vvEPA
United States
Environmental Protection
Agency
Research and Development
Environmental Research
Laboratory
Athens GA 30605
»„,«,, I/
'V 1978
Fate and Impact
of Pentachlorophenol
in a Freshwater Ecosystem
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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-
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describes research on the effects of pollution on humans, plant and animal spe-
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ences Investigations include formation, transport, and pathway studies to deter-
mine the fate ol pollutants and the*r effects This work provides the technical basis
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This document is available to the public through the National Technical informa-
tion Service, Springfield, Virginia 22161
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EPA-600/3-78-063
July 1978
FATE AND IMPACT OF PENTACHLOROPHENOL
IN A FRESHWATER ECOSYSTEM
by
Richard H. Pierce, Jr.
Institute of Environmental Science
University of Southern Mississippi
Hattiesburg, Mississippi 39401
1
-S
s
\
0
Grant Number R-803-82-0010
*
~j
i
Project Officer
N. Lee Wolfe
Environmental Processes Branch
Environmental Research Laboratory
Athens, Georgia 30605
U.S. Environmental Protection
Region 5, library tf»HZft
77 West befcsoft Boufwwj, 12th Flow
l 60604-3590
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ATHENS, GEORGIA 30605
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
U.S. Environmental Protection Agency, Athens, Georgia, and approved for
publication. 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
prevention of adverse health and ecological 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 envi-
ronmental contaminants, the Environmental Processes Branch studies the
microbiological, chemical, and physico-chemical processes that control
the transport, transformation and impact of pollutants in soil and water.
Human illness and death have occurred from exposure to pentachlorophenol
(PGP) in industrial and agricultural applications. In 1974 and 1975,
accidental release of wood-treatment wastes containing PGP in fuel oil
caused extensive fish kills in a freshwater lake in Mississippi. The study
reported here examines the persistence and distribution of PCP and PGP-
degradation products in this lake to provide information on the effects
of PCP contamination of aquatic systems.
David W. Duttweiler
Director
Environmental Research Laboratory
Athens, Georgia
iii
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ABSTRACT
This investigation was undertaken to determine the fate of pentachloro-
phenol (PCP) that caused extensive fish kills in a freshwater lake in December
1974 and again in December 1976. The kills resulted from the accidental
release of wood-treating wastes containing PCP in fuel oil. Samples of water,
suspended particulates, sediment, leaf litter and fish were collected from
February 1975 through April 1977 in an attempt to determine the persistence
and distribution of PCP and PCP-degradation products in the aquatic environment,
Food chain relationships were investigated in the lake and the
accumulation and elimination of sublethal concentrations of dissolved PCP
were studied under laboratory conditions for the bluegill (Lepomis
macrochirus). Also investigated were the solubility of PCP in water
at various pH levels and the release of PCP from contaminated watershed
material.
Concentrations of PCP well above background levels were found in the
water and in fish for over six months following the first spill with another
increase observed fourteen months after the spill (February 1976) following
a period of heavy rain. After the second spill, high PCP concentrations
were observed in samples collected in January 1977, and samples collected
in April 1977 showed that PCP still remained in water and fish four months
after the spill. The highest concentrations in fish were observed in the
bile followed by liver, gills, and muscle. Lake sediment and leaf litter
contained high concentrations of PCP throughout the two-year study. Studies
of leaf litter from the contaminated watershed area showed it to be a source
for chronic pollution of the aquatic ecosystem.
The major degradation products observed were pentachloroanisole
(PCP-OCH3) and the 2,3,5,6- and 2,3,4,5-tetrachlorophenol (TCP) isomers.
These products were found to persist in sediment and fish along with PCP.
The methyl ethers (anisoles) of both TCP isomers and 2,3,4,6-TCP isomer
were observed in some samples but the small amounts were difficult to
quantitate. The results suggested that PCP-OCH,, was formed within the
aquatic environment, whereas much of the TCP appeared to have been formed
before entering the lake, perhaps by photoreduction in the fuel oil solution.
At the pH of the lake water, PCP existed primarily as the phenate
anion and the distribution of PCP throughout the water column was enhanced
by the solubility. The acute toxicity to fish observed immediately after
each spill occurred by uptake of the phenate anion dissolved in water.
Food chain relationships within the lake showed that game fish populations
depended ultimately upon benthic organisms as a food source. The accumula-
tion of PCP in sediments, therefore, provided a source for chronic pollution
of fish caught for human consumption.
iv
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This report was submitted in fulfillment of grant no. R-803-82-0010
by the University of Southern Mississippi, Institute of Environmental Science
under the sponsorship of the U.S. Environmental Protection Agency. This
report covers a period from January 1975 to April 1977 and the work was
completed as of August 31, 1977.
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CONTENTS
Foreword ill
Abstract iv
Figures viii
Tables ix
Acknowledgement xi
1. Introduction 1
2. Conclusions 5
3. Recommendations 7
4. Materials and Methods 9
Environmental samples 9
PCP release from contaminated leaf litter 13
Solubility of PCP in water 13
Accumulation and elimination of PCP in fish 16
Food chain study 17
5. Results and Discussion 19
Environmental samples 19
PCP release from contaminated leaf litter 34
Solubility of PCP in water 36
Accumulation and elimination of PCP in fish 42
Food chain study 47
References 56
Appendix 59
vii
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FIGURES
Dumber Page
1 Sample sites in contaminated stream and lake 2
2 Extractipn of PCP and PGP-degradation products from environmental
samples 12
3 Titration of PCP in aqueous solution 38
4 Ultraviolet spectra of PCP and SPCP in 0.1 M buffer 39
5 Accumulation of PCP in tissue during exposure to 0.1 ppm PCP .... 44
6 Elimination of PCP from Group 1 (after 4 days exposure) 45
7 Elimination of PCP from Group II (after 16 days exposure) 46
viii
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TABLES
Number Page
1 PCP and PGP-Degradation Products in Control Samples 20
2 PCP in Water Samples Collected February 1975 - May 1976 21
3 PCP in Water Samples Collected August 1976 - April 1977 22
4 PCP in Sediment Samples Collected February 1975 - May 1976 24
5 PCP in Sediment Samples Collected August 1976 - April 1977 25
6 PCP in Fish Collected February 1975 - May 1976 25
7 PCP in Fish Collected August 1976 - April 1977 26
8 PCP in Leaf Litter 27
9 PCP and Degradation Products in Technical Grade PCP, Industrial
Waste Holding Pond and Oil Slick from Stream 29
10 PCP-Degradation Products in Lake Water (Dissolved) Collected
August 1976 - April 1977 30
11 PCP-Degradation Products in Lake Sediment Collected August 1976 -
April 1977 31
12 PCP-Degradation Products in Fish Collected August and October 1976. 32
13 PCP-Degradation Products in Fish Collected January and April 1977 . 33
14 PCP-Degradation Products in Leaf Litter Collected August 1976 -
April 1977 35
15 PCP Release from Contaminated Leaf Litter 36
16 Dissociation of PCP in Water 37
17 Spectrophotometric Characteristics in the Ultraviolet of PCP and
SPCP 40
18 Solubility of 26°C of PCP and SPCP at Three pH Values Using Two
Analytical Methods 41
IX
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19 LC-50 of PCP to Lepomis machrochirus 43
20 Accumulation of PCP in Fish Tissue after Exposure to 0.1 mg
PCP/1 Water 43
21 Elimination of PCP from Group II Fish, Placed in a Clean Environ-
ment after Sixteen Days Exposure to PGP-Contaminated Water .... 47
22 Benthic Organisms Collected During August and October 1976 49
23 Benthic Organisms Collected During January and April 1977 50
24 Stomach Contents of Sunfish Collected from August 1976 - April 1977. 52
25 Stomach Contents of Catfish Collected from August 1976 - April 1977. 53
26 Stomach Contents of Non-game Fish Collected from August 1976 -
April 1977 54
A-l Physical Parameters from Water Sampling Sites from February 1975
to May 1976 59
A-2 Physical Parameters from Water Sampling Sites from August 1976
to April 1977 61
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ACKNOWLEDGEMENTS
Special acknowledgement is given to Dr, C. R. Brent, Institute of
Environmental Science, and Dr. H. P. Williams, Department of Chemistry,
University of Southern Mississippi, for their involvement as coinvestigators
during the first year grant project and for their continued assistance.
throughout the project period. Several people were involved with various
aspects of the investigation. The major contributers were: Dr. D. M.
Victor, postdoctoral research fellow, Institute of Environmental Science,
analysis of PCP and degradation products in environmental samples;
Dr. M. S. Torrey, chemical consultant, solubility and pH studies;
S. R. Reeves, graduate assistant in the Department of Chemistry, analysis
of environmental samples; G. W. Pruitt, graduate assistant in the Department
of Biology, study of the accumulation and elimination of PCP in bluegills;
and T. C. Modde, graduate assistant in the Department of Biology, study
of food chain relationships in the contaminated lake.
Fish used for the accumulation and elimination studies were supplied
by the Mississippi Game and Fish Commission Lyman Fish Hatchery, Lyman,
Mississippi. The Department of Biology, University of Southern Mississippi,
provided boats and equipment for the collection of environmental samples
and the gas chromatograph with accessories was supplied by the Department
of Chemistry.
The cooperation of the project director, Dr. N. L. Wolfe, of the U.S.
Environmental Protection Agency's Environmental Research Laboratory, Athens,
Georgia, throughout the investigation and in obtaining GC-MS analysis of
selected samples is gratefully acknowledged. The GC-MS analysis of other
samples was performed by C. A. McDaniel of the U.S. Department of Agriculture,
Gulfport, Mississippi.
XI
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SECTION 1
INTRODUCTION
In December 1974, an undetermined amount of wood-treating wastes contain-
ing pentachlorophenol (PCP) in fuel oil overflowed the banks of a wood-treat-
ment company's waste water holding pond and soaked into soil and leaf litter
throughout the spill area (Figure 1). The oil and PCP waste entered a small
creek and traveled about one kilometer to a sixty-acre freshwater lake near
Hattiesburg, Mississippi where the resulting fish kill was described as
extensive to total (Mississippi Air and Water Pollution Control Commission,
1975). In December 1976 another extensive fish kill was observed in the lake
and subsequent analyses showed that the fish died from acute PCP poisoning„
The investigation reported here was undertaken to study the persistence and
distribution of PCP and major PCP-degradation products and impurities in the
aquatic ecosystem.
Pentachlorophenol (I) and the sodium salt, sodium pentachlorophenate (II),
are pesticides that are used extensively for many industrial and agricultural
applications. The most important uses include: as a fungicide and bacteri-
cide in processing textiles, paints, rubber and food; as a mulluscacide to
control snails; as a herbicide; and, most extensively, as a wood preservative
(Bevenue and Beckman, 1967). It is highly toxic to fish and other aouatic
organisms with a median lethal concentration (LC-50) of 0.2 to 0U6 ppm (Cote",
1972; Cardwell et al., 1976) and accumulation also has been observed in live-
stock and in humans (Plimmer, 1973; Shafik, 1973; Dougherty and Pitrowska,
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1976). Human illness and death have occurred from improper handling of PCP-
treated lumber, from breathing PCP-contaminated sawdust, and from exposure
when spraying solutions of PCP in fuel oil as a herbicide (Bevenue and Beckman,
1967; Shafik, 1973; Arsenault, 1976)„
fl)
In addition to acute PCP poisoning, there is concern for contamination
and biological magnification of PCP in aquatic organisms resulting from chronic
exposure (Rudling, 1970; Stark, 1969; Buhler et al., 1973; Zitko et al., 1974;
Kobayashi et al., 1976). Although the photo- and microbial-degradation of PCP
has been observed to occur rapidly under controlled laboratory conditions
(Crosby and Wong, 1976; Kirsch and Etzel, 1973), the pesticide has been found
to persist in natural aquatic environments. The problem is magnified by the
persistence of PCP-impurities and degradation products, many of which are also
highly toxic, such as tetrachlorophenol (TCP), tetrachlorobenzo-p-quinone
(chloranil), tetrachlororesorcinol, polychlorinated dibenzo-p-dioxins (PCDD)
and polychlorinated dibenzofurans (PCDF) (Munakata and Kuwahara, 1969; Crosby
et al., 1973; Crummett and Stehl, 1973; Rappe and Nilsson, 1972; Buser, 1976)„
This two-year investigation has focused on the concentration of PCP and
major PCP-impurities and degradation products in water, sediment, and fish in
the contaminated ecosystem. In addition to investigating the fate of PCP,
other studies have been performed to gain insight into the overall effect of
PCP contamination. Food chain relationships were studied by observing benthic
invertebrate populations in sediment and the stomach content of fish caught
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in the lake. The accumulation and elimination of PCP by one of the major
species of fish in the lake, the bluegill (Lepomis macrochirus), was observed
under controlled laboratory conditions„ Leaf litter, contaminated with PCP-in-
oil, was washed in water to determine the rate at which PCP might be released
from a contaminated water shed area. The solubility of PCP in water at various
pH levels was also observed.
Routine collection of environmental samples was initiated in February
1975, two months after the PCP spill and fish kil!0 The project received
funding in July 1975 and analysis of the environmental samples was initiated
in November 1975.
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SECTION 2
CONCLUSIONS
Pentachlorophenol concentrations in the water column and in fish remained
high for several months following each fish kill; leaf litter from the contam-
inated water shed area and lake sediment contained high concentrations through-
out the two-year study periodo
The major degradation products observed were PCP-OCH 2,3,5,6-TCP, and
2,3,4,5-TCP, The TCP isomers appeared to have been formed from PCP in the oil
solution, prior to entering the lake, whereas PCP-OCH seemed to have been
o
formed within the aquatic ecosystem, and within leaf litter along the stream
bank.
Fish rapidly accumulated PCP and PCP-degradation products from the contam-
inated lake with the largest concentrations observed in bile, followed by
liver, gills, and muscle tissue. Laboratory experiments showed that the LC-50
for the bluegill, Lepomis macrochirus, was 0.3 ppm. Fish exposed to sublethal
concentrations rapidly accumulated PCP, but were abTe to elimirate most of it
within sixteen days when placed in a clean environment. Thus, the persistence
of PCP in lake fish for several months following each spill indicates that the
fish were subjected to chronic PCP contamination over an extended period of time.
At the pH observed for the lake water, PCP existed in the soluble phenate
anion form, thus it was readily available for uptake by aquatic organisms, A
small amount was associated with particulate matter v/hich mav have provided an
important means for incorporation into sediments.
5
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Food chain studies showed that all gamefish depended upon benthic
organisms either as a direct or indirect food source. Therefore, PCP and
PCP-degradation product accumulation in sediments provided a continuous source
for contamination of fish caught for human consumption,, Questions remain
regarding the persistence of PCP in sediment, since a continuous influx of PCP
from the contaminated water shed could have given the appearance of PCP per-
sistence in the sediment. In either case the sediment received continuous
exposure to PCP and further study is needed to determine the long-term fate
of PCP in the sedimentary environment.
In general, the results indicate that PCP released into the aquatic
ecosystem was not rapidly assimilated by photo- or microbial-degradation.
Acute toxicity to fish occurred by rapid uptake of the water soluble phenate
anion, whereas chronic exposure occurred by leaching of PCP from the contam-
inated water shed into the lake and by incorporation of PCP into the benthic
food chain. Thus, once contaminated with PCP, the sediment and water shed
area provided a source for chronic PCP pollution of the aquatic ecosystem.
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SECTION 3
RECOMMENDATIONS
Having ascertained the persistence of PGP in various parts of the ecosystem
and observed major PCP-degradation products, it would now be advantageous to
study individual aspects of the project more intensely both in the contaminated
lake and under controlled laboratory conditions to gain a better understanding
of the factors involved in the accumulation and degradation of PCP in the aquatic
environments.
Of primary concern would be the partitioning, of PCP between oil and water
and the adsorption of PCP to suspended participate matter. The photodegrad-
ation of PCP in fuel oil and volatilization of resulting products would be of
concern to companies utilizing evaporation as their method of waste water
disposal. The formation and persistence of minor degradation products which
have been reported from previous laboratory experiments (i.e. tetrachloro-
hydroxyquinone5 tetrachlororesorcinol, PCDD and PCDF) should be monitored in
the contaminated ecosystem and their accumulation in fish should be studied
to determine if they present a possible hazard to the ecosystem or to people
using the Iake0
Further study is needed on the accumulation and metabolism of PCP in fish
including uptake via food as well as through the gills0 Associated with this
would be the accumulation of PCP in benthic organisms and their role in PCP
bioaccumulation0 The relatively high concentration of PCP-like compounds in
the liver of supposedly non-contaminated fish should also receive more
7
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attention. Additional study of PCP in fish should include the conjugated
forms of PCP that are known to exist in liver and bile,,
Questions remain regarding the composition and amount of the original
spill and the frequency with which additional PCP-containing waste was released
from the holding pond,, The apparent persistence of PCP in lake sediment
alternatively could have resulted from a continuous influx of PCP from the
timber-treating company's operation. Frequent monitoring of the waste water
holding pond and of runoff from the holding pond area would have been necessary
to determine the extent of PCP influx to the lake. Further study is needed of
the persistence of PCP in lake sediment, of degradation products, and of the
potential for continuous contamination of the aquatic ecosystem via the benthic
food chain, resuspension, and dissolution in the water.
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SECTION 4
MATERIALS AND METHODS
ENVIRONMENTAL SAMPLES
Samples of the oil slick and a few random environmental samples were
obtained immediately after the spill in December 1974. Routine sampling was
initiated in February 1975 and samples were stored frozen until analysis
began in November 1975, after the necessary equipment had been received and
the reagent blanks and efficiency of the extraction and analysis procedures
had been established. Extraction and analytical techniques were developed
and improved throughout the first year. These procedural improvements along
with information about the major PCP-degradation products helped to develop an
improved study for the second year. Analytical procedures were modifications
of those described by the U.S. Environmental Protection Agency (1974).
During the first year of the study, samples of water and sediment were
obtained bi-monthly from six sites (Figure 1). Leaf litter was collected along
the stream bank at site 1 near the spill area and fish were collected by seine
along the lake shore near sites 3 and 5. Water quality parameters observed
were temperature, hydrogen ion activity (pH), dissolved oxygen, turbidity,
and total organic carbon.
Water samples were collected in 4-liter glass jugs with aluminum-lined
caps. Dissolved and particulate PCP were separated by filtering the water
through Reeve Angel grade 934-AH glass-fiber filter pads. Dissolved PCP was
recovered by acidifying 2 liters of the filtrate to pH 2 with HC1 and extract-
-------
ing with 2 x 50 ml of beizene. Participate PCP was recovered from the filter
pads by washing with 0.1 N HC1, extracting the pads in benzene with ultra-
sonication for IS mi .lutes, followed by rinsing the pads with hexane. The
benzene and hexane -;nlutions were then combined. Sediment samples were
collected with three ^rabs using an Eckman dredge to provide at least 1 kg
of wet sediment from each site. The samples were stored frozen in aluminum-
lined freezer containers, 'xtracted by ultrasonication in benzene, and analyzed
as described above for particulatc PCP. Leaf litter was air-dried, blended to
small pieces, and analyzed as described above for sediment„
Fish collected by seine from the two sites were combined, chopped into
small pieces, and air-J-'ied. A 25-g sample of air-dried, chopped fish was
blended with 25 g Na_SO., extracted into 100 ml benzene with ultrasonication,
and filtered. The benzene extraction was repeated, the filtered residue washed
with hexane, and the benzene and hexane solutions were combined.
The resulting benzene and hexane solution from each of the above sample
extractions was washed with 0..1 N_ NaOH to separate phenolic compounds from
base-insoluble components. The aqueous solution was then acidified to pH 2
with HC1 and the phenols were extracted into hexane. The PCP content of the
resulting hexane solution was determined by gas chromatographic analysis
utilizing electron capture (EC-GC) detectors. Samples were analyzed before
and after methylation with diazomethane according to the procedure of Schlenk
and Gellerman (I960).
During the second year of the study (July 1976 to July 1977), improved
sample collection and analysis techniques were utilized allowing more involved
analyses to be performed on the samples. Second-year samples were collected
from four sites on a quarterly basis (Figure 1)„ Fish were collected near
site B by gill net to obtain large specimens so that various organs of the fish
10
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could be analyzed separately. Fish tissue was analyzed by lysing 1 g (wet
weight) in 2 ml of 50% H?SO., adding 2 ml acetonitrile, and extracting with
2 x 5 ml hexane. The hexane solution (hexane-I) was then washed with dis-
tilled water and the base-soluble components (phenols) were extracted with
2 x 5 ml of 0.1 N^NaOH, the aqueous HaOH solution was then acidified to pH
2 with HC1 and the phenols were extracted with 2 x 5 ml hexane (hexane II)„
Both hexane-I (containing base-insoluble components) and hexane-II (con-
taining base-soluble components) were then analyzed by GC-EC before and
after methylation with diazomethane. The basic extraction procedure used
during the second year of the study is shown in Figure 2.
Water samples were analyzed in triplicate by acidifying filtered
1-liter samples to pH 2 and extracting with 2 x 25 ml hexane. The hexane
extract was separated into hexane-I and hexane-II and analyzed as described
above for fish samples. Sediment samples were air-dried, washed with 0.1 N_
HC1 and extracted with a solution of acetone/hexane (60/40, v/v) under
reflux for 20 hours. The acetone was removed by washing with water and the
hexane solution recovered and treated as described above,, Particulate
matter, collected on filter pads, and leaf litter were analyzed by the same
method as sediments.
The base-insoluble components (hexane-I solution) from selected samples
were further fractionated by elution through an alumina micro-column (Buser,
1975) in an attempt to isolate and identify dioxins and dibenzofurans. Control
samples of water, sediment, leaf litter, and fish were concurrently collected
from an isolated 5-acre pond which received no industrial or agricultural
drainage. Levels of PCP and PCP-degradation products in the control pond
are considered to represent background concentrations in this area. Efficiency
11
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of the extraction procedures was determined by the addition of known amounts
of PCP to clean samples and subjecting the "spiked" samples to the extraction
and analysis procedures.
Samples were analyzed with a Varian model 2700 gas chromatograph with
Sc H electron capture detectors. Two 3 mm x 2 m stainless steel columns were
used for identification: a non-polar 3% SP-2100 on 80/100 Supelcoport and
a polar 10% SP-1000 on 80/1000 chromosorb W.A.W. Injector temperature was
HEXANE-I
BASE-INSOLUBLE
COMPOUNDS
SAMPLE
HEXANE EXTRACT
0.1 N NaOH
BASE-SOLUBLE
COMPOUNDS
Acidify
pH 2
HEXANE EXTRACT
HEXANE-II
AQUEOUS SOLUTION
(Discard)
METHYLATE, CH =N=N
ANALYZE, GC-EC
VERIFY, GC-MS
Figure 2. Extraction of PCP and PCP-degradation products
from environmental samples0
12
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175°C, column 200°C, and detector 250°C. The carrier was N at a flow rate of
25 ml/min. Compound identity was verified from representative samples utiliz-
ing a Hewlett-Packard 5933 gas chromatography-mass spectrometry (GC-MS) data
system. All solvents were reagent grade, redistilled in a glass distillation
apparatus and tested for purity by concentrating 100 fold, methylating and
analyzing by GC-EC.
PCP RELEASE FROM CONTAMINATED LEAF LITTER
Leaf litter collected from the stream bank at site 1 on February 27, 1975,
was used to study the release of PCP from contaminated leaf litter into water.
Three replicate experiments were performed in which 1 g of air-dried leaf
litter was placed in 100 ml of distilled water and shaken at 60 rpm for 24
hourso The water was then decanted through medium-porosity glass fiber filter
pads and saved for PCP analysis.
The filter pad was then back-washed with 100 ml of distilled water and
the water added to the leaf litter for an additional 24-hour period of shaking.
The process was repeated and after the third 24-hour equilibrium period, the
three separate water samples and the leaf litter were analyzed for PCP content
according to the procedures described above for the first year study of
environmental samples.
SOLUBILITY OF PCP IN WATER
Solubility in Distilled Water
The solubility of PCP in distilled water was determined by adding excess
reagent grade PCP to 250 ml distilled water and enhancing dissolution by ultra-
sonication or stirring up to 62 hours„ The concentration of the molecular
(phenol) and ionic (phenate) forms of PCP in aqueous solution was determined
13
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by titrating a solution of PCP with HC1 and obtaining the spectrophotometric
curve of the solution from 400 to 200 nm as increments of HC1 were added. To
calculate the concentration of the above forms at equilibrium, the absorbance
maxima at 219 nm was observed after the addition of 0.09 M HC1. Assuming that
the concentration of the phenate anion is represented by the absorbance maximum
(A at pH 12, then the quantity of phenol may be represented by
(A - A ) where A_,g is the absorbance at a lower pH. The dissociation
constant is then calculated according to equation (1).
Ka = A319 (!)
The pH was monitored with a Corning Model 12 pH meter with glass vs. calomel
electrodes, and spectral data was obtained with a Gary 17 spectrophotometer
utilizing 1-cm and 10-cm cells.,
Solubility in Buffered Solutions
Since natural aquatic systems usually have some buffer capacity (i.e.
alkalinity), the study was expanded to determine the solubility of PCP in buf-
fered solutions at pH 2.2, 606, and 10.1. The pH 2.2 buffer solution was
prepared by mixing 0.1 M H PO. and 0.1 M KH-PO. until the desired pH was
obtained. Buffer solutions at pH 606 were prepared from 0.1 f4 KH_PO. and 0.1
M NaOH, whereas pH 10.1 buffers were made from 0.1 M NaOH and 0.1 M H-BO,,.
To determine absorption maxima in the ultra-violet (u.v.), buffered
samples were prepared in pH 2.2 buffer for PCP and in pH 10.1 buffer for
sodium pentachlorophenate (SPCP). Spectrophotometric curves from 200 to 360 nm
were determined using a Bausch § Lomb Spectronic 710.
Two methods were used to prepare PCP solutions for solubility studies. In
the first, a quantity of PCP was weighed out; to this was added a known volume
14
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of prepared buffer. Each flask, including blanks and standard recoveries, was
then closed with an aluminum-foil covered stopper; wrapped with foil to exclude
light; and shaken for 18 hours or more at 120 rpm and 25-28°C on a New Brunswick
Scienctific Controlled Environment Incubator (New Brunswick, N.J.).
In the second method, 0.1, N^NaOH was added to dissolve a known quantity
of PCP. Acid was then added to the SPCP solution to bring the pH to the desired
level. Flasks were then covered with aluminum foil and treated as in the first
method.
Flasks were removed from the shaker, and aliquots transferred to glass
centrifuge tubes to be spun down in either a Model CL International Clinical
Centrifuge for 10 minutes or a Sorvall General Laboratory Centrifuge at 4700
rpm for 10 minutes. Unwetted, floating PCP was removed from the solution sur-
face and sample aliquots were then taken from beneath the surface.
Sample aliquots of 50 ml each were extracted using 125-ml separatory
funnels, and 1 ml aliquots were extracted in 15 x 150 mm test tubes using a
vortex mixer. Sulfuric acid was added to each sample, where necessary, to
bring the pH to 2. Samples were then extracted with two aliquots of hexane,
and the aqueous phase was discarded. The combined hexane portions were then
washed with distilled water, and the water was discarded. Then the hexane
phase was washed with two aliquots of 0.1 N_ NaOH and the hexane was set aside
as hexane-I. The combined basic solutions were then acidified to pH 2 with
sulfuric acid and extracted with two aliquots of hexane. These two aiiquots
were combined and set aside as hexane-II„
For gas chromatographic analysis, samples containing phenols were
concentrated to about 5 ml under a stream of N and then subjected to methyl-
ation according to the method of Schlenk and Gellerman (1960). Samples were
15
-------
allowed about 5 minutes to react and then were brought to an appropriate volume
for gas chromatographic analysis as described above for environmental samples.
ACCUMULATION AND ELIMINATION OF PCP IN FISH
Six-month-old bluegill, Lepomis machrochirus, were obtained from the
Lyman Fish Hatchery, Lyman, Mississippi, The fish were acclimated to laboratory
conditions for a two-week period, randomly divided into test groups of ten to
thirty in 30-liter glass aquaria and re-acclimated for 24 hours with no feeding
before initiating toxicity and exposure studies„
The studies were performed as static bioassays in dechlorinated tap water.
Each aquaria was monitored at the beginning and end of each 24-hour period for
pH, temperature, conductivity, and dissolved oxygen. The PCP content was mea-
sured in each aquarium after the initial 24-hour period to verify the desired
exposure level„ The test solutions were changed every 24 hours thereafter to
compensate for PCP loss by evaporation, adsorption, and decomposition,, Control
aquaria were treated in the same manner, without PCP.
Standard stock solutions of PCP were prepared by two procedures. A PCP
standard was made by dissolving 1 g reagent grade PCP in 1 liter of acetone.
An SPCP standard (for sodium pentachlorophenate) was prepared by dissolving
1 g PCP in an aqueous NaOH solution at pH 12 and bringing the pH to 7.4 with
the addition of 0.1 M H PO.. All exposure studies were performed with the
PCP standard solution.
Before initiating exposure studies, the toxicity of PCP for L_^ machrochirus
was verified using 96-hour median lethal concentration (LC-50) static bio-
assays according to the procedure in Standard Methods (American Public Health
Association, 1971). Ten fish were placed in each test aquaria and ten for
control„ Percent survival was observed at the end of each 24-hour period.
16
-------
Accumulation studies were performed in three replicate aquaria containing
thirty fish each along with a control aquaria. The fish were exposed to a
sublethal concentration of 0.1 ppm PCP and two fish from each aquarium were
removed for analysis on day 1, 2, 4, 8, and 16 of the exposure period. A 1-g
sample of muscle tissue and the combined gills, liver, and digestive tract
were analyzed for PCP content for each fish. On days 4, 8, and 16, three
additional fish were killed for separate analysis of gills, liver, and digestive
tract.
Elimination studies were initiated by placing fish exposed to 0.1 ppm PCP
in clean water, which was changed every 24 hours. These studies were performed
in triplicate aquaria versus control for each of two groups of fish. Group-I
fish (short-term exposure) were placed in clean water after 4 days exposure
and group-II fish (long-term exposure) were placed in clean water after 16
days exposure.
Group-I fish were collected on day 1, 2, 4, 8, and 16 and the muscle and
combined gills, liver, and digestive tract were analyzed. Group-II fish were
collected on day 2, 4, 8, and 16 for muscle and combined tissue analysis of
the muscle, gills, liver, and digestive tract„
Fish Collected for PCP analysis were weighed and measured prior to dis-
section. Tissues to be analyzed were weighed and placed in 30-ml vials. The
PCP content was determined in a manner similar to that described above for
fish tissue analysis of environmental samples.
FOOD CHAIN STUDY
Benthic collections were taken quarterly at four stations during the
second year of the project« Collections consisted of three (15,5 cm x 15.5 cm)
samples from an Eckman dredge at each station. Samples were placed in plastic
17
-------
bags and packed in ice. After returning to the laboratory, samples were fixed
in a ten percent formalin solution. Organisms were separated from the substrate
by passing samples through a wire sieve (U.S. Standard No. 50). Benthic
invertebrates were then separated from the strained material, identified and
volume determined.
Fish analyzed in food habit studies were captured by both trawl and gill
net.. Immediately after capture fish were placed on ice and returned to the
laboratory. Stomachs were removed and the species and length of fish recorded.
Stomachs were then placed in ten percent formalin until examination. Food
items were identified in the same manner as the benthic samples. Analysis of
the stomach contents includes the determination of the number, volume and
frequency of occurrence of each food item.
18
-------
SECTION 5
RESULTS AND DISCUSSION
ENVIRONMENTAL SAMPLES
Low levels of PCP and PCP-degradation products were observed in the con-
trol pond samples (TABLE 1) exhibiting concentrations of 0.3 ppb in water, 3
ppb in sediment, 7 ppb in fish muscle, and 70 ppb in fish liver. Reagent
blanks were well below the PCP concentration observed in control samples
indicating that a slight background concentration of PCP did exist in the
supposedly non-contaminated environment samples. This background contamination
could be due to atmospheric transport of PCP from treatment sites. It has
also been suggested (Arsenault, 1976), that a naturally occurring fungal
metabolite, p-methoxytetrachlorophenol could be mistaken for low levels of
PCP in some samples.
Procedures utilized during the first year study provided the following
percent recoveries ± one standard deviation: water, 90 ± 15; sediment, 90 ± 40;
fish, 62 ± 14 0 Second-year procedures provided the following extraction
efficiencies: water, 95 ± 10; sediment 110 ± 25; fish, 90 + 8.
PCP Persistence and Distribution
The investigation during the first year of sampling, concerned primarily
with the distribution and persistence of PCP in the lake, has been discussed
by Pierce et al. (1977) and will be summarized here in relation to the rest
of the study. Within two months after the spill, PCP was uniformly distrib-
uted throughout the lake at a concentration of 10 ppb in February 1975
19
-------
TABLE 1. PCP AND PCP-DEGRADATION
PRODUCTS IN CONTROL SAMPLESOa
Sample
Waterb
Dissolved
Particulate
Sediment
PCP
003±001
001±0005
3.0±0_4
PCP-OCH3 2,3,5,6-TCP 2,3,4,5-TCP
<0001C O.liO.Ol <0001
<0.1 <0.1 <0.1
<0.1 1.010.2 <0.1
Fish6
Muscle 7.0±3 <1 <1 <1
Liver 7000±40 <1 <1 <1
Leaf Litter£ 500±1 5.0±2 700±2 300±1
Average of duplicate analyses ± % the range,,
Concentrations reported as ug/liter0
£
Lower limit of detection,,
Concentrations reported as pg/kg air-dry sediment.
Concentrations reported as ug/kg wet-weight tissue,,
f
Concentrations reported as yg/kg air-dry leaf litter,,
(TABLE 2)o The concentration decreased to background levels (<1 ppb) by
October 1975, increased again to about 15 ppb in February 1976, and steadily
declined through May 1976. Pentachlorophenol associated with particulate
matter usually represented less than ten percent of the dissolved PCP (TABLE 2)
Water samples collected during the second year (TABLE 3) showed background
20
-------
TABLE 2. PCP CONCENTRATION IN WATER SAMPLES
COLLECTED FEBRUARY 1975 THROUGH MAY 1976.
Date Sample
Feb. 27, 1975
Dissolved
Particulate
Apr. 24, 1975
Dissolved
Particulate
June 28, 1975
Dissolved
Particulate
Aug. 5, 1975
Dissolved
Particulate
Oct. 11, 1975
Dissolved
Particulate
Dec. 6, 1975
Dissolved
Particulate
Feb. 7, 1976
Dissolved
May 3, 1976
Dissolved
1
9
0.2
11
3
13
2
N.A.
N.A.
10
2
76
5
29
18
2 3
Pg
11 6
0.4 0.
6 8
NaA0a 0.
8 2
0.2 1
N.A. 3
N.A. 0.
5 0.
0.3 <0.
25 1
0.4 <0.
N0A0 15
N.A. 2
Site
4
PCP/ liter
9
3 0.4
8
2 0.1
3
2
2
3 0,4
8 0.1
lb <0.1
2
I <0.1
10
N.A.
5 6
9 8
0.6 0.2
9 15
0.2 N.A.
2 82
0.2 0.4
3 1
0.2 N.A.
1.0 0.2
0.1 <0.1
2 N.A.
<0.1 N.A.
10 26
2 2
,N0A0 = Not Analyzed,
Lower limit of detection.
21
-------
levels in lake water in August and October 1976, high concentrations in January
and February 1977, and decreasing somewhat, yet still well above background in
April 1977. Although suspended particulate matter contained less than ten
percent of the PCP in the water column throughout the study, it may be important
as a transport mechanism from the water column to sediment.
TABLE 3. PCP IN WATER SAMPLES COLLECTED
AUGUST 1976 THROUGH APRIL 1977,
Date Sample
Site
B C
yg PCP/liter
Aug, 11, 1976
Dissolved3
Parti culate
Oct., 22, 1976
Dissolved
Part icu late
Jan, 5, 1977
Dissolved
Particulate
Feb. 22, 1977
Dissolved
Particulate
Apr. 27, 1977
Dissolved
Particulate
11.0±0.4
0,2
N,A.C
N.A,
82.0±32
0,4
146,0 7
0,18
16oO±0.4
0,33
0,1+0,02
0006
0,1+0,03
0,04
24,0±13
N.A,
N.A.
N.A.
5,0±0,5
0,29
0,110,03
0,05
0,2±0,05
0,04
25,0±7
0.2
29,0±2
0,14
5.0+0,7
0,35
0.1±0.
0,06
1,0±0,
0.2
16.0+6
0.2
N.A.
N.A.
5,0±0,
0,29
01
1
2
wean of triplicate analyses ± sandard deviation.
Composite of all filter pads from three one-liter samples,
Q
N,A0 = Not Analyzed,
22
-------
Water quality parameters monitored for February 1975, through May 1976,
(TABLE A-l) show that the pH remained slightly acidic in the lake, generally
between pH 6.0 to 6.8 but was more acidic in the shallow stream (4.2 to 6.2).
Near-surface water was well oxygenated, except in the shallow stream when the
water was stationary and varied seasonally with temperature. Total organic
carbon (TOC) remained fairly constant throughout the study period except for
anomolously low concentrations observed in April 1975.
During the second year of the study, dissolved oxygen (D.O.) and tempera-
ture were monitored near the surface and near the bottom at each site, but pH
was recorded for surface samples only (TABLE A-2). Surface samples exhibited
conditions similar to the previous year, but the bottom samples revealed
anoxic conditions existing at the bottom of site C for the summer, fall, and
spring sample periods.
Sediment samples retained PCP concentrations well above background
throughout the first year of the study (TABLE 4). The variability observed
among these samples may be attributed to the inefficiency of the sonication
extraction procedure, compounded by variation in the environmental samples.
The second-year sediment samples (TABLE 5) show that large concentrations of
PCP remained in sediments throughout the study period.
Sediment contained high PCP concentrations in August and October 1976
(500 ppb average) and exhibited a slight decrease in January 1977 (200 ppb),
after the second fish kill, indicating a residence time of over a week in the
water column before incorporation into sediment. Lake sediment showed an
increase near the mouth of the stream in February 1977 (1,500 ppb) but in
April 1977 the concentration returned to the January levels (250 ppb).
23
-------
TABLE 4» PCP CONCENTRATION IN SEDIMENT SAMPLES
COLLECTED FEBRUARY 1975 THROUGH MAY 1976.a
Date
Feb.
Apr,
June
Aug.
Ont.
Dec,
Feb.
May
27, 1975
24, 1975
28, 1975
5, 1975
11, 1975
6, 1975
7, 1976
3} 1976
1
800
1,160
1,300
N.A,C
927
163
100
96
Site
2 345
yg PCP/kg air-dry sediment
lb 22
36 21
1 1.4
Nr,A0 10
471 48
900 11
N.A. 313
NoA. 20
26
98
583
205
91
97
10
N.A.
119
180
860
207
56
97
84
21
6
2
34
92
45
24
N.A.
81
7
o
Composite of three grab samples„
Lower limit of detection,.
£
Not analyzed.
Small fish collected by seine during the first year showed high whole-
body PCP concentrations in February (2,500 ppb dry-weight) and April 1975
(TABLE 6), The concentration was diminished yet still above background in
June 1975, increased some in December 1975 and February 1976, and then decreased
again to background by May 1976 (TABLE 6). Improved sampling and analytical
procedures used for the second year of the study provided a more complete
description of the accumulation of PCP in fish. These results (TABLE 7) show
that fish contained only background levels of PCP in October 1976 but rapidly
accumulated very high concentrations in January 1977 immediately after the
24
-------
TABLE 5. PCP IN SEDIMENT SAMPLES COLLECTED
AUGUST 1976 THROUGH APRIL 1977.
Date A
Augo 11, 1976 857±57
Oct. 22, 1976 166+38
Jan. 5, 1977 277153
Feb. 22, 1977 N.A0b
Apr, 27, 1977 4+1
0
Average of duplicate analyses 1
Not analyzed.
TABLE 60
FEBRUARY
Collection Date
Feb. 27, 1975
Apr, 24, 1975
June 23, 1975
Oct. 11, 1975
Deco 6, 1975
Feb. 2, 1976
May 3, 1976
Site
B C a D
ug/kg air-dry sediment
429+169 520+151 142144
9941394 389+12 212+8
239175 150±13 170178
1, 518187 NoAo N.A.
250114 238+3 132121
^ range0
PCP IN FISH COLLECTED
1975 THROUGH MAY 1976.
ng PCP/g air-
dry fish tissue
2,500+200
1,380120
130+70
<50b
651+650
87122
<50
Average of replicate analyses 1 % the range.
Lower limit of detection.
25
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spill. Concentrations decreased somewhat by April 1977 but were still well
above background levels. Bass collected freshly dead or dying in January 1977
shortly after the spill, contained PCP in concentration of 2,100,000 ppb in
bile; 230,000 ppb in liver tissue; 42,000 ppb in gills; and 13,000 ppb in
muscle. These values represent concentration factors over the PCP content in
water of 500 for muscle; 1,500 for gills; 8,000 for liver; and 80,000 for bile.
Leaf litter collected near the spill site contained very high concentrations
of PCP throughout the investigation (TABLE 8). The variability observed between
sampling dates probably reflects the heterogeneous nature of the collection
area, but the large concentrations observed overall indicate that contaminated
TABLE 8. PCP IN LEAF LITTER.
Date
PCP
ng/g air-dry leavesc
Feb. 27, 1975
Apr. 24, 1975
June 23, 1975
Aug. 11, 1975
Dec. 6, 1975
Feb. 10, 1976
May 3, 1976
Aug. 11, 1976
Oct. 22, 1976
Jan. 5, 1977
Apr. 27, 1977
6,4001250
2,5501650
5,2001300
5,800+1,200
3,470
1,680+12
6,00011,000
10,300+1,000
3,1491380
15,900+180
2,9701800
Average of duplicate analyses 1 % the range.
27
-------
leaf litter provided a potential for long-term pollution of the aquatic
ecosystem.
PGP Degradation Products
Since the source of PCP contamination was the oil and water from the
industrial waste water holding pond, samples of oil and water from the pond
and oil from the surface of the stream near the spill site were collected and
analyzed for PCP and PCP-degradation products. The results (TABLE 9) show that
the PCP concentration was 617,000 ppb in the oil slick from the stream;
220,000 ppb in holding pond oil; and 8,700 ppb in holding pond water. The
major degradation products observed in the oil slick were the 2,3,4,5- and
2,3,5,6-TCP isomers, each approximately 13% of the PCP concentrate (TABLE 9)=
These percentages far exceeded the percent TCP observed in a sample of technical
grade PCP (1 to 2%) suggesting that both isomers of TCP were formed from PCP in
the oil solution, probably by photodegradation as suggested by Crosby (1972).
The major degradation products observed in the contaminated lake were
pentachloroanisole (PCP-OCH ) and the two isomers of TCP. Varying quantities
of the methyl ether (anisole) of both TCP isomers were also observed but
proved difficult to quantitate, due to low concentrations and interference
from naturally occurring substances. The 2,3,4,6-TCP isomer was not observed
but may have been present in small quantities. Trichlorophenol eluted with the
solvent front under the chromatographic conditions used, thus its concentration
was not determined. The presence of chloranil, PCDD, and PCDF were indicated
in sediment and holding pond oil samples in ug/kg quantities, utilizing the
procedure described by Buser (1975), but our analytical system did not provide
adequate quantitation. The gas chromatographic retention of the methylated
TCP isomers relative to methylated PCP was observed to be 0.5 for 2,3,5,6-TCP
28
-------
TABLE 9. PCP AND DEGRADATION PRODUCTS IN TECHNICAL GRADE
PCP, INDUSTRY WASTE-HOLDING POND AND OIL SLICK FROM STREAM.
PCPa 2,3,5,6-TCPb 2,5,4,5-TCPb
Technical-Grade PCP Solution 1,000 1.2% <0001%
Holding Pond
Oil Slick
Water
Stream Oil Slick6
220,000
8,700
617,000
13%
9%
12%
N.A.
N.A.
13%
ang PCP/ml solution (ppb).
Reported as percentage relative to PCP concentration.
Q
Not analyzed.
Collected from industrial waste holding pond, September 8, 1975.
g
Colelcted from spill area, December 18, 1974.
and 0.80 for 2,3,4,5-TCP on 3% SP-2100, and 0.46 for 2,3,5,6-TCP and 1.21 for
2,3,4,5-TCP on 10% SP-1000o The relative response factor for peak height was
1.0 for 2,3,5,6-TCP and 0.25 for 2,3,4,5-TCP on 3% SP-2100; and 1.5 for 2,3,5,6-
TCP and 0.25 for 2,3,4,5-TCP on 10% SP-1000.
Degradation products in control pond samples are given in TABLE 1. The
concentrations were found to be essentially at reagent blank lavels in all
samples except leaf litter, which exhibited background levels of all products
in the 3-7 ug/kg range.
29
-------
The concentration of degradation products dissolved in the water column
are shown in TABLE 10. The PCP-OCH_ remained at background levels throughout,
reflecting the low solubility in water. The 2,3,5,6-TCP was below background
in August and October, but increased to almost 1 ppb in January 1977 and re-
mained above background through April 1977. The 2,3,4,5-TCP isomer remained
below or near background throughout the sampling period.
Sediment retained high concentrations of all the degradation products
throughout the August 1976 through April 1977 study period (TABLE 11). The
TCP isomers were both more concentrated than PCP-OCH from August through
o
February, but the latter increased in April. This observation, along with
the absence of PCP-OCH3 in technical PCP or the oil slick suggests that PCP-
OCH was formed in the aquatic environment, probably by microbial action on
PCP as reported by Cserjesi and Johnson (1972). The changes in TCP con-
centration followed a pattern similar to that for PCP, suggesting that TCP
was formed before it reached the sedimentary environment„
TABLE 10. PCP-DEGRADATION PRODUCTS IN LAKE WATER
(DISSOLVED) SAMPLES COLLECTED AUGUST 1976 THROUGH APRIL 1977.
Date
Aug.
Oct.
Jan.
Feb.
Apr.
PCP-OCH
•J
11,
22,
5,
22,
27,
1976
1976
1977
1977
1977
0.
0.
0.
0.
0.
06±0
04±0
08±0
07±0
03±0
.01
.01
.02
.03
.01
2,3
ug
0.
Oo
0.
0.
0.
,5,6-TCP
PCP/liter
08±0.02
06±0.02
9+0.1
3±0.02
9±0.1
2,
0
0
0
0
0
3,4,5-TCP
005±0
.03+0
.07±0
.05±0
.3±0.
.04
.01
.02
.02
1
aAverage of sites B, C, and D ± standard deviation,
30
-------
TABLE 11. PCP-DEGRADATION PRODUCTS IN LAKE SEDIMENT
COLLECTED AUGUST 1976 THROUGH APRIL 1977.
Date
Aug.
Oct.
Jan.
Feb.
Apr.
PCP-OCH3a
ug
11,
22,
5,
22,
27,
1976
1976
1977
1977
1977
18.
6.
16.
1.
60.
0±5
0±6
Oil
5±0.2
0±20
2,3,5,6-TCPa 2,3,4,5-TCPb
PCP/kg air-dried sediment
120±60
62±5
50±20
340115
53±25
129
63
24
N.A.C
15+3
o
Average o£ values for sites B, C, and D ± standard deviation.
Site B only.
c
N.A. = not analyzed.
The concentrations of PCP-degradation products in various fish tissues
are shown in TABLE 12 for samples collected in August and October 1976, and
in TABLE 13 for fish collected in January and April 1977. Fish collected in
August and October contained low concentrations. Samples collected in January
1977, shortly after the second fish kill, showed very high concentrations
indicating that fish rapidly accumulated all products from the environment.
The TCP isomers were found in increasing concentration in muscle, gills, liver,
and bile, in the same manner as was observed for PCP. The PCP-OCH was general-
ly less concentrated than TCP and showed a marked reduction in bile, probably
as a result of the insolubility in aqueous solution. The high concentration
of PCP-OCH_ in fish obtained from water containing very low concentrations
indicates a very high partition coefficient for PCP-OCH from water to fish.
It is also possible that the fish received some of the PCP-OCH in their diet.
31
-------
The persistence of PCP and PCP-degradation products observed in fish indicate
that the fish were exposed to high levels over an extended period of time and
that the toxic chemicals were not rapidly eliminated but were retained, perhaps
in a conjugated form, as reported by Kobayashi, et a!0 (1976).
TABLE 12. PCP-DEGRADATION PRODUCTS IN FISH
COLLECTED AUGUST AND OCTOBER 1976.
Fish
(No.)
Size
cm
PCP-OCH3 2,3,5,6-TCP 2 3,4,5-TCP
ng PCP/g wet-weight tissue
Sunfish (2)
5-10
muscle
i • b
liver
August 11, 1976
41±20
600
5±2
92
130
October 22, 1976
Sunfish (2)
Catfish (2)
Sucker (1)
12-15
muscle 3±1 <1
liver 11±1 40±10
25-26
muscle <1 1±0.6
liver 17+5 50±15
20
muscle N.A. <1
liverb 30 260
<1
5±1
<1
150±100
285
Average of replicate samples ± % the range.
Analysis of single, composite sample.
'Lower limit of detection.
Not analyzed.
32
-------
TABLE 13. PCP-DEGRADATION PRODUCTS IN
FISH COLLECTED JANUARY AND APRIL 1977.
Fish (No.)
Sunfish6 (2)
e
Bass (3)
Catfish6 (1)
Suckerf (1)
f
Sunfish (2)
£
Catfish (2)
Size
cm
15-20
muscle
gills*
liver
36-44
muscle
gills
liver
bile
25 b
muscle
liver
22
muscle
gills'
liver
14-20
muscle
liver
40
muscle
liver
bile
PCP-OCH3
January 6,
60130
230
560
170 80
60+8
600+200
208±8
164
1,200
84
90
490
April 27,
29±1
155±35
140±35
350+200
210+20
2,3,5,6-TCP
ng PCP/g wet-weight
1977
75±15
360
950
230 96
335±35
5,600+2,000
114,000±10,000
219
8,500
45
450
11,360
1977
20±2
200±50
60±20
720±60
1,000±500
2.3,4,5-TCP
tissue
/•»
-------
Since leaf litter contained very high concentrations of PCP, some of the
minor degradation products (i.e., TCP-OCH ) were observed which were not dis-
tinguishable in other environmental samples. The large concentration of the
anisoles of PCP and both TCP isomers (TABLE 14) throughout the study period
indicates that the process by which anisoles are formed occurred within the
dried grass and leaf litter along the banks of the stream0 The persistence of
all products throughout the study period showed that the leaf litter was a
source for continuous environmental pollution.
PCP RELEASE FROM CONTAMINATED LEAF LITTER
This experiment was devised to determine the extent to which contaminated
leaf litter might release PCP to the water, thus providing a source for con-
tinuous pollution of the aquatic ecosystem. The results (TABLE 15) show that
approximately ten percent of the PCP in the leaf litter was leached out over
each 24-hour peiod of shaking in water. The original concentration of PCP
in the leaf litter was 6.4 Mg/g and that in the water after equilibrium
was about 0.5 yg/g. Since the reported solubility of PCP in water is 15 ppm
(Bevenue and Beckman, 1967), solubility would not have been a limiting factor.
A mass balance (TABLE 15) shows that all of the PCP was accounted for in the
experiment.
These data indicate that PCP associated with leaf litter and vegetation
along the bank of a stream would be released slowly over a period of time.
Therefore, contaminated leaf litter serves as a source for chronic PCP contami-
nation of the aquatic environment. This finding is supported by the persistence
of PCP in leaf litter throughout the study.
34
-------
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35
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TABLE 15. PCP RELEASE FROM CONTAMINATED LEAF LITTER
Sample
1
2
3
Average
Original
Day 1
0.9
0,6
0,3
0,6
leaf litter
Water
Day 2 Day 3
mg PCP /liter
003 0,3
0.3 0.2
0.8 1.1
0.5 0,5
sample
Leaf Residue
mg PCP/k?
4.9
3,9
5.9
408
Total
6.2
5oO
8.1
6,4±1
6.4±0,3
SOLUBILITY OF PCP IN WATER
The solubility of PCP in unbuffered, distilled water was found to be in
the range of 10 to 14 mg/1 (ppm) at 23°C with a resulting pH of 5.1. These
values are similar to those (14-19 mg/1 in water) reported by Bevenue and
Beckman C1967). Curves representing the titration of a solution of 9.2 ppm
PCP in distilled water with 0,09 M HC1 along with the back titration of the
acidified solution with 0<,1 M NaOH are shown in Figure 3. Concentrations of
the phenate and phenol forms calculated for various pH values are given in
TABLE 16 along with the pKa values which were found to be an average of 4.5.
The fact that the curves in Figure 3 are not congruent indicates that a
nonreversible process occurred upond acidification of PCP and that the solu-
bility of PCP was suppressed. This nonreversible process could result from
difficulty in dissolving and dissociating the solid phenol before equilibrium
could be attained.
36
-------
TABLE 16. PCP DISSOCIATION IN WATER AT VARIOUS pH LEVELS
A" a
0,77
0.716
0.605
0.514
Oo444
0.390
HA
1.08
Iol34
1,245
1.34
1.41
1.46
pH
5.06
4.61
4.32
4.41
3.40
3.39
pKa
5.17
4.80
4.63
4.30
4.50
4.46
Phenate concentration (dissociated form).
Phenol concentration (undissociated form),
£
pKa calculated from equation (1), p. 14.
Ultraviolet scans of PCP and SPCP in buffer solutions are presented in
Figure 4 and summarized in TABLE 17. The absorbance maxima for SPCP agree
with the maxima (218.5, 248, and 319 nm) presented in Sadtler (1972), although
conditions of analysis, such as pH or ionic strength, were not indicated in
Sadtler. However, the molar absorptivities calculated from Sadtler (218.5 nm,
1.3 x 106; 248 nm, 4.4 x lO4; 319 nm, 2.7 x lO1*) are much higher than those in
TABLE 1. Without a description of conditions under which the Sadtler data
were obtained, it is not possible to speculate on the causes of these dif-
ferences. The molar absorptivity of pentachlorophenate ion at 319 nm agrees
well with that reported above from the data for distilled water.
Absorbance was linear with concentration in the mg/1 range at 320 nm for
SPCP in 0.1 M borate buffer at pH 10.1 and in 0.1 M phosphate buffer at pH 6.6.
37
-------
For PCP, absorbance was also linear at pH 2.2 at 2.4 nm in 0.1 M phosphate
buffer.
A preliminary study indicated that solubility, as determined by adding
prepared buffer to known amounts of PCP, was complete within one day. All
subsequent samples were shaken overnight before centrifugation and analysis.
A Titration of PCP with 0.09 M HC1
O Back titration with 0.1 M NaOH
0 10 20 30 40 50 60 70 80 90 100 110 120 130
ul HC1
140 130 120 110 100 90 80 70 60 50 40 30 20 10
ul NaOH
Figure 3. Titration of PCP in aqueous solution.
38
-------
All samples analyzed by gas chromatography were prepared by adding
buffer solutions to known quantities of crystalline PCP. This method was
plagued by PCP crystals floating on the surface tension of the solution.
Transfer of floating PCP during sample preparation of sample aliquots for
extraction may account for at least part of the great variability (i.e.,
large standard deviations) in solubilities determined by this method (TABLE 18)
w
ca
oi
o
219
SPCP
200 220 240 260 280 300 320
WAVELENGTH (nm)
340 360 380
Figure 4. Ultraviolet spectra of pentachlorophenol (PCP) and sodium-
pentachlorophenate (SPCP) in 0.1 M buffer.
39
-------
TABLE 17. SPECTROPHOTOMETRIC CHARACTERISTICS IN THE ULTRAVIOLET
OF PENTACHLOROPHENOL AND SODIUM PENTACHLOROPHENATE
IN 0.1 M BUFFER
X max(nm-> Molar Absorptivity
Pentachlorophenol
214 7.5 x 104
229 (shoulder) 1.7 x 104
Sodium Pentachlorophenate
219 4.6 x 104
249 1.0 x 104
320 5.0 x 103
Another factor contributing to this variability could be dilution errors,
especially in light of the fact that samples had to be diluted as much as
250,000-500,000 times to get them within the working range of the instrument.
At the dilutions needed to use the gas chromatograph, there were no
evidences of contaminants in either the hexane-I fraction or the hexane-II
fraction prior to methylation0 Blanks at pH 2.2 and 6.6 were less than 10 ug/1,
and at pH 10.1 less than 200 yg/1. Recoveries ranged from 80% to 130%.
Samples analyzed by absorption in the u.v. were prepared by dissolving
PCP in base and acidifying the SPCP solutions to the desired pH. With this
method, the problem of PCP floating on the surface tension was considerably
reduced, although not entirely eliminated. The higher values for solubilities
at pH 2.2 and 6«6 (TABLE 18) for spectrophotometry compared with gas chroma-
tography may indicate that true saturation had not been achieved in samples
40
-------
analyzed by gas chromatography. Another possibility is that the high dilution
ratios needed for samples prepared for gas chromatography consistently
produced lower concentrations than samples analyzed spectrophotometrically;
for example, pH 10.1 samples were diluted at most only 1000 times before
spectrophometric analysis., Or, the difference may indicate that samples
analyzed spectrophotometrically were supersaturated, although this possiblity
seems remote since there were copious amounts of PCP and SPCP precipitated
in the pH 2.2 and 6.6 sample flasks. A further check on this question is
needed. In the case of pH 10»1, reproducibility within individual experiments
was fairly good, but reproducibility between runs was poor. Apparently, PCP
at high pH's readily forms SPCP by reacting with the NaOH in the buffer. Thus,
a crude titration was effected by adding PCP to a 0.1 M NaOH-H_B03 buffer.
Evidences of this titration effect were found in the marked drops in pH, to as
low as 8.5, in solutions of 0.1 M basic buffer to which PCP had been added in
what was thought initially to be excess. As a rough estimate from data devel-
oped in this study, it seems possible that the solubility of SPCP at pH 10.1
TABLE 18. SOLUBILITY AT 26 C OF PENTACHLOROPHENOL AND
SODIUM PENTACHLOROPHENATE AT THREE pH VALUES
USING TWO ANALYTICAL METHODS
pH mg
Determination by
Gas Chromatography
2.2 300±200
6.6 550 ±240
10.1 17,400 ±800
PCP/litera
Determination by
Spectrophotometry
7.2
660
11.900 ±900
o
± one standard deviation.
-------
exceeds 0.05 M» A more valid estimate could be obtained using 1 M buffer;
however, such a concentrated buffer is outside the realm of what can be found
in all but the most extraordinary aquatic environments. Thus, it is perhaps
best, albeit vague, to say that SPCP is freely soluble at pH 10.1.
The pKa of PCP is variously reported to be 4.8 (Blackman et al., 1955),
4.82 (Robinson and Bates, 1966), 502 (Dyer, 1959), 5.26 (Tiessens, 1929), and
5.3 (Griffith et a!0, 1938) and 4.5 (above) for distilled water. Even allowing
for the variation in reported pKa values, all PCP in solution at pH 2,5 or less
is present in the acidic, non-dissociated form. Taking the solubility of PCP
at pH 2.2 to be 5,3 mg/1 (TABLE 18), one can calculate a Ksp for PCP ranging
from 1.0 x 10~10 (for pKa = 5»3) to 6.3 x 10~10 (for pKa = 4.5).
ACCUMULATION AND ELIMINATION OF PCP IN FISH
These results have been reported previously by Pruitt et al. (1977), and
are summarized here.
The 96-hour LC-50 value for the PCP Standard was 0.26 ± 0.01 mg/1 and
that for the SPCP Standard was 0.33 ± 0.01 mg/1 (TABLE 19). These values agree
with those reported for various aquatic organisms by Goodnight (1942) , Holmbert
et al. (1972), and Cardwell et al. (1976). The pH of the test solutions
ranged from 7.2 to 7.7, indicating that the active form of either standard
was the phenate anion.
Exposure of six-month old bluegill sunfish (Lepomis macrochirus) to a
sublethal concentration of PCP (0.1 mg/1) resulted in the accumulation of PCP in
various tissues. The liver was found to concentrate the largest amount (35,000
ng/g) followed by the digestive tract (21,000 ng/g), gills (6,000 ng/g), and
42
-------
TABLE 19. LC-50 OF PCP TO LEPOMIS MACROCHIRUS.
Time
(hours)
24
48
72
96
PCP LC'5°a SPCP
mg/liter
0.42 ± .02 0.33
0.32 ± .02 0.32 ± .02
0.28 ± .03 0.35 ± .01
0.26 ± 0.1 0.33 ± .01
aAverage of three replicate samples ± one-half the range.
muscle (1,000 ng/g) (TABLE 20). Pentachlorophenol was rapidly accumulated by
all tissues with equilibrium apparently being established by day 8 (Figure 5).
The efficiency of the extraction procedure was determined to be 93 percent
with a standard deviation of 6 percent by the addition of standard PCP to fish
TABLE 20. ACCUMULATION OF PCP IN FISH TISSUE
AFTER EXPOSURE TO 0.1 mg PCP/1 WATER.
Tissue
Muscle
Gills
Digestive
Tract
Liver
4
0.5±0.1
2.6+0.5
9 ±2
35 ±20
Days
yg PCP/g wet
1.
6
21
25
ft
Exposure
8
weight tissue
3+0.2
±2
±4
±20
16
0.4±002
5 ±3
13 ±5
23 ±13
Q
Average of triplicate samples ± standard deviation.
43
-------
tissue and subjecting these "spiked" tissues to the extraction procedures in
six replicate analyses. Reagent blanks were obtained by subjecting control
fish from PCP-free water to the above extraction and analysis procedure and
the PCP content was less than 0.01 ppm in all control samples.
The elimination of PCP from muscle and combined gills, liver, and intesti-
nal tract from Group I fish (4 days exposure) is shown in Figure 6. Similar
data for fish from Group II (16 days exposure) are shown in Figure 7. The
elimination of PCP from the individual organ tissues from Group II fish is
shown in TABLE 21.
16
Q Combined tissues
O Muscle tissue
Figure 5.
2 4
Days of Exposure
Accumulation of PCP in tissue during exposure to 0.1 ppm PCP.
Average of six replicate samples ± standard deviation.
44
-------
A comparison of Group I with Group II elimination data (Figures 6 and 7)
reveals a lag period of 4 days in Group I that was not observed in Group II,
indicating that it took the fish about 8 days to develop a mechanisms for PCP
elimination. This supports the results of the accumulation study which showed
a leveling off and apparent decrease in PCP concentration in tissue after 8
days of continued exposure, possibly representing the time necessary for the
fish to develop an enzyme system capable of eliminating PCP. Kobayashi and
bo
t»
Cu
u
CL,
3 •
2 •
D Combined tissue
O Muscle tissue
Figure 6.
2 4 8 16
Days of Elimination - Group I
Elimination of PCP from Group I (after 4 days exposure)
of six replicate samples ± standard deviation.
Average
45
-------
Akitake (1975) reported that loss of PCP in goldfish (Carassius auratus) was
caused by the metabolic transformation of PCP to pentachlorophenylsulfate.
This was identical to the conjugate form found in the shortnecked clam by
Kobayashi et al. (1970). It has also been reported that fish dispose of such
lipid-soluble substances by passive diffusion through the gills (Brodie et al.,
1962).
(8.1)
W)
I?
a,
u
2 -
O Combined tissue
O Muscle tissue
2 4 8 16
Days of Elimination - Group II
Figure 7. Elimination of PCP from Group II (after 16 days exposure)
of six replicate samples ± standard deviation.
Average
46
-------
TABLE 21o ELIMINATION OF PCP FROM GROUP II FISH,
PLACED INTO A CLEAN ENVIRONMENT AFTER
SIXTEEN DAYS EXPOSURE TO PCP-CONTAMINATED WATER.
Tissue
Muscle
Gills
Digestive Tract
Liver
4
0.06±0002
0.16±0007
2 ±0.9
0.7 ±005
r\
Days Exposure
8
yg PCP/g wet weight tissue
0.03±0.01
0.13±0.09
0,3 ±0.1
4 ±5
16
0.03+0.01
0.08±0003
0.13±0.02
0.6 ±0.5
o
Average of triplicate samples ± standard deviation.
After 16 days of elimination in PCP-free water, the fish still contained
PCP residues (TABLE 21). The liver contained the greatest amount (600 ng/g)
followed by the digestive tract (130 ng/g), gills (80 ng/g), and muscle (30
ng/g)• By the end of the 16-day elimination study, both Group I fish and
Group II fish had eliminated PCP to about the same level (Figures 6 and 7).
Thus, after the initial 8-day lag period, the fish were able to eliminate PCP
to the same degree regardless of exposure time, yet a longer period would be
required for complete depuration.
FOOD CHAIN STUDY
Benthic Organisms
Station A, nearest the spill area, was represented by a slow stream
environment with a bottom chracterized by gravel, coarse sand, and sediment.
However, stations B, C, and D were lentic habitats characterized by soft, silty,
clay substrates. Benthic invertebrates at all four stations were dominated by
47
-------
the larvae of the two insect families, Chironomidae and Culicidae, and
Oligochaeta (TABLE 22 and TABLE 23).
Chironomid larvae appeared as the dominant benthic organisms within inter-
mediate depths of the lake (stations B and D). The dominant chironomid
collected was the genus Chironomus. Other genera present were Harnischia,
Coelotanypus, and Tanypus. Chironomids were least abundant both near the spill
area and in the deepest area of the lake. Undesirable sediment at station A
in August and low oxygen levels produced by thermal stratification during
August and April at station C may have affected chironomid abundance„
Culicidae, exclusively Chaoborus, present in consistent numbers within
the intermediate depths of the lake but were most abundant at the deepest
station, C. Due to its tolerance to low levels of oxygen, Chaoborus was the
only benthic invertebrate collected at station C during August. Numerically,
Chaoborus larvae were dominant at station C throughout the study. Oligochates,
primarily tubeficidae, were common only at station B in the upper region of
the lake, nearest the head waters.
Diversity of the benthos appeared low throughout the lake. Little seasonal
change in the composition of the benthos within each station was observed.
Following the pentachlorophenol spill occurring in December 1976, no
organisms were observed in the sample collected from station A nearest the
spill area. Absence of organisms may be attributed to the dry conditions pre-
ceding the sampling period. Although an unusually large number of chironomid
head capsules were collected at station B, its benthic composition following
the spill did not appear adversely altered. Subsequent collections in April
1977 did not reflect a numerical decrease among the dominant invertebrates
present.
48
-------
TABLE 22. BENTHIC ORGANISMS COLLECTED
DURING AUGUST AND OCTOBER 1976.
Organisms
Stations
B C
Diptera
Chironomidae
August
L"
Pd
Culicidae
Chaoborus L
Ceratopogonidae L
Oligocheata
Nematoda
Gastropoda
Chironomidae
L
P
Chaoborus
L
P
Oligocheata
Nematoda
Copepoda
15 290
5
87
1
30 87
1
1
October
62
222
72
1
204
6
787 90
1
2
8 215
3
1350 359
12 1
11
2
Total number of organisms<• from three (15.5 cm x 15.5 cm) Eckman dredge samples.
Stream was dry in October.
Larvae.
Pupae.
49
-------
TABLE 23. BENTHIC ORGANISMS COLLECTED
DURING JANUARY AND APRIL 1977.a
Stations
Organisms
Chironomidae
Lc
Pd
Ceratopogonidae L
Cuclicidae
Chaoborus L
Oligocheata
January
65
1
170
132
April
56
1
3024
158
1
286
Chironomidae
L
P
Chaoborus
L
P
Ceratopogonidae L
Dytiscidae L
Nematoda
Oligocheata
379
2
15
2
1
1
1
4
271
1
298 238
6 15
1 3
2 4
1
number of organisms from three (15.5 cm x 15.5 cm) Eckman dredge samples,
No invertegrates were observed at site A.
"Larvae.
Pupae.
50
-------
Food Habits
Results indicate that small sunfish (Lepomis spp.) and intermediate sized
bluegill (L_._ macrochirus) fed predominantly on small bottom organisms, primarily
chironomid larvae (TABLE 24). Benthic oraganisms together consitute 77.3% of
the diet of the smaller sunfish (<50 mm) collected. Larger sunfish also
appeared to utilize benthic prey, largely in the form of chironomid larvae.
In addition to insect larvae, larger warmouth (JU_ gulosus) also utilized smaller
fish as foods
Larger game fish, collected with the aid of a gill net were observed to
feed on fewer numbers of larger food items (TABLE 25). Crayfish constituted
the bulk of the observed channel catfish (Ictalurus punctatus) ration, however,
large numbers of chironomid larvae and the presence of a smaller fish within
the stomachs suggests a diverse dieto The black bullhead (!„ melas) and yellow
bullhead (I0 natal is) appeared to feed on a wide range of food items indicated
by both insect larvae, vegetation, and fish scales found in the gut.
Rough fish, golden shiner (Notemigonus crysoleucas) and sharpfin chub-
sucker (Erimyzon tenuis), appeared to be grazers with their respective rations
consisting primarily of filamentous algae and microcrustaceans (TABLE 26).
Among the three dominant benthic groups, chironomid larvae, Chaoborus
larvae, and Oligochaetes, chironomids appeared as the most utilized food source
by fishes„ Although chironomid larvae were represented in the stomachs of
most fish species, they comprised the dominant food source of small and inter-
mediate length sunfish (Lepomis spp.)0 Larger sunfish, particularly warmouth
(L, gulosus) and green sunfish (L. cyanellus), also appeared to prey on smaller
fish (Calhoun, 1966)„ Although no largemouth bass (Micropterus salmoides) were
collected with identifiable stomach contents, Snow (1971) and Bennett (1962)
51
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contend that fish and larger invertebrates constitute the major portion of
their diet.
The black bullhead (Ictalurus melas), yellow bullhead (I0 natalis) and
channel catfish (I0 punctatus) appeared as omnivorous feeders in which much
of their ration was also derived either directly or indirectly from the benthos.
Bullheads appeared to feed on both aquatic dipteran larvae and smaller fishes
with aquatic vegetation also utilized by black bullhead. Crayfish appeared to
constitute the bulk of the channel catfish ration with chrionomid larvae and
smaller fish also present.
Both non-game species collected, golden shiner (Notemigonous crysoleucas)
and sharpfin chubsucker (Erimyzon tenuis) appeared as discriminant grazers
upon non-benthic bood items. Filamentous algae appeared to constitute the
major component of the larger golden shiner ration with chironomid larvae
representing a minor portion. Sharpfin chubsucker appeared to graze primarily
upon microcrustaceans with chironomid larvae also constituting a secondary
importance.
In general it appears that the basis of the food chain within Country
Club Estates Lake is channeled through the benthic invertebrate community,
particulary in regard to game fish species, The benthic food source is of
immediate significance to the younger components of both the sunfish and
catfish populations (Calhoun, 1966)0 Smaller sunfish in turn furnish forage to
larger predaceous game species (i<>e., larger sunfish, largemouth bass, catfish).
Non-game species appear less dependent upond a benthic food source, utilizing
prey items suspended in the water column.
55
-------
REFERENCES
1. American Public Health Association., 1971. Standard Methods for the Exam-
ination of Water and Waste-water. 13th ed. American Public Health Asso-
ciation, Inc., New York. p. 562-576.
2. Arsenault, R. D. Pentachlorophenol and Contained Chlorinated Dibenzo-
dioxins in the Environment. Proc. Amer. Wood Preser. Assoc. Annual
Meeting, Atlanta, Ga0, April, 1976, pp. 1-25.
3. Bennett, G. W. Management of Artifial Lakes and Ponds„ Reinhold Pub-
lishing Corp., New York, 1972, 283 pp.
40 Bevenue, A., and H. Beckman. Pentachlorophenol: A Discussion of Its
Properties and Its Occurrence as a Residue in Human and Animal Tissues.
Residue Rev., 19: 83-134, 1967.
50 Blackman, G. E., M. H. Parker, and G. Carton, The Physiological Activity
of Substituted Phenols. II. Relations Between Physical Properties and
Physiological Activity. Arch,, Biochem. Biophys. 54, 55-71, 1955.
6. Brodie, B. B. and R. P. Maickel. Mode of Action of Drugs0 Proco Inst.
Intern. Parmacol. Meeting, 1962, pp. 299-324.
7. Buhler, D. R0, M. E. Rasmusson, and H. S. Nakave. Occurrence of Hexachlo-
rophene and Pentachlorophenol in Sewage and Water. Env. Sci. Tech,,, 7,
1973. pp 929-934.
8. Buser, H. R. Analysis of Polychlorinated dibenzo-p-dioxins and Dibenzo-
furans in Chlorinated Phenols by Mass Fragmentography. J. Chromatogr0
107, 1975. pp 295-310.
9. Buser, H. R. High-Resolution Gas Chromatography of Polychlorinated Dibenzo-
p-dioxins and Dibenzofurans. Anal0 Chem., 48, 1976,, pp 1553-1557.
10. Calhoun, A. Inland Fisheries Management. Calif. Dept. of Fish and Game.,
1966. pp 546.
11. Cardwell, R. D., D. G. Foreman, T. R. Payne, and D. J. Wilbur. Acute
Toxicity of Selected Toxicants to Six Species of Fish. U.S. EPA-600/3-76-
008, 1976. pp 1-117.
12. Cote, Ro P, A Literature Review of the Toxicity of Pentachlorophenol and
Pentachlorophenates. Environmental Protection Service Manuscript Report,
72-2, Halifax, N.S., 1972. pp 1-14.
56
-------
13. Crosby, D. G. Photodegradation of Pesticides in Water. Amer. Chem. Soc.,
Advances in Chemistry Ser., Ill, 1972. pp 173-188,,
14. Crosby, D. G., K0 W0 Moilanen, and A0 S0 Wong. Environmental Generation
and Degradation of Dibenzodioxins and Dibenzofurans. Env. Health Per-
spect., 5, 1973. pp. 259-266.
15. Crosby, D. G,, and A. So Wong. Photochemical Generation of Chlorinated
Dioxins. Chemosphere, 5, 1976. pp. 327-332.
16. Grummet, W. B. and R. H. Stehl. Determination of Chlorinated Dibenzo-
p-dioxins and Dibenzofurans in Various Materials. Env. Health Perspect.,
Issue #5, HEW, Pub. #NIH 74-218, 1973. pp. 15-25.
17. Cserjesi, A. J., and E. L. Johnson. Methylation of Pentachlorophenol by
Trichoderma virgatum. Canadian J. Microbiol., 18, 1972,, pp» 45-49.
17(a)Dougherty, R.C., and K. Pitrowska. Screening by Negative Chemical loni-
zation Mass Spectrometry for Environmental Contamination with Toxic
Residues. Proc. Natl. Acad. Sci. 73(6): 1777-1781, 1976.
18. Dyer, D. L. The Effect of pH on Solubilization of Weak Acids and Bases.
J. Colloid Sci., 14, 19590 pp. 640-645.
19. Goodnight, C. J. Toxicity of Solium Pentachlorophenate and Pentachloro-
phenol to Fish. Ind. Eng. Chem., 34, 1942. pp. 868.
20. Griffith, R. ()„, A. McKeown, and W. J. Shutt. Annual Tables of Constants
and Numerical Data, No. 22, Vol. 13. Herman, Paris, 1938.
21. Holmbert, B.,. Jensen, A. Larsson, K. Lewander, and M. Olsson. Metabolic
Effects of Pentachlorophenol (Penta) on the Eel Anguilla anguilla L.
Comp. Biochem. Physiol. 43(B), 1972. pp. 171-183.
22. Kirsch, E. J. and J. E. Etzel. Microbial Decomposition of PCP. Journal
WCPF, Vol. 45, No. 2, 1973. pp0 359-364.
23. Kobayashi, K., H. Akitake, and T. Tomiyama. Studies on the Metabolism
of Pentachlorophenate, A Herbicide, In Aquatic Organisms-II. Biochemical
Change of PCP in Sea Water by Detoxification Mechanisms of Tapes philip-
pinarum. Bull. Jap. Soc. Sci0 Fish, 36, 1970. pp. 96-102.
24. Kobayashi, K. and H. Akitake. Studies on the Metabolism of Chlorophenols
in Fish--III. Isolation and Identification of a Conjugated PCP excreted
by Goldfish. Bull. Jap. Soc0 Sci. Fish., 41, 1975. pp 321-327.
25. Kobayashi, K. S. Kimura, and H. Akitake. Studies on the Metabolism of
Chlorophenols in Fish--VII, Sulfate Conjugation of Phenol and PCP by
Fish Livers. Bull. Jap. Soc. Sci. Fish., 42, 1976,, pp. 171-177.
26. Mississippi Air and Water Pollution Control Commission, Court Hearing,
Jackson, Mississippi, January 14, 1975.
57
-------
27. Munakata, K. and M. Kuwahara. Photochemical Degradation Products of
Pentachlorophenol. Residue Rev., 25, 1969. pp. 13-23.
28. Pierce, R. H., Jr0, C. R. Brent, H. P. Williams, and S. G. Reeves. Penta-
chlorophenol distribution in a Fresh-water Ecosystem. Bull. Env. Contam.
Toxicol., 18(2): 251-257, 1977.
29. Plimmer, J. R. Technical Pentachlorophenol: Origin and Analysis of
Base-insoluble contaminants. Env. Health Perspect., U.S0 Dept. of Ag»,
1973. pp. 41-47.
30. Pruitt, G. W., B. J. Grantham, and R0 H. Pierce, Jr. Accumulation and
Elimination of Pentachlorophenol in the Bluegill, Lepomis macrochirus,
Trans. Amer. Fisho Soc0, 1977. In press.
31. Rappe, C., and C. A. Nilsson. An Artifact in the Gas Chromatographic
Determination of Impurities in Pentachlorophenol. J. Chromatogr0 67,
1972. pp. 247-2530
32. Robinson, R. A., and R0 G0 Bates. Dissociation of Some Substituted
Phenols in 50% Aqueous Methyl Alcohol. J. Res. Nat. Bur. Stand. A 70,
1966. pp. 553-556.
33. Rudling, L. Determination of PCP in Organic Tissues and Water. Water
Research, 4, 1970, pp. 533-537.
34. Sadtler Research Laboratories, Inc. The Sadtler Standard Spectra, UV-
112, Vol. 1, 1970.
35. Schlenk, H., and J. Gellerman. Esterification of Fatty Acids with Diazo-
methane on a Small Scale. Anal. Chem., 32, 1960. pp. 1412-1414.
36. Shafik, T. M. Determination of PCP and Hexachlorophene in Human Adipose
Tissue. Bull. Environ. Contam. Toxicol, 10, 1973. pp. 57-63.
37. Snow, H. Harvest and Feeding Habits of Largemouth in Murphy Flowage,
Wisconsin. Wise, Dept. Nat. Res. Tech. Bull. No. 50, 19710 pp. 25.
38. Stark, A. Analysis of Pentachlorophenol in Soil, Water, and Fish. J.
Agric. Food Chem., 17: 871-873. 1969.
39. Tiessens, G. J. Trichloro- and the Higher Chlorophenols and Their
Electrical Conductivity in Water. Rec. Trav. Chem., 48, 1929. pp. 1066-
1068.
40. U. S. Environmental Protection Agency. Manual of Analytical Methods for
the Analysis of Pesticide Residues in Human and Environmental Samples,
Chapter 5, U.S. EPA, Env. Toxicol. Div* , Research,, Triangle Park, North
Carolina, 1974.
41. Zitko, V., 0. Hutzinger, and P.M.K. Choi. Determination of Pentachloro-
phenol and Chlorobiphenyls in Biological Samples. Bull. Env. Contam.
Toxicol., 12, 1974. pp. 649-653.
58
-------
TABLE.A-l PHYSICAL PARAMETERS FROM WATER SAMPLING SITES
FROM FEBRUARY 1975 TO MAY 1976
Date Station
2-27-75 1
2
3
4
5
6
4-24-75 1
2
3
4
5
6
6-28-75 1
2
3
4
5
6
8-2-75 1
2
3
4
5
6
Temp
°C
15
16
15
16
16
16
19
19
22
22
22
22
23
23
30
30
30
30
NA
NA
28
29
29
29
Do00
pH mg/1
8,8
8,4
906
904
8.6
9.4
6.4
6.0
9o8
9,5
9.5
9.3
0,6
4.3
9,0
9.8
906
9o4
NA
NA
6,2 7,8
6.3 6.6
6.3 702
5.8 6.6
TOC
mg/1
12o6
14,2
12.4
NA&
14.0
17.6
6.3
6,2
7.9
7.9
7.9
704
NA
2009
17.5
17.4
NA
16o2
NA
NA
16,9
18.1
17.2
15.5
(continued)
59
-------
TABLE A-l. (CONTINUED)
Date
10-11-75
12-6-75
2-7-76
5-3-76
Station
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
Temp
°C
20
20
22
23
24
24
12
10
15
15
15
14
NA
11
12
11
11
18
NA
22
NA
23
22
pH
5.1
5o2
NA
NA
6,2
5.0
4.8
4o6
5,1
6.5
606
6,0
NA
6.4
6.5
6,5
6,3
6.2
NA
6,8
NA
6.7
6.6
D000
mg/1
8.0
6,0
4.0
8.0
9,0
8.8
5.9
6.6
10.2
10.4
10.6
8.2
NA
8.9
9.7
9.7
10.0
5,9
NA
8.0
NA
8.7
8.2
TOC
mg/1
24.9
24.5
18o5
17.2
19.6
17.7
24 .1
NA
16.6
16.7
16.9
20.0
NA
15.8
19.8
19.8
22.2
21.7
NA
15.6
NA
15.4
17.9
TMot analyzed.
60
-------
TABLE A-2 PHYSICAL PARAMETERS FROM WATER SAMPLING SITES
FROM AUGUST 1976 - APRIL 1977
Date
August, 1976
October, 1976
January, 1977
April, 1977
Station
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
(depth)
meters
0.3
0.3
2.0
0,3
5.0
0.3
2.0
Dry
0.3
108
0.3
4.0
Oo3
1.5
0.3
0.3
1.8
0.3
6.0
0.3
2.6
0.3
0.3
2,0
0.3
5.0
0.3
1.5
Temp
°C
27
26
26
26
20
27
25
N0A.
16
12
15
12
17
12
12
12
7
12
6
12
7
25
24
18
26
15
24
20
D.O.
mg/1
N.A.a
8.0
7.4
8.7
0.2
7.5
5.0
N.A.
10.4
10.2
10.6
2,7
10.5
10o6
N.A.
11.8
9.5
12.2
6.2
12.8
6.0
N.A.
6.5
5.0
6.7
0.3
6.6
3.0
pH
5.8
5,7
6.1
6oO
NoA.
6.6
6.5
6.5
6.8
6.5
6.5
6.5
6.5
6.5
6.6
6.6
Not analyzed.
61
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1 REPORTNO.
EPA-600/3-78-063
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Fate and Impact of Pentachlorophenol in a
Freshwater Ecosystem
5. REPORT DATE
July 1978 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Richard H. Pierce, Jr.
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Institute of Environmental Science
University of Southern Mississippi
Hattiesburg, MS 39401
10. PROGRAM ELEMENT NO.
1HE775
11. CONTRACT/GRANT NO.
R-803820
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory - ATHENS
Office of Research and Development '
U.S. Environmental Protection Agency
Athens, GA 30605
GA
13. TYPE OF REPORT AND PERIOD COVERED
Final 7/75-11/77
14. SPONSORING AGENCY CODE
EPA/600/01
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This investigation was undertaken to determine the fate of pentachloro-
phenol (PCP) that caused extensive fish kills in a freshwater lake in
December 1974 and again in December 1976. The kills resulted from the
accidental release of wood-treating wastes containing PCP in fuel oil.
Food chain relationships were investigated in the lake and the accumu-
lation and elimination of sublethal concentrations of dissolved PCP
was studied under laboratory conditions for the bluegill (Lepomis
macrpchirus). The highest concentrations of PCP in fish were observed
in the bile followed by liver, gills, and muscle.
Lake sediment and leaf litter contained high concentrations of PCP
throughout the two-year study. Studies of leaf litter from the contami-
nated water shed area showed it to be a source for chronic pollution
of the aquatic ecosystem. The major degradation products observed were
pentachloroanisole (PCP-OCH3) and the 2,3,5,6- and 2,3,4,5-tetrachloro-
phenol (TCP) isomers. These products were found to persist in sediment
and fish along with PCP. The methyl ethers (anisoles) of both TCP iso-
mers and the 2,3,4,6-TCP isomer were observed in some samples but the
small amounts were difficult to quantitate.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Phenol
Toxicology
Degradation
Water pollution
Chlorinated phenols
Bioaccumulation
Environmental fate
Aquatic degradation
Environmental per-
sistence
06F
06T
68D
13. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report}
Unclassified
21. NO. OF PAGES
74
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 22?0.'i 15-73}
62
MlEM PRINTING OFFICE I978-- 7-140/1395
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