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Order Number 8726585
Soils containing 2,3,7,8-Tetrachlorodibenzo-p-dioxin: Aspects of their microbial activity and the potential for their microbially-mediated decontamination
Arthur, Mickey Francis, Ph.D.
The Ohio State University, 1987
UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106
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SOILS CONTAINING 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN:
ASPECTS OF THEIR MICROBIAL ACTIVITY AND THE POTENTIAL
FOR THEIR MICROBIALLY-MEDIATED DECONTAMINATION
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of the Ohio State University
By
Mickey Francis Arthur, B.S., M.S.
The Ohio State University
1987
Dissertation Committee: Approved by
J .I . Frea
R.M. Pfister
W .R. Strohl
0. H . Tuovinen Department of Microbiology ACKNOWLEDGEMENTS
I express sincere appreciation to the many people who helped make the completion of this work possible. My thanks go to Dr. James I. Frea for his guidance, insight, and patience throughout this research. I also thank Drs.
Robert M. Pfister, William R. Strohl, and Olli H.
Tuovinen for their advice and suggestions during the
General and Final Examinations. I express gratitude to
Drs. Barney W. Cornaby, James A. Fava, L. Barry Goss, and
Danny R. Jackson at Battelle-Columbus Division for their constant support and encouragement. The technical assistance of Thomas C. Zwick, G. Kelly O ’Brien, Thomas deJolsvay, John Steichen, Dan Aichele, and Lisa M.
Hendrickson is gratefully acknowledged. Finally, I am indebted to my wife, Vicki, and my children, Melissa,
Thomas, and Natalie, for their patience and love during this long effort.
ii VITA
November 18, 1950 ...... Born - Columbus, Ohio
1976 ...... B.S., The Ohio State University, Columbus, Ohio
1978 ...... M.S., The Ohio State University, Columbus, Ohio
1978-1983 ...... Researcher, Bi©environmental Sciences, Battelle-Columbus Division, Columbus, Ohio
1983-Present ...... Research Scientist, Environmental Technology and Assessment, Battelle-Columbus Division, Columbus, Ohio
PUBLICATIONS
Litchfield, J.H. and M.F. Arthur. 1982. Growth of selected ectomycorrhizal fungi in aerated liquid culture. Dev. Indust. Microbiol. 24:289-293.
Arthur, M.F., C.K. Wagner, and P. Van Voris (eds.). 1983. Response of agricultural soils to acid deposition. Environ. Experiment. Botany 23:197-284.
Arthur, M.F. and C.K. Wagner. 1983. Response of agricultural soils to acid deposition: supplemental literature review. Environ. Experiment. Botany 23:259-279.
Tolle, D.A., M.F. Arthur, and P. Van Voris. 1983. Microcosm/field comparison of trace element uptake in crops grown in fly ash-amended soil. Sci. Total Environ. 31:243- 261.
Arthur, M.F., T.C. Zwick, D.A. Tolle, and P. Van Voris. 1984. Effects of fly ash on microbial C02 evolution from an agricultural soil. Water Air Soil Pollution 22:209-216.
iii Zwick, T.C., M.F. Arthur, D.A. Tolle, and P. Van Voris. 1984. A unique laboratory method for evaluating agro ecosystem effects of an industrial waste product. Plant Soil 77:395-399.
Tolle, D.A., M.F. Arthur, J. Chesson, and P. Van Voris. 1985. Comparison of pots versus microcosms for predicting agroecosystem effects due to waste amendment. Environ. Toxicol. Chem. 4:501-509.
Van Voris, P., D.A. Tolle, M.F. Arthur, and J. Chesson. 1985. Terrestrial microcosms: applications, validation and cost-benefit analysis, pp. 117-142. In: J. Cairns (ed.). Multispecies Toxicity Testing. Pergamon Press, New York.
DeGraeve, G.M., W.H. Clement, M.F. Arthur, R.B. Gillespie, and G.K. O ’Brien. 1987. Environmental persistence/ degradation of toxicity in complex effluents: laboratory simulations of field conditions. In: Tenth Symposium on Aquatic Toxicology, American Society for Testing and Materials (in press).
Arthur, M.F. and J.I. Frea. 1987. Microbial activity in soils contaminated with 2,3,7,8-tetrachlorodibenzo-p- dioxin. Environ. Toxicol. Chem. (in press).
FIELDS OF STUDY
Major Field: Environmental Microbiology
Studies in
Biochemistry: L. Johnson; G. Means Plant Physiology: C. Swanson Soil Chemistry: E.O. McClean Soil Fertility: T.A. Arscott Soil Microbiology: R.H. Miller
iv TABLE OF CONTENTS
ACKNOWLEDGEMENTS ...... ii
VITA ...... ill
LIST OF TABLES ...... vii
LIST OF F I G U R E S ...... xii
INTRODUCTION ...... 1
SCOPE AND OBJECTIVES...... 5
APPROACH ...... 5 Y CHAPTER PAGE
I. LITERATURE REVIEW ...... 9
Physicochemical Properties of TCDD .... 9 Sources of TCDD...... 12 Biological Properties of TCDD...... 13 Environmental Properties of TCDD ..... 30 Potential Microbial Metabolism: TCDD and Other Chlorinated Hydrocarbons ...... 34 Summary...... 53
II. MATERIALS AND METHODS...... 55
Soil Characterization...... 55 Microbial Activity in TCDD Soils ...... 68 Microbial Mineralization of TCDD ...... 74 Surfactant Experiments ...... 76 HCB Degradation Experiments...... 84 Surfactant-Mediated TCDD Degradation Experiments...... 88
III. RESULTS AND DISCUSSION...... 90
Soil Characterization...... 90 Microbial Activity in TCDD Soils ...... 94
v Surfactant Experiments ...... 116 HCB Biodegradation Experiments ...... 138 TCDD Biodegradation Experiments...... 153
IV. GENERAL DISCUSSION ...... 164
Soil Characterization...... 164 Microbial Activity in TCDD Soils ...... 166 Surfactant Experiments ...... 179 HCB Biodegradation Experiments ...... 184 TCDD Biodegradation Experiments...... 186 V. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS...... 191
Summary...... 191 Conclusions...... 194 Recommendat ions...... 195
APPENDIX ...... 199
BIBLIOGRAPHY ...... 210
vi LIST OF TABLES
TABLE PAGE
1. Possible chlorinated compounds of dibenzo-p-dioxin...... 10
2. Physicochemical properties of 2,3,7,8- tetrachlorodibenzo-p-dioxin ...... 11
3. Acute toxicity of TCDD compared to other toxic materials, and the relative toxicity of TCDD among species ...... 16
4. TCDD Bioconcentration Factors (BCF) in aquatic organisms grown in model ecosystems in the laboratory...... 27
5. Physicochemical parameters of experimental soils...... 91
6. Mean microbial numbers in TCDD soils and a non-TCDD control soil (n=5). Within columns, means followed by a common letter are not significantly different at the 99% confidence level...... 96
7. Mean activity of selected enzymes in TCDD soils and a non-TCDD control soil (n=3). Within columns, means followed by a common letter are not significantly different at the 99% confidence level .... 100
8. Mean cumulative evolution of C02-C (mg) from Times Beach soil (8 ng TCDD/g) diluted and undiluted with non-TCDD soil (n=3). Within rows, means followed by a common letter are not significantly different at the 95% confidence level...... 105
9. Mean cumulative evolution of CO2-C (mg) from Piazza Road soil (1.1 ug TCDD/g) diluted and undiluted with non-TCDD soil
vii (n=3). Within rows, means followed by a common letter are not significantly different at the 95% confidence level . . . . 107
10. Mean cumulative evolution of CO2-C (mg) from Shennendoah Stables soil (2.4 ug TCDD/g) diluted and undiluted with non-TCDD soil (n=3). Within rows, means followed by a common letter are not significantly different at the 95% confidence level . . . . 109
11. Mean cumulative evolution of CO2-C (mg) from New Jersey soil (26.3 ug TCDD/g) diluted and undiluted with non-TCDD soil (n=3). Within rows, means followed by a common letter are not significantly different at the 95% confidence level . . . . 113
12. Mean solubility of hexachlorobenzene (HCB) in surfactant solutions (n=3). Within columns, means followed by a common letter are not significantly different at the 95% confidence level . . . . 118
13. Mean extraction of hexachlorobenzene (HCB) from soil with surfactant solutions (n=3). Within columns, means followed by a common letter are not significantly different at the 95% confidence level . . . . 120
14. Effects of surfactant solutions on the development of colony forming units (CFU) of Pseudomonas aeruginosa on nutrient agar ...... 123
15. Relative toxicity of surfactant solutions to Photobacterium nhosphoreum in the Microtox assay. Results are expressed as the effective concentration that inhibits 50% of control bioluminescence (ECso), within 5 minutes at 15 C ...... 125
16. Mean cumulative evolution of CO2-C (mg) from West Jefferson soil treated with Morwet 425 and 2000 ug/g Cd (n=3). Within rows, means followed by a common letter are not significantly different at the 99% confidence level ...... 127
17. Mean cumulative evolution of CO2-C (mg) from West Jefferson soil treated with
viii Agrimul 70 and 2000 ug/g Cd (n=3). Within rows, means followed by a common letter are not significantly different at the 95% confidence level ...... 129
18. Mean cumulative evolution of C02-C (mg) from West Jefferson soil amended with 1% (v/w) Agrimul 70, 10 ug/g hexachlorobenzene (HCB), or 1000 ug/g Cd (n=3). Within rows, means followed by a common letter are not significantly different at the 95% confidence level...... 132
19. Mean cumulative evolution of CO2-C (mg) from West Jefferson soil treated with 2% (v/w) Agrimul 70, 10 ug/g hexachlorobenzene (HCB), or 1000 ug/g Cd (n=3). Within rows, means followed by a common letter are not significantly different at the 95% confidence level...... 133
20. Mean cumulative evolution of CO2-C (mg) from West Jefferson soil treated with 0.1% (w/w) Morwet 425, 10 ug/g hexachlorobenzene (HCB), or 1000 ug/g Cd (n=3). Within rows, means followed by a common letter are not significantly different at the 95% confidence level...... 135
21. Mean cumulative evolution of CO2-C (mg) from West Jefferson soil treated with 2% (v/w) Agrimul Jl, 10 ug/g hexachlorobenzene (HCB), or 1000 ug/g Cd (n=3). Within rows, means followed by a common letter are not significantly different at the 95% confidence level...... 136
22. Recovery of hexachlorobenzene (HCB) from West Jefferson soil amended with 10 ug/g HC-HCB and incubated aerobically at 22-25 C for one year. In the last column, means followed by a common letter are not significantly different at the 95% confidence level...... 139
23. Recovery of H C O 2 from combustion of West Jefferson soil amended with i*C- hexachlorobenzene. Soil originally contained 4310 disintegrations per minute/g (DPM/g). Within the last column, means followed by a common
ix letter are not significantly different at the 95% confidence level ...... 143
24. Mean hexachlorobenzene (HCB) remaining in septic anaerobic sediments following the addition of 10 ug/g HCB on day 0 (n=2). Within columns, means followed by a common letter are not significantly different at the 95% confidence level .... 146
25. Mean hexachlorobenzene (HCB) in autoclaved sediments following addition of 10 ug/g HCB on day 0 (n=2). Within columns, means followed by a common letter are not significantly different at the 95% confidence level...... 149
26. Comparison of mean hexachlorobenzene (HCB) in Morwet 425-treated septic and autoclaved sediments. Sediments were dosed with 10 ug/g HCB on day 0 (n=2). Within columns, means followed by a common letter are not significantly different at the 95% confidence l e v e l ...... 151
27. Comparison of mean hexachlorobenzene (HCB) in Agrimul 70-treated septic and autoclaved sediments. Sediments were dosed with 10 ug/g HCB on day 0 (n=2). Within columns, means followed by a common letter are not significantly different at the 95% confidence level ...... 152
28. Surfactant-mediated extraction of 14C from Piazza Road (PR) and Shennendoah Stables (SS) soils amended with 14C-TCDD. Surfactant concentrations were 2% (v/w) compared to extraction with 100% t o l u e n e ...... 156
29. TCDD in soils (ng/g) initially, after one year of bioaugmentation, and following six months of incubation with 2% (v/w) Agrimul J1...... 158
30. Combustion of HC-TCDD soils for recovery of HC02. TCDD soils initially were amended with 4400 DPM/g additional HC-TCDD prior to incubation. Within the last column, means followed by a common letter are
x not significantly different at the 95% confidence level...... 160
31. Mean microbial numbers in TCDD and non-TCDD soils after treatment with Agrimul J1 and aerobic incubation for 6 months (n=5). Within columns, means followed by a common letter are not significantly different at the 95% confidence level...... 162
v
xi LIST OF FIGURES
FIGURE PAGE 1. (aj Structure of 2,3,7,8-tetrachloro- dibenzo-p-dioxin; (b) Formation of dioxins from ortho-substituted benzenes; (c) Formation of predioxin from 2,4,5- trichlorophenol; (d) Formation of isopredioxin from 2,4,5-trichloro- phenol...... 14
2. Evolution of C02-C from TCDD soils (TB, PR, SS), a non-TCDD soil (WJ), and a TCDD soil amended with Cd (SS/Cd) . . . 111
3. Sample GC results of analysis for hexachlorobenzene (HCB) in soils; (a) 1 ug/ml HCB standard; (b) Soil treated with HCB and 5% (v/w) Agrimul 70; (c) Soil treated with HCB but no surfactant...... 141
4. GC/MS results of the TCDD response factor solution, indicating the peak locations of 2,3,7,8-; 13C-2,3,7,8-; and 1 3 C-l,2,3,4-tetrachlorodibenzo-p- dioxin. The peak identities are the same in Figures 5 through 13...... 200
5. GC/MS results for soil TB plus Agrimul Jl. The recovery factor for i3C-l,2,3,4-TCDD (used for calculating the final concentration of 2,3,7,8- TCDD) was 88.8%. The original (Day 0) TCDD concentration was 0.008 ug/g. The two top chromatograms in Figures 5 through 12 represent unspiked (native) TCDD soil and the two lower chromatograms represent *3C spiked TCDD soils...... 201
6. GC/MS results for soil TB with no Agrimul Jl but after one year of
xii Incubation with fertilizer and moisture contrbl. The recovery factor for 13 C-1,2,3,4-TCDD was 91.4%. The initial (Day 0) TCDD concentration was 0.008 ug/g . . 202
7. GC/MS results of soil PR plus Agrimul Jl. The recovery factor for i 30-1,2,3,4-TCDD was 47.5%. The initial (Day 0) TCDD concentration was 1.1 ug/g . . . 203
8.. GC/MS results of soil PR with no Agrimul Jl but after one year of incubation with fertilizer and moisture control. The recovery factor for i3C-l,2,3,4- TCDD was 53.5%. The initial (Day 0) TCDD concentration was 1.1 ug/g ...... 204
9 . GC/MS results of soil SS plus Agrimul Jl. The recovery factor for 13 0-1,2,3,4- TCDD was 37.3%. The initial (Day 0) TCDD concentration was 2.4 ug/g ...... 205
10. GC/MS results of soil SS with no Agrimul Jl but after one year of incubation with fertilizer and moisture control. The recovery factor for 13 0- 1,2,3,4-TCDD was 6%. The initial (Day 0) TCDD concentration was 2.4 ug/g . . . 206
11. GC/MS results of soil WJ plus Agrimul Jl. The recovery factor for 13 0-1,2,3,4- TCDD was 77.1%...... 207
12. GC/MS results of soil WJ with no Agrimul Jl but after one year of incubation with fertilizer and moisture control. The recovery factor for 13C-l,2,3,4-TCDD was 77.8% . . . . 208
13. GC/MS results of the method blank ...... 209
xiii INTRODUCTION
A recent and widely publicized incident of environmental pollution was the contamination of soils in
Missouri with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
Near Verona, Missouri in the early 1970’s, TCDD was produced as an unwanted by-product during the manufacturing of hexachlorophene from 2,4,5-trichlorophenol (Powell,
1984). Approximately 68,000 liters of waste oils and still bottoms containing TCDD and other contaminants were spread on roadways, dirt lots, and in horse arenas in eastern
Missouri for the control of dust. Approximately 95,000 liters of TCDD-containing wastewater were transported to southwestern Missouri for disposal. At Shennendoah Stables in eastern Missouri, undiluted still bottoms containing approximately 350 mg/1 TCDD, representing nearly 3 kg of
TCDD, were sprayed on arena floors. Within one day, horses in the arena became sick and within one week, sparrows in the rafters of the arena began dying. Eventually, 75 horses from the stables either died or were destroyed.
Shennendoah Stables represents a small area of high
TCDD concentration. On the other hand, the Times Beach site in eastern Missouri is a site of widespread TCDD contamination. Roads around Times Beach were sprayed with
TCDD-containing oil in 1972 and 1973, and the town now is known to contain over 60% of all TCDD-contaminated soil in
Missouri (Powell, 1984). Because TCDD is a known toxicant and a suspected carcinogen and teratogen, the Environmental
Protection Agency decided to purchase the entire town. In
1982, the area was evacuated, secured, and designated as a
"Superfund" site under the Comprehensive Environmental
Response Compensation and Liability Act (CERCLA, or
"Superfund"). Investigations concerning the degree of TCDD contamination and the potential effects on wildlife in the area are continuing.
It is now estimated that more than 500,000 cubic yards of soil in Missouri are contaminated with TCDD (Powell,
1984). The contamination is spread over at least 38 sites and possibly 200 additional sites widely distributed throughout eastern and southwestern Missouri. To date, no viable solution to the problem of TCDD contamination in
Missouri has been developed and concern is increasing over the potential for groundwater and biota contamination with
TCDD.
The situation in the Times Beach area is not the only example of environmental contamination with TCDD. In 1976 in Seveso, Italy, an explosion at a pesticide manufacturing facility resulted in the contamination of more than 2000 hectares of soil with TCDD (Connell and Miller, 1984; Young, 1983). The distributions of TCDD in soils near
Seveso were found to range from less than 0.750 to 5000 ug/m2. Despite widespread death of plants and animals, no human deaths were or have been attributed to TCDD contamination in Seveso, although several cases of chloracne were reported. Chloracne is a temporary skin ailment that has also been a frequent complaint among veterans of the Vietnam war who were exposed to the TCDD- containing herbicide "Agent Orange," a mixture of 2,4- dichlorophenoxacetic acid (2,4-D) and 2,4,5- trichlorophenoxyacetate (2,4,5-T). The concentration of
TCDD in Agent Orange applied in Vietnam ranged from 1 to 47 mg/1 (Connell and Miller, 1984). In the United States,
Agent Orange and other TCDD-contaminated herbicides were applied repeatedly between 1962 and 1970 to test areas at
Eglin Air Force Base in Florida. This was done in order to evaluate the aerial spraying equipment to be used in
Vietnam and the herbicides’ effectiveness (Harrison, 1979).
The long-term environmental and health implications associated with TCDD contamination are still unclear. What is clear, however, is that TCDD is toxic, carcinogenic, and teratogenic to many laboratory animals, and is environmentally persistent. As a result, the U.S.
Environmental Protection Agency (EPA), in its guidelines for disposal of hazardous materials, considers any release of TCDD to the environment to be unacceptable, standards which are among the most stringent promulgated by the agency. In addition, the cleanup of TCDD-contaminated sites has become a major problem, due to the costs incurred in using available technology. For example, the two methods currently most suitable for the clean-up of TCDD soils are incineration and removal by excavation (Esposito et al., 1980). Unfortunately, it is neither economically nor ecologically feasible to incinerate large quantities of widely dispersed soil, and excavation with stockpiling of soil is not an ultimate solution to the presence of TCDD.
If the areas around Times Beach and the other TCDD sites are to be reclaimed, an alternative method of soil decontamination needs to be developed.
One potential decontamination method that has received limited attention is jja situ biodegradation of TCDD. The reason for the inattention to biodegradation is due to the observed persistence of TCDD in soils and sediments, and the lack of significant biodegradation of the compound in laboratory tests. Studies that have addressed this topic have generally shown that, like many other chlorinated hydrocarbons, TCDD is resistant to microbial attack.
According to Esposito et al. (1980), however, biodegradation may be an important method for the ultimate safe and cost-effective clean-up of TCDD soils. Compared to other options, such as excavation and incineration of large volumes of soil, In situ biodegradation of TCDD by indigenous soil microorganisms may offer economic and ecological advantages over other disposal methods, but only if the problems inherent to this approach can be overcome.
SCOPE AND OBJECTIVES The objectives of this research were to examine levels of microbial activity in soils that have been contaminated with TCDD for many years, and to develop and evaluate a potential approach for stimulating the biodegradation of
TCDD in soils. Throughout this research, the emphasis was on evaluating the potential for stimulating jja situ TCDD biodegradation by the microflora indigenous to contaminated soils. The hypothesis was that, if TCDD-contaminated soils proved to be microbially active, and the availability of
TCDD could be enhanced, then in situ biodegradation by indigenous microorganisms may be possible. Biodegradation by indigenous microorganisms could offer a cost-effective remedial alternative to more expensive decontamination methods, such as incineration and excavation. In addition, if such an approach proved to be successful with TCDD, then a similar approach might be valid at numerous other waste disposal sites containing chlorinated hydrocarbons.
APPROACH
This project was carried out in discrete stages designed to systematically address the problems associated with in situ biodegradation of TCDD in a complex environmental matrix (soil). Each phase of this research depended on the results of the previous phase, as summarized below.
1. The level of activity of selected microbial groups and metabolic processes in TCDD soils was determined and compared to a similar but non-TCDD soil. This was done to assess the potential for effecting the biodegradation of
TCDD by the indigenous microflora; That is, if microbial activity in TCDD soils was nonexistent or severely restricted due to toxicity of the compound or associated co-contaminants, the potential for jjQ situ decontamination by biodegradation could be low.
2. The potential for stimulating TCDD biodegradation in Missouri soils by using relatively simple ia situ management techniques was evaluated in a year-long study.
Soils containing different levels of TCDD were variously diluted with non-TCDD soils, or were left undiluted, and were either amended or unamended with a complete inorganic nutrient solution. In addition, HC-TCDD was added to all soils, incubated aerobically, and monitored for the evolution of H C O 2 . These steps were done to determine if a) the addition of soil microorganisms unexposed to TCDD, b) the dilution of TCDD concentrations in Missouri soils, c) the provision of inorganic nutrients, d) the optimization of moisture conditions, or e) some combination of these factors, would stimulate the mineralization of
TCDD, either through direct or co-metabolic processes.
3. Despite the fact that TCDD concentrations in soil were well in excess of those reported to be toxic to many laboratory animals, microbial activity in TCDD soils was comparable to a fertile non-TCDD soil. Even with high levels of microbial activity in TCDD’ soils, however, no mineralization of TCDD was detected. This suggested that
TCDD either was not toxic to the soil microflora, that it was metabolized to intermediates instead of to CO2 , or that
TCDD was not bioavailable. The next phase of this research was to investigate ways to improve the bioavailability of
TCDD in soils. This was done through the use of surfactants formulated to solubilize chlorinated pesticides. Because surfactants themselves may be toxic to soil microorganisms, numerous surfactants were screened for their microbial toxicity, and their ability to solubilize soil-bound TCDD or a soil-bound TCDD surrogate compound, hexachlorobenzene (HCB).
4. HCB was added to aerobic soil and anaerobic sediment, treated with surfactants, and monitored for biodegradation. Anaerobic conditions were investigated in order to determine if reductive dechlorination played a role in surfactant-mediated biodegradation of chlorinated hydrocarbons. Soils and sediments were monitored for loss of parent compound using gas chromatography. In addition, aerobic soils were monitored for evolution of H C O 2 , and a mass balance of the fate of the added i*C was determined.
5. Finally, TCDD soils were amended with a non-toxic concentration of a surfactant that had been shown to be effective in solubilizing soil-bound TCDD and HCB.
Biodegradation of TCDD was evaluated by monitoring for the evolution of 1*002, ^or ^ ie loss of parent compound using high resolution gas chromatography-mass spectroscopy, and for mass balance of i*C by soil combustion.
The rationale for this experimental approach is developed more thoroughly in the following sections. A review of the literature describes the previous research in this and related areas, and discusses some of the problems in dealing with a highly recalcitrant and toxic compound like TCDD. CHAPTER I
LITERATURE REVIEW
Dioxins constitute a family of isomers and congeners of substituted dibenzo compounds in which the two benzene rings are joined in a six-membered ring that contains two oxygens in the 1,4- (para) position (Connell and Miller,
1984). The benzene rings may be substituted with halogens, hydrogens, or organic groups at positions 1 to 4 and 6 to
9, resulting in the possibility for numerous isomers and congeners. For example, seventy-five chlorinated compounds are possible (Table 1). Merz and Weith prepared the first chlorinated dioxin, the octochloro isomer, in 1872; TCDD was first synthesized in the laboratory in 1957 (Rappe,
1978). Since then, as many as 40 of these compounds have been prepared in the laboratory (Esposito et al., 1980).
PHYSICOCHEMICAL PROPERTIES OF TCDD
Selected physicochemical properties of TCDD are shown in Table 2 (Esposito et al., 1980; NIOSH, 1984; Marple et al., 1986). TCDD is nearly insoluble in water, only slightly soluble in many organic solvents and, as evidenced by its high octanol/water partition coefficient, is 10
Table 1. Possible chlorinated compounds of dibenzo-p- dioxin.
Number of
Congener Isomers
Monochloro 2
Dichloro 10
Trichloro 14
Tetrachloro 22
Pentachloro 14
Hexachloro 10
Heptachloro 2
Octochloro 1
Sum 75 Table 2. Physicochemical properties of 2,3,7,8- tetrachlorodibenzo-p-dioxin.
Empirical formula C12H4CI4O2
Molecular weight (g/mol) 322
Percent by weight C 44.7
0 9.95
H 1.25
Cl 44.1
Melting point 305 C
Decomposition temperature >700 C
Solubilities (g/1)
o-dichlorobenzene 1.4
chlorobenzene 0.72
benzene 0.57
chloroform 0.37
acetone 0.11
n-octanol 0.048
methanol 0.01
water 2 x 10-10
Partition coefficients
Octanol/water 4.4 x 106
Soil/water 2.3 x 10<
Vapor pressure (mm Hg) 10-6 - 10-7 12 extremely lipophilic. It is stable in acid, alkali, and heat, but will decompose when exposed to sunlight and other sources of ultraviolet radiation. At room temperature TCDD is a colorless, crystalline solid.
SOURCES OF TCDD
The main source of TCDD is the manufacturing of 2,4,5- trichlorophenol (TCP) from tetrachlorobenzene (NIOSH, 1984;
Esposito, et al., 1980); TCDD is not known to be produced biologically. Because TCP was used as a feedstock for the production of chlorinated phenoxy acetic acid pesticides, such as 2,4,5-T, 2,4-D, and silvex (2-[2,4,5- trichlorophenoxy]propionic acid), and is currently used in the production of hexachlorophene, TCDD has become a generally widespread trace environmental pollutant.
Dioxins (not necessarily TCDD) also occur in fly ashes and other combustion products, such as cigarette smoke and incinerator emmissions, illustrating the generally high temperature requirements (180-400 C) for dioxin formation
(Esposito et al., 1980; Connell and Miller, 1984).
Combustion of many materials, such as polyvinylchloride in the presence of naturally occurring phenols, vegetation treated with phenoxy acetic acid herbicides, paper and wood treated with chlorophenols, and pesticide-treated wastes, may lead to dioxins and dioxin precursors. Czuczwa and
Hites (1984, 1986) have proposed that waste combustion followed by long-range airborne transport is a major source of dioxins in the environment. Using dated sediment cores from Lake Huron, these researchers found that chlorinated dioxins and dibenzofurans began to accumulate in sediments around 1940, which corresponds to the period of production of aromatic compounds and their disposal by incineration.
A compound may serve as a dioxin precursor if 1) it is an ortho-substituted benzene in which one of the substituents includes an oxygen atom attached directly to the benzene ring, and 2) the ortho-substituents, excluding the oxygen atom, are able to react with each other to form an independent compound (Esposito et al., 1980). The generalized reaction is shown in Figure 1. The formation of TCDD from 2,4,5-T is a two-step process that leads to a predioxin intermediate (Figure 1). In the process of TCDD formation, isopredioxin may also be formed. Polymerization of isopredioxin may lead to other toxic condensation products, such as chlorinated dibenzofurans. As a result of these competing reactions, TCDD is generally found in trace quantities with many other possible chlorinated products.
BIOLOGICAL PROPERTIES OF TCDD
The biological properties of TCDD include toxicity, suspected carcinogenicity and teratogenicity, possible bioaccumulation, and metabolism to some degree by various 14
PREDIOXIN
ISOPREDIOXIN
Figure 1. (a) Structure of 2,3,7,8-tetrachlorodibenzo-p- dioxin; (b) Formation of dioxins from ortho-substituted benzenes; (c) Formation of predioxin from 2,4,5- trichlorophenol; (d) Formation of isopredioxin from 2,4,5- trichlorophenol. 15 species. These biological properties manifest in species- specific manners, as discussed below.
TCDD is considered to be one of the most toxic substances known, based on its extremely low LDso (the dosage lethal to half of the test subjects) in some experimental animals. Table 3, adapted from Esposito et al. (1980), Connell and Miller (1984), and Tschirley
(1986), shows both the relative acute toxicity of TCDD compared to other toxicants and TCDD toxicity among various species. It is clear that TCDD acute toxicity, while substantial compared to well-known toxicants, is highly species-specific, as well as dependent on the sex of the animal and the route of injection. For example, the male guinea pig is the most TCDD-susceptible mammal tested, while frogs and hamsters are relatively resistant to TCDD acute toxicity. In addition, the acute toxicity of TCDD is orders of magnitude greater than for any other chlorinated dioxin. The basis for species-, sex-, and route-specific acute toxicity has not been determined.
TCDD also exhibits chronic toxicity and represents, along with potential teratogenicity and carcinogenicity, issues of great concern in terms of environmental contamination of soils. Chronic effects are more difficult to discern than are acutely toxic effects (measured by death of the test species). For example, in four-week old specific-pathogen-free male mice, daily TCDD doses of 16
Table 3. Acute toxicity of TCDD compared to other toxic
materials, and the relative toxicity of TCDD among species.
Minimum
Lethal Dose
Substance Species(a) Route (mol/kg)
Botulinum toxin A Mouse IP< b) 3.3 :k lO-i?
Tetanus toxin Mouse IP 1 :ic 10-15
Diptheria toxin Mouse IP 4.2 :ic 10-12
TCDD Guinea Pig (m) Oral 1.9 x 10-9
TCDD Guinea Pig (f) Oral 6.5 X 10-9 TCDD Rat (m ) Oral 6.8 X 10-8
TCDD Monkey (f) Oral <2.2 X 10-7 TCDD Rabbit(m,f) Oral 3.5 X 10-7
TCDD Mouse (m) IP 3.7 X 10-7
TCDD Mouse (m) Oral <4.7 X 10-7
TCDD Rabbit (m,f) Dermal 8.5 X 10-7
Strychnine Mouse IP 1.5 X 10-6
TBrDD( c) Rat Oral 2 X 10-6
TCDD Frog Oral 3.1 X 10-6
TCDD Hamster (m,f) Oral 3.6 X 10"6
TCDD Hamster (m,f) IP 9.3 X 10-6
Sodium cyanide Mouse IP 2.0 X 10-4
1,2,3,4-TCDD Rat Oral >3.1 X 10-3
OCDD( d) Rat Oral 4.3 X 10-3 Table 3 (continued)
(a) m = male; f = female
(b) IP = intraperitoneal
(c) TBrDD = 2,3,7,8-tetrabromodibenzo-p-dioxin
(d) OCDD = octachlorodibenzo-p-dioxin 0.14 ug/kg of body weight for four weeks, followed by infection with Salmonella berne. led to significantly increased mortality and decreased post-infection survival times, compared to non-TCDD control animals exposed to
Salmonella (Kimbrough et al., 1984). The mortality of mice infected with Herpes virus suis. however, was not affected by chronic exposure to TCDD. At daily TCDD doses of 2.86 and 1.43 ug/kg of body weight, thymic atrophy and liver pathology were observed; at lower doses, these effects were not seen. On the other hand, male mice fed weekly for four weeks with 50 ug of TCDD and challenged with Listeria monocytogenes showed no impaired macrophage function or increased spleen counts of viable Listeria.
Other low-dose TCDD-induced immunotoxic effects in rodents include increased sensitivity to endotoxin from
Escherichia coli or Salmonella typhimurium. decreased cell- mediated immunity (as measured by lymphocyte responses to phytohemagglutinin or concanavalin-A), reduced graft- versus-host reactions, and suppressed delayed hypersensitivity (Kimbrough, 1984). Whether chronic exposure to low doses of TCDD may have significant effects on immunological function in humans is not known, but recent work by Stehr et al. (1986) (see below) has not detected significant alterations of immune function in residents from TCDD-contaminated areas of Missouri. Chronic exposure of rats to low doses of TCDD also elicited fetotoxicity, decreased body weight gain, increased mortality, changes in hematological structure and function, increased urine concentrations of porphyrins and delta-aminolevulinic acid, increased serum enzyme activity
(alkaline phosphatase, gamma-glutamyl transferase, and glutamic-pyruvic acid transaminase), and morphological changes in respiratory, vascular, and lymphoid tissues
(Kociba et al., 1978). Daily TCDD doses as low as 0.01 or
0.1 ug/kg body weight for two years were effective in eliciting these responses.
Ascertaining TCDD-induced chronic toxicity effects in humans is difficult because known exposures have usually been associated with exposure to co-contaminants such as
2,4,5-T. The major chronic effect in humans appears to be chloracne that may persist for ten years or more after exposure (Kimbrough, 1984). Other symptoms in humans exposed to TCDD have included weight loss, insomnia, muscle pain, increased weariness, irritability, tender and enlarged livers, decreased libido, and increased total serum lipids content.
In a recent study, Stehr et al. (1986) provide some interesting insights into chronic environmental exposure of humans to TCDD. These researchers conducted a- pilot epidemiological study of persons living in Missouri where
TCDD has been present in soils since 1971. The subjects were divided into high-risk and low-risk groups. High-risk subjects had lived or worked for at least six months in areas where TCDD levels in soil exceeded 100 ug/g or for at least two years in areas where soil TCDD levels were between 20 and 100 ug/g; or on the average had participated more than once per week in activities involving close contact with soil (gardening, sports, horseback riding, playing in the soil, etc.) for at least six months in areas where soil TCDD exceeded 100 ug/g or for at least two years where soil TCDD levels were between 20 and 100 ug/g. The longest exposure in the high-risk group was for 12 years; the highest soil TCDD level was 33,000 ug/g. In contrast, the low-risk group had no regular contact with contaminated soil and was matched with the high-risk group (2:1 ratio, high-risk:low-risk) on the basis of type of exposure site, age, sex, race, and socioeconomic status. There were 68 high-risk and 36 low-risk subjects, and each individual received thorough physical, neurological, dermatological, immunological, and blood chemistry examinations.
Few differences were found between the two groups regarding indicators of chronic toxicity. There were no statistically significant signs of general disorders in either group, but statistically non-significant trends toward diminished peripheral pulses, minor musculoskeletal abnormalities, minor pulmonary abnormalities, and increased platelet counts (but within the normal range) occurred in 21 the high-risk group. No significant differences were found between the two groups with respect to renal/urinary tract indicators, dermatology, reproductive health, or immunological function. The low-risk group had significantly higher incidences of palpable axillary nodes and other liver diseases compared to the high-risk group.
These results suggest that risks to the general population, which normally would be exposed to concentrations of TCDD orders of magnitude lower than the high-risk group studied, are low. In an earlier study,
Kimbrough et al. (1984) had suggested that human exposure to soils containing 1 ug/g TCDD constituted an unacceptable risk. Their risk assessment was based on extrapolations from animal toxicity studies. Stehr et al. (1986), however, failed to demonstrate that chronic exposures of humans to TCDD constituted unacceptable risks, in contrast to the suggestions of Kimbrough et al. (1984).
Nevertheless, Stehr et al. (1986) concluded from their study that even though the results failed to show differences attributable to TCDD exposure in the high-risk and low-risk groups, further and more refined epidemiological studies need to be designed that follow larger sample sizes chronically exposed to TCDD.
In addition to toxic effects, TCDD elicits teratogenic and carcinogenic effects in laboratory test animals.
Teratogenicity in mice reportedly is induced at lower doses than for most teratogens (Kimbrough, 1984). Smith et al.
(1976) treated CF-1 mice with TCDD between days 6 and 15 of
gestation and found statistically significant teratogenic
effects (cleft palate and kidney abnormalities) at daily
TCDD doses of 1 or 3 ug/kg body weight; lower doses had no
effects. Murray et al. (1979) found that through three
generations of rats, daily TCDD doses of 0.01 ug/kg body
weight resulted in decreased fertility, reduced litter
size, decreased survival of fetuses and neonates, and
decreased neonatal growth. In humans exposed to TCDD-
contaminated 2,4,5-T, reports of increased spontaneous
abortions have not been substantiated (Kimbrough et al.,
1984). In the epidemiological study of residents from
Missouri discussed above (Stehr et al., 1986), no
reproductive abnormalities were found in the high-risk
group exposed to TCDD soils.
TCDD is mutagenic in Salmonella typhimurium strain TA
1532 and coli strain Sd-4, but not in several other
strains, and is positive for prophage induction in JL. coli
K-39 (Kimbrough et al., 1984). Cell transformation of
hamster kidney cells (Hay, 1982) and mouse teratoma cells
(Knutson and Poland, 1982) (similar to the transformation
that occurs in chloracne in humans) has been reported.
While not proving the carcinogenicity of TCDD, these
results suggest a possible relationship between exposure to
TCDD and tumor development. 23
In other studies, however, the tumorogenic potential
of TCDD has been documented. In male and female rats dosed
daily for two years with TCDD at 0.01 or 0.1 ug/kg body
weight, Kociba et al. (1978) reported significantly
increased incidences of several cancers: hepatocellular
neoplastic nodules, hepatocellular carcinoma, stratified
squamous-cell carcinoma of the hard palate or nasal
turbinates, keratinizing squamous-cell carcinoma of the
lung, adenoma of the adrenal cortex, and pheochromocytoma
of the adrenal gland. In the same study, significantly
fewer incidences of mammary carcinoma, pituitary adenoma,
and benign tumors of the uterus and mammary gland occurred
in the group receiving the highest TCDD dose.
Other studies have also documented the carcinogenicity
of TCDD in laboratory animals. Significantly increased
incidences of thyroid follicular-cell adenoma, liver
neoplastic nodules, hepatocellular carcinoma, ear duct
carcinoma, lymphocytic carcinoma, kidney'adenocarcinoma,
and skin angiosarcoma have been observed after exposure to
TCDD (Kimbrough et al., 1984; NIOSH, 1984).
Establishing the carcinogenicity of TCDD in humans is
plagued by the same problems as those involved in studying the toxicity of the compound, that is, concomitant exposure
to other potentially harmful materials. Nevertheless,
several studies have inferred an increased incidence of
soft-tissue sarcoma in humans exposed to 24
TCDD-contaminated phenoxyacetic herbicides (NIOSH, 1984).
Stehr et al. (1986), however, found no incidences of soft- tissue sarcoma in residents from TCDD-contaminated areas of
Missouri; five cases of cancer in the high-risk group, compared to three cases of cancer in the low-risk group, were considered normal, and none were soft-tissue sarcoma.
The metabolism of TCDD and other dioxins in laboratory animals has not been worked out, although metabolites have been isolated in some species. The tissue distribution of administered HC-TCDD varies among species, but the liver appears to be the site of greatest concentration of the chemical in most species. Accumulation of TCDD in the rat liver follows first-order kinetics and is 5, 12, and 50 times greater in the liver than in fat, kidney, and thymus tissue, respectively (Esposito et al., 1980). In monkeys, however, TCDD tends to accumulate in the skin, fat, and muscles instead of the liver. In an autopsied woman who died from a pre-existing cancer seven months after she was exposed to high concentrations of TCDD in the Sevaso accident, the highest concentration of tissue TCDD was found in fat, followed in order by the pancreas, liver, thyroid, brain, lung, kidney, and blood (Young, 1983).
It has been suggested that chlorinated dibenzodioxins
(and polynuclear aromatic hydrocarbons) bind to specific receptors in the cytosol and then enter the cell nucleus, where a number of enzymes may be induced (Esposito et al., 1980). Depending on the species and the target organ, the
induced enzymes include hepatic aryl hydrocarbon
hydroxylase, other mixed function oxidases of the liver,
UDP glucuronyl transferase, benzopyrene hydroxylase, DT
diaphorase, glutathione transferase, ornithine
decarboxylase, and aldehyde dehydrogenase (Kimbrough et
al., 1984; Esposito et al., 1980). The activity of other
enzymes decreases in the presence of TCDD. These include
UDP-glucuronic acid pyrophosphatase, D-glucuronolactone
dehydrogenase, and L-gluconate dehydrogenase (Esposito et
al., 1980). Enzyme systems unaffected by TCDD exposure
have included NADPH cytochrome, B-glucuronidase, UDP-
glucose dehydrogenase, epoxide hydrase, and glycine N-
acetyl transferase.
Whereas the enzyme activities mentioned above require
specific receptor/dioxin binding, dioxin-induced lipid
peroxidation does not appear to be mediated by binding to a
cytosol receptor (Kimbrough et al., 1984). Lipid
peroxidation precedes the formation of polymeric
lipofuscins and may damage lysosomes and other cell
organelles. Lysosome damage in rats treated with lethal
doses of TCDD has been documented and may explain the
incidences of fatty livers, increased serum cholesterol
esters, free fatty acids, and decreased acid lipase
activity in autopsied rats (Esposito et al., 1980).
Similar lipid effects have been observed in humans exposed 26 to TCDD or TCDD-containing materials (Kimbrough et al.,
1984; NIOSH, 1984), but were not found in the study by
Stehr et al. (1986).
The final biological property of concern regarding
TCDD is its potential for bioaccumulation. Like lipophilic pesticides (such as DDT), TCDD may bioaccumulate in both aquatic and terrestrial biota. The bioaccumulation of TCDD should be less than that for DDT because of the relatively low fat solubility of TCDD compared to DDT. For example, the solubilities of TCDD and DDT in corn oil are approximately 47 and 86,000 mg/1, respectively, and the corresponding ratios of oil- to water-solubility are 0.2 x
106 and 80 x 106 (Norris, 1981). Nevertheless, TCDD has been found to bioaccumulate in some organisms.
The Bioconcentration Factor (BCF) for an environmental pollutant is its tissue concentration divided by its concentration in the environmental matrix (e.g., soil or water). The BCFs for TCDD in some aquatic organisms grown in various laboratory model ecosystems are summarized in
Table 4 (Connell and Miller, 1984). Danhnia. a common aquatic invertebrate that serves as an important food source for fish species, had TCDD BCFs that ranged between
1,762 and 48,000, which was the highest BCF determined among the species tested. It is clear from Table 4 that
TCDD has the potential to bioaccumulate in aquatic plants and animals. The ranges of reported BCFs for individual Table 4. TCDD Bioconcetration Factors (BCF) in aquatic organisms grown in model ecosystems in the laboratory.
Organism BCF
Algae 6 - 2,083
Northernbrook silverside fish 54
Ostracoda 107
Mosquito fish 676 - 24,000
Snails 735 - 24,000
Daphnids 1,762 - 48,000
Catfish 2,000
Mosquito larvae 2,846
Duckweed 4,000 28 species are considerable, indicating the need for definitive analyses of field-grown organisms in order to determine the factors controlling the potential for bioaccumulation.
In terrestrial ecosystems, the data on TCDD bioaccumulation are also somewhat equivocal. Fanelli et al. (1980) examined terrestrial wildlife from the area around Seveso, Italy for the presence of TCDD. Tissue concentrations of TCDD in field mice, rabbits, toads, snakes, and earthworms averaged 4.5, 7.7, 0.2, 2.7, and 12 ng/g, respectively; livers from birds in the area were negative for TCDD. The average soil concentration for TCDD in the area was 3.5 ng/g, suggesting bioaccumulation of
TCDD in rabbits and earthworms. In several other studies reviewed by Esposito et al. (1980), positive results for bioaccumulation of TCDD were found for beach mice, rats, lizards, sparrows, doves, meadowlarks, some insects, and in cow's milk. In other studies, no TCDD bioaccumulation was found in dear, opossum, grasshoppers, Maine birds, sea lions, bald eagles, mountain beavers, or cattle exposed to
TCDD. Recently, Ryan et al. (1986) detected bioaccumulation of TCDD (and 2,3,4,7,8- pentachlorodibenzofuran) in snapping turtles from the St.
Lawrence River; the TCDD was relatively concentrated in fat compared to liver tissue. The authors suggested that bioaccumulation in turtles was due to their predominantly 29
fish diet. On the other hand, O ’Keefe et al. (1986) failed
to detect bioaccumulation of TCDD in goldfish raised in
aquaria with bottom substrates of TCDD-contaminated fly ash
or sediments. The authors suggested that much of the
previous research on fish bioaccumulation of TCDD had made
the compound unrealistically bioavailable through the use
of solvents to make TCDD water-miscible.
Uptake and bioaccumulation of.dioxins in plants have
also been studied. Plant uptake of pesticides and other
chemicals may occur through root absorption from soil or
through leaf translocation of foliarly applied chemicals.
Helling et al. (1973) reviewed several dioxin-related plant
studies and concluded that root uptake of TCDD is not
likely to occur, largely because of the strong adsorption
of TCDD to soil particles. Similarly, translocation of
TCDD through soybean leaves was not detected.
Nevertheless, TCDD adsorption to leaf surfaces was
suggested as a route for the chemical to enter the food
chain (Helling et al., 1973). In later studies of plants
from areas near Seveso, Italy, Cocucci et al. (1979)
reported the presence of TCDD in ug/kg levels in all plants
examined, including fruits, new leaves, twigs, and cork.
Because these studies were conducted a year after the TCDD
accident at Seveso, the presence of TCDD in new tissue
suggests that TCDD was absorbed from soil and/or
translocated to new tissue from storage in other plant parts. The same researchers examined root food crops and found less TCDD in the aerial portions than in the tubers, which in turn had less TCDD than present in the soil. When the plants were transplanted to uncontaminated soil, the
TCDD slowly disappeared, suggesting elimination of the chemical by transpiration, volatilization, metabolism, photodegradation, or some other process.
Norris (1981) has concluded that bioaccumulation of
TCDD is likely to occur only if the compound is present in a bioavailable form. The degree of bioaccumulation also depends on the persistence of the chemical, and Norris
(1981) concluded that significant bioaccumulation of TCDD
(residue levels in excess of 10 ng/kg) has not occurred in forest animals exposed to relatively high concentrations of
2,4,5-T because of natural mechanisms of degradation and dilution of TCDD. However, in an area such as Times Beach, where considerably high levels of TCDD in soils have apparently persisted for more than a decade, the issue of bioaccumulation needs to be investigated.
ENVIRONMENTAL PROPERTIES OF TCDD
TCDD is an environmentally persistent compound, with an environmental half-life possibly as great as 10 to 12 years (Kimbrough et al., 1984). However, the persistence of TCDD in the environment depends on several processes, including photodegradation, mobility, volatility, and 31 chemical and biological degradation. Thus, depending on conditions, the half-life of TCDD would be expected to vary widely among different environments.
For example, in the presence of a proton donor, photodegradation may be the major environmental degradative mechanism for TCDD (Helling et al., 1973; Connell and
Miller, 1984). However, when proton donors are not present, TCDD may be resistant to photodegradation, depending on other environmental conditions. Crosby and
Wong (1977) reported, rapid (within 6 hours) photodegradation of TCDD to dechlorination products when applied in a proton-donating herbicide formulation to soil, leaves, or glass plates and exposed to natural sunlight.
Crosby et al. (1971) reported rapid photodegradation of
TCDD in a methanol solution irradiated with UV light, with the degradation products being 2,3,7-trichlorodibenzo-p- dioxin and smaller quantities of dichlorodibenzodioxin.
However, when aqueous suspensions of TCDD or crystalline
TCDD spread on glass plates were irradiated, no decomposition occurred. These results suggest that TCDD on inert surfaces may be resistant to photodegradation. On the other hand, Gebfuegi et al. (1977) (reviewed in Norris,
1981) found the extent of TCDD degradation under simulated environmental conditions (presumably no proton, donors present) to be 92 and 98% when irradiated for 7 days with longwave or shortwave UV, respectively. 32
These results suggest that environmental
photodegradation of TCDD occurs in sunlight or other UV
radiation sources, but that the rate of decomposition may
vary depending on the presence of proton donors.
Degradation occurs rapidly when proton donors are present,
but may be retarded in their absence. In addition, the
major degradation products are dechlorinated dibenzo
compounds.
Compared to photodegradation, other processes, such as
volatility, mobility, and degradation by chemical or
biological means, appear to be relatively minor fate paths
for TCDD. The vapor pressure of TCDD is on the order of
10-6 mm Hg at 25 C (see Table 1) and thus is not likely to be a major route for the dissipation of TCDD. Recently,
the levels of TCDD detected in urban air samples have been
reported to be the result of combustion of organic wastes,
rather than volatility (C2uczwa and Hites, 1986).
TCDD has been reported to be only slightly mobile in
soils because of its low solubility in water and its
affinity for soil particles, especially clay and organic matter. When 2,7-dichlorodibenzo-p-dioxin or TCDD were added to different types of soil varying in organic matter content, the mobility of both dioxins was inversely related to the soil organic matter content (Kearney et al., 1973).
Many other studies have suggested that the potential for groundwater contamination with TCDD is minimal because of the immobility of the compound in soils (Helling et al.,
1973; Esposito et al., 1980; Kimbrough et al., 1984). Di
Domenico et al. (1982) found that TCDD mobility in soil from the Seveso, Italy site was limited to the upper 8 cm within one year of the TCDD release. Recent work by
Jackson et al. (1986), in which ten different soils from the Times Beach and New Jersey areas were studied, confirmed the low mobility of TCDD in soils, but also found that soil organic matter was not as important in controlling TCDD mobility as had been previously suggested.
The results suggested that for jya situ soils exposed for many years to TCDD, as opposed to clean soils spiked with
TCDD in the laboratory, the factors controlling TCDD mobility were soil electrolytes and chlorinated co contaminants; the presence of soil electrolytes inhibited
TCDD mobility, while the presence of chlorinated co contaminants increased TCDD mobility.
In soils, an accurate TCDD half-life has not been determined, but has been estimated to be between 1 and 3 years (Norris, 1981; Connell and Miller, 1984); the half- life of TCDD in sediments has been reported to be 550 to
590 days (Connell and Miller, 1984). Biodegradation of
TCDD in the natural environment is slow or non-existent, and the major degradative mechanism may be decomposition by ultraviolet light when a proton donor is present. 34
POTENTIAL MICROBIAL METABOLISM: TCDD AND OTHER CHLORINATED
HYDROCARBONS
Carbon cycling in soils and natural waters depends largely on the biodegradation of organic compounds. Non- enzymatic degradative mechanisms are usually of secondary importance compared to biologically mediated transformations of organic compounds, and the role of microorganisms is usually more important than that of higher organisms (Alexander, 1981). Microorganisms have evolved that are effective biodegraders of most aliphatic, heterocyclic, and aromatic compounds, often mineralizing the organics to inorganic compounds such as CO2 , NH3, and
H2O. In other cases, complete mineralization does not occur, yet the organic compound may be biotransformed to intermediates that may or may not be further transformed.
In the environment, as opposed to the laboratory, biodegradation of organic compounds is usually effected by indigenous mixed populations of microorganisms interacting with each other within the limits imposed by their physicochemical surroundings (Alexander, 1981; 1985).
Biodegradation of halogenated organic compounds, such as TCDD, polychlorinated biphenyls (PCBs), hexachlorobenzene (HCB), and halogenated pesticides, is generally more difficult to effect than biodegradation of non-halogenated compounds. As a result, many halogenated organic compounds are environmentally persistent. Hill 35
(1978) reviewed the factors most likely responsible for the environmental persistence of pesticides and other compounds, as follows:
1. The organisms present in the particular environment
may lack the enzymatic or biological potential to
degrade the compound.
2. The compound may be unable to penetrate the cell.
3. The concentration or the physical nature of the
compound in the environment may be inhibitory to
organisms or their enzymes (either intracellular or
extracellular).
4. The compound may be inaccessible due to adsorption
or is coated with impenetrable materials.
5. The steric configuration of the compound may
prevent or hinder enzymatic attack.
6 . The environment may be toxic or deficient in some
factor essential to support the growth of
degradative organisms.
All of these factors may play a role in the environmental persistence of TCDD, as discussed below.
That degradative microorganisms may lack the biological potential to attack TCDD has been examined, the conclusion being that such potential is limited. For example, Matsumura and Benezet (1973) screened approximately 100 strains of pesticide-degrading microorganisms for their ability to metabolize TCDD. The organisms were maintained in liquid axenic culture and the
production of metabolites from ring-labeled HC-TCDD was
measured by thin-layer chromatography and autoradiography.
Five strains were identified that showed some ability to
biotransform TCDD to metabolites, but mineralization was not observed. The degradative organisms included a fungus,
Trichoderma vlride. a bacterium, Pseudomonas pirtMjt, and
three organisms referred to only by coded numbers. Hutter
and Philippi (1982) also screened pure and mixed cultures
of microorganisms for their ability to metabolize TCDD in
liquid culture. Although metabolism was weak despite the
organisms or the cultural conditions used, approximately 1%
of the added material was recovered as a metabolite, but
the production of 14C02 from HC-TCDD did not occur. In
other studies (Philippi et al., 1981), the major metabolite
was identified as l-hydroxy-2,3,7,8-tetrachlorodibenzo-p-
dioxin.
Other studies have confirmed the relatively limited
ability of microorganisms to metabolize TCDD. Quensen and
Matsumura (1983) studied the biodegradation of HC-TCDD by
a pure culture of Bacillus megat^riwn from the ATCC
collection and a soil-isolated Nocardiopsis spp. Again,
limited biodegradation was detected using thin-layer
chromatography and autoradiography. Two unidentified bacteria that showed some ability to degrade TCDD were
isolated from farm soil enriched with napthalene during the 37 same experiments, but metabolism was not extensive. It was suggested, but not demonstrated, that organisms capable of biotransforming TCDD possess a unique mono-oxygenase system for hydroxlating an unsubstituted position on one of the aromatic rings of TCDD.
Recently, Bumpus et al. (1985) reported that a ligninolytic white-rot fungus, Phanerochaete chrysosporium. converted i *C-TCDD to h C O 2 when grown in aerated nitrogen- deficient liquid culture. The organism had been previously found to metabolize chlorinated lignin-like aromatic compounds using extracellular ligninases. When 1.25 nmol of TCDD were incubated for 30 days with IL. chrysosporium. a total of 27.9 pmol of the substrate were converted to CO2 .
In addition to mineralizing TCDD, the organism also mineralized DDT [1,l-bis(4-chlorophenyl)-2,2,2- trichloroethane], lindane (1,2,3,4,5,6- hexachlorocyclohexane), benzo[a]pyrene, and polychlorinated biphenyls (2,4,5,2’,4’,5’-hexachlorobiphenyl and 3,4,3’,4’- tetrachlorobiphenyl) under nitrogen-deficient conditions.
The authors suggested that the same extracellular enzyme system that effects lignin degradation was responsible for degradation of the organohalides, and that other white-rot
Basidiomycetes might possess similar abilities.
In addition to studies with pure cultures of microorganisms, investigators have determined that the potential for biological degradation of TCDD in a wide variety of environmental samples is low. For example,
Kearney et al. (1972) found that TCDD persisted in two
different soil types for at least a year. One of the
soils, Hagerstown silty clay loam, contained 2.5% organic
matter and was initially microbially active. The other
soil, Lakeland loamy sand, contained 0.9% organic matter,
implying relatively limited initial microbial activity.
TCDD was added to both soils at concentrations of 1, 10, or
100 ug/g. After one year, between 54 and 71% of the added
TCDD was recovered from the soils, with little difference between the two soils. Ward and Matsumura (1978) examined
the fate of TCDD in sediment and water from two lakes in
Wisconsin. After incubation periods of up to 589 days,
little metabolism of TCDD was detected by a combination of
TLC, autoradiography, and liquid scintillation counting.
The slight metabolism that was detected was stimulated by the presence of sediment and the addition of nutrients. It
was concluded that the half-life of TCDD in the model aquatic ecosystem, in which photolysis was not expected to be a factor, was approximately 600 days.
Matsumura et al. (1983) showed that TCDD was metabolized slightly in artificial terrestrial and aquatic ecosystems and in an outdoor pond. As in earlier studies
(Ward and Matsumura, 1978), metabolism in artificial aquatic ecosystems was stimulated by the addition of nutrients, and the presence of sediment was necessary for significant metabolism. In artificial terrestrial
ecosystems, nutrients also enhanced metabolism, but the
addition of napthalene to stimulate aromatic-degrading
microorganisms had no effect on TCDD degradation.
Degradation of TCDD in an outdoor pond was also detected, with the TCDD half-life calculated to be approximately one year (Matsumura and Ward, 1976). In this case,
photodegradation may have been a major fate path.
These attempts to stimulate the metabolism of added
TCDD in soils, water, and laboratory media indicate that
organisms in environments previously unexposed to TCDD may
lack the enzymatic or biological potential to degrade the
compound. An alternative approach to studying the
potential for TCDD metabolism in the environment was taken by Camoni et al. (1982). They collected soil containing
TCDD at a concentration of 0.1 mg/kg from the accident site
in Seveso, Italy. Although not explicitly stated in their
paper, presumably the soil was collected at least 2 or 3
years after the TCDD contamination at Seveso. It was
reasoned that degradative organisms may have adapted to the
presence of TCDD and with stimulation, may effect enhanced
metabolism of the compound. A control soil similar in
structure to the Seveso soil was dosed with TCDD to a final
concentration of 0.1 mg/kg. An additional treatment was the presence or absence of extra organic compost with a
total initial microbial count of 2x109 cells/g. The soils 40 were aerobically incubated for 480 days. Periodic analysis for TCDD confirmed the persistence of the compound in all treatments; no differences in TCDD metabolism occurred in the Seveso soil compared to the control soil, or due to the presence or absence of extra organic matter. The slight loss of TCDD in all treatments was attributed to a combination of volatilization, photolysis, irreversible binding to soil, and slow biodegradation.
These studies suggest that microorganisms with the specific ability to metabolize TCDD are not widespread in the environment. Even after relatively long-term exposure to the pollutant, indigenous microorganisms were not effective at degrading soil-bound TCDD.
The second criterion described by Hill (1978) that affects environmental persistence of compounds, i.e., that the compound may not penetrate the cell membrane, also may be important in explaining the apparent resistance of TCDD to microbial metabolism. As noted earlier, TCDD is extremely hydrophobic, has high octanol/water and soil/water partition coefficients, and like many other chlorinated organic compounds, is non-polar. A unique factor about TCDD, however, is that despite its propensity for the solvent phase in solvent/water systems, it is only slightly soluble in most solvents (Quensen and Matsumura,
1983; Connell and Miller, 1984). This suggests that cell membrane penetration may be inhibited because of the low lipid solubility of TCDD and that the limiting factor for
TCDD metabolism in the environment may be the lack of microbial uptake, as suggested by Quensen and Matsumura
(1983). The discovery by Bumpus et al. (1985) that TCDD was enzymatically degraded to CO2 by Phanerochaete chrysosporium also suggests that enzymatic metabolism of
TCDD is possible but limited by membrane transport.
Apparently in this case, the initial attack on the TCDD molecule occurred extracellularly, followed by transport of more polar degradates across the cell wall and membrane for subsequent metabolism.
Matsumura et al. (1983) and Quensen and Matsumura
(1983), in their studies on the metabolism of TCDD by pure cultures of EL megaterlum and Nocardiopsls spp., found that the nature of the solvent carrier was the most important factor affecting TCDD metabolism. The solvents compared included corn oil, ethanol, dimethyl sulfoxide (DMSO), and ethyl acetate. Corn oil inhibited TCDD metabolism, while
DMSO and ethyl acetate significantly stimulated its metabolism compared to ethanol. Ethyl acetate was the most effective solvent in stimulating TCDD metabolism in the presence of additional bacterial nutrients.
Therefore, the second criterion that may result in environmental recalcitrance of organic compounds, that is, the inability of the molecule to cross the cell membrane
(Hill, 1978), may be critical in limiting the potential for 42
TCDD degradation. According to Quensen and Matsumura
(1983), additional research to mobilize TCDD for microbial attack needs to be performed.
A third factor that may cause persistence, the potential inhibition of organisms or their enzymes due to the concentration or the physical nature of the compound in the environment, has received little attention with respect to TCDD. While numerous studies of TCDD toxicity have confirmed its highly toxic nature in higher organisms
(Kimbrough et al., 1984; Esposito et al., 1980), almost no work has been reported on TCDD toxicity to microorganisms.
This is strange considering the attempts to stimulate its microbially mediated biodegradation. That is, if environmental concentrations of. TCDD are highly toxic to aquatic and soil microorganisms, then efforts to effect jja situ biodegradation of the compound may not be feasible.
The studies that have been reported on biodegradation of
TCDD are usually concluded with a statement to the effect that almost no degradation by environmental organisms occurred, yet no effort was reported to ascertain that the microflora was still active after treatment with TCDD. The distinct possibility exists that biodegradation is limited by the toxicity of the TCDD (or in the environment, by co contaminants ).
Bollen and Norris (1979), in possibly one of the only studies published that deals with potential TCDD toxicity to environmental microorganisms, examined forest floor (L,
F, and H horizons from a mixed stand of Douglas fir and red alder) and soil respiration (CO2 evolution) in the presence of TCDD. The concern was that TCDD in 2,4,5-T applied to forests could upset carbon and nutrient cycling in forest soils. TCDD was applied at very low doses (13.1x10-9,
13.1x10-7, or 13.1x10-5 ug/g to forest floor; 5.2x10-9,
5.2x10-7, or 5.2x10-5 ug/g to soil), equivalent to doses expected due to spraying forests with different levels of
2,4,5-T containing 0.1 ug/1 TCDD. The treatments were incubated aerobically for 28 days; evolved CO2 -C was trapped in alkali and determined gravimetrically after precipitation. TCDD had no effect on forest floor respiration but stimulated soil respiration. The authors concluded that the stimulation of soil respiration agreed with what they termed the Heuppe principle (after a nineteenth century bacteriologist): compounds which are lethal at some concentration will inhibit development at certain sub-lethal concentrations and will stimulate biological activity at even smaller concentrations.
Furthermore, the authors suggested that TCDD at higher concentrations than those used in their experiments would have inhibited soil and forest floor respiration, but this hypothesis was not tested.
At a recent workshop on dioxin bioavailability, the working group on ecosystems concluded: "Data on the impact 44 of dioxins [on ecosystems] are essentially nonexistent, and relatively few data are available describing the effects of these chemicals on single species.... Toxic effects of
[dioxins and related compounds] in actual aquatic ecosystems have not been studied" (Dagani, 1984). Clearly, much more work needs to be done on TCDD toxicity to environmental organisms, including microorganisms.
The fourth factor described by Hill (1978) that may limit biodegradation potential is that the compound may be inaccessible due to adsorption or to being coated with impenetrable materials. Adsorption has been proposed as one of the major factors contributing to the recalcitrance of TCDD (Quensen and Matsumura, 1983; Dagani, 1984). It has been shown that TCDD is tightly adsorbed to soils and sediments and thus may be unavailable for microbial attack.
For example, at the accident site in Seveso, TCDD mobility in soil was limited to the upper 8 cm one year after spil contamination (di Domenico et al., 1982). Helling et al.
(1973) found that TCDD was immobile in five different soils with widely differing physicochemical properties. This limited mobility of TCDD is due to its low water solubility
(0.2 ng/1) and relatively high solubility in non-polar solvents such as octanol, giving it a propensity for soil particles. For hydrophobic compounds like TCDD, the octanol-water partition coefficient (Kow) is one of the major factors controlling adsorption to soils and sediments 45
(Weber et al., 1983). When the Kow is greater than 10, the compound is highly partitioned onto soils (Hassett et al.,
1983; Jackson et al., 1986). The average log Kow for TCDD was determined by Marple et al. (1986) to be 6.64, with individual replicates varying between 3.48x106 and
8 .8 7 x 1 0 6 , indicating the great affinity of TCDD for soils and sediments compared to water.
Quensen and Matsumura (1983, 1984) consider the binding of TCDD to soil and sediment to be a major factor limiting the bioavailability of TCDD in the environment.
When TCDD is dislodged from soil, however, it may retain its biological activity. Earthworms fed TCDD-contaminated soils were killed when the TCDD concentration exceeded 5 ug/g; at lower concentrations, no deaths were observed
(Reinecke and Nash, 1984). In the same experiments, direct external contact with up to 199 ug of TCDD did not cause abnormalities in behavior or reproduction through the 85 day observation period. On the other hand, Umbreit et al.
(1986) fed TCDD-contaminated soil from Missouri and New
Jersey to guinea pigs, the most TCDD-susceptible mammal known. No deaths occurred in animals fed any dose of TCDD- soil, while controls fed equivalent doses of TCDD showed typical signs of TCDD-induced wasting disease (thymic atrophy, absence of body fat, loss of body weight, liver and heart enlargement, death). Even though TCDD-soils did not cause death, bioavailability of TCDD from Missouri soil 46
was greater than that from New Jersey soils, based on TCDD
concentrations in autopsied livers.
These results indicate that the nature of the
environmental matrix is critical in regulating the
bioavailability of TCDD, including the potential for
toxicity and biodegradation (Quensen and Matsumura, 1983;
Tsushimoto et al., 1982). Quensen and Matsumura (1984)
have proposed that one of the most important areas of
dioxin research is the development of methods to enhance
its environmental bioavailability in order to effect in
situ biodegradation.
Another factor described by Hill (1978) that may
contribute to the environmental persistence of organic
compounds is that steric configuration may prevent or
hinder enzymatic attack. A classic example of this effect
is the persistence of 2,4,5-T in soil compared to that of
2,4-D. Depending on the soil, the biodegradative half-life
of 2,4-D is on the order of 4 to 5 days (Norris, 1981). In
the same type of soils, the biodegradative half-life of
2,4,5-T is approximately 14 to 21 days. The difference in
half-lives is due to the extra chlorine in 2,4,5-T
substituted meta to the phenoxyacetate group. One pathway
for biodegradation of 2,4-D involves hydroxylation at the
C-5 position of the aromatic moiety due to mixed function oxidases (Hill, 1978; Ghosal et al., 1985). Molecular
oxygen and NADH are required for the initial attack of the 47
aromatic moiety, which generally requires adjacent
unsubstituted carbons. However, in the case of 2,4,5-T,
the meta-substituted chlorine interferes with the
hydroxylation, resulting in retarded biotransformation
compared to 2,4-D.
In the case of TCDD, adjacent positions normally
subject to hydroxylation by oxygenases are occupied by
chlorines. This is also true in 1,3,6,8 tetrachloro-
dibenzo-p-dioxin, which has been shown to undergo minimal
metabolic transformations in soils and sediments (Miur et
al., 1985). Dibenzodioxins chlorinated to a lesser degree
apparently are more susceptible to enzymatic attack. For
example, nc-labeled 2,7-dichlorodibenzo-p-dioxin was
oxidized to i*C0 2 and polar metabolites by soil samples, while TCDD was not extensively oxidized (Kearney et al.,
1972). Klecka and Gibson (1979) examined the potential for metabolism of nonchlorinated dibenzo-p-dioxin in a
Pseudomonas spp. and found that the compound was co oxidized to cis-1.2-dihvdroxy-l.2-dihydrodibenzo-p-dioxin
and 1,2-dihydroxydibenzo-p-dioxin. In later studies using a biphenyl-degrading Bei.ierinokia spp. , Klecka and Gibson
(1980) found that when the organism was grown with
succinate and biphenyl, it co-oxidized nonchlorinated and chlorinated dibenzo-p-dioxins. Monochlorinated dioxins (1- chlorodibenzo- and 2-chlorodibenzo-p-dioxin) were oxidized more extensively than the nonchlorinated compound, but further chlorination substantially inhibited oxidation.
The relative rate of oxidation for monochlorinated dibenzo- p-dioxins was greater than 72% (compared to biphenyl), while for 1,2,4-trichlorodibenzo-p-dioxin the relative rate was only 8.9%. As in earlier studies with Pseudomonas. the first detectable metabolite of the oxidation of nonchlorinated dibenzo-p-dioxin was the cis-dihydroxy compound. However, this compound was converted to 1,2- dihydroxydibenzo-p-dioxin, which proved to be a strong mixed-type inhibitor of 2,3-dihydroxybiphenyl and catechol oxygenases in Bei.lerinckia.
The authors concluded from these studies that nonchlorinated and monochlorinated dibenzo-p-dioxins induced the synthesis in Bei.lerinckia of enzymes able to oxidize the aromatic nucleus. However, higher chlorinated isomers did not induce enzyme synthesis, indicating the inhibitory effect of increasing chlorination on the potential for biotransformation. While the 2,3,7,8 congener was not examined in these studies, it can be concluded from investigations on TCDD and other halogenated compounds, such as polychlorinated and polybrominated biphenyls (Hankin and Sawhney, 1984), halogenated organic pesticides (Dagley et al., 1967), and halogenated organic pollutants (Baker et al., 1980), that tetrachloro- and higher chlorinated dioxin congeners inhibit biotransformation potential to progressively greater 49 degrees. Thus, steric hindrance, coupled with other characteristics of TCDD (low water solubility, high partitioning onto soils and sediments, potential toxicity of the parent compound or metabolites, low bioavailability) probably contributes to its environmental persistence.
The final factor described by Hill (1978) that may contribute to persistence is that the environment itself is either toxic or deficient in some factor essential to support the growth of degradative organisms. Whether this is the case for TCDD is difficult to determine for a couple of reasons. First, even under presumably optimum cultural conditions in the laboratory, TCDD metabolism is slow or nonexistent, as described above. This implies that even if conditions in the environment were optimized for degradative organisms, TCDD might actually be recalcitrant, similar to some plastics (Alexander, 1981). If this is true, manipulation of environmental physicochemical conditions could have no effect on TCDD biodegradation or biotransformation. Second, the overall microbial activity in environments containing TCDD has not been evaluated.
That is, TCDD usually occurs with a number of co contaminants, often including other chlorinated dioxins and dibenzofurans, PCBs, chlorinated pesticides, and other organic wastes (Hay, 1982). Jackson et al. (1986) reported that TCDD-soils from the Times Beach area contain high solvent-extractable contents (0.3 to 19 mg/g) apart from TCDD, indicative of chlorinated and nonchlorinated
semivolatile co-contaminants. This is not surprising
considering that TCDD was applied to Missouri soils along
with used crankcase oils and chemical still bottoms from
the manufacture of trichlorophenols. Similarly, TCDD-soil
from New Jersey contained 22 mg/g solvent-extractable
organic matter (Jackson et al., 1986). Many of the co
contaminants associated with TCDD may be microbially toxic
and could limit the biodegradation potential for TCDD in
the environment. This possibility should be evaluated.
An additional possibility is that co-contaminants act
as preferred substrates and thus act to prevent TCDD
metabolism in the environment. This phenomenon, known as i diauxie, has long been recognized in the preferential
metabolism of particular carbohydrates (for example,
glucose is often preferred over lactose) by pure cultures
of bacteria in the laboratory. Recently, Meyer et al.
(1984) presented evidence that diauxie is also applicable
in the utilization of aromatic hydrocarbons. When either
benzene, phenol, or napthalene was supplied as a sole
carbon source to a mixed microbial inoculum from an oil
refinery settling pond, rapid biodegradation of the parent
compound was detected. However, when benzene and phenol,
or napthalene and phenol, were presented together, phenol was preferentially utilized; metabolism of the additional
substrate did not occur until phenol degradation was nearly complete. The authors pointed out the potential significance of such diauxie growth in the biodegradation of complex organic mixtures, such as complex effluents and chemical spills. That is, if diauxie is environmentally important, laboratory predictions of biodegradation of single organic pollutants may be unrealistic, unless relevant co-contaminants are included in the experimental design. More importantly in the case of TCDD, because of its recalcitrant nature under somewhat optimum laboratory conditions, the presence of co-contaminants that are used preferentially in the environment may further limit its biodegradation potential. This potential phenomenon deserves much more attention.
Obviously, the factors affecting environmental persistence of organic compounds are not mutually exclusive. Factors such as concentration, toxicity, bioavailability, steric configuration, etc., probably interact to control biodegradation potential. Alexander
(1981) has commented on the factors responsible for environmental recalcitrance, and the relationship to TCDD persistence is interesting. According to Alexander, only a narrow range of metabolic pathways have evolved in response to particular chemical structures, and if a xenobiotic compound or its metabolites are too far removed from these pathways, the potential for their degradation is low. In particular, molecules that cannot penetrate the microbial 52 cell in order to induce enzyme synthesis and are not subject to extracellular enzymatic attack will persist. As discussed above, TCDD is only slightly soluble in water and lipids, and is tightly bound to soil and sediment particles. Because of these properties, it is possible that specific TCDD-degradative enzymes have not evolved — not because TCDD metabolism is intrinsically impossible, but because TCDD is not available for recognition by the microbial cell.
Consider, for example, that when TCDD is presented in soluble form to mammalian systems, such as mouse hepatoma cells, a twenty-fold increase in the transcription of the cytochrome Pi-450 gene is induced (Jones et al., 1985).
The translation product is aryl hydrocarbon hydroxylase
(AHH), which functions in the detoxification of TCDD.
Apparently, TCDD binding to specific intracellular protein receptors is required to induce AHH. Detoxification enzymes are induced in other mammals also (Kimbrough et al., 1984).
These results suggest that TCDD, if presented in soluble form, can be biologically recognized and induce specific enzymatic activity. The implication for environmental biodegradation is that TCDD needs to be solubilized and made bioavailable to induce degradative enzymes, as suggested by Quensen and Matsumura (1983).
However, very little work concerning this area has been 53 reported.
SUMMARY
This literature review has illustrated that TCDD is a widely dispersed and persistent environmental pollutant that is produced only by anthropogenic means. It is extremely toxic in many laboratory animals and manifests its toxicity in both acute and chronic ways, although the specific mechanism of TCDD toxicity has not been determined. TCDD toxicity in microorganisms has not been widely examined. TCDD is a suspected carcinogen and teratogen, but biological effects are more difficult to ascertain in humans than in laboratory animals, as evidenced by recent epidemiological studies.
Because of its known and suspected biological effects, the environmental persistence of TCDD has become a subject of concern to both legislators and the public. The widespread contamination of areas such as Missouri in the
United States and Seveso, Italy, have stimulated considerable research into the ultimate environmental fate of TCDD. The evidence reviewed above indicates that TCDD is extremely long-lived, due in part to its microbial recalcitrance. The reason for its microbial recalcitrance has not been fully elucidated, but a distinct possibility is that TCDD is unavailable to enzymatic attack except by extracellular means. Another possibility is that 54
environmental levels of TCDD and or its co-contaminants are
microbidlly toxic and thus inhibit biodegradation.
The clean-up of TCDD-contaminated soils is not going
to be easily solved because the available means of soil
decontamination include incineration and excavation with
stockpiling. At the present time, these options are not
economically feasible on a very large scale.
An attractive but unproven alternative could be to
manage decontaminated soils in a manner that stimulates in
situ biodegradation of TCDD. Research discussed in this
literature review has shown limited solvent-dependent
microbial transformation of TCDD in axenic bacterial
cultures. In soils or in situ, microbial transformation of
TCDD has not been detected. However, no attempts to
mobilize TCDD in soils in order to stimulate biodegradation
have been reported, possibly because of the potential
toxicity of bioavailable TCDD and the potential to increase
groundwater contamination. The development of a cost-
effective method to enhance TCDD bioavailability to
indigenous microorganisms without toxifying the soil system may lead to large-scale soil decontamination through biological means. Such an attempt is the subject of the
research described in the following chapters. CHAPTER II
MATERIALS AND METHODS
SOIL CHARACTERIZATION
Surface soils (top 15 cm) from locations in and near
Times Beach, Missouri were collected in labeled, plastic- lined cans using shovels. Several kilograms of soil were obtained at each location. Immediately after collection, the cans were sealed, shipped to the laboratory, and refrigerated in the dark at 4 C under field-moist conditions. The soils were designated as Times Beach (TB),
Piazza Road (PR), or Shennendoah Stables (SS). In addition, a soil sample was collected in a similar manner from a pesticide manufacturing plant in New Jersey and stored in the same way as the Times Beach soils. This sample was designated as soil NJ.
The soils were characterized for several physicochemical parameters. The field moisture content of the soils was determined gravimetrically. Triplicate samples of moist soil in tared aluminum pans were weighed, dried overnight at 105 C, and weighed again after cooling in a desiccator. The moisture content was calculated as follows:
55 56
% moisture =[(A - B)/(B - C)] x 100 where:
A = moist weight of soil and pan,
B = dry weight of soil and pan, and
C = weight of empty pan.
The field capacity (FC) of the soils was determined using a procedure developed by the U.S. EPA at Corvallis,
Oregon (personal communication). The tare weight of a 0.95
1 glass canning jar containing a small hole drilled in its side, the jar lid-band, a 7 cm diameter piece of Whatman no. 1 filter paper, and a 7 cm diameter piece of copper mesh was determined. Approximately 0.5 1 of air-dry, sieved (<2.00 mm mesh) soil was added to the jar, the jar was closed with the filter paper, then the copper mesh, and finally the lid-band, and the weight of the soil and the closed jar was determined. The jar was inverted, tapped gently on the lab bench several times to settle the soil, and placed inverted in a pan of distilled water until the soil surface was visibly moist. The jar was removed from the water, allowed to drain in the inverted position for 24 hours, and weighed. The percent FC was calculated as follows:
FC =([(A-B)+(B-C)D]/[(B-C)-(B-C)D]) x 100 57 where:
A = weight of drained (moist) soiland jar,
B = weight of dry soil and jar,
C = weight of jar, and
D = moisture content of air-dry soil.
D was determined in a manner similar to the determination of the field-moisture content described above.
TCDD concentrations in the soils were determined by high-resolution gas chromatography-mass spectrometry, after extraction of the soil in the following manner. One- hundred microliters of 13C-TCDD spiking solution were added to 5.0 g of air-dry sieved (<2.00 mm mesh) TCDD soil. The spiking solution was added in small portions at several sites on the soil surface in the extracting jar.
Approximately 20 g of purified anhydrous ammonium sulfate were mixed thoroughly with the spiked TCDD soils using a stainless steel spatula. The mixtures were incubated at room temperature for 2 hours, mixed again to break any visible lumps, and incubated an additional 4 hours. After mixing again, 20 ml of methanol were mixed with each sample, followed by the addition of 150 ml of hexane. The extraction jars were then shaken at room temperature for 3 hours, allowed to settle overnight, and the solvent phase was decanted from each jar and passed through a glass funnel containing hexane-rinsed filter paper (Whatman no. 58
4). The jars, solid residues, and filter residues were rinsed with four 5-ml portions of hexane.
The extract volumes were combined and concentrated in evaporator flasks to approximately 2 to 3 ml in a Kuderna-
Danish apparatus. Concentrated extracts were transferred to 8-ml glass culture tubes. Evaporator flasks were rinsed with three 5-ml portions of hexane, which were then added to the culture tubes. Between additions of the hexane rinses, the extract volumes in the culture tubes were reduced enough to allow addition of another 5-ml volume of rinse, by incubation in a water bath held at 80 C. After addition of the final rinse, the extract volume was reduced to approximately 1 m l .
Column chromatography was used to separate the extracts. The first column consisted of, in order 1.0 g of activated silica gel, 2.0 g of sodium-impregnated silica gel, 1.0 g of activated silica gel, 4.0 g of sulfuric acid- impregnated silica gel, and 2.0 g of activated silica gel, in a 1 cm x 20 cm glass column. The second column contained 18.0 g of basic alumina, activated at 300 C for
90 min, in a 1 cm x 30 cm glass column. During the preparation of the columns, hexane was added until the packing was free of air bubbles and channels. The hexane extracts of the samples from the culture tubes' were quantitatively transferred to the top of column 1, the culture tubes were rinsed with two 0.5 ml portions of hexane, and the rinses were added to column 1. The extracts were then eluted from the first column with 90 ml of hexane, and the eluates were concentrated with filtered nitrogen and transferred to the top of column 2 along with three 2-ml hexane rinses of the concentrating vial. Fifteen milliliters of a mixture of 3% (v/v) methylene chloride in hexane were added to the top of column 2 and eluted with 40 ml of 50% (v/v) methylene chloride in hexane. The entire eluate was concentrated to 1.0 ml in a conical mini-vial using a gentle stream of filtered nitrogen.
The capillary column gas chromatography/high resolution mass spectrometry (HRGC/HRMS) system used for sample analyses consisted of a Carlo Erba Model 4160 gas chromatograph interfaced directly into the ion source of a
VG Model 7070H high resolution mass spectrometer. The chromatographic column was a 60 m CP Sil-88 fused silica capillary column. Helium was used as a carrier gas at a flow rate of approximately 30 cm/sec. The mass spectrometer was operated in the electron impact ionisation mode (El) at a mass resolution of 9000-12000 (M/Mdeita, 10% valley definition). All HRGC/HRMS data were acquired by multiple-ion-detection (MID) using a VG Model 2035 Data
System. The exact masses monitored for 2,3,7,8-TCDD were
319.8965/321.8936, and for 13C-1,2,3,4- and -2,3,7,8-TCDD were 331.9367/333.9338. A standard solution containing 60
2,3,7,8-TCDD; 13C-l,2,3,4-TCDD; and i 3C-2,3,7,8-TCDD was analyzed to obtain the response factor. The formula used for native response factor determination was:
Native response factor = [ (Ac ) (Qi s ) ]/[ (Ai s ) (Qc ), where:
Ac = sum of integrated areas for the native isomer,
-Qis = quantity of internal standard,
Ais = sum of integrated areas for the internal standards,
Qc = quantity of target isomer.
An internal standard (quantification compound) of 2,3,7,8-
TCDD and 13C-2,3,7,8-TCDD was used to quantify the native
TCDD congener. The formula for quantification was:
Quantity (pg/g) = [ (Ac ) (Qi s ) ]/[ (Ai s ) (Rf ) (wt) ] , where:
Ac = sum of integrated areas for the target congener,
Qis = quantity of internal standard in pg,
Ais = sum of integrated areas for the internal standard,
Rf = response factor,
wt = soil weight, in grams.
The criteria used to identify the TCDD isomer were:
1) Simultaneous responses at both ion masses,
2) Chlorine isotope ratio within 15 percent of theoretical value,
3) Signal-to-noise ratio equal to or greater than 2.5
to 1, and
4) Chromatographic elution times within 2 seconds of
the isotopically labelled analog.
Recovery response factors were calculated by comparing the internal standard responses to the responses of 13C-
1,2,3,4-TCDD, according to the following formula:
Recovery response factor = [(Ais)(Qrs)]/[(Ars)(Qis)], where:
Ais = sum of integrated areas for the internal standard,
Qrs = quantity of recovery standard in ng,
Are = sum of integrated areas for the recovery standard,
Qis = quantity of selected internal standard in ng.
Recovery of the internal standard was calculated as follows:
Recovery (%) = [ (Ai s ) (Qr s ) ]/[ (Ar a ) (Qi a ) (RRF) ] (100), where:
Ais = sum of integrated areas for the internal standard,
Qrs = quantity of recovery standard,
Ars = sum of integrated areas for the recovery standard,
Qis = quantity of internal standard,
RRF = recovery response factor. 62
Soil pH was determined according to the method of
Mclean (1980). Air-dried, sieved soil (<2.00 mm mesh) was mixed with distilled water in a 1:1 (weight:volume) soil:water ratio. The pH of the constantly stirred mixture was determined to the nearest 0.1 pH unit using a glass indicating electrode paired with a calomel reference electrode in combination with an Orion model 601A pH/millivolt meter. The meter and electrodes were calibrated with standardized buffer solutions prior to the analysis.
The organic matter of the soils was determined according to the method of Schulte (1980). One gram of air-dry, 2.00 mm mesh sieved soil was mixed with 10 ml of
2N K2 Cr2 07 and 20 ml of concentrated H2 SO4 . After 1 hour,
100 ml of 0.5% (w/v) BaCl2 were added to the flasks, which were then incubated overnight at room temperature. The absorbance at 611 nm (A6ii) of the supernatant solution due to Cr3+ was determined on a Varian model 634 spectrophotometer. The results were compared to a standard curve at Aen that was prepared by oxidizing 0, 0.5, 1.0,
1.5, 2.0, and 2.5 g portions of a soil of a standard organic matter content, in the same manner as described for the TCDD soils. The standard soil was obtained from The
Ohio State University Department of Agronomy.
The electrical conductivity (EC) of the soils, which is an indication of the soluble salts content, was (
63 determined using the method of Watson (1978). Twenty grams of air-dry, 2.00 mm mesh sieved soil were equilibrated for
30 min with 40 ml of deionized water. The supernate was filtered through Whatman no. 1 paper and pipetted into a calibrated conductivity cell (Yellow Springs Instrument
[YSI] model 3401 platinized platinum-iridium electrode with a cell constant of K=1.0/cm). The EC was determined with a
YSI model 32 digital display conductance meter.
The cation exchange capacity (CEC) of the soils, an indication of the capacity of the soils to bind important nutrient ions such as Mg, Ca, and K, was determined using the method described by Allen et al. (1974). Five grams of air-dry, sieved (<2.00 mm) soil were mixed vigorously for 1 hour with 125 ml of 1M ammonium acetate in order to displace other cations and saturate exchange sites with
NH4+. The mixture was vacuum filtered through Whatman no.
44 paper and washed repeatedly with portions of an industrial ethanol solution (60 ml of denatured ethanol plus 40 ml of distilled water); the washings were discarded. Next, to displace sorbed NEU+, the soils were leached with 20-30 ml portions of 5% (w/v) KC1 until a total of 100 ml of leachate in a volumetric flask were collected. The concentration of the NH4 + -N in the leachate was determined using an ammonium ion-specific electrode attached to an Orion model 601A pH/millivolt meter and compared to a standard curve of 1, 10, 100, and 1000 ug/ml 64 NH4+-N prepared from a 0.1M NHkCl Orion standard. The CEC was expressed as milliequivalents (meq) per 100 g of soil as follows:
CEC=[(ug/ml NH4+-N)(100 ml leachate)(1 mg/1000 ug)]/ [(5 g air-dry soil)(1-moisture content)(18 mg/meq)].
The specific surface area of the soils was determined using an ethylene glycol monoethyl ether method (EGME) adapted from Carter et al. (1965), Heilman et al. (1965), and Cihacek and Bremner (1979). In triplicate, 1.1 g air- dry, sieved (<2.00 mm) soil was added to aluminum weighing pans that were tared to the nearest 0.001 g. After the pans of soil were vacuum desiccated to a constant weight
(to the nearest 0.001 g), 3.0 ml of reagent grade EGME were added to each pan and allowed to equilibrate for 1 hour.
The pans of soil were vacuum desiccated to constant weight
(nearest 0.001 g) over several days. The total surface area (TSA) was calculated by dividing the grams of EGME adsorbed per gram of soil by 0.000286 g/m2 , the Dyal-
Hendricks value for the weight of EGME required to form a monolayer on a surface area of 1 m2 . That is,
TSA=(a-b)/[(0.000286 g/m2 )(b-c)]' where:
a = weight of desiccated soil, EGME, and pan; 65
b = weight of desiccated soil and pan; and
c = tare weight of pan.
Soil particle size distribution was determined using a combination of sieving and sedimentation, according to methods described by the American Society for Testing and
Materials (ASTM) (1972, 1978). A weighed portion of air- dry soil that was gently ground and mixed with a mortar and pestle was separated into three fractions using sieves of the following pore size: >4.75 mm, <2.00 mm, and between
4.75 and 2.00 mm. The fractions were weighed and the portion passing the 2.00 mm sieve was fractionated further.
Fifty grams of the less-than-2.00-mm soil were mixed with
125 ml of sodium hexametaphosphate solution (40 g/1) and allowed to stand at room temperature for at least 16 hours.
The soil mixture was then vigorously dispersed with a high speed electric mixer, transferred to a 1000 ml glass graduated cylinder, and brought to volume with distilled water. The cylinder was sealed with parafilm, inverted repeatedly, and placed on a benchtop to settle. At 2, 5,
15, 30, 60, 250, and 1440 min, a sedimentation reading was obtained using an ASTM hydrometer. After the last reading, the suspension was poured onto a 75 urn sieve, washed with several changes of distilled water, transferred to a beaker, and dried overnight at 105 C. The dried material was then fractionated on the following sieves: 850, 425, 250, 106, and 75 um pore sizes, and <75 um. The portions greater than 4.75 mm (A), between 4.75 and 2.00 mm
(B), and less than 2.00 mm (C) were calculated as follows:
[Fraction of interest (in grams)/(A + B + C)] x 100.
The percentage (P) of soil remaining in suspension at the level at which the hydrometer measured the density of the suspension was calculated as follows:
P = [1-(Ra/W)] x 100 where:
R = hydrometer reading minus a hydrometer-specific
composite correction factor,
a = a correction factor for the model of hydrometer
used, and in most cases equals 1.00 for a specific
gravity of 2.65 for soil,
W = oven-dry mass of soil dispersed in the hydrometer
test.
Finally, the diameter (D) of the particles corresponding to the percentage indicated by a particular hydrometer reading was calculated as follows:
D = K (L/T)i/2 where: 67
K = a constant that is dependent on temperature and
specific gravity (equal to 0.01332 at 22 C and
2.65 specific gravity; other values are in ASTM,
1972),
L = distance from the surface of the suspension to
the level at which the density of the suspension
is measured, in cm; this is known as the
effective depth and was determined from ASTM
(1972),
T = interval of time from the beginning of
sedimentation to the taking of the reading,
in minutes.
To determine the total solvent-extractable content
(TSEC) in the TCDD soils, the method of Warner (1978) was
used. Thirty grams of soil were mixed for 16 hours with 30 ml of methyl tert-butyl ether (MTBE) and 30 ml of a
solution containing 2% (w/v) NaCl and 30% (w/v) KH2 PO4 .
After allowing phase separation of the settled mixture to
occur, the MTBE and aqueous layers were transferred to a separatory funnel, allowed to settle further, and the aqueous layer was discarded. The MTBE was mixed with 2 g of anhydrous MgS04, allowed to stand for a few minutes, and
25 ml of the clear extract were concentrated to 0.5-0.8 ml in a Kuderna-Danish apparatus in a water bath at 80-90 C.
The concentrated extract was filtered through a 0.5 um 68
membrane filter and brought to volume with MTBE in a 100 ml
volumetric flask. Finally, 100 ul of the extract were
evaporated to dryness in a tared aluminum weighing pan and
weighed to determine the residue weight. The TSEC (in ug/g
soil) was calculated as follows:
TSEC = (Wr x 10)/25
where:
Wr = residue weight (ug) of the 100 ul of
concentratred extract.
MICROBIAL ACTIVITY IN TCDD SOILS
Microbial Enumeration
Enumeration of microorganisms was carried out by the
soil dilution spread-plating method. Ten-fold dilutions of
the soils were prepared in sterile water blanks according
to Wollum (1982). Ten grams of field-moist soil were added
to a 90-ml sterile water blank and dispersed by vigorous
shaking. Subsequent ten-fold dilutions were prepared by
transferring 1.0 ml into sterile 99-ml blanks, through a final dilution of 10-8. From each of five appropriate
dilutions, 0.1 ml was plated in replicates of five on the media specific to the microbial group of interest.
Eutrophic bacteria were enumerated on nutrient agar plates incubated aerobically at 25 C for 4 to 7 days.
Oligotrophic bacteria were enumerated 'on 100-fold dilution nutrient agar plates (Ohta and Hattori, 1983) incubated aerobically at 25 C for 20 to 25 days. Actinomycetes were cultured on starch-casein agar plates incubated aerobically at 25 C for 15 days (Williams and Wellington, 1982).
Starch-casein agar consisted of (per liter): soluble starch, 10 g; vitamin-free casein, 0.3 g; KN03, 2.0 g;
NaCl, 2.0 g; K2 HPO4 , 2.0 g; MgS04, 0.01 g; CaC03, 0.02 g;
FeS04-H2 O, 0.02 g, agar, 15 g; adjusted to pH 7 after autoclaving. Fungi were enumerated on rose-bengal agar incubated aerobically at 25 C for 4 to 7 days (Parkinson,
1982). Rose-bengal agar consisted of (per liter): glucose,
10 g; peptone, 5.0 g; K2 HPO4 , 0.5 g; MgS04, 0.24 g; rose- bengal, 0.03 g; yeast extract, 0.5 g; agar, 15 g, and 0.03 g of streptomycin sulfate (added as a filter-sterilized solution after the agar had been autoclaved and cooled to approximately 50 C). Enumeration results were analyzed for statistically significant effects using one-way Analysis of
Variance (ANOVA) and mean separation by Duncan’s Multiple
Range Test.
Soil Enzyme Activity
Soil enzymes, including acid phosphatase, alkaline phosphatase, arylsulfatase (arylsulfate sulfohydrolase), rhodanese (thiosulfate-cyanide sulfurtransferase), and dehydrogenase were analysed according to the methods of
Tabatabai (1982). In all cases, results were analyzed for 70
statistically significant differences using one-way ANOVA
and Duncan’s Multiple Range Test.
For an acid phosphatase (AP) assay, 1.0 g of field-
moist, sieved (<2.00 mm) soil was combined with 0.2 ml of
toluene, 4.0 ml of pH 6.5 modifed universal buffer (MUB),
and 1.0 ml of p-nitrophenol phosphate solution (PNP) for 1
hour at 37 C. Controls received no PNP. MUB stock
solution contained 12.1 g of tris(hydroxymethyl)amino-
methane (THAM), 11.6 g of maleic acid, 14.0 g of citric
acid, 6.3 g of H3 BO4 , and 488 ml of 1.0N NaOH, in a final
volume in distilled water of 1.0 1. The MUB for the assay
was prepared by titrating 200 ml of MUB stock solution to
pH 6.5 with 0.1N HC1, bringing the final volume to 1.0 1
with distilled water. PNP consisted of 0.420 g of p-
nitrophenyl phosphate tetrahydrate (Sigma 104, Sigma, St.
Louis) in 40 ml of pH 6.5 MUB and diluted to 50.0 ml with
distilled water. The 1-hour incubation was stopped by the
addition of 1 ml of 0.5M CaCl2 , 4 ml of 0.5N NaOH, and
filtration of the mixture through Whatman no. 2 filter
paper. Controls received PNP just prior to filtration.
The p-nitrophenol (PN) released to the filtrate due to AP
activity was determined colorimetrically at 410 nm and compared to standards of PN. Alkaline phosphatase activity
in the soils was analyzed in a similar manner,' except that
MUB was adjusted to pH 11 using 0.1N NaOH. All phosphatase activity measurements were carried out in triplicate. 71
For the analysis of arylsulfatase, 1 g of field-moist, sieved (<2.00 mm) soil, 0.25 ml of toluene, 4.0 ml of acetate buffer (0.5M, pH 5.8), and 1.0 ml of p-nitrophenyl sulfate (PNS, Sigma) (0.025M in acetate buffer) were mixed and incubated at 37 C, After 1 hour, the reaction was stopped and the PN released determined colorimetrically as described for AP. For the controls, PNS was added just prior to filtration. All assays were done in triplicate.
Rhodanese was analyzed by incubating 4 g of soil with
0.5 ml of toluene, 8.0 ml of THAM-H2 SO4 buffer (0.05M THAM, pH 6.0), 1.0 ml of 0.1M Na2S03, and 1.0 ml of 0.1M KCN for
1 hour at 37 C. The incubation was stopped by the addition of 10.0 ml of CaS04-formaldehyde (900 ml of 0.01M
CaS04- 2 H2 O and 100 ml of formaldehyde). The soil was then filtered through Whatman no. 2 paper and 5.0 ml of the filtrate were combined with 1.0 ml of ferric nitrate reagent [0.25M Fe(NOs)3- 9H2 0-3.IN HNOs]. The SCN" formed as a result of rhodanese activity was determined by measuring the absorbance at 460 nm, compared to standards of thiocyanate. Sterile soils were analyzed simultaneously as controls for non-biological production of SCN". All assays were done in triplicate.
For dehydrogenase activity, 20 g of air-dry, sieved
(<2.00 mm) soil were combined with 0.2 g of CaC03. Three
6-g portions of this mixture were each incubated at 37 C with 1.0 ml of 3% (w/v) 2,3,5-triphenyltetrazolium chloride 72
and 2.5 ml of distilled water. After 24 hours, the soils
were removed from incubation, mixed with 10 ml of reagent-
grade methanol, and quantitatively filtered through
absorbent cotton. The red color resulting from the
production of triphenyl formazan (TPF) by soil dehydrogenase was washed from the cotton into a 100 ml
volumetric flask using 10-ml portions of methanol; the
flask was brought to volume with additional methanol. TPF
was determined colorimetrically at 485 nm and compared to a
standard curve.
Soil Respiration
Soil respiration (C02 evolution) was measured on the
TCDD soils and a fertile, non-TCDD agricultural soil from
West Jefferson, Ohio (soil WJ) using the method of Arthur et al. (1984). The soil respiration test was done to test
for microbial activity, i.e, the potential to metabolize an organic substrate in the presence of TCDD, and thus measures potential soil toxicity. Soil WJ was added to vary the concentration of TCDD in each treatment and to
introduce different strengths of a mixed microbial inoculum unexposed to TCDD.
In triplicate, 50 g (oven-dry basis) of field-moist, sieved (<2.0 mm) soil were amended with 0.5 g of finely ground alfalfa meal and placed in 0.5 1 acid-washed glass canning jars. The treatments were as follows (all weights 73
are on an oven-dry basis; soils were at field-moisture
content):
Treatment TCDD Soil lai Non-TCDD Soil (g)
1 0 50
2 12.5 37.5
3 25.0 25.0
4 37.5 12.5
5 50.0 0
Distilled water was added to each jar to bring the soils to * 70% of their field capacity, while taking into account the
moisture already in the field-moist soils. An alkali trap,
consisting of 10.0 ml of 0.6N NaOH in a small nalgene
container, was placed in each jar. The alkali trap did not
sit on the soil surface but rested on a nalgene platform
that was designed to minimize the inhibition of gas
exchange between the soil surface and the container head
space. Blank jars contained alkali traps but no soil; positively inhibited controls contained undiluted TCDD soil
that was amended with 1000 ug/g of Cd as CdCl2 . Each jar was loosely capped with a canning lid and incubated at 22 C
in the dark. Throughout the incubation, the soil moisture was maintained at 70% field capacity by monitoring the loss
in weight of the jars, returning them to their original weights with distilled water. Periodically over 50 days, the jars were aerated and the traps were recharged with fresh alkali. Absorbed CO2 was precipitated by the addition of 5.0 ml of 1.3N BaCl2- 2H2O, and the used alkali was titrated to pH 9.0 with tris-standardized 0.6N HC1 using a Fisher Titralyzer II autotitrator. Evolution of
CO2-C for each replicate was calculated as follows:
CO2-C = (B-V)NE where:
B = ml of acid to titrate method blanks,
V = ml of acid to titrate treatment traps,
N = normality of the standardized acid, and
E = equivalent weight of CO2-C, i.e. 6 meq/mg.
Cummulative evolution of CO2-C was determined by summing the results of individual replicates. These results were analyzed for statistically significant differences using one-way ANOVA and Duncan’s Multiple Range Test.
MICROBIAL MINERALIZATION OF TCDD
A design similar to that described above was used to examine the mineralization of HC-TCDD in TCDD soils that had been shown to be microbially active. In addition, the influence of inorganic fertilizer and dilution with fresh, fertile soil was examined. A stock solution containing 0.1 uCi/ml of 14C-TCDD in acetone was prepared from a solution containing 0.1936 mg of HC-TCDD in 0.3 ml of toluene and having a specific activity of 33.24 mCi/mmol. The TCDD was a gift from Monsanto (St. Louis, MO). Five treatments (in triplicate) of soils TB, PR, and SS, containing 0.008, 1.1, and 2.4 ug/g TCDD, respectively, were used for this experiment. The TCDD soils were diluted with soil WJ as described earlier in order to provide a range of TCDD concentrations. In addition, an identical set of triplicate treatments was amended with 5.0 ml of Hoagland’s nutrient solution, a complete inorganic fertilizer.
After the soils were thoroughly mixed in the jars in the appropriate proportions as described above, 0.1 uCi of
14C-TCDD in a total of 10.0 ml of acetone was added to each jar except background controls (equivalent to 4400 disintegrations/min/g [DPM/g]). The soils were mixed by stirring, allowed to stand uncovered for approximately 1 hour to evaporate the acetone, and were mixed again., The soils were then adjusted to 70% field capacity with either distilled water or, in the case of the fertilized treatments, with Hoagland’s solution and water. An alkali trap containing 10.0 ml of 0.6N NaOH in a small nalgene container was added to each jar; the trap rested on a small nalgene platform off of the soil surface. The jars of soil were dark-incubated at 22 C for approximately 1 year.
Periodically, the jars were aerated, the weights of the jars were returned to the original with distilled water, 76
and the alkali traps were recharged with fresh alkali.
Whenever the traps were changed, a 1-ml aliquot of the used
trap was mixed with 10.0 ml of Aquasol 2 liquid
scintillation cocktail (New England Nuclear, Boston, MA) in
a liquid scintillation vial. Each vial was counted for 10 minutes in a Searle model Mark III refrigerated liquid
scintillation counter. Counting efficiency was determined
using quenched external i*C standards (New England
Nuclear).
SURFACTANT EXPERIMENTS
Methods to increase the bioavailability of TCDD in
soils were investigated. The treatments to improve TCDD bioavailability needed to be relatively mild to prevent poisoning the soil system. Therefore, several surfactants were evaluated for their usefulness as non-toxic TCDD- solubilizing agents.
Potential surfactants were first identified by selecting those recommended as solubilizers and eulsifiers of chlorinated pesticides. Of the more than 10,000 surfactants listed in McCutcheon’s Manual of Sufactants and
Emulsifiers, approximately 30 were selected as potential candidates, based on their suggested use with chlorinated pesticides and their reported low toxicity. After discussions with technical representatives from several manufacturers, nine surfactants were chosen for further investigations. The surfactants included the following:
Jjuri.agtaat Agrimul A-300 Alkyl aryl polyether alcohol (nonionic)
Agrimul J-l Alkyl aryl polyether alcohol (nonionic)
Agrimul 70 Alkyl aryl polyether alcohol (nonionic)
Agriwet Alkyl aryl polyether alcohol (nonionic)
Cetiol HE Polyethylene glycol 7 glyceryl cocoate
Conco sulfate 3B Sodium decyl diphenyl ether disulfonate
Morwet 425 Sodium napthalene formaldehyde (anionic)
Morwet EFW A proprietary anionic emulsifier
Petrodispersant Sodium napthalene sulfonate (anionic)
Surfactant Toxicity Experiments
The toxicity of the surfactants to microorganisms was evaluated in order to avoid poisoning the TCDD-soil system with toxic surfactants. Three toxicity screening methods were used: 1) plate counts of viable Pseudomonas aeruginosa exposed to various concentrations of the surfactants; 2) concentration-dependent inhibition of bioluminescence by
Photobacterium phosphoreum; and 3) concentration-dependent inhibition of soil respiration.
To conduct the toxicity test with JL. aeruginosa, five concentrations of each of the surfactants were- prepared in sterile distilled water in test tubes, using ten-fold dilutions. The surfactant concentrations were 0, 0.01, 0 .1, 1.0, and 10% (v/v for liquid surfactants, w/v for
powdered surfactants) in a final volume of 9.0 ml. P.
aeruginosa was reconstituted from a lyophilized culture
(American Type Culture Collection, Baltimore, MD) according
to the supplier’s instructions and cultured in nutrient
broth at 25 C. One-tenth ml of a 24-hour culture of P.
aeruginosa was added to each concentration of surfactant,
and the test tubes were mixed by vortexing for several
seconds and were incubated overnight at 25 C in the dark.
Then, 0.1 ml of each tube was pipetted onto triplicate
plates of nutrient agar and spread to dryness with a
sterile glass rod. The plates were inverted and incubated
at 25 C in the dark for 48 hrs. Plates that developed
between 30 and 300 colonies were counted with the aid of an
AO Quebec model 3327 darkfield colony counter. The
dilution factor was taken into account when expressing the
results.
The Microtox assay was used to test surfactant toxicity to bioluminescence by phosphoreum. The
Microtox analyzer (Beckman Instruments, Los Angeles, CA) was operated according to the manufacturer’s instructions
for determining the ECso for each surfactant, i.e., the effective concentration that causes a 50% reduction in bioluminescence by phosphoreum.
Soil respiration was also used to test for surfactant toxicity to soil microorganisms. To conduct surfactant toxicity tests using soil respiration (CO2 evolution) a
method similar to that described earlier was used. In
triplicate, 50 g (oven-dry weight basis) of West Jefferson
soil (2.00 mm mesh sieved, 14.6% field-moisture content,
amended with 0.5 g of finely ground alfalfa meal) in 0.95-1
glass canning jars were amended with surfactant solutions
in order to bring the soil moisture content to 70% of field
capacity. The final soil concentrations of surfactants
were 0.1, 1.0, and 5.0% (w/w) of Morwet 425 and similar
concentrations (v/w) of Agrimul 70. These surfactants were
chosen because of their ability to solubilize
hexachlorobenzene (HCB) (see below), a surrogate compound
for TCDD. Controls included no surfactant and the 1.0 and
5.0% surfactant concentrations amended further with 2000 ug/g Cd as CdCl2 . The jars were incubated in the dark at
25 C. Periodically over 40 days, jars were aerated and the alkali traps (10 ml of 0.6N NaOH) in each jar were changed
and titrated as before with standardized 0.6N HC1.
Cummulative evolution of CO2-C was calculated and
statistically analyzed as described before.
Evaluation of Solubilizing Potential of Surfactants
In an effort to increase the bioavailability of soil- bound chlorinated organics, four different experiments were
conducted to evaluate the potential of surfactants to mobilize HCB and TCDD. These included the ability of 80 surfactants to 1) solubilize HCB, 2) extract HCB from soil,
3) extract i^C-HCB from soil, and 4) extract HC-TCDD from soil. HCB was used as a surrogate compound because its soil binding properties and solubilities in various solvents are similar to that of TCDD, and it is readily available for experimentation, unlike TCDD.
The solubility of HCB in 0.1, 0.5, 1.0, and 5.0% (w/v or v/v, as appropriate) concentrations of surfactants was evaluated. In triplicate, 0.05 g of HCB was added to 5.0 ml of surfactants in acid-washed glass test tubes. This was excessive HCB in all cases, as evidenced by a precipitate in all tubes. Controls included distilled water with or without 0.05 g of HCB. The tubes were stoppered, repeatedly vortexed for several minutes, and incubated in the dark overnight at room temperature. Then, the tubes were vortexed again and centrifuged for 10 min at
600 rpm on an IEC HN-SN II tabletop centrifuged. The supernates were filtered through 0.22 um membrane filters into acid-washed glass test tubes. A total of 6 ml of benzene was added in 2-ml portions to the filtered supernates in order to extract the dissolved HCB. After each portion of benzene was added, the tubes were vortexed and centrifuged as before. The benzene layers were removed with a Pasteur pipet, combined for each replicate treatment, and evaporated to 1.0 ml over nitrogen in
Supelco 3-3297 micro reaction vials. HCB in the benzene extracts was analyzed by electron- capture detection gas chromatography using a Varian model
3700 gas chromatograph (GC) attached to a Varian model 4270 peak integrator. The column used was a 2.0 m x 0.25 inch
OD x 2 mm ID TightSpec glass column containing 1% SP-1000 on 100/200 Supelcoport. GC operational conditions were as follows: injector temperature, 250 C; detector temperature,
200 C; column temperature, 150 C for 5 minutes, raised to a final temperature of 200 C at 10 C/min; gas flow rate, 40 ml/min N2 ; injection volume, 1.8 ul. A standard curve was prepared using HCB dissolved in benzene. HCB retention times were determined using a standard mixture of chlorinated hydrocarbons (Supelco) containing 1,3- dichlorobenzene; hexachloroethane; 1,4-dichlorobenzene;
1,2-dichlorobenzene; hexachlorobutadiene; 1,2,4- trichlorobenzene; 2-chloronapthalene; and HCB.
The ability of the surfactants to extract HCB from soil was evaluated next. For each concentration of surfactant to be tested, 1.0 ml of a solution containing
200 ug/ml of HCB in benzene was added in triplicate to 2.0 g of air-dry West Jefferson soil in acid-washed glass tubes, giving a final soil concentration of HCB equal to
100 ug/g. After the tubes had evaporated to dryness
(several days), 5.0 ml of the appropriate surfactant solution were mixed thoroughly with the soil by vortexing.
The surfactant treatments included 1.0 and 5.0% solutions 82
(v/v) of either Agrimul Jl, Agrimul 70, or Petrodispersant, or 1.0 and 5.0% solutions (w/v) of Morwet 425. Controls included extractions of HCB-treated soil with either benzene or water.
After settling overnight at room temperature, the tubes were centrifuged at 600 rpm on an IEC HN-S II tabletop centrifuge, and the supernates were withdrawn with
Pastuer pipets and filtered through 0.22 um membrane filters. Each filtered supernate was extracted three times by adding 2.0 ml of benzene with vortexing. The benzene layers from the extractions for each replicate were combined and evaporated over a stream of nitrogen to a final volume of 1.0 ml. HCB was analyzed by gas chromatography as described earlier. The results were expressed as the mean amount of HCB extracted (ug/ml) and as a percent of the HCB extracted with the benzene control.
The third experiment concerning the potential to increase the bioavailability of chlorinated organic compounds in soil was the surfactant-mediated extraction of
HC-HCB from soil. Two milliliters of a solution containing 100 ug/ml of HCB in benzene were added to 2.0 g of air-dried, sieved (<2.00 mm) West Jefferson soil in acid-washed glass tubes. The HCB solution contained 0.37 ug/ml of HC-HCB, having a specific activity of 15.3 mCi/mmol (equivalent to 44,000 DPM/g of soil). The benzene was allowed to evaporate over several days until the soils 83 were visibly dry. Five milliliters of either a 2% (v/v) solution of Agrimul Jl, water, or benzene were added to duplicate tubes of HCB-amended soil. The tubes were mixed by repeated vortexing and were shaken for 48 hours on an orbital shaker at 250 rpm.
When the tubes were removed from the shaker, they were vortexed repeatedly and centrifuged for several hours at
750 rpm on the IEC tabletop centrifuge. The supernate from each tube was filtered through a 0.2 urn membrane filter into clean tubes. One milliliter of the filtered supernatate was combined with 10 ml of scintillation cocktail and each vial was counted for 10 min in a Beckman
LS3801 liquid scintillation counter. As before, quenched external standards were used to determine the quench curve for conversion of results to DPM. For the Agrimul Jl and water extracts, the scintillation cocktail was Aquasol 2; for the benzene extract, Instafluor was the cocktail.
The fourth experiment to test the extraction efficiency of the surfactants involved TCDD soils to which
14C-TCDD (0.002 uCi/g, equivalent to 4440 DPM/g) had been added more than one year earlier, as described previously.
Using a standard soil scoop, 1 g of soil PR or SS, containing 1.1 and 2.4 ug/g, respectively, of unlabeled
TCDD, was added to acid-washed glass tubes. In duplicate,
5.0 ml of surfactant solution were added to the soils and mixed thoroughly by repeated vortexing and shaking as described above. The surfactants included 2% (v/v)
solutions of Agrimul Jl or Agrimul 70. These were selected
based on HCB solubility trials and toxicity tests described
earlier. In addition, benzene and water served as
controls. After shaking overnight, the tubes were processed and 1.0-ml aliquots of the supernates were
counted for radioactivity as described previously.
HCB DEGRADATION EXPERIMENTS
The potential for surfactant-mediated biodegradation
of HCB under aerobic and anaerobic conditions was
evaluated. This was done because, as mentioned earlier,
HCB and TCDD share similar environmental properties.
Successful surfactant-mediated HCB biodegradation would be
an important development in its own right, but might also
yield insight to surfactant-mediated TCDD biodegradation.
An additional goal was to evaluate a potentially general method for in situ decontamination using surfactants to
improve the bioavailability of soil- or sediment-bound
halogenated pollutants. Anaerobic conditions were included because of the potential to effect reductive
dehalogenation.
Surfactant-mediated metabolism of HC-HCB in aerobic
soil was examined, using 0.1, 1.0, and 5.0% (v/v) solutions
of Agrimul 70, and 0.1 and 1.0% (w/v) solutions of Morwet
425. These surfactants and concentrations were chosen 85 based on previous toxicity and solubility tests. Controls included HCB-amended soil with no surfactants, surfactant- amended soils with no HCB, and Cd-inhibited (1000 ug/g) systems containing filter-sterilized HCB and either 0.1%
(w/v) filter-sterilized Morwet 425 or 1.0% (v/v) filter- sterilized Agrimul 70. All treatments were carried out in triplicate.
To initiate the tests, 50 g (oven-dry basis) of field- moist, sieved (<2.00 mm) West Jefferson soil in acid-washed
0.5 1 glass canning jars received 500 ug of HCB (final soil concentration of 10 ug/g) in a total of 5.0 ml of benzene. * The HCB dosing solution contained 0.1 uCi of HC-HCB, to give a total of 4400 DPM/g of soil. After dosing, the benzene was allowed to evaporate from the jars and the soil moisture content was adjusted to 70% field capacity using the appropriate surfactant solutions or distilled water.
Alkali traps to absorb H C O 2 (10 ml of 0.6N NaOH) were placed in the jars as described previously, and the jars were loosely capped and incubated in the dark at 22 C.
Periodically over 6 months, the jars were aerated, the moisture content was readjusted to the initial level using surfactant solutions or water as appropriate, the traps were recharged with fresh alkali, and a 1.0-ml aliquot of the used alkali was mixed with 10 ml of Aquasol 2 and counted as described previously. Sterile controls were handled similarly except that they were opened for aeration 86 under a UV-decontaminated laminar flow hood.
At the conclusion of the experiment, the mass balance of the added *4C was determined. First, 2.0 g of soil from each jar were extracted twice with 5.0-ml portions of distilled water. This was done to test the effectiveness with which the surfactants solubilized soil-bound HCB. As described previously, soil was separated from the extraction water by centrifugation and filtration, and a
1 .0-ml aliquot of the water extract was mixed with 10 ml of
Aquasol 2 and counted. The extracted soil was then combusted at 900 C in a Harvey 0X400 Biological Oxidizer
(Harvey, Hillsdale NJ) and the resulting 14C02 was trapped in Harvey *4C Cocktail and counted in the Beckman LS3801 liquid scintillation counter.
Surfactant-mediated metabolism of HCB in anaerobic sediment was evaluated. This was done to test for the possible removal of HCB due to reductive dehalogenation or some other anaerobic mechanism of biodegradation, as a surrogate for anaerobic degradation of TCDD and other chlorinated organics. To set up the test, 11.2 ml of sediment (equivalent to 3.0 g oven-dry sediment) from a pond near West Jefferson, Ohio, were added to acid-washed
35-ml capacity glass bottles. Half of the bottles were capped and autoclaved twice for 60 minutes, to provide for sterile controls. Two surfactants (Morwet 425 and Agrimul
70) at three concentrations (0.1, 1.0, and 5.0%, w/v or 87 v/v, as appropriate) were used. Each bottle was filled to capacity with the appropriate filter-sterilized surfactant solution, which also contained 20 mmol of ammonium acetate.
To initiate the experiments, 1.0 ml of a benzene solution containing 30 ug of HCB was aseptically injected to the bottom of each bottle, using a 2 ml glass syringe and long
20 gauge needle. The completely filled bottles were closed with screw-cap lids and were incubated in the dark at 37 C.
At 30, 60, and 365 days, replicate bottles of each treatment were removed from incubation and analyzed for total HCB remaining as follows. Each bottle to be harvested was shaken thoroughly to mix the sediment and liquid portions. Immediately, a 10.0-ml aliquot was transferred to a 30-ml teflon screw-cap vial, along with
0.2 g of CaCl2- 2H2 0, 1.0 g of NaCl, and 4.0 ml of benzene.
The vial was shaken for approximately 2 min and centrifuged in the IEC tabletop centrifuge at 750 rpm for 10 min. The benzene layer was then pipetted into a 4-ml reaction vessel where it was evaporated nearly to dryness with nitrogen.
The extraction of the 10.0-ml aliquot was repeated two more times using 4.0-ml portions of benzene, which were combined with the previous extracts and were evaporated as before.
The final volume of the extractant was 1.0 ml. HCB in the extracts was analyzed by electron-capture detection gas chromatography as described earlier. The results were expressed as the percentage of added HCB remaining and were 88 statistically evaluated by one-way ANOVA and Duncan’s
Multiple Range Test.
SURFACTANT-MEDIATED TCDD DEGRADATION EXPERIMENTS
Surfactant-mediated biodegradation of TCDD in Missouri soils was evaluated using the TCDD soils that had been previously amended with additional HC-TCDD, incubated for one year, and monitored for evolution of i*C02. This was done to determine whether treatments with a surfactant that tended to solubilize soil-bound TCDD would stimulate mineralization of the compound and thus provide a potential in situ soil decontamination method.
Nine jars of TCDD soil from Missouri were selected for this experiment. These included triplicate jars of soils originally containing 0.008, 1.1, or 2.4 ug/g of TCDD. In addition, three jars of the non-TCDD soil from West
Jefferson, Ohio were included. All twelve jars contained
50 g (dry-weight basis) of soil that had been previously amended with Hoagland’s nutrient solution as a fertilizer.
Previous incubation for one year had failed to reveal any mineralization of added HC-TCDD from the Missouri soils.
The soils were air-dried overnight under an exhaust hood and then adjusted to 70% field capacity using a solution of 2% (v/v) Agrimul Jl. One replicate jar of each soil was then capped and stored at 4 C in the dark, to be analyzed later for total TCDD. An alkali trap containing 10.0 ml of 0.6N NaOH was added to the remaining jars as described previously, and each jar was loosely capped and incubated at 22 C in the dark. Periodically over six months the jars were aerated, the traps were collected and recharged with fresh alkali, and the collected alkali was assayed for H C O 2 by liquid scintillation counting, as described previously.
At the end of the incubation period, samples of the incubated treatments and the refrigerated samples were analyzed for total TCDD using GC/MS as described earlier.
Water-extractable 14C-TCDD was determined in additional samples of incubated soils to indicate the extent to which the surfactant treatment solubilized soil-bound TCDD.
Samples of the soils were also combusted in a Harvey
Biological Oxidizer and the 14(302 was trapped and counted as before. Finally, the soils were assayed for eutrophic and oligotrophia bacteria, fungi, and actinomycetes, as described previously. CHAPTER III
RESULTS AND DISCUSSION
SOIL CHARACTERIZATION
The initial characterization of soils for several
physicochemical parameters is shown in Table 5. The soils
were characterized not only in order to determine initial
TCDD concentrations, but also to examine other parameters
that influence soil microbial activity, such as organic
matter content, pH, CEC, and moisture capacity.
The TCDD soils were highly variable with respect to most parameters. TCDD concentrations ranged from 0.008 to
26.3 ug/g. Soil from the town of Times Beach (TB) had the
lowest TCDD concentration, followed by Piazza Road (PR),
Shennendoah Stables (SS), and New Jersey (NJ) soils. A
similar trend was seen for total solvent-extractable
content, which indicates the level of organic contaminants other than TCDD. Therefore, these TCDD soils contained
other uncharacterized co-contaminants generally in proportion to the levels of TCDD.
Soil TB was a loamy sand with a relatively low organic matter content (1.7%) and slightly alkaline pH'. Its cation exchange capacity (CEC), which is related to soil fertility
90 91
Table 5. Physicochemical parameters of experimental soils.
Soil( a)
Parameter TB PR SS NJ WJ
TCDD (ug/g) 0.008 1.1 2.4 26.3 0
Organic matter (%) 1.7 2.7 6.0 8.0 3.0
TSEC (mg/g)(b) 0.3 5.2 7.1 21.5 ND( e)
Surface Area (m2/g) 19.7 27.6 35.7 22.3 ND
CEC (meq/100g)( °) 23.2 35.2 39.0 12.7 10.0
PH 7.7 6.7 7.3 7.5 6.5
EC (umhos/cm)( *) 107.1 95.0 195.2 1.3 190.0
Bulk density (g/cm3 ) 1.22 1.34 1.30 1.27 1.40
Field capacity (%) 25.0 37.0 47.0 ND 25.7
Sand (%) 52.4 57.4 42.3 81.2 24.2
Silt (%) 43.2 30.1 51.6 17.9 66.3
Clay (%) 4.4 12.5 6.1 1.0 9.6
Texture Loamy Loamy Silty Sandy Silty
sand sand loam loam loam
(a) TB: Times Beach; PR: Piazza Road; SS: Shennendoah
Stables; N J : New Jersey; WJ: West Jefferson.
(b) Total solvent-extractable content.
(c) Cation exchange capacity.
(d) Electrical conductivity.
(e) Not determined because it is an indication of the ability of a soil to bind nutrient cations such as Mg, Ca, and K, was the lowest of the Missouri soils (23.2 meq/lOOg). Soil PR was also a loamy sand with an organic matter content of 2.7%, a near neutral pH, and a CEC of 35.2 meq/lOOg. Soil SS, a silty loam with a pH near neutral, had a CEC of 39.0 meq/lOOg and the highest organic matter content of any of the Missouri soils (6.0%). Finally, soil NJ, which was the most heavily contaminated soil examined, was a sandy loam with a relatively high organic matter content (8.0%), a slightly alkaline pH, and a low CEC (12.7 meq/lOOg) compared to the other TCDD soils. NJ also had a very low electrical conductivity (EC) compared to the other TCDD soils. EC is an indication of soluble salts content, which affects the osmotic potential of a soil and thus the water availability for plants and microorganisms. The EC for all TCDD soils was low compared to a typical saline soil (EC>4 mmhos/cm).
By comparison, soil WJ contained no detectable level of TCDD. It was a siIt-loam with a circumneutral pH, an organic matter content of 3.0%, and a CEC of 10.0 meq/lOOg.
The results shown in Table 5 indicate that all four
TCDD soils contained TCDD at concentrations potentially lethal to many test organisms ia vitro (see Table 3). Soil
WJ contained no detectable level of TCDD, despite the fact that it was collected from an agricultural field subject to pesticide application. Soils PR, SS, and NJ contained very 93 high TCDD concentrations compared to levels normally expected as a result of spraying soils with herbicides.
Obviously, the TCDD soils (especially soil NJ) also contained substantial levels of other uncharacterized organic (presumably halogenated) pollutants. These results suggest that microbial activity in TCDD soils should be depressed compared to the non-TCDD soil, if TCDD in soils is bioavailable and as toxic to microorganisms as it is to many other species.
On the other hand, if the presence of TCDD and co contaminants is not a factor in controlling microbial activity in these soils, then some general predictions on expected microbial parameters can be made based on physicochemical conditions. Soil SS would be expected to have the highest overall microbial activity because of its organic matter content and CEC, both of which indicate a relatively fertile soil. Soil NJ also had a high organic matter content but a lower CEC than soil SS. The pH of both of these soils is near neutral to slightly alkaline, conditions that favor eubacteria as opposed to actinomycetes or fungi. Soil SS also had a relatively high moisture capacity, due to its organic matter and silty loam texture. These conditions also favor microbial growth.
Soils TB and PR, with lower organic matter contents than the other soils, normally would be expected to contain fewer organisms per gram of soil compared to soil SS. Soil 94
TB, being slightly alkaline, may tend to favor actinomycetes, while soil PR is not acidic enough to favor fungi over bacteria.
MICROBIAL ACTIVITY IN TCDD SOILS
Three methods were used to indicate whether TCDD soils were microbially active compared to the non-TCDD soil.
These included enumeration of selected microbial groups, determination of the activity of certain soil enzymes, and measurement of soil respiration (CO2 evolution) in TCDD soils both diluted and undiluted with non-TCDD soil. These measurements were taken to determine whether TCDD in soils, at concentrations known to be toxic to other organisms in vitro, is toxic to soil microorganisms; and whether TCDD soils contain active microbial populations that potentially 4 may be stimulated for An situ microbially mediated soil decontamination. All results were analyzed by one-way analysis of variance and Duncan’s Multiple Range Test at 95 and 99 percent confidence levels.
Microbial Enumeration
Four groups of soil microorganisms were enumerated in the TCDD soils and a fertile non-TCDD silty loam soil from
West Jefferson, Ohio (soil WJ): eutrophic aerobic bacteria, oligotrophic aerobic bacteria, actinomycetes, and filamentous fungi. Soil NJ contained a mean of 1.72x106 95 nutrient agar-platable bacteria per gram of soil (dry basis), which was significantly (p<0.01) fewer eutrophic bacteria than all other soils (Table 6). The other soils averaged between 6.00x106 and 11.7x106 eutrophic bacteria per dry gram of soil, but no significant differences (p>0.05) in numbers of eutrophic bacteria occurred among all other soils, including the non-TCDD soil.
Oligotrophic bacteria were enumerated on 100-fold dilute nutrient agar. The non-TCDD soil WJ contained a mean of 21.2xl07 oligotrophic bacteria per dry gram of soil, which was significantly (p<0.01) greater than all
TCDD soils (Table 6). Soils PR and SS did not differ significantly (p>0.05) with respect to mean numbers of oligotrophic bacteria, but they had significantly (p<0.01) more oligotrophs than soils TB and NJ; the mean number of oligotrophs in soils TB and NJ did not differ significantly
(p>0.05).
Actinomycetes were enumerated on starch-casein agar.
As in the case of eutrophic bacteria, soil NJ, containing greater than 2 6 .3 ug TCDD/g, also had significantly
(p < 0 .0 1 ) fewer actinomycetes than all other soils (Table
6); the mean number of actinomycetes per dry gram of soil was only 4.80x104 in NJ, compared to the next lowest mean of 3.76x106 (soil SS). On the other hand, soil PR, which contained 1.1 ug TCDD/g, had significantly (p<0.01) more actinomycetes than all other soils, including the non-TCDD Table 6. Mean microbial numbers in TCDD soils and a non-TCDD control soil (n=5). Within columns, means followed by a common letter are not significantly different at the 99% confidence level.
Colony Forming Units/g< a)
Eutro Oligo Actino Fungi Sum
Soil( b) (xlO®) (xlO®) (xlO®) (Xl04) (xl07)
TB 6.60 a 17.6 c 4.72 b 2.10 b 2.89 c
PR 11.70 a 80.0 b 6.64 a 8.02 b 9.84 b
SS 9.88 a 111.0 b 3.76 b 48.4 b 12.5 b
NJ 1.72 b 38.0 c 0.05 c 1.86 b 3.98 c
WJ 9.32 a 212.0 a 4.10 b 946 a 23.5 a
(a) Eutro = eutrophic bacteria; Oligo = oligotrophic
bacteria; Actino = actinomycetes.
(b) TB = Times Beach; PR = Piazza Road; SS = Shennendoah
Stables; NJ = New Jersey; WJ = West Jefferson. 97 soil. The mean numbers of actinomycetes in soils TB, SS, and the non-TCDD soil were not significantly (p>0.05) different.
The final microbial group examined, filamentous fungi, was enumerated on rose-bengal agar. The mean number of fungi in the non-TCDD soil was 9.46xl06 per gram of dry soil, which was significantly (p<0.01) greater than all
TCDD soils (Table 6 and Figure 4). None of the TCDD soils differed significantly (p>0.05) with respect to mean numbers of filamentous fungi.
Finally, in terms of total aerobic microorganisms plated on the four media, the non-TCDD soil contained significantly (p<0.01) more total organisms per gram of dry soil than all TCDD soils (Table 6), due largely to the number of oligotrophic bacteria in soil WJ. Soils TB and
NJ, containing the lowest and highest TCDD concentrations, respectively, contained significantly (p<0.01) fewer total organisms than all other soils, while the number of total organisms in soils PR and SS did not differ significantly
(p>0.05).
These results suggest that the presence of TCDD and/or co-contaminants in soils may suppress total microbial numbers and specific physiological groups of microorganisms. Numbers of actinomycetes and eutrophic bacteria were lowest in the soil with the greatest TCDD and co-contaminant concentrations, while oligotrophic bacteria 98 and fungal numbers were suppressed in all TCDD soils compared to a non-TCDD soil. In no case among the TCDD soils was a clear dose-dependent effect of TCDD concentration on microbial numbers apparent. While a cause-and-effect relationship is not shown by these data, the results do suggest that other indicators of microbial activity that depend on viable microbial biomass in TCDD soils should be similarly affected. This was examined in the following experiments.
Enzymatic Activity in TCDD Soils
As described in detail in Chapter II, five different enzymes were examined in soils WJ, TB, PR, and SS; soil NJ was not analyzed because of the limited quantity available.
The enzymes included dehydrogenase, acid phosphatase, alkaline phosphatase, arylsulfatase, and rhodanese. These were analyzed because they function in the cycling of nutrients in terrestrial and aquatic ecosystems by mediating organic matter mineralization. In all enzymatic assays, appropriate controls were included to account for non-biological product formation.
Soil dehydrogenases represent a family of oxidation- reduction enzymes that are particularly important in organic carbon turnover and overall soil microbial metabolic potential. Dehydrogenases were measured by the reduction of an alternative electron acceptor, 99
2,3,5-triphenyltetrazolium chloride, to triphenyl formazan
(TPF). As indicated in Table 7, the mean amount of TPF produced by soil PR was 468 ug/g, which was significantly
(p<0.01) more dehydrogenase activity than any other soil.
The mean amount of TPF produced in the remaining soils varied from 232 (soil TB) to 319 (soil WJ) ug/g and did not differ significantly (p>0.05) from one another.
Microbial acid phosphatases (AP) in soils hydrolyze phosphate esters at acidic pH and thus function in the mineralization of organic soil phosphorus to bioavailable inorganic phosphate. The assay for AP is based on the enzymatic production of p-nitrophenol (PN) from p- nitrophenyl phosphate at a pH of 6.5. The mean production of PN from soil SS was 150 ug/g (Table 7), which was significantly (p<0.01) greater than any other soil. Mean production of PN in soil PR was 111 ug/g, which was significantly (p<0.05) more PN than in soils TB and WJ.
The latter two soils did not differ significantly (p>0.05) in AP activity.
Alkaline phosphatases (A1P) in soil microorganisms also function to hydrolyze phosphate esters to inorganic phosphate but are active at higher pH than acid phosphatases. The activity of A1P was assayed by measuring the production of PN from p-nitrophenyl phosphate at a pH of 11. As in the case of soil dehydrogenases, A1P activity was significantly (p<0.01),greater in soil PR, containing 100
Table 7. Mean activity of selected enzymes in TCDD soils and a non-TCDD control soil (n=3). Within columns, means followed by a common letter are not significantly different at the 99% confidence level.
Enzyme Activity(a)
Dehy AP A1P AS Rhoda
Soil( b> (ug/g) (ug/g) (ug/g) (ug/g) (nmol/i
TB 232 b 91.8 c 115 b 20.6 c 630 b
PR 468 a 111 b 197 a 49.2 a 909 a
SS 234 b 150 a 110 b 16.3 c 509 c
WJ 319 b 95.3 be 134 b 37.2 b 502 c
(a) Dehy = dehydrogenase; AP = acid phosphatase; A1P =
alkaline phosphatase; AS = arylsulfatase; Rhoda =
rhodanese.
(b) TB = Times Beach; PR = Piazza Road; SS = Shennendoah
Stables; WJ = West Jefferson. 101
1.1 ug TCDD/g, than in any other soil; in soil PR the mean production of PN was 197 ug/g (Table 7). The mean production of PN in soils WJ, TB, and SS was 134, 115, and
110 ug/g, respectively, results that were not significantly
(p>0.05) different from one another.
Microbial arylsulfatases (AS) in soils hydrolyae sulfate esters and thus convert unavailable organic sulfur to bioavailable sulfate for plant and microbial uptake.
The activity of AS was determined by the production of PN from p-nitrophenyl sulfate at pH 5.8. As was true with soil dehydrogenases and A1P activity, AS activity was significantly (p<0.01) greater in soil PR than in all other soils (Table 7); the mean production of PN due to AS activity in soil PR was 49.2 ug/g of soil. Soil WJ, the non-TCDD soil, contained the next highest mean AS activity,
37.2 ug/g, which was significantly (p<0.01) greater than AS activity in soils TB and SS. Mean activity of AS in the latter two soils did not differ significantly (p>0.05) between one another.
The final indicator of soil microbial enzymatic activity was the measurement of rhodanese (thiosulfate- cyanide sulfurtransferase). Microbial rhodanese in soils is important in the cycling of soil sulfur because thiosulfate (S2O32-) is an intermediate compound in the oxidation of sulfur. The activity of microbial rhodanese was determined by measuring the production of thiocyanate 102
(SCN-) from KCN and Na2 S2 03.
Piazza Road soil, as In the case of arylsulfatase,
alkaline phosphatase, and soli dehydrogenases, had
significantly (p<0.01) more rhodanese activity than any
other soil (Table 7); the mean production of SCN" in soil
PR was 909 nmol/g. Rhodanese activity in soil TB (a mean
production of SCN" of 630 nmol/g) was significantly
(p<0.01) greater than in soils WJ and SS, which were not
significantly different from one another.
These results for microbial enzymatic activity in TCDD
soils are interesting because they indicate that soil PR,
containing TCDD at a level of 1.1 ug/g, is generally the most microbially active of the soils examined. This is in
contrast to the microbial enumeration results reported earlier, in which soil WJ, the non-TCDD soil, had the
greatest total number of viable organisms. This is also in contrast with the physicochemical characterization reported
earlier, because soil SS, with a higher organic matter content than soil PR, would be expected to have the higher
level of total microbial activity. However, the enumeration results showed that oligotrophs accounted for the largest proportion of total microorganisms in all soils and were significantly greater in soil WJ than in all TCDD soils. Because metabolic rates of oligotrophs- are lower than in eutrophic bacteria, the results of enzyme activity measurements may reflect the status of non-oligotrophic 103 microorganisms, such as eutrophic bacteria, fungi, and actinomycetes. Compared to soil WJ and the other Missouri soils, the mean number of eutrophic bacteria was highest in soil PR, and the mean number of actinomycetes was significantly higher in soil PR. Therefore, these combined results suggest that soil PR is the most metabolically active of the Missouri soils, even though soil SS would be predicted to be the more microbially active soil, based strictly on physicochemical characteristics irrespective of
TCDD and co-contaminants. It is not clear, however, whether the relatively suppressed activity in soil SS is due to the high concentration (2.4 ug/g) of TCDD and co contaminants. The results of experiments to address this question are reported in the following section.
Soil Respiration
As described in Chapter II, TCDD soils were tested for overall microbial metabolic activity by monitoring over several days the evolution of CO2 -C from alfalfa meal- amended soils. All four TCDD soils (TB, PR, SS, and NJ) and the non-TCDD soil were examined. In addition, all four
TCDD soils were variously diluted with non-TCDD soil in order to provide a wide range of TCDD concentrations. This was done to test for a dose-dependent effect of TCDD on soil microbial metabolic activity. The original hypothesis was that TCDD, being toxic to many laboratory test animals 104 at very low concentrations, would also prove to be toxic to soil microorganisms and would inhibit the microbially mediated mineralization of a biodegradable substrate
(alfalfa meal) in soils in a dose-dependent manner.
Soil TB was diluted with soil WJ to give five concentrations of TCDD: 0, 2, 4, 6, and 8 ng/g. The final quantity of soil in each case was 50 g (dry weight basis) in triplicate, amended with 0.5 g of finely ground alfalfa meal. The soils were incubated aerobically for 50 days at approximately 22 C and monitored for evolution of C02-C titrametrically as described in Chapter II. At each subsequent day in the experiment, the cumulative evolution of CO2 -C was statistically analyzed by one-way ANOVA and mean separation by Duncan’s Multiple Range Test at 95 and
99 percent confidence levels. The results for soil TB are shown in Table 8.
Significant differences in cumulative evolution of
CO2 -C occurred as early as day 3, however, the differences were not as expected. At days 3, 5, 6, and 8, treatments containing 0 and 2 ng TCDD/g had evolved significantly
(p<0.05) less CO2 -C than the other treatments. By days 12 and 16, the 0 TCDD soil had evolved significantly (p<0.01) less CO2 -C than all TCDD soils, and this remained consistent through day 20. By day 33, the 0 TCDD soil had evolved significantly (p<0.05) less CO2 -C than all other soils except the soil containing 8 ng TCDD/g. Finally, by 105
Table 8. Mean cumulative evolution of CO2 -C (mg) from
Times Beach soil (8 ng TCDD/g), diluted and undiluted with non-TCDD soil (n=3). Within rows, means followed by a common letter do not differ significantly at the 95% confidence level.
TCDD Concentration (ng/g)
Day 0 2 4 6 8
1 7.83 a 7.13 a 7.73 a 8.04 a 8.04 a
2 17.6 a 16.6 a 18.3 a 18.4 a 18.2 a
3 25.0 b 24.8 b 27.9 a 29.0 a 29.8 a
5 40.9 b 43.8 b 48.3 a 49.4 a 50.0 a
6 46.4 b 48.7 b 53.0 a 55.1 c 54.7 a
8 56.6 c 57.5 be 61.2 ab 63.6 a 62.6 a
12 62.8 c 75.2 b 78.5 ab 84.5 a 78.7 ab
16 65.6 c 77.3 b 80.2 ab 86.0 a 79.0 ab
20 77.1 c 88.3 ab 91.1 ab 96.4 a 87.4 b
26 85.6 c 96.2 ab 99.0 ab 104 a 92.6 be
33 91.7 c 103 ab 104 ab 113 a 96.4 be
50 106 d 120 be 124 b 137 a 109 cd
50( a) 45.4
(a) Mean cumulative evolution of CO2 -C (mg) from
Cd-treated Times Beach soil through 50 days. 106 day 50, the soil containing 6 ng TCDD/g had produced a mean of 137 mg of CO2 -C, which was significantly (p<0.01) more than the 0 and 8 ng/g treatments, and significantly
(p<0.05) more than the soils containing 2 and 4 ng TCDD/g.
Although these results are variable from day to day, they do illustrate some important points. First, contrary to what was expected, the evolution of CO2 -C from alfalfa meal-amended soils was apparently not inversely proportional to increasing concentrations of TCDD. Thus, the toxicity of soil-bound TCDD to soil microorganisms is questionable. Second, even though soil TB had been exposed to TCDD for at least a decade and contained a relatively low organic matter content, its level of microbial activity as measured by the soil respiration assay was comparable to a fertile non-TCDD soil from Ohio that had significantly more platable microorganisms per gram of soil. These combined results suggest that TCDD in soils is either not toxic to the soil microflora at the concentrations tested, or is not bioavailable to the soil microflora.
Similar results were seen with soil PR, which contained 1.1 ug TCDD/g. When diluted with non-TCDD soil and tested for soil respiration, the final soil concentrations of TCDD were 0, 0.28, 0.56, 0.83, and 1.1 ug
TCDD/g. Cumulative evolution of CO2 -C followed a pattern similar to that of soil TB (Table 9). However, less day to day variation among treatments occurred with soil PR 107
Table 9. Mean cumulative evolution of CO2 -C (mg) from
Piazza Road soil (1.1 ug TCDD/g), diluted and undiluted with non-TCDD soil (n=3). Within rows, means followed by a common letter are not significantly different at the
95% confidence level.
TCDD Concentration (ug/g)
Day 0 0.28 0.56 0.83 1.1
1 9.38 a 10.7 a 10.4 a 11.0 a 11.9 a
2 18.7 a 21.1 a 21.5 a 20.5 a 20.9 a
3 26. 9 c 29.1 be 31.3 b 30.9 b 32.5 a
5 44.2 c 47.3 be 50.6 b 50.0 b 57.1 a
6 50.4 c 53.0 be 57.3 b 55.7 b 63.0 a
8 59.6 c 61.7 be 65.5 b 63.7 be 71.8 a
12 79.3 a 78.3 a 77.0 a 78.2 a 84.7 a
16 86.6 a 85.7 a 83.2 a 82.0 a 88.4 a
20 99.1 a 98.3 a 96.1 a 93.7 a 101 a
26 109 a 110 a 107 a 105 a 109 a
33 115 a 117 a 114 a 112 a 114 a
50 132 a 139 a 134 a 133 a 134 a
50(a) 37.8
(a) Mean cumulative evolution of CO2 -C (mg) from
Cd-treated Piazza Road soil through 50 days. 108 compared to TB, as indicated by few significant differences in CO2 -C evolution. After 50 days of incubation, the mean evolution of CO2 -C among all soil PR treatments ranged from
132 mg (no TCDD) to 139 mg (0.28 ug TCDD/g), with no significant (p>0.05) differences among treatments. The only significant differences in soil respiration occurred on days 3, 5, 6, and 8. At each of these days, undiluted soil PR produced significantly (p<0.05) more CO2 -C than all other treatments. The lowest mean cumulative production of
CO2 -C at each of these days was from the non-TCDD soil, and on days 3, 5, and 6 these results were significantly
(p<0.05) different from those in treatments containing 0.56 and 0.83 ug TCDD/g.
At higher TCDD concentrations than those reported above, soil respiration from alfalfa meal-amended soils was still as great or greater in TCDD soils compared to a non-
TCDD soil (Table 10). That is, soil SS, which contained
2.4 ug TCDD/g, was diluted with non-TCDD soil to give final
TCDD concentrations ofO, 0.6, 1.2, 1.8, and 2.4 ug/g. No significant (p>0.05) differences in cumulative production of CO2 -C occurred among all treatments until day 12, when all TCDD treatments were significantly (p<0.05) greater than the non-TCDD treatment. By the end of the incubation
(day 50), the non-TCDD soil had a mean production of 122 mg
CO2 -C, compared to TCDD soils, which produced from 137 to
151 mg CO2 -C. The results for the non-TCDD soil were 109
Table 10. Mean cumulative evolution of CO2 -C (mg) from
Shennendoah Stables soil (2.4 ug TCDD/g), diluted and undiluted with non-TCDD soil (n=3). Within rows, means folowed by a common letter are not significantly different at the 95% confidence level.
TCDD Concentration (ug/g)
Day 0 • 0.6 1.2 00 2.4
1 10.9 a 10.6 a 10.3 a 10.6 a 9.39 a
£n 20.8 a 20.8 a 21.3 a 20.9 a 18.2 a
3 27.8 a 28.9 a 29.9 a 30.5 a 28.0 a
5 45.5 a 47.1 a 49.3 a 48.7 a 47.1 a
6 52.1 a 53.6 a 55.8 a 54.9 a 54.2 a
8 61.4 a 63.6 a 65.9 a 64.8 a 64.3 a
12 73.4 b 78.0 a 81.1 a 80.1 a 77.2 a
16 79.1 b 87.6 a 89.3 a 89.3 a 85.3 a
20 90.7 b 96.6 a 98.4 a 99.0 a 93.7 ab
26 101 c 109 ab 113 a 113 a 103 be
33 106 b 115 ab 122 a 122 a 112 b
50 122 c 139 ab 151 a 151 a 137 b
50(a) 17.2
(a) Mean cumulative evolution of CO2 -C (mg) from
Cd-treated Shennendoah Stables soil through 50
days. 110
significantly (p<0.05) different from the TCDD soils.
Finally, in order to compare the production of CO2 -C
from each of the undiluted TCDD soils with the non-TCDD
soil, results from alfalfa meal-amended non-TCDD WJ soil
were compared to each of the alfalfa-meal amended TCDD
soils from Missouri (Figure 2). After 50 days of
incubation, the two soils with the lowest TCDD
concentrationsy soils WJ (no TCDD) and TB (0.008 ug
TCDD/g), did not differ significantly (p>0.05) in
production of CO2 -C. Soils PR and SS also were not
significantly (p>0.05) different from one another, but they
produced significantly (p<0.05) more CO2 -C than soils TB
and WJ.
These results, as mentioned previously, were not as
expected. The original hypothesis was that microbial
activity in TCDD soils would be less than in a non-TCDD
soil. By diluting TCDD soils with non-TCDD soil, it was
expected that a concentration that inhibited 50 percent of
the microbial activity, analogous to the LCso reported
commonly in other areas of environmental toxicology, could
be identified. Instead, there was no apparent affect of
TCDD concentrations up to 2.4 ug/g on the level of soil
respiration.
Therefore, in a separate experiment, soil NJ was
diluted with non-TCDD soil, amended with alfalfa meal, and tested for CO2 -C evolution. In this experiment the final Ill
140 130
120 - PR TB 110
100
00 -
80 -
70 -
60 - WJ SO 40 SS/Cd 30 20
0 20 40 DAYS
Figure 2. Evolution of C02-C from TCDD soils (TB, PR, SS), a non-TCDD soil (WJ), and a TCDD soil amended with Cd
(SS/Cd). concentrations of TCDD were 0, 6.6, 13.2, 19.8, and 26.3 ug/g (dry weight basis). Thus, the lowest TCDD concentration was approximately three times greater than the highest concentration tested previously. Nevertheless, production of CO2 -C was apparently unaffected by these high
TCDD concentrations (Table 11). Throughout 53 days of incubation, undiluted soil NJ (26.3 ug TCDD/g) and the non-
TCDD soil WJ did not differ significantly (p>0.05) from each other in terms of cumulative production of CO2 -C.
Nevertheless, beginning at day 15 and continuing through day 53, the mean production of CO2 -C from the undiluted NJ soil was significantly (p<0.05) greater than from soils containing 6.6 or 13.2 ug TCDD/g.
Because of these results, it was not only impossible to calculate a TCDD concentration in soil that inhibited soil respiration by 50 percent, it was also not possible to identify a TCDD concentration that had any substanstial impact on soil microbial mineralization of alfalfa meal.
These results were unexpected. The fact that TCDD concentrations ranging from 2 ng/g to 26.3 ug/g had no apparent impact on microbial activity in soils suggested that TCDD is not toxic to the soil microflora or is not bioavailable. Because TCDD is extremely toxic to many laboratory animals at test concentrations much, lower than those found to have no effect in the microbial activity assays described above, it is possible that instead of 113
Table 11. Mean cumulative evolution of CO2 -C (mg) from New
Jersey soil (26.3 ug TCDD/g) diluted and undiluted with non-TCDD soil (n=3). Within rows, means followed by a common letter are not significantly different at the 95% confidence level.
TCDD Concentration (ug/g)
Day 0 6.6 13.2 19.8 26.3
2 23.6 a 24.1 a 20.4 a 20.8 a 17.2 a
4 44.2 a 42.4 a 40.5 a 39.5 a 38.8 a
9 65.3 a 63.6 a 59.1 a 60.2 a 64.6 a
11 71.0 a 69.1 ab 63.3 b 66.5 ab 72.7 a
15 81.0 ab 77.4 abc 69.8 c 75.0 be 83.5 a
18 91.3 ab 85.6 be 77.4 c 82.9 be 95.1 a
22 99.6 ab 91.4 be 82.5 c 89.4 be 104 a
28 107 ab 96.2 be 86.1 c 95.8 be 113 a
34 115 a 101 b 88.9 b 99.2 b 118 a
47 128 ab 109 c 97.1 c 111 be 135 a
53 132 ab 114 cd 101 c 118 be 143 a 114 being non-toxic to soil microorganisms, soil-bound TCDD is
not bioavailable. If this is true, then the question that
arises is whether TCDD would be toxic if it were made
bioavailable to the soil microflora. This question was
addressed in experiments described later in this chapter.
The other possible explanation for the lack of
observed impacts on soil microbial activity due to high
concentrations of TCDD is that the TCDD was metabolized by
the soil microflora. The fact that the soils examined were
microbially active and yet had TCDD concentrations that may
have remained unchanged for a decade, initially suggested
that TCDD was not subject to biotransformation or
biodegradation. Nevertheless, an experiment was
established to determine whether TCDD mineralization could be stimulated by management practices that could be
incorporated is situ. These management practices included
fertilization, adjustment of moisture and aeration status,
and dilution of TCDD soils with fertile non-TCDD soils.
Soils TB, PR, and SS were diluted with non-TCDD soil
WJ in the same manner as described earlier for the toxicity experiments. This gave a range of TCDD concentrations from
2 ng/g to 2.4 ug/g (dry weight basis). All the treatments were amended with additional uniformly ring-labelled n c -
TCDD as described in Chapter II. In addition, triplicate treatments received a complete (macro- and micronutrients) mineral fertilizer in order to stimulate soil microbial 115 activity, and triplicate treatments were left unfertilized.
The moisture content was adjusted to 70 percent field capacity in all treatments. These combined treatments are referred to as bioaugmentation. The soils were incubated at 22-25 C in the manner described previously in order to trap respired 14(302. Periodically the jars were aerated and the alkali traps were sampled for H C 0 2 v*a liquid scintillation counting. After one year of incubation, no evolution of 14C02 was detected from any sample. The results in all treatments were always equivalent to background counts of approximately 40-60 disintegrations per minute (DPM).
These results confirmed that TCDD was not mineralized in microbially active soils, even when amended with a complete fertilizer and diluted with fertile soil. The results do not indicate whether TCDD underwent biotransformation to stable or volatile daughter products.
Therefore, this initial set of experiments on microbial activity in TCDD soils confirmed that 1) TCDD soils are microbially active; 2) high concentrations of
TCDD in soils are apparently not microbially toxic or inhibitory to most indicators of microbial activity; and 3)
TCDD is not mineralized in microbially active soils even when conditions for microbial activity are optimized. In addition, the results suggested that TCDD may not be bioavailable, because research with other organisms has 116 shown that bioavailable TCDD exerts significant biological
effects.
SURFACTANT EXPERIMENTS
Because TCDD in soils apparently was not bioavailable, and TCDD soils contained active indigenous microbial populations, it was determined that microbial attack of
TCDD by indigenous microorganisms could possibly be stimulated if the bioavailability of TCDD could be enhanced. On the other hand, bioavailable TCDD might also prove to be toxic to indigenous organisms. Methods to enhance the bioavailability of TCDD, with the ultimate goal being to develop a treatment to stimulate the in situ biodegradation of TCDD, were investigated.
The methods chosen were treatments with various surfactants to increase the solubility of TCDD in soils.
Surfactants were investigated because of their widespread use in agriculture to emulsify and solubilize halogenated organic pesticides, and their potential cost-effectiveness for large-scale in situ use compared to other potential
TCDD solubilizing agents, such as acetone or some other solvent. Potential surfactants were chosen initially based on their use in chlorinated aromatic pesticide formulations. Subsequently, potential surfactants were screened based on their ability to solubilize hexachlorobenzene (HCB), a hydrophobic, highly chlorinated, 117 and environmentally recalcitrant surrogate compound for
TCDD. Because many surfactants are themselves microbially toxic at certain concentrations and may poison the soil system if used in excess, toxicity experiments were conducted to identify non-toxic concentrations of potentially useful surfactants.
HCB Solubility Experiments
As described in Chapter II, assays were conducted to assess the solubilizing potential of the surfactants. In the first set of assays, several surfactants were screened for their ability to solubilize HCB apart from soil. Of the surfactants tested, three in particular were shown to be relatively effective compared to other surfactants or water in solubilizing HCB and these were examined further.
The three surfactants included Agrimul Jl, Agrimul 70, and
Morwet 425. The ability of these surfactants to solubilize
HCB apart from soil is shown in Table 12. The results were analyzed by one-way ANOVA with mean separation by Duncan’s
Multiple Range Test.
The most effective HCB solubilizing agent was 5% (v/v, in water) Agrimul 70. The mean solubility of HCB in this surfactant was 235 mg/1, which was significantly (p<0.05) greater than any other treatment. Agrimul Jl at a 5% (v/v, in water) concentration solubilized 165 mg of HCB/1, which was significantly (p<0.05) greater than all other 118 i Table 12. Mean solubility of hexachlorobenzene (HCB) in
surfactant solutions (n=3). Within columns, means followed
by a common letter are not significantly different at the
95% confidence level.
HCB Solubilized
Surfactant (ug/ml)
5% Agrimul 70 235 a
5% Agrimul Jl 165 b
1% Agrimul 70 38.9 c
5% Morwet 425 26. 9 c
1% Agrimul Jl 25.9 c
1% Morwet 425 11.3 c
Water 0 d
Y 119 surfactants except 5% Agrimul 70. With all three surfactants, the solubility of HCB generally increased significantly (p<0.05) with increasing surfactant concentration.
These results suggested the possibility to solubilize HCB (and possibly other chlorinated aromatic pollutants such as TCDD) in soils by using relatively inexpensive surfactant treatments. Therefore, the second assay to evaluate the effectiveness of surfactants was their ability to solubilize HCB in soil. Two grams of WJ soil containing
100 ug HCB/g were extracted with surfactant solutions, and the HCB in solution was analysed by electron capture gas chromatography (see Chapter II).
The relative efficiencies of extraction of HCB from soil differed from the results for HCB solubility in surfactants apart from soil. In the presence of soil, the most effective HCB solubilizing surfactant was 5% (w/v, in water) Morwet 425 (Table 13). The mean extraction of HCB from soil with this surfactant was 155 ug, out of a maximum possible amount of 200 ug. This was significantly (p<0.01) greater than any other surfactant treatment and was 82% of the HCB extracted from soil using benzene. Agrimul 70 at a concentration of 5% (v/v, in water), which was the most effective HCB solubilizing surfactant from the earlier assays in the absence of soil, extracted a mean of 108 ug of HCB, which was significantly (p<0.01) more HCB than all 120
Table 13. Mean extraction of hexachlorobenzene (HCB) from soil with surfactant solutions (n=3). Within columns, means followed by a common letter are not significantly different at the 95% confidence level.
HCB Extracted Percent of :
Surfactant (ug) Control
5% Morwet 425 155 a 82.0
5% Agrimul 70 108 b 57.1
1% Agrimul 70 72.9 c 38.6
5% Agrimul Jl 38.5 d 20.4
5% Petrodispersant 19.9 e 10.5
1% Agrimul Jl 18.7 e 9.89
1% Morwet 425 17.6 e 9.31
1% Petrodispersant 0 f 0
Distilled water 0 f 0 121 other surfactants except 5% Morwet 425. Agrimul 70 at a 1%
(v/v, in water) concentration extracted a mean of 72.9 ug of HCB, which was also significantly (p<0.01) more than the remaining surfactants. Finally, 5% Agrimul Jl, which was a fairly effective HCB solubilizer from the earlier assays, extracted a mean level of 38.5 ug HCB from the soil. This level was significantly (p<0.05) more than the remaining surfactants tested, which did not differ significantly
(p>0.05) among themselves.
These results for surfactant extraction of HCB from soil were encouraging because of the tenacity with which
HCB binds to soil. The results suggested that it may be possible to enhance the bioavailability of chlorinated hydrophobic organics in soil through the use of aqueous solutions of surfactants. However, surfactants themselves and/or surfactant-solubilized hydrophobic organics may be toxic to soil microorganisms. If this were the case, the potential cure might be worse than the original problem.
Therefore, the toxicity of surfactants with and without HCB was evaluated extensively, as described in the next section.
Surfactant Toxicity Experiments
As described in Chapter II, surfactant toxicity experiments consisted of plate counts of viable Pseudomonas aeruginosa, the Microtox assay using Photobacterium 122
phosphoreum. and concentration-dependent inhibition of soil
respiration.
Results of plate counts of fL. aeruginosa in the
presence of several surfactants are shown in Table 14.
From an overnight culture in nutrient broth, 0.1 ml was added to 9.0 ml of surfactant solution and incubated
overnight. Next, 0.1 ml from each tube was plated in
triplicate on nutrient agar and counted for colony
development. Although not analyzed statistically, the data
in Table 14 show that only Agriwet and Morwet 425 inhibited
growth of the bacteria. Agriwet, which exhibited growth
inhibition at a concentration of 0.1% (v/v), was the most
toxic of the surfactants tested. The only concentration of
Morwet 425 that inhibited growth was 10% (w/v). Morwet 425
was one of the surfactants that proved to be relatively
effective at solubilizing HCB, as described in the previous
section. The other two surfactants that were effective HCB
solubilizers, Agrimul Jl and Agrimul 70, were not
inhibitory to JL. aeruginosa at the concentrations tested.
The relative toxicity of the surfactants was also
evaluated using the Microtox assay of luminescence by
Photobacterium phosphoreum. Microtox results are expressed as ECso, that is, the concentration of the test material necessary to inhibit 50% of the light output of P. phosphoreum, compared to undosed controls. Therefore, the lower the ECso, the higher the toxicity. 123
Table 14. Effects of surfactant solutions on the development of colony forming units (CFU) of Pseudomonas aerucinosa on nutrient agar.
Percent Surfactant
Surfactant Rep 0.01 0.1 1.0 10 Agriwet 1 275 33 0 0
2 311 34 0 0
3 329 45 0 0
Agrimul 70 1 300 300 300 300
2 300 300 300 300
3 300 300 300 300
Conco Sulfate 1 300 300 300 300
2 300 300 300 300
- 3 300 300 300 300
Agrimul 300 1 300 300 300 300
2 300 300 300 300
3 300 300 300 300
Morwet 425 1 300 300 300 0
2 300 300 300 1
3 300 300 300 0
Cetiol HE 1 300 300 300 300
2 300 300 300 300
3 300 300 300 300
Agrimul Jl 1 300 300 300 300
2 300 300 300 300
3 300 300 300 300 124
The ECsos for the surfactants that were tested previously with Pseudomonas plate counts varied from 0.013 to 27.8% (Table 15). The most toxic surfactant with the
Microtox assay was Agriwet (ECso = 0.013%), which was also the most toxic surfactant in the Pseudomonas assay. On the other hand, Agrimul Jl, which was an effective HCB solubilizer, showed no toxicity in the Pseudomonas assay but was relatively toxic (ECso = 0.016%) in the Microtox assay. Morwet 425, another effective HCB solubilizer, was fairly toxic to both the Microtox assay (ECso = 0.049%) and the Pseudomonas assay. The most effective HCB solubilizer,
Agrimul 70, was relatively non-toxic in both assays.
The results of the Microtox and the Pseudomonas assays show wide variation in surfactant toxicity. Thus, the use of surfactants in soils needs to be approached carefully in order to avoid toxifying the soil system. Because it was necessary to select non-toxic surfactants for later experiments with TCDD, a third series of assays with HCB solubilizing surfactants was done. This third set of assays, soil respiration from alfalfa meal-amended soils, was done in order to determine if the surfactants would exhibit toxic effects on soil microbial activity at concentrations necessary to solubilize HCB (and presumably
TCDD).
The two surfactants that were most effective at
1 solubilizing HCB, Morwet 425 and Agrimul 70, were selected 125
Table 15. Relative toxicity of surfactant solutions to
Photobacterium phosphoreum in the Microtox assay. Results are expressed as the effective concentration that inhibits
50% of control bioluminescence (ECso), within 5 minutes at
15 C.
Microtox ECso
Surfactant (%)
Agriwet 0.013
Agrimul Jl 0.016
Cetiol HE 0.034
Agrimul 300 0.048
Morwet 425 0.049
Agrimul 70 0.49
Conco Sulfate 27.8 126 for initial tests. As described in Chapter II, evolution of CO2 -C from soils amended with alfalfa meal and various doses of surfactants was monitored for 42 days. Controls that were dosed with 2000 ug/g Cd as CdCl2 were included to indicate respiration rates under chemically stressed conditions. The results were analyzed for daily differences by one-way ANOVA and mean separation at 95 and
99% confidence levels by Duncan’s Multiple Range Test.
After 42 days of incubation, Morwet 425 inhibited soil respiration in a dose-dependent manner (Table 16). The treatment that received no surfactant was significantly
(p<0.05) more microbially active than all surfactant treatments, as indicated by cumulative evolution of CO2 -C
(83.7 mg) after 42 days. The lowest concentration of
Morwet 425 was 0.1% (w/w, soil dry weight basis) and did not inhibit soil respiration significantly (p>0.05).
However, the 1.0 and 5.0% Morwet 425 treatments inhibited soil respiration significantly (p>0.05), similar to the Cd control. A similar pattern of significant inhibition of soil respiration was observed as early as day 1 and continued daily throughout the incubation.
These results confirmed the toxicity of Morwet 425 indicated in the Pseudomonas plate count test and the
Microtox assay. Therefore, despite its ability to solubilize HCB in soil, especially at concentrations of
1 .0% (w/w) or greater, the highest safe concentration of 127
Table 16. Mean cumulative evolution of CO2 -C (mg) from
West Jefferson soil treated with Morwet 425 and 2000 ug/g
Cd (n=3). Within rows, means followed by a common letter
are not significantly different at the 99% confidence
level.
Morwet 425 Concentration (%, w/w)
Day 0 0.1 1.0 5.0 1.0+Cd
1 4.78 a 4.46 a 1.72 b 1.45 b 1.52 b
2 10.6 a 10.3 a 5.98 b 4.76 b 5.12 b
3 15.6 a 14.5 a 8.17 b 7.44 b 7.93 b
6 26.8 a 25.2 a 13.3 b 12.2 b 13.9 b
8 33.4 a 30.8 a 15.6 b 14.3 b 17.0 b
10 38.0 a 34.9 a 17.7 b 15.7 b 18.5 b
13 42.2 a 39.2 a 19.6 b 16.9 b 21.1 b
16 46.3 a 42.6 a 21.3 b 18.3 b 23.5 b
20 51.7 a 47.8 a 24.4 b 20.2 b 27.1 b
28 63.7 a 56.8 a 28.1 b 23.5 b 32.8 b
42 83.7 a 73.4 a 31.8 b 29.4 b 39.3 b 128
Morwet 425 for use in subsequent experiments appeared to be
0 .1% (w/w, dry soil basis); higher concentrations are
potentially toxic to the soil microflora and may result in
inhibition of the metabolic activity for which stimulation was being sought. The results of a similar assay with Agrimul 70 (Table
18) were substantially different than those observed with
Morwet 425. After 42 days of incubation, the greatest amount of cumulative CO2 -C was produced in the treatment
receiving the highest dose of Agrimul 70, that is, 5% (v/w, dry soil basis). The soil treated with this level of
surfactant produced 104 mg of CO2 -C, while the soil with no
surfactant produced 83.7 mg of CO2 -C; these results are
significantly (p<0.05) different. The remaining surfactant treatments were not significantly (p>0.05) different from
the untreated soil. The soil dosed with 1% (v/w, dry soil basis) surfactant plus Cd produced significantly (p<0.05)
less CO2 -C than all other soils. This pattern of significantly greater microbial activity from the soil with the greatest amount of surfactant and no inhibition of microbial activity by any dose of Agrimul 70 occurred throughout the experiment, beginning as early as day 1.
Therefore, unlike Morwet 425, non-toxic concentrations of
Agrimul 70 appeared to be potentially useful for solubilizing HCB in soils. 129
Table 17. Mean cumulative evolution of CO2 -C (mg) from
West Jefferson soil treated with Agrimul 70 and 2000 ug/g
Cd (n=3). Within rows, means followed by a common letter are not significantly different at the 95% confidence level.
Agrimul 70 Concentration (%, v/w)
Day 0 0.1 1.0 5.0 1.0+Cd
1 4.78 c 6.66 b 4.80 c 8.18 a 3.80 c
2 10.6 c 13.9 b 11.2 c 17.9 a 10.9 c
3 15.6 c 19.6 b 16.1 c 24.5 a 15.3 c
6 26.8 d 31.8 be 28.3 cd 38.8 a 25.5 d
8 33.4 d 39.1 be 34.9 cd 46.4 a 30.8 d
10 38.0 cd 43.8 b 40.0 c 52.4 a 34.2 d
13 42.2 be 48.9 b 45.2 b 56.0 a 38.8 c
16 46.3 be 53.3 b 49.3 b 60.7 a 41.9 c
20 51.7 cd 60.1 b 54.4 be 67.7 a 46.6 d
28 63.7 be 71.1 b 67.1 be 84.2 a 57.5 c
42 83.7 b 90.7 b 85.5 b 104 a 72.8 c 130
As a result of the three toxicity screens— Pseudomonas plate counts, Photobacterium bioluminescence, and microbial production of CO2 -C from alfalfa meal-amended soils— non toxic levels of HCB-solubilizing surfactants were identified. These results suggested that non-toxic concentrations of surfactants may be useful as soil amendments for solubilizing soil-bound halogenated organic compounds, possibly resulting in improved bioavailability of the pollutants. This could lead to a cost-effective mechanism to effect in situ soil decontamination via biodegradation.
On the other hand, surfactant-mediated solubilization of soil-bound halogenated organic compounds potentially may toxify a soil system due to increased bioavailability of the pollutants. To test this possibility, additional experiments to evaluate microbial activity in soils amended with HCB plus HCB-solubilizing surfactants were conducted.
Alfalfa meal-amended WJ soil was further amended with
10 ug of HCB/g (dry soil basis) and either 1.0 or 2% (v/w, dry soil basis) Agrimul 70, 2.0% (v/w, dry soil basis)
Agrimul Jl, 0.1% (w/w, dry soil basis) Morwet 425, or no surfactant. These concentrations were identified earlier as potentially effective at solubilizing HCB in soils.
Controls included alfalfa meal-amended soil with: no HCB; no HCB or surfactant; and 1000 ug Cd/g (as CdCl2). In addition, aseptic controls of the surfactant plus HCB 131 treatments were prepared using autoclaved soil and filter- sterilized HCB and surfactants. All treatments were prepared in triplicate and were maintained at 10% field capacity over several weeks of dark, aerobic incubation at
22-25C. As before, cumulative evolved CO2 -C was determined titrametrically. The cumulative results were analyzed for each sampling period using one-way ANOVA and Duncan’s
Multiple Range Test.
After 26 days of incubation, soil with no surfactant and soil amended with 1% Agrimul 70 but no HCB produced significantly (p<0.05) more CO2 -C than treatments containing HCB (Table 18). However, production of CO2 -C from soil amended with HCB plus surfactant was not significantly (p>0.05) different than HCB-soil with no surfactant. These trends started early in the incubation and persisted.
The results suggested that treatment with 1% Agrimul
70 did not magnify the potential microbial toxicity of HCB, even though the surfactant treatment should have enhanced the solubility of HCB. That is, HCB in the absence of surfactant was sufficient to depress soil respiration compared to unamended controls, but HCB was not as toxic as the Cd control. This experiment was repeated after doubling the concentration of surfactant (Table 19).
Throughout 54 days of incubation, the same pattern of soil respiration was observed in terms of relative magnitude of 132
Table 18. Mean cumulative evolution of CO2 -C (mg) from
West Jefferson soil amended with 1% (v/w) Agrimul 70,
10 ug/g hexachloroberizene (HCB), or 1000 ug/g Cd (n=3).
Within rows, means followed by a common letter are not significantly different at the 95% confidence level.
Agrimul Concentration (%, v/w)
Day 0 0+HCB 1.10 1.0+HCB 1.0+HCB 0+Cd
(autocl)
1 5.96 ab 0.28 c 7.81 a 4.27 b 0 c 0.97 c
4 25.5 ab 12.7 c 29.9 a 23.4 c 0.76 d 14.0 c
7 35.6 ab 22.1 c 39. 9 a 33.8 b 0.76 e 19.4 d
11 45.8 a 33.9 b 50.6 a 44.2 a 5.12 d 24.3 c
15 51.6 a 41.1 b 56.6 a 49.7 a 12.0 d 26.6 c
18 58.2 ab 48.8 c 63.5 a 56.0 b 17.6 e 30.2 d
21 62.9 b 53.7 c 69.4 a 60.3 b 20. 9 e 33.3 d
26 67.4 ab 57.7 c 73.4 a 63.8 c 23.8 e 33.8 d 133
Table 19. Mean cumulative evolution of CO2 -C (mg) from
West Jefferson soil treated with 2% (v/w) Agrimul 70,
10 ug/g hexachlorobenzene (HCB), or 1000 ug/g Cd (n=3).
Within rows, means followed by a common letter are not significantly different at the 95% confidence level.
Agrimul 70 (%, v/w)
Day 0 0+HCB 2.0 2.0+HCB 2.0+HCB 0+Cd
(autocl)
1 5.45 a 2.99 c 4.71 b 0.10 e 0.12 e 1.57 d
3 17.4 a 11.9 b 17.0 a 6.54 c 0.93 e 3.28 d
6 30.7 a 25.7 b 28.9 a 18.0 c 7.34 d 7.10 d
10 42.9 a 36.6 be 39.2 ab 30.9 c 14.3 d 9.75 e
14 50.6 a 44.4 ab 46.1 a 39.5 b 18.5 c 11.9 d
17 55.2 a 49.3 ab 51.3 ab 44.7 b 21.4 c 13.7 d
25 67.3 a 62.3 a 63.7 a 57.1 a 26.3 b 17.0 b
39 79.1 a 74.6 a 75.3 a 68.2 a 31.5 b 21.4 b
54 91.3 a 83.4 a 93.4 a 86.3 a 35.9 b 32.1 b 134
C02-C production among treatments. That is, the surfactant-treated soil with no HCB produced the most
C02-C, followed in order by the unamended control, the surfactant plus HCB, and the HCB with no surfactant.
However, these results were not statistically different (p>0.05) from one another. The autoclaved and Cd controls were significantly (p<0.01) different from other treatments.
With 0.1% Morwet 425, after 26 days the surfactant- treated soil with no HCB produced significantly (p<0.05) more cumulative C02-C than any other treatment (Table 20).
The soils containing HCB, either with or without surfactant, produced statistically similar (p>0.05) amounts of CO2 -C, and these were significantly (p<0.01) greater than in Cd-treated soil. As before, the presence of HCB with or without surfactant was sufficient to suppress soil respiration to some degree.
The third surfactant tested, 2% Agrimul Jl, was not previously tested for its effects on soil respiration.
Nevertheless, it was included because of its ability to solubilize HCB (and potentially TCDD) in soils. As in the results reported above, soil respiration through 54 days was significantly (p<0.01) greater from the surfactant- treated soil containing no HCB than from any other treatment (Table 21). Significant differences occurred as early as day 6. Soil respiration in the unamended control 135
Table 20. Mean cumulative evolution of CO2 -C (mg) from
West Jefferson soil treated with 0.1% (w/w) Morwet 425,
10 ug/g hexaclorobenzene (HCB), or 1000 ug/g Cd (n=3).
Within rows, means followed by a common letter are not significantly different at the 95% confidence level.
Morwet Concentration (%, w/w)
Day 0 0+HCB 0.1 0.1+HCB 0.1+HCB 0+Cd
(autocl)
1 5.96 ab 0.28 c 7.37 a 3.69 b 0 c 0.97 c
4 25.5 a 12.7 c 27.5 a 18.2 b 1.48 d 14.0 be
7 35.6 a 22.1 be 38.7 a 27. 3 b 1.48 d 19.4 c
11 45.8 a 33.9 b 50.3 a 37.9 b 1.48 d 24.3 c
15 51.6 b 41.1 c 57.3 a 45.0 c 7.38 e 26.6 d
18 58.2 b 48.8 c 64.3 a 52.0 b 11.7 e 30.2 d
21 62.9 a 53.7 b 69.3 a 56.4 b 15.2 d 33.3 c
26 67.4 b 57.7 c 73.7 a 61.4 be 18.2 e 33.8 d 136
Table 21. Mean cumulative evolution of CO2 -C (mg) from
West Jefferson soil treated with 2% (v/w) Agrimul Jl,
10 ug/g hexachlorobenaene (HCB), or 1000 ug/g Cd (n=3).
Within rows, means followed by a common letter are not significantly different at the 95% confidence level.
Agrimul Jl Concentration (%, v/w)
Day 0 0+HCB 2.0 2.0+HCB 2.0+HCB 0+Cd
(autocl)
1 5.45 a 2.99 c 3.65 b 1.54 d 0.27 e 1.57 d
3 17.1 a 11.9 b 18.0 a 10.8 b 4.07 c 3.28 c
6 30.7 a 25.7 b 32.4 a 23.1 b 14.2 c 7.10 d
10 42.9 a 36.6 b 46.1 a 34.5 b 23.1 c 9.75 d
14 50.6 a 44.4 b 55.7 a 43.2 b 26.3 c 11.9 d
17 55.2 ab 49.3 b 61.7 a 48.3 b 29.5 c 13.7 d
25 67.3 b 62.3 b 80.8 a 62.9 b 38.6 c 17.0 d
39 79.1 b 74.6 b 97.0 a 78.0 b 46.6 c 21.4 d
54 91.3 b 83.4 b 115 a 93.0 b 56.3 c 32.1 d 137 and the two treatments containing HCB was not significantly
(p>0.05) different, but was significantly (p<0.01) greater than in the aseptic or the Cd controls.
These results illustrate three points. First, 2%
Agrimul Jl was not toxic to soil microbial respiration.
Therefore, it may prove useful in efforts to enhance the soil bioavailability of chlorinated organic pollutants.
Secondly, as with the other surfactants, the presence of
HCB with or without surfactant was sufficient to suppress soil respiration, compared to the surfactant alone. This was unexpected because it was suspected that HCB toxicity would not manifest itself unless the compound were solubilized. Even in the absence of surfactant, however,
HCB appeared to be bioavailable, unlike TCDD (based on soil respiration experiments reported earlier). Thus, thirdly, the soil respiration assay appears to be sensitive enough to detect chemically induced stress in the soil system, as evidenced by the statistically significant effects due to
HCB described above.
Because microbial activity in soils containing HCB was substantial relative to Cd controls and unamended controls, and surfactants were available that solubilized HCB, the next logical experiment was to monitor for the biodegradation of HCB in surfactant-amended soils. This is described in the next section. 138
HCB BIODEGRADATION EXPERIMENTS
Biodegradation experiments with HCB encompassed two major activities. The first set of experiments examined the potential for HCB biodegradation in surfactant-amended
WJ soils. The second set of experiments looked at the biodegradation of HCB in surfactant-amended anaerobic sediments.
The biodegradation of HCB in WJ soil, described more fully in Chapter II, was monitored by incubating HC-HCB in surfactant-amended soils. The evolution of H C O 2 was monitored for 1 year and the loss of parent compound as determined by electron-capture gas chromatography was monitored at the end of the incubation. Surfactants included 0.1, 1.0, and 5.0% (v/w, dry soil basis) Agrimul
70, and 0.1 and 1.0% (w/w, dry soil basis) Morwet 425.
At no time during the year-long aerobic incubation period was the production of H C O 2 detected. Liquid scintillation results consistently were no different than background counts (40-60 DPM). These results indicate that
HCB was not mineralized in any surfactant-treated or non surfactant WJ soil.
However, at the end of the incubation, the analysis for parent compound using benzene extraction of soils and electron capture GC revealed the disappearance of HCB in some treatments (Table 22). That is, significantly
(p<0.01) less parent compound was present in soils 139
Table 22. Recovery of hexachlorobenzene (HCB) from West
Jefferson soil amended with 10 ug/g of i*C-HCB and incubated aerobically at 22-25 C for one year. In the last column, means followed by a common letter are not significantly different at the 95% confidence level.
Hexachlorobenzene
Sample Mean ug/g St. Dev Mean Percent
(n=2) (ug/g) Recovery
Agrimul 70 (%, v/w)
0.1 0.28 0.054 2.84 d
1.0 0.87 0.003 8.73 cd
5.0 3.77 0.668 37.7 a
1.0 + Cd(a> 0.61 0.050 6.15 cd
Morwet 425 (%, w/w)
0.1 0.81 0.007 8.12 cd
1.0 2.92 0.176 29.2 b
0.1 + Cd<»> 1.07 0.043 10.8 c
No Surfactant 0.74 0.084 7.37 cd
(a) Cd controls (1000 ug/g) became septic during the
year-long incubation. 140
containing low concentrations of surfactants (0.1 and 1%
Agrimul 70; 0.1% Morwet 425) or no surfactant compared to
high surfactant treatments (5% Agrimul 70; 1% Morwet 425).
Significantly (p<0.05) more parent compound (37.7% of the
original dose) was recovered from the 5% Agrimul 70
treatment compared to the highest treatment with Morwet 425
(29.2% recovery of the original dose). Because no
mineralization of HCB was detected, as indicated by the
lack of HC02 production, the parent compound may have been
transformed either to other stable products or to volatile
compounds other than C02. However, this transformation
apparently was inhibited c epressed by the highest
concentrations of surfactants used. These results were the
opposite of the expected results.
The gas chromatograms from the analyses for HCB in the
soil extracts indicated the production of unidentified metabolites with retention times near HCB (Figure 3). None
of the unidentified peaks matched the retention times of
any of the standards that were chromatographed for
comparison: 1,2-, 1,3-, or 1,4-dichlorobenzene, or 1,2,4- trichlorobenzene.
To determine whether the significantly greater recovery of parent compound from soils with the highest
concentrations of surfactants was due simply tb surfactant- enhanced recovery during soil extraction, or actually was
the result of a loss of parent compound from soil, soil 141
HCB
HCBj
HCB
Figure 3. Sample GC results of analysis for hexachloro benzene (HCB) In soils; (a) 1 ug/ml HCB standard; (b) Soil treated with HCB and 5% (v/w) Agrimul 70; (c) Soil treated with HCB but no surfactant. 142
samples were combusted for recovery of total 14 CO2 .
Duplicate soil samples from each jar were combusted, and
because each treatment was originally done in duplicate,
the results represent the mean of four combustions per
treatment. The data were analyzed for statistically
significant differences using one-way ANOVA and Duncan’s
Multiple Range Test.
The results for Agrimul 70 confirm that soil with the
highest concentrations of surfactant actually had more
remaining radiolabel than either the non-surfactant soil or
the soil with the lower doses of surfactants (Table 23).
That is, significantly more H C O 2 was recovered from soil
treated with the 5% dose of Agrimul 70 than from remaining
Agrimul treatments (p<0.01) or the non-surfactant control
(p<0.05). The mean percent recovery of added radiolabel as
HCO 2 was 37.6% in the case of soil treated with 5% Agrimul
70 (Table 23). On the other extreme, the mean recovery of
radiolabel was 11.7% from the soil treated with 0.1%
Agrimul 70. This was significantly less radiolabel
remaining than in either the non-surfactant control
(p<0.05) or the high-surfactant treatment (p<0.01).
Cadmium controls (1000 ug/g as CdCl2 , filter-sterilized 1%
Agrimul 70, filter-sterilized HCB) were included in this
experiment in order to distinguish between loss of compound due to biological activity versus physical-chemical mechanisms. However, the controls became septic during the 143
Table 23. Recovery of 14C02 from combustion of West
Jefferson soils amended with i 4C-hexachlorobenzene. Soil
originally contained 4310 disintegrations per minute/g
(DPM/g). Within the last column, means followed by a
common letter are not significantly different at the 95% confidence level.
HC02
Sample Mean DPM/g St. Dev. Mean Percent
(n=4) (DPM/g) Recovery
Agrimul 70 (%, v/w)
0.1 504 185 11.7 d
1.0 691 105 16.0 c
5.0 1622 417 37.6 a
1.0 + Cd(a) 303 23 7.03 d
Morwet 425 (%, w/w)
0.1 550 75 12.8 d
1.0 1491 83 34.6 a
1.0 + Cd 669 126 15.5 c
No Surfactant 1050 587 24.4 b
(a) Cd controls (1000 ug/g) became septic during the
year-long incubation. 144
Incubation as evidenced by the growth of fungi on the soil surface.
By comparing Tables 22 and 23 it can be seen that, in the case of 5% Agrimul 70, the remaining radiolabel was present as HCB. That is, the mean percent recovery of HCB was 37.7% (Table 22) and the mean percent recovery of total radiolabel by combustion was 37.6% (Table 23). With the other concentrations of Agrimul 70, however, only a portion of the remaining radiolabel was present as parent compound.
For example, with the 0.1% Agrimul 70, 2.84% of the original dose of HCB was recovered as parent compound.
However, 11.7% of the original radiolabel was still in the soil (Table 23), apparently as metabolites of HCB. In addition, significantly (p<0.05) less radiolabel remained in the soils with the two lowest doses of Agrimul 70 compared to the soil with no surfactant. Therefore, the low dpses of surfactants may have stimulated the biotransformation of HCB or particularly the metabolites of
HCB, compared to no surfactant.
In the case of Morwet 425, similar results were obtained. The mean recovery of added i«C-HCB as 14C02 from the high-surfactant soil after combustion was 34.6%, which was significantly (p<0.01) greater than the low-surfactant soil (a mean recovery of 12.8%) and significantly (p<0.05) greater than the non-surfactant control (mean recovery of
24.4%) (Table 23). The mean recovery of radiolabel from 145
the low-surfactant soil was significantly (p<0.05) less
than the mean recovery from the non-surfactant control. As
with Agrimul 70, comparison with the results in Table 22
indicated that the recovered radiolabel in the 5% Morwet
dosed soil was present as parent compound.
The second HCB biodegradation experiment included the
addition of non-radiolabeled HCB to sediment in order to
test for anaerobic degradation. Fresh pond sediment in
screw-cap bottles was amended with 10 ug HCB/g (dry weight
basis), 20 mmol ammonium acetate, and 0.1, 1, or 5%
solutions of either Agrimul 70 (v/v basis) or Morwet 425
(w/v basis). The bottles were filled to the very top with
surfactant solution and capped. After 30, 60, and 365 days
of incubation at 35 C, the sediment was resuspended and an
aliquot of the suspension was extracted and analyzed for
HCB using electron-capture gas chromatography as described
in Chapter II. The percent loss of parent compound was
analyzed using one-way ANOVA and Duncan’s Multiple Range
Test.
The results were similar to the previous results with
HCB-amended soil in that surfactants, especially at the
highest concentrations tested, tended to inhibit the loss
of HCB. This is most obvious in the one-year treatments
(Table 24). That is, the non-surfactant control sediments contained 12.4% of the initial concentration of HCB, which
was significantly (p<0.05) less than in any surfactant 146
Table 24. Mean hexachlorobenzene (HCB) remaining in septic anaerobic sediments following the addition of 10 ug/g HCB on day 0 (n=2). Within columns, means followed by a common
letter are not significantly different at the 95% confidence level.
Hexachlorobenzene (ug/g)
Day 30 Day 60 Day 365 Sample
Agrimul 70 (%, v/v)
0.1 5.27 b 3.48 be 3.44 be
1.0 6.93 ab 6.73 ab 3.39 be
5.0 9.23 ab 7.93 a 4.07 ab
Morwet 425 (%, w/v)
0.1 9.79 a 5.22 abc 2.74 c
1.0 7.52 ab 6.04 ab 2.69 c
5.0 7.66 ab 8.23 a 4.69 a
No Surfactant 1.18 c 1.24 c 1.24 d (n=3) 147
treatment. The mean percent remaining HCB in the two
highest surfactant treatments (5% Morwet 425 and Agrimul
70) were 46.9 and 40.7%, respectively. These were not
significantly (p>0.05) different from each other but the 5%
Morwet 425 treatment contained significantly (p<0.05) more
HCB than any other treatment except the 5% Agrimul 70
treatment. In the other surfactant treatments, the mean
percent of the initial HCB varied from 26.9 (1% Morwet 425)
to 34.4% (0.1% Agrimul 70), results that were not
significantly (p<0.05) different. In addition, the results
for all three concentrations of the Agrimul 70 treatments
ranged from 33.9 to 40.7% of the initial HCB and did not
differ significantly (p<0.05).
The pattern of HCB persistence in the presence of
surfactants was also evident in the 30- and 60-day samples.
In the 30-day control samples, the mean level of HCB had
already declined to 11.8% of the initial amount added
(Table 24), which was significantly (p<0.05) less HCB than
in any surfactant treatment. For example, the least amount
of initial HCB remaining in the surfactant-treated
sediments was 52.7% (0.1% Agrimul 70), and the greatest
amount was 97.9% of the initial HCB added (0.1% Morwet
425). By day 60, the pattern described above for the one- year samples was apparent, in that the greatest mean levels of HCB were found in the sediments containing the highest concentrations of surfactants (Table 24). In all the treatments discussed above, the production
of gas was obvious, indicating anaerobic microbial
activity. Substantial gas production was observed within a
few days of the initiation of the experiment. Although the
gas was not qualitatively or quantitatively analyzed, gas production was most likely the result of methanogenesis
because of the prior addition of ammonium acetate and the
anaerobic nature of the sediment and incubation. In
contrast to the septic treatments, aseptic treatments were
also included in an attempt to differentiate between
biological and non-biological degradation of HCB. The
sterile controls were prepared using autoclaved sediments
and filter-sterilized ammonium acetate and HCB. Initially,
the sterility of the sterile controls was maintained as
indicated by the visual absence of gas production. At the
30-day sampling, no significant differences (p=0.08)
occurred in the amount of HCB recovered from any of the
sterile sediments (Table 25). The sterility of the
sediments, however, apparently was not maintained, as
visually indicated by the production of gas in the
autoclaved treatments by day 60. When the 60-day samples
were analyzed for HCB, the amount of HCB in the non
surfactant aseptic controls was 29.5% of the initial level.
This was not significantly different (p>0.05) from three of the aseptic surfactant treatments but was significantly
different (p<0.05) from three other treatments (Table 25). 149
Table 25. Mean hexachlorobenzene (HCB) in autoclaved
sediments following addition of 10 ug/g HCB on day 0 (n=2).
Within columns, means followed by a common letter are not
significantly different at the 95% confidence level.
Hexachlorobenzene (ug/g)
Day 30 Day 60 Day 365 Sample
Agrimul 70 (%, v/v)
0.1 5.64 a 3.06 b 2.11 de
1.0 5.67 a 5.74 ab 5.79 c
5.0 8.33 a 8.43 a 7.64 b
Morwet 425 (%, w/v)
0.1 5. 30 a 5.30 ab 3.27 d
1.0 7.97 a 7.97 a 8.14 b
5.0 10.0 a 10.0 a 10.2 a
No Surfactant 3.57 a 2.95 b 1.32 e (n=3) 150
By the one-year sampling, a pattern of HCB persistence had developed that was similar to the one-year septic samples.
That is, the non-surfactant controls contained the least amount of HCB (13.2% of the initial level) and the higher surfactant treatments generally contained the most HCB. Despite the failure of the sterile treatments, a comparison with the septic treatments indicated that the one-year septic samples were significantly different than the one-year "sterile" samples. For example, in the Morwet
425 experiments, significantly (p<0.01) more HCB was recovered from the one-year 1 and 5% surfactant "sterile" treatments than from any other treatments (Table 26).
Similarly, the one-year 1 and 5% "sterile" treatments of
Agrimul 70 contained significantly (p<0.05) more HCB than any other treatment (Table 27).
The results described above indicate that HCB was biodegraded (or biotransformed) in anaerobically incubated sediments, but that surfactants inhibited the degradation process apparently in a concentration-dependent manner.
Loss of parent compound in sediments with no surfactants was fairly rapid, in that over 85% of added HCB was degraded or transformed within 30 days in non-surfactant septic sediments. These were not the expected results.
Therefore, the original hypothesis that surfactants can be used to stimulate the anaerobic biodegradation of HCB must be rejected, at least with respect to the two surfactants 151
Table 26. Comparison of mean hexachlorobenzene (HCB) in
Morwet 425-treated septic and autoclaved sediments.
Sediments were dosed with 10 ug/g HCB on day 0 (n=2).
Within columns, means followed by a common letter are not significantly different at the 95% confidence level.
Hexachlorobenzene (ug/g)
Morwet Dose Day 30 Day 60 Day 365 (%, w/v)
Septic
0 1.18 d 1.24 c 1.24 e
0.1 9.79 a 5.23 abc 2.74 d
1.0 7.52 ab 6.04 abc 2.69 d
5.0 7.67 ab 8.23 a 4.70 c
Autoclaved
0 3.57 cd 2.95 be 1.32 e
0.1 5.26 cb 5.30 abc 3.27 d
1.0 7.97 ab 7.97 ab 8.14 b
5.0 10.0 a 10.0 a 10.2 a 152
Table 27. Comparison of mean hexachlorobenzene (HCB) in
Agrimul 70—treated septic and autoclaved sediments.
Sediments were dosed with 10 ug/g HCB on day 0 (n=2).
Within columns, means followed by a common letter are not significantly different at the 95% confidence level.
Hexachlorobenzene (ug/g)
Agrimul Dose Day 30 Day 60 Day 365 (*. v/v)
Septic
0 1.18 c 1.24 c 1.24 e
0.1 5.27 abc 3.49 be 3.44 cd
1.0 6.93 ab 6.73 ab 3.39 cd
5.0 9.24 a 7.94 a 4.07 c
Autoclaved
0 3.57 be 2.95 be 1.32 e
0.1 5.64 abc 3.06 be 2.11 de
1.0 5.68 abc 5.75 ab 5.80 b
5.0 8.33 a 8.43 a 7.65 a 153 tested. The results also cast doubt on the utility of surfactants for stimulating the anaerobic biodegradation of other halogenated hydrophobic organic pollutants (discussed more fully in Chapter IV).
TCDD BIODEGRADATION EXPERIMENTS
Several experiments to test for the biodegradation of
TCDD in soils from the Times Beach area were conducted.
The hypothesis was that TCDD biodegradation can be stimulated in microbially active soils if the soils are managed in a manner conducive to the stimulation of general microbial activity. Positive results could lead to methods to stimulate the ip situ biodegradation of TCDD in soils.
The first set of experiments included the further amendment of the three TCDD soils with HC-TCDD, complete mineral fertilizer, and various amounts of fresh, non-TCDD soil. These amendments, described more fully in Chapter
II, were added in order to 1) determine whether the mineralization of HC-TCDD to H C O 2 would occur in microbially active soils previously acclimated to TCDD, 2) determine whether complete inorganic fertilization along with proper aeration and moisture was sufficient to stimulate the indigenous microflora to metabolize TCDD
(bioaugmentation), 3) determine if the dilution of TCDD soils with microbially active non-TCDD soil would stimulate
TCDD biodegradation, and 4) examine the combination of the 154 above factors in stimulating the biodegradation of TCDD.
Throughout a one-year incubation of TCDD soils, in which triplicate jars of each treatment were periodically aerated and monitored for evolution of H C O 2 , no mineralization of HC-TCDD was detected in any treatment. That is, all liquid scintillation counts were similar to background counts of 40-60 DPM. Despite the considerable microbial activity in the Missouri soils and the apparent lack of toxicity of TCDD to soil microorganisms (as shown in results described earlier), apparently TCDD remained recalcitrant, even when soil microorganisms presumably were stimulated by the addition of inorganic nutrients. The fact that no adjustment of TCDD soil resulted in mineralization of added HC-TCDD leads to the rejection of the original hypothesis that TCDD mineralization can be effected by manipulating soil conditions. However, the lack of TCDD mineralization did not rule out the possibility that TCDD may have been biotransformed to volatile or non-volatile daughter products.
The second TCDD biodegradation experiment involved the addition of a surfactant to the three Missouri soils containing HC-TCDD. This was done to test the hypothesis that TCDD biodegradation in soils is limited by its unavailability to the soil microflora, and that if rendered bioavailable with a nontoxic treatment, TCDD would be mineralized. If the hypothesis proved to be true, then a 155
method for stimulating the jya situ microbially mediated
decontamination of TCDD soils may be possible, based on the
metabolic diversity of the indigenous soil microorganisms.
The surfactant, Agrimul Jl, was chosen because it was
shown to be relatively effective at extracting TCDD from
soils, compared to other treatments tested. That is, two
surfactants, Agrimul Jl and Agrimul 70, which had been
tested for their ability to extract HCB from soil and their
toxicity to soil respiration, were subsequently tested for
their ability to extract H C from two Missouri soils that
had been amended one year earlier with 14C-TCDD. The
Missouri soils originally contained either 1.1 or 2.4 ug
TCDD/g. A 2% (v/v) solution of Agrimul Jl was 88% as
effective as toluene in extracting *4C (Table 28). At the
time of the extraction, the assumption was made that the
extracted 14C represented i4C-TCDD, even though the soils
had been incubated for one-year in an effort to stimulate
TCDD biodegradation (as described earlier). Subsequent
analysis of soils by GC/MS suggested the possibility that
14 C-TCDD was converted to daughter products in at least one
of the soils (PR). Thus, surfactant-extracted 14C may not
have represented only i 4C-TCDD. Nevertheless, Agrimul Jl was chosen as the surfactant to test the hypothesis that
TCDD mineralization in soils can be stimulated by enhancing
its solubility. 156
Table 28. Surfactant-mediated extraction of H C from
Piazza Road (PR) and Shennendoah Stables (SS) soils amended with 14C-TCDD. Surfactant concentrations were 2% (v/w) compared to extraction with 100% toluene.
Mean DPM/g Percent of
Soil Treatment (n=2) SD( ») toluene control
PR Agrimul 70 36.1 1.76 51.1
Agrimul Jl 37.4 0.58 53.0
Ag 70 + Ag Jl 32.3 1.78 45.7
Toluene 70.6 6.25 NA( b)
SS Agrimul 70 142.0 54.5 65.1
Agrimul Jl 192.7 11.8 88.3
Ag 70 + Ag Jl 64.8 4.95 29.7
Toluene 218.3 162.3 NA
(a) Standard deviation of the mean
(b) Not applicable 157
Missouri soils originally containing TCDD at 0.008,
1.1, or 2.4 ug/g had been amended with i* C-TCDD and
complete inorganic fertilizer and tested previously for the
evolution of *4C02, as described earlier. Subsequently,
duplicate samples of these soils were amended with 2% (v/w,
dry soil basis) Agrimul J1 and monitored for the evolution
of i4C02. No production of 14 002 was detected through 6
months of incubation at 22 C; as in other experiments,
liquid scintillation counts were always comparable to
background (40-60 DPM). The soils were then extracted as
described in Chapter II and analyzed for total TCDD using
high-resolution GC/MS. Soils that had not received Agrimul
J1 served as controls. The soils were also analyzed for
microbial enumeration and were combusted for total recovery
of 140 as 14 002, as described in Chapter II.
Approximately 50% of the original TCDD was lost from
the soil with the lowest original TCDD concentration, that
is, soil TB (Table 29; GC/MS results are shown in the
Appendix). This was true in both Agrimul Jl-treated and
non-surfactant control soils. On the other hand, soil PR
that initially contained 1.1 ug TCDD/g, lost over 90% of
the original parent compound in both surfactant and non
surfactant treatments (Table 29). These results occurred
despite the fact that no evolution of 14C02 was detected,
indicating that TCDD was converted to volatile or non volatile daughter products. 158
Table 29. TCDD in soils (ng/g) initially, after one year of bioaugmentation, and following six months of incubation with or without 2% (v/w) Agrimul Jl.
Initial TCDD TCDD post- TCDD post-
Soil(a) (Day 0) bioaugmentation surfactant
TB 8.0 4.2 4.8
PR 1100 54 52
SS 2400 2000 987
WJ 0 0 0
(a) TB = Times Beach; PR = Piazza Road; SS = Shennendoah
Stables; WJ = West Jefferson 159
Soil SS initially contained 2.4 ug TCDD/g and also
showed an approximately 50% loss of the parent compound
after Agrimul J1 treatment (Table 29). However, the
concentration of TCDD in the non-surfactant, bioaugmented
soil was essentially unchanged from the initial level. As
before, no production of 14C02 was detected.
Although no evolution of 14CJ02 was detected from any
treatment, the combustion of nc-TCDD soils and subsequent
capture of 14(302 showed losses of the 14C-TCDD added
initially (Table 30). No significant (p>0.05) differences
occurred between similar TCDD soils treated or untreated
with Agrimul Jl. For example, surfactant-treated and
untreated soils TB contained 60.5 and 49.0% of the original
radiolabel, respectively, results that were not
significantly (p>0.05) different from one another.
However, the results for the surfactant-treated TB were
significantly (p<0.01) different from remaining treatments.
The least amount of added radiolabel recovered (31.6%) was
from soil PR soil containing no surfactant.
These results suggest that between approximately 40
and 70% of the originally added radiolabel had escaped from
the soils. Because no evolution of 14C02 was detected, the
indication is that the loss of added radiolabel occurred as
non-C02 volatiles.
Finally, the TCDD soils were plated for microbial enumeration as had been done at the beginning of this 160
Table 30. Combustion of KC-TCDD soils for recovery of i*C02. TCDD soils initially were amended with 4400 DPM/g additional HC-TCDD Prior* to incubation. Within the last column, means followed by a common letter are not significantly different at the 95% confidence level.
Mean Mean
Soil Plus 2% (v/w) Agrimul J1 TB 2662 264. 3 60.5 a PR 1876 275.2 42.6 be SS 1993 362.6 45.3 b WJ 61.9 2.4 NA( O No Agrimul J1 TB 2162 13.4 49.0 ab PR 1388 206.7 31.6 c SS 1690 61.2 38.4 be WJ 61.9 0.41 NA (a) TB = Times Beach; PR = Piazza Road; SS = Shennendoah Stables; WJ = West Jefferson. (b) Standard deviation of the mean. (c) Not applicable. 161 project. This was done to determine if presumably solubilized and bioavailable TCDD had been inhibitory to indigenous soil microorganisms. Soil SS (the highly TCDD contaminated Missouri soil) treated with Agrimul J1 had 1.29x10® eutrophic bacteria per gram of soil, which was significantly (p<0.05) more eutrophic bacteria than any other soil (Table 31). The soil with the least original amount of TCDD, soil TB, had significantly (p<0.05) fewer eutrophic bacteria than the other TCDD soils. These results also coincide with the amount of organic matter in each of the TCDD soils (see Table 5). In general, all soils treated with Agrimul J1 had significantly (p<0.05) more eutrophic bacteria than their non-surfactant counterpart soils. Similar results were observed with respect to oligotrophic bacteria (Table 31). Agrimul Jl-treated SS had significantly (p<0.05) more oligotrophic bacteria than any other soil (41.8x10®/g); soil TB had the fewest oligotrophs (19.2x10®/g); and the number of oligotrophic bacteria was significantly (p<0.05) greater in all Agrimul Jl-treated soils than in their non-surfactant counterparts. Compared to the results for eutrophic and oligotrophic bacteria, the results for actinomycetes and fungi are substantially different (Table 31). Among the Agrimul Jl- treated soils, soil TB (low TCDD) contained significantly (p<0.05) more actinomycetes and fungi than the other TCDD 162 Table 31. Mean microbial numbers in TCDD and non-TCDD soils after treatment with Agrimul J1 and aerobic incubation for 6 months (n=5). Within columns, means followed by a common letter are not significantly different at the 95% confidence level. Colony Forming Units/g( «) Eutro Oligo Actino Fungi Sum Soil( b) (xlO®) (xlO® ) (Xl04) (X104) (xlO®) Agrimul TB 14.2 cd 19.2 c 115 a 9.46 b 19.4 c PR 118 b 26.0 b 11.0 b 2.34 c 27.2 b SS 129 a 41.8 a 0.45 c 1.07 d 43.1 a WJ 32.6 c 13.0 c 562 a 900 a 13.5 c No Agrimul TB 4.50 e 1.60 d 196 a 2.90 c 1.66 d PR 6.06 e 1.40 d 542 a 1.90 c 1.51 d SS 10.6 d 2.00 d 0.70 c 4.80 b 2.11 d WJ< O 9.32 d 2.12 d 410 a 946 a 2.35 d (a) Eutro = eutrophic bacteria; Oligo = oligotrophic bacteria; Actino = actinomycetes. (b) TB = Times Beach; PR = Piazza Road; SS = Shennendoah Stables; WJ = West Jefferson. (c) Microbial enumeration in West Jefferson soil prior to aerobic incubation (from Table 6). 163 soils. Soil SS (high TCDD) had significantly (p<0.05) fewer actinomycetes and fungi than other TCDD soils. The number of fungi in SS was also significantly (p<0.05) lower than in any of the non-surfactant soils. Finally, in terms of total organisms plated per gram of soil, the Agrimul Jl-treated soils contained significantly (p<0.05) more organisms than the non surfactant soils. Among the surfactant-treated soils, SS had significantly (p<0.05) more total organisms than the other soils, due to the number of eutrophic and oligotrophic bacteria. These results suggest that the presence of TCDD may have suppressed the numbers of actinomycetes and fungi. This is seen in Table 31, in which the numbers of these organisms appear to decrease with increasing TCDD concentration. This trend was also indicated earlier (see Table 6) with respect to soil NJ, which was highly contaminated with TCDD and other co-contaminants. CHAPTER IV GENERAL DISCUSSION SOIL CHARACTERIZATION Soils from the Times Beach area of Missouri and from a pesticide manufacturing plant in New Jersey contained relatively high levels of TCDD compared to levels normally expected in soils (see Table 5). A soil from an agricultural field near West Jefferson, Ohio, to which various pesticides had been previously applied, contained no detectable TCDD. TCDD in the Missouri and New Jersey soils is the result of unintentional contamination by chemical wastes more than 10 years ago and has probably remained largely unchanged since (Jackson et al., 1986). Complicating the issue of potential environmental effects due to TCDD is the presence of co-contaminants, as indicated by the solvent-extractable contents of the TCDD soils (see Table 5). The co-contaminants are the result of the matrix in which TCDD was applied to the soil ( e.g., chemical still bottoms) and the competing reactions during TCDD formation (Esposito et al., 1980) (Figure 1). Compared to other TCDD soils, soils TB, PR, SS, and NJ were heavily contaminated with TCDD. For example, Norris 164 165 (1981) calculated that forest soil sprayed repeatedly with 2,4,5-T at a rate of 2.24 kg/ha would accumulate TCDD over several years to levels around 0.001 ng/kg. Young (1983) reported that soils from Eglin Air Force Base in Florida, where TCDD-contaminated herbicides had been heavily applied for several years, contained at most 1.5 ug TCDD/g; most soils had less than 0.5 ug TCDD/g. At Seveso, Italy, soils reportedly contain on the average approximately 3.5 ng TCDD/g (Fanelli et al., 1980). Therefore, considering the concentrations of TCDD that affect many organisms (see Table 3), it is reasonable to assume that if TCDD soils present potential environmental problems such as microbial toxicity, these effects would manifest in the soils used in the current investigations. Also, the soils used in this research contained TCDD along with numerous co-contaminants, as compared to many studies that relied on dosing soils with neat TCDD or other neat chlorinated materials (e.g., Baker et al., 1980; Bumpus et al., 1985; Matsumura and Benezet, 1973; Muir et al., 1985). Jackson et al. (1986) found that TCDD in soils contaminated by complex mixtures of co-contaminants and subjected to years of weathering is less mobile than neat TCDD added to soils. The purpose of characterizing the TCDD soils was not only to determine TCDD levels, but also to measure other parameters that may influence microbial activity in the 166 soils. A factor that exerts considerable influence on microbial activity is the soil organic matter content. That is, microbial numbers in soils are generally roughly proportional to the soil organic matter content because organic matter is a source of reduced carbon and nitrogen, and improves soil tilth and moisture capacity (Alexander, 1977). Thus, soils SS and NJ would be expected to have the greatest microbial activity among the TCDD soils, other factors being equal. Also, the relatively high CEC of soil SS compared to the other soils indicates a fertile soil with potentially large microbial populations. The results of the soil characterization suggest that if TCDD is bioavailable and microbially toxic, and/or if co-contaminants are microbially toxic, then microbial activity should be greatest in soils WJ or TB and least in soils SS or NJ. On the other hand, if TCDD and/or co contaminants have no or minimal impact on microbial activity, then soil SS should have the greatest level of microbial activity (based on organic matter content and CEC), followed by NJ, WJ, PR, and TB. The experiments to ascertain the levels of microbial activity in TCDD soils are discussed below. MICROBIAL ACTIVITY IN TCDD SOILS The initial objective for measuring microbial activity in TCDD soils was to determine whether TCDD and/or 167 co-contaminants are toxic to soil microorganisms. Little work on the potential toxicity of TCDD to microorganisms has been reported. According to Hill (1978), the environmental persistence of many chlorinated organic compounds is due to the toxicity of the compound or associated compounds. Bollen and Norris (1979) predicted that relatively high concentrations of TCDD in soils would inhibit microbial respiration. Although the highest TCDD concentration that they tested (5.2x10“5 ug/g) had no effect on soil respiration, their prediction that higher concentrations would be inhibitory was based on TCDD toxicity to other organisms (e.g., Table 3). In studies on the metabolism of nonchlorinated dibenzo-p-dioxin and chlorinated dioxins other than TCDD, Klecka and Gibson (1980) found that succinate-grown Beiierinckia was inhibited after 4 hours in the presence of 0.05% (w/v) dibenzo-p-dioxin. The inhibition was attributed to a metabolite, 1,2-dihydroxydibenzo-p-dioxin, which proved to be a mixed-type inhibitor of two ring-fission enzymes: 2,3- dihydroxybiphenyl oxygenase and catechol oxygenase. Further information on microbial toxicity of TCDD is not available. The second objective for measuring microbial activity in TCDD soils was to assess the potential for stimulating indigenous microorganisms to metabolize soil-bound TCDD. 168 That is, in situ bioremediation of contaminated soils requires viable mixed populations of indigenous microorganims in order to metabolize or co-metabolize pollutants (Dagley, 1975, 1983; Hankin and Sawhney, 1984). Anderson (1984) found that the rates of degradation of thiocarbamate herbicides in soil were directly related to soil microbial biomass. In addition, complex non biochemical pollutants (such as TCDD) apparently are rarely attacked by a single organism. For example, degradation may rely initially on extracellular attack by fungi followed by further metabolism of polar degradates by mixed bacterial populations (Dagley, 1975; 1983). In the case of TCDD soils, no information was available in the literature concerning the presence or proportions of viable microorganisms. If microbial populations were suppressed in TCDD soils compared to non- TCDD soils, the possibility to effect in situ biodegradation could be severely limited. The results of the microbial activity measurements indicated that soil-bound TCDD and/or co-contaminants had some impact on microbial numbers and activity, but not to the degree expected based on the toxicity of TCDD to other organisms. For example, despite its high organic matter content, soil NJ was the only soil with a significantly reduced number of eutrophic bacteria (see Table 6). On the other hand, NJ had very high TCDD and solvent-extractable 169 concentrations. Therefore, low numbers of eutrophic bacteria in NJ may be a response to the high level of contamination. Another interesting point concerning NJ was the significantly lower numbers of actinomycetes compared to other soils. When NJ was monitored for soil respiration, however, no suppression of microbial activity was observed compared to soil WJ (see below and Table 11). The number of fungi was significantly less in all TCDD soils compared to the non-TCDD soil (see Table 6). Based on the level of organic matter in the soils, WJ would not normally be expected to have the greatest population of fungi. Instead, soil SS or NJ would normally be expected to contain the greatest number of fungi. These results suggest that fungi were inhibited in TCDD soils. The numbers of fungi were not significantly different among TCDD soils and did not vary in proportion to TCDD or solvent-extractable concentrations. Therefore, it is not clear whether the low numbers of fungi in TCDD soils were due to TCDD, co-contaminants, or combined effects. Similar results were seen concerning oligotrophic bacteria. Significantly more oligotrophic bacteria were plated from the non-TCDD soil than from any of the TCDD soils, suggesting inhibition in the TCDD soils. However, the numbers of oligotrophic bacteria in TCDD soils did not vary in proportion to TCDD or solvent-extractable contents. 170 It is well known that enumeration methods for viable microorganisms in soils tend to underestimate the actual number of viable microorganisms (Wollum, 1982). Soil dilution plating underestimates viable micoorganisms largely because media are unavailable which fully satisfy the nutritional needs of all soil microorganisms. Nevertheless, when performed consistently, enumeration by the soil dilution method yields relative results among different soils (Wollum, 1982). Therefore, while the enumeration results of this study should not be interpreted in absolute terms, they do indicate relative numbers of organisms among the soils. Total numbers of microoganisms and specific physiological groups were suppressed in TCDD soils relative to a similar but non-TCDD soil. Another indicator of soil microbial activity in TCDD soils was the analysis of certain soil enzymes important in nutrient cycling. Soil enzyme activity has been suggested as a measure of biological activity and productivity in soils (Skujins, 1967), and work is continuing in this area (Tabatabai, 1982). Soil enzymes have been analyzed in other toxicity studies with both inorganic and organic pollutants, but not with TCDD. For example, Tyler (1974) examined the response of soil urease, acid phosphatase, B- glucosidase, and soil respiration to Cu and Zn contamination. Acid phosphatase, urease, and soil 171 respiration activity decreased in response to increasing metal concentrations, while B-glucosidase was unaffected. Lindemann and Ryder-White (1983) found that dehydrogenase and soil respiration in forest soil were unaffected by normally applied levels of 2,4,5-T. Tabatabai (1982) discussed the potential applications of soil enzyme measurements in environmental toxicology and called for more work in this area. Although effects of TCDD on soil enzymes have not been reported, studies with higher organisms have shown numerous effects of TCDD on enzyme systems (see Chapter I). Some enzymes are stimulated, some are inhibited, and some are unaffected by TCDD (Esposito et al., 1980). In some cases, there appear to be specific receptors that bind TCDD prior to enzyme induction. In the current research, the activity of five different enzyme systems was variable among the soils tested and did not correlate with enumeration results or soil properties. Soil PR, which contained the intermediate levels of TCDD (1.1 ug/g) and organic matter (2.7%) among the Missouri soils, had the greatest activity of four out of the five enzymes examined (see Table 7). This is in contrast to the microbial enumeration results discussed above, in which the non-TCDD soil WJ had the greatest number of viable microorganisms. The soil with the greatest TCDD and organic matter concentrations tested in 172 the enzyme assays, soil S3, had the lowest activity of arylsulfatase and rhodanese, but the greatest acid phosphatase activity. Even though the enzyme results are variable, they indicate some interesting points compared to the enumeration results. Oligotrophic bacteria accounted for the greatest proportion of total platable microorganisms in all soils and were significantly greater in soil WJ than all TCDD soils. On the other hand, soil PR generally had the greater enzyme activity among all soils, as well as the greatest number of eutrophic bacteria and actinomycetes. These results suggest that soil enzyme assays may indicate the activity of non-oligotrophic organisms, possibly because of the lower metabolic rates of oligotrophs compared to non-oligotrophs. Thus, the lack of correlation of enzyme results with enumeration data would be expected. In addition, these results indicate that soil PR is generally the most metabolically active soil among the TCDD soils, despite the higher organic matter content of soil SS. It is possible that the relatively lower microbial activity indicated in soil SS compared to PR is due to TCDD and/or co-contaminants, because specific microbial groups appeared to be suppressed in other experiments in which TCDD bioavailability was improved (see below). On the other hand, the soil respiration results did not indicate that soil SS was microbially supressed compared to other TCDD soils. In fact, despite differences in microbial numbers in TCDD soils compared to the non-TCDD soil, no suppression of soil respiration was observed in any TCDD soil. Soil respiration was not inhibited in soils containing TCDD between 2 ng/g and 26.3 ug/g. These results are in contrast to the prediction of Bollen and Norris (1979), that soil respiration would be inhibited by TCDD. The efficacy of the soil respiration assay was shown by the inclusion of Cd controls, which inhibited the evolution of CO2 -C from alfalfa meal-amended soils. In previous work, the soil respiration assay used in this study was shown to be a sensitive indicator of toxicity due to fly ash amendment, and the results compared well with other indicators of soil toxicity, such as field-observed toxicity of fly ash on crops (Arthur et al., 1984). Other researchers have used variations of soil respiration to assay toxic effects of organic compounds (e.g., Lindemann and Ryder-White, 1983; Brunner et al., 1985) and in biodegradation studies (e.g., Laskowski et al., 1983; Hankin and Sawhney, 1984; Boatman et al., 1986). The soil respiration assay is designed to indicate heterotrophic microbial activity by measuring the degree to which alfalfa meal is mineralized to CO2 . Previous research has shown that the majority of the CO2 -C that evolves from soils amended with alfalfa meal or other organic substrates (e.g., glucose) is the result of 174 biological activity (Arthur et al., 1984); approximately 80% of the C02-C evolved is the result of microbial activity (Alexander, 1977). Thus, this assay is a test for viable and active microorganisms. Soil enzyme assays, on the other hand, may measure a combination of extracellular and viable intracellular activities. Skujins (1967) pointed out that soil enzymes originate from a number of sources: viable microbial cells, soil-bound cell-free enzymes, cell-free enzymes released from lysed cells, enzymes* present in dead but not lysed cells, plants, and soil animals. These differences in the origin of soil enzyme activities compared to soil respiration have complicated previous efforts to directly correlate the two assays (Skujins, 1967; Tabatabai, 1982). Therefore, it is not suprising that enzyme and respiration assays in the TCDD soils did not agree exactly, and illustrates the need to consider more than one assay for micobial activity in soils. The combined results of enumeration, enzyme activity, and soil respiration were not as expected. Because of the reported toxicity of TCDD to other organisms, it was expected that TCDD soils would prove to be clearly toxic to indicators of microbial activity. This would have been in concurrence with predictions of Bollen and Norris (1979). Especially in the case of soil respiration, where TCDD concentrations were varied by dilution with non-TCDD soil, 175 it was expected that the calculation of an ECso (the concentration of TCDD that suppressed soil respiration by 50%) would be possible. In summary, using three indicators of microbial activity in TCDD soils, the soils proved to be microbially viable. Even though TCDD was present at concentrations expected to be clearly inhibitory, most indicators of microbial activity were comparable to those in a similar but non-TCDD soil. Lower numbers of oligotrophic bacteria, fungi, actinomycetes, and total microorganisms in contaminated versus non-contaminated soils were not reflected in suppressed soil respiration. Even in the case of soil NJ, in which enumeration results were consistently low relative to the other soils, soil respiration was not suppressed. These results led to the question: if TCDD is present at what should be toxic levels, but microbial activity appears to be largely unaffected in TCDD soils, what accounts for TCDD persistence? That is, if these soils are microbially active, why is TCDD apparently not biodegradable? As outlined in Chapter I, Hill (1978) described six reasons for the persistence of organic compounds in soils. Besides toxicity of the compound, the other reasons for environmental persistence are: microorganisms may lack necessary degradative enzymes, the compound may not penetrate the cell, the compound may be 176 adsorbed to soil and rendered unavailable, its steric configuration may prevent enzymatic attack, or the environment may be toxic or deficient in its ability to support microbial activity. The last possibility described above seemed unlikely based on the results of the microbial activity assays. That is, microbial activity in TCDD soils containing a number of co-contaminants was not greatly different than in a fertile, agricultural, non-TCDD soil. Also, soil-bound TCDD toxicity seemed unlikely, because no inhibition of soil respiration occurred at TCDD concentrations from 2 ng/g to 26.3 ug/g. Therefore, because the soils were microbially active, an effort was made to determine if TCDD biodegradation could be stimulated by further enhancing microbial activity in the soils. That is, Missouri TCDD soils were amended with additional HC-TCDD and complete inorganic fertilizer, diluted to various levels with non-TCDD soil, and were incubated under favorable conditions of moisture and aeration. This approach, sometimes referred to as bioaugmentation, is recommended by the U.S EPA for in situ decontaminantion efforts because it relies on relatively simple soil management practices and indigenous microorganisms. TCDD was not mineralized after one year of incubation, as evidenced by the lack of evolution of HC02. Similar results were reported in previous attempts to mineralise TCDD and other dioxins in soils and broth cultures (Klecka and Gibson, 1980; Hutter and Phillipi, 1982; Quensen and Matsumura, 1983). However, subsequent analysis of soils by high-resolution GC/MS indicated that in soils TB and PR, the parent TCDD was converted to daughter products (see Table 29). The conversions apparently were co-metabolic in nature, although the direct metabolism of TCDD to metabolites can not be ruled out. Some of the daughter products may have been stable and would have accumulated in the soil; others may have been volatile organics that would not be absorbed by the NaOH traps used to collect H C O 2 . The conversion of TCDD to daughter products in soils was not specifically analyzed. These results suggest that in some TCDD soils (e.g., TB and PR), TCDD biotransformation may be enhanced by simple bioaugmentation. In other soils (e.g., SS), more drastic measures may be necessary to stimulate TCDD degradation. This may be due to the nature of the soil, the presence of co-contaminants that affect the availability of TCDD, or the concentration of TCDD. The results also illustrate that even in soils in which TCDD can be transformed to daughter products, no mineralization of TCDD occurred. Nevertheless, conversion of - TCDD to any daughter products is probably desirable, considering the toxicity of the parent compound to many test species. 178 The results of this phase of the experiments do not indicate a rigorously derived half-life of TCDD in soil subjected to bioaugmentation. This is because samples of soil were not periodically extracted and analyzed for TCDD over the year-long incubation. That is, in order to determine the biodegradation half-life of organic compounds, the normal procedure is to determine the degradation rate constant, k, from the slope of the degradation curve (Larson, 1984). In first order kinetics (apparently applicable to many organic pollutants), the half-life is then ln2/k. In addition, approximately 95% of a material is degraded within five half-lives. Even though a rigorous kinetic approach was not used in the current experiments, in the case of soil PR, the concentration of TCDD decreased by approximately 95% within one year. This is equivalent to approximately five half- lives within one year of incubation, indicating a maximum bioaugmented half-life of approximately 2-3 months. The apparent degradative half-life may be less than 2-3 months in bioaugmented soil PR if 95% of the parent TCDD was transformed in less than one year. However, this was not determined. In the case of soil TB, approximately 50% of the initial parent compound was transformed to daughter products. Using the same rationale described above, the apparent TCDD degradative half-life is 6 months in 179 bioaugmented soil TB. As mentioned previously, no degradation of TCDD occurred within one year in bioaugmented soil SS. SURFACTANT EXPERIMENTS The objective of the surfactant experiments was to attempt to identify a non-toxic, cost-effective treatment to enhance the bioavailability of TCDD in soil. As discussed previously, one of the properties responsible for the environmental persistence of organic compounds is their unavailability (Hill, 1978; Knezovich et al.,1987). Unavailability implies that the compound is not only unexposed to constitutive degradative enzymes in the indigenous microflora, but inducible enzymes may never be stimulated. Improving the bioavailability of xenobiotic compounds for biodegradation by indigenous microorganisms has been proposed by the U.S EPA as a mechanism to effect in situ decontamination. In the case of TCDD, its high affinity for soil and extremely low water solubility render it unavailable to potentially degradative organisms (Esposito et al., 1980). However, Quensen and Matsumura (1983) found that TCDD metabolism could be stimulated depending on the solvent. That is, limited metabolism of TCDD by Bacillus megaterium and Nocardiopsis spp. was stimulated in the presence of dimethyl sulfoxide or ethyl acetate; corn oil inhibited 180 TCDD metabolism. No mineraliaation of TCDD was detected in any case. Quensen and Matsumura (1983) concluded from this and related work (Matsumura et al., 1983) that research on methods to mobilize TCDD for biodegradation is critical. However, little work has been reported in this area. Surfactants were chosen for the current work because of their usefulness in solubilizing chlorinated organic pesticides and their relative non-toxicity compared to potential solvents. For example, literally hundreds of anionic and nonionic surfactants are used routinely to solubilize highly water-insoluble chlorinated agricultural pesticides for application to soils (Rosen, 1985). On the other hand, cationic surfactants are often too toxic to employ routinely in agriculture (Huddleston and Allred, 1967). The potential microbial toxicity of surfactants is one of the factors limiting their use in soils. Also, some surfactant treatments have been shown to inhibit the biodegradation of biodegradable compounds (Urano and Saito, 1985), illustrating that surfactants for use in in situ reclamation must be chosen carefully. HCB Solubility Experiments Potential surfactants for solubilizing TCDD were initially chosen based on their ability to solubilize hexachlorobenzene (HCB), a surrogate compound for TCDD. Like TCDD, HCB is highly chlorinated; nearly insoluble in 181 water, and Is partitioned onto soils and sediments (Ausmus et al., 1979; Courtney, 1979). Unlike TCDD, HCB is easily obtained for research purposes and also is detectable using electron-capture gas chromatography. Nevertheless, by using a surrogate compound (HCB) in place of the compound of interest (TCDD), the assumption is made that the two compounds have similar environmental properties (biodegradability, toxicity, volatility, availability, fate and effects, etc.). This assumption may or may not be valid. The results of the HCB solubility trials in the absence of soil suggested that sufactants could be used to solubilize soil-bound HCB (and possibly TCDD and other chlorinated organics). In the presence of soil, however, the relative efficiencies of HCB extraction with the surfactants differed compared to the absence of soils. This illustrates the influence that soils can have on the fate of chemicals, especially those that partition onto soils and sediments. In addition, it is likely that surfactants behave differently in different soils, compared to their behavior in the one soil (WJ) used for the HCB experiments. Thus, if surfactants are to be used to mobilize soil-bound pollutants for jjj situ decontamination, tests of surfactant-mediated solubility should be conducted with the specific soil in question. This will aid in the selection of the proper surfactants from among the 182 thousands available. In the current research, surfactants were identified that solubilized soil-bound HCB up to 82% as effectively as benzene. However, the most effective HCB solubilizing surfactant was not the most effective TCDD solubilizing surfactant. That is, while the screens with HCB were helpful in identifying surfactants potentially useful with TCDD, specific solubility trials with TCDD were necessary to identify the best surfactant for use in TCDD biodegradation experiments. Therefore, the use of chlorinated surrogate compounds to represent other pollutants has its limitations. Surfactant Toxicity Experiments As mentioned previously, the toxicity of surfactants to soil microorganisms can limit their potential usefulness in soils for mobilizing pollutants. The toxicity experiments showed that surfactants varied in their toxicities. That is, some concentrations of surfactants that extracted HCB from soil were toxic to the three microbial indicators tested: development of colony forming units of Pseudomonas aeruginosa, bioluminescence of Photobacterium Phosphoreum. and soil respiration from alfalfa meal-amended soils. For example, Morwet 425, an anionic sodium napthalene formaldehyde, was very effective at solubilizing and extracting HCB from soil, but at 183 effective concentrations was toxic to all three indicators. On the other hand, Agrimul 70, a nonionic alkyl aryl polyether alcohol, was fairly effective at solubilizing and extracting HCB and was nontoxic in all three toxicity tests. In fact, Agrimul 70 may have been biodegraded in the soil respiration assay, as evidenced by greater evolution of C02-C from treated versus control soils. These results illustrate that surfactants may be microbially toxic and need to be chosen with care for large-scale use in soils. The results also support the use of the soil respiration assay as an indicator of microbial toxicity compared to other indicators. Compared to the other assays, the soil respiration test indicates the interactions of many microbial species and numerous microbial processes going on simultaneously. A disadvantage of the soil respiration assay compared to the other assays is the length of time required (e.g., one month or more). The alfalfa meal-amended soil respiration assay was also used to determine the potential toxicity of solubilized HCB in soils. That is, by improving the availability of normally unavailable xenobiotic compounds, toxicity rather than biodegradation may be stimulated. This situation could lead to increased environmental problems, such as plant and animal deaths due to uptake of xenobiotic compounds. Another potential problem with 184 mobilized pollutants is the possibility of increased groundwater contamination. The results indicated that HCB plus surfactants was no more toxic than HCB alone, despite sufactant concentrations that should have enhanced the bioavailability of HCB. The toxicity of HCB was substantially less than Cd induced toxicity, indicating the efficacy of the soil respiration assay. Whether surfactants plus other normally unavailable xenobiotics (TCDD, PCBs, etc.) would show similar results is not clear from the present results. HCB BIODEGRADATION EXPERIMENTS The objective of biodegradation experiments with HCB was to determine if nontoxic surfactants could be used to enhance the availability and thus the potential for biodegradation of a highly chlorinated, recalcitrant xenobiotic compound. Success with HCB could serve as a model for work with other chlorinated compounds. Both aerobic and anaerobic surfactant-mediated biodegradation were examined. Aerobic conditions were examined because the degradation of aromatic rings normally requires attack by oxygenases and molecular oxygen (Cerniglia, 1984; Ghosal et al., 1985). Anaerobic conditions were also employed because reductive dehalogenation of aromatic rings may be required prior to ring cleavage by oxygenases (Chapman, 1979; Mikesell and 185 Boyd, 1985). There is also evidence that anaerobic degradation of aromatic rings may occur after dehalogenation (Evans, 1977). Switching from anaerobic to aerobic conditions, which has been found useful in some degradation studies, was not attempted in the current experiments. No mineralization of C-HCB was detected in aerobically incubated soils. Nevertheless, GC analysis and soil combustion revealed that the parent compound disappeared in soils treated with low levels of surfactants. While the loss of the parent compound was confirmed, most of the compound was apparently lost as organic volatiles. This is indicated by the failure to account for all the added-radiolabel, either in the alkali traps or in the soil combustion. This problem could have been avoided if additional traps (e.g., ethylene glycol) had been included to collect organic volatiles. On the other hand, the highest concentrations of surfactants appeared to inhibit the loss and/or biotransformation of HCB. The radiolabel recovered from these soils apparently was present as HCB rather than daughter products, as revealed by comparing combustion and GC results. Urano and Saito (1985) reported that two cationic surfactants at 10 mg/1, and an anionic surfactant at 30 mg/1, inhibited the biodegradation of synthetic sewage sludge. These authors suggested that other 186 pollutants may be similarly affected but pointed out that no additional literature on surfactant-mediated inhibition of degradation was available. Therefore, the current results concerning HCB may be the first such report with a specific environmental pollutant. In the anaerobic sediments amended with HCB, surfactants also tended to inhibit degradation. This was apparent at all concentrations of surfactants tested and began within 30 days of anaerobic incubation. Loss of the parent compound in non-surfactant septic sediments was fairly rapid (85% loss within 30 days). Thus, no benefit with respect to HCB biodegradation was obtained by treating anaerobic sediments with Agrimul 70 or Morwet 425 surfactants. Whether other surfactants or other halogenated pollutants would give similar results requires additional research. TCDD BIODEGRADATION EXPERIMENTS The objective of the TCDD biodegradation experiments was to examine the feasibility of using a surfactant to stimulate the bioreclamation of TCDD-contaminated soil. While such a possibility has been speculated in the literature (e.g., Esposito et al., 1980; Quensen and Matsumura, 1983), no research in this area has been reported. 187 As discussed previously, TCDD concentrations decreased in soils TB and PR after fertilisation and one year of aerobic incubation. On the other hand, the TCDD concentration in soil SS was unchanged with the same treatment. As in the case of HCB, no mineralisation of added HC-TCDD was detected. Subsequent treatment with Agrimul Jl, a nonionic alkyl aryl polyether alcohol, appeared to stimulate the degradation of TCDD in soil SS, while no further degradation of TCDD occurred in soils TB and PR. In soil SS, approximately 50% of the original TCDD was degraded within six months of surfactant treatment. These results suggest the possibility to effect soil decontamination of TCDD using the indigenous (and/or added) microflora. The potential success of this approach, however, appears to depend on soil type and the original concentration of soil-bound TCDD. For example, parent TCDD in soils TB and PR disappeared within one year due to simple bioaugmentation. These soils originally contained levels of TCDD of 0.008 and 1.1 ug/g, respectively. Both soils had relatively low contents of organic matter and total solvent extractable material, and had electrical conductivities near 100 umhos/cm. On the other hand, simple bioaugmentation was not sufficient to stimulate degradation of parent TCDD in soil SS. This soil originally contained TCDD at a level of 2.4 ug/g, had relatively high contents of organic matter and 188 total solvent extractable materials, and an electrical conductivity approximately twice that of soils TB and PR. In soil SS, TCDD degradation was stimulated by the addition of Agrimul Jl, unlike the results for soils TB and PR. The influence of soil characteristics on TCDD behavior was examined by Jackson et al. (1986). They reported that TCDD mobility in soils from Missouri was very complex and was affected by total solvent extractables and soil electrolytes. Organic co-contaminants (solvent extractables) were shown to enhance the solubility and transport of TCDD in soil. On the other hand, soil electrolytes inhibited TCDD mobility and appeared to be the factor controlling TCDD mobility; when electrolytes were leached from the soils, TCDD mobility in subsequent leauhings was significantly enhanced. Others have reported that soil organic matter retards TCDD mobility in soils (Norris, 1981; Young, 1983; Umbreit et al., 1986). Jackson et al. (1986), however, found no influence of soil organic matter on TCDD mobility. There have been no reports of the interactions of these soil properties with surfactants and the possible influence on TCDD mobility. The results of the current research suggest some interesting possibilities concerning the effects of surfactants on TCDD bioavailability. For example, the fact that TCDD was degraded in soils TB and PR, but not SS, when subjected to bioaugmentation with fertilizers but no surfactant, suggests that the high electrolyte content of SS may have prevented TCDD from being mobilized for degradation. In soil TB and PR, the electrolyte contents may have been low enough that when the soil microflora was stimulated by fertilizers, TCDD mobility.was sufficient enough to be bioavailable. Subsequent treatment of the soils with the surfactant may have neutralized the electrolyte effect in soil SS, resulting in TCDD mobility. These possibilities deserve more research, especially considering that TCDD mobility in soils is poorly understood (Jackson et al., 1986), and surfactants can be tailored to specific soil chemical conditions. In addition, sterile soils should be examined to ascertain that TCDD degradation in the presence or absence of surfactants is biologically mediated. However, it is highly unlikely that the degradation of TCDD in soils TB and PR (no surfactant) and SS (plus surfactant) was strictly by physical-chemical mechanisms. The only report in the literature of chemical degradation of TCDD in soils is by Klee et al. (1984), using alkali-based polyethylene glycol reagents. In addition, all incubations in the current research were conducted in the dark, eliminating the possibility of UV degradation of TCDD. At the conclusion of the incubation of surfactant- amended TCDD soils, specific groups of soil microorganisms were again enumerated. The objective was to determine 190 whether presumably mobilized TCDD inhibited the indigenous soil microflora. Numbers of fungi and actinomycetes were suppressed in TCDD soils, apparently in proportion to the level of TCDD contamination (see Table 31). Interestingly, this same trend was observed in the initial microbial enumeration of the TCDD soils (see Table 6). The apparent suppressive effect in TCDD soils was enhanced by the addition of surfactant. On the other hand, whereas oligotrophs were initially fewer in TCDD soils compared to the non-TCDD soil, oligotrophs were greater in number following surfactant treatment. In general, the addition of surfactant appeared to stimulate the numbers of soil microorganisms. These results suggest that mobilized TCDD (and/or co contaminants) may be inhibitory to specific groups of microorganisms, but not to others. Eutrophic and oligotrophic bacteria apparently were not detrimentally impacted, while filamentous organisms (actinomycetes and fungi) appeared to be impacted. The reason for the apparent effect on filamentous organisms is not clear. In the case of actinomycetes, however, the highly hydrophobic nature of their aerial mycelia may result in a route of entry for lipophilic pollutants like TCDD. CHAPTER V SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS SUMMARY In Chapter I, it was explained that TCDD is an anthropogenic compound whose environmental and biological properties are poorly understood. TCDD occurs in soils as a co-contaminant with other halogenated organic compounds, is immobile in soils and sediments, and is recalcitrant; a definitive environmental half-life has not been determined. TCDD is an acute and chronic toxicant that is a mutagen and a suspected carcinogen and teratogen. The toxicity of TCDD is species-specific and the mechanism of toxicity is not understood. It was also explained in Chapter I that despite intensive efforts to demonstrate TCDD toxicity and other biological effects in many laboratory species and higher organisms, including man, very little work has been reported on the effects of TCDD on microorganisms. Considering that the fate of TCDD appears to be soils and sediments, it is of interest to learn something of the microbial toxicity of TCDD and its possible effects on microbially mediated nutrient cycling. 191 All reported efforts to stimulate TCDD biodegradation by microorganisms have shown that the compound is recalcitrant; limited co-metabolism has been reported, especially extracellularly by the white rot fungus, Phanerochaete chrysosporium. The literature reviewed in Chapter I suggested that the recalcitrance of TCDD may be due to the limited bioavailability of the compound. In some experiments, it was found that limited TCDD metabolism was stimulated by certain solvents. It was suggested further that a high research priority is to develop methods to enhance the bioavailability of TCDD for biodegradation, because alternative methods to decontaminate soils are prohibitively expensive and inefficient. However, little additional work on mobilizing TCDD for biodegradation or the effects of mobilized TCDD on the soil ecosystem has been reported. In Chapter II, methods for examining the potential toxicity of TCDD to microorganisms were described. When it was found that indigenous microbial activity was substantial in soils highly contaminated with TCDD compared to a non-TCDD soil, yet TCDD persisted in these soils, a method to improve the bioavailability of TCDD was developed. The hypothesis was that the recalcitrance of TCDD was due to its immobility and unavailability in soils, not necessarily due to its toxicity or inherent non- biodegradability. Surfactants rather than laboratory , 193 solvents were chosen to enhance TCDD bioavailability because the emphasis was on developing methods to effect large-scale in situ biodegradation. The ability of surfactants to mobilize a TCDD surrogate compound, hexachlorobenzene, and the potential toxicity of surfactants were investigated. The results described in Chapters III and IV were very interesting. Some suppression of microbial activity in TCDD soils was noted, but soil microbial respiration in alfalfa meal-amended soils was not inhibited by TCDD concentrations between 2 ng/g and 26.3 ug/g. Thus, the microbial LCso for TCDD in soil could not be determined. Although microbial activity in soils was high, no mineralization of TCDD was detected in any treatment. Nevertheless, in the two Missouri soils containing the low and intermediate concentrations of TCDD and subjected to bioaugmentation, that is, fertilization and moisture adjustment, the initial TCDD concentrations decreased by at least 50% within one year of aerobic incubation. Because no mineralization was detected, the results suggest the production of daughter compounds of TCDD. Using the same treatments, the TCDD concentration in the most highly contaminated Missouri soil was unchanged. When all three soils were subsequently treated with a non-toxic concentration of a TCDD-solubilizing surfactant, the TCDD concentration in the highly contaminated soil decreased by 194 approximately 50% within six months; TCDD concentrations in the two soils with initially low and intermediate levels of contamination were not reduced further. Finally, the numbers of actinomycetes and fungi in surfactant-amended TCDD soils were suppressed, apparently in proportion to the initial TCDD concentrations. CONCLUSIONS The results of this research suggest that the biologically mediated in situ decontamination of TCDD soils is feasible. Even though the mineralization of TCDD may not be likely, the results of the current research suggest that it is possible to stimulate the indigenous soil microflora to attack TCDD. In two soils, simple bioaugmentation was apparently sufficient to stimulate TCDD degradation to daughter products; in another soil, the addition of a TCDD-solubilizing surfactant was necessary to stimulate degradation. These results need to be confirmed in other soils using additional surfactants, and with more thorough analyses for parent TCDD and daughter products. In addition, the current experimental design did not allow for sterile controls in TCDD soils. While it is unlikely that degradation of TCDD was due to strictly physical-chemical reactions, inclusion of sterile controls would improve the experimental design. 195 It will also be necessary to examine the potential fate and effects of TCDD, daughter products, and co contaminants in surfactant-amended soils. One of the major concerns in TCDD soils is the potential for groundwater contamination with toxic, recalcitrant, halogenated compounds. If soils are amended in order to mobilize TCDD for degradation, there is a possibility of increasing the migration of TCDD and/or co-contaminants through the soil and into groundwater. Another possibility is to enhance the bioaccumulation of TCDD in terrestrial plants and animals. Both of these situations would be environmentally unacceptable. Therefore, the results of this research establish a framework for additional studies on the bioreclamation of TCDD soils. A similar approach could be considered in order to decontaminate other polluted soils. While considerable research needs to be done, bioreclamation of soils contaminated with TCDD and other halogenated organics may offer a cost-effective alternative to strictly physical and chemical methods of decontamination. RECOMMENDATIONS Additional research needs to be conducted in order to demonstrate unequivocally that bioreclamation of TCDD soils is feasible. 196 • Only one surfactant was evaluated in the TCDD degradation experiments described in this report. There are hundreds of surfactants that have been designed to solubilize halogenated organic compounds, and some of these may be useful for mobilizing soil-bound TCDD. These need to be identified and evaluated for their microbial toxicity and efficacy for use in TCDD soils. An improved experimental design should consider using a laboratory solvent (e.g., dimethyl sulfoxide) as a control. • A more elaborate experimental design than the one described above should be employed to thoroughly evaluate the biodegradation of TCDD in surfactant-amended soils. The design should include sterile and septic soils that are amended with HC-TCDD an(* incubated with constant C02-free aeration that exhausts through traps to collect both C02 and organic volatiles. Enough replicates should be included to allow for periodic (for example, bi-weekly) removal of replicate soil samples for extraction and complete analysis for TCDD and daughter products by high- resolution gas chromatography/mass spectroscopy. This would allow for the determination of degradative half-lives for TCDD and daughter products, and may also reveal the metabolic or co-metabolic pathways of TCDD degradation in soils. The design would also demonstrate biological versus physical-chemical degradation mechanisms. 197 • A similar design could be employed using anaerobic sediments. Periodically through the course of the anaerobic incubation, sacrificed sediments could be extracted and analyzed for TCDD and daughter compounds. With sufficient replication, the design could facilitate the following of anaerobic incubation with aerobic incubation. This would demonstrate whether reductive dehalogenation followed by aerobic (oxygenase) attack of the aromatic rings would stimulate the mineralization of TCDD. • If TCDD biodegradation can be ascertained in soils, either through metabolic or co-metabolic mechanisms, it would be useful to isolate and identify the microorganisms responsible for the degradation. The purpose would be to further elaborate degradative pathways in axenic cultures, and to begin genetic analysis of these pathways. Because xenobiotic degradation is often encoded on plasmids, it may be possible to increase degradative plasmid copy numbers or transfer plasmids to other organisms. • The results of the current research indicated that TCDD or daughter products may suppress actinomycetes and fungi compared to eutrophic and oligotrophic bacteria. It would be interesting to examine the mechanism of apparent toxicity and determine the relationship between filamentous 198 growth and TCDD toxicity. • Additional experiments on the mobility, fate, and effects (toxicology) of TCDD and daughter products in surfactant-amended soils need to be conducted. This will confirm the current results, provide a clearer role of the potential impacts of TCDD on nutrient cycling, and will allow for assessing the potential for mobilized TCDD to flow to groundwater and/or to bioaccumulate. These experiments could be conducted using soil cores collected from contaminated sites. An additional treatment could be to plant the soil cores and monitor for bioaccumulation of TCDD in plants as a result of surfactant treatments. APPENDIX DATA RELATIVE TO HIGH RESOLUTION GC/MS OF TCDD SOILS Y 199 200 RARF1 13-KC-BB Sir Voltage 70H Rent Systen NEUSYS Sample 1 Injection 1 Group 1 Hass 319.8965 Textl.BUL, R.A. RESPONSE FACTOR SOLUTION m Norn- 23S6 2.3.78 40. 20. -L - i - JL_i_ 12 00 H O B 16-OB 18 00 20 00 22 00 24:00 26 00 20:00 30 00 A0RF1 I3-0EC-BB SirVoltage 7011 Rent: SystenHEUSYS Sanple 1 Injection 1 Group 1 Aass 321.0336 Text’l.OUL, A.A. RESPONSE FACTOR SOLUTION 100, 8 12 00 14:00 16 08 10 00 20 00 22:00 24H 26:00 20:00 30:00 AARF1 13-DEC-86 Sir Voltage 7BH Rent: Systen-NEUSYS Sanple 1 Injection 1 Group 1 Hass 331.3367 T e x M . O U L , R.A. RESPONSE FACTOR SOLUTION 180. Norn: 2825 88. 13 Cr -2,3,7,8 - 68. 48. 13,C-l,2,3,4 2 0 . 8 1 1 1 . \ £ i-J —«,—L 12 80 14:00 16:00 18 00 20 00 22:00 24:00 26:00 28:00 30:0 AARF1 13-DEC-86 Sir Voltage 78H Rent: Systen:NEUSYS Sanpl? 1 Injection 1 6roup 1 Aass 333.3333 T e x W . B U L , R.A. RESPONSE FACTOR DILUTION 108. Norn: 3401 8 8 . 60. 40. 2 0 . I 4 ■! 4iII « h in'T" 12 00 14 0 16 00 18-8 20:00 “ 22-80 24 0 26:00 20 00 38 0 Figure 4. GC/MS results of the TCDD response factor solution, indicating the peak locations of 2,3,7,8-; * C- 2,3,7,8-; and 1 3C-l,2,3,4-tetrachlorodibenzo-p-dioxin. The peak identities are the same in Figures 5 through 13. 201 HBSPB 13-DEC-0S Sir Voltage 78H Rent: Systen NEUSYS Sanple 1 Injection I Group 1 llass 319.6365 Text l.BUL, 6 PPB SIMPLE, 120 166. Norn: 23EB5 46. 26. 12 66 16 8 8 28 66 24 68 28:86 RRSPB 13-BEC-86 S i r M t a g e 78H Rent: Systen:NEUSYS Sanple 1 Injection 1 Group 1 Hass 321.6938 Textl.BUL, G PPB SBJIPLE, 120 166. Norn: 23718 46. 12 86 16 66 28 66 24:86 26:86 32:66 R6SPB 13-0EC-8G Sir Voltage 7BH Rent: Systen NEUSYS Sanple 1 Injection 1 Group 1 Rass 331.9367 Text=2.0UL, 8 PPB SIMPLE, 120 186. Norn: 3254 86 66 46 J I i.i -i-kJL 12 68 1 6 8 8 26 66 24 60 32 86 H6SP8 13-DEC-86 Sir Voltage 78H flcnt: Systen:NEHSYS Sanple 1 Injection 1 Group 1 Hass 333.9339 Text-B.aUL, 8 PPB SIMPLE, 120 N o m: 4265 12 86 16 86 28 86 24 BB 28 68 32 68 Figure 5. GC/MS results for soil TB plus Agrimul Jl. The recovery factor for 1 3c-l,2,3,4-TCDD (used for calculating the final concentration of 2, 3, 7, 8-TCDD) was 88.836. The original (Day O) TCDD concentration was 0.008 ug/g. The two top chromatograms in Figures 5 through 12 represent unspiked (native) TCDD soil and the two lower chromatograms represent 13C spiked TCDD soils. 202 HRSP12 13-OEC-ee SLr VoLtage 70H flcnt= Systen NEUSYS Sanple 1 Injection I Group 1 Hass 31S.8SG5 Text fl.R. 8 PPB SIMPLE, PUL 8 F 14 100. N o r r 4PE2 40. 12 00 16 80 20 00 2 4 * 28 80 3 2 :80 D0SP12 13-0EC-8B Sir Voltage 70H ficnt= Systen'NEUSYS Sanple 1 Injection 1 Group 1 Mass 321.693G TentR.fl. 8 PPB SIMPLE, 2UL OP 14 N o r r 5294 40. 2 0. 12 08 16:00 20 00 24 0 26 00 32:00 NflSPIP 13-DEC-8G Sir Voltage 70H Rent: SystenNEHSYS Sanple 1 Injection 1 Group 1 Hass 331.9367 Text R.R. 8 PPB SRItPLE, 2UL OF 14 i0e„ Norn- 655 48 20 J- 1_L 1200 1 6 8 0 20:00 24:00 28:00 32*1 RRSP12 13-DEC-86 Sir Voltage 78H Rent: Systen:NEUSYS Sanple 1 Injection 1 Group 1 Hass 333.9339 Text R.R. B PPB SRflPLE, 2UL OF 14 Norn: 794 2 0. 12 80 16 00 28 00 24 00 28 00 32:00 Figure 6. GC/MS results for soil TB with no Agrimul J1 but after one year of incubation with fertilizer and moisture control. The recovery factor for 13c-l,2,3,4-TCDD was 91.4%. The initial (Day 0) TCDD concentration was 0.008 ug/g. 203 tlflSPM 13-DEC-B6 Sir Voltage 70H flcnt^ Systen NEUSYS Sanple 1 Injection 1 Group 1 Bass 319.8965 Text n.R. ieee ppb srnple, i.8ul or ko 9 100 , Norr 23327 46. 26. 12 08 16 60 26 66 24 66 26:66 32 06 KRSP14 13-DEC-B6 SirVoltage 76H Re n t Systen:NEUSYS Sanple 1 Injection 1 Group 1 Itass 321.6936 TextV.R. 1666 PPB SANPLE, 1.0UL OF NO 9 Norn: 32314 46. 26. e 12:6 16:66 26 66 24 00 26 66 32:86 NRSP14 13-DEC-B6 SirVoltage 76H Rent: SysterNEHSYS Sanple 1 Injection 1 Group 1 Hass 331.9367 Text n.R. 1666 PPB SRNPLE, 1.6UL OF NO 9 100 . Nom: 241 II 26 J_L_i I I -l.l 12:66 1680 26:66 24:66 28:88 32:86 HRSP14 13-DEC-86 SirVoltage 76H Rent: SysterNEHSYS Sanple 1 Injection 1 Group 1 Itass 333.9339 Text n.R. 1668 PPB SRNPLE, 1.611L OF HO 9 Norn; 296 2 6 . -i L- 1 2 8 8 1680 20:06 24 v T 28:08 32:86 Figure 7. GC/MS results of soil PR plus Agrimul Jl. The recovery factor for 13C-1,2,3,4-TCDD was 47.5%. The initial (Day 0) TCDD concentration was 1.1 ug/g. 204 HRSP15 13-DEC-B6 Sir Voltage 78H D e n t Systen'NEUSYS Sanple 1 Injection 1 Group 1 Hass 319.B9G5 Text n.fi. 1000 PPB SRNPLE, l.BUL OF NO IB Horn- 23094 4B. 20 12 BB 14 BB 1E:B0 18 BB 20 BB 22 BB 24 00 26^00 20.00 30:00 BR5P15 13-0EC-B6 Sir Voltage 78H flcnt= SystenNEUSYS Sanple 1 Injection 1 Group 1 Hass 321.B93S Text n.R. 1000 PPB SRHPLE, l.BUL OF NO 10 100. Norn- 25375 12 BB 14 BB 16: BB 16 00 20 BB 22 8B 24 BB 26 BB 28 BB 30 BB HRSP15 13-DEC-86 Sir Voltage 70H Rent: Systen:NEUSYS Sanple 1 Injection 1 Group 1 Bass 331.9367 TexLB.R. 1000 PPB SRNPLE, l.BUL OF NO IB Norn: 2B7 _L i ' M ii i i • 12 00 14 00 16:80 10:00 20 80 2^00 24 :0026:00 28:00 3 B B B RRSP15 13-DEC-86 Sir Voltage 70H Rent: Systen:NEUSYS Sanple 1 Injection 1 Group 1 Rass 333.9339 TexLH.R. 18BB PPB SRRPLE, l.BUL OF NO IB Nom: 372 - r — k . 12 0 14 88 16:80 1 B B B 20 00 22fi 24:00 28:00 28:08 38 B Figure 8. GC/MS results of soil PR with no Agrimul J1 but after one year of incubation with fertilizer and moisture control. The recovery factor for 13C-l,2,3,4-TCDD was 53.5%. The initial (Day 0) TCDD concentration was 1.1 ug/g. 205 HHSP1E 13-OEC-BB Sir Voltage 7BH Re n t Systen:NEUSYS Sanple 1 Injection I Group 1 Itass 319.8S6S TextH.R. 2000 PPB SRNPLE, l.BUL OF NO 5 Norr 58972 68. 48. 28. 8 1 2 8 8 1 4 8 16:88 IB 0 28 00 22:88 24 88 26:88 28 88 38 88 RRSP16 13-DEC-86 Sir Voltage 78H Rent: Systen:NEUSYS Sanple 1 Injection 1 Group 1 Nass 321.8936 Text-n.fi. 2888 PPB SRNPLE, 1.8UL Of NO 5 180, Norn: 57826 88 68 48 28 8 12 14 88 16 88 18 88 28 88 22 8B 24 88 26:88 2 B 8 RRSP16 13-DEC-86 Sir Voltage 7BH Rent Systen NEUSYS Sanple 1 Injection 1 Group 1 Hass 331.9367 Text n.R. 288e PPB SRNPLE, l.BUL OF NO 5 188, Norn: 225 48 28 J J 12 88 14:88 161 18 88 281 26:88 26 88 30:00 NRSP16 13-OEC-06 Sir Voltage 78H Rent: SystenMEUSYS Sanple 1 Injection 1 Group 1 Rass 333.9339 Text n.R. 2800 PPB SRNPLE, l.BUL OF NO 5 Norn: 274 48. 28. -4— T.-t.J.I. i -U- 12 88 14 8B 16 88 181 28 00 22 88 24 00 26 88 28 88 30 80 Figure 9. GC/MS results of soil SS plus Agrimul Jl. The recovery factor for 13C-1,2,3,4-TCDD was 37.3%. The initial (Day 0) TCDD cohcentration was 2.4 ug/g. 206 N0SP1B 13-DEC-86 Sir Voltage 7BH Rent Systen NEUSYS Sanple 1 Injection 1 Group 1 Hass 319.0965 Text fl.fl. 2000 PPB SIMPLE, l.BUL OF NO 7 160L N o r * 6514 60. 6 6 . 48. 26. 12 66 14 00 16 60 18 00 20 60 22 60 24 00 26-60 20 89 30:00 NHSP16 13-DEC-06 SirVoltage 76N Bcnt= Systen:NEISVS Sanple 1 Injection 1 Group 1 Nass 321.893G Text n.R. 2000 PPB SfltlPLE, l.BUL OF NO 7 100 , Nor*: 1587S 00. 46. 26. 6 12 00 14 00 16:00 lBflB 20 00 22:0 24:0 26:00 2 B :0 NBSP1B 13-DEC-86 Sir Voltage 76H Rent: SystenNEVSYS Sanple 1 Injection 1 Group 1 Nass 331.9367 Text n.R. 2660 PPB SflnPLE, 1.6UL OF NO 7 100 , H o r * 76 -- ,-- ,-- f J - L 12:60 14 60 16:00 18:00 28 66 22:06 24 60 26:00 s e e RRSPie 13-DEC-06 Sir Voltage 766 Rent: SystenNEVSYS Sanple 1 Injection11 Group 1 Hass 333.9339 Text n.R. 2600 PPB SIMPLE, 1.6UL OF NO 7 No r * 36 46. 26. ■ I * r J A j 4 ± " "i1"1 "I 28:06 36:8612 68 14 6616:06 1 6 6 20:06 22:1 24 60 26:66 28:06 36:8612 Figure 10. GC/MS results of soil SS with no Agrimul J1 but after one year of incubation with fertilizer and moisture control. The recovery factor for i3C-l,2,3,4-TCDD was 6%. The initial (Day 0) TCDD concentration was 2.4 ug/g. 207 RRBK5 13-DEC-BB Sir Voltage 7BH R e n t Syste* HEHSVS Sanple 1 Injection 1 Group 1 flass 3I9.B9G5 Text 2.6UI, B PPB SRRPLE, H E IBB. Norn- 284 BB. 6B. 48. EB. j — u Wfil l 12 80 16=80 20 00 E 4 :BB 28 08 32=1 HRBK5 13-DEC-8G Sir Voltage 78H Rent SgsterNEBSVS Sanple 1 Injection 1 Group 1 Hass 3S1.B936 Text 2.8UL, B PPB SRRPLE, H E IBB. Norn: E15 48. EB. JL JJ. IE 80 1 6 B B 28 08 24=80 EBBB 32=80 RRBK3 13-DEC-BG Sir Voltage 7BH Rcnt= SystenNEUSYS Sanple 1 Injection 1 Group 1 Hass 331.9367 TexLE.BUL, B PPB SRRPLE, H E 100. Horn- 2306 4B. EB. X J_-L 12 80 16=88 E B B B 24=8B 28=80 32=80 RRBK5 13-DEC-86 SirVoltage 70H Rcnt= Systen=NEHSyS Sanple 1 Injection 1 Group 1 Hass 333.9339 Text=2.8UL, 8 PPB SRRPLE, H E 180. Horn= 3871 48. 28. 12=88 16=80 28=88 24=88 28=88 32=88 Figure 11. GC/MS results of soil WJ plus Agrimul J1 The recovery factor for 13C-l,2,3,4-TCDD was 77.1%. 208 HRBK4 13-DEC-8G Sir Voltage 78H Rent: SysterNENSYS Sanple 1 Injection 1 Group 1 Hass 319.8965 Text 2.8IIL, B PPB SRRPLE teeL Nor*: ieg ee. 6 8 . 48. 28. i | — L jLL l J j_L 1 2 8 8 16:88 26 88 24 88 26:88 32:8 HRBK4 13-0EC-B6 Sir:Voltage 7BH Rent: SysteoNEUSYS Sanple 1 Injection 1 Group I Hass 321.8936 Text 2.8UL, 8 PPB SRRPLE HRBK4 13-DEC-86 Sir Voltage 78H Sanple 1 Injection 1 Group 1 Rass 331.9367 Text:2.BUL, 8 PPB SRRPLE 180L Nor*: 2294 B8. 68. 48. 28. _Ll_ 12:88 16:88 28 88 2 4 8 2 B 8 S 32:88 HRBK4 13-DEC-B6 SirVoltage 78H Rent: SysterNEUSYS Sa*ple 1 Injection 1 Group 1 Rass 333.9339 Text:2.8i)L, 8 PPB SRRPLE 188. Nor*: 2979 48. 28. 1 2 8 8 16:88 28 88 24 88 28:88 32 88 Figure 12. GC/MS results of soil WJ with no Agrimul J1 but after one year of incubation with fertilizer and moisture control. The recovery factor for *3C-l,2,3,4-TCDD was 77.8%. HRRB2 13-DEC-BB SLr Voltage 7BH Rent: SystenNENSYS Sanple 1 Injection 1 Group 1 Rass 319.0965 Text 2.BUL, RETHOO BLANK, H.R. 1 ieeL No r r 13-DEC-86 SLr VoLtage 78H Sanple 1 Injection 1 Group 1 Rass 321.8936 Text 2.8UL, HETHOD BLRNK, H.R. 1 N o m : JL 14 BB 16:00 18 00 20 00 22:08 24:00 26:00 28:00 30:00 RBRB2 13-DEC-B6 Sir Voltage 70H Rent: SysterNEMSYS Sanple 1 Injection 1 Group 1 Rass 331.9367 Text:2.0UL, RETROD BLRNK, R.R. 1 iool 13-DEC-86 Sir:Voltage 7flH Rent: Sanple 1 Injection 1 Group 1 Hass 333.9339 Text 2.0UI, RETROD BLANK, H.R. 1 1B0L Norr 63 40. 2 0 . L rtiii 12 08 14 00 16'oe 18'ee 2 0 :ee 22'eo ' 24:B0 2 6 4 0 20:00 3 H Figure 13. GC/MS results of the method blank. BIBLIOGRAPHY Alexander, M. 1977. Introduction to Soil Microbiology (2nd ed.). John Wiley and Sons, New York. 467 pp. Alexander, M. 1981. Biodegradation of chemicals of environmental concern. Science 211:132-138. Alexander, M. 1985. 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