xvEPA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Athens GA 30605
EPA-600/3 78 068
July 1978
Research and Development
Fate of 3,3' -Dichloro
benzidine in Aquatic
Environments
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9 Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-78-068
July 1978
FATE OF 3,3'-DICHLOROBENZIDINE IN AQUATIC ENVIRONMENTS
by
Harish C. Sikka
Henry T. Appleton
Sujit Banerjee
Life Sciences Division
Syracuse Research Corporation
Syracuse, NY 13210
Grant No. R 804-584-010
Project Officer
William C. Steen
Environmental Processes Branch
Environmental Research Laboratory
Athens, GA 30605
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ATHENS, GA 30605
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
U.S. Environmental Protection Agency, Athens, GA, and approved for publica-
tion. Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
ii
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FOREWORD
Environmental protection efforts are increasingly directed towards pre-
vention of adverse health and ecolgical effects associated with specific
compounds of natural or human origin. As part of this Laboratory's research
on the occurrence, movement, transformation, impact, and control of environ-
mental contaminants, the Environmental Processes Branch studies the microbio-
logical, chemical, and physico-chemical processes that control the transport,
transformation, and impact of pollutants in soil and water.
The discharge of 3,3'-dichlorobenzidine (DCB), which is regarded as a
human carcinogen, into the aquatic environment is of great concern to human
health because of possible exposure through drinking contaminated water or
eating fish in which the chemical or its metabolites have accumulated. The
potential hazard of the chemical may be compounded by its biological or non-
biological conversion to compounds of even greater toxicity or persistence
than the parent chemical. This study assesses the role of several environ-
mental processes affecting the fate of the chemical in natural waters to pro-
vide information needed for establishing effluent guidelines for DCB.
David W. Duttweiler
Director
Environmental Research Laboratory
Athens, Georgia
iii
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ABSTRACT
Several aspects of the aquatic environmental fate of 3,3'-dichlorobenzi-
dine (DCS), a suspected human carcinogen, were examined. Greater than 95%
of dichlorobenzidine present was adsorbed to natural pond and lake sediments
in aqueous suspensions. Only a portion of the adsorbed chemical could be
extracted from the sediments, with this amount decreasing over time,
suggesting chemical reaction of DCB with sediment constituents. Dichloro-
benzidine was rapidly degraded by natural and artificial light in aqueous
solution, with a half-life of the order of 90 seconds in natural sunlight.
Monochlorobenzidine and benzidine were found to be intermediate products of
this process. In contrast, DCB appeared recalcitrant to degradation by
naturally occurring aquatic microbial communities with only a minor loss
of chemical detected over a 30-day incubation period. Dichlorobenzidine
was rapidly bioconcentrated in bluegill sunfish, with mortality occurring
prior to establishment of a chemical equilibrium between water and fish.
Bioconcentration factors of 132-554 were achieved at this point. The only
metabolite detected in the fish was an acid-labile conjugate of DCB. Based
on these observations, chemical and physical processes, rather than bio-
logical ones, appear to be the important factors governing the fate of DCB
in the aquatic environment. The ability of DCB to concentrate in aquatic
organisms may pose a direct hazard to human health through consumption of
contaminated fish.
This report was submitted in fulfillment of EPA Grant No. R 804-584-010
by Syracuse Research Corporation under the sponsorship of the U.S. Environmental
Protection Agency. This report covers the period October 1, 1976 to
December 15, 1977, and work was completed as of December 15, 1977.
iv
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CONTENTS
Foreword iii
Abstract iv
Figures vi
Tables vii
Abbreviations viii
Acknowledgment ix
1. Introduction 1
2. Conclusions 3
3. Recommendations 4
4. Materials and Methods 5
5. Results and Discussion 13
6. General Discussion 41
Appendix A 43
References 46
v
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FIGURES
Number Page
1 Rate of adsorption of DCB by aquatic sediments 14
2 DCB adsorption as a function of DCB concentration . 16
3 DCB adsorption isotherms 17
14
4 Disappearance of C-DCB from water in the presence of
bluegills 31
5 Fractionation of fish tissue extract for DCB and
metabolite verification ... 36
vi
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TABLES
Number Page
1 Distribution Coefficients (K,) of Sediment Sorption of DCB ... 13
d
2 Effect of pH on Sorption of DCB by Sediment 15
3 Sorption of DCB by Sediment 15
4 Desorption of DCB as a Function of Pre-exposure Time 18
5 Extraction of DCB from Sediment 19
6 Photolysis of DCB.2HC1 21
14
7 Photodegradation of C-labeled DCB 22
8 Photodegradation of DCB in Natural Sunlight 23
9 Kinetics of Disappearance of Transients Generated from
the Photolysis of DCB and MCB 25
10 Disappearance Quantum Yields for DCB, MCB and Benzidine .... 25
11 pH Dependence of the Rate of Disappearance of DCB 26
12 Persistence of DCB in Lake Water 27
13 Degradation of DCB by Activated Sludge 29
14 Microbial Release of 14C-DCB from 14C-Yellow Pigment 12 .... 29
14
15 -,C Residues in Bluegills after 24 hours Exposure to
C-DCB.2HC1 32
16 14C Distribution in Bluegills Exposed to 2.0 ppm 14C-DCB.2HC1 . 33
14 14
17 C Distribution in Bluegills Exposed to 0.5 ppm C-DCB.2HC1 . 33
18 Elimination of 14C from Bluegills Exposed to 2 ppm1 C-DCB.2HC1 . 34
14
19 Elimination of C from Bluegills Exposed to 0.5 ppm
C-DCB.2HC1 34
vii
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Tables (continued)
Number Page
20 Relative Abundance of DCB and Metabolite in Bluegills
Exposed to 2.0 ppm C-DCB.2HC1 38
21 Relative Abundance of DCB and Metabolite in Bluegills
Exposed to 0.5 ppm C-DCB.2HC1 39
LIST OF ABBREVIATIONS
DCB — 3,3'-dichlorobenzidine
GC-MS — gas chromatography-mass spectrometry
GLC — gas-liquid chromatography
HPLC — high performance liquid chromatography
MCB — 3-monochlorobenzidine
MLSS — mixed liquor suspended solids
NMR — nuclear magnetic resonance
TLC — thin-layer chromatography
viii
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ACKNOWLEDGMENTS
We wish to thank the staff of the Environmental Research Laboratory,
U.S. Environmental Protection Agency, Athens, GA, and in particular
Dr. W.C. Steen (project monitor) for suggestions and information that were
useful in completion of this work.
We also acknowledge the technical assistance of Mr. E. Pack and
Mr. R. Gray.
ix
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SECTION 1
INTRODUCTION
3,3f-Dichlorobenzidine (3,3*-dichloro 4,4'-diaminobiphenyl), hereafter
referred to as DCS, is widely used as an intermediate in the manufacture of
azo pigments. It is of considerable commercial importance; total DCS
production in the United States in 1972 was about 4.6 million pounds (1).
With current work practices, effluents containing this chemical are dis-
charged directly into receiving waters. Moreover, the discharge of dichloro-
benzidine-pigment wastes into receiving waters constitutes an additional
source of DCB contamination in the environment since free, unreacted DCB is
reported to be present in these pigments.
Dichlorobenzidine is known to induce cancer in animals (2) and is
regarded by the Occupational Health and Safety Administration (OSHA) as
being carcinogenic to man (3). The discharge of DCB into the aquatic
environment is of great concern to human health because of possible exposure
to the chemical through drinking-water supplies. Also, DCB and its meta-
bolites may accumulate in fish and could pose a health hazard if fish from
contaminated waters were to be used as human food. The potential hazard of
DCB may be compounded by its biological or non-biological conversion to
compounds of even greater toxicity and/or persistence than the parent
chemical. For instance, it is believed that in the case of carcinogenic
aromatic amines, it is the metabolites of the chemicals that produce the
carcinogenic response (4). Furthermore, potential degradation products of
DCB, such as benzidine, may constitute an even greater carcinogenic hazard.
Therefore, in order to fully evaluate the hazards associated with the release
of DCB into the environment, it becomes important to study the environmental
fate of the chemical because its persistence, disappearance, or partial
transformation will determine the degree of its hazard.
A number of physical, chemical and biological factors determine the
fate of a chemical in the aquatic environment. These include adsorption to
sediment, chemical hydrolysis, photodegradation, microbial degradation, and
uptake and metabolism by aquatic organisms. Currently nothing is known
about the effect of these factors on the persistence and transformation of
DCB in the aquatic environment. This study was undertaken to assess the
role of some of the processes that may determine the environmental behavior
of the chemical. The overall objective of this investigation was to obtain
information needed for establishing effluent guidelines for DCB.
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Specific Objectives:
1. To determine the sorption-desorption of DCB by aquatic sediments.
2. To study the photodegradation of DCB in simulated and natural
sunlight.
3. To study the biodegradation of DCB by microorganisms in lake
water and activated sludge.
4. To determine whether aquatic microorganisms can liberate DCB
from DCB-based azo dyes.
5. To determine the uptake, elimination, and metabolism of DCB
by fish.
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SECTION 2
CONCLUSIONS
1. DCB adsorbs extensively to a variety of aquatic sediments and becomes
more tightly bound to the sediment with the passage of time.
2. DCB is very rapidly degraded in aqueous solutions by the action of
natural or simulated sunlight. 3-Chlorobenzidine (MCB) and
benzidine are intermediates generated in this process.
3. DCB is resistant to degradation by naturally occurring aquatic
microbial communities.
4. DCB is rapidly bioconcentrated into both the edible and non-edible
portions of bluegill sunfish. Mortality occurred prior to
establishment of a chemical equilibrium between water and fish with
bioconcentration factors of 132-554 achieved at this point. The
only DCB metabolite detected in the fish was an acid-labile conjugate
of DCB.
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SECTION 3
RECOMMENDATIONS
1. Based on available data, DCB, a suspected human carcinogen, should be
viewed as a significant hazard to human health and environmental
well-being when present in aquatic ecosystems. It has demonstrated
properties common to many hazardous environmental contaminants, such
as resistance to biodegradation and the ability to bioconcentrate in
aquatic organisms. The major pathway of DCB destruction, photo-
degradation, increases the hazard through generation of benzidine, a
carcinogen in humans. The discharge of DCB-containing effluents into
water systems should therefore be subject to strict regulation.
2. Studies should be undertaken to determine the ability of DCB to
undergo biomagnification through trophic levels. Bibconcehtration
studies in phytoplankton and zooplankton are also indicated.
3. The toxicity of DCB to aquatic organisms should be studied to determine
its adverse effects on aquatic biota.
4. The mechanism of photodegradation should be examined, and the photo-
products should be tested for toxicity.
5. The relative contribution of various mechanisms governing DCB fate
(adsorption, photodegradation, biodegradation, and bioconcentration)
acting in concert should be studied in model environmental microcosms.
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SECTION 4
MATERIAL AND METHODS
CHEMICALS
3,3'-Dichlorobenzidine dihydrochloride (ring-UL-1 C)(DCB), 5mCi/mMole,
was obtained from California Blonuclear Corp., Sun Valley, CA. Radiochemical
purity was judged to be greater than 99% by TLC on silica gel 60 (Merck) in
systems of etherrhexane (3:1 v/v) and benzene:ethyl acetateracetic acid
(7:3:0.1 by volume). Only one radioactive peak, chromatographing with non-
labeled DCB, was detected upon scanning the developed plates with a Nuclear
Chicago Actigraph II.
Non-radioactive recrystallized DCB.2HC1 was provided by Fine Chemicals
Division of Upjohn Co., North Haven, CT. Under a uv lamp, only one spot was
detected on thin-layer chromatograms of DCB developed in the above solvent
systems. The dihydrochloride melted in stages between 173-210°C. Because
of the absence of published melting point data for the dihydrochloride salt,
DCB-free amine was prepared and found to melt at 131-132°C (literature m.p.
132-133°). High performance liquid chromatography did not reveal the
presence of benzidine or mono-chlorobenzidine (MCB) in freshly prepared stock
solutions of DCB.2HC1. The purity of DCB was further confirmed by NMR and
combined GC-MS analyses.
All DCB stock solutions were refrigerated and protected from light
during storage. Unless specifically stated, the dihydrochloride of DCB was
used in this investigation, and calculations of DCB equivalents were based
on the salt form.
Benzidine (dihydrochloride salt) was obtained from J.T. Baker Chemical
Co., Philipsburgh, NJ, and 3-chlorobenzidine (MCB) was synthesized according
to Branch (5).
All other chemicals used were of reagent grade.
ANALYTICAL METHODS
Thin-Layer Chromatography
All TLC analyses were performed on silica gel 60 (Merck). Non-radio-
active DCB and benzidine were visualized under a hand-held uv lamp. Radio-
graphic analyses were performed with a Nuclear Chicago Actigraph II
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chromatogram scanner. Systems utilized in this study included: (1) ether:
hexane (3:1 v/v)-Rf DCB = 0.37, Rf benzidine = 0.17; (2) benzene:ethyl
acetate:gl. acetic acid (7:3:0.l)-Rf DCB = 0.60, Rf benzidine = 0.07.
Gas-liquid Chromatography
f O
A MicroTek Model 220 gas chromatograph equipped with a Ni electron-
capture detector, and a Hewlett-Packard Model 5730A gas chromatograph with
flame ionization detector were employed. Systems employed are listed below.
Column Material Inlet Column N2flow tr
1. 3% OV-1 on Chromsorb WHP 220 210 50 ml/min 4.4 min
(100-120 mesh)
2. 1.5% OV-17, 1.95% QF-1 on
Chromosorb-W (80-100 mesh) 275 225 60 ml/min 12,8
3. 3% SE-30 on gas chrom Q
(80-100 mesh) 275 235 60 ml/min 4.8
4. 10% UCW 932 on Chromosorb W
(80-100 mesh) 250 225 50 ml/min 9.5
Columns were 180 cm in length.
The lower limit of detection in system 1 was 2 ng, and 10 ng in systems
2 and 3. At these lower levels, DCB appeared to decompose, with chromato-
grams showing several peaks in addition to DCB.
The utility of GLC in this study was limited by the presence of electron-
capturing materials in pond and lake waters which interfered with DCB analysis.
Liquid Scintillation Counting
Radiometric determinations were made with a Model 3255 Packard Tri-Carb
liquid scintillation spectrometer. Radioactivity in alkaline solutions
containing llfC02 was determined using Instagel phosphor solution (Packard
Instruments, Downers Grove, IL.). All other determinations were done with
a phosphor solution containing 5.5 g 3,5-diphenyloxazole (PPO), 0.1 g 1,4-
bis[2-(5-phenyloxazolyl)]benzene (POPOP), 667 ml toluene, and 333 ml Triton
X-100. Up to 1 ml of aqueous samples were mixed with 10 ml of the phosphor.
Strongly acidic samples were neutralized prior to counting, to prevent
quenching.
High Performance Liquid Chromatography
Analysis of DCB solutions by HPLC was performed with a Waters Associates
(Milford, MA) liquid chromatograph (model M6000A) equipped with a uv detector
(Schoeffel Instrument Corp., Westwood NJ, model GM770). A 4 mm (i.d.) X 30cm
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column packed with y Bondapak CIS reversed phase medium (Waters Associates)
was utilized. DCB was detected by its absorbtion at 282 nm.
Two solvent systems were utilized. The first, consisting of 1% (pH 5.5)
ammonium phosphate buffer:acetonitrile (70:30 v/v) gave retention volumes for
benzidine, monochlorobenzidine, and dichlorobenzidine of 6.9, 12.3, and 30 ml,
respectively. One hundred nanograms or more of DCB gave a quantifiable
response. The second system utilized was more suitable for routine analysis
and consisted of acetonitrile:5% gl. acetic in water (70:30 v/v). Retention
volumes for benzidine, monochlorobenzidine, and dichlorobenzidine were 6.8,
4.6, and 3.3 ml, respectively. Five nanograms of DCB were quantifiable in
this system at maximum sensitivity. In addition, changing the relative
proportion of acetonitrile and acetic acid affected the elution pattern seen.
For example, with a 50:50 mixture of acetonitrile:acetic acid, the order of
elution observed was the reverse of that from the 70:30 solvent mixture.
Prefiltration of samples prior to HPLC analysis is a recommended pro-
cedure to prevent clogging of the column by particulate matter in the samples.
It was determined that up to 30% of DCB in distilled water solution was
retained on 0.45 y cellulose acetate filters (Millipore Corp., Bedford, MA)
and over 60% on Fluoropore (Teflon over a polyethylene support) filters.
In turbid lake water samples (from the microbial metabolism experiments),
over 95% retention was noticed on both filter types. This problem was
alleviated by addition of an equal volume of acetonitrile to samples prior
to filtration with the Fluoropore filters. Filter retention of DCB from
distilled and lake water was reduced to 1% or less (determined radio-
metrically) by this procedure. Samples containing ltfC-DCB were analyzed by
scintillation counting before and after filtration to maintain a check on
the efficiency of filtration. Subsequent studies revealed that the addition
of acetonitrile also reversed the association of DCB with materials in lake
water (Section 5), which probably caused the higher filter retention of DCB
in lake water samples compared to distilled water samples.
HPLC proved to be the preferred method of analysis of DCB and analogs
because it permitted direct determination in aqueous solution, thus
eliminating a possible loss of the chemical through photodegradation during
extraction and concentration steps required with other procedures. Also,
the selectivity offered by the uv detector eliminated the need for sample
clean-up prior to analysis.
EXPERIMENTAL PROCEDURES
Adsorption/Desorption of DCB to Aquatic Sediments
Bottom sediments from USDA pond, Hickory Hill pond, and Doe Run pond of
the Athens, GA area were provided by the Athens Environmental Research
Laboratory. The composition of these sediments is listed below.
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Sediment Composition
Pond PH % Organic % Clay % Silt % Sand
carbon
USDA 6.4 0.8
Doe Run 6.1 1.4 <1% 44% 56%
Hickory Hill 6.3 2.4 <1% 45% 55%
In addition, sediments from Lake Theriot, LA (an organic muck) and Eagle Lake,
MS (a silt, reduced clay sediment) were obtained from the U.S". Army Corps of
Engineers, Waterways Experiment Station, Vicksburg, MS. The sediments were
passed through a screen to remove pebbles, leaves,and other detritus. After
centrifugation to remove excess water, the sediment was thoroughly mixed.
Moisture content was determined by drying portions of sediment at 90°C over
dessicant until a constant weight was obtained.
To determine sorption, a solution of lltC-DCB.2HCl was mixed with
sediment to give a water:sediment (dry-weight equivalent) ratio of 100:1.
The sediment suspension was contained in screw-cap centrifuge tubes with
aluminum foil-lined caps and shaken on a wrist-action shaker, or in Erlenmeyer
flasks shaken at 250 rpm on a rotary shaker. All samples were protected from
light and maintained at 22°C. The shaking rate was adjusted to maintain the
sediment in complete suspensions. At the appropriate time and at equilibrium,
samples were centrifuged at 15000 rpm for 10 minutes, and 11+C content in
the supernatant was determined by scintillation counting. The amount of
14C-DCB disappearing from the solution was assumed to be sorbed by the
sediment. Adsorption of DCB to glass was not detected over 24 hours in
centrifuge tubes or Erlenmeyer flasks containing a solution of 1£tC-DCB in
distilled water. The effect of pH on sorption was studied in appropriately
buffered solutions: pH5 (0.01M acetate buffer), pH7 (0.01M phosphate buffer),
pH 9 (O.OlMborate buffer). All other sorption studies were done in
distilled water-sediment suspensions. Except where otherwise noted, sorption
solutions contained an initial DCB.2HC1 concentration of 2.0 ppm.
The effect of DCB.2HC1 concentration on the degree of sorption was
measured for the Hickory Hill pond and Lake Theriot sediments. Sediments
were suspended in solutions containing 0.5 - 2.0 ppm DCB and sorption
determined as described above.
Desorption was studied in sediments that were suspended in 2 ppm
DCB.2HC1 for either 24 hours or 7 days, at which time sorption was measured
and desorption initiated. After centrifugation, the supernatant solution
was removed, and its 1HC content determined. The sediment pellet was washed
once by suspension in distilled H20, to remove DCB from the solution that may
have been trapped in interstitial spaces of the pellet. After ltfC determina-
tion, the wash was discarded, an appropriate volume of distilled water was
added (maintained a liquid/sediment ratio of 100), a 0 time sample was taken,
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and the samples were then shaken vigorously for up to 72 hours. During this
period, the suspension was centrifuged at appropriate intervals and the
supernatant was counted for 1I+C to determine the amount of desorbed DCB.
Extraction of DCB from sediment by organic solvents and other agents was
performed by similar procedures.
Photodegradation
Studies on the photodegradation of DCB were carried out in a preparative
photochemical reactor (.Ace Glass Company) system. The reaction system
consists of a jacketed borosilicate glass vessel and is equipped with a side
arm for withdrawing samples. A double-walled water-cooled quartz well,
housing the light source and filter, is fitted into the vessel and is immersed
in the solution to be irradiated. The aqueous solution of DCB was irradiated
with a 400 watt Hanovia medium pressure mercury lamp fitted with a Pyrex 7740
filter to exclude light of wavelength less than 300 nm. Aliquots of the
photolyzed solution were withdrawn at appropriate intervals and analyzed for
DCB and its degradation products. Quantum yield was measured using a
Rayonet "merry-go-round" apparatus.
Analysis of Photoproducts —
Photolyzed aliquots were basified and extracted with ether, and the
dried (MgSOi+) ether concentrate was spotted on a silica-gel TLC plate and
developed in etherrhexane (2:1). A spot which co-chromatographed with
benzidine was scanned with a spectrofluorimeter, and its spectrum was found
to be identical to that of pure benzidine. The presence of benzidine was
further confirmed by comparison of its GC (20 in. UCW 982 on Chromosorb W
and OV-1) and HPLC retention times with an authentic sample and by GC-MS
analysis. MCB was identified by GC-MS and by comparison of GC and HPLC
retention times with an authentic sample (synthesized from nitrobenzene and
o-chloro-aniline according to Branch et al. (5).
Photodegradation of DCB in Natural Sunlight —
To determine the rate of photodegradation of DCB in natural sunlight,
a 3.6 ppm aqueous solution of DCB.2HC1 was divided into eight 5 ml portions
in tightly stoppered quartz tubes that were exposed to sunlight on a clear
day (Aug. 24, 1977, 10 A.M. EST) on the roof of our laboratory. The tubes
were covered with aluminum foil every 30 sees, until a maximum exposure
time of 4 minutes was reached.
Degradation of DCB by Aquatic Microorganisms
These studies were done using samples of lake water and sediment and
in activated sludge.
Microbial Degradation in Lake Water and Sediment —
Samples of water and sediment were obtained from the following sources:
(i) Oneida Lake - a large mesotrophic lake approximately 20 miles northeast
of Syracuse, NY, (ii) Jamesville Reservoir - a small eutrophic lake about
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10 miles southeast of Syracuse. Microbial degradation of DCB was examined
in water alone and water + sediment. Prior to starting the biodegradation
studies, microbial counts in the water samples were made using the standard
serial dilution and plating techniques (6).
To determine the rate of biodegradation of DCB, non-radioactive DCB was
added to the water in Erlenmeyer flasks at a concentration of 2 ppm. The
flasks were stoppered with foam plugs and incubated on a gyrotary shaker in
the dark at 21 + 1°C. Sterilized water samples containing DCB were included
as controls to account for any non-biological degradation. At appropriate
intervals, the contents of the flasks were analyzed for DCB by HPLC as
described previously. The rate of degradation of DCB in the water was also
examined in the presence of sediment. DCB was added to flasks containing
50 ml water and 0.5 g lake sediment (dry wt. equivalent). At suitable
intervals, samples of the aqueous solution above the sediment "were removed
and centrifuged to remove suspended material and the concentration of DCB
in the water was determined. The sediment was shaken with methanol to
remove adsorbed material for analysis.
Carbon-14-labeled DCB was used to determine the products resulting from
the biodegradation of the chemical. Carbon-14-DCB was added to the water
samples at a concentration of 2 ppm. At periodic intervals, the water samples
were adjusted to pH 11, shaken with ether, and the 11+C distribution between
the aqueous and organic phases was determined. The ether extracts were
analyzed by TLC. Additional search for metabolic products was performed by
HPLC coupled with scintillation counting of the eluted fractions.
The evolution of 1'tC02 was measured using the biometer flask described
by Bartha and Pramer (7). The water samples were incubated with 2 ppm of
1 C-DCB in the biometer flask and 0.1N KOH was used as the C02~trapping
solution in the side arm. The solution in the side arm was removed and
replaced at appropriate intervals. The amount of ltfC in the CC>2-trapping
solution was measured by liquid scintillation counting. The radioactivity
collected in the KOH trap was verified as 1<+C02 by acidifying with HC1.
Microbial Degradation in Activated Sludge —
Activated sludge for this study was obtained from a local sewage treat-
ment plant. The sludge was adjusted to a MLSS concentration of 1000 mg/1.
Carbon-14-DCB was added to the sludge at a concentration of 2 ppm. The
flasks containing the sludge and DCB were incubated on a rotary shaker at
21°C + 1°. Periodically, aliquots were removed, diluted with acetonitrile,
and centrifuged. The precipitated solid sludge was also extracted with
methanol, and both the water and methanol extracts analyzed by HPLC.
To minimize the loss of the chemical because of adsorption on the sludge
solids and to determine whether the microorganisms in activated sludge would
adapt to degrading DCB, a separate experiment on the biodegradation of DCB
was conducted using activated sludge as a microbial inoculum (8). In this
test, 10 ml of settled activated-sludge supernatant was added as a source of
microbial inoculum to 90 ml of a mineral medium (8) containing 50 or 100 mg
of yeast extract and 2 ppm of DCB. After an incubation period of 7 days,
10
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10 ml of the contents from the inoculated flask were withdrawn and trans-
ferred to a fresh medium containing the DCB and the procedure was repeated
for three consecutive weeks. After incubation for 7 days, each sub-culture
was analyzed for DCB.
Release of Free DCB from DCB-based Pigments —
Carbon-14-Yellow Pigment 12 was prepared by the coupling of llfC-(UL)-
DCB.2HC1 with two equivalents of acetoacetanilide by a diazotization pro-
cedure provided by the chemists of Sun Chemical Corp., Staten Island, NY.
The purity of the product was not determined because of the insolubility of
the pigment in water or organic solvent. The product was repeatedly washed
with methanol and no lftC-DCB was detected in the final wash.
Carbon-14-Yellow Pigment 12 was mixed with test water (added as a con-
centrated acetone suspension) to give a theoretical pigment concentration of
4 ppm. The pigment was incubated with the following types of water: (i)
sterilized distilled water, (ii) Muskegon County, Michigan Waste Treatment
Lagoon water (this lagoon receives the effluent of a DCB-utilizing factory),
(iii) Jamesville Reservoir water, and (iv) Oneida Lake water. The samples
were shaken in the dark at a temperature of 21 + 1°C.
Aliquots of water were sampled weekly, adjusted to pH 10, and shaken
with ether. Because of the low quantity of 1IfC detected in these extracts,
further analysis was not done until after 28 days of incubation. At this
time, the entire water sample was sacrificed, pH adjusted to 10, and shaken
with ether. The extracts were fortified with non-radioactive DCB, divided
into two portions, and concentrated. One portion was applied to thin-layer
chromatographic plates and developed in a system of etherrhexane (3:1 v/v)
along with a known DCB spot. The other portion was developed in benzene:
ethyl acetate:acetic acid (7:3:0.1). After development, the DCB-containing
area of the plates was located under a uv lamp and removed from the plate.
The ltfC was eluted from the silica gel with methanol, and determined by
scintillation counting. Methanol eluted 84% of authentic 14C-DCB from
silica gel, which was considered in calculating recovery of DCB in authentic
samples.
Uptake, Elimination and Metabolism of DCB in Bluegills
Bluegills (Lepomis macrochirus Raf.) (2 to 3 in. in length) were obtained
from the National Fish Hatchery, Orangeburg, SC, and from a commercial
hatchery. Fish were acclimatized to laboratory conditions for 30 days under
a 14 hr light/10 hr dark cycle and fed a pelleted diet.
Exposures were conducted at 21°C in all-glass containers protected from
light. Solutions were mildly aerated during exposures. A preliminary exper-
iment showed no loss of DCB over 7 days under these conditions, in the
absence of fish. Because this solution was prepared from water taken from
fish-holding aquaria, contribution to DCB metabolism under test conditions
by fish associated microbes appears negligible.
11
-------
The fish were removed from the treated water at appropriate intervals,
rinsed with clean water, and sacrificed. The content and distribution of
14C-DCB and derived materials in exposed fish was measured in the following
manner. After sacrifice, the fish were dissected into two portions prior to
analysis: the head plus viscera fraction (consisting of the head, gills, and
internal organs), and the edible flesh fraction (flesh and skin). After
weighing, the tissue fractions were homogenized with methanol (5 ml/g fresh
weight) in a blender. The slurry was centrifuged and the supernatant was
decanted. The residue was reextracted with methanol. After centrifugation,
the two extracts were combined and the amount of 1IfC in the pooled extract
was determined by liquid scintillation counting. The amount of 1£tC in the
tissue residue was determined by solubilizing it in the NCS tissue solubilizer
(Amersham Searle Corp.) for 48 hours at 50°C, as described by Sikka et^ al^. (9).
Glacial acetic acid (0.02 ml/ml of solubilizer) was added to the solubilized
tissue and the solution was counted for llfC using scintillation fluid
containing Triton X-100. The samples were stored overnight at 4°C in the
dark before counting. The radioactivity in the methanol extract and in the
tissue residue was combined to calculate the ltfC concentration in the fish.
The whole fish 14C residues were calculated from the combined values of the
edible flesh and head plus viscera fractions. To study the metabolism of
DCB by the fish, the methanol extracts of the tissues were analyzed by
partition extraction, TLC and HPLC (Figure 5, Section 5).
The nature of the 1£|C remaining in the water bathing the fish was also
determined. After removing the fish, the water was adjusted to pH 10 and
extracted with diethyl ether. The amount of ll|C in the organic and aqueous
phases was determined. The ether extract was concentrated and aliquots were
chromatographed on thin-layer silica gel plates.
12
-------
SECTION 5
RESULTS AND DISCUSSION
ADSORPTION AND DESORPTION OF DCB IN AQUATIC SEDIMENTS
Results
The rate of DCB adsorption to all sediments studied was initially very
rapid (Figure 1). The end-point of the sorption was generally achieved
within the first 24 hours of the experiments. The comparative distribution
coefficients (K^ = 100 x ug DCB sorbed/ug DCB in solution) listed in Table 1
indicate the high affinity of DCB for a variety of sediments. In addition,
sorption was reduced significantly at pH 9, compared to values obtained at
pH values of 5 or 7 (Table 2).
TABLE 1. DISTRIBUTION COEFFICIENTS (Krf) OF SEDIMENT SORPTION OF DCB
Sediment
US DA Pond
Hickory Hill Pond
Doe Run Pond
Lake Theriot
Eagle Lake
A
Kd
2850 (666)
3210 (328)
2670 (310)
8866 (166)
12800 (2776)
*Values are the average of three replications. Figures
in parentheses are standard deviations.
13
-------
CJ
-a-
100
80
40 _
20 _
10
Sediment
• Lake Theriot
A Hickory Hill Pond
Q Eagle Lake
Time (hours)
Figure 1. Rate of adsorption of DCB by aquatic sediments (Ig sediment
per 100 ml solution).
14
-------
TABLE 2. EFFECT OF pH ON SORPTION BY DCB BY SEDIMENT
Sediment
Doe Run Pond
Hickory Hill Pond
USDA Pond
pH5
3325 (655)
2225 (252)
3783 (1254)
*
Kd
pH7
2417 (295)
2122 (545)
3083 (445)
pH9
206 (18)
'269 (98)
95 (2)
*The values are the average of three replications. Values in
parentheses are standard deviation.
Equilibrium adsorption isotherms for Hickory Hill pond and Lake Theriot
sediments are presented in Figures 2 and 3. Using the equation of Freundlich,
/ v 1/n
x/m = kc
e
where x = ug adsorbed DCB, m = dry weight of sediment, and ce = equilibrium
(or end-point) concentration of DCB in solution, the constant 1/n was
obtained from the slope of the plot of log x/m against log ce and was found
to be near unity. The constant k was obtained from the slope of a plot of
x/m against ce- Slopes were obtained by a least squares method, with
correlation coefficients exceeding 0.98. These values are given in Table 3.
The experimental Kd values obtained with 2 ppm DCB.2HC1 in separate studies
approximate the k values obtained by this approach, although the K0.98).
15
-------
Sediment
200
•o
01
FQ
O
G
100
A Hickory Hill Pond
I I I | I I I I I I I I | I I I I I I I I | I I I I I I I 1 | i >
Equilibrium concentration (ppm)
Figure 2. DCB adsorption as a function of DCB concentration.
-------
(U
•H
•8
0)
oc
CO
Q
00
3.
t>0
O
Sediment
• Lake Theriot
A Hickory Hill Pond
2.5
2.0
1.5
r i i i
I i
-3
-2
I
-1
log Equilibrium concentration (ppm)
Figure 3. DCB adsorption isotherms.
-------
The desorption of DCB from Lake Theriot sediment is listed in Table 4.
In the 24 hour samples, desorption rapidly approached an apparent equilibrium
as indicated by lkC recovered in the washes and 0 times of the desorption
phase. This 14C is not due to DCB from the adsorption solution trapped in
the interstitial spaces of the centrifuged pellet, because lkC amounts
similar to wash values were obtained in the first and second 0 time desorp-
tion determinations. K
-------
TABLE 5. EXTRACTION OF DCB FROM SEDIMENT
Percent ^C extracted*
Period of sediment
exposure to ^
Agent 24 Hours 7 Days
5N NHifCl
IN HC1 f
IN NaOH
Methanol
2.1
9.0
31.3
36.2*
0.7
3.1
16.7
21.9
*Values are the average of three replicates.
tHCl extraction data were obtained from Lake Theriot sediment;
all other data were obtained from Hickory Hill sediment.
^Extraction of these samples with a second portion of methanol
liberated an additional 3.8% of adsorbed ^C.
Discussion
No obvious relationship between organic carbon content of the sediments
and the degree of sorption was observed, even though organic matter is
believed to be the major site of sorption of most hydrophobic chemicals (10).
Given that the distribution equilibria so heavily favor sorption, it may be
that even sediments relatively deficient in organic matter possess sufficient
adsorbant to favor nearly complete DCB sorption under the experimental con-
ditions. In preliminary experiments, sediment concentrations of DCB of
700 ppm or more could be achieved by repeated exposure of sediment to 2 ppm
DCB solutions or by suspending 1 g of sediment in 500 ml of a 2 ppm DCB
solution.
The linear isotherms obtained from the sediments studied show that DCB
sorption may be described by the mathematical treatment of Freundlich,
within the concentration limits of this study.
Studies have shown that desorption of lipophilic substances such as
toxaphene (11) from sediment in aqueous solution is extremely slow or insig-
nificant. This condition was seen in the 7-day DCB-sediment samples,
although the 24 hour sediments studied rapidly established a DCB distribution
with the water similar to that obtained by an adsorption approach. There-
fore, extrapolation on the role of absorption and, especially, desorption in
determining the long-term environmental fate of DCB, should be made with
caution until further work establishes the time-dependent nature of the DCB-
sediment complex.
The inability of 5N NHi^C! or IN CaClz to appreciably enhance desorption
of DCB from the sediments studied would indicate the cation-exchange
19
-------
mechanisms may play a minimal role, if any, in sorption of DCB under normal
environmental conditions (12), Because the pKa of DCB isj.ess than 4, DCB
is predominantly in a neutral state under our experimental conditions, thus
minimizing any potential ionic interactions between DCB and sediment. For
the same reason, it is unlikely that the decrease in sorption seen under
alkaline conditions in this study is caused by repression of ionic mechanisms.
As another possible contributing factor, alkaline conditions may change the
sorptive properties of the sediments. For instance, it is known that treat-
ment of sediments with NaOH releases humic and fulvic acids from soil or
sediment (13). In this light, it is interesting to note that sorption at
pH9 was reduced by approximately 30-50% (Table 2), and that treatment of
DCB-sediment with 1N_ NaOH released over 30% of sorbed lkC from the sediment
(Table 5), indicating the possibility that alkaline conditions may solubilize
or otherwise modify a portion of the sediment components responsible for
adsorption of DCB.
In this study, a high degree of difficulty was encountered in extracting
more than a fraction of adsorbed DCB from sediment. Also, the efficiency of
desorption and extraction decreased according to the length of time DCB had
been in contact with the sediment. These observations are consistent with
the occurrence of chemical reactions between DCB and sediment constituents.
This process has been noted with other chemicals, including aromatic amines.
For example, Hsu and Bartha (14,15) have demonstrated that chloroanilines
react with organic constituents of soil, forming covalent complexes, by
condensation of the amine moiety with carbonyl and other functions in the
soil. Portions of these complexes are resistant to acid or base hydrolysis,
and the amount of the complex increases with time. This mechanism may apply
to other amines as well. For instance, it was recently shown that amino-
parathion is very highly bound to soils, a condition not seen with the
parent chemical, parathion (16). It is likely that this process would lead
to formation of humic-like materials that presumably would be non-hazardous
(13). These findings for chemicals that are structurally similar to DCB
are consistent with our observations. Although there is little doubt that
the rapid decrease in free DCB from aqueous suspensions of sediment is pre-
dominantly the result of a phycial adsorptive process; it seems likely that,
once adsorbed, DCB may covalently bind to the sediment and become increas-
ingly resistant to desorption and extraction as this process occurs over time.
PHOTODEGRADATION OF DCB
Results
Products of DCB Photodegradation —
In our preliminary studies, aqueous solutions of DCB.2HC1 (approx.
5 x 10~5M) were photolyzed for periods ranging from 5-30 min with a 450W
medium pressure mercury lamp equipped with a Pyrex filter. A red-brown
material was found to deposit on the outside surface of the well. The uv
spectrum of the resulting solution showed a decrease in absorbance with
respect to the original solution as well as a slight hypsochromic shift.
20
-------
Analysis of the solution showed that it contained benzidine. The red material
that deposited on the walls of the photoreactor was dissolved in ether and
the ether concentrate was worked up and spotted on a silica gel TLC plate
and developed with etherrhexane (2:1). Five to eight brightly colored bands
were observed.
Finally, a solution of DCB in hexane was photolyzed for 30 min, and
concentrated. TLC analysis showed only one spot corresponding to DCB, and
no degradation products were observed.
To determine the time course of DCB photodegradation and the appearance
of products, an aqueous solution of DCB.2HC1 was photolyzed for 45 minutes.
Aliquots withdrawn at periodic intervals were analyzed for DCB, MCB, and
benzidine by HPLC. The results presented in Table 6 clearly demonstrate
that DCB is readily degraded by light; more than half of the chemical had
disappeared within four minutes of exposure. On the basis of these findings,
we conclude that DCB (in part) is photolyzed sequentially to MCB and
benzidine.
TABLE 6. PHOTOLYSIS OF DCB.2HC1
Minutes of
irradiation
0
1
2
3
4
5
10
15
45
DCB.2HC1*
x 107M
20
18
17
13
7.4
2.4
—
—
—
MCB.2HC1*
x 107M
—
1.2
2.1
2.3
3.9
1.5
1.5
—
1.2
Benzidine.2HCl*
x 107M
—
—
—
—
—
1.7
2.8
3.3
2.8
*Average of two determinations.
In order to have some information on the mass balance of the products
resulting from the photodegradation of DCB, preliminary studies using
^C-DCB^HCl were carried out. An aqueous solution of 14C-DCB.2HC1 was
irradiated for an hour, and samples were periodically withdrawn, basified
and extracted with benzene. The ltfC measured in the benzene and aqueous
fractions are listed in Table 7. It is evident that a significant portion
of substrate is converted to acidic materials which have a high affinity for
the aqueous phase. Furthermore, a large fraction of the original material
was converted to insoluble products that were retained on the walls of the
21
-------
photoreactor. This was confirmed in a subsequent experiment in which an
aqueous solution of 14C-labeled DCB.2HC1 was irradiated for one hour. As
before, the resulting solution was found to contain less radioactivity than
the starting solution. The photoreactor was rinsed with ether, and the
missing 1UC was found to be in the ethereal solution.
TABLE 7. PHOTODEGRADATION OF ^C-LABELED DCB
Time
(minutes)
2
5
7
12
15
30
60
135
150
Percent of initial llfC
in benzene phase
97.4
80.9
54.6
40.2
30.4
23.4
12.4
5.6
4.6
Percent of initial lkC
in aqueous phase *
3.08
12/4
21.2
16.4
13.9
16.5
12.2
16.4
22.2
*After extraction with benzene.
Photodegradation of DCB in Natural Sunlight —
To determine the rate of photodegradation of DCB in natural sunlight,
a 3.6 ppm aqueous solution of DCB.2HC1 was photolyzed for 4 minutes. The
DCB and MCB levels were measured by HPLC and are presented in Table 8. No
benzidine could be detected. However, in an earlier preliminary experiment
in which the solution was exposed to sunlight for 19 minutes to 5 hours, the
presence of benzidine was observed.
Lifetime of Transients Generated from the Photolysis of Chlorobenzidines —
During our experiments on the photodegradation of dichlorobenzidine, we
noticed the appearance of a green color that disappeared when the photolyzed
solution was allowed to stand in the dark. In an attempt to understand the
mechanism involving the disappearance of the green intermediates,Oan experi-
ment was conducted in which DCB and MCB were irradiated with 2537A light in
a Rayonet "merry-go-round" photoreactor.
Irradiation of aqueous solutions of DCB and MCB with a 2.2W mercury arc
at 2537A (Rayonet minireactor) for approximately one minute led to the forma-
tion of green intermediates which decayed in the dark. The rate of decay,
which appeared to be slower in acidic solutions, was monitored spectrophoto-
metrically at the absorption maximum of the transient and was of the first
22
-------
order with respect to substrate (correlation coefficient of the linear
regression line >0.999). The results are presented in Table 9.
TABLE 8. PHOTODEGRADATION OF DCB IN NATURAL SUNLIGHT
Time
(seconds)
0
30
60
90
120
150
180
210
DCB.2HC1*
x 106M
11
9.8
6.7
5.7
4.8
4.4
3.3
2.6
MCB.2HC1*
x 106M
0
1.5
2.4
2.3
3.2
3.7
3.6
3.4
*Average of two determinations.
It is difficult to interpret these results in an absence of a knowledge
of the structure of the transients. It is evident, however, that the rate
is dependent upon initial substrate concentration to a limited extent, and
more importantly that the similarities of the rates for DCB and MCB point to
a common mechanism. In other experiments conducted in solutions of higher
pH, the rate of disappearance of the transients increased markedly.
Measurement of Quantum Yields
The disappearance quantum yields of benzidine and MCB were measured in
a merry-go-round apparatus with respect to a ferrioxalate actinometer at
2537A and 3000&. The wavelength dependence of the quantum yields was minimal,
and the results obtained at 3000A are reported in Table 10. The limited
solubility of DCB in water made direct determination of the quantum yield at
30001 difficult, and consequently the measurement was made with respect to
MCB at 2537A. The high quantum yields for DCB and MCB confirm the photo-
lability of these substrates whereas the relatively low value for benzidine
is in keeping with Metcalf's finding that the disappearance half-life of
benzidine in methanol at 254 nm. is about 2 hours (17).
A number of experiments were also conducted to determine the pH depend-
ence of the quantum yield for DCB. These results, presented in Table 11,
indicate that the rate of degradation is acid catalyzed. The appearance
quantum yields for MCB, although less accurate due to the reactivity of this
material, also appear to be acid catalyzed. In a future, more mechanistic-
ally oriented study, we will determine the rate profile for the conversion
of MCB to the relatively more stable benzidine.
23
-------
Photolysis in Organic Solvents —
To determine the photoreactivity of DCB in solvents other than water, a
number of preliminary experiments were carried out in hexane, isopropanol and
methanol. Irradiation of these solutions under approximately the same con-
ditions under which disappearance of DCB in aqueous solution is virtually
complete showed that very little degradation had occurred. For example,
irradiation of a 15 ppm solution of D.CB.2HC1 in iso-propanol at 2537& for
one hour led to 28% degradation. Under similar conditions, a 4 ppm aqueous
solution of DCB is decomposed to an extent of 42% in 0.3 minutes. It is
therefore evident that photodegradation of DCB in water proceeds through a
mechanism different from that in organic solvents.
Mechanistic Considerations —
Recent studies on aromatic dehalogenation in aprotic solvents has shown
that the mechanism involves homolysis of the carbon-chlorine bond, followed
by hydrogen abstraction from solvent (18-20). In aqueous media, irradiation
of chloroaromatics frequently leads^to photosubstituted products, presumably
through the nucleophilic photosubstitution mechanism advocated by Havinga
and coworkers (21). While most of the photochemistry of chloroaromatics may
be rationalized in terms of these two mechanisms, examples do exist where
other pathways must be considered. Nordblum and Miller (22) observed that
irradiation of 4,4'-dichloro-biphenyl in ether containing 2% trifluoroacetic
acid gave 4-chloro-biphenyl. In hexane, cyclohexane, acetonitrile or pure
ether, complex product mixtures were obtained. Furthermore, the reduction
is accompanied by a kinetic isotope effect, as determined by the use of
deuterated trifluoroacetic acid. Simple bond homolysis does not explain
the acid catalysis or the large isotope effect.
The photoreduction of DCB would also appear to involve a mechanism
different from simple bond homolysis. The relative inertness of the material
in isopropanol or hexane, both of these solvents being appreciably better
hydrogen atom donors than water, implies the specific involvement of water
in the transition state. While the limited scope of our work does not allow
further mechanistic speculation at present, it appears that proton transfer
is involved. We are presently investigating the mechanism of the reaction
in somewhat greater detail.
Conclusions
DCB is very rapidly photodegraded under enviornmental conditions
through reductive dechlorination and other processes. In organic solvents,
the reaction is considerably slower. The mechanism of dechlorination while
as yet unknown, nevertheless does not involve carbon-chlorine bond homolysis.
24
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TABLE 9. KINETICS OF DISAPPEARANCE OF TRANSIENTS GENERATED FROM THE
PHOTOLYSIS OF DCB AND MCB
[DCB.2HC1] x 105*
2.15
2.15
2.15
44.5
44.5
5.61
11.2
16.8
22.4
28.0
[MCB.2HC1] x 104*
7.63T
"
"
n
7.63*
"
HC1(N) A(nm.) T(°C)
1.00 425 22.5
1.00 " 22.2
1.00
0.12
0.12
1.00
1.00
1.00
1.00
1.00
1.00 435 22.5
1.00
1.00
1.00 " "
1.00 435 22.5
1.00
-1 2
k(min ) x 10
8.50
8.87
7.61
12.8
13.1
12.8
15.8
16.5
18.9
14.1
19.1
18.6
18.3
18.6
18.3
16.8
* Initial concentration (molar).
t Photolyzed for 1 minute.
* Photolyzed for 2 minutes.
TABLE 10. DISAPPEARANCE QUANTUM YIELDS FOR DCB, MCB AND BENZIDINE
Benzidine
MCB
DCB
0.
0.
0.
+
012
70
43
X
2537,
2537,
2537
(A)
3000
3000
7.
7.
6.
PH
o,
o,
7,
8.
8.
8.
2
2
1
25
-------
TABLE 11. pH DEPENDENCE OF THE RATE OF DISAPPEARANCE OF DCB
PH
*rel. (DCB) +rel. (MCB)
1.96
3.96
6.01
8.28
1.0
0.71
0.42
0.50
1.0
0.73
0.49
0.56
BIODEGRADATION OF DCB BY AQUATIC MICROORGANISMS
Results
Microbial Degradation In Lake Water and Sediment —
A preliminary investigation utilizing Oneida Lake and Jamesville
Reservoir water showed a negligible loss of DCB over a 3-week period. This
study was initiated during May, 1977, when microbial populations were
relatively low (less than 500 cells/ml). In order to assess the degradation
of DCB under conditions of greater microbial activity, an additional study
was initiated in mid-July when microbial assay of Jamesville Reservoir water
showed approximately 5 x 106 cells/ml and Oneida Lake water 900 cells/ml.
As shown in Table 12 (first experiment), the DCB assayed decreased with
time in both lake water sources. No degradation products were detected by
either HPLC or TLC of the samples, and ^C trapped in biometer flasks was
less than 0.1% over 14 days in both lake water and sterile samples.
During the course of incubation, the samples became increasingly turbid,
probably as a result of microbial growth and the accumulation of metabolic
by-products (lipid, protein) and dead microbes. This accumulated material
might have provided a surface for DCB adsorption. However, the specific
activity of ltfC-DCB in this experiment was too low to provide an accurate
determination of the amount of lkC in the organic matter itself. Therefore,
Jamesville Reservoir water (5 x 106 cells/ml) was incubated with 14C-DCB.2HC1
of higher specific activity to facilitate determination of the distribution
of 14C in the incubation medium. As in the previous study, a progressive
loss of DCB was observed by HPLC over time (Table 12, second experiment),
but no degradation products were detected. Also, as before, sample turbidity
increased over time.
Portions of the 30-day samples prior to acetronitrile treatment were
centrifuged, with 64.4% of original 14C detected in the supernatant. In
contrast, the distribution seen in acetonitrile-treated samples was 94.7% in
the supernatant and 3.3% in the precipitate. Because there was a discrepancy
between the amount of 14C-labelled material equivalent to DCB in the
26
-------
TABLE 12. PERSISTENCE OF DCB IN LAKE WATER
Time (days) DCB concentration (ppm)*
First Experiment
0
7
21
28
Second Experiment
0
10
20
30
«• '
Jamesville Reservoir Oneida Lake
2.07
1.81
1.55
1.47
Sterile Control
2.07
2.02
2.03
2.04
2.26
2.22
1.66
1.68
Jamesville Reservoir
1.96
1.68
1.56
1.52
As analyzed by high performance liquid chromatography. Values
are the average of three replicates.
supernatant acetonitrile diluted samples (1.86 ppm) and the amount of DCB
assayed in the samples by HPLC (1.52 ppm), portions of the 30-day samples
containing known amounts of 1!*C were applied to the HPLC column, eluted
fractions collected, and the lltC content of the fractions determined. The
eluted fraction containing DCB showed 85.6 + 3.8% of the applied radio-
activity. Carbon-14 in the fractions before and after DCB, although present,
was too low to quantify. It appears, therefore, that a portion of 14C-
material in the lake water samples does not chromatograph distinctly with the
HPLC systems employed. In addition, portions of the 30-day samples were
treated with acetonitrile and centrifuged. The supernatant was then basified
and partitioned with ether. Only 0.9% of the original supernatant ll+C
remained in the aqueous phase after ether partition. The extracts were
analyzed by TLC. No distinct materials other than DCB were seen, under
conditions capable of detecting components that represented 5% or more of
the material applied to the plate. It seems likely, therefore, that the
^C-material unaccountable by HPLC as DCB is indistinguishable by TLC as well.
After 4 weeks, samples containing water + sediment showed only 0.7%
(Oneida Lake) to 3.6% (Jamesville Reservoir) of the original llfC in the
water after centrifugation. Values were 1.3% (Oneida Lake) and 2.0%
(Jamesville Reservoir) for sterile control samples. Exhaustive extraction
with methanol removed only 29 to 42% of the original lltC from the sediment.
The extracted ll+C was shown to be DCB by TLC. A further experiment in which
Jamesville Reservoir water/sediment was incubated with 1I+C-DCB.2HC1 in
biometer flasks did not show the evolution of 1£tC02, indicating that the
non-recoverable portion of the DCB is adsorbed to sediment. The difficulty
27
-------
in extraction of DCB from sediment precluded the further use of sediment-
fortified water samples because of the inability to recover more than a
fraction of the original material.
Microbial Degradation in Activated Sludge —
The fate of DCB in activated sludge was studied because waste treatment
systems may represent an important site of entry of DCB into the environment
and because the sludge microbial community may have a higher biodegradation
capacity than lake water communities. In the samples containing sludge and
solids, only 34.7% of the original DCB (analyzed by HPLC) was recovered in
the acetonitrile-water and methanol extracts of the non-sterile samples, as
opposed to 29.8% in the sterile samples after a 7-day incubation. In the
sludge supernatant incubations, 83.6% of DCB was recovered from the non-
sterile samples, but only 28.8% from the sterile controls. This difference
might be caused by autoclaving, which resulted in a heavy residue, even in
the settled supernatant sludge, and possibly changed the adsorptive proper-
ties of the solid matter and efficiency of extraction. Carbon-14 values
agreed with DCB (as analyzed by HPLC) within 5%, and no materials other than
DCB were detected. Less than 0.1% of the llfC incubated in biometer flasks
was detected as evolved ^CC^ over a 10-day period. Much of the unaccount-
able 14C appeared to be in the remaining sludge residue, although variable
quenching and heterogenous samples prevented an accurate determination of
this portion by scintillation counting. It is likely that adsorption to the
solids in activated sludge was responsible for the decrease in free DCB.
To minimize the loss of DCB from adsorption on the sludge solids and
microorganisms and to determine whether activated sludge microorganisms would
adapt to degrading DCB, we examined the biodegradation of DCB in a test
system that utilized settled activated sludge as a source of microbes, rather
than as an incubation medium and included repeated weekly subcultures into
fresh medium.
The results shown in Table 13 show that the subculture enrichment method
through weekly subcultures into fresh medium did not increase the biodegra-
dation of DCB. The decrease of DCB seen in this system was comparable to
that seen in lake water. As in the previous studies, a considerable amount
of the il*C unaccounted for in the analyzed water samples was detected in the
organic material formed during incubation. For example, 14C in the isolated
organic material of the third culture accounted for 12 to 17% of original
llfC in the incubations. Again, no DCB metabolites were detected by HPLC
analysis.
Microbial Release of DCB from Yellow Pigment 12 —
The amount of DCB detected in the Yellow Pigment 12 incubations after
28 days was extremely low (Table 14). In addition, the DCB values from the
control incubations indicate that the release might be non-biological or the
results of a trace DCB impurity in the starting material.
28
-------
TABLE 13. DEGRADATION OF DCB BY ACTIVATED SLUDGE
(weekly subculture enrichment)
Treatment
Percent DCB remaining after 7 days
of incubation
DCB (% 0 time)
A
Subculture
Sterile Control
Activated Sludge
100 mg/1 Yeast Extract
Activated Sludge
50 mg/1 Yeast Extract
100.6
84.6
87.3
101.2
87.3
73.6
99.6
90.5
82.5
* Sampling and culturing procedures are explained in Section 4.
The data represents one experiment per treatment.
TABLE 14. MICROBIAL RELEASE OF 14C-DCB FROM 14C-YELLOW PIGMENT 12
Sample
14
C-DCB-ppb*
Sterile Control
Muskegon Co. Lagoon
Jamesville Reservoir
Oneida Lake
0.12
0.14
0.16
0.06
* Values are the average of two replicates, each
analyzed by two different TLC systems.
Discussion
Our findings indicate that DCB is not readily degraded by microorganisms
under the experimental conditions. There was some indication of biodegrada-
tion in the lake water, although no distinct metabolites were detected. We
speculate that the loss of the chemical noticed was primarily the result of
adsorption and/or accumulation by microorganisms in the water.
The adsorption of hydrophobic chemicals such as methoxychlor and toxa-
phene to aquatic microbes has been well-documented (23,24). The affinity of
29
-------
DCS for aquatic sediments and many organic materials was repeatedly observed
in this study. Our results show that, during the course of incubation, a
portion of DCB becomes associated with organic matter in the water. The
addition of acetonitrile releases or solubilizes the majority of this bound
C-material, although a portion remains associated with the organic debris.
This suggests that the progressive decrease in DCB detected during the
incubation period is probably related to the increase of organic material
formed during incubation, resulting in a DCB/organic matter complex that
cannot be analyzed by chromatographic techniques. In this regard, Wilson (25)
showed that incomplete recoveries of DDT from natural water were the result
of adsorption to suspended matter in the water. The results of the studies
utilizing whole and settled activated sludge indicate that adsorption, rather
than metabolism, may predominantly determine DCB fate in an activated sludge
unit. No evidence was seen to indicate that sludge microbial populations
may be induced to metabolize DCB. This is in contrast to benzidine, which
is rapidly degraded in sludge systems (26), even without a period of
acclimation.
Even in the event that DCB loss was the result of active microbial
degradation, however, the rate of this process is slow. The rate of loss of
detected DCB in the lake water systems tested indicates half-lives greater
than 40 days, based on the initial rate of DCB loss. In contrast, the half-
life of DCB loss in the sediment sorption and photodegradation experiments
was measured in minutes. Also, the stability of DCB in sterile samples
protected from light indicates that hydrolytic, oxidative, or other chemical
mechanisms do not significantly degrade DCB in aqueous solution.
The resistance of Yellow Pigment 12 to microbial metabolism, releasing
free DCB, can be attributed in part to its extreme insolubility in water, in
which it forms clumped aggregates. It is unlikely that the pigment can be
absorbed by microbes for intracellular metabolism. The enzymes necessary for
DCB release from the pigment may not be of the extracellular type. Because
of the inability to directly measure the amount of pigment in the incubations,
we cannot exclude the possibility that DCB was released and subsequently
degraded or adsorbed to organic material in the water. Our studies showed,
however, that DCB is resistant to microbial metabolism, and the extraction
procedures employed should have removed the majority of DCB bound to organic
material.
UPTAKE, ELIMINATION, AND METABOLISM OF DCB IN BLUEGILLS
Results
Uptake of DCB —
A preliminary study was conducted to assess the effect of DCB concen-
tration and fish loading ratios upon DCB uptake in a static system. The
loss of llfC from the water was monitored for 24 hours, after which time the
fish were sacrificed for lkC analysis. The water bathing the fish was also
analyzed for DCB and possible metabolites.
30
-------
As shown in Figure 4, DCB uptake is dependent on both initial DCB con-
centration and the biomass of fish present. Uptake was initially fast,
followed by a relatively slower rate, perhaps indicating the approach of a
near-equilibrium in the system. After 24 hours of exposure, an appreciable
concentration of ltfC-materials was detected in the fish (Table 15), mainly
in the non-edible portions. In order to determine whether any metabolites
resulting from DCB metabolism in fish were excreted into the water, the
nature of the lkC remaining in the water bathing the fish was examined.
Essentially, all of the radioactivity in the water was extractable with
diethyl ether at pH 10. Thin-layer chromatographic analysis of the ether
extract indicated the presence of only one spot with an Rf value correspond-
ing to that of authentic DCB.
100
60
A 2g fish/liter, 2 ppm DCB
• 2g fish/liter, 0.5 ppm DCB
o 12g fish/liter, 2 ppm DCB
T~
10
20 24
Time (hours)
14
Figure 4. Disappearance of C DCB from water in presence of bluegills.
31
-------
TABLE 15. 14C RESIDUES IN BLUEGILLS AFTER
24 HOURS EXPOSURE TO 14C-DCB.2HCl
Edible flesh Head and viscera Whole fish
2g
12g
2g
fish/1,
fish/1,
fish/1,
2
2
0
ppm
ppm
DCB
DCB
. 5 ppm DCB
220.
89.
68.
4
5
2
448.
228.
215.
6
3
2
316.
149.
131.
8
5
3
In subsequent studies, loading ratios of 2 to 3g fish/liter were main-
tained wherever possible. Continuous-flow exposure experiments were not
attempted because of problems entailed in disposal of large volumes of water
containing carcinogenic material. In experiments that exceeded 24 hours,
the fish were placed in fresh water containing DCB at each 24-hour interval.
This step minimized effects on uptake kinetics caused by depletion of DCB
in the water bathing the fish. Also, the exchange of exposure water pre-
vented deterioration of water quality that might contribute to fish mortality.
A more detailed study was conducted to determine equilibrium values of
DCB and derived materials achieved in fish exposed to 2 ppm DCB.2HC1. As
shown in Table 16, 14C uptake was very rapid, and residues in the fish
increased through 48 hours of exposure. The 14C-residues were concentrated
predominantly in the non-edible portion. By 24 hours, however, an equil-
ibrium was apparently reached in this portion, after which concentration into
the edible portion increased significantly. By 48 hours, the fish exhibited
symptoms of intoxication and within several hours, the entire remaining test
population had succumbed. Control fish held under the same conditions
showed no mortality.
Because mortality in the fish exposed to 2 ppm of DCB may have been a
result of the inability of fish to detoxify or eliminate DCB at a rate
exceeding the rate of uptake, a similar study was conducted at 0.5 ppm to
determine ultimate equilibrium concentrations of DCB in bluegills. Through
the first 48 hours of exposure, the fish responded well to food and external
stimuli and, overall, appeared to be in excellent condition. After 72 hours,
however, only about 10% of the test fish responded to food or stimulus.
Between 96 and 120 hours, approximately one-half of the remaining test
population died, and the survivors exhibited extreme toxic symptoms, as noted
in the 2 ppm studies. These symptoms included non-responsiveness (even to
the capture net), depressed opercular movements, reddened gills, and
hemorrhagic areas on the body surface. The uptake of DCB in this study is
shown in Table 17. As in the 2 ppm experiments, residues in the head and
viscera fraction appeared to reach near-equilibrium levels prior to the
edible flesh fraction. Residue levels in the fish dead at 120 hours were
essentially the same as those in the live fish sampled at 96 hours. However,
levels in fish surviving through 120 hours were substantially higher.
32
-------
TABLE 16. 14C DISTRIBUTION IN BLUEGILLS EXPOSED TO 2.0 ppm 14C-DCB.2HC1
ppm-DCB
Exposure time (hours)
3
6
24
48
Edible
92.
83.
75.
192.
9 ±
6 ±
6 ±
5 ±
flesh
1.9
3.5
2.2
4.9
Head
151
179
368
358
Equivalent
and
.1
.1
.7
.8
+
+
+
+
viscera
2
13
9
6
.3
.0
.5
.1
Whole
119.2
123.1
187.0
265.3
fish
+
+
+
+
1.8
7.3
3.8
7.3
*
The values are the average of two experiments, with three fish per
experiment samples at each exposure intervals. Non-extractable 14C
residues were less than 0.5% of total ll*C (<0.1 ppm).
TABLE 17. 14C DISTRIBUTION IN BLUEGILLS EXPOSED TO 0.5 ppm 14C-DCB.2HC1
*
ppm-DCB Equivalent
Exposure time (hours)
24
48
72
96
120
Edible
20.5
39.0
118.5
98.3
213.2
flesh
± 1.3
± 0.3
± 6.6
± 7.1
± 9.4
Head and viscera
98.2 ± 6.4
193.5 ± 13.4
207.1 ± 27.6
212.6 ± 15.3
372.4 ± 29.6
Whole fish
59.4 ± 2.8
109.7 ± 4.1
164.1 ± 13.7
151.3 ± 10.3
277.0 ± 14.8
Values are the average of two experiments, with two fish per experiment
sampled at each exposure interval. Non-extractable C residues were
less than 0.5% of total ll*C (<0.1 ppm).
Elimination of DCB —
Some of the fish exposed to 2 ppm DCB.2HC1 for 24 hours were trans-
ferred to water free of the chemical to determine the rate of 14C-residue
elimination (depuration). In the 0.5 ppm study, surviving fish at 120 hours
were chosen for depuration studies. The fish were placed in fresh water,
flowing at a rate to give 12 complete turn-overs of water per 24-hour period.
During this period, both water and the fish were periodically sampled and
analyzed for ll*C content.
The depuration of DCB and derived materials is shown in Tables 18 and 19.
Although the rate of elimination is extremely rapid initially, DCB levels in
33
-------
the fish remained virtually constant after this excretion phase, on a whole
fish basis. Also symptoms of toxicity disappeared within several hours of
initiating the depuration.
TABLE 18. ELIMINATION OF 14C FROM BLUEGILLS EXPOSED TO 2 ppm 14C-DCB.2HC1
Percent of initial c remaining
Depuration time (hours)
24
48
72
Edible flesh
45.6 ± 3.2
37.2 ± 4.4
35.1 ± 4.1
Head and viscera
51.5 ± 0.7
42.3 ± 8.0
34.8 ± 11.7
Whole fish
42.2 ± 7.4
33.6 ± 2.1
28.9 ± 0.2
* 1 U
Initial 1HC residues were 60.7 ppm, 233.7 ppm, and 170.4 ppm for edible
flesh, head and viscera, and whole fish, respectively (average of 3
experiments). Depuration data are the average of duplicate experiments
(4 fish per experiment).
TABLE 19. ELIMINATION OF C FROM BLUEGILLS EXPOSED TO 0.5 ppm 14C-DCB.2HC1
14 *
Percent of initial C remaining
Depuration time (hours) Edible flesh Head and viscera Whole fish
24
120
240
5.
2.
2
6
-
± 0.3 14.
± 0.1 19.
4
7
-
± 0
± 0
.1
.1
56t
11.
13.
6
3
± 0.
± 0.
2
1
*Initial
1<4C residues were
213.
2
ppm, 372.4 ppm,
and
277.0 ppm
for
edible
flesh, head and viscera, and whole fish, respectively (average of 2
experiments). Depuration data are the average of duplicate experiments
(4 fish per experiment).
tCalculated from the 1Jfc in depuration water over the first 24 hours of
the experiment.
Metabolism of DCB —
Preliminary evidence of metabolism of DCB by fish was obtained by TLC
of methanol extracts of the edible flesh or head and viscera fractions.
Chromatography in two solvent systems showed the presence of at least two
materials. One radioactive peak co-chromatographed with DCB whereas another
remained at the origin, indicating that it was of a polar nature. To confirm
this possibility, the fish extracts were fractionated according to parti-
tioning behavior. The lkC material that partitioned into ether from basic
34
-------
(pH 11) water (fraction A) was found to contain only DCB as shown by TLC.
The water-soluble material remaining (fraction B) did not partition into
ether when the aqueous medium was acidified to pH 1 with HC1. Because
authentic DCB is almost quantitatively recovered into ether from basic water
in a one-step partition (Appendix 1), the water-solubility of the metabolic
derivative might be attributed to a zwitterionic nature (such as hydroxylated
products), or the conjugation of DCB to a material that retains a free
acidic functional group and liberates free DCB upon exposure to mildly
acidic conditions. The latter possibility was investigated by rebasification
and partitioning of the acid-treated aqueous phase with ether. The 14C was
quantitatively recovered into ether, and co-chromatographed with DCB in two
thin-layer chromatographic systems. This indicates that a highly acid-
labile conjugate of DCB is formed in the fish. The conjugate was
hydrolyzed to form free DCB in as little as 30 seconds at pH 1, but was
stable at pH 9 or higher. The identity of the fraction A material and the
material produced by acid treatment of fraction B was further confirmed to
be DCB, both qualitatively and quantitatively, by HPLC. In addition to
identical retention times, uv absorbance of the eluted unknown materials
was scanned with the chromatograph uv detector. The uv spectra of both
samples was virtually the same as authentic DCB, including identical A max.
= 282 nm. The identification procedures and results are summarized in
Figure 5. Therefore, the degree of metabolism seen in the uptake and elim-
ination studies is reported as the distribution of extracted materials into
the fraction A (DCB), and fraction B (metabolite) as determined radiometri-
cally.
The partition behavior of the metabolite in basic aqueous media indicates
that the DCB nucleus was modified by addition of at least one ionizable
acidic group. Mono- or bis-N-sulfate or N-glucuronide conjugates of DCB
would be expected to behave in such a manner. Because of the limited amounts
of free sulfate available in vertebrate systems, sulfate conjugates rarely
constitute a major portion of metabolism of foreign chemicals (27). In
contrast, glucuronidation is well-documented as an important metabolic path-
way in vertebrates, including fish (28). Futhermore, previous studies of
aromatic amine metabolism indicates the occurrence of _N-glucuronidation (29).
These conjugates are highly labile under mildly acidic conditions (30) and
may be formed in the absence of the enzymes required for synthesis of
0-glucuronide conjugates (31).
To check this possibility, DCB was mixed with glucuronic acid according
to the procedure of Boyland et^ al. (32). The crystalline product obtained
was washed with ethanol and dried. The synthetic material obtained was
stable in pH 10 media, exhibiting a A max. of 285 nm (as opposed to 282 nm
for DCB under identical conditions). When the pH was adjusted to 2 with HC1,
a maximum absorbance at 248 nm (corresponding to DCB) rapidly appeared. The
acid-lability of the synthesized product, producing free DCB, was confirmed
by both thin-layer and high performance liquid chromatography. Without the
acid treatment, both the synthetic and biological materials appeared to
elute with the column void volume. Bovine liver B-glucuronidase (P-L
Biochemicals, Milwaukee, WI) was inactive in catalysis of the conjugate,
relative to non-enzymic controls (pH 7 and 9). The inactivity of g-glucuro-
nidase towards N-glucuronide conjugates of aromatic amines has been noted
previously (30).
35
-------
Head plus viscera methanol extract from four fish exposed to DCB for 24 hours
14
TLC assay: 2 C spots
53.8 pg DCB equivalent ( C assay)
extract concentrated
add ether, pHll aqueous
shake, centrifuge
LO
ON
Fraction A
ether
14
C assay:24.9 pg DCB equivalent
HPLC assay:25.0 pg DCB
TLC assay:one C spot; Rf = DCB
14
Fraction B
water
C assay: 24. 4 Pg DCB equivalent
acidify to pH
5 min.
pH to 11 with NaOH
add ether, shake, centrifuge
ether
14C assay:22.5 pg
HPLC assay: 24.0 pg
TLC assa'y: one C spot, R
water
14
C assay:2.6 Pg DCB equivalent
DCB
Figure 5. Fractionation of fish tissue extract for DCB and metabolite verification.
-------
As shown in Tables 20 and 21, the amount of metabolite present was
dependent on both time and DCB concentration, indicating a rate-limiting
factor in biosynthesis. This might be a limited amount of conjugating
material (such as glucuronie acid), and/or that the rate of biosynthesis is
less than the rate of DCB uptake, particularly in the early stage of exposure.
Relatively less metabolite is present at 24 hours in the 2.0 ppm exposures
than at 0.5 ppm. Within 48 hours, an equilibrium between the relative
amounts of DCB and metabolite in the edible and non-edible fractions appears
to have been reached, except that metabolite in the edible fraction of fish
exposed to 2.0 ppm DCB may be increasing at this point. This equilibration
is probably reached through stabilization of factors including rates of DCB
uptake and elimination, and conjugate synthesis, hydrolysis, and elimination.
Distributional mechanisms between edible and non-edible tissues probably
contribute as well.
Discussion
Dichlorobenzidine is bioaccumulated by fish to considerable levels,
apparently limited by the toxicity of the chemical. This is indicated by
the presence of nearly equal levels of 14C residues in both the 2.0 and
0.5 ppm exposures (approximately 250 ppm on a whole fish basis) at the point
at which the experiments were terminated due to mortality. Except for a
brief period of stabilization in the 0.5 ppm exposures, residues increased
through the course of the experiments. The high residue levels seen in fish
surviving at 120 hours in the 0.5 ppm exposures, compared with the 96-hour
samples, might be due to the fact that mortality reduced the biomass of the
systems, resulting in a greater availability of DCB on a per fish basis.
Also, the fish surviving at 120 hours represent a biased sample in the sense
that survival might be the result of metabolic and/or physiological factors
unique to the survivors that, in turn, allowed higher concentrations of
residues to be achieved. This effect could have been masked when random
samples of the general test population were taken at previous time intervals.
It should be reiterated that the surviving fish were severely intoxicated
and would have succumbed if exposure had been continued beyond 120 hours.
The nature of elimination of DCB from contaminated fish after transfer
to fresh water was different from many previous observations. Other lipid-
soluble chemicals such as lindane and methoprene are bioconcentrated to a
large degree but are progressively eliminated - after removal to non-contamina-
ted water (33,34). In this investigation, however, an extremely rapid
initial rate of elimination was followed by a very low or negligible rate,
with appreciable residues, primarily in the non-edible portions, remaining
even after 10 days of exposure to fresh water. This observation suggests
the possibility of an enterohepatic circulation of DCB and metabolite or
that a portion of the residues are tightly bound to lipoproteins or other
substances and resistant to elimination. The former possibility is indicated
by the preponderance of conjugated DCB in the non-edible fraction of the
fish after the rapid elimination phase has ceased. The likelihood of biliary
excretion of a large molecule such as the DCB conjugate, plus the labile
nature of this material under acid conditions, such as found in the digestive
tract, also favor this event.
37
-------
u>
QO
TABLE 20. RELATIVE ABUNDANCE OF DCB AND METABOLITE IN BLUEGILLS
EXPOSED TO 2.0 ppm 14C-DCB.2HC1
Edible flesh
Exposure (hours)
3
6
24
48
Depuration
24
48
72
94.4
93.8
70.2
59.8
(hours)
35.2
28.2
27.5
A
(87.7)
(78.4)
(53.1)
(115.1)
(16.1)
(10.5)
(9.6)
B
5.6 (5.2)
6.2 (5.2)
29.8 (22.5)
40.2 (77.4)
64.8 (29.5)
71.8 (26.7)
72.5 (25.4)
85.5
86.3
59.0
60.5
28.4
21.3
26.6
Percent
*
abundance
Head and viscera
A
(129.2)
(154.6)
(217.5)
(217.1)
(14.6)
(9.0)
(9.3)
15.5
13.7
41.0
39.5
71.6
78.7
73.4
B
(23.4)
(24.5)
(151.2)
(141.7)
(36.9)
(33.3)
(25.5)
88.8
89.5
61.9
60.2
21.0
23.0
26.4
Whole fish
A
(105.8)
(110.2)
(115.8)
(159.7)
(8.9)
(7.7)
(7.6)
11.2
10.5
38.1
39.8
79.0
77.0
73.6
B
(13.4)
(12.9)
(71.2)
(105.6)
(33.5)
(25.9)
(21.3)
Values are the average of duplicate analyses of 6 fish pooled for analysis,
The values in parentheses are the data expressed as ppm-DCB equivalent.
A - Refers to DCB
B - Refers to the acid-labile conjugate of DCB
-------
u>
\o
TABLE 21. RELATIVE ABUNDANCE OF DCB AND METABOLITE IN BLUEGILLS
EXPOSED TO 0.5 ppm 14C-DCB.2HCl
Edible flesh
Exposure (hours) A
24
48
72
96
120
Depuration
120
240
37.1 (7.6)
42.2 (16.5)
50.9 (60.3)
45.8 (45.0)
45.2 (96.4)
(hours)
51.2 (2.7)
28.5 (0.7)
62.9
57.8
49.1
54.2
54.8
48.8
71.5
B
(12.9)
(22.5)
(58.2)
(53.3)
(116.8)
(2.5)
(1.9)
18.0
22.3
43.3
42.3
39.2
29.2
13.0
Percent
Head
A
(17.7)
(43.2)
(89.7)
(89.9)
(146.0)
(4.2)
(2.6)
abundance
and viscera
82.0
77.7
56.7
57.7
60.8
70.8
87.0
B
(80.5)
(150.3)
(117.4)
(122.7)
(226.4)
(10.2)
(17.1)
21.3
26.1
46.0
43.5
41.9
33.1
14.2
Whole fish
A
(12.7)
(28.6)
(75.5)
(65.8)
(116.1)
(3.8)
(1.9)
B
78.7
73.9
54.0
56.5
58.1
66.9
85.8
(46.7)
(81.1)
(88.6)
(85.5)
(160.9)
(7.8)
(11.4)
Values are the average of duplicate analyses of 4 fish pooled at each time intervals.
The values in parentheses are the data expressed as ppm-DCB equivalent.
A - Refers to DCB
B - Refers to acid-labile conjugate of DCB
-------
The identity of 'the acid-labile conjugate was not directly determined
because of inability to recover the material intact into non-aqueous media.
In all aspects examined, the material showed properties similar to the
synthetic N-glucuronide of DCB and other aromatic amines (29). These
properties include a free acidic functional group, high lability under acidic
conditions, chromatographic behavior, and resistance to hydrolysis by
0-glucuronidase. The ease of synthesis of the conjugate may indicate that
the reaction proceeds non-enzymatically in vivo. It is unlikely that the
product is an artifact caused by reaction of DCB during the extraction
procedures, because the relative and absolute amounts increase with time
of exposure.
No evidence of alteration of the DCB nucleus, such as ring hydroxylation,
was found in this study. Hutzinger et^ a^l. (35) did not detect hydroxylation
in the metabolism of a series of polychlorinated biphenyls by brook trout,
although a hydroxylated product was found to be a minor metabolite of tetra-
chlorobiphenyl in rainbow trout (36). Although microsomal enzymes catalyzing
hydroxylation and other oxidative modifications of foreign chemicals are
known to occur in fish (28), in vivo importance of these enzymes may be less
in fish than in mammals, because of the much smaller relative size of the
liver (the major site of these enzymes) and the lower temperatures involved.
Equilibrium bioconcentration factors (based on total C residue) of
38 to 44 in edible flesh were reported in a study of the uptake of benzidine
in bluegills (37). Levels were 12- to 43-fold higher in the non-edible
portions. No evidence of toxicity was seen. Elimination of residues was
progressive in the depuration phase of the studies, with 70 to 73% of the
residues in the edible portion and 96% of those in the non-edible portions
eliminated after 14 days exposure to fresh water. There appears to be a
higher concentration into the non-edible portions achieved by benzidine
than dichlorobenzidine, although concentration into edible portions was much
higher for DCB. The lack of toxic symptoms seen for benzidine might be the
result of the continuous-flow exposures utilized. It is possible that static
conditions potentiated DCB toxicity through accumulation of waste products.
It is equally likely, however, that DCB is more toxic to fish than benzidine.
The action of toxicants is often well-correlated to lipophilicity and water
solubility (38). This situation may apply to the benzidine series as well,
as dichlorobenzidine is much less water soluble and more lipophilic than
benzidine.
4
Bioconcentration factors in fish of greater than 10 have been recorded
for such chemicals as DDT (39) and Arachlor 1254 (40), under equilibrium
conditions. In comparison, the bioconcentration factor for DCB of 132 to 554
(for DCB and its conjugate) are rather low. The DCB values, however, do not
represent equilibrium values and could potentially have been higher if
mortality had not occurred. Also, the presence of even relatively low
concentrations of DCB must be regarded as significant in view of the carcino-
genic hazard posed by the chemical.
40
-------
SECTION 6
GENERAL DISCUSSION
Based on the results of this study, it is apparent that physico-chemical
processes will largely determine the persistance of DCB in the aquatic
environment and, ultimately, the associated health and ecological hazards.
The degree of adsorption to sediments and other suspended matter may dictate
the ultimate fate of DCB in aquatic environments. For example, sorption of a
chemical may have a negative effect upon rates of biodegradation and photo-
decomposition. Sorption may also lead to a wide translocation of the chemi-
cal from its original site of entry, and possibly to accumulation through
trophic levels by the action of detritus-feeding organisms. In contrast,
sorption may serve as a mechanism of DCB inactivation if the affinity between
the sediment and chemical is sufficiently strong. In the case of DCB,
further research is required to determine the ultimate role of sorption
mechanisms in the fate of DCB. The difficulty in extraction of more than a
fraction of sorbed DCB suggests that very strong interactions, perhaps
covalent, are involved. By analogy with chloroanilines and other amines,
incorporation of DCB into humic-like materials might serve as a major means
of DCB degradation. The affinity of DCB for organic materials also dictates
that the adsorption to biological entities such as microbes and plankton be
assessed because bioaccumulation of DCB by this route might lead to a direct
threat to human health.
In a similar fashion, the photodegradation of DCB presents a paradoxical
situation. Ultimately, the photolysis of DCB may lead to innocuous products.
However, in the process, monochlorobenzidine and benzidine are generated,
which may pose a greater carcinogenic hazard than the parent chemical. The
degree of photolysis in a natural aquatic system is dependent on many factors;
depth, turbidity, latitude, season, and climate (41). As a result, the
ultimate role of photolysis must be assigned on a case by case basis.
Compared to sorption and photolysis, biodegradation appears to be a
minor component governing the fate of DCB. Hydroxylation of the ortho
position of benzidine appears to be the major site of transformation of that
chemical. It may be that substitution of the benzidine nucleus with chlorine
at the 3-carbon positions decreases the susceptibility of the molecule to
enzymatic attack.
The release of DCB from DCB-based pigments does not appear to be an
important source of entry of DCB into the environment. This process may be
more important with water-soluble dyes.
41
-------
DCB is bioconcentrated by fish to a significant degree directly from
contaminated water. Toxic levels of the chemical may be achieved, although
further studies at lower concentrations are indicated. No evidence of
direct metabolism of the DCB nucleus was seen. However, the fish are capable
of conjugating DCB. This conjugate, apparently an N-glucuronide, is labile
to hydrolysis under -very mildly acidic or neutral conditions and, therefore,
should be considered the toxicological equivalent of DCB. In addition, a
portion of the DCB in contaminated fish, mainly situated in the non-edible
portions, remains within the fish after placement in a non-contaminated
environment, giving an indication of a long residual life of DCB in higher
life forms.
As a final note, workers involved in monitoring DCB in the environment
should consider the photolability and high affinity for organic matter
displayed by DCB. Exhaustive extractions are indicated, and" benzidine and
monochlorobenzidine should be monitored as well.
Future work designed to determine the hazard potential of DCB should
focus upon the relative contributions of these several mechanisms governing
DCB fate as they operate in concert.
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APPENDIX
WATER SOLUBILITY AND PARTITION ANALYSIS OF DCB
Water Solubility of DCB
The solubility of DCB.2HC1 in aqueous buffers of pH 4.6-8.9 at 22 + 1°
was determined spectrophotometrically from a knowledge of the extinction
coefficients-of its uv absorption maxima. All experiments were conducted
in the dark or with glassware wrapped with aluminum foil to exclude light.
To measure "extinction coefficients, a weighed amount of DCB.2HC1 (typically
5 to 30 mg) was introduced into a 50- or 100-ml volumetric flask, shaken with
methanol and made up to the mark. Aliquots (0.1 to 1 ml) were pipetted out,
introduced into volumetric flasks of 25 to 100 ml capacity, concentrated to
about 1.1 ml by a stream of air and made up to the mark with the appropriate
buffer. The uv spectra of these solutions were recorded, and the values
derived from at least two separately weighed methanol solutions, and from
4 to 6 spectral determinations.
An excess of DCB.2HC1 was mechanically shaken with the appropriate
buffer for up to 7 days. Samples were withdrawn at daily intervals beginning
from 10 hours after mixing and were centrifuged at 17,000 rpm for 30 minutes,
and the uv spectra were then recorded.
Recovery of DCB from Water
To determine the recovery of DCB from water with organic solvents,
aqueous solutions of 2 ppm ^C-DCB^HCl were shaken vigorously with equal
volumes of organic solvent and centrifuged. The distribution of ll+C in the
phases was measured by scintillation counting.
Results
Water Solubility of DCB —
Ultraviolet spectra of the surface layer of the supernatant liquid showed
essentially no change in DCB concentration over the 7-day period. In
separate experiments, DCB.2HC1 was warmed at 60°C with the appropriate buffer,
cooled to ambient temperature and worked up as above. The uv spectrum of the
centrifuged solution indicated that supersaturation to the extent of about
three times the solubility at room temperature had occurred. Furthermore,
the solution remained stable for several hours and returned to the solubility
limit only after it was mechanically shaken overnight. The spectral and
solubility data are listed below. The similarity of the solubility results
43
-------
is not surprising since DCB remains in its free base form throughout the pH
range studied (the first pKa of benzidine is 4.66, and that of DCB is there-
fore lower). The variation in solubility in the three media is possibly a
consequence of salt effects.
Spectral and Solubility Data of DCB.2HC1 at 22°C
PH*
8.9
6.9
4.6
26
28
27
E
,200
,200
,000
(Anm)
2%
1%
4%
(282)
(282)
(283)
53
57
52
E (Xnm)
,600
,800
,000
0.
1%
4%
4% (212)
(211)
(212)
Solubility (ppm)"1*
4
3
5
.59
.99
.36
4%
2%
4%
*
These refer to borate (8.9), phosphate (6.9) and acetate (4.6) buffers.
Averaged from at least 4 determinations. Calculations are based on the
peak at 282 nm. Results based on the maximum at 212 nm give essentially
identical results.
Recovery of DCB from Water
The partition coefficients obtained are listed below. The data show
that DCB partitioned into organic solvents is pH dependent and efficient at
pH 7 or greater. Based on the pH-dependence of partition of DCB into ether
reported by Classman and Meigs (42) , the first and second pKa of DCB are less
than 4. In general, extraction of DCB from aqueous media was performed after
adjusting pH to 10, to permit detection of transformation products which
might also partition into ether at intermediate conditions of pH. The
organic solvent partition coefficients obtained are lower than the distribu-
tion coefficients obtained from aquatic sediments (Section 5). This differ-
ence may relate to the solvents used, since n-octanol is often preferred for
correlating partition properties to biological or environmental events. Also,
the above determinations were done primarily to test DCB extraction from
water, using lltC-DCB of low specific activity (relative to sediment partition
experiments). Therefore, the partition coefficients probably represent
minimum values since the difference between an observed Kp of 199 (99.5%
recovery into organic solvent under the experimental conditions) and a Kp of
499 (99.8% recovery), for example, represents only a very small difference
in measured radioactivity which lies within the range of experimental error,
particularly in measuring the 1JtC remaining in the aqueous phase.
44
-------
Recovery of DCB from Water
Phase K
P
Organic Aqueous
benzene pH 11 124
ether pH 11 199
ether pH 7 199
ether pH 1 0.04
hexane pH 11 82.3
Data are the average of triplicate determinations.
K = [DCB] organic/[DCB] aqueous
45
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/3-78-068
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
-t
Fate of 3,3-Dichlorobenzidine in Aquatic Environments
5. REPORT DATE
July 1978 issuing date
6. PERFORMING ORGANIZATION CODE
'. AUTHOR(S)
Harish C. Sikka, Henry T. Appleton, and Sujit Banerjee
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Syracuse Research Corporation
Life Sciences Division
Syracuse, NY 13210
10. PROGRAM ELEMENT NO.
1HE775
11. CONTRACT/GRANT NO.
R804584
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency-Athens, GA
Environmental Research Laboratory
College Station Road
Athens, GA 30605
13. TYPE OF REPORT AND PERIOD COVERED
Final 10/76-12/77
14. SPONSORING AGENCY CODE
EPA/600/01
15. SUPPLEMENTARY NOTES
Project Officer: William C. Steen, ERL-Athens, GA
16. ABSTRACT
Several aspects of the aquatic environmental fate of 3,3'-dichlorobenzidine (DCB), a
suspected human carcinogen, were examined. Greater than 95% of dichlorobenzidine
present was adsorbed to natural pond and lake sediments in aqueous suspensions. Only
a portion of the adsorbed chemical could be extracted from the sediments, with this
amount decreasing over time, suggesting chemical reaction of DCB with sediment con-
stituents. Dichlorobenzidine was rapidly degraded by natural and artificial light in
aqueous solution, with a half-life of the order of 90 seconds in natural sunlight.
Monochlorobenzidine and benzidine were found to be intermediate products of this pro-
cess. In contrast, DCB appeared recalcitrant to degradation by naturally occurring
aquatic microbial communities with only a minor loss of chemical detected over a
30-day incubation period. Dichlorobenzidine was rapidly bioconcentrated in bluegill
sunfish, with mortality occurring prior to establishment of a chemical equilibrium
between water and fish. Bioconcentration factors of 132-554 were achieved at this
point. The only metabolite detected in the fish was an acid-labile conjugate of DCB.
Based on these observations, chemical and physical processes, rather than biological
ones, appear to be the important factors governing the fate of DCB in the aquatic
environment. The ability of DCB to concentration in aquatic organisms may pose a
direct hazard to human health through consumption of contaminated fish.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COS AT I Field/Group
Aromatic compounds
Dyes
Azo dyes
Pigments
3,3 '-Dichlorobenzidijne
(DCB)
Benzidine
Aromatic amine
DCB Fate and Transport
06J
06E
06T
11C
18B
68D
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report)'
Unclassified
21. NO. OF PAGES
22. PRICE
EPA Form 2220-1 (9-73)
50
ftUSGPO: 1978 — 757-140/1407 Region 5-11
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