain ASS", Seap Chem. Special tie«, 40(2),
51-53, 106. :.[p. 167, 169, 170, 171] -
Fund«rburkf H-H, (1969), "Bitjuafe sti4 fafaqual", in Daflgadation ot Hfttbietdft
(lteA«i«y, F,C, afl4 Kau£Mfl, D.B,, ed.) Dekket , New York. [p,. 37» 484]
Furakawa, K.T.) Suzuki, f.i sn4 fottoauird, Ki (1969), "BfeGeapoiitioft of
Organie Itefeuf ial e§sg§uttd8 by Mefeuty Resietant laetttria",
. Ch4B§., M' 128-30, tp, SI2, 399]
Gage, J.C, (1961), "The ffa§e Deteftfliflafeien @I t»henyl- and MethylWeuey
Salts ia.Sieiegieal Msiefial", A»aly§fi, Mi 4S7. [p. ,198] '
Gatuum,
-------
German Government (1962), "Ordinance on the Degradability of Detergents in
Washing and Cleaning Agents", Bundesgesetzblatt (Bonn), Fart I,
No. 49, 698. [p. 101, 131]
Gerolt, P. (1961), "Investigation into the Problem of Insecticide Sorption
by Soil", World Health Org. Bull., 24, 577. [p. 270]
Getzln, L.W. (1968), "Persistence of Diazinon and Zinophos in Soil:
Effects of Autoclaving, Temperature, Moisture and Acidity", J. Econ.
Entomol., 61(6), 1560. [p. 265]
Getzin, L.W. and Rosefield, I. (1968), "Organophosphorus Insecticide
Degradation by Heat-labile Substances in Soil", J. Agr. Food Chem.,
16(4), 598-601. [p. 277]
Getzin, L.W. and Rosefield, I. (1971), "Partial Purification and Properties
of a Soil Enzyme that Degrades the Insecticide Malathion", Bloch.
Bioph. Acta 235. 442-53. [p. 277]
Gibson, D.T. (1972), "Initial Reactions in the Degradation of Aromatic
Hydrocarbons", in Degradation of Synthetic Organic Molecules in
the Biosphere. National Academy of Sciences Conference, San Francisco,
Calif., June 12-13, 1971. [p. 37]
Gilmour, C.M., Damsky, L. and Bollen, W.B. (1958), "Manometric Gas Analysis
as an Index of Microbial Oxidations and Reductions in Soil", Can. J.
Microbiol. 4., 287-293. [p. 226, 255]
Gitchell, A.; Simonaitis, R. and Heicklen, J. (1974), "The Inhibition of
Photochemical Smog. I. Inhibition by Phenol, Benzaldehyde and Aniline",
J. Air Pollut. Contr. Fedr., 24_, 357. [p. 318]
Glasson, W.A. and Tuesday, C.S. (1970a), "Inhibition of Atmospheric Photo-
oxidation of Hydrocarbons by Nitric Oxide", Environ. Sci. Technol.,
4., 37. [p. 286, 287, 306, 307, 317, 330]
Glasson, W.A. and Tuesday, C.S. (1970b), "Hydrocarbon Reactivities in the
Atmospheric Photooxidation of Nitric Oxide", Environ. Sci. Technol.,
4., 916. [p. 286, 307, 317, 323, 330, 467, 468]
Glasson, W.A. and Tuesday, C.S. (1970c), "Hydrocarbon Reactivity and the
Kinetics of the Atmospheric Photooxidation of Nitric Oxide", J. Air
{ Pollut. Contr. Assoc., 20, 239. [p. 286, 307, 330]
Glasson, W.A. and Tuesday, C.S. (1971), "Reactivity Relationships of
Hydrocarbon Mixtures in Atmospheric Photooxidation", Environ. Sci.
Technol., 5., 151. [p. 286, 306, 317, 483]
507
-------
Gledhill, W.E. (1974), "Biodegradation of 3,4,4'-trichloro-carbanilide,
:TCC, in Sewage and Activated Sludge", submitted .to Water Resi
[p. 99, 139, 140, 154, 213] \
Gloyna, E.F.; Comstock, R.F. and Renu, C.E. (1952) "Rotary Tubes as Experi-
mental Trickling Filters", Sewage Ind. Wastes, 24, 1355. ,tp; '145]
Golding, B. (1959), Polymers and Resins, D. van Nostrand Co., Inc., N.Y.,
N.Y. [p. 443, 444]
Gomaa, W.M. arid Faust, S.D. (1972), "Chemical Hydrolysis and Oxidation of
Parathion.and .Paraoxon in Aquatic Environments". Fate of iOrganic
Pesticides in the Aquatic Environment, Advan. in Chem. Ser. Ill,
American Chem. Soc., Washington, B.C. Ghpt. 10, p. 188. [p.;354]
Corner, R. andtttoyes, W.A. (1949), "Photochemical Studies XII", J. Amer.
Chem. Soc. 71, 3390. [p. 382]
Gonzalez, C.F.; Taber, W.A. and Zeitoun, M.A. (1972), "Biodegradation of
Ethylene i.Glycol by a Salt-Requiring Bacterium", Appl. Micrdbiol,
24(6), 911-919. [p. 106]
Goodman, G.T. (1973), "How Do Chemical Substances Affect the Environment",
Proc. Roy. Soc. London, Ser. B, 185(1079), 127-48. [p. 23, 461]
Goodnow, ,R.A.,and Harrison, A.P. (1972), "Bacterial .Degradation of,/Deter-
gent Compounds", Appl. Microbiol., 24(4), 555-60. [p. 104] !
Goring, C.A.I. (1967), "Physical Aspects of Soil in Relation to the Action
of Soil Fungicides",,Ann. Rev. Phytopathol., _5, 285.
Goswami, K.P. and Green, R.E. (1971), "Microbial Degradation of .:the Herbi-
cide Atrazine and its 2-hydroxy Analog in Submerged Soils",,. Environ.
Sci. Technol. 1(5), 426-429. [p. 228, 252, 253]
Gould, E. (1962), "An Extractant for Microamounts of Anionic Surfactant
-Bound to.Large Amounts of Protein, with Subsequent Spectrophotometric
Determination", Anal. Chem., 34. 567-571. [p. 152]
Gould, R.F. (editor) (1972) Biological Correlations - The Hansch Approach,
.Advances.in Chemistry Series 114, American Chemical Society.,
Washington, D.C. [p. 463]
Grady, L. and ,Busch, A.W. (1963), "B.O.D. Progression in Soluble Substrates
VI. Cell Recovery Techniques in the TBOD Test", Proc. 18th Ind. Waste
Conf., Purdue Univ., Eng. Bull., Ext. Ser. 115, 194-203. [p.. 72]
Graetz, D..A.; Chesters, G.; Daniel, T.C.; Newland, L.W.; and Lee, 6.B. (1970),
"Parathion Degradation in Lake Sediments", J. Water Pollut. Contr. Fed.,
_2(Pt. 2), R76-R94. [p. 101]
Graham, R.E.; Buram, K.R.; Hammer, C.F.; Hansen, L.B. and Kenner, C.T. (1973),
"Photochemical Decomposition of Heptachlor Epoxide", J. Agr. Food Chem.,
21(5), 824-834. [p. 337, 352, 356]
508
-------
Gray, F.H.H. and Thornton, H.G. (1928), "Soil Bacteria that Decompose
Certain Aromatic Compounds", Zentra. Bakteriol. Parasitenk,. Abst. II,
73, 74-96. [p. 96]
Gray, W.F.; Pomerantz, J.H. and Ross, R.D. (1972), "UV Irradiation of
6-Methyl-2,3-Quinoxalinedithiol Cyclic Carbonate (Morestan)", J.
Heterocyclic Chem., £, 707. [p. 337]
Greathouse, G.A.; Weasel, C.J. and Shirk, H.G. (1951), "Microbiological
Deterioration of Manufactured Materials", Annual Rev. Microbiology,
1, 333. [p. 434, 475]
Griffiths, D.C. (1966), Rept. Rothamsted Exptl. Sta. for 1965, 170.
[P. 265]
Grunwell, J.R. and Erickson, R.H. (1973), "Photolysis of Parathion (0,0-
Diethyl-0-(4-Nitrophenyl)-thiophosphate) New Products", J. Agr. Food
Chem., 21, 929. [p. 337, 347]
Guenzi, W.D. and Beard, W.D. (1967), "Anaerobic Biodegradation of DDT to ODD
in Soil", Science 156(3778), 1116-1117.
Guenzi, W.D. and Beard, W.E. (1968), "Anaerobic Conversion of DDT to DDD
and Aerobic Stability of DDT in Soil", Soil Sci. Soc. Amer. Proc.,
32, 522. [p. 228, 268]
Gunner, H.B. and Zuckerman, B. M. (1968), "Microbiology. Degradation of
Diazinon by Synergistic Microbial Action", Nature 217/5134), 1183-1184.
[p. 192, 277]
Gutfreund, K. (1971), "Feasibility Study of the Disposal of Polyethylene
Plastic Waste", G.P.O. Stock No. 5502-0036, Govern. Printing Office,
Washington, D.C. [p. 429, 444, 446, 454, 477, 478, 479, 480]
Haagen7Smit, A.J. and Wayne, L.G. (1968), "Atmospheric Reactions and
Scavenging Processes", in, Air Pollution, ed. Stern, A.C., Vol. I,
Academic Press, N.Y. [p. 46, 283, 284]
Haines, J.R. and Alexander, M. (]974), "Microbial Degradation of High-
Molecular-Weight Alkanes", App. Microbiol., 28(6), ]084-85. [p. 474]
Halvorson, H.; Ishaque, M.; Solomon, J.; and Grussendorf, O.W. (1971),
"Biodegradability Test for Insecticides", Can. J. Microbiol., 17_(5) ,
585-91. [p. 100, 175, 484]
Hamaker, J.W. (1972), "Decomposition: Quantitative Aspects", in, Organic
Chemicals in the Soil Environment, Vol. 1 (C.I. Goring and J.W. Hamaker,
eds.), p. 253-340. [p. 270, 271]
Hammerton, C. (1955), "The Decay of Synthetic Anionic Detergents, in Natural
Waters", J. Appl. Chem., ^, 517-24. [p. 73]
509
-------
Hammond, M.W. and "Alexander, M. (1972), "Effect of Chemical Structure on
Microbial'Degradation of Methyl-Substituted Aliphatic Acids", Environ.
Sci. Technol., »(8), 732-735. Ip. 69, 232, 238, 254, 464, 465]
Hance, R.J. (1967), "Decomposition of Herbicides in the Soil by Nonrbiological
Chemical Processes", J. Sci. Food Agr., 18(11), 544-7. [p. 35.4]
Hahc'e, R.J. (1967), "Decomposition of Herbicides in Soil", J. Sci. Food Agr.,
20(3), 144-5., [p. 226] v
Hanst, P.L.; Stephens, E.R.; and Scott, W.E. (1956), "Reactions Involving
Ozone, Nitrogen Dioxide, and Organic Compounds at Low;Concentrations
in Air",, Air .Repair, J> 219-25. [p. 285]
''"'"' - ' '
Harris, C.I. (1969), "Movement of Pesticides in Soil", J. Agr. Food Chem.,
JL7, 80-2. [p. 226, 246, 258]
t
Harris, C.I.; Woolson, E.A.; and Hummer, B.E. (1969), "Dissipation'of
Herbicides at Three Soil Depths", Weed Sci., 17_, 27.
Hartmann, L. (1963), "The Removal of Alkylbenzenesulfonate (ABS):'by Micro-
organisms", Biotechnol. Bioeng., 5., 331-45. [p. 152]
Hatch, W.R. and Ott, W.L. (1968), "Determination of Sub-microgram Quantities
of Mercury by Atomic Absorption Spectrophotometry", Anal. Chem., 40^, 2085.
Hatfield, R. '(1957), "Biological Oxidation of Some Organic Compounds",
Ind. Eng'. Chem., 49, 192-6. [p. 465]
Hattihgh, W;H.J. (1963), "Activated Sludge Studies. I. The Nitrogen and
Phosphorus Requirements of the Microorganisms. II. Influence; of
Nutrition on Bulking", Water Waste Treat. J., £, 380-386, 424.-426,
476-480. [p. 165]
Hawkes, H'.A. (1963), The Ecology of Waste Water Treatment, MacMillan^ N.Y.
, [P- 171].
Hazeu, I.W. (d967), "Results of the First Interlaboratory Experiment on Bio-
degradation of Plastics", Intern. Biodeterior. Bull., 3^(1), 15-19.
[p. 424, 438, 448, 455]
Hazeu, I.W. and Waterman, H.A. (1965), "Evaluation of the Microbial Degradation
by the Vibrating-Reed Method", Int. Biodetn. Bull., l.(i)-, 43f49. [p. 436]
Heap, W.M. and Morrell, S.H. (1968), "Microbiological Deterioration of Rubbers
and Plastics", J. Appl. Chem., 18_, 189-93. [p; 456]
Heck, W.W;; Fox, F.Li,; Brandt, C.S.; and Dunning, J.A. (1969), "Tobacco,
A Sensitive Monitor for Photochemical Air Pollution", Nat. Air
Pollut..'Contr. Admin. Public No. AP-55. [p. 312]
510
-------
Hegeman, G.D. (1972), "The Evolution of Metabolic Pathways in Bacteria", in,
Degradation of Synthetic Organic Molecules in the Biosphere. Proc. Conf.,
San Francisco, Calif., June 11-13, 1971, National Academy of Science,
Washington, D.C.
Hemmett, R.B., Jr. (1972), "The Biodegradation Kinetics of Selected Phenoxyacetic
Acid Herbicides and Phenols by Aquatic Microorganisms", Dis. Abst., 7098-B.
[p. 97, 185]
Henderson, G.L. and Crosby, D.G. (1967), "Photodecomposition of Dieldrin and
Aldrin", J. Agr. Food Chem., 15, 888. [p. 358, 366, 367]
Henderson, G.L. and Crosby, D.G. (1968), "The Photodecomposition of Dieldrin
Residues in Water", Bull. Environ. Contain. Toxicol., 3_(3) , 131-134.
[p. 335, 337, 347, 358, 367]
Herring, J.L.; Hannan, E.J.; and Bills, D.D. (1972), "The UV Irradiation of
Aroclor 1254", Bull. Environ. Contain. Toxicol., JJ (3), 153-7. [p. 269,
328, 337]
Hester, N.E.; Stephens, E.R.; and Taylor, O.C. (1973), "Fluorocarbons in the
Los Angeles Basin", Air Pollution Association Meeting, Chicago, June 24-28.
Heukelekian, H. (1949), "Aeration of Soluble Organic "Waste with Non-floeculent
Growths", Ind. Eng. Chem., 4^, 1412. [p. 55]
Heukelekian, K.H., and Gelman, I. (1951), "II. Effect of Certain Environmental
Factors on the Biochemical Oxidation of Wastes", Sewage Ind. Waste,
23., 1546. [p. 171]
Heuss, J.M. and Glasson, W.A. (1968), "Hydrocarbon Reactivity and Eye Irritation",
Environ. Sci. Technol., 2, 1109. [p. 302, 325, 469]
Heyman, J.J. and Mo'lof, A.H. (1967), "Inititation of Biodegradation of
Surfactants", J. Water Pollut. Contr. Fed., .3£(1), 50-62. [p. 60, 63,
158]
Heyman, J.J. and Molof, A.H. (1968), "Biodegradation of Linear Alkylated
Sulfonates", Environ. Sci. Technol., .2(10), 773-778. [p. 114, 238]
Hill, D.W. and McCarty, P.L. (1967), "Anaerobic Degradation of Selected
Chlorinated Hydrocarbon Pesticides", J. Water Pollut. Contr. Fed.,
39(8), 1259-77. [p. 148, 170]
Hilton, B.D. and O'Brien, R.D. (1964), "A Simple Technique for Tritiation of
Aromatic Insecticides", J. Agr. Food Chem., 12, 236-8. . [p. 154]
Hinkel, M.E. and Learned, R.E. (1969), "Determination of Mercury in Natural
Waters by Collection on Silver Screens", U.S. G.S. Prof. Paper No. 650D.D
251. [p. 407]
511
-------
Hirwe, A.S., Metcalf, R.L. and Kapoor, I.P. (1972), "«-Trichloromethyl-
benzylanilines and ^-Trichloromethylbenzyl Phenyl Ethers with DDT-Like
Insecticidal Action", J. Agr. Food Chem., 20, 818-24. [p. 120]
Hitztnan, D.O. (1964), "New Accelerated Test for Rapid Measurement of
Detergent Bibdegradability", J. Am. Oil Chemists' Soc., 41(9),
593^5. [p. 77]
f
Hitz, H.R. and.Zinkernagel, R. (1967), "Test-tube Method for Evaluation
of Biodegradation of Plasticized PVC [poly(vinyl chloride)] by Pseudomonas
Aeruginosa NTCC 8060", Intern. Biodeterior. Bull., 3(1), 21-3. [p. 438,
441, 448, 450]
i
Hpadley, A.W. and McCoy, E. (1965), "Characterization of Certain .Gram
Negative Bacteria from Surface Waters", Appl. Microbiol., 13,
575-578. [p. 163]
Hobbs, P.V.; Harrison, H.; and Robinson, E. (1974), "Atmospheric^Effects of
Pollutants",'Science, 183, 909. [p. 482] .*
HoIden, A.V. (1970), "International Cooperative Study of Organochlorine
Pesticide Residues in Terrestrial and Aquatic Wildlife, 1967/68",
Pest. Monitoring J., 4^, 117. [p. 35]
Hoogerhyde, T.C. (1965), "A Differential Manometer for the Measurement
of Microbial,Growth in Sewage", Thesis, Michigan State University,
East Lansing, Michigan, [p. 65] '
Horvath, R.S. (1970), "Microbial Cometabolism of 2,4,5-trichlorophenoxy-
acetic Acid", Bull. Environ. Contamin. Toxicol., 5_, 537-541." '[p. 279]
Horvath, R.S. (1971), "Cometabolism of the Herbicide 2,3,6-Trichlorb-
benzoate", J. Agr. Food Chem., 19(2), 291-293. [p. 279]
Horvath, R.S. (1972a), "Microbial Cometabolism and the Degradation of
Organic Compounds in Nature", Bacteriol. Rev., ^6(2), 146-55L
[p. 34, 107, 168, 278, 462]
Horvath, R.S. (1972b), "Cometabolism of the Herbicide, 2,3,6-Trichlorob.enzoate
by Natural Microbial Populations", Bull. Environ. Contam. Tpx^col.,
.7(5), 273-6. [p. 79, 168, 279]
Horvath, R.S. ,(1973), "Enhancement of Co-Metabolism of Ch.lorobenzoa.tes
by the Co-Substrate Enrichment Technique", Appl. Microbiol.,
.25(6), 961-963. [p. 108, 279]
Horvath, R.S. and Alexander, M. (1970), "Cometabolism of m-Chlorobenzoate
by an Arthrobacter". Appl. Microbiol., 20(2), 254-258. [p. 10.8]
Horvath, R.S. and Koft, B.W. (1972), "Degradation of Alkyl Benzene
Sulfonate by Pseudomonas Species", Appl. Microbiol., 23(2),
407-414. [p. 109, 168]
512
-------
House, R. and Fries, B.A. (1956), "Radioactive ABS in Activated
Sludge Sewage Treatment", Sewage Ind. Wastes, 28, 492. [p. 141]
Houston, C.A. (1963), "Foreign Requirements and Developments in
Biodegradability", J. Am. Oil Chemists Soc., 40(11), 659-61.
[p. 134, 136, 212]
Howard, P.H. (1974), "Establishing Environmental Priorities for Synthetic
Organic Chemicals: Focusing on the Next PCB's", Seminar on Early
Warning Systems for Toxic Substances, Jan. 30 - Feb. 1, Seattle,
Washington, Battelle Memorial Institute. Ip. 481]
Huber, L. (1962), "Investigations on the Decomposition of a New Anionic
Surfactant in an Oxidation Channel", Miinchen Beitrag yur Abwasser-
Fisch-, Flussbiol., j)» 235-265. [p. 152]
Huber, L. (1968), "Degradation Tests with Alkylbenzene Sulfonates in
an Oxidation Ditch", Tenside, j>, 65-76. [p. 152]
Huddleston, R.L. and Allred, R.C. (1963), "Microbial Oxidation of
Sulfonated Alkylbenzenes", Dev. Ind. Microbiol., 4_, 24.
[p. 83, 84, 85, 86, 104, 153, 166, 185]
Huddleston, R.L. and Allred, R.C. (1964), "Evaluation of Detergents by
Using Activated Sludge", J. Am. Oil Chem. Soc., 41, 732-5. [p. 127,
131, 132, 133]
Huddleston, R.L. and Allred, R.C. (1965), "Determination of Nonionic Surfactant
Biodegradability: Physical Properties vs. Colorimetry", J. Amer. Oil
Chem. Soc., 42/11), 983-986. [p. 84, 160]
Hueck, H.J. (1960), "Biological Deterioration of Plastics", Plastics, (Lond.),
Z5, 419-22. [p. 456]
Hueck, H.J. and van der Toorn, J. (1965), "An Interlaboratory Experiment With
the Soil Burial Test [of Microbial Degradation]", Int. Biodeterioration
Bull., 1(1), 31-40. [p. 427, 450, 453, 455]
Hummel, D.O. (1966), "II Experimental: Infrared Spectra of Polymers in the
Medium and Long Wave Regions", Polymer Rev., 14, 14-7. [p. 430, 444]
Hunter, J.V. and Heukelekian, H. (1964), "Determination of Biodegradability
with Warburg Respirometric Techniques", Purdue Univ., Eng. Bull., Ext.
Ser. No. 117 (Pt. 2), 616-27. [p. 59, 60, 158]
Husmann, W.F.; Malz, W.F.; and Jendreyko (1963), "Removal of Detergents From
Wastewaters and Streams", Forschungsber Landes Nordhein-Westfalen No.
1153, 40. [p. 131, 145]
Hutzingeri 0.; Safe, S.; and Zitko, V. (1972), "Photochemical Degradation
of Chlorobiphenyls (PCBs)", Environ. Health Perspectives, 1, 15. [p. 369]
Huyser, H.W. (1960), "Relation Between the Structure of Detergents and Their
Biological Degradation", Vortraege Originalfassung Intern. Kongr.
Grenzflaechenaktive Stoffe, 3., Cologne, 3., 295-301. [p. 164]
513
-------
Iliff, N. (1972), "Organic Chemicals in the Environment", New Scij.,
531(781), 263^5. [p. 26, 465]
Imura,. N.: Sukegawa, E.; Pan, S.; Nagoa, N.; Kim, J.; Kwan, T.; and Ukita,
T. (1971), "Chemical Methylation of Inorganic Mercury, with Metshyl-
cobalamin, a Vitamin B12 Analog", Science, 171, 1248. [p. 414]
Inoue, M. (1973),'"Regeneration of Plastics by Microorganisms", plastics
Industry'News, pp. 17-26. :
Isensee, A.R. (1974), personal communication. [p. 189]
;
Isensee, A.R. .and. Jones, G.E. (1974), "Distribution of 2,3,7,8-^-Tetra-
chlorodibenzo-p-dioxin (TCDD) in an Aquatic Model .Ecosystem'', Environ.
Sci. Tech. (in press). [p. 122, 189] .:
Isensee, A.R.; Kearney, P.C.; Woolson, E.A.; Jones, G.E.; and Williams,
V.P. (1973), "Distribution of Alkyl Arsenicals in Model Ecosystem",
Environ. Sci. Technol., _7 (9), 841-5. [p. 122, 395, 399, 413]'
Iskander, I.K.; Syers, J.K.; Jacobs, L.W.; Keeney, D.R. and Gilmour, J.T.
(1972), "Determination of Total Mercury in Sediments and Soil",
Analyst, 97.,^ 388-393. [p. 400]
Ivie, G.W. and Casida, J.E. (1971a), "Sensitized Photodecomposition and
Photosensitizer Activity of Pesticide Chemicals, Exposed to. Sunlight
on Silica Gel Chromatoplates", J. Agr. Food Chem., 19(3), 405-9.
[p. 337,' 351]
Ivie,- G.W. and Casida, J.E. (1971b), "Photosensitizers for the Accelerated
Degradation of Chlorinated Cyclodienes and Other Insecticide^Chemicals
Exposed to Sunlight on-Bean Leaves", J. Agr. Food Chem.,. '19(3),
410r-16. [p.' 337, 352, 362, 366, 367]
^_
Ivie, G.W.; Dorough, H.W.; and Cardona, R.A. (1973), "Photodecompo.sition
of the-Herbicide Methazole", J. Agr. Food Chem., 21(3), 386-91.
[p. 337, 351]
Iwata, Y.; Westlake, W.E.; and Gunther, F.A. (1973), "Persistence;of
Parathion in Six California Soils Under Laboratory Conditions",
Arch. Environ. Contam. Toxicol., _!, 84. [p. 228, 244]
Jacobs, L.W. and Keeney, D.R. (1974), "Methylmercury Formation in
Mercury-r-Treated River Sediments during in situ Equilibration",
J. Environ. Qual., ^, 121. [p. 391, 400]
Jaegers, K. and Niemitz, W. (1952), "BOD Detection of Sewage", Stadtehygiene,
9_, 246. . [p. 63]
Jaffe, R.J. (1974), personal communication, July 10.
Jaffe, H.H. and Orchin, M. (1962), Theory and Applications of Ultraviolet
Spectroscopy, J. Wiley and Sons, N.Y. [p. 43]
514
-------
Jaffe, R.J. and Smith, F.C., Jr. (1974), "Factors Affecting Reactions
in Smog Chambers", Paper presented at 67th Ann. Meeting of APCA,
Denver, Colorado, June 13. [p. 308, 309, 311, 313, 314, 320, 321,
322, 323]
Jagnow, G. and Haider, K. (1972), "Evolution of lkC02 from Soil Incubated
With Dieldrin-14C and the Action of Soil Bacteria on Labelled
Dieldrin", Soil Biol. Biochem., 4.(1), 43-49. [p. 253, 268]
Janicke, W. (1971), "Behavior of Synthetic Organic Substances During the
Treatment of Waste Liquors. IV. Progress on the Biodegradation of
"soft" Anionic Synthetic Detergents", Water Res., 1(10), 917-31.
[p. 203]
Jannasch, H.W. (1967), "Enrichments of Aquatic Bacteria in Continuous Culture",
Archiv. fur Mikrobiol., 59, 165-173. [p. 117]
Japar, S.; Pitts, J.N., Jr.; and Winer, A.M. (1974), "The Photostability
of Fluorocarbons", Environ. Sci. Technol., (in press). [p. 287, 306,
307, 327, 330]
Jendreyko, H. and Ruschenberg, E. (1963), "The Behavior of Biologically
Soft ABS in Trickling Filters and Activated Sludge Plants",
Gas-Wassenfach, 104. 391. [p. 131]
Jen-hao, L. and Scwartz, A. (1961), "Action of Bacterial Mixtures on
Polyethylene of Various Molecular Weights", Kunstsloffe, 51, 317-9.
Jenkins, S.H.; Harkness, N.; Lennon, A.; and James, K. (1967), "The
Biological Oxidation of Synthetic Detergents in Recirculating
Filters", Water Res., j.(l), 31-53. [p. 144, 145, 146, 147]
Jensen, H.L. (1957), "Decomposition of Chloro-substitute Aliphatic Acids
by Soil Bacteria", Can. J. Microbiol., 3_, 151-66. [p. 232, 236]
Jensen, H.L. (]963), "Carbon Nutrition of Some microorganisms Decomposing
Halogen-Substituted Aliphatic Acids", Acta. Agr. Scand., 13, 404-412.
[P. 107]
Jensen, H.L. (1964), "Studies on Soil Bacteria (Arthrobacter globiformis)
Capable of Decomposing the Herbicide Endothal", Acta. Agr. Scan.,
14, 193-204. [p. 177]
Jensen, S. and Jernelov, A. (1969), "Biological Methylation of Mercury
in Aquatic Organisms", Nature, 223. 753-4. [p. 382, 386, 398, 404,
405, 413, 414]
Jernelbv, A.; Lien, E.L.; and Wood, J.M. (1972), Unpublished report
prepared for the Government of Ontario for Analysis of St. Glair
River Sediments, (1972). [p. 409]
Jobson, A.; Cook, F.D.; and Westlake, D.W.S. (1972), "Microbial Utilization
of Crude Oil", Appl. Microbiol., ^3, 1082-1089. [p. 247, 268, 269]
515
-------
Johnson, G.W. and Vickers, C.W. (1970), "The Examination of Organomercury
Compounds and Their Formulation by Thin-Layer Chromatography",
Analyst,'25, 356. [p. 402]
' . ' i
Johnson, B.T. (1974), "The Biodegradation of Di-n-butyl Phthalatejand
Di-2-ethylhexyl Phthalate by Freshwater Hydrosol.", ASM Annual
Meeting, Chicago. [p. 123]
i
Jones, E.S.L.: (1968), "Some Problems Posed by Quality Screening for
Biodeterioration", in Biodeterioration of Materials; Micro-
biological and Allied Aspects, (A.H. Walters;and J.J. Elplnick,
Editors), Elsevier, N.Y., p. 188. [p. 427, 440, 449] I
Jones, A.S. and Hodges, C.S. (1974), "Persistence of Mirex and Its Effect
on Soil Microorganisms", J. Agr. Food Chem., 22, 435. [p. 247, 251]
Jordan, L.S.;.Coggins, C.W. Jr.; Day, B.E.; and Clerx, W.A. .(d964),
"Photodecomposition of Substituted Phenylureas", Weeds, 12(1;, 1-4.
[p. 337] .
Kaiser, K.L.E. and Wong, P.T.S. (1974), "Bacterial Degradation of' Poly-
chlorinated Biphenyls. I. Identification of Some Metabolic*Products
from Arochlor 1242", Bull. Environ. Contam. Toxicol., 11, 291.
[p. 109, 176, 371]
Kamen; M.D. (1957), "lostopic Tracers in Biology, An Introduction to
Tracer Methodology", p. 133, Academic Press, N.Y. [p. -;193] ;
Kania, H.J.; Ferens, M.C. and Beyers, R.J. (1973), "The Fate of Mercury in
Artificial Stream Systems", Interim Report for EPA Project No. R800510.
[p. 391, 398, 415]
Kapoor, I.P;. Metcalf, R.L.; Nystrom, R.F. and Sangha, G.K. (d970), ''Compara-
tive Metabolism of Methoxychlor, Methiochlor, and DDT in Mouse, Insects,
and in a Model Ecosystem", J. Agric. Food Chem. 48, 1145. [p. 120,
-154]
Kapoor, I.P,; Metcalf, R.L.; Nystrom, R.L.; and Sangha, G.K. (197,2), "Compara-
tive Metabolism of DDT, Methylchlor and Ethoxychlor in Mquse, Insects,
and in a Model Ecosystem", J. Agric. Food Chem., 2J), 1-6. [p. 120,
154]
Kapoor, I.P.; Metcalf, R. L.; Hirwe, A.S.; Coats, J.R.; and Khalsa, M.S.
(1973), "Structure Activity Correlations of Biodegradability of DDT
Analogs", Ji Agr. Food Chem., ^1(2), 310-15. [p. 461, 462]
Karasek, F.W. and Laub, R.J. (1974), "Detection Limits in Instrumental
Analysis", Research/Develop., 25, 36, June. [p. 254, 355, 400, ,403]
Kator, H. (1973), "Utilization of Crude Oil Hydrocarbons by Mixed .Cultures
of Marine Bacteria", in Microbial Degradation of Oil Pollutants.
(D.G. Ahearn and S.P. Meyers, Eds.), Louisiana State University
Publication No. SG-73-01. [p. 112, 152]
516
-------
Kaufman, D.D. (1965), "Structure of Pesticides and Decomposition by Soil
Microorganisms", Pestic. Their Eff. Soils Water. Symp. Pap., Columbus,
Ohio, 85-94. [p. 465]
Kaufman, D.D. (1966), "An Inexpensive, Positive Pressure, Soil Perfusion
System", Weeds, 14, 90. [p. 230, 231, 256]
Kaufman, D.D. and Kearney, P.C. (1965), "Microbial Degradation of
Isopropyl-N-3-Chlorophenylcarbamate and 2-Chloroethyl-N-3-
Chlbrophenylcarbamate", App. Microbiol., 13, 443-6. [p. 231, 234, 249,
263, 264]
Kaufman, D.D.; Plimmer, J.R.; Kearney, P.C.; Blake, J. and Guardia, F.S.
(1968), "Chemical Versus Microbial Decomposition of Amitrole in Soil",
Weed Sci., 16, 266. [p. 227, 253, 261, 271, 272, 353]
Kazano, H.; Kearney, P.C. and Kaufman, D.D. (1972), "Metabolism of Methyl-
Carbamate Insecticides in Soils", J. Agric. Food Chem., 20, 975-9.
[p. 226, 251, 253, 260]
Kearney, P.C. and Woolson, E.A. (1973), "Persistence and Reaction of 1'*C-
Cacodylic Acid in Soil Environments", Environ. Sci. Technol., _7_, 47-50.
[p. 384]
Kearney, P.C.; Kaufman, D.D. and Beall, M.L. (1964), "Enzymatic Dehalogena-
tion of 2,2-Dichloropropionate", Biochem. Biophys. Research Comm.,
14, 29. [p. 394]
Kearney, P.C.; Woolson, E.A.; Plimmer, J.R. and Isensee, A.R. (1969),
"Decontamination of Pesticides in Soils", Residue Reviews, 2^, 137.
[p. 346]
Kelly, R.J.; Konecky, M.S.; Shewmaker, J.E. and Bernheimer, R. (1965),
"Physical and Biological Removals of Detergent Activities in a Full
Scale Sewage Plant", Surface Activity and the Living Cell, Soc. Chem.
Ind. (London), Monograph No. 19, pp. 286-304. [p. 152]
Kempton, A.C.; Maisel, H. and Kaplan, A.M. (1963), "A Study of the Deteriora-
tion of Fungicide Treated Fabric in Soil Burial", Text. Res. (J.)> 33(2),
87-93.
Kenaga, E.E. (1968), "Guidelines for Evaluating the Properties of Pesticides
for Safe Use in the Wildlife Environment", Down to Earth, 23(4), 11-18.
Kenaga, E.E. (1972), "Factors Related to Bioconcentration of Pesticides",
Environ. Toxicol. Pestic., Proc. U.S.-Jap., 193-228. [p. 35]
Kendeigh, S.C. (1974), Ecology with Special Reference to Animals and Man,
Prentice Hall, Inc., N.J.
Klausmeier, R.E. (1967), "Deterioration of Vinyl Resin Systemsthe Current
Plasticizer Deterioration Study", Develop. Ind. Microbiol., £, 218-21.
517
-------
Klein, S.A. (1965)j "Travel of Synthetic Detergents with Percolating Water",
Fourth Annual Report," SERL 65-4. Univ. of Calif., Berkeley. ,[p. 149]
i '*' , \'
Klein, W. and Korte, F. (1971), "Conversion of Pesticides Under Atmospheric
Conditions and in Soil", in Trace Substances in Environmental"
ttealth-V. (ed. D.D. Hemphill), U. of Missouri, [p. 284] .
Klein, S.A. and McGaughey, P.H. (1965), "Degradation of Biologically Soft
Detergents by'Waste Water Treatment Processes", J. Water Poll.
Contr. Fedr.,:37.(6), 857-66. [p. 146]
Kleopfer, R.D.'and Fairless, B.J. (1972), "Characterization of Organic
Components in a Municipal Water Supply", Environ. Sci. Techno1.,
6, 1036. <[p. 27] !
Klug, M.J. and'Markovetz, A.J. (1967), "Degradation of Hydrocarbons''by
Members of the Genus Candida. I. Hydrocarbon Assimilation", Appl.
Microbiol*. 15(4), 690-693. [p. 104]
Y
Knaggs, E.A.; Yeager, J.A.; Varenzi, L. and Fischer, E. (1965), "Alpha-
Sulfo Fatty Esters in Biologically Soft Detergent Formulations",
J. Amer. Oil Chem. Assoc., 42, 805-810. [p. 178]
Knowles, C.O. and Sen Gupta, A.K. (1969), "Photodecomposition of the
Acaricide N'-(4-chloro-o-tolyl)-N,N-dimethylf6rmamfdine", Jf Econ.
Entomol., 62_(2), 344-8. [p. 337, 351]
Roller, L.R. (1965), "Solar Radiation" in Ultraviolet Radiation. J. Wiley
and Sons, N.Y., 2nd Edit. [p. 42]
Konrad, J.G.; Armstrong, D.E. and Cheaters, G. (1967), "Soil Degradation of
Diazinon, a Phosphorothioate Insecticide", Agron. J. 59(6), 591-594.
[p. 265]
Kopcznski, S.; Lonneman, W.; Sutterfield, F.D. and Darley, P.E. (1972),
"Photochemistry of Atmospheric Samples in Los Angeles", Environ. Sci.
Technol., £(4), 342-7. [p. 290, 308, 315, 321, 325, 326]
Korth, M.W.; Rose, A.H., Jr. and Stahmen, R.C. (1964), "Effect of Hydro-
carbons to Oxides of Nitrogen Ratios on Irradiated Auto Exhaust",
J. Air Pollut. Contr. Assoc. 14(5), 168-175. [p. 287, 298, 299, 300,
312]
Krone, M. and Schneider, G. (1968), "Biodegradability of Secondary'AlkaWe
Sulfonates Under Laboratory and Practice Conditions", Fette-Sekfen-
Anstrichmittel, 7£, 753-757. [p. 178]
518
-------
Kuhr, R.J. and Casida, J.E. (1967), "Persistent Glycosides of Metabolites
of Methylcarbamate Insecticide Chemicals Formed by Hydroxylation
in Bean Plants", J. Agr. Food Chem., 15, 814. [p. 36]
Kuntz, R.L.; Kopczynski, S.L. and Bufalini, J.J. (1973), "Photochemical
Reactivity of Benzaldehyde-NOx and Benzaldehyde-Hydrocarbon-NOx
Mixtures", Environ. Sci. Technol, 1(13), 1119. [p. 300, 318]
Kuwahara, M.; Kato, N. and Munakata, K. (1966a), "The Photochemical
Reaction of Pentachlorophenol. Part I. The Structure of the Yellow
Compound", Agr. Biol. Chem., _30, 232. [p. 335, 337]
Kuwahara, M.; Kato, N. and Munakata, K. (1966b), "The Photochemical Reaction
of Pentachlorophenol. Part II. The Chemical Structures of Minor
Products", Agr. Biol. Chem., 30, 239. [p. 335, 337]
Kuwahara, M.; Shindo, N.; Kato, N. and Munakata, K. (1969), "The Photo-
chemical Reaction of Pentachlorophenol: III. The Chemical Structure
of a Yellow C18-compound", Agr. Biol. Chem., 33(6), 892-899. [p. 337]
Kuwatsuka, S. (1972), "Degradation of Several Herbicides in Soils Under
Different Conditions", Environ. Toxicol. Pestic. Proc. U.S.-Jap. 1971.
385-400.
Lagerwerff, J.W. (1972), "Lead, Mercury and Cadmium as Environmental
Contaminants", in Micronutrients in Agr.. Proc. Symp. 1971 (John J.
Mortredt, Ed.), pp. 593-636. [p. 407]
Laity, J.L. (1971), "A Smog Chamber Study Comparing Blacklight Fluorescent
Lamps with Natural Sunlight", Environ. Sci. Technol., 5, 1218.
[p. 295, 314]
Laity, J.L. and Maynard, J.B. (1972), "The Reactivities of Gasoline Vapors
in Photochemical Smog", J. Air Pollut. Contr. Assoc., 22^ 100.
[p. 293, 302, 320, 322]
Laity, J.L.; Burstain, I.E.; and Appel, B.R. (1973), "Photochemical Smog and
the Atmospheric Reactions of Solvents", in Solvents Theory and
Practice, Adv. in Chem. Series #124, American Chemical Society.
[p. 27, 302, 312, 323]
Lamb, J.C.; Westgarth, W.C.; Rogers, J.L.; and Vernimmen, A.P. (1964),
"A Technique for Evaluating Biological Treatability of Industrial
Wastes", J. Water Pollut. Control Fed., 36, 1263. [p. 70]
Lang, D.; Trotter, C; Long, L., Jr. and Hasselstrom, T. (1965), "Synthesis
and Biodegradability of Some Isomeric Sodium p_-(Thia-n-dodecyl)-
benzenesulfonates", J. Amer. Oil Chem. Soc., ^2_(12), 1095-1097.
[p. 178]
519
-------
.Langford, C.H.; Wingham, M. and Sastri, V.S. (1973), "Ligand Photooxidation
in Copper (II) Complexes of Nitrilotriacetic Acid. Implications for
Natural Waters", Environ. Sci. Technol. , 7_(9) , 820. [p. 337]
i
.Langley, W.D. ,(1970), "Intermediate Products in the Bacterial Decomposition
of Hexadecano'l and Octadecanol" , Texas A&M Univ. Water- Resources Inst .,
Tech. Kept. TR-29. U.S. Nat.. Tech. Inform. Serv. , PB194237, 59 pp.
[p. 94] , .,
Langley, D.G. (1971), "Mercury Methylatidn in an; Aquatic ^Environment" ,
-paper presented at the 44th Annual Conference of the Water Pollution
Control Federation, Oct. 6, 1971 and the 162nd ACS National Meeting,
>Washington, D.C., Sept. 15. [p. 406, .409]
Larock, P.A. andvOppenheimer, C.H. (1970), "Growth of a Hydrocarbon
Bacterium in-? .a Nonaqueous Fluid", .Bacterial Proceedings. [p. 113]
Lashen, E.S. and Booman, K.A. (1967), "Biodegradability and Treatability
of Alkyl "Phenol Ethoxylates; a Class of Nonionic Surfactants", Water
Sewage Works , 114 (Nov . ) , 155- 63 .
Lashen, E.S. ; Blankenship, F.A.; Booman, K.A. and Dupre, J. (1966),
"Biodegradation Studies on a p_, ter t . -octylphenoxypolyethoxyethanol" ,
J. -.Water f Oil Chem. Soc., .43(6), 371-376. [? 73, 154, 160, 1'64, 183]
Lashen, E.S. ; 'Trebbi, G.F. ; Booman, K.A. and Dupre, J. (1967), "Biodegrad-
;ability of Nonionic Detergents", Soap Chem. Specialties,. 43(1) , 55-58,
122-129. [p. 78, 148, 149]
Lassiter, R:R. and Kearns, O.K. (1973), "Phytoplankton Population Changes
and Nutrient Fluctuations in a Simple Aquatic Ecosystem -Model", in
Proceedings of the Workshop in Modelling the Eutrophication? Process
PRWG 136rl. p. 131-138. [p. 124]
Lavy, T.L.; Roeth, F.W. ; and Fenster , C.R. (1973), "Degradation of 2, 4-D
and Atrazine at Three Soil Depths in the Field", J. 'Environ. 'ijual.,
.^(1), 132-7. [p. 257, 261, 262, 272]
Leigh, G.M. (1969), "Degradation of Selected -Chlorinated Hydrocarbon
Insecticides", J. Water Pollut. Contr. Fed., 41(11) (Pt. 2),;R450.
[p. 87, .152, 175, 484]
Leighton, P,.H. (1961), "Photochemistry of Air Pollution", Academic Press,
New York. [P- 42, 283]
Leutritz, J. and Hermann, D.B. (1946), "The Effect of High Humidity and Fungi
on the Insulation Resistance of Plastics", ASTM Bull. No. 138, p. 25-32.
[p. 477]
i
Levy, A. (1973), "Photochemical Smog Reactivity of Organic Solvents", Advan.
Chem. Ser., 124, 70-94. [p. 27, 304, 313]
520
-------
Lewicke, O.K. (1972), "Cleveland Opts for Physical-Chemical", Environ. Sci.
Technol., <6, 782. [p. 126]
Liang, T.T. and Lichtenstein, E.P. (1972), "Effect of Light, Temperature and
pH on the Degradation of Azinphosmethyl", J. Econ. Entomol., 65(2),
315-321. [p. 337, 351, 358, 363]
Lichtenstein, E.P. and Schultz, K.R. (1959), "Persistence of Some Chlorinated
Hydrocarbon Insecticides as Influenced by Soil Types, Rate of Application,
and Temperature", J. Econ. Entomol., _52, 124-31. [p. 226, 242, 243,
245, 266, 271]
Lichtenstein, E.P.; Schultz, K.R.; Fuhremann, J.W. and Liang, J.T. (1970),
"Degradation of Aldrin and Heptachlor in Field Soils During a Ten-
year Period; Translocation into Crops", J. Agr. Food Chem., 18,
100-6. [p. 242]
Lichtenstein, E.P.; Fuhremann, T.W.; and Schulz, K.R. (1971), "Persistence
and Vertical Distribution of DDT, Lindane, and Aldrin Residues, 10
and 15 Years after a Single Soil Application", J. Agr. Food Chem.,
19, 718-721.
Liebmann, H. and Offhaus, K. (1966), Abwassertechnic, 17/3), iv-vi. [p. 66]
Lightbody, A.; Roberts, M.E. and Wessel, C.J. (1954), "Cpt #9. Plastics
and Rubber", Deterioration of_ Materials; Causes and Prevention,
Reinhold Publ. Corp., N.Y., pp 537-595. [p. 435, 456, 474, 475, 476,
477, 478]
Lijmbach, G.W.M. and Brinkhus, E. (1973), "Microbial Degradation of Secondary
n-Alkyl Sulfates and Secondary Alkanols", J. Microbiol. Serol., 39(3),
415-23. [p. 115, 193, 196]
Lisk, D.J. (1974), "Recent Developments in the Analysis of Toxic Elements",
Science, 184. 1137. [p. 399]
Little, A.D., Inc. (1970), "Water Quality Criteria Data Book Volume 1:
Organic Chemical Pollution of Freshwater", 18010 DPV 12/70 Govern.
Printing Office, Washington, D.C. [p. 27]
Lloyd, A.O. (1955), "Testing Textiles for Resistance to Microbiological
Attack", J. Textile Inst., 46, T653-61. [p. 425, 450]
Loeffler, J.E. and Van Overbeek, J. (1971), "Metabolism of Herbicides",
in Pestic. Environ.. _l(Pt. 1)(White-Stevens, R., edit.) Dekker, N.Y.,
237-70. [p. 35]
Lombardo, P., Pomerantz, I.H. and Egry, I.J. (1972), "Identification of
Photoaldrin Chlorohydrin as a Photoalteration Product of Dieldrin",
J. Agr. Food Chem., ^0, 1278. [p.337, 352]
521
-------
Loos, M.A.; Roberts, R.N. and Alexander, M. (1967a), "Phenols as Inter-
mediates in the Decomposition of Phenoxyacetates by an Arthrobaicter,
sp.", Can. J. Microbiol., 13, 679-689. [p. 196, 197] ---,.
Lobs, M.A.; Bollag, J.M. and Alexander, M. (1967b), "Phenoxyacetate
Herbicide Detoxication by Bacterial Enzymes", J. Agr. Food Chem.,
15(5), 858-860. [p. 196, 197]
Lovelock, J.E.; Maggs, R.J. and Wade, R.J. (1973), "Halogenated Hydrocarbons
In and Over the Atlantic", Nature, 241, 19.4. [p. 328] !
Ludzack, F.J. (1960), "Laboratory Model Activated Sludge Unit", J. Water
Pollut. Contr. Fedr., .32, 605-609. [p. 98, 131, 133]
Ludzack, F.J. and, Ettinger, M.B. (1960), "Chemical Structures Resistant to
Aerobic Biochmeical Stabilization", J. Water Pollut. Contr'. Fedr.,
32, 1173-1200. [p. 465] \ '
Ludzack, F.J. and'Ettinger, M.B. (1963), "Estimating Biodegradability and
Treatability of Organic Water Pollutants", Biotechnol. Bioeng., 5,
309-30. [p.,130]
Ludzack, F.J.; Schaffer, R.B. and Ettinger, M.B. (1961), "Temperature and
Feed as Variables in Activated Sludge Performance", J. Water Ppllu.
Gbnt. Fed., 33_, 141-156. [p. 202]
Lykken, L. (1972), "Role of Photosensitizers in Alteration of Pesticide
Residues in Sunlight", in Environmental Toxicology of Pesticides
(ed. Matsumura, F., Boush, G.M. and Misato, T.), Academic Pr>|:s, N.Y.
[p. 349, 361]
Lyons, W.S. (ed.) (1964), Guide to Activation Analysis, Van Nostrand Co.,
Princeton, N.J. [p. 401]
McBride, B.C. and Wolfe, R.S. (1971), "Biosynthesis of Dimethylarsine
by Methanobacterium", Biochemistry, 10(23), 4312. [p. 381, 3$4, 389,
391, 397, 398, 399, 404, 406, 413, 416]
McCarty, P.L- and Speece, R.G. (1963), "Nutrient Requirements in "Anaerobic
Digester", Stanford Univ., Dept. of Civil Engineering, Stanford,
Calif., Tech. Rept. No. 25. [p. 387] *
McClure, G.W. (1970), "Accelerated Degradation of Herbicides in Soil by
the Application of Microbial Nutrient Broths", Contrib. Boyce
Thompson Inst., 24(11), 235-240. [p. 226, 271]
McClure, G.W. (1972), "Degradation of Phenylcarbamates in Soil by-Mixed
Suspension of IP6-adapted Microorganisms", J. Environ. Qual. 1(2),
177-180. [p. 239, 257]
522
-------
V
McCormick, L.L. and Hiltbold, A.E. (1966), "Microbiological Decomposition
of Atrazine and Diuron in Soil", Weeds, 14(1), 77-82. [p. 227, 271]
McGauhey, P.H. and Klein, S.A. (1959), "Removal of ABS by Sewage Treatment",
Sewage Ind. Wastes, 31, 877. [p. 131]
McGuire, R.R.; Zabik, M. J.; Schuetz, R.D.; and Flotard, R.D. (1972),
"Photochemistry of Bioactive Compounds: Photochemical Reactions of
Heptachlor: Kinetics and Mechanisms", J. Agr. Food Chem., 20(4),
856-861. [p. 337]
McKenna, E.J. and Kallio, R.E. (1964), "Hydrocarbon Structure: Its Effect
on Bacterial Utilization of Alkanes", in Principles and Applications
in Aquatic Microbiology, H. Henkelskian and N.C. Dondero, ed. (Wiley
and Sons, N.Y.). [p. 103, 162, 465]
McKenna, E.J. and Kallio, R.E. (1971), "Microbial Metabolism of the
Isoprenoid Alkane Pristance", Proc. Nat. Acad. Sci. U.S.A., 68,
1552. [p. 38]
McKinney, R.E. and Donovan, E.J. (1959), "Bacterial Degradation of ABS.
II. Complete Mixing Activated Sludge", Sewage Ind. Waste, 31, 690.
[P. 131]
McKinney, R.E. and Symons, J.M. (1959), "Bacterial Degradation of ABS.
I. Fundamental Biochemistry", Sewage Ind. Wastes, 31, 549-56.
[p. 59]
McKinney, R.E. and Wetchlein, R.G. (1955), "Isolation of Floe-Producing
Bacteria from Activated Study1', Appl. Microbiol., I, 259-261.
[P. 163]
Macfadyen, A. (1957), Animal Ecology, Aims and Methods, Pitman and Sons,
London. [p. 39]
Macfadyen, A. (]963), ibid., 2nd edition, [p. 39, 40]
MacRae, I.C. and Alexander, M. (1963), "Metabolism of Phenoxyalkyl
Carboxylic Acids by a Flavorbacterium Species", J. Bacteriol., 86,
1231-35. [p. 195, 236, 237, 245, 248]
MacRae, I.C. and Alexander, M. (1965), "Microbial Degradation of Selected
Herbicides in Soil", J. Agr. Food Chem., 13(1), 72-6. [p. 226, 236,
253, 254, 257, 466]
Mancy, K.H.; Westgarth, W.C.; and Okun, D.A. (1963), "The Application
of the Galvonic Cell Oxygen Analyzer to Waste Control Programs",
Purdue Univ. Ext. Ser., 112, 508. [p. 68]
523
-------
Mandels, G.R. and Siu, R.G.H. (1950), "Rapid Assay for Growth: Determination
of Microbiological Susceptibility and Fungistatic Activity", J. Bacteriol.,
60, 249-62. [p. 423, 438, 439, 452] ,
Manganelli, R.M;; Heukelekian, H.; and Henderson, C.N. (1960), "Persistence
and Effect of an Alkylaryl Sulfonate in Sludge Digestion", Purdue
Univ. Ex. Ser., J.5, 199. [p. 149] j.
Mann, A.H. and Reid, V.W. (1971a), "Biodegradation of Synthetic Detergents
Evaluation by. Community Trials. II. Alcohol and Alkylphenol
Ethoxylates",'J. Am. Oil Chem. Soc., 48, 794. [p. 204,,205]
Mann, A.H. and Reid, V.W. (1971b), "Biodegradation of Synthetic Detergents
Evaluation by. Community Trials: III. Primary Alcohol Sulphates!',
J. Am. Oil Chem. Soc., 48(12), 798-799. [p. 204] ,
Marshall, K.C. (1971), "Sorptive Interactions Between Soil Particles and
Microorganisms", in Soil Biochemistry. Vol. 2 (McLaren, A.D. and
Skujins, J. edit.) Dekker, New York. [p. 260] (
Matsumura, F. and Boush, G.M. (1967), "Dieldrin: Degradation by Soil
Microorganisms", Science, 156. 959. [p. 367] (
Matsumura, F.; Khanvilkar, V.G.; Patil, K.C.; and Boush, G.M. (1971),
"Metabolism of Endrin by Certain Soil Microorganisms", J. Agr. Food
Chem., 19(1), 27-31. [p. 106, 252]
Matsuo;, H. and Casida, J.E. (1970), "Photodegradation of Two Dinitrophenolic
Pesticide Chemicals, Dinobuton and Dinoseb, Applied to Bean Leaves",
Bull. Environ. Contam. Toxicol., 5(1), 72-8. [p. 337, 352]
""" i
Maurer, E.W.; Cordon, T.C.; Weil, J.K.; Nunez-Ponzoa, M.V.; Ault,,W.C.; and
Stirton, A.J. (1965), "The Effect of Tallow-Based Detergents on
Anaerobic Digestion", J. Am. Oil Chem. Soc., ^2_, 189-192. [p. 152]
Maurer, E.W.; Cordon, T.C.; and Stirton, A.J. (1971), "Microaerophilic
Biodegradation of Tallow-Based Anionic Detergents in River Water",
J. Amer. Oil Chem. Soc., .48(4), 163-165. [p. 81, 82, 170, 172]
I.
Mazzocchi, P.H. and Rao, M.P. (1972), "Photolysis of 3-(p-chlorophenyl)-
1,1-dimethylurea (Monuron) and 3-phenyl-l,l-dimethylurea (Fenuron)",
J. Agr. Food Chem., 20, 957. [p. 337]
Meagher, W.R. (1966), "A Heat-Labile Insoluble Conjugated Form of 2,4-
Dichlorophenoxyacetic Acid and 2-(2,4,5-Trichlorophenoxy)propionic
Acid in Citrus Peel", J. Agr. Food Chem., 14, 599- [? 3fi]
Meddings, P.J. and Potter, O.E. (1972), "The Degradation of Linear and
Branched Polymers: A Quantitative Study Using Simulation
Techniques", Adv. Chem. Ser. No. 109, 96-8, Amer. Chem. Society.
524
-------
Meinck, F. and Bringmann, (1961), "The Behavior of Tetrapropylene-benzene-
sulfonate in Sewage Treatment", Schriftenreiche Ver. Wasaer-Boden-
Luftug, No. 19, 68. [P. 149]
Mendel, H.G. and Mayersak, J.S. (1962), "The Metabolism and Actions of
Arsenite in Microorganisms", Federation Pro., 21, 179. [p; 381]
Merrell, J.C.; Jopling, W.F.; Bott, R.F.; Katko, A.; and Pintler, H.E.
(1967), The Santee Recreation Project, Santee, California, Final
Report. Water Pollution Control Research Series, Publication No.
WP-20-7, Fed. Water Pollution Control Administration, Cincinnati.
[P. 177]
Messersmith, C.G.; Burnside, O.C.; and Lavey, T.L. (1971), "Biological
and Non-biological Dissipation of Trifluralin from Soil", Weed Sci.,
19, 285-90. [p. 226, 256, 257, 263]
Metcalf, R.L. (1973), "An Academic Look At Environmental Regulatory Require-
ments", Pest Ch. ACS, in Am. Chem. Soc. Ann. Meeting, [p. 154]
Metcalf, R.L. (1974), "Laboratory Model Ecosystem to Evaluate Compounds
Producing Biological Magnification", Essays Toxicol., 5_, 17-38.
[p. 120, 122]
Metcalf, R.L. and Lu, P-Y. (1973), "Environmental Distribution and
Metabolic Fate of Key Industrial Pollutants and Pesticides in a Model
Ecosystem", Univ. 111. Water Resources Center, UILU-WRC-0069. U.S.
Nat. Tech. Inform. Service. PB Rpt. No. 225479. [p. 120, 122, 154,
174, 175, 176, 188, 484]
Metcalf, R.L.; Sangha, G.K.; and Kapoor, I.P. (1971), "Model Ecosystem
for the Evaluation of Pesticide Biodegradability and Ecological
Magnification", Environ. Sci. Technol., ,5(8), 709-13. [p.-7, 118, 119,
120, 122, 179, 395]
Metcalf, R.L.; Kapoor, I.P.; and Hirwe, A.S. (1972), "Development of
Biodegradable Analogues of DDT", Chem. Tech., 2(2), 105. [p. 120]
Metcalf, R.L.; Booth, G.M.; Schuth, C.K.; Hansen, D.J.; and Lu, P-Y.
(1973a), "Uptake and Fate of Di-2-ethylhexyl Phthalate in Aquatic
Organisms and in a Model Ecosystem", Environ. Health Perspectives,
Expl. Issue No. 4, p. 27-34. [p. 120]
Metcalf, R.L.; Kapoor, I.P.; Lu, P-Y.; Schuth, C.K.; and Sherman, P.
(1973b), "Model Ecosystem Studies of the Environmental Fate of Six
Organochlorine Pesticides", Envir. Health Perspectives, Expl. Issue
No. 4, p. 35-44. [p. 120, 175]
Miget, R.J.; Oppenheimer, C.H.; Kator, H.I.; and LaRock, P.A. (1969),
"Procedings API/FWPCA Joint Conference on Prevention and Control
of Oil Spills", New York, Dec. 15, 327-331. [p. 112]
525
-------
Miles, J.R.W.; Tu, C.M.; and Harris, C.R. (1969), "Metabolism of Hejptachlor
and its Degradation Products by Soil Microorganisms", J. Econ.
Entomol., 62(6), 1334-1338. [p. 245]
Miles, J.R.W.; Tu, C.M.; and Harris, C.R. (1971), "Degradation of Heptachlor
Epoxide and Heptachlor by a Mixed Culture of Soil Microorganisms",
J. Econ. jEntomol., j64(4), 839-41. [p. 229] :
Miller, S.S. (1973), "Are You Drinking Biorefractories Too?", EnvdLron.
Sci. & Technol., _7, 14. [p. 27]
Miller, L.L. and Narang, R.S. (1970), "Induced Photolysis of DDT"', Science,
169. 368-370J [p. 45]
Mills, E.J. and Stack, V.T. (1954), "Biological Oxidation of Synthetic
Organic Chemicals", Proc. 8th Ind. Waste Conf., (Purdue Univ. Exten.
Ser.), 8^, 492-517. [p. 55, 465]
Mitchell, -L..C. (19^61), "The Effect of Ultraviolet Light (2537A) oh 141
Pesticide Chemicals by Paper Chromatqgraphy", J. Assoc. Offie. Anal.
Chem., 44, 643. [p. 337, 351, 355] ,
Miyazaki, S.; Boush, G.M. and Matsumura, F. (1969), "Metabolism of ll*C-
chloropropylate by Rhodotorula Gracilis", App. Microbiol., ,18,
972-6. [p. 247, 253, 271, 272]
Miyazaki, S.; Boush, G.M.; and Matsumura, F. (1970), "Microbial Degradation
of Chlorobenzilate (ethyl 4,4-dichlorobenzilate) and Chloropropylate
(isopropyl 4,4-dichlorobenzilate)", J. Agr. Food Chem., 18(1), 87-91.
[p. 194, 235, 245, 250, 252]
Mohanrao, G.J. and McKinney, R.E. (1962), "A Study of the(Biochemical
Characteristics of Quaternary Carbon Compounds", Int. J. AirLWater
Ppllut., 6, 153-168. [p. 465]
Moilanen, K.W-. and Crosby, D.G. (1972),, "Photodecomposition of 3' ^4'-Dichloro-
propionanilide (Propanil)", J. Agr. Food Chem., 20, 950. [p. 358]
Moilanen, K.W. and Crosby, D.G. (1974), "The Photodecomposition.of Bromacil",
Arch. Environ. Contamin. Toxicol., 2^, 3. [? 337, 358]
Montgomery, H.A.C. (1967), "The Determination of BOD by Respirometric
Methods",, Water Res., !_, 631-662. [p. 71, 171]
Montgomery, M.L.; Klein, D.; Goulding, R.; and Freed, V.H. (1972),
"Biological Degradation of Pesticide Wastes", Pestic. Chem...Proc.
Int. Congr. Pestic. Chem., 2nd, ^, 117-25, (Tahori, A.S., edit.)
Gordon and Breach, New York. [p. 226, 244, 245, 267]
526
-------
Hosier, A.R. and Guenzi, W.D. (1973), "Picloram Photolytic Decomposition",
J. Agr. Food Chem., 21, 835. [p. 337]
Hosier, A.R.; Guenzie, W.D.; and Miller, L.I. (1969), "Photochemical
Decomposition of DDT by a Free-Radical Mechanism", Science, 164. 1083.
[p. 337, 352]
Munakata, K. and Kuwahara, M. (1969), "Photochemical Degradation Products
of Pentachlorophenol", Residue Rev., 25_, 13-23. [p. 368]
Muraoka, J.S. (1966), "Effect of Deep Sea Micro-organisms on Rubber and
Plastic Insulation", Materials Protection. 1(4), 35-37. [p. 426, 429,
434, 453, 454]
Muraoka, J.S. (1969), "Effect of Deep-Ocean Environments on Plastics",
in Materials Performance and the Deep Sea, ASTM Spec. Public 445,
Amer. Soc. for Testing Materials, Philadelphia, p. 5. [p. 427, 428, 432,
433, 437, 440, 452, 453, 454]
Murray, D.S.; Rieck, W.L.; and Lynd, J.Q. (1970), "Utilization of Methylthio-
S-Triazine for Growth of Soil Fungi, Appl. Microbiol., 19(1), 11-13.
[p. 103]
Naik, M.N.; Jackson, R.B.; Stokes, J. and Swaby, R.J. (1972), "Microbial
Degradation and Phytotoxicity of Picloram and Other Substituted
Pyridines", Soil Biol. Biochem, 4. (3), 313-323. [p. 229]
Nash, R.G. and Woolson, E.A. (1967), "Persistence of Chlorinated Hydro-
carbon Insecticides in Soils", Science, JL5£(3791), 924-7. [p. 242]
Nelson, J.D.; Blair, W.; Brinckman, F.E.; Colwell, R.R. and Iverson, W.P.
(1973), "Biodegradation of Phenylmercuric Acetate by Mercury-resistant
Bacteria", Appl. Microbiol., 26(3), 321-6. [p. 379, 392, 393, 399]
Nelson, J.F.; McKinney, R.E.; McAteer, J.H. and Konecky, M.S. (1961),
"Biodegradability of Alkylbenzenesulfonates", Develop. Ind. Microbiol.,
2, 93-101. [p. 60, 64, 158]
Neujahr, H.Y. and Varga, J.M. (1970), "Degradation of Phenols by Intact
Cells and Cell-free Preparations of Trichosporon Cultaneum". Eur. J.
Biochem., 13(1), 37-44. [p. 255]
Newsom, H.C. and Woods, W.G. (1973), "Photolysis of the Herbicide Dinitramine
(N3,N3-diethyl-2,4-dinitro-6-trifluoromethyl-m-phenylenediamine)",
J. Agr. Food Chem., 21(4), 598-601. [p. 337, 351]
Nickerson, W.J. (1971), "Decomposition of Naturally Occurring Organic
Polymers" in Organic Compounds in Aquatic Environments, ed. S.D. Faust
and J.V. Hunter, Marcel Dekker, Inc., N.Y. [p. 419, 453]
527
-------
Niemer, H. and .Bucherer, H. (1961), "Uber den Abban von Atropin durch
.Corynebacterium belladonnae, II", Hoppe-Seylers Z. Physiol. Chem. ,
326. 9-12. [p. 38]
Niemer, H.; Bucherer, H. ; and Kohler, H. (1959), "Uber den Abban yon
Atropin durch Corynebacterium belladonnae. I", Hoppe-SeylerslZ.
. Chem., 317, 238-242. [p. 38]
Nisbet, I..C.T. ,and Sarofim, A.F. (1972), "Rates and Routes of Transport of
P.CB's in the Environment", Environ. Health 'Perspectives, Exptl. Issue
No. 1, April, 1972, pp. 21-38. [p. 175]
Nordbl,opm, G.D. and Miller, L.L. (1974), "Photoreduction of 4,V-
Dichlorob^phenyl", J. Agr. Food Chem., 22, 57. [p. 337, 347, 360,
370]
Oba, K.; Yoshida, Y. and Tomiyama, S. (1968), "Studies on Bibdegradation of
Synthetic Detergents. II. Determination of Straight and Branched
.Qhain Alkybenzene Sulfonates in Waters", Ayukagaku, 17, 455-460.
[pi 178]
pgden, C.P.; Webster, H.L. and Halliday, J. (1961), "Determination 'of
Biologically Soft and Hard ABS in Detergents and Sewage", Analyst,
86," 22-29. .[p. 156] ,
!
Ohmori, T. ; Ikai, T. ; Minoda, Y. and Yamada, K. (1973), "Utilization of
Polyphenyl and Polyphenyl-related Compounds by Microorganisms. Part I",
Agr. Biol. Chem. Tokyo, 37/7) , 1599. [p. 232, 235]
Okey, R.W. and Bogan, R.H. (1965), "Apparent Involvement of Electronic
Mechanisms in Limiting Microbial Metabolism of Pesticides", J. Water
Pollut. Contr. Fedr., 37/5), 692^712. [p. 60, 63, 466]
Omori, T. and Yamada, K. (1973), "Utilization of Hydrocarbons by Micro-
organisms. 24. Relation Between Electronic Structure and Hydroxylation
of Aromatic Compounds by Microorganisms", Agr. Biol. Chem., 37(8) ,
1809-11. [p. 463]
Orford, H.E. ; Rand, M.C. and Gellman, I. (1953), "A Single Dilution Technique
for BOD Studies", Sewage Industr. Waste, 25. 283. [p. 56]
Orpin, C.G.; Knight, M. and Evans, W.C. (1972), "The Bacterial Oxidation
of N-methyLisonicotinate, a Photolytic Product of Paraquat", Biochem. J.,
127. 833-844. [p. 234, 237, 238, 252, 255]
Ornston, L.N. and Stanier, R.Y. (1966), "The Conversion of Catechol and'
Protocatechuate to g-oxoadipate by Pseudomonas putida", J. Biological
Chemistry, 241, 3776-3786. [p. 198]
528
-------
Osburn, Q.W. and Benedict, J.H. (1966), "Polyethoxylated Alkyl Phenols:
Relation of Structure to Biodegradation Mechanism", J. Am. Oil Chem.
Soc., 43(3), 141-6. [p. 74, 152, 156]
Owen, E.D. (1971), "Principles of Photochemical Reactions In Aqueous Solution",
In Organic Chemicals in Aquatic Environments. (S.D. Faust and J.D.
Hunter, ed.), M. Dekker, N.Y. [p. 42, 44]
Pady, S.M., Kelly, C.D.; and Polunin, N. (1948), "Arctic Aerobiology, II",
Nature, 162, 379-81.
Paigen, K. and Williams, B. (1970), "Catabolic Repression and Other Control
Mechanisms in Carbohydrate Utilization", in: Adv. Microbial Physiol
(Eds: A.H. Rose and J.F. Wilkinsons), 4_, Academic Press, N.Y. [p. 169]
Painter, H.A. (1973a), "Biodegradability", Proc. Roy. Soc. London, Ser. B.,
185(1079), 149-58. [p. 25, 103, 162, 462, 464, 465]
Painter, H.A. (1973b), "Organic Compounds in Solution in Sewage Effluents",
Chem. Ind. (London), (17), 818-22. [p. 103]
Panel on Hazardous Trace Substances, 1972, "PCS Environmental Impact:
Chpt. 2; Transport and Transformation in the Environment", Environ.
Res., 5_, 273. [p. 370]
Pankhurst, E.S. and Davles, M.J. (1968), "Investigations into the Effect
of Microorganisms on PVC Pressure-Sensitive Adhesive Tape and its
Constitutents", in Biodeterioration of Materials; Microbiological and
Allied Aspects, pp. 302-315, (A.H. Walters and J.J. Elphick, editors),
Elsevier, N.Y. [p. 424, 425, 436, 437, 438, 440, 441, 448, 449]
Pape, B.E. and Zabik, M.J. (1972a), "Photochemistry of Bioactive Compounds.
Solution Phase Photochemistry of a Symmetric Triazln-5(4H)-rones",
J. Agr. Food Chem., 2(), 72. [p. 337, 344]
Pape, B.E. and Zabik, M.J. (1972b), "Photochemistry of Bioactive Compounds:
Solution-phase Photochemistry of Symmetrical Triazines", J. Agr.
Food Chem., 20(2), 316-320. [p. 337, 344]
Pape, B.E.; Papa, M.F.; and Zabik, M.J. (1970), "Photodecomposltion of
Bioactive Compounds: Photodecomposition of 2-(l,3-dioxolane-2-yl)-Penyl-
N-Methyl Carbamate", J. Agr. Food Chem., 18, 490. [p. 338, 344]
Parochettl, J.V. and Hein, E.R. (1973), "Volatility and Photodecomposition
of Trifluralin, Benefin, and Nitralin", Weed Sci., 21(5), 469-73.
[p. 338, 351, 356, 361]
Parr, J.F. and Smith, S. (1973), "Degradation of Trifluralin Under Laboratory
Conditions and Anaerobiosis", Soil Sci., 115(1), 55-63. [p. 228, 244]
529
-------
Parr, J.F.; Willis, G.H.; and Smith, S. (1970), "Soil Anaerobiosis: II.
Effect of Selected Environments and Energy Sources on the Degradation
of DDT", (Soil* Sci., 110(5), 306-312. [p. 227, 228, 256] ' *
Parsons, A.M. »and Moore, D.J. (1966), "Some Reactions of Dieldrin and Proton
Magnetic 'Resonance Spectra of the Products", J. Chem. Soc., Sec. C,
Part II, '2026;. [p. 365] '.'.'. ;V
.'.'':': '('"
Patterson, R.S. (1-962), "Use of Phorate and Di-syston for Potato; ; ;
Insect Control and a Study of .Factors .Which Influence :phorate ',.,,
Absorption by Plants and Loss in the Soil", Dissertation Abstr., 23_,
1471. [p. 266] '_.';:
Patterson, S.J.; Scott, C.C. and Tucker, K.B.E. (1967), ^NonionictDeter-
gent Degradation. I. Thin-Layer Chromatography-and JP^ainlng
Properties of Alcohol Polyethoxylates", J. Amer. Oil Ghem.: Soc.,-44,
407. [p. -89] ' ' " . :-$$?:?&.
Patterson, S.J.; Scott, C.C.; and Tucker, R.B.E. (1968), "Nonionic
Detergent Degradation. II. Thin-Layer Chromatqgraphy and Foaming
Properties of Alkyl Phenol Polyethoxylates", J. -Amer. Oil .Chem. Soc.,
45, 528. [p., 89]
i
Pauli, 0. and Frarike, G. (1971), "Behavior and Degradation of Technical
.Preservatives' in the Biological Purification of Sewage". Bjodeterior.
Mater., Proc. Int. Biodeterior. Symp., .2nd, pp. 52-r60. [p.'^66^ 67]
Pawlowsky, U. and Howell, J.A. (1973a), "Mixed.Culture JBipoxidatiop of
Phenol. II. Steady State Experiments in Continuous (-Culture",
Bioteehnol. Bioeng., _15_(5), 897. .[p. 99]
Pawlowsky, U. and .Howell, J.A. (1973b), "Mixed.Culture Biooxidatibn of
Phenol. 'Ill/ Existence of.Multiple Steady States in Continuous
Culture .With Wall Growth", Bioteehnol. Bioeng., 15(5),: 9.05.
[P. 99]
Payne, W.J. (1963), "Pure Culture Studies of the Degradation of Detergent
Compounds", Bioteehnol. fBioeng., 5^, .355-.6S. [p. 109]
Payne, W.J. and Feisal, V.E. (1963), "Bacterial Utilization of- Dodecyl Sulfate
and Dodeeyl Benzene Sulfonate", Appl. Microbiol., _11, 339-344. [? 99,
109, 114, 159]
Payne, -W.J.; Wiebe, W.J.; and Christian, R.R. (1970), "Assays for [Biodegrad-
ability Essential to Unrestricted Usage of Organic Cqmpqunds", Bio-
Science,, 20(15), 862-5. [p. 103, 462]
Perry, J.J. and Cerniglia, C.E. (1973a), "Studies of the Degradation
of Petroleum by Filamentous Fungi" in, The Microbial Degradation of
Oil Pollutants, (D.G. Ahern and S.P. Meyers, edit.) Louisiana State
Univ. Public, No. LSU-SG-73-01. [p. Ill, 465]
530
-------
Perry, J.J. and Cerniglia, C.E. (1973b), "Crude Oil Degradation by
Filamentous Fungi", J. Gen. Appl. Microbiol., 19, 151-153.
[p. Ill]
Pfaender, F.K. and Alexander, M. (1972), "Extensive Microbial Degradation
of DDT in vitro and DDT Metabolism by Natural Communities", J. Agr.
Food Chem., .20(4), 842-846. [p. 37, 115, 121]
Pfeil, B.H. and Lee, G.F. (1968), "Blodegradation of Nitrilotriacetic Acid
in Aerobic Systems", Environ. Sci. Technol., 21(7), 543-6. [p. 54, 164, 167]
Pickhardt, W.P., Center, A.N.; and Mitchell, T. (1955), "Determination
of Total Carbon in Organic Materials by a Wet-Dry Combustion Method",
Anal. Chem., 27_, 1784. [p. 159]
Pike, E.B. and Curds, C.R. (1971), "The Microbial Ecology of the Activated
Sludge Process", in Microbial Aspects of Pollution, Sykes, G. and
Skinner, F.A. (edit.), Academic Press, N.Y. [p. 129]
Pitter, P. (1964a), "Biodegradability of Sulfated and Nonsulfated Nonionic
Detergents", Prumysl Potravin, 15(12), 649-50.
Pitter, P. (1964b), "Synthetic Surfactants in Wastewaters. VIII. Relation
Between Molecular Structure and the Susceptibility of Anionic
Surfactants to Biochemical Oxidation (Summary of Results)", Sb.
VSCHT, £, 13. [p. 149]
Pitter, P. (1964c), "Alkyl Sulfates Resistant to Biodegradation and
Persistence of n-Alkylbenzene Sulfonates Under Anaerobic Conditions",
Surf. Cong. #4, 3, 861. [p. 149]
Pitter, P. and Tutko, J. (1964), "Synthetic Surfactants in Wastewaters.
VI. Experiments With Hard and Soft Alkylbenzensulfonates in Laboratory
Models of Aeration Tanks", Sb. VSCHT, 8., 167. [p. 131]
Plimmer, J.R. (1970), "Photochemistry of Halogenated Herbicides", Residue
Rev., 33, 47-74. [p. 42, 348, 359, 360, 371]
Plimmer, J.R. (1972a), "Principles of Photodecomposition of Pesticides", in
Degradation of Synthetic Organic Molecules in the Biosphere, Nat.
Acad. Sciences, Washington, D.C. [p. 45]
Plimmer, J.R. (1972b), "Photochemistry of Pesticides. Discussion of the
Influence of Some Environmental Factors", Pestic. Chem.. Proc. Int.
Congr. Peatic. Chem., 2nd. £, 47-76, (Tahori, A.S., edit.) Gordon and
Breach, N.Y. [p. 44, 351, 360, 367]
Plimmer, J.R. and Hummer, B.E. (1969), "Photolysis of Amiben and its
Methyl Ester", J. Agr. Food Chem., 17_, 83-5. [p. 338]
531
-------
Plimmer, J.R. and Klingebiel, U.I. (1971), "Riboflavin Photosensitized
Oxidation of 2,4-Diqhlorophenol: Assessment of Possible Chlorinated
Dioxin Formation", Science, 174. 407. [p. 338]
Plifflmer, J.R..and Klingebiel, U.I. (1974), "Photochemistry of N-sec^Butyl-
4-tert-Butyl-2.6-Dinitroaniline". J. Agr. Food Chem., 22, 689.
Plimmer, J.R.; Klingebiel, U.I.; Crosby, D.G.; ,and Wong, A.S. (1973),
"Photochemistry of Dibenzo-£-dioxins", Advan. Chem. Series, 120, 44,
Amer. Chem. Society, [p. 360] /
Plimmer, J.R.; Klingebiel, U.I.; and Hummer, B.E. (1970), "Phptopxidation
of DDT and DDE", Science, 167. 67. [p. 338, 344, 350]
Plimmer, J.R.; Kearney, P.C., Kaufman, D.D.; and Guardia, F.S. (1967),
"Amitrole Decomposition by Free Radical Generating Systems and by
Soils", J. Agr. Food Chem., 15(6), 996-9. [p. 338, 344, 354]
Popel, F.; Huhken, K.H.; and Steinecke, H. (1958), "Gas-V. .Wass F^ch
(Wassen-Abwasser), 99, 897. [p. 71]
Porter, J.J. (1973), "A Study of the Photodegradation of Commercial Dyes",
Government Printing Office (EPA-R2-73-058), Washington, D.C.'
[p. 338/356]
Potts, J.E.; Clendinning, R.A.; and Ackart, W.B. (1972), "An Investigation
of Biodegradability of Packaging Plastics", EPA-R2-72-046,
Contract # CPE-70-124, U.S. Nat. Tech. Inform. Serv.. PB 2134,88.
[p. 27, 419, 424, 425, 427, 429, 431, 438, 440, 443, 444, 446, 447,
474] '
Powers, G.W.; Martin, R.L.; Piehl, F.J.; and Griffin, J.M. (1959), "Arsenic
in Naphthas", Anal. Chem., 31. 1589. [p. 397, 401]
Pritchard, P.H. and Starr, T.J. (1973), "Mlcrobial Degradation of^Oil and
Hydrocarbons in Continuous Culture", in Microbial Degradation of Oil
Pollutants, (D.G. Ahearn and S.P. Meyers, edts.), Louisiana S,tate
Univ., Publication No, S.G.-73-01. [p. 116, 117,]
Prochazka, G.J. and Payne, W.J. (1965), "Bacterial Growth as a Practical
Indicator of Extensive Biodegradability", Appl. Microbiol., 13(5),
702-5. [p. 2, 26, 107, 109, 159, 462]
Procter &, Gamble Co., personal communication, cited in Hitzman, 1964.
[P- 77]
Rabson, R. and Plimmer, J.R. (1973), "Photoalteration of Pesticides: Summary
of Workshop", Science, 180. 1204. [p. 357, 374]
Raiziss, G.W.- and Garron, J.L. (1923), "Organic Arsenical Compounds",
The Chemical Catalog Co., N.Y. [p. 397]
532
-------
Rawls, C.R. (1966), "Providing Estuarine Fauna for Residue Analysis of
Field-Applied Herbicides", South Weed Conf. Proc., 19th Annu.
Meeting. 397. [p. 35]
Read, D.C. (1969), "Persistence of Some Newer Insecticides in Mineral
Soils Measured by Bioassay", J. Econ. Entomol., 62(6), 1338-42.
[p. 241, 258]
Redemann, C.T. and Youngson, C.R. (1968), "The Partial Photolysis of
6-Chloropicolinic Acid in Aqueous Solution", Bull. Environ. Contam.
Toxicol., _3» 97. [p. 338, 356, 358]
Reisfeld, A.; Rosenberg, E.; and Gutnick, D. (1972), "Microbial
Degradation of Crude Oil: Factors Affecting the Dispersion in
Sea Water by Mixed and Pure Cultures", Appl. Microbiol., .24(3), 363-368.
[p. 112, 161]
Renn, C.E. (1964), "Techniques of Determining Biodegradability of Detergent
Bases", Detergent Age, 1(2), 24-28. [p. 131, 178]
Renn, C.E. (1965), "Degradation of Ucane LAS Alkylate in Laboratory Trickling
Filters", Annual Convention, Soap and Detergent Assoc., New York.
[P. 145]
Renn, C.E.; Kline, W.A.; and Orgel, G. (1964), "Destruction of Linear Alkylate
Sulfonates in Biological Waste Treatment by Field Test", J. Water
Pollut. Contr. Fedr., ^6, 864-79.
Reynolds, J.F. (1969), "Comparison Studies of Winkler vs. Oxygen Sensor",
J. Water Pollut. Contr. Federation, 41, 2002-2009. [p. 69]
Ripin, M.J.; Noon, K.F.; and Cook, T.M. (1970), "Kinetic Studies of the
Oxidative Degradation of Detergent and Related Species in Aqeuous
Solution", Univ. of Maryland Water Resources Research Cent., Completion
Report, U.S. Nat. Tech. Inform. Serv., PB 199365, 69 pp. [p. 109]
Ripperton, L.A. and Lillian, D. (1971), "The Effect of Water Vapor on Ozone
Synthesis in the Photooxidation of Alpha-pinene", J. Air Pollut.
Contr. Assoc., 21, 629. [p. 315]
Risebrough, R.W. (1969), "Chlorinated Hydrocarbons in Marine Ecosystems" in,
Chemical Fallout; Current Research on Persistent Pesticides, Miller,
M.W. and Berg, G.G (ed.), Thomas Publishing, Springfield, 111.
[p. 283]
Risebrough, R.W.; Huggett, R.J.; Griffin, J.J.; and Goldberg, E.D. (1968),
"Pesticides: Transatlantic Movements in the Northeast Trades",
Science, 159. 1233. [p. 283, 284]
Robeck, G.G.; Cohen, J.M.; Sayers, W.T.; and Woodward, R.L. (1963),
"Degradation of ABS and Other Organics in Unsaturated Soils", J. Water
Pollut. Contr. Fedr., 35, 1225-36. [p. 154, 239, 240] ;
533
-------
Roberts, F.W. and Lawson, G.R. (1958), "Some Determination of the Synthetic
Detergent Content of Activated Sludge", Water Waste Treat, jl, T_: 14-17.
[P. 152]
Robinson, J.; Richardson, A.; Bush, B; and Elgar, K.E. (1966), "A.Phbto-
isomerization Product of Dieldrin", Bull. Environ. Contam. Toxicol.,
if 127. [p. 365, 366]
Roburn, J. (1963), "Effect of Sunlight and Ultraviolet Radiation on
Chlorinated Pesticide Residues", Chem. Ind., 3£, 1555. [p. 365]
Rodriguez, F. (1971a), "the Prospects of Biodegradable Plastics", Chem.
Tech., it 409-15. [p. 420]
Rodriguez, F. (1971b), "Prospects for Biodegradable Plastics", Modern
Plastics, 48, 92-94. [p. 420]
Rogers, M.R. and Kaplan, A.M. (1970), "Identification and Surfactant Degrading
Ability of Bacteria - "Standard" Shake Flask Inoculum", J. Water
Pollut. Contr. Fed., ^2(8 part 2), R263-R269. [p. 109]
Rogers, M.R. and Kaplan, A.M. (1971), "Effects of Penicilium janthinellum
on Parachute Nylon Is There Microbial Deterioration?", Int.
Biodeterior. Bull., T_, 1524. [p. 422, 423, 432, 438, 451]
Rose, A.H. Jr., and Brandt, G;S. (1960), "Environmental Irradiation Test
Facility", J. Air. Poll. Contr. Assoc., 10, 331-5. [p. 292, 296, 297,
298, 299, 313, 319]
Rosen, J.D. (1967), "The Photolysis of Diphenamid", Bull. Envir. Contam.
Toxicol., 2^, :349. [p. 338, 358] ,
Rosen, J.D. (1971), "Photodecomposition of Organic Pesticides", in 6r%. Com-
pound Aquatic Environ., Rudolfs Res. Conf., 5th, 425-38. Edited by
Faust, Samuel, J., Dekker, New York. [p. 367]
Rosen, J.D. (1972a), "Coversion of Pesticides Under Environmental Conditions",
Environ. Qual. Saf., _!, 85-86. [p. 45, 46, 371]
Rosen, J.D. (1972b), "Photochemistry of Several Pesticides", Environ!.
Toxicol. Pestic.. Proc. U.S.-Jap..435-47. [p. 356, 360]
Rosen, J.D. and Gary, W.F. (1968), "Preparation of the Photoisomers of
Aldrin and Dieldrin", J. Agr. Food Chem., 16, 536. [p. 366, 367]
Rosen, J.D. and Siewierski, M. (1970), "Sensitized Photolysis of Heptachlor",
J. Agr. Food Chem., 18, 943. [p. 349, 350]
Rosen, J.D. and Siewierski, M. (1971), "Photolysis of 4-Amino-3-Methylthio-
6-Phenyl-l,2,4-Trlazin-5-one", Bull. Environ. Contam. Toxicol., £, 406.
[p. 338, 344]
534
-------
Rosen, J.D. and Siewierski, M. (1972), "Photolysis of 5-Amino-4-Chloro-2-
Phenyl-3(2H)-Pyridazinone", J. Agr. Food Chem., 20, 434. [p. 338,
344]
Rosen, J.D. and Strusz, R.F. (1968), "Photolysis of 3-(p-Bromophenyl)-l-
Methoxy-1-Methylurea", J. Agr. Food Chem., 16, 568. [p. 338]
Rosen, J.D.; Sutherland, D.J.; and Lipton, G.R. (1966), "The Photochemical
Isomerization of Dieldrin and Endrin and Effects on Toxicity", Bull.
Environ. Contain. Toxicol., 1^, 133. [p. 365, 366]
Rosen, J.D.; Strusz, R.F.; and Still, C.C. (1969), "Photolysis of Phenylurea
Herbicides", J. Agr. Food Chem., 17_, 206. [p. 338]
Rosen, J.D.; Siewierskl, M.; and Winnet, G. (1970), "FMN - Sensitized
Photolysis of Chloroanilines", J. Agr. Food Chem., 18, 494.
[p. 388, 349]
Ross, R.D. and Crosby, D.G. (1973), "Photolysis of Ethylenethioures", J.
Agr. Food Chem., 21, 335. [p. 338, 361]
Ross, W.D.; Sievers, R.E.; and Wheeler, G. (1965), "Quantitative Ultratrace
Analysis of Mixtures of Metal Chelates by Gas Chromatography", Anal.
Chem., 37., 598. [p. 398]
Ruschenberg, E. (1963), "The Biological Behavior of Surfactants. I. The
Relation Between the Constitution and the Biological Degradation of
Alkylbenzenesulfonates", Fette, Seifen, Anstrichmittel, 65, 810-14.
Ruzo, L.O.; Zabik, M.J.; and Schuetz, R.D. (1972), "Polychlorinated
Biphenyls: Photolysis of 3,4,3',4'-Tetrachlorobiphenyl and 4,4'-
Dichlorobiphenyl in Solution", Bull. Environ. Contain. Toxicol.,
£, 217. [p. 369]
Ruzo, L.O.; Zabik, M.J.; and Schuetz, R.D. (1973), "Photochemistry of
Bioactive Compounds. Kinetics of Selected S_-Triazines in Solution",
J. Agr. Food Chem., 21, 1047. [p. 338]
Ruzo, L.O.; Zabik, M.J.; and Schuetz, R.D. (1974), "Photochemistry of
Bioactive Compounds: Photoproducts and Kinetics of Polychlorinated
Biphenyls", J. Agr. Food Chem., 22, 199. [p. 338, 370]
Ryckman, D.W. (1956), "The Significance of Chemical Structure in Biodegradation
of Alkyl Benzene Sulfonates", Sc. D. Thesis, MIT. [p. 56, 178]
Ryckman, D.W. and Sawyer, C.N. (1957), "Chemical Structure and Biological
Oxidizability of Surfactants", Purdue Univ., Eng. Bull., Ext. S«r.,
No. 94.; Proc. Ind. Waste, Conf., 12th, 270-84. [p.. 54, 56, 60, 178]
Ryckman, D.W.; Prabnakara Rao, A.V.S.; and Buzzell, J.C., Jr. (1966),
"Behavior of Organic Chemicals in the Aquatic Environment: A
Literature Critique", Manufacturing Chemists Association, Washington,
D.C. [p. 464, 465]
535
-------
Sachs, R.M. ; Michael, J.T.; Anastasio, F.B.; and Wells, W.A. (1971), "Determina-
tion of Arsenical Herbicide Residue in Plant Tissues", Weed Sci., 19, 412.
[p. 397, 401] s '
t
Safe, S. and Hutzinger, 0. (1971), "Polychlorinated Biphenyls: Photolysis of
", Nature 232, 641. [p. 338, 368]
Saltzman, B.E.; Coleman, A. I.; and demons, C.A. (1966), "Halogehated Compounds as
Gaseous Meteorological Tracers", Anal. Chem, 38, 753. [p. 289, 308, 327]
Sanborn, J.R. ; and Yu, C.-C. (1973), "Fate of Dieldrin in a Model Ecosystem",
Bull. Environ.. Contain. Toxicol. 10(6), 340-6. [p. 120]
Sandell, E.B. (1959), Colometric Determination of Metals, Interscience
Publishers, Inc., New York. [p. 401]
Sanders, W.M. ; and Falco, J.W. (1973), "Ecosystem Simulation for Water Pollution Re-
search", Advances in Water Pollution Research, 6th Inter. Conf.. 1972, S.H.
Jenkins (Ed.) ,", Pergamon Press, [p. 124, 125]
Sawyer, C.N.; Bogan, R.H. ; and Simpson, J.R. (1956), "Biological Behavior of
Synthetic Detergents", Ind. Eng. Chem. 48, 236-40. [p. 54, 59, ISO,
63, 74] '
SCEP (1970), Man's Impact on the Global Environment, MIT Press, Boston.
[P- 41] ','
Schbhborn, W. (1962a) , "Investigations of the Conditions Permitting Degradation
of Detergents in Conventional Sewage Treatment Plants", Gas-Wasserfach,
103. 1133. [p. 145] ,
Schonborn, W. (1962b) , "Methods for Testing Biodegradability of Detergents",
Seiferi-Ole-Fette-Wachse, 88, 870-875.
Schulz', K.R. ; and Lichteiistein, E.P. (1971), "Persistence and Movement of
Dyforiate in Field Soils", J. Econ. Entomb 1. 64(1), 283-7. [p. 242,
243, 244, 272]
Schultz, K.R. ; Llchtenstein, E.P.; Liang, T.T.; and Fuhremaim, T.W. (1970),
"PersistencB and Degradation of Azinphosmethyl in Soils as Affected by
Formulation and Mode of Application", J. Econ. Entomol. j63_(2) , 432-8.
[p. 245, 247, 252]
Schulze, K.L. and Hoogerhyde, T.C. (1967), "Bio-Degradability as Measured
by Differential Manometer," Develop. Ind. Microbiol., £, 284-297.
[P- 64, 65]
Schuth, C.K.; Isensee, A.R. ; Woolson, E.A. ; and Kearney, P.C. (1974), "Dis-
tribution of ll*C and Arsenic Derived from [^C] Cacodylic Acid in an
Aquatic Ecosystem", J. Agr. Food Chem. (in press), [p. 395, 413]
536
-------
Schwartz, H.6., Jr. (1967), "Microbial Degradation of Pesticides in Aqueous
Solutions", J. Water Pollut. Contr. Fed. 39(10 Pt. 1), 1701-1714,
[p. 96, 185]
Scott, G. (1970), "Vanishing Plastics?" New Sci. 47_(713) , 293. [p. 419, 453,
478]
SDA (1965)(Soap and Detergent Association), "A Procedure and Standards for the
Determination of the Biodegradability of Alkyl Benzene Sulfonate and
Linear Alkylate Sulfonate", J. Am. Oil Chem. Soc. 4£, 986-993. [p. 2, 6,
26, 85, 142, 143, 156, 184, 199, 206, 208, 213]
SDA (1969)(Soap and Detergents Association Biodegradation Subcommittee) "The
Status of Biodegradability Testing of Nonionic Surfactants", J. Amer.
Oil Chem. Soc. 46, 432. [p. 144, 154, 160, 209]
Searle, N.Z. (1971), "Analytical Methods for the Study of the Photodegradation
of Polymers", Anal. Phdtochem. Photochem. Anal.; Solids, Solutions, Polym.,
249-36. [p. 479]
Sethunathan, N. (1972), "Diazinon Degradation in Submerzed Soil and Rice-
Paddy Water", in Fate of Organic Pesticides in the Aquatic Environment,
Advance in Chemistry Series 111 266.
Sethunathan, N. (1973), "Microbial Degradation of Insecticides in Flooded
Soil and in Anaerobic Cultures", Residue Rev. 47, 143-65.
Sethunathan, N. and MacRae, I.C. (1969), "Persistence and Biodegradation
of Diazinon in Submerged Soils", J. Agr. Food Chem. 17_(2), 221-225.
[p. 227, 233, 237, 268]
Sethunathan, N. and Yoshida, T. (1969), "Fate of Diazinon in Submerged Soil.
Accumulation of Hydrolysis Product", J. Agr. Food Chem. 17.(6), 1192-5.
[p. 228, 244, 247, 252}
Sethunathan, N.; Bautista, E.M.; and Yoshida, T. (1969), "Degradation of
Benzene Hexachloride by a Soil Bacterium", Can. J. Microbiol 15(2),
1349-1354. [p. 247]
Setzkorn, E.A.; and Carel, A.B. (1963), "The Analysis of Alkyl Aryl Sulfonates
by Mlcrodesulfonation and Gas Chromatography", J. Amer. Oil Chem Assoc.
40, 57-59. [p. 153]
Setzkorn, E.A. and Huddleston, R.L. (1965), "Ultraviolet Spectroscppic
Analysis for Following the Biodegradation of Hydrotropes", J. Amer. Oil
Chem. Soc. .42(12), 1081-1084. [p. 85, 156, 157]
Setzkorn, E.A.; Huddleston, R.L.; and Allred, R.C. (1964), "An Evaluation of
the River Die-Away Technique for Studying Detergent Biodegradability",
J. Am. Oil Chem. Soc. 41, 826-30. [p. 74]
537
-------
Sharman, S.H. (1964), "Extensive Biodegradation of Synthetic Detergents",
Nature 201(4920), 704-5.
Sharp, R.F. and Eggins, H.O.W. (1970), "Biodeterioration Appraisal of Silicones",
Int. Biodeterior. Bulled), 19-26. [p. 449, 451, 452]
i
Sharp, R.F.; and Taylor, B.P. (1968), "A New Soil Percolator for the Elective
Culture of Soil Organisms", Soil Bio. Biochem. 1^, 191-194. ;
Sharpe, A.N.; and Woodrow, M.N. (1971), "A Rapid Test for Biodegradability
of PVC [Polyvinyl-Chloride] Film by Pseudomonads", J. Appl. Bacteriol.
34(2), 485-489. [p. 424, 438, 440, 441, 449, 452] ;
Sheers, E.H.; Wehner, D.C.; and Sauer, G.F. (1967), "Biodegradation of a Sulfonated
Amide Surfactant", J. Water Pollut. Contr. Fed. 39_(8), 1410-16. [p. 78]
Sheets, T.J.; Smith, J.W.; and Kaufman, D.D. (1968), "Persistence of Benzoic
and Phenylacetic Acids in Soils", Weed Sci. 16(2), 217-222. [p. 241, 257]
Shortridge, R. and Heicklen, J. (1973), "The Photooxidation of 2-Bromoethanol",
J. Air Pollut. Contr. Fedr. 23, 714.
Sierp, F. and Thiele, H. (1954), "Influence of Surfactants on Sewage Purifica-
tion and on the Self-Purification of Rivers", Vom Wasser 21, 197. [p. 141]
l
Sikka, B.C. and Rice, C. (1973), "Persistence of Endothall in Aquatic Environ-
ment as Determined by Gas-Liquid Chromatography", J. Agr. Food them.
21, 842. [p. 37, 121, 176]
Sikka, H.C. and Saxena, J. (1973), "Metabolism of Endothall by Aquatic Micro-
organisms", J. Agr. Food Chem. 21, 402. [p, 37, 109, 169, 177,
194, 195]
Sikka, H.C.; Ford, D.; and Lynch, R.S. (1974), "Uptake, Distribution and
Metabolism of Endothall in Fish", (in press), [p. 37]
Skipper, H.D. and Volk, V.V. (1972), "Biological and Chemical Degradation
of Atrazine in Three Oregon Soils", Weed Sci. .20(4), 344-347.
Slade, P. and Smith, A.E. (1967), "Photochemical Degradation of Diqu'at",
Nature 213. 919. [p. 338, 351]
Smith, A.E. and Grove, J. (1969), "Photochemical Degradation of Diquat in
Dilute Aqueous Solution and on Silica Gel", J. Agr. Food Chem. 17(3),
609-613. [p. 338, 351]
Smith, G.E. and: Isom, B.E. (1967), "Investigation of Effects of Large-Scale
Applications of 2,4-D on Aquatic Fauna and Water Quality", Pest. Monitor-
ing J. .1(3), 16. [p. 35]
538
-------
Snaddon, X.V.M. and Jenkins, S.H. (1964), "Biological Oxidation in a Self-
Operating Respirometer", Advans. in Water Pollution Research 2, 389.
[p. 71]
Snell, F.D. and Snell, C.T. (1949), Colorimetric Method of Analysis. Vol. II,
3rd Ed., Van Nostrams Company, New York. [p. 401]
Snoke, L.R. (1957), "Resistance of Organic Materials and Cable Structures to
Marine Biological Attack", Bell System Tech. J. 3_6, 1095-1127.
[p. 427, 428, 434, 438, 440]
Soli, G. (1973), "Hydrocarbon-Oxidizing Bacteria and Their Possible Use As
Controlling Agents of Oil Pollution in the Ocean", U.S. Nat. Tech.
Inform. Serv.. AD Rep. No. 763655, 14 pp. [p. 106]
Spangler, W.J.; Spigarelli, J.L.; Rose, J.M.; and Miller, H.M. (1973a),
"Methylmercury: Bacterial Degradation in Lake Sediments", Science
180(4082), 192-193. [p. 383, 392, 397, 398, 473]
Spangler, W.J.; Spigarelli, J.L.; Rose, J.M.; Flippin, R.S.; and Miller,
H.H. (1973b), "Degradation of Methylmercury by Bacteria Isolated from
Environmental Samples", Appl. Microbiol., 25(4), 488-493. [p. 383,
392, 397, 398, 414, 473]
Stahl, W.H. and Pessen, H. (1953), "Microbiological Degradation of
Plasticizers. I. Growth on Esters and Alcohols", Appl. Microbiol.,
1, 30-5. [p. 113]
Stanier, R.Y. (1947), "Simultaneous Adaption: A New Technique for the
Study of Metabolic Pathways", J. Bacteriol., 54, 339-348. [p. 195,
196]
Stanley, C.W.; Barney, J.E.; Helton, M.R.; and Yobs, A.R. (1971), "Movement
of the Atmospheric Levels of Pesticides", Environ. Sci. Techno1.,
5_, 430. [p. 283]
Steinle, E.C.; Myerly, R.C.; and Vath, C.A. (1964), "Surfactants
Containing Ethylene Oxide: Relationship of Structure to Biodegradabllity",
J. Am. Oil Chem. Soc., 41, 804-807.
Stennett, G.V. and Eden, G.E. (1971), "Assessment of Biodegradabllity of
Synthetic Surfactants by Tests Simulating Sewage Treatment", Water
Res., _5(8), 601-609. [p. 127, 131, 135, 136, 206, 212, 214]
Stephens, E.R. (1958), "Long-Path Infrared Spectroscopy for Air Pollution
Research", Appl. Spectroscopy, 12. 80-84. [p. 285, 306, 307]
Stephens, E.R. (1973), "Hydrocarbons in Polluted Air", Project CAPA-5-68,
Statewide Air Pollution Research Center, U. of Calif., Riverside,
U.S. Nat. Tech. Inform. Serv.. PB-230-993. [p. 293, 320, 326, 327,
471, 483]
539
-------
Stephens, E.R. and Burleson, P.P. (1967), "Analysis of the Atmosphere
for Light Hydrocarbons", J. Air. Poll. Contr. Assoc., 17(33), 147-53.
[p. 293, 308, 325]
Stewart, D.K.R. and Chisholm, D. (1971), "Long-term Persistence of BHC,
DDT, and Chlordane in Sand Loam Soil", Can. J. Soil Sci., 51, 379-383.
[p. 242, 244]
'Stewart, D.K.R.; Chisholm, D.; and Ragab, M.T.H. (1971), "Long-term
Persistence of Parathion in Soil", Nature, 229, 47. [p. 242]
Stiff, M.J. and Rootham, R.C. (1973), "Effect of Temperature on the
Removal of Nonionic Surfactants During Small-Scale Activated Sludge
Sewage Treatment. II. Comparison of a Linear Alkylphenol Ethoxylate
With Branched Chain Alkylphenol Ethoxylates", Water Res., 1(10),
1407-15. [p. 136, 202, 314]
Stiff, M.J.; Roothan, R.C.; and Culley, G.E. (1973), "The Effect of
Temperature on the Removal of Non-Ionic Surfactant's^ During Small-Scale
Activated Sludge Sewage Treatment. I. Comparison of Alcohol Ethoxylates
With A Branched-Chain Alkylphenol Ethoxylate", Water Research, 7_,
1003-1010. [p. 136, 202, 205]
Stockii, A. (1950), "Die Eruahrung der Pflanze und ihre Abhangigkeit von
Kleinlebewelt des Bodens", Z. Pflanzernahr Dung Bodenkde., 48, 264.
[p. 41]
Straus, A.E. (1963), "Biodegradation of Alkylbenzene Sulfonate in a Simulated
Septic Tank and Drain Field", Science, 142(3589), 244-245. [p. 148, 149,
154]
Strzelczyk, E. ; Donderski, W. and Lewosz, W. (1972), "Utilization of Aromatic
Compounds by Benthic Microorganisms of an Eutrophic Lake", Acta
Microbiol. Pol., Ser. B., 4^(4), 191-200. [p. 106]
Sturm, R.N. (1973), "Biodegradability of Nonionic Surfactants. Screening
Test for Predicting Rate and Ultimate Biodegradation", J. Amer. Oil
Chem. Soc., .50(5), 159-67. [p. 54, 92, 93, 157]
Su, C-W. and Goldberg, E.D. (1973), "Chlorofluorocarbons in the Atmosphere",
Nature, 245. 27. [p. 328]
Su, G.C.C. and Zabik, M.J. (1972a), "Photochemistry of Bioactive Compounds:
Photolysis of m-(N,N-dimethylmethylformamidine) phenyl N-methyl-
carbamate Hydrochloride in Water", J. Agr. Food Chem., 2(3(3), 642-644.
[p. 338]
Su, G.C.C. and Zabik, M.J. (1972b), "Photochemistry of Bioactive Compounds:
Photolysis of Arylamidine Derivatives in Water", J. Agric. Food Chem.,
20(2), 320-323. [p. 338]
540
-------
Summer, W. (1964), "Microbial Degradation of Plastics", Corros. Techno1.,
11, 19-21.
Swanson, C.L.; Throp, F.C.; and Friend, R.B. (1954), "Adsorption of
Llndane by Soils", Soil Sci., 78_, 379. [p. 265]
Sweeney, W.A. and Foote, J.K. (1964), "A Rapid Accurate Test for Surfactant
Aerobic Biodegradability", J. Water Pollut. Contr. Fedr.,
3j6» 14-37. [p. 131, 136, 137, 138, 139, 154, 172, 213, 221]
Swisher, R. D. (1963), "Biodegradation Rates of Isomeric Deheptylbenzene
Sulfonates", Dev. Ind. Microbiol., .4, 39-45. [p. 153]
Swisher, R.D.; O'Rourke, J.T.; and Tomlinson, H.D. (1964), "Fish
Bioassays of LAS and Intermediate Biodegradation Products", J. Amer.
Oil Chem. Soc., 4.L, 746-752. [p. 131, 136, 138, 139]
Swisher, R.D. (1966), "Shake Culture Biodegradation of LAS Without
Inoculation", Develop. Ind. Microbiol., ]_, 271-278. [p. 100, 101,
164, 185]
Swisher, R.D. (1967a), "Biodegradation of Surfactant Benzene Rings",
Ind. Chim. Beige, 12, 719-22. [p. 74, 114, 131, 136, 138, 139, 213,
221]
Swisher, R.D. (1967b), "Biodegradation of LAS Benzene Rings in Activated
Sludge", J. Amer. Oil Chem. Soc., 44(12), 717-724. [p. 114, 131,
136, 138, 139, 144, 157, 213, 221]
Swisher, R.D. (1968), "Biodegradation of Surfactant Benzene Rings in
Shake Cultures", Develop. Ind. Microbiol., £, AIBS, Washington, [p. 101]
Swisher, R.D. (1969), "Benzene Rings Biodegradation in Quaternary Alkyl-
benzenesulfonates", Tenside, £(3), 135-9. [p. 144]
Swisher, R.D. (1970), Surfactant Biodegradation. Marcell Dekker, Inc., N.Y.
[p. 25, 27, 51, 76, 88, 128, 129, 130, 134, 139, 140, 142, 144,
148, 149, 150, 156, 163, 173, 178, 199, 201, 204, 205, 206, 211,
212, 213, 214, 276, 462, 464, 483]
Swisher, R.D. and Gledhill, W.E. (1973), "Biodegradation of o-Benzyl-£-chloro-
phenol", Appl. Microbiol., 26(3), 394. [p. 74]
Swisher, R.D.; Taulli, T.A.; and Malec, E.J. (1973), "Biodegradation of
NTA Metal Chelates in River Water", Trace Metals and Metal-Organic
Interactions in Natural Waters, Ann Arbor Science Publishers.
[p. 385, 392]
Swisher, R.D.; O'Rourke, J.T.; and Tomlinson, H.D. (1964), "Fish Bioassays
of Linear Alkylated Sulfonates (LAS) and Intermediate Biodegradation
Products", J. Am. Oil Chem. Soc., 41(11), 746-52.
541
-------
Swisher, R.D.; Crutchfield, M.M.; and Caldwell, D.W. (1967), "Biodegradation
of Nitrilotriacetate in Activated Sludge [Bacterial]", Environ. Sci.
Technol., _1(10), 820-827. [p. 144] :
Tanabe, M.; Dehn, R.L.; and Bramhall, R.R. (1974), "The Photochemistry of
Imidan in Diethyl Ether," J. Agr. Food Chem., 22_, 54. [p. 338, 347]
Tatton, J.O.G. and Wagstaffe, P.H. (1969), "Identification of and
Determination of Organomercurial Fungicide Residues in Thin-Layer
and Gas Chromatography", J. Chromatography, 44, 284. [p. 402]
Thimann, K.V. (1966), The Life of Bacteria. The MacMillan Co., N.Y.
[p. 38, 39]
Thorn, N.S. (1971), "Nitrilotriacetic Acid: A Literature Survey", Water
Res., ,5, 391. [p. 385]
Thomas, T.H. and Kendrick, T.C. (1969), "Gel-permeation Chromatography of
Poly (dimethylsiloxane)", J. Polymer Sci., Part A-2, 7.(2) , 297-307.
[p. 442]
Thompson, J.E. and Duthie, J.R. (1968), "The Biodegradability and Treatability
of NTA", J. Water Pollut. Contr. Fed., 40(2), 306-319. [p. 54, 97,
157]
Thomson, W.T. (1967), Agricultural Chemicals Book 1, Thomson Publications
Davis, California, [p. 41]
Tiedje, J.M.; Duxbury, J.M.; Alexander, M.; and Dawson, J.E (1969), "2,4-D-
metabolism: Pathway of Degradation of Chlorocatechols by Arthrobacter sp.
J. Ag. Food Chem., 17, 1021-1026. [p. 250]
Tiedje, J.M.; Mason, B.B.; Warren, C.B.; and Malec, E.J. (1973), "Metabolism
of Nitriloacetate by Cells of Pseudomonas Species", Applied Microbiol.,
25_(5), 811-818. [p. 195, 232, 236, 237, 254, 255]
Titus, J.B. (1973), Environmentally Degradable Plastics; A Review, Plastics
Technical Evaluation Center (Pub.) Plastic Note N24, U.S. Nat. Tech.
Inform. Serv.. AD Rep. No. 760-718. [p. 419, 427, 428, 454, 45,6,
475, 478, 479]
Tomiyama, S.; Takao, M.; Mor, A.; Sekiguchi, H.; and Oba, K. (1968),
"Studies of Alpha-olefin Sulfonate. III. Biochemical Behavior of
Alpha-olefin Sulfonate", Annual Meeting, Amer. Oil Chem. Soc.,
New York, October. [p. 152]
Tomiyama, S.; Takao, M.; Mori, A.; and Sekiguchi, H. (1969), "New.
Household Detergent Based on AOS", J. Amer. Oil Chem. Assoc., 4.6,
208-212. [p. 178] ^~
542
-------
Tonomura, K.T.; Nakayami, T.; Fuai, F.; and Maeda, K. (1968), J. Ferment.
Technol., ^6, 506. {p. 393]
Tonomura, K.; Furukawa, K.; and Yamada, M. (1972), "Microbial Conversion
of Mercury Compounds", Environ. Toxicol. Pestic.. Proc. U.S.-Jap.t
115-33. [p. 389, 406, 408, 410]
Torgeson, D.C. (1971), "Interaction of Herbicides and Soil Microorganisms",
Water Pollution Control Research Series 16060 BMP 03/71, EPA, Government
Printing Office, Washington, D.C. [p. 463, 466]
Truesdale, G.A.; Jones, K.; and Vankyke, K.G. (1959), "Removal of Synthetic
Detergents in Sewage Treatment Processes: Trials of a New Biological
Attackable Material", Water Waste Treat. J., 7., 441-444. [p. 131,
144, 145]
Truesdale, G.A.; Stennett, G.V. and Eden, G.E. (1969), "Assessment of
Biodegradability of Synthetic Detergents: A Comparison of Methods",
Surf. Cong. #5, JL, 91. [p. 88, 89, 144, 146, 206, 207, 210, 212]
Tuesday, C.S. (1961), "The Atmospheric Photooxidation of Trans-Butene-2 and
Nitric Oxide", in Chemical Reactions in the Lower and Upper Atmosphere,
R.D. Cadle (ed.), J. Wiley and Sons, N.Y. [p. 286, 306]
Tuesday, C.S. (1963), "Atmospheric Photooxidation of Olefins", Arch. Environ.
Health, .7, 188-201. [p. 286]
Turner, A.W. (1954), "Bacterial Oxidation of Arsenite: I. Description of
Bacteria Isolated from Arsenical Cattle-dipping Fluids", Aust. J.
Biol. Sci., ]_, 452. [p. 381]
Turner, A.W. and Legge, J.W. (1954), "Bacterial Oxidation of Arsenite. II.
The Activity of Washed Suspensions", Aust. J. Biol. Sci., ]_, 479.
[p. 381]
Turro, N.J. (1967), Molecular Photochemistry, W.A. Benjamin, Inc., New York.
[p. 361]
Ueda, K.; Gaughan, L.C.; and Casida, J.E. (1974), "Photodecomposition of
Resmethrln and Related Pyrethroids", J. Agr. Food Chem., 22, 212.
[p. 338, 351, 356]
Urban, P.J.; Stander, G.J.; Osborn, D.W.; Theron, P.F.; and Walker, S.M.
(1965), "Experiments to Establish the Degradability of a New
Biologically Soft Detergent", CS1R Research Report, 231, South
African Council for Scientific and Industrial Research, Pretoria.
[p. 131, 177]
Urone, P.; Lutsep, H.; Noyes, C.M.; and Parcher, J.F. (1968), "Static
Studies of Sulfur Dioxide Reactions in Air", Environ. Sci. Technol.,
2., 611. [p. 295]
543
-------
U.S..Tariff Commission, "Synthetic Organic Chemicals: U.S. Production
and Sales", annual. [p. 26, 27]
Vaicum, L. and Ilisescu, A. (1967), "Biodegradability of Detergents and
Determining Methodology", Rev. Chem. (Bucharest), 18, 6. [p. -141]
Van Alfen, N.K. and Kosuge, T. (1974), "Microbial Metabolism of the
Fungicide 2,6-dichloro-o-4-nitroaniline", J. Agr. Food Chem., 22/2),
221-4. [p. 104]
Van Der Linden, A.C. and Thijsse, G.J.E. (1965), "The Mechanisms of
Microbial Oxidations of Petroleum Hydrocarbons", Adv. Enzymol.,, 27,
469. [p. 465]
Van Hall, C.E. and Stenger, V.A. (1964), "Use of Infrared Analyzer!for
Total Carbon Determination", Water Sewage Works, 3_, 266-270.
[p. 159]
Van Middelem, C.H. (1966), "Fate and Persistence of Organic Pesticides
in the Environment", Advan. Chem. Ser., No. 60, 228-49. [p. 33,]
Vath, C.A. (1964), "A Sanitary Engineer's Approach to Biodegradation of
Nonionics", Soap Chem. Specialties, 40(2), 56-58. [p. 59, 149, 154]
Veen, H. (1966), "Transport, Immobilization, and Localization of
l-Naphthaleneacetic-l-ltfC Acid in Coleus Explants", Acta. Bot.
Neer, 15_, 419. [p. 36]
Veith, G.D. (1972), "Fluctuations of Chlorobiphenyls (PCB's) in the
Green Bay, Wisconsin Region", Environ. Health Perspectives, Exptl.
issue, No. 1, 51-54. [p. 175]
Von Endt, D.W.; Kearney, P.C.; and Kaufman, D.D. (1968), "Degradation
of Monosodium Methane Arsonic Acid by Soil Microorganisms",
J. Agr. Food Chem., 16(1), 17. [p. 379, 393, 394, 399, 402, 411,
414]
Vonk, J.W. and Sijpesteijn, A.K. (1973), "Studies on the Methylation
of Mercuric Chloride by Pure Cultures of Bacteria and Fungi",
Anton Leeuwenhock J. Microbiol., 39/3), 505-513. [p. 390, 398,
406, 410, 413]
Walchi, 0. (1968), "Biodegradation Test Methodology", in Biodeterior.ation
of Materials; Microbiological and Allied Aspects, (A.H. Walters and
J.J. Elphick, editors), Elsevier, N.Y., p. 242. [p. 421, 425, ,426,
440, 449, 451]
Walker, J.D. and Colwell, R.R. (1973), "Microbial Ecology of Petroleum
Utilization in Chesapeake Bay", in Microbial Peg, of Oil Pollutants,
D.G. Ahearn and S.P. Meyers, (Eds), Center for Wetland Res., LSU
Pub. //LSU-SG-73-01, pp. 185-197. [p. 106]
544
-------
Walker, J.D. and Cooney, J.J. (1973a), "Aliphatic Hydrocarbons of
Cladosporium resinae Cultured on Glucose, Glutamic Acid and Hydro-
Carbons", Appl. Microbiol., .26(5), 705-8. [p. 60, 158]
Walker, J.D. and Cooney, J.J. (1973b), "Pathway of n-Alkane Oxidation in
Cladosporium resinae". J. Bact., 115(2), 635-39. [p. 60]
Walker, W.W. and Stojanovic, B.J. (1973), "Microbial Versus Chemical
Degradation of Malathion in Soil", J. Environ. Qual., .2(2), 229-32.
[p. 226]
Walker, J.D.; Cofone, L.; and Cooney, J.J. (1973), "Microbial Petroleum
Degradation: The Role of Cladosporium resinae", AP1/EPA/USCG Conf.
on Prevention and Control of Oil Spills, Washington, D.C., 821-825.
[p. 106]
Wallace, R.A.; Fulkenson, W.; Shultz, W.; and Lyon, W.S. (1971), "Mercury
in the Environment, The Human Element", Oak Ridge National Laboratory,
ORNL, NSF-EP-1, Oak Ridge, Tenn. [p. 400, 402]
Warren, C.B. and Malec, E.J. (1972), "Biodegradation of Nitrilotrlacetic
Acid and Related Imino and Amino Acids in River Water", Science
176(4032), 277-279. [p. 74]
Water Pollution Research Laboratory (1972), Leaflet No. 58, Dept. of
Environment, Herts, England, [p. 170]
Wayman, C.H. and Robertson, J.B. (1963), "Biodegradation of Surfactants
in Synthetic Detergents Under Aerobic and Anaerobic Conditions
at 10°", U.S. Geol. Surv., Profess. Papers, 475-C. 224-7. [p. 79,
149, 170, 178]
Wayne, L.G. and Romanovsky, J.C. (1961), "Rates of Reaction of the Oxides
of Nitrogen in Photooxidation of Diluted Automobile Exhaust Gases",
in Chemical Reactions in the Lower and Upper Atmosphere, R.D. Cadle
(ed.), J. Wiley and Sons, N.Y. [p. 298]
Weaver, P.J. (1962), "Synthetic Detergents - A Look into the Past, the
Present and the Future", 24th Annual Conference, Ohio Section, American
Water Works Assoc., Cincinnati, November, [p. 145]
Weaver, P.J. (1965), "Testing Detergent Biodegradability", Soap Chem.
Specialites, 41(7), 45-9, 95-7. [p. 203]
Weaver, P.J. and Coughlin, F.J. (1964), "Measurement of Biodegradability",
J. Am. Oil Chem. Soc., 41(11), 738-41. [p. 74, 172]
Weber, W.J. and Morris, J.C. (1964), "Determination of Carbon in Waste-
waters by High Temperature Wet Oxidation", J. Water Pollut. Contr. Fed.,
.36, 573. [p. 159]
545
-------
Wedemeyer, G. (1967a) , "Dechlorination of l,l,l-Trichloro-2,2-bis(p-
chlorophenyl) ethane by Aerobacter Aerogenes; Metaboblic Product's",
Appl. Mlcrobiol., 15, 569-74. [p. 279]
Wedemeyer, G. (1967b) , "Biodegradation of Dichlorodiphenyltrichloro-
ethane: Intermediates in Dichlorodiphenylacetic Acid Metabolism by
Aerobacter aerogenes", Appl. Microbiol., 15. 1494-5. [p. 279]
Wei, Y.K. and Cvetanovic, R.J. (1963), "A Study of the Vapor Phase
Reaction of Ozone With Olefins in the Presence and Absence of
Molecular Oxygen", Can. J. Chem., 41, 913. [p. 291, 468]
Weil, J.K. and Stirton, A.J. (1963), "Biodegradation of Some Tallow-Based
Surface Active Agents in River Water", J. Amer. Oil Chem. Soc.,
40(9), 24. [p. 74]
Wessel, C.J. (1964), "Biodeterioration of Plastics. Plastics Attacked by
Fungus, Bacteria, Termites, Insects, Rodents, and Marine Organisms",
SPE (Soc. Plastics Eng.) Trans., 4_, 193-207. [p. 420, 425, 447,
451, 456, 475]
Westberg, K. ; Cohen, N.; and Wilson, K.W. (1971), "Carbon Monoxide: Its
Role in Photochemical Smog Formation", Science, 171, 1013. [p. 318]
Westoo, G. (1967), "Determination of Methylmercury Compounds in Food
Stuff", Acta Chemica Scandinavia, 21, 1790. [p. 398]
Westoo, G., (1969), "Methylmercury Compounds in Animal Foods", in Chemical
Fallout. (Editors, M.W. Miller and G. Berg), p. 75. [p. 402]
White, W.L. and Siu, R.G.H. (1947), "Resistance of Resin-Impregnated Cotton
Fabrics to Microorganisms", Ind. Eng. Chem., 39, 1628-30. [p. 422,
427, 437]
Wiebe, H.A. ; Villa, A.; Hellman, T.M. ; and Heicklen, J. (1973), "Photolysis
of Methyl Nitrite in the Presence of Nitric Oxide, Nitrogen Dioxide,
and Oxygen", J. Amer. Chem. Soc., 95, 7. [p. 291, 311]
Wiener, H.A. (1966), "Synthetic Sewages for Bench-scale Activated Sludge
Units", M.S. Thesis, Washington University, St. Louis, Mo. [p. 90]
Wildung, R.E.; Chesters, G. ; and Armstrong, D.E. (1968), "Chloramben
(amiben) Degradation in Soil", Weed Res., 1(3), 213-225. [p. 246, 253]
Willis, G.H.; Wander, R.C.; and Southwick, L.M. (1974), "Degradation
of Trifluralin in Soil Suspensions as Related to Redox Potential",
J. Environ. Qual. , _3> 262- [? 269, 270]
Wilson, J.S. (1967), "Problems in the Treatment of Effluents from the
Manufacture of Organic Chemicals", Chem. Ind., 30, 1278. [p. 71]
546
-------
Wilson, W.E., Jr. and Levy, A. (1970), "A Study of Sulfur Dioxide in
Photochemical Smog. I. Effect of S02 and Water Vapor Concentration
In the 1-Butene/NO /S02 System", J. Air. Pollut. Contr. Assoc.,
20, 385. [p. 316]x
Wilson, W.E.; Merryman, E.L.; Levy, A.; and Taliaferro, H.R. (1971),
"Aerosol Formation in Photochemical Smog. I. Effect of Stirring",
J. Air Pollut. Contr. Assoc., _21, 128. [p. 320]
Wilson, W.E., Jr.; Levy, A.; and Wimmer, D.B. (1972a), "A Study of Sulfur
Dioxide in Photochemical Smog. II. Effect of Sulfur Dioxide on
Oxidant Formation in Photochemical Smog", J. Air Pollut. Contr.
Assoc., 22., 27. [p. 304, 318, 320]
Wilson, W.E.; Schwartz, W.E.; and Kinzer, G.W. (1972b), "Haze Formation
Its Nature and Origin", U.S. Nat. Tech. Inform. Serv., PB Rep.
No. 212-609. [p. 304, 311, 331, 470]
Wolfe, H.R.; Staiff, D.C.; Armstrong, J.F.; and Comer, S.W. (1973),
"Persistence of Parathion in Soil", Bull. Environ. Contain. Toxicol.,
10(1), 1-9. [p. 241, 242, 263]
Wong, P.T.S.; Liu, D.; and Dutka, B.J. (1972), "Rapid Biodegradation of
NTA by a Novel Bacterial Mutant", Water Res., £(12), 1577-1584.
[p. 114]
Wood, J.M. (1972), "A Progress Report on Hg.", Environment, 14(1), 33-39.
[p. 415]
Wood, J.M. (1973), "Metabolic Cycles for Toxic Elements in Aqueous Systems",
Rev. Intern. Oceanogr. Med., 31-32, 7. [p. 415]
Wood, J.M. (1974), "Biological Cycles for Toxic Elements in the Environment",
Science, 183(4129), 1049-52. [p. 23, 283, 381, 382, 383, 384,
472, 473]
Wood, J.M.; Kennedy, F.S.; and Rosen, C.G. (1968), "Synthesis of Methyl-
mercury Compounds by Extracts for Methanogenic Bacterium", Nature
220. 173-75. [p. 382, 389, 390, 391, 396, 408, 414]
Wood, W.A. (1971), "Assay of Enzymes Representative of Metabolic Pathways".
In: Methods In Microbiology, (J.R. Norris and D.W. Ribbons, Editors),
6A, 411-424, Academic Press, N.Y.
Woodwell, G.M.; Craig, P.P.; and Johnson, H.A. (1971), "DDT in the
Biosphere: Where Does It Go?", Science, 174, 1101. [p. 41]
Woolson, E.A. and Kearney, P.C. (1973), "Persistence and Reactions of
ll»C-Cacodylic Acid in Soils", Environ. Sci. Techno 1., 7.(1), 47.
[p. 393, 399, 410]
547
-------
WPCF (1967) Water Pollution Control Federation, Biodegradability Subcommittee,
"Require Characteristics and Measurement of Biodegradability", J.
Water Pollut. Contr. Fedr., .39, 1232-1235. [p. 1, 2, 25, 200]
Wright, S.J.L. (1971), "Degradation of Herbicides by Soil Microorganisms",
in Microbial Aspects of Pollution, Sykes, G. and Skinner (edit.)>
Academic Press, N.Y. [p. 37, 277]
Wright, W.L. and.Warren, G.F. (1965), "Photochemical Decomposition of
Trifluralin", Weeds, 1.3(4), 329-331. [p. 338, 351, 352, 356]
Wuhrmann, K. (1964), "River Bacteriology and the Role of Bacteria in Self-
Purification of Rivers", in Principles and Applications in Aquatic
Microbiology, ed. Heukelekian, H. and Dondero, N.C., J. Wiley and
Sons, Inc., N.Y. pp. 167-192. [p. 49]
1
Yanagi, M. and Onishi, G. (1971), "Assimilation of Selected Cosmetic
Ingredients by Microorganisms", J. Soc. Cosinet. Chem., 22(13),
851-865. [p. 113]
Yaron, B.; Heuer, B. and Birk, Y. (1974), "Kinetics of Azinphosmethyl
Losses in the Soil Environment", J. Agr. Food Chem., 22, 439.
[p. 270] .
Yasuno, M.; Hirakoso, M.; Sasa, M.; and Uchida, M. (1966), "Inactivation
of Some Organophosphorus Insecticides by Bacteria in Polluted
Water", Japan. J. Exptl. Med., 35_(6), 545-63. [p. 80, 159]
Yellow Spring Instrument Co. (1970), YSI Biological Oxygen Monitor,
Yellow Springs, Ohio. [p. 69]
Young, R.H.F.; Ryckman, D.W.; and Buzzell, J.C. (1968), "An Improved
: Tool for-Measuring Biodegradability", J. Water Pollut. Contr. Fed.,
40(8 Part 2), 354-368. [p. 56, 57]
Yu, C-C.; Booth, G.M.; Hansen, D.J.; and Larsen, J.R. (1974), "Fate of
Carbofuran in a Model Ecosystem", J. Agr. Food Chem., 22, 431.
[p. 120]
Zabik, M.J.; Schuetz, R.D.; Burton, W.L.; and Pape, B.E. (1971), "Photo-
chemistry of Bioactive Compounds. Studies of a Major Photolytic
Product of .Endrin", J. Agr. Food Chem., 19, 308. [p. 338]
Zepp, R.C.; Wolfe, N.L.; and Gordon, J.A. (1973), "Photodecomposition of
Phenylmercury Compounds in Sunlight", Chemosphere, 2_(3), 93-9.
[p. 338, 348, 356, 395]
Zepp, R.G.; Wolfe, N.L.; Baughman, G.L.; and Gordon, J.A. (1974), "Dynamics
of 2,4-D Esters in the Aquatic Environment: Hydrolysis and Photo-
degradation", 167th ACS Meeting, Los Angeles, [p. 363]
548
-------
Zimdahl, R.L.; Freed, V.H.; Montgomery, M.L.; and Furtick, W.R. (1970),
"The Degradation of Triazine and Uracil Herbicides in Soil", Weed
Res., 10(1), 18-26. [p. 226, 244, 267]
ZoBell, C.E. (1946), "Action of Microorganisms on Hydrocarbons", Bact. Revs.,
10, 1-49.
ZoBell, C.E. and Beckwith, J.D. (1944), "The Deterioration of Rubber Products
by Microorganisms", J. Am. Water Works Assoc., 36, 439-53.
ZoBell, C.E. and Grant, C.W. (1942), "Discussion: The Bacterial Oxidation
of Rubber", Science, 9£, 379-80. [p. 452]
549
------- |