AN ECOLOGICAL STUDY OF
HEXACHLOROBENZENE (HCB)
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EPA 560/6-76-009
AN ECOLOGICAL STUDY OF HEXACHLOROBENZENE (HCB)
ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF TOXIC SUBSTANCES
WASHINGTON, O.C. 20460
April 1976
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This report is available from:
National Technical Information Center
U.S. Department of Commerce
Springfield, Va. 22151
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EPA 560/6-76-009
AN ECOLOGICAL STUDY OF HEXACHLOROBENZENE (IICB)
by
John L. Laseter, Ph.D.
Clelmer K. Bartell, Ph.D.
Anthony L. Laska, Ph.D.
Doris G. Holmquist, Ph.D.
Donald B. Condie, B.S.
Jean W. Brown, Med. Tech. A.S.C.P.
Robyn L. Evans, B.S.
EPA Contract No. 68-01-2689
EPA Project Officer: William A. Coniglio
for
Environmental Protection Agency
Office of Toxic Substances
4th and M Streets, S.W.
Washington, D.C. 20460
April 1976
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ACKNOWLEDGMENTS
The authors wish to express their appreciation to William
Coniglio and Robert Carton, EPA Project Officers, for their guidance
and constructive criticism. We thank Dempsey Thomas for developing
algal cultures for this study. We also express appreciation to Douglas
Carlisle, Michael lovine, Bud Schuler, Harry Rees and Frank Stone for
their assistance in the construction of instrumentation. Technical
assistance was provided by several students at the University of New
Orleans. For their enthusiastic support, we thank Robert Albares,
James Boogaerts, Brian Bayer, Daniel Deane, Fred Gaupp, Susan Huffman
and James Long. For their patience in preparation of several drafts
of the manuscript we thank Diane Sloan and Carolyn Jas.
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This report has been reviewed by the authors,
EPA, and approved for publication. Approval
does not signify that the contents necessarily
reflect the views and policies of the Environ-
mental Protection Agency, nor does mention of
trade names or coniiiercial products constitute
endorsement of reconm endation for use.
1 - 14 -
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Page
TABLE OF CONTENTS
List of Tables Vi
List of Figures vii
I. Summary and Conclusions 1
II. Introduction 2
LII. Field Studies, Methodology 4
A. Overview
B. Area of Higher Concentration 7
IV. Laboratory Methods and Materials 9
A. Test Compound 9
B. Culture Techniques 10
C. Static and Flow-Through Assay Systems 11
0. Gas Chromatography 17
E. Mass Spectrometry 18
F. Corticosteroid Analysis 18
S. Fish Blood Hematocrit 18
H. Respirometry 19
I. Metabolic Fate 0 4 C) 19
J. Histology 20
K. Photochemistry 20
V. Results 21
A. Field Studies 21
1. Overview 21
2. Area of Higher Concentration 21
B. Acute Toxicity 25
1. Crayfish Injections . 25
2. Fundulus Injections . 25
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3. Crayfish LC 50 s .
4. Fish LC 50 s
C. Chronic Toxicity
1. Crayfish Tissue Morphology - Normal
2. Bass Tissue Morphology - Normal and
5. Fish Blood Hematocrit
6. Fish Corticosteroid Level
D. Accumulation and Clearance
1. Crayfish
2. Mollies
3. Bass
4. Algae
5. Bottom Sediment .
6. Effect of Food Chain
7. Crayfish Uptake of HCB
E. Respirometry
1. Crayfish
2. Mollies
F. Metabolic Fate
G. Photochemistry
VI. Discussion of Results . .
Literature Cited
and Pathological.
Pathological.
31
32
35
35
36
37
39
40
43
44
44
46
48
48
49
52
53
54
60
Page
• 26
• 26
26
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3. Retention and Distribution of HCB in Crayfish
4. Retention and Distribution of HCB in Bass .
in Fiel
d Environment
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LIST OF TABLES
Page
1 Field localities for overview collections 6
2 Concentrations of HCB in water and soil samples from sites
removed from Mississippi River transect 23
3 Mean concentrations of HCB in Mississippi River mosquitofish
( Gambusia affinis ) in comparison with levels measured in water
and soil 23
4 Mean concentrations of HCB in crayfish ( Procambarus ja.) from
ditches in comparison with levels measured in soil 23
5 HCB residues in water, mud and organism samples from area of
higher concentration 24
6 Concentrations of HCB (in pg/g) in crayfish bodies and tissues
of animals exposed to HCB in aqueous solution for 8 days . . . 31
7 Whole-body HCB residue concentrations in jig/Kg (ppb) in males
and female crayfish ( Procambarus clarki ) during uptake and
depuration
8 Whole-body HCB residue concentrations in largemouth bass
( Micropterus salmoides ) during uptake and depuration 42
9 Concentration and depuration of HCB by green alga, Oedogonium
cardiacum exposed to a flowing solution of 11.5 ppb (ug/L) HCB
in water
10 HCB concentrations in largemouth bass ( Micropterus salmoides )
feeding on contaminated sailfin mollies ( Poecilia latipinna ) . 44
11 HCB concentrations in sunfish ( Lepomis macrochirus ) feeding
on contaminated mollies ( Poecilia latipinna ) 45
12 Short-term exposure and depuration of crayfish introduced into
a contaminated environment (43.5 ppb HCB) 46
13 Effect of time of exposure to HCB on respiration of juvenile
crayfish (u 02/ mg/ hr) 48
14 Effect of time of exposure to HCB on respiration rate of
juvenile mollies (iiL 2/ mg/ hr) 50
15 Activity (cpm/g) of extracts of bass ( Micropterus salmoides )
fed C 14 -HCB 52
16 Minimum concentrations of HCB tested that resulted in an
observed response in organisms 59
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LIST OF FIGURES
No. Page
1 Localities sampled for presence of HCB in soil and water . . . . 5
2 Location of Geismar, Louisiana 8
3 Location of Geismar field sites (area of higher concentration) . 8
4 Mass spectrometry plot of HCB 9
5 Gas chromatographic traces of HCB and HCBD (chromatographic
conditions as described in Section IV. D) 10
6 Water treatment system 12
7 Modified proportional diluter 13
8 Major test tank 15
9 Model ecosystem 15
10 Distribution of HCB in soil along the Mississippi River,
Louisiana. Baton Rouge - Port Sulphur transect 22
11 Photomicrographs of bass tissue sections 29
12 HCB concentrations in organ samples from bass exposed to HCB
for 15 days 33
13 HCB concentrations in organ samples from bass exposed to HCB
for 10 days
14 Mean plasma cortisol levels (pg/lOOml) in the Gulf killifish
( Fundulus grandis ) exposed to HCB for ten days 36
15 Uptake and depuration of HCB in crayfish ( Procambarus clarki )
exposed to a mean concentration of 31.7 ppb (pg/ f l 38
16 Uptake and depuration of HCB in sailfin mollies ( Poecilia
latipinna ) exposed to 55 ppb (pg/ i) HCB 39
17 Uptake and depuration of HCB in sailfin mollies ( Poecilia
latipinna ) exposed to 7.9 and 62.2 ppb (pg/I) FICB 40
18 Uptake and depuration of HCB in bass ( Micropterus salmoides )
exposed to 2 ppb and 9.5 ppb (pg/I) 43
19 Levels of HCB in crayfish during depuration following 10 days’
exposure to a contaminated environment (74.9 ppb HCB) 47
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No. Page
20 HCB concentrations in crayfish exposed to a contaminated
environment (43.5 ppb) 47
21 The effect of time of exposure to HCB (1Q03 ppb, . ; 2437 ppb o)
on respiration rate of juvenile crayfish ( Procambarus clarki ) . 49
22 The effect of time of exposure to HCB (2783 ppb, • and 0;
1587 ppb, a ) on respiration rate in juvenile mollies
( Poecilia latipinna ) 51
23 Gas chromatographic separation of photoproducts resulting
from ultraviolet irradiation of HCB 53
Viii
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I. SUMMARY AND CONCLUSIONS
purpose of the study suninarized in this report was to determine
the distribution and toxic effects of hexachlorobenzene (HCB) in selected
aquatic systems and organisms Field collections of soil and water were
made in southeastern Louisianaç and were supplemented by samples of aquatic
organisms from sites where available. In laboratory experiments, the
principal animals used were the red swamp crayfish, Procambarus clarki , the
sailfin molly, Poecilia latipinna , and the largemouth black bass, Micropterus
salmoides . Both acute and chronic effects of HCB were studied.
Tests aimed at establishing LC 0 1 s for organisms were carried out in
flow-through experiments which utilized a modified proportional diluter. HCB,
at concentrations near its upper limit of solubility in water (20 ppb), did
not produce mortalities in animals exposed for 10 days. In other acute studies
the substance dissolved in oil, was injected directly into animals. No signi-
ficant acute ethal effects were seen following dosages of 125 g/g body weight
in fishes or crayfish.
Although HCB was not lethally toxic to juvenile bass, HCB concentration
reached a maximum of 88 pg/g (ppm) following 15 days’ exposure to 2 ppb. This
is a concentration factor in excess of 44,000X. Highest levels were found
in extracts prepared from the gut, in which some concentration factors exceeded
l00,000X. Kidney, gills and liver contained substantial proportions of HCB
as well. Light microscope observation of histological slides of tissues from
chronically exposed fish and crayfish revealed damage at the cellular and organ
level. Changes were observed in kidney, liver and gall bladder of bass exposed
to 25 ppb HCB. Hepatopancreas of crayfish was affected by 10 days’ exposure to
36 ppb HCB.
Analysis of blood samples taken from freshly-sacrificed fish revealed no
differences in hematocrit following chronic exposure to HCB. Serum cortisol
levels were observed as indicators of stress. HCB elevated cortisol level,
but the differences were not statistically significant (5%, F-test).
Oxygen consumption rates were not significantly altered by HCB exposure
within three hours after exposure. Chronic exposures produced complex response
patterns which were not resolved satisfactorily in the present experiments
with fish and crayfish.
A flow-through system was used to determine uptake of the compound by
algae ( Oedogonium cardiacum ) and sediment. Algae concentrated HCB to a level
more than 600 times that measured in aquarium water within seven days. Sediment
accumulated 40 times the quantity of HCB in experimental water in one day of
exposure. A food-chain study compared the relative accumulation effects on a
predator feeding upon HCB-contaminated food fish and a similar predator taking
up the compound both through its food and through contaminated water. Bass took
up more than 20 times the HCB from water than they did from food. In another
feeding experiment with Carbon 14 -labelled HCB, radioautography of thin-layer
chromatographic plates showed that recovered material from bass tissue extracts
had not been altered by metabolic processes in fish.
Laboratory experiments were compared with patterns of accumulation under
field conditions. HCB-free crayfish were caged, placed at a contaminated field
site and removed periodically for GC analysis, or for laboratory depuration
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and subsequent GC analysis. Crayfish exposed to an average level of 44 ppb
HCB had a concentration factor of l,l64X HCB in whole-body tissue. For
specimens left in the field site for 10 days, 40 to 60% of the maximum
attained level of HCB was lost after 25 days of depuration.
Since HCB has been found as a contaminant in the environment, it is
possible that it is degraded or altered by ambient UV light. The environment
was simulated in the laboratory and the resultant photoproducts formed following
short exposure to UV light were analyzed by GC. Irradiation of HCB resulted
in its gradual disappearance and a steady increase of lower molecular weight
products.
II. INTRODUCTION
The present study was initiated following the observation, in recent
years, of excessive levels of HCB in adipose tissue and milk of cattle being
raised in the vicinity of an industrialized region bordering the Mississippi
River between Baton Rouge and New Orleans, Louisiana. The field portion of
this study was undertaken to measure HCB levels in the environment in south-
eastern Louisiana. Laboratory experiments were aimed at observing acute and
chronic effects of the compound under controlled conditions.
Hexachlorobenzene, in its pure form, is a crystalline powder which has
been used as a seed dressing for prevention of fungus disease in wheat. It
is both a starting material and by-product of the chemical industry (Gilbertson
and Reynolds, 1972). The occurrence and effects of HCB have been reported in
many organisms, birds (Vos et al., 1971, Crornartie etal., 1975) sheep
(Avrahami and Stë Te, 1972), ratsTM line eta]., 1973), man (Cam and
Nigogosyan, 1963) and fishes (Holden, 1970; Johnson et al., 1974, Zitko, 1971).
Magnification in the natural food chain is indicatecfFy Gilbertson and Reynold’s
(1972) observation of HCB in the eggs of common terns, which had apparently
eaten contaminated fish. This compound has also been found in samples of
ocean water and its persistence in the environment has been acknowledged
(Seltzer, 1975).
In the present study, field work was divided into two distinct phases.
First, wide-ranging collections of water, soil and organisms were made to
establish levels and distribution of HCB in the southern central portion of the
state of Louisiana, extending eastward to Mississippi and following the Missis-
sippi River from Baton Rouge to Port Sulphur. Results from this work provided
an overview of environmental reality against which gross exposures could be
compared. The study of the extreme in environmental exposure was concentrated
in the imediate vicinity of the Vulcan Materials Company at Geismar, La. Here
a periodic monitoring of HCB concentrations in soil, water and selected aquatic
organisms at various trophic levels was conducted. During the course of field
work, localities in this area were contaminated at a fairly constant level and
afforded the opportunity for observation in an environment having otherwise
relatively natural conditions.
In an effort to define acute and chronic effects of HCB upon local fauna
more precisely, laboratory experiments were designed to expose aquatic biota
to the test compound. Histological preparations, behavioral observations and
gas chromatographic analysis were used to obtain data and gain a greater under-
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standing of the significance of the presence of HCB in the environment.
Tests of acute toxicity have been central to studies of contaminants
introduced into environmental systems. Tarzwell (1966, 1971) had discussed
the use of acute toxicity levels and of application factors setting safe
standards for levels of toxic substances in natural waters. It has been
obvious for a long time that the concept of application factors is more of
a convenient means of dealing with the complicated problems of water quality
than an accurate scientifically established criterion. Long term effects of
toxic substances are difficult to determine in actual practice. The impor-
tance of the problem of water pollution, however, is sufficiently great to
justify attempts to approximate a relationship between short term lethal
effects relatively simple to measure and long term effects which are far more
difficult to determine.
Because of the relative ease with which the experiments can be conducted,
there have been numerous experiments designed to determine the level of a
lethal factor that can be tolerated by a given percentage of animals for a
given period of time. Warren (1971) has discussed this subject and also has
reviewed the use of application factors in conjunction with tolerance studies.
The rationale for incorporating tolerance studies into the present project
is that because of the physiological studies included in the project there is
a preliminary basis for suggesting application factors for HCB. The physiolo-
gical and morphological indications of long term effects of sublethal concen-
trations of HCBa-re difficult to establish and such studies are relatively
rare. As Warren points out it is extremely important to establish application
factors which can be applied with some scientific basis.
Three comonly-used methods of introducing a potential toxicant into an
organism are: (1) direct injection, (2) oral feeding, and (3) contaminating
its air or water environment. In some experiments during the present study,
animals were injected with the test compound and in others they were subjected
to a range of concentrations of HCB in aquatic systems. Condition of animals
was observed regularly, and any abnormal behavior or appearance was noted for
inclusion as possible pathological effects of HCB.
Mortalities give the most positive, visible evidence that a substance
is toxic to organisms. In contaminated natural systems, however, concentrations
of toxic substances are normally below lethal levels. The variable conditions
in nature often preclude positive determination of chronic effects of a given
substance upon resident flora and fauna. Controlled laboratory conditions are
helpful, therefore, in assessing various responses of an organism to low-level
exposures. Results of such work can provide a basis for field observations
and augment acute tests in establishing application factors.
Chronic tests and observations reported here dealt with a variety of
physiological and morphological parameters. Uncontaminated organisms were
brought into the laboratory and adapted to in vitro conditions for periods of
from two days to as much as two months before being used in tests. Most animals
were subjected to HCB in the flow—through system, the diluter, which is discussed
in Section IV. C. Following exposure for specified periods, whole organisms, or
excised organs served as material for GC analysis of uptake and differential
distribution in chronic studies. Light microscopic examination of histological
slides prepared from excised organs provided further information on chronic
effects of HCB. Samples of blood taken from fish were used in determinations
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of hematocrit and cortisol levels. Cortisol levels were determined by
competitive protein binding radioassay (Murphy, 1967, 1971) which has been
successfully employed in teleost plasma (Hargreaves and Porthë -Nibelle,
1974). Alterations in these two levels would be considered an indication
of stress upon the animals. Significant changes in these parameters might
provide a further means of monitoring conditions in a given area of potential
contamination.
Rate of oxygen utilization by an organism may vary in response to stress.
In order to study possible effects of HCB in ambient water of fish and crayfish,
respirometry experiments were carried out in the laboratory.
Accumulation and clearance of HCB in whole-body samples of fish and
crayfish and samples of algae and mud are best accomplished in a flow-through
system. The modified proportional diluter was used for these exposures and
depurations. Concentrations of HCB in diluter tanks were comparable to levels
found in contaminated natural systems, except for some experiments, in which
higher concentrations were used. Samples of fish or crayfish being tested
were removed from all tanks according to schedules outlined in discussions of
the various experiments in the text which follows. Handling of specimens and
preparation of extracts for GC analysis are described in the Gas Chromatography
unit (IV.D.) in the Laboratory Methodology section of this report.
A series of field experiments with crayfish provided comparative information
to results from laboratory studies. Uptake and clearance of HCB was determined
in animals, initially free of the compound, which were placed in an HCB
contaminated field locality.
Two types of experiments were designed to observe possible breakdown
products of HCB. In one, a metabolic fate study, extracts of bass tissue were
analyzed following digestion of a HC’ 4 B-labelled food fish by the bass. Other
experiments subjected HCB to UV light in order to determine the resulting
photoproducts.
III. FIELD STUDIES, METHODOLOGY
A. Overview
To develop a transect of contamination along the Mississippi River,
collections of samples were made at five-mile intervals between Baton Rouge
and New Orleans and at greater intervals from New Orleans south to Port Sulphur
(Fig. 1). These samples were collected between March and May, 1975. At each
site approximately 1 liter of water was taken from about 15 cm beneath the
surface of the Mississippi River near the river’s edge. A specimen of levee
soil was also collected from the river’s edge. These samples were taken from
beneath the water surface whenever possible. Mud samples were taken from the
bottom of ditches running parallel to the levee on the inland side. Fish and
aquatic invertebrates were collected from these ditches whenever they were
present. All animal specimens were wrapped in aluminum foil and frozen
immediately on dry ice for later analysis. The localities for these field
sites are shown in Table 1
In addition to the samples taken along the Mississippi River, collections
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were maae at several inland sites. these samples were used to provide
baseline information on geographic distribution of the compounds. These
localities are also shown in Table 1, designated by numbers in italics.
Figure 1. Localities sampled for presence of HCB in soil and water.
Sites
A-I
B 1 B 2 E 1 , U—Z
i, ii, iii, V
iv
vi
Vii
Description
East bank of Mississippi River
West bank of Mississippi River
Inland, east of Mississippi River
Pass Manchac, inland tidal lake
Spiliway of Mississippi River, connected to Lake
Pontchartrain
Bay
Legend to Figure 1
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Table 1. Field localities for overview collections.
Type of Sample Type of
Location Description Water* Soil* Blota* Organi iii
A Hwy. 90, North of Baton Rouge (E) X X X F
B Intersect, River Rd., La. Hwy 327 CE) X X
B 1 Addis at La. Hwy. 990 & La. 998 (W) X X X F
B2 Plaquemine at La. Hwy. 988 & La. 1148 (w) X X X C, F
C 5 Mi. South of B (E) X X X C, S
D Sunshine CE) X X X C, F
E Carville CE) X X X C, F
E 1 La. 405, 4 mi. South of White Castle (W) X x x C, F
F Ashland Plantation (E) X X X F & C
G Darrow(E) X x x C
H Intersect, La. 44 & La. 942 CE) X X X C, F
I Romeville CE) X X X F & C
J Convent (E) X X X C
K Lutcher (E) X X X C
L Garyville CE) X X X F
M Reserve (E) X X X Sh
N Laplace CE) X X X C, F, S
0 South of Bonnet Carré Spiliway (E) X x
P Destrehan (E) X X X C, F
Q St. Rose (E) X X X C, Sh
R River Ridge (E) X x x C
S New Orleans, River Rd. at Causeway CE) X X X F, Sh, S
I New Orleans, Audubon Park (E) X X
U Lower Algiers (W) X X
V Belle Chase (W) X X X C, F
W Myrtle Grove (W) X X X C, F
X Mile Marker 42 (W) X X X C
Y .8 Mi. South of X (W) X X
Z Port Sulphur (W) X x x C
i Walker(I) X x x c
ii Hammond (I) X X X F, Sh, Cl
iii Covington (I) X x x c
iv Pass Manchac (1) X
v Sorrento (I) X x x C
vi Spiliway CS) X x x c, F
vii Lake Grand Ecaille (B) X x
E: East bank of Mississippi River C: Crayfish
W: WpcL bank of Mississippi River Cl: Clam
I: Inldnd, east of Mississippi River F: Fish
1: Tidal lake S: Snail
S: Spil Iway Sh: Shrimp
B: [ y
* : GC analyses performed on soil and water samples from all locations.
**: tiC analyses performed on biota from selected locations (Ref. Tables 3 & 4).
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B. Area of Higher Concentration
The site of known contamination was near the Mississippi River, between
New Orleans and Baton Rouge, at Geismar, La. (Fig. 2). The locations of
samples taken on the property of the Vulcan Materials Company at Geismar are
presented in Figure 3. These samples were collected during September and
December, 1974, and May, August and October, 1975. Water and mud samples were
taken regularly, and aquatic organisms were collected whenever available. These
specimens were preserved by freezing for analysis in the laboratory.
The first site, an impoundment referred to as Recreation Pond, covers
approximately one-fourth of an acre and is located in the north sector of the
property (Fig. 3). The man-made pond is 20 feet deep with steep edges
characterized by scattered patches of rooted and floating vascular plants.
The ecosystem here more closely resembled a natural one than the other sites
sampled at the contaminated locale. Zooplankton were abundant in the water column.
Oedogonium sp., a conunon green alga provided food and substrate for micro-
crustacea. Two common aquatic plants support and protect higher organisms.
These are Chara and Najas , in whose mats we collected snails, crayfish,
dragonfly larvae and small fishes. These animals feed on the plants and
their epiphytic and epizoic components. The small fishes, breeding populations
of mosquitofish ( Gambusia affinis ) and sunfish ( Lepomis macrochirus) , are food
for largemouth black bass ( Micropterus salmoides ) which were stocked as sport
fish. At the Recreation Pond, samples of soil, water, aquatic plants, inverte-
brates and fishes were collected and analyzed to determine HCB content in
abiotic and biotic components of this environment.
The second sampling site was adjacent to the hex waste disposal area where
waste containing HCB and HCBD are now buried. This sampling site was a newly
dug pond measuring approximately 50 by 100 feet, 20 feet in depth. Ground water
keeps this pond approximately 2/3 full of water. Eventually this pond is
destined for waste disposal and burial. During the study, little vegetation
was found in the water, but populations of mosquitofish were common there and
were sampled regularly along with soil and water. Clean crayfish were brought
in, caged, and left in this pond in a field study of HCB uptake.
The third sampling site was located along a small stream carrying runoff
water from the field adjacent to the plant to a small stream in the South ditch.
Rainfall determined water level in the south effluent ditch, and the only fishes
living there were the mosquitofish and a few mollies ( Poecilia latipinna) .
Crayfish ( Procambarus !p.) were taken occasionally, along with regular samples
of water, mud and fish.
Organisms were collected on several occasions, and their HCB content
determined and tabulated. Concentration factors were computed as a function of
HCB level in water at the time they were taken. When organisms from several
dates of collection were combined, their concentration factor was computed by
taking a weighted average of their HCB content and dividing it by the mean
concentration in water samples taken on the same collection dates.
Plankton samples resulted from a tow of a 21 cm plankton net across the
Recreation Pond, just below the surface. The material was refrigerated at 3°C
in pond water after collection. All organisms settled to the bottom of the
holding jar, and were pipetted off. They were then blotted on Whatman #1
filter paper, and subsequently treated with the same methodology as described
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for algae samples in the Methodology Section (IV.D.) of this report.
Concentration is expressed in terms of wet weight of solid material.
Figure 2.
Louisiana.
Location of Geismar Field Sites (area of higher concentration).
Location of Geismar,
Figure 3.
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IV. LABORATORY METHODS AND MATERIALS
A. Test Compound
In order to guarantee the composition of test compounds following their
absorption into organisms it is necessary to analyze their purity before
application in various test systems. Zone refined hexachlorobenzene (HCB)
was obtained from B. Pauric, Philadelphia, Pa. 19120. A purity check via
gas chromatography was made using a standard of 0.1 ppm of the pesticideTh
benzene solvent. The compounds found to be “pure” were used throughout all
experiments.
Mass spectra were obtained for the compound using a duPont 21-491 double
focusing 900 magnetic sector mass spectrometer (MS) equipped with a Bell and
Howell Datagraph 5-134 galvanometer driver recording oscillograph. Hexachioro-
benzene was introduced directly into the mass spectrometer via the direct
injection probe. All spectra were obtained at 70 eV at a source temperature
of 200°C. Computer plots of the spectra were obtained by use of a PDP-12
LDP computer and a D1100 Versatec electrostatic printer plotter. A mass
spectrometry plot of HCB starting material is given in Figure 4. Figure 5
represents gas chromatographic traces of the two compounds, HCB and HCBD.
Figure 4. Mass spectrometry plot of HCB.
C 6 CIe
264
20 40 60 60
m/e
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Figure 5.
Gas Chromatographic traces of HCB and HCBD (chromatographic
conditions as described in Section IV.D.).
TIME ( NUTES)
B. Culture Techniques
Due to seasonality of some forms or habitat preferences of others,
experimental organisms used in laboratory studies were acquired from several
sources. In a given experiment, however, organisms were all from the same
source.
The original stock of filamentous green algae, Oedogonium cardiacum
(IU #39), was received from the Indiana University Culture Collection at
Bloomington, Indiana. Subsequent cultures were maintained under controlled
conditions in aerated flasks containing Bold’s Basal Medium in the algae
culture room at the contractor’s institution.
Crayfish ( Procambarus clarki ) were received as available from commercial
facilities at The Crayfish Farm, Sorrento, Louisiana. They were maintained
in fiberglass tanks filled with enough water to keep gill regions wet. Cray-
fish were kept at room temperature and fed HCB-free chicken meat and small
fish.
Small estuarine fishes, including sailfin mollies ( Poecilia latipinna )
and sheepshead minnows ( Cyprinodon variegatus ) were collected by seines and
dipnets as needed for experiments and food. These were taken in small canals
and ditches at Irish Bayou, inland from the southeastern shore of Lake
Pontchartrain, Orleans Parish, Louisiana. Grass shrimp (Palaemonetes sp.)
and small crayfish ( Procambarus p.) were also found at this locality. These
organisms were maintained in filtered, aerated aquaria at room temperature.
Salt levels were held at 4 to 5 ppt with Rila Marine Mix (Rila Products,
Teaneck, N.J.) artificial sea salts.
STANDARD
HCBD
KCB
0 10 20
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Largemouth bass ( Micropterus salmoides ) were provided by the Louisiana
Wildlife and Fisheries Commission through their hatchery at Alexandria,
Louisiana. The fish were held in 300 - liter tanks in filtered, aerated,
room-temperature water at a level of .8 to 1 ppt salinity. Initially,
bass were fed a commercial fish food (Purina Trout Chow) until it was
found to contain HCB residues at levels up to 80 ppb. Small fish from
Irish Bayou collections served as bass food during subsequent work.
Experimental animals and the water from which they were taken were
analyzed for HCB content prior to acceptance for routine use. All fish and
crayfish were maintained in the same quality water, with salinity adjustments
as needed. Water for stock holding tanks and experiments was prepared in the
following manner. Chemical residues in New Orleans tap water, including
chlorine, were reduced by passing water through an activated carbon bed and
deionizer tanks. Deionized water was aged approximately 24 hours in holding
tanks, diagranmed in Figure 6. Ionic balance was restored and standardized
with the addition of Rila Marine Mix to a level of 1 ppt. An automatic salt
mix dosing apparatus assured constant salinity level. This device also
administered sodium bicarbonate which maintained the pH between 6.65 and 7.9.
C. Static and Flow-Through Assay Systems
Aqueous experiments were of two basic types, static and flow-through.
Static tests with mollies ( Poecilia latipinna ) were carried out in five-
gallon glass jugs filled approximately half full with ioe of prepared water
at 1 ppt salinity. The test compound, dissolved in nanograde acetone was
pipetted into the jugs concurrently with pouring water to effect mixing.
Test fish were added and air above the water’s surface was saturated with
flowing oxygen before the covers were screwed shut. In this type of
experiment, water was left unchanged but oxygen was added periodically during
the test. Later static tests involved replacement of water and toxicant
no more frequently than once daily.
Static tests with crayfish ( Procanibarus clarki ) involved placing
the animals in individual finger bowls. Water and toxicant in acetone carrier
were replaced daily. The animals were held in environmental chambers with
light and temperature controls for the duration of experiments.
The flow-through aqueous system more closely simulated the natural
environment because water with a predetermined and constant load of test
compound and acetone carrier was flowing at regular intervals into tanks
containing the organisms. This system received water from the tank source
discussed in Methodology section IV.B. and functioned as a modified pro-
portional diluter. The design for this apparatus was based upon developments
by DeFoe (1975) and preceding workers (Benoit and Puglisi, 1973, and Mount and
Brungs, 1967). In the modified proportional diluter (Fig. 7), water from a
single source fills a series of seven glass chambers in stepwise fashion.
Once full, a self-priming siphon in the final chamber initiates a flow of
water in the venturi vacuum system. The partial vacuum thus created empties
a pre-set, constant volume of water from each of the filled chambers through
a siphon whose action is started by the partial vacuum.
11
-------
Figure 6. Water treatment system.
-I
-------
Figure 7. Modified proportional diluter.*
water metering
cells
teflon toxicant
tube from injector
venturi vacuum
f low-
splitting
* one of six similar units schematically represented.
to waste
tc
13
-------
The flowing water from each chamber fills a flask (mixing chamber)
mounted upon a lever extending from an injector apparatus developed by
George J. Frazer . The flasks are suspended by spring tension. Weight
of the water filling the flask depresses the lever, actuating an advance
rachet whose pressure hub bears upon the plunger of a 50 ml syringe. A
known volume of test compound and acetone carrier is injected into the flask
(mixing chamber) through an elongate teflon needle. Once filled, the mixing
chambers each empty automatically into flow-splitting chambers through self-
priming siphons. Other siphons within each flow-splitting chamber deliver
the water to respective test tanks through glass tubing.
Each of the six major test tanks (Fig. 8) have a filled capacity of 70e,
but were usually filled to a level of 30 as controlled by an adjustable over-
flow standpipe. The smaller tanks (Fig. 9) are model ecosystems in which water
enters a larger section containing an inoculated algae culture. Next, water
flows into the smaller space, containing mollies, through a gap at the base
of the partition. Eventually water exits at the discharge tube which maintains
a volume of 30 in the tanks. Flow rate was fixed such that each experimental
tank received 60 to l20 of water daily.
The protocol for most tests provided for two duplicate test tanks of each
of two compound concentrations in addition to a water control and an acetone
carrier control. The number of organisms per tank was usually determinedby
the number and type of samples needed for analysis. Availability of animals
and their tolerance to conditions were other factors contributing to this figure.
HCB levels in tanks were checked by analysis of water samples
siphoned from beneath the surface. In certain experiments, organisms were
removed according to a fixed schedule during both phases, uptake and depuration.
Each phase usually lasted 10 or 15 days for each test. In other experiments,
all animals were sacrificed at the same time, 10 days after initiation of
exposure. Variations in routine will be discussed in appropriate sections of
this report. Temperature, pH, and dissolved oxygen content of tank water were
monitored regularly during flow-through experiments. These ranged as follows:
temperature, 22.2 to 23.9°C; pH, 6.5 to 7.9; and oxygen, 7.6 to 8.5 ppm.
The greater portion of acute experiments utilized the flow-through system,
with HCB in the test animals’ aquatic medium. Some injection experiments,
however, were carried out, with HCB dissolved in peanut oil. A dosage of .01
cc/u body weight was injected directly into the hemocoel of each animal, at the
base of its second walking leg on the left side. Several concentrations of HCB
were utilized.
In an attempt to obtain a more uniform distribution of injected material
throughout crayfish bodies, a series of emulsions was prepared with the HCB-peanut
oil mixture. Lecithin was added as an emulsifier. In the first of the two
emulsion tests reported, the final solution was made up of 40% spring water and
40% 2X saline, by volume. The second solution contained 40% spring water and 40%
2X sucrose, by volume. Prior to injection, each solution had been sonicated while
*: 4528 Pitt St., Duluth, Minn. 55804
14
-------
Figure 8. Major test tank.
Figure 9. Model ecosystem.
15
-------
chilled in an ice bath.
Fish were also used in injection experiments. Injections of HCB
dissolved in peanut oil were administered intraperitoneally to a series
of 10 Gulf killifish ( Fundulus grandis) . The same volume of oil, .01 cc/gram
body weight, was injected into 10 control fish. Fish were held in HCB-free
water in diluter tanks for four weeks following injection.
The flow-through conditions of the diluter were useful for two other types
of experiments; specifically, those in which algae and sediment were tested.
To test uptake of HCB by a common filamentous green alga, a culture of
Oedo onium cardiacum (I.IJ. #39) was prepared. Original stock was grown in
41 flasks in 31 of 1:1 3N BBM: H 2 0 solution. Aeration was not provided.
Illumination in the culture room was provided by Sylvania lifeline fluorescent
bulbs (output 3150 lumens) operating on a 12:12 light: dark photoperiod.
Approximately 40 days after inoculation of the stock flask, the algae was
divided into four relatively equal portions, three of which were placed into
open glass jars and submerged in three tanks (model ecosystems) of the diluter.
The fourth was used in an HCBD experiment.
Protocol for diluter tanks consisted of acetone and water controls and
11.5 ppb HCB solution. Illumination to the tanks was provided by three Sylvania
lifeline fluorescent bulbs (40 watt; output 3150 lumens) placed at right angles
to the tanks’ long axes: one lamp located centrally 20 cm above water surface,
and two lamps 10 cm behind tanks, +8 cm and -16 cm from water surface. Trace
elements present in 1 ppt Rila marine mix-reconstituted deionized water were
complemented by metabolic products of four mollies in a separate portion of
each tank.
Samples of the algae were collected following one day of exposure and
subsequently on every other day through a period of two weeks. Samples were
removed from each tank by pipet and placed in a cone of filter paper to drain
off excess water. Damp clumps of algae were stored in glass vials and refrig-
erated at approximately 3°C. Preparation of samples for GC analysis is discussed
in Section III.D. of this report.
The sediment experiment was designed to measure uptake of HCB by soil in
the form of sediment in the bottom of a test container. For both test and
control flasks 200 g samples of dry soil collected at a farm in Talisheek,
Louisiana, and determined by GC to be free of HCB, were screened through 1.6
niii mesh aluminum screening and poured into the bottom of 1000 ml aspirator flasks.
At the flow rate of 3 1/hr. water originating in the diluter passed through a
glass tube into the bottom of the stoppered flasks. Circulated water left
through a waste tube attached to the aspirator arm at the neck of the flask.
Samples of 40 g (wet wt) of mud were removed from the flasks periodically and
stored in glass jars at approximately 3°C until they were extracted for analysis
on GC.
Laboratory experiments with crayfish were supplemented with a series of
field experiments. Animals initially free of HCB were placed in cages and
set on the bottom of a pond adjacent to the hex waste disposal area at the
highly elevated field site discussed in Section III.B. HCB and HCBD content of
the water was monitored. Periodically, crayfish were removed from the cages
16
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and either frozen for later analysis of uptake or returned to the laboratory
and maintained in clean water in environmental chambers. Samples of crayfish
were removed at intervals from the environmental chambers, and extracts made
from them provided depuration data for animals exposed to both compounds in
an environment further complicated by the natural products of the industrial
wastes.
D. Gas Chromatography
Preparation methods for samples prior to gas chromatographic (GC) analysis
depended upon the substance being extracted. In preparation of water samples,
aliquots of 350 ml were shaken with 20 ml of benzene for 3 hr on an Eberback
reciprocating action shaker. Following passage through a separatory funnel an
aliquot from the benzene layer was ready for injection into the GC. In preparing
mud, aliquots of approximately 20 g of mud were shaken with 20 ml of acetone for
20 minutes, after which 20 ml of benzene was added. Following 24 hr of shaking,
the benzene-acetone extract was injected into the GC.
Samples of algae were scraped from walls of the tanks and blotted on filter
paper. Each sample was weighed, sonicated in acetone in an ice bath to disrupt
cell walls, and subjected to the same procedure used for animal tissue. Concen-
trations of HCB were expressed in terms of wet weight of the algae.
Samples of animal tissue were weighed and homogenized with anhydrous sodium
sulfate and acetone. The liquid was filtered into a separatory funnel and the
residue homogenized twice with acetone which was then added with filtration to
the separatory funnel. After adding sodium chloride to the combined acetone
extracts in a separatory funnel, the acetone-sodium chloride mixture was extracted
three times with hexane and the hexane evaporated to near dryness on a rotary
evaporator. This residue was dissolved in hexane and placed on a Florisil column
washed previously with 50 ml of elution solvent (95% hexane, 5% ether). Following
elution with 100 ml of elution solvent the eluent was evaporated on a rotary
evaporator. The residue was dissolved in 10 ml of benzene and an aliquot was
prepared for injection into the GC.
All extracts of water, soil, algae and tissue samples were analyzed by a
Hewlett Packard 5719A gas chromatograph (GC) equipped with an electron capture
detector utilizing ° 3 N1 foil. Extracts were introduced by a 7671A automatic
sampler. This system was attached to a Hewlett Packard 3352B Laboratory Data
System. Separation was accomplished with a 91.44 cm X 4 mm I.D. 10% OV—l column
maintained at either 150° or 165°C depending upon the integration method used.
Argon-methane 95:5 was employed as the carrier gas at a flow rate of 35 mi/mm.
The injection port temperature was held at 250°C and the detector was set at
300°C.
Quantification was accomplished using external standards of hexachlorobenzene
at concentrations of 1 ppm and 0.1 ppm in a benzene solvent. Concentrations of
HCB in water were computed in terms of pgf e of sample, and expressed in parts
per billion (ppb). Other samples reported in pg/g are expressed in parts per
million (ppm).
17
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E. Mass Spectrornetry
A double focusing duPont 21-491 mass spectrometer was employed which
was attached to a Hewlett-Packard 5750 gas chromatograph and coupled to a
PDP-12-LDP computer. Separations were carried out isothermally on a 9 m X
4mm ID glass column packed wtth 10% OV-l stationary phase on acid washed
chromosorb. Transfer lines were maintained at 200°C. All spectra were
obtained at 70 eV at a source temperature of 200°C.
F. Corticosteroid Analysis
The fish were maintained on a 12:12 (light:dark) photoperiod (light on at
0730) for the entire duration of exposure to HCB. On the final day, blood was
collected between 2 and 3 hours after “dawn.” Blood was taken from the sinus
venosus into heparinized hematocrit tubes which were then sealed with clay and
centrifuged. The tubes were cut and the plasma separated and stored in a freezer
until used.
Plasma corticosteroid concentration was determined using a modification of
the competitive protein-binding radioassay technique described by Murphy (1971)
which has been used successfully in teleost plasma (Hargreaves and Porth -Nibel1e,
1974, Meier and Srivastava, 1975). A 10 p1 aliquot of plasma was expelled into
a centrifuge tube containing 1.0 ml absolute ethanol. The ethanol precipitates
plasma proteins which might interfere with the assay (including any endogenous
corticosteriod binding globulin) and also extracts the cortisol. The tubes were
centrifuged and a 0.5 ml aliquot of the supernatant ethanol containing the
cortisol was transfered to a reaction vessel and evaporated to dryness under
N 2 . Each sample was then incubated for 5 minutes in a water bath at 45°C with
1.0 ml of corticosteroid binding solution. Each 100 ml of this solution
contained 0.5 pCi of l,2H 3 -cortisol (obtained from New England Nuclear Carp)
and 5 ml of pooled male horse serum (obtained from the ISU Veterinary School)
with distilled water to volume. Horse serum was used as a source of cortico-
steroid binding globulin (CBG) since Ficher etal. (1973) have found that it
gives greater specificity for cortisol.
After inclubation, the samples were transfered to an ice bath for 30 mm.
and then 40 mg of Florisil was added to absorb the unbound cortisol. A 0.5
ml aliquot of each sample was counted and compared to a standard curve obtained
by measuring known amounts of cortisol.
G. Fish Blood Hematocrit
The animals used in these studies were exposed to various concentrations
of toxicant. They were bled on the tenth day of exposure, first being stilled
by immersion in ice cold distilled water. Blood was taken from the sinus
venosus into heparinized hematocrit tubes which were then sealed with clay and
centrifuged. Packed cell volumes were read using a Critocap® micro-
hematocrit tube reader.
18
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H. Respirometry
The effect of exposure of juvenile crayfish and mollies to HCB was
measured using a Gilson Respirometer (Unbreit, etal., 1972). Each set
of tests using juvenile crayfish (average weight =10 mg) employed animals
from one hatching of one female. Juvenile mollies (average weight = .3 g)
were collected from uncontaminated environments and used immediately after
collection.
Five crayfish were placed in each respirometer vessel in 3 ml of
water. In the experiments using mollies a single fish was placed in each
vessel in 7 ml of water.
Animals were placed in the respirometer, and following 15 minutes
of acclimation, oxygen consumption was monitored. In crayfish studies,
respiration rates were determined every 15 minutes for two consecutive
1 hour periods. The system was flushed with air after the first hour.
The rates of respiration of mollies were determined every 10 minutes
over two thirty-minute periods. The system was flushed with air following
the first thirty-minute period.
Animals were placed in covered 8-inch finger bowls in either control
or experimental conditions during exposure periods. The culture solution
was changed dialy. Mollies were kept in deionized water reconstituted to
2.5 ppt with artificial sea salt. Crayfish were kept in deionized water
reconstituted to 1 ppt. The experimental groups were exposed to water that
had been stirred with appropriate amounts of HCB for at least 24 hours before
the animals were placed in the solutions.
The solution in each respiration vessel was the same as that to which
the individual animal or group of animals had been exposed. Therefore,
respiration rates were determined during exposure. Respiration rates for
HCB were determined immediately following exposure and after several longer
intervals of exposure. The concentrations are given in the “Results” section
(V.E.). Oxygen consumption rates were expressed in terms of wet weight of the
animal s.
I. Metabolic Fate ( 14 C )
Thin layer chromatography using Kodak Chromogram silica gel sheets
was used to determine if any metabolites of HCB had formed. The HCB used
was carbon-l4 labelled (New England Nuclear Corp.) and followed by means of
autoradiography using Kodak no—screen X—ray film (Estar base).
The HC 14 B was incorporated into the bass by means of a freshlX -killed
molly whose peritoneal cavity had been injected with 0.1 ml of HCl B in a
peanut oil carrier. This amount of HC 14 B contained 1.8 x 102 i.iCi. Injected
fish were readily consumed by the bass. The experimental animals were allowed
either 24 or 48 hours to digest the HC 14 B—fed bass were then killed and
dissected. Five separate body samples were examined for evidence of metabolic
products: (1) feces, (2) stomach and intestines, (3) liver, (4) kidney,
19
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and (5) remaining body. These samples were extracted separately and the
extracts applied to the thin layer autoradiography system described above.
J. Histology
Fingerling largemouth bass and crayfish for histological examination
were selected at random from the inhabitants of each tank of the modified
proportional diluter system to represent water control, acetone control,
and each nominal level of HCB utilized for GC analysis. Animals were
sacrificed on the day of termination of toxicant accumulation. Brain, green
gland, hepatopancreas, one gill anda sample of abdominal muscle were excised
from each crayfish.
Largemouth bass were stilled by chilling, weighed, and total and standard
lengths measured. The liver was excised and weighed as another measure of
size and nutritional condition and fixed. The right kidney, the first right
gill arch, approximately 5 mm of epaxial muscle caudal to the right operculum,
and a segment of intestine immediately caudal to the stomach were excised and
fixed. Organs were examined under dtssecting microscope magnifications and
any grossly damaged areas noted. In one experiment, each animal utilized
for histological study was also subjected to GC analysis.
Tissue samples were fixed in neutral buffered formalin, Bouin’s or
Zenker—acetic, appropriately washed, dehydrated, cleared in toluene, and
infiltrated and embedded in 56°C - 58°C paraffin. Tissue blocks were
serially sectioned and mounted. Slides representing step sections were
stained in Harris’ hematoxylin and eosin or Lillie’s modification of Weigert’s
iron hematoxylin and eosin. All mounted sections were kept to permit study
of serial sections of any areas found on microscopic examination to be of
specific interest.
K. Photochemistry
Solutions of HCB in both hexane and benzene (1 jig/b ml) were irradiated
at varying time intervals. HCB was irradiated for 30 minutes, 65 minutes
and 120 minutes. Irradiations at 253.7 nm were conducted in serum capped
quartz test tubes employing a Rayonet RPR-lOO Chamber Reactor equipped with
16 8-watt lamps and a “merry-go-round” apparatus (The Southern New England
Ultraviolet Co., Middletown, Conn.) All samples were degassed by purging
with nitrogen for a period of 10—15 minutes prior to irradiation.
20
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V. RESULTS
A. Field Studies
Field work consisted of two distinct phases. The first,a regional overview,
dealt with HCB residue determinations in water, mud and aquatic organism samples
collected along a Mississippi River transect from Baton Rouge to Port Sulphur,
and other parts of southeastern Louisiana. The second phase, at an area of
higher concentration, was located on property of the Vulcan Materials Company in
Geismar, Louisiana. Specific localities and protocol are discussed in the
Field Methodology section of this report.
1. Overview
Figure 1 shows localities in the immediate vicinity of the Mississippi
River sampled for HCB residues in collections of water, mud, crayfish and
fish made during the first phase of field work. Concentrations of HCB in mud
and soil are presented in Figure 10. Supplementary sites are outlined in
Table 1 in the Field Methodology section. Comparisons of HCB residues in soil,
water and organisms from various sites can be seen in Tables 2 throuqh 4.
2. Area of Higher Concentration
The area near the Vulcan Materials Company plant in Geismar, Louisiana,
was the site of a series of field collections during 1974 and 1975. Three
specific localities were selected for sampling of water, mud and organisms.
These sites, designated Recreation Pond, South Effluent, and Landfill Pond
(pond adjacent to hex landfill) are discussed in detail in Section III of
this report. Table 5 gives concentrations of HCB residues in water, mud, and
organism samples taken during several seasons of the year.
Chara and Najas are two plants found in the pond. The former, an alga, is less
plentiful, has a high calcium carbonate content and attaches to the bottom.
The latter occurs as floating, unattached masses in all parts of the pond.
Eleocharis , an emergent plant,was taken in shallow water less than .3 meter
deep, near the shoreline. The snail Physa was common in masses of Najas .
Anisoptera were represented by dragonfly larvae taken while dipnetting in
floating vegetation near the shore. Procambarus p, the crayfish,was taken
periodically during scoops of the dipnet that took up some bottom sediment
as well. Gambusia, the mosquitofish, was more frequently found near the
surface in shallow water, while juvenile sunfish, Lepomis , less than 4 cm
in length, lived in the protective cover of aquatic vegetation. Micropterus ,
the single bass collected, is a common sport fish in this region.
21
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rO l E LOCALITY
A HwY. 90. NORTH OF BATON ROUGE
B INTERSECT RIVER RD.. 1*. Hwy. 327
B, ADDISATLA HWY. 990*IA 998
B. PLAOUEIIINE AT 1*. HWY. 988 & 1*. 1148
C 5 MI. SOUTH OF B
0 SUNSHINE
( CARVILLE
1, I i 405. 4 HI SOUTH OF WHITE CASTLE
F ASHLAND PLANTATION
6 OURRON
CO l E LOCALITY
H INTERSECT. LA. 44 $ l.A 942
I ROMEVILLE
J CONVENT
K LUTCHEN
L GARVVILLE
N RESERVE
N LAPLACE
o SOUTH OF BONNET CARRI SPILLNAT
P DESTRENAN
o ST. ROSE
CO D E LOCALITY
R RIVEN RIDGE
S Rn. ORLEANS. RIVER Ro. AT CAUSEWAY
I NEW ORLEANS. AUGURON PARE
U LOWER ALGIERS
V BELLE CHASSE
W MYRTLE GROVE
H MILE NAREER 42
V .8 MI. OOUTH OF B
2 PORT SULPHUR
Fiqure 10. Distribution of HCB in soil along the Mississippi River,
Louisiana. Baton Rouge - Port Sulphur transect.
BITCH 1W
F ’
900
800
t 700
U
.e 600
500
-j
490
0.
i 300
l IE
BOO
700
600
500
- I
-a
400
300
U,
C
U.
100
LEVEE SOIL
n linFlilA
n It
I ]
I I
11
LOCALITY A B B. B. C 0 C 1, F G H I J K L N N 0 P 0 R S I U V U 0 V 2
22
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Table 2. Concentrations of HCB in water and soil samples from sites
removed from Mississippi River transect.
Location
Walker
Code
(1)
Water
HCB in ugh (ppb )
.8
Soil
HCB in ug/Kg (ppb)**
*
Hammond
Covi ngton
Pass Manchac
Sorrento
(ii)
(iii)
(iv)
Cv)
.9
*
*
*
*
*
*
*
Spiliway (vi)
Lake Grand
Ecaille (vii)
1.5
171.7 (231.2)
<.7 ppb
**: Figures in
parentheses are corrected for dry weight of sample.
Table 3. Mean concentrations of HCB in Mississippi River mosquitofish
( Gambusia affinis ) in comparison with levels measured in water
and coil
Location
Garyville
Code
CL)
HCB
in
Water
ig/1
(ppb)
HCB in
Soil
ig/Kg (ppb)**
HCB
Fish
in pg/Kg (ppb)
*
*
71.8
Romeville
(I)
*
107.0
(135.0)
136.8
Baton Rouge
(A)
2.2
134.9
(167.0)
379.8
< .7 ppb
**: Figures
in parentheses are
corrected for
dry weight
of
sample.
Table 4. Mean concentrations of HCB in crayfish ( Procambarus from
ditches in comparison with levels measured in soil.
Crayfish
HCB in ua/Ka (imb)
Soil
Location Code HCB in jig/Kg (ppb)**
Walker
(1)
*
*
Rorneville
(I)
160.6
(217.7)
22.2
Ashland
(F)
440.3
(884.5)
192.3
Darrow
(G)
874.4
(1676.8)
194.3
*
*
*: < .7 ppb
**: Figures in parentheses are corrected for dry weight of sample.
23
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Table 5 .HCB residues in water, mud and organism samples from highly elevated sampling area.
(Number of organism samples in brackets; figures in parentheses corrected for dry weight
_____ of mud samples).
Mean HCB
Abiotic Dec. April May Aug Oct
Organism # of ana1yses concentration Concentration Component 1974 1975 1975 1975 1975
in jig/Kg (ppb) factor
Recreation Pond
Plankton 1 561. 3,573x Water(ppb) * ** 2.2 .04 .16
Chara 1 563. 13,093x Mud (ppb) * 290 170. 570. 130.
(410.) (220.) (660.) (170.)
Najas 1 147. 66x -
Eleocharis 1 423. 192x
Physa [ 3] 1 294. 6,837x
Gambusia [ 27] 9 3,291. 2,789x
Lepomis [ 45] 15 3,170. 2,690x
Mjcropterus [ 1] 2 liver 23,506. 10,684x
muscle 3,362. l,528x
South Effluent
Anisoptera 1 4,699. 49,998x
Procambarus [ 9) 3 48,669. 17,382x
Gambusia [ 9] 3 41,353. 16,408x
Water(ppb) 3.9 2.8 72.8 .10 9.981-
Mud (ppb) 11,960 2,520. 13,800. 2,150 6001-
(19,000.) (4,200.) (19,000.) (3,000.) (1,320.)
Landfill Pond
Gambusia [ 24] 9 82,891. 2,060x Water(ppb) 4.8 12.1 74.9 * 33.72
Mud (ppb) * 10,500. 29,080. * 53,130.
(13,000. (38,800. (15,000.)
* : no sample taken
**: below detectable levels.
t : site changed due to construction.
-------
B. Acute Toxicity
1. Crayfish Injections
Several experiments were carried out in which HCB, dissolved in peanut oil
alone, or prepared as an emulsion, was injected into crayfish. Concentration
of HCB in stock emulsion of the first experiment was measured at 1.67 mg/ml (ppt).
At the highest concentration, emulsion was injected (full strength) at the rate
of .02 cc/g body weight, which resulted in a dose of 33 pg HCB/g body weight, or
33 ppm. The stock emulsion was diluted to .1, .01 and .001 its original
concentration, and five crayfish each were injected with these dilutions, at the
same rate of .02 cc/g body weight. At the highest concentration, one animal
died on day 2, 4 and 56 after injection. The remaining two survived until the
experiment was terminated after 61 days. One death occurred after 8 days in
the group injected with the lowest concentration and another occurred in the
emulsion control group after 9 days. In summary, of the 20 crayfish injected
in this experiment, 16, or 80% survived two months after the injections, at
which time they were sacrificed. HCB at the concentrations injected did not
appear to exert a significant critical toxic effect, with the exception of the
most highly concentrated series.
The second emulsion was measured at 423 pg/mi (423 ppm). A total of 20
experimental and control animals was injected. Five of these received .004
cc HCB emulsion/g body weight and another five received .04 cc/g body weight.
Sucrose and emulsion controls received .04 cc/g body weight of their respective
solutions. During the 24 day period prior to sacrificing only one death was
recorded; that of one crayfish injected with .04 cc/g HCB emulsion, which was
a dosage of 17 pg HCB/g body weight, or 17 ppm.
A third crayfish injection experiment used only HCB dissolved in peanut oil.
One solution was composed of 5 mg HCB/ml oil; the second contained 12.5 mg/mi and
was a saturated solution at 35°C. Experimentais and oil control groups were
composed of five males and five females each, and were injected with .01 cc oil/g
body weight. At this rate, crayfish injected with the HCB—saturated oil were
receiving 125 pg HCB/g body weight, or 125 ppm.
Of the 30 crayfish reported in this test, all but two survived the seven days
of the experiment in individual finger bowls in the environmental chamber. One
specimen, a 23g male, died 24 hours after injection, and the second, a l6.5g
female, died 4 1/2 days (103 hours) after injection. Both mortalities were in
the group receiving the highest concentration of HCB in oil.
2. Fundulus Injections
HCB, dissolved in peanut oil, was injected intraperitoneally into Gulf
Killifish ( Fundulus grandis ). The dosage of 125 ppm, or 125 pg/g body weight,
had no acute lethal effects during the four-week experimental period. During
the first 24 hours following injection, both experimentals and controls had
blotches of dark pigment dorsally, which disappeared by the second day. These
spots may have been in response to the excitation of being handled.
25
-------
3. Crayfish LC 50 s
To determine possible acute toxicity of HCB in aqueous solution to crayfish,
both static and flow-through experiments were carried out. Initially, a
static test exposed eight juvenile crayfish ( Procambarus sp.)(3-6 cm rostrum-
telson length) to each of four different conditions. These were 5.2 ppb,
2.6 ppb, acetone control and water control. Animals were kept individually
in finger bowls with approximately 50 cc of water. The water was replaced
daily. Toxicant solution was prepared by dissolving HCB in acetone to
produce a concentrated stock solution. A portion of this stock was further
diluted in acetone and added to spring water with vigorous mixing. All crayfish
in this test survived the entire eight days of the experiment, with no
observable behavioral alterations.
In one experiment in the diluter 32 mature crayfish ( Procambarus clarki )
were exposed in each of four tanks to 27.3 ppb HCB, 36 ppb HCB, water control
and water plus acetone control conditions. HCB was flowing in test tanks for
10 days, followed by 10 days of depuration. A total of four (6%) experimental
animals and two controls (3%) died during the entire 20-day period. These
deaths in diluter tanks were the result of cannibalism of vulnerable
individuals following molt.
4. Fish LC 50 s
Two separate experiments were carried out in the diluter in an effort to
test acute toxic effects of HCB. In the first test, twenty bass were
placed in each of six tanks containing water alone, acetone and water, HCB
concentrations of 2, 2, 9 and 10 ppb, respectively. Exposure to HCB lasted
15 days, followed by a 13-day depuration period. No mortalities or abnormal
behavior patterns could be attributed to HCB during this experiment.
The second exoeriment exposed five bass in each of four tanks to water
and water plus acetone controls and 21.6 and 25.8 ppb HCB, respectively. All
animals survived the 10 days of the test, and showed no overt negative effects
due to exposure to HCB. Exposure to HCB in aquatic systems did not result
in overt behavioral responses by fishes or crayfish at the concentrations
or during the time periods of our tests.
C. Chronic Toxicity
1. Crayfish Tissue Morphology - Normal and Pathological
The hepatopancreas of Procambarus clarki and closely related species
has been the subject of histological study since the late nineteenth century.
(Huxley, 1880.) Morphological and physiological studies indicate that the
hepatopancreas is analogous to vertebrate liver, pancreas and gut. It
functions in enzyme secretion, digestion, and absorption of food and in
glycogen, lipid and mineral storage (Fingerman etal. 1967.) These facts
coupled with reports of liver damage in other species following HCB exposure
led to priority being given to histological analysis of this organ.
26
-------
The hepatopancreas develops as a bilateral evagination of the midgut.
Each unilateral component consists of a short common duct which gives rise to
longitudinally oriented central canals of the anterior and posterior lobes.
The common duct and central canals are lined with tall, simple columnar
epithelium with a striated border. From the central canal numerous
diverticula of the epithelium are derived. These are surrounded by a “basket”
of myoepithelial cells and are bound one to another by fine connective tissue.
The lining of each diverticulum is a simple columnar epithelium which
shows a variety of cell types from distal to proximal extent. Four or five
distinct cell types have been described; but a discussion of the merits of
various classifications is not pertinent to the present report (for
references see Loizzi, 1971). There is general agreement that the germinative
center for the epithelium lies in the distal-most region of each d verticulum
where the epithelium consists of relatively small, slender columnar cells
with basophilic cytoplasm (E-cells). These cells apparently give rise to
all other cell types, including absorptive, secretory and fibrillar cells
which vary cytologically and stain differentially with routine hematoxylin-
eosin preparations following Zenker-acetic fixation. Also seen are inclusions
which have been described as metals (iron and copper) in the deeply basophilic
fibrillar cells (Fe-cells) and in eosinophilic cells (Cu-cells) (Ogura, 1959,
Miyawaki etal., 1961). All cell types are found intermingled within the
epithelium gradually giving way to large vacuolated columnar cells in the
proximal region of each diverticulum. Cells in the proximal region of the
diverticulum exfoliate into the lumen and are removed from the gland as a
component of the secretion.
Each green gland (antennal gland) is a nephron-like unit consisting of
coelomosac, labyrinth, and nephridial tubule. The tubUle leads to a bladder
and ultimately to an excretory pore. In Procambarus blandingi light and
electron microscope studies (Peterson and Loizzi, 1975) reveal that the
epithelial cells of the coelomosac are similar to the podocytes of the
vertebrate glomerulus. Epithelium with well-defined brush border comprising
the labyrinth indicates a reabsorptive function for this region; whereas the
nephridial tubule is lined with epithelial cells lacking a brush border but
with basal plasmalemmal invaginations (Beams etal., 1956, Peterson and
Loizzi, 1975) such as are found in the distal tubule of the mammalian kidney
(Pease, 1955). Thus the organ is considered as excretory and osmoregulatory
and in many respects analogous to the vertebrate kidney. For this reason
the green glands have been preserved for histological analysis from water
and acetone controls, and HCB exposed crayfish.
Judicious assessment of tissue health or disease related to toxicant
exposure requires consideration of the feeding of the animal, time elapsed
between feeding and sacrifice, and possibly the relationship of time of
molt. In a diluter experiment in which crayfish were exposed for 10 days to
36.1 ppb, minimal gross pathology was noted as the crayfish were sacrificed;
however, occasional, hardened, brown-pigmented areas involving one to several
hepatopancreatic diverticula were observed. The color of the organ varied
from gray to a bright yellow and this may be related to that cell type
predominating in any one gland.
27
-------
Careful study of numerous serial microscopic sections of hepatopancreas
indicates that despite the generally normal gross appearance, change at the
cellular and tissue level is taking place in HCB—exposed crayfish. In static
experiments with nominal HCB concentrations of 5 ppb and 10 ppb as well as the
diluter experiment above (36.1 ppb) there appears to be a heightened exfoliation
of epithelium in the central, lobar canals as well as in proximal levels of
the diverticula, resulting in empty sacs. The mitotic rate does not appear to
be increased, and therefore epithelial loss is not compensated. The various
cell types are represented but the majority of the cells are large, with
numerous fine vacuoles (“foam cells”) unlike the single larger vacuoles in
cells of controls. There is additional material in preparation and the analysis
of this should lead to a better understanding of the more subtle cytological
changes.
2. Bass Tissue Morphology - Normal and Pathological
Cursory histological examination of the various excised organs (see
Methodology) has indicated that liver, gall bladder, and kidney were of
prime importance for histological study in bass.
In the largemouth bass, ( Micropterus salmoides) , the liver is elongate,
conforming to the shape of the pleuro—peritoneal cavity, and a gall bladder
is present. The gland parenchyma is seen to be lobulated with the classical
lobule easily discerned. Large hepatic portal veins, hepatic arteries, and
bile ducts are seen in the interstices of the lobules (Fig. hA).
The gall bladder lies cupped by a lobe of the liver. It is lined by a
simple columnar epithelium with a rather dense fibrous connective tissue
layer beneath. The muscularis is distinct. (Fig. llC).
The present study reveals that in this species of fish, acini of exocrine
pancreas are situated in the connective tissue around the hepatic portal vein
and around the major bile ducts in the hilus of the liver and accompany the
hepatic portal vein, hepatic artery, and bile ducts in their distribution within
the liver parenchyma (Fig. hA, arrow). Exocrine pancreas also is situated
between the muscularis and serosa of the gall bladder (Fig. llC, arrow).
Exocrine and endocrine cells are intermixed to form small nodules of pancreas
in the gastro-hepatic ligament. Variability among fish species has been noted
previously in the location of endocrine and exocrine pancreas (Bengelsdorf and
Elias, 1950, Bertolini, 1965, Falkmer and Windbladh, 1964, Fujita,1964, Weis, 1972).
The kidney, an opisthonephros, shows relatively large renal cortuscles and
short proximal and distal tubules. A thin loop of Henle is not present (Fig. liE).
Other bass tissues (gill, brain, and muscle) have been excised, examined
grossly, and scanned histologically. One hundred and ninety blocked specimens
are available for further detailed study.
The fish were normal in appearance and behavior at the time of sacrifice;
however under dissecting microscope magnifications some abnormalities were noted
in the appearance of various organs. Thus, in 9 of 19 HCB-exposed bass in one
series, the liver was noted to be pale and the gut white or empty. In one fish
the gall bladder was noted to be white and in another, was absent. In the fish
in the highest exposures of HCB, (10 ppb) a marked gold irridescence of the
parietal peritoneum was also observed.
28
-------
Figure 1 I, A-F.
Photomicrographs of bass tissue sections.
-
IE
29
-------
Explanation of Figures
(All figures are photomicrographs at the same magnification, taken of
sections stained with H & E.)
A. A microscopic field of fingerling largemouth bass ( Micropterus
salmoides ) liver shows the normal lobulation pattern. Cells
interpreted as exocrine pancreas (arrow) are seen in the connective
tissue around a large vessel. (H 2 0 Control Bass)
B. A microscopic field of
after ten days diluter
liver architecture and
exposed to 3.5 ppb HCB
the liver removed from a fingerling bass
exposure to HCB. Note the loss of normal
the beginning necrosis (arrow). (Bass
in diluter; 26.8 ig/g HCB in tissue).
C. Normal liver may be seen on the left and a small segment of the
gall bladder wall in section is seen on the right. The arrow
indicates the location of pancreatic cells. (H 2 0 Control Bass)
0. A section through the necrotic liver hilus also
of the wall of the gall bladder in which almost
of the epithelium, muscle, and pancreatic acini
exposed to 25.8 ppb HCB for 10 days in diluter;
tissue. See section V.C4.)
E. A section of fingerling bass kidney illustrates
Note the size of the glomeruli and the tubules.
includes a portion
complete destruction
was noted. (Bass
69.9 pg/g HCB in
the normal histology.
(H 2 0 Control Bass).
F. A microscopic field from the kidney of another I-ICB-exposed bass in
which distended tubules and a large damaged glomerulus (arrow) may
be seen. (Bass exposed to 3.5 ppb HCB for 10 days in diluter; 2.4
pg/g HCB in tissue. See section V.C4.)
30
-------
The minor alterations in gross morphology were in no way indicative of the
degree of pathological change , the extent of which could not have been
appreciated without thorough microscopic examination. Histopathological change
was noted in the liver, gall bladder, and the kidney in HCB-exposed bass.
In the liver, areas of necrosis were found to be most pronounced in the
region of the hilus (Fig. liD), but existed also deeper in the liver parenchyma
and just beneath the serosa (Fig. liB). The necrotic areas seemed to follow
the portal canal distribution. Different stages of necrotic change from
early hepatocyte death to hepatocyte-barren stroma and Dhagocytic infiltration
were seen. In non-necrotic areas of affected livers, the typical pattern of
lobulation was lost and there was an increased intensity of cytoplasmic
staining (Fig. 11B).
Gall bladder damage was striking. Both the epithelium and the smooth
muscle were destroyed, leaving a dense connective tissue bag (Fig. liD).
Remnants of cords of deeply basophilic pancreatic acini show that this tissue
too is affected in its site in the wall of the gall bladder; although it
persisted within the liver parenchyma.
In animals with liver damage, kidney damage was also found. Some glomeruli
were destroyed, resulting in distended balls of stagnant blood (Fig. hF).
Necrotic areas of tubule destruction were present also. The liver and kidneys
from H 2 0-controls thus far sectioned were normal histologically. Among
acetone controls some liver and kidney necrosis was noted but was not as
extensive as that found in HCB-exposed animals.
3. Retention and Distribution of HCB in Crayfish
Crayfish exposed to HCB in a nominal concentration of 5 and 1 ) pDb
(GC values at 5.2 ppb and 2.6 ppb respectively), in aqueous solution in finger
bowls, changed daily, showed no readily observable adverse response to the
compound. All experimentals and controls survived and were sacrificed after
8 days. Dissected tissue samples were grouped and frozen together. Subsequent
GC analysis gave the results seen in Table 6.
Table 6. Concentrations of HCB (in pg/g) in crayfish bodies and
tissues of animals exposed to HCB in aqueous solution for
8 days.
N** 5 ppb N 10 ppb N Acetone N H 2 0
— HCB — HCB — Control — Control
Whole body Cx) 2 .45 3 .68 7 * 7 *
Hepatopancreas ( ) 5 * 4 24.59
Gills (i) 5 * 4 12.35
Muscle ( ) 5 1.17 4 2.63
Remaining body ( ) 5 .89 4 .60
*: below detectable levels
**: number of specimens
31
-------
4. Retention and Distribution of HCB in Bass
Two separate tests with bass were conducted to determine the distribution
of HCB in organs as a function of dosage administered, size (weight) of
specimens, and time of exposure. Figure 12 gives results of an early
experiment in which very small fingerling bass were exposed to HCB for 15 days.
During this first test, GC analysis of the comercial food revealed a concentra-
tion of 80 ppb HCB. Both the controls and experimentals, therefore, were
exposed to HCB in their food, It is interesting to note that, in the controls,
living in clean water and eating contaminated food, the highest concentration
of FICB was found in the kidney. In contrast, the organisms living in a
contaminated environment and eating contaminated food had the highest HCB
concentration in the gut (the content of the lumen of the gut was not included
in the analysis). The relatively high concentration in the kidney of both
experimental and control animals may be correlated with its excretory
function and the filtration of blood plasma, thus establishing a system which
allows the hydrophobic HCB to partition into the lipid-rich components of the
kidney cells.
Results of the second experiment are given in Figure 13 . The relative
importance of various tissue types in concentrating the compound is reasonably
consistent with the previous experiment. Lower overall levels in the second
experiment are due to larger average size (weight) of specimens and a shorter
period of exposure. Concentration factors tend to be considerably higher in
fish exposed to lower concentrations of HCB than those in higher test compound
concentrations.
In these two experiments, bass showed the highest concentration of HCB in
the gut, kidney and gills. After fifteen days’ exposure to a mean concentration
of 10 ppb HCB, these organs concentrated the compound to levels of 1811, 1441,
and 748 lig/g (ppm) respectively. These are concentration factors of 181 ,100X,
144,100X and 74,800X.
32
-------
Figure 12 . HCB concentrations in organ samples from bass exposed to HCBt
for 15 days. Concentration factors in parentheses.
C)
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Tank A:
Tank B:10 ppb HCB
STank C: 2 ppb HCB
Tank D: 2 ppb HCB
JTank E: Acetone control
Tank F: H 2 0 control
N=2, X wt = 4.46 g
N=2, Xwt = 1.66 g
N=2, X wt = 3.62 g
N=2, Xwt = 1.40 g
t All specimens maintained on
comercial fish food, containing
80 jig/Kg (ppb) HCB.
C
4-) -
CD
*Sample lost in processing
**Below detectable levels
33
-------
Figure 13. HCB concentrations in organ samples from bass exposed to
- HCB for 10 days. Concentration factors in parentheses.
U) W
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(D
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In ug/g
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300 ug/g
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UTank A and B:
•Tank C:
UTank D:
*
below detectable levels
CONTROL
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23.7 ppb HCB
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34
-------
5. Fish Blood Hematocrit
Percent hematocrit in blood is considered an effective means of estimating
red blood cell counts in an organism. In the present study, three experiments
with three different fish species were carried out in an effort to find
differences in hematocrit between experimentals and controls, which could be
interpreted as a stressful effect of HCB.
In the first experiment, a total of 24 bass was exposed to control
conditions and HCB concentrations up to 25.8 ppb for 10 days. In the second
experiment, 6 sunfish ( Lepomis macrochirus ) were exposed to 2.7 ppb HCB and
6 were held in HCB-free water for 10 days. The third experiment used a total
of 75 Gulf killifish, maintained in control conditions and 5.7 ppb HCB for
10 days. In all of the above experiments the individual variation and mean
values did not indicate an effect upon hematocrit by HCB at the concentrations
tested.
6. Fish Corticosteriod Level
Three experiments were carried out to observe possible cortisol level
changes in fish blood as a response to stress imposed by exposure to HCB. In
the first two experiments, bass were exposed to HCB for 10 days at the nominal
concentrations of 1 and 10 ppb. In neither case did a competitive protein-
binding radioassay of plasma cortisol levels show a significant difference
between the means of the experimental and control groups. These results were
rendered inconclusive for the following reasons: (1) the naturally high
variability of any physiological parameter (here plasma cortisol level),
(2) small sample sizes necessitated by the limited availability of bass, (3)
the tendency to excitability of a predator species, accentuated by capture,
(4) inability to match animals by size with consequent variation in cortisol
level due to age and not stress endured.
When bass became unavailable from the hatchery because of the season, the
Gulf killifish ( Fundulus grandis ) was substituted in order to continue studies
of possible toxic (stressful) effects of HCB. This animal was chosen because
it is readily available in the numbers required for good statistical analysis
and also because it is a far more docile fish which is less excited by the
process of capture. By minimizing stress of capture, matching the fish closely
with regard to size and employing far greater numbers than was possible with
the bass, extraneous variations in plasma cortisol determinations were
eliminated and a significant stressful effect of HCB was found.
Figure 14 shows the results of an assay of plasma corticosteroid 1eve s
(pg/lOO ml) in the Gulf killifish. Statistical analysis has shown that the
control groups have significantly lower plasma cortisol levels than do the
experimental groups, indicating a significant stressful effect of HCB.
35
-------
I-
E
CD
I
Figure 14. Mean plasma cortisol levels (ug/lOOml) in the Gulf killifish
( Fundulus grandis ) exposed to HCB for ten days.
water
control
N=20
acetone
control
N=20
nominal l5ppb HCB
5.7 ppb HCB
N=1 7
15
nominal l5ppb HCB
5.9 ppb HCB
N=20
RAN GE
An analysis of variance (F-test) was performed which showed that the
experimental groups do not differ significantly from the controls at the 5%
level, but do differ significantly at the 6.5% level. Statistical analysis
(F-test) of control groups indicated that they were not different from each
other. The same results were found between experimental groups.
D. Accumulation and Clearance
It is an established fact that many contaminants accumulate at different
levels in different abiotic substances and in organisms at various trophic
levels in an ecosystem. Biomagnification has been observed numerous times.
With this in mind, the present study was designed to analyze HCB levels from
several parts of the natural system. Levels of accumulation were observed in
field samples, and laboratory tests were carried out for comparative purposes
and to establish rates of accumulation. Components of the simulated ecosystem
included sediment, algae, crayfish and fish. The algae, Oedogonium , is a genus
found at the contaminated study site, and serves as food for such grazers as
snails (Ph,ysa), crayfish ( Procambarus ) mollies ( Poecilia latipinna) , and
mosquitofish ( Gambusia affinis) . These invertebrates and fishes are common
food items of larger predators, including sunfish ( Lepomis ) and bass
( Micropterus) . Several organisms named above were used in a number of lab-
oratory experiments discussed here, in an effort to increase available
knowledge of accumulation of HCB in the food chain.
40
35
30
25
10
5
36
-------
1. Crayfish
In an experiment to determine rates of uptake and depuration of HCB by
crayfish, 32 test animals were held in each of two experimental diluter tanks.
Measured HCB concentrations were 27.3 ppb (iig/ e) and 36.1 ppb, respectively.
The controls were 32 crayfish in acetone in water and 32 others in uncontaminated
water. A low level of contamination was measured in both control systems
(.03 ppb and .005 ppb, respectively).
Two males and two females were removed from each tank following 1, 5 and 10
days of exposure and 1, 5 and 10 days of depuration. Whole bodies of males
and females were processed and analyzed separately for each removal date.
Data from this experiment are presented in Table 7 and Figure 15.
.0
0.—
•
tø —
I -C •4
.0
0.—
L
C •
cø 0
I—c ’ .,
—
L) 0.-- C 0
c J5..
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0. 0 %..
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a,
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t: two specimens per data point.
*: below detectable levels.
Table 7 Whole-body
and female
depurati on
HCB residue concentrations in jig/Kg (ppb) in male
crayfish (Procambarus clarki)t durin j uptake and
Days
exposure
Days
depuration
1
5
10
1
5
10
Males
657.
3,164.
4,056.
2,675.
6,638.
2,347.
Females
824.
5,467.
2,277.
5,802.
2,219.
1,864.
Males
1,287.
5,754.
4,038.
3,134.
4,319.
1,483.
Females
871.
4,810.
2 7l7.
5,115.
4,484.
l,523
Males
33.
12.
*
11.
32.
38.
Females
101.
27.
15.
77.
15.
18.
Males
21.
17.
29.
13.
20.
Females
*
13.
17.
26.
5.
4.
37
-------
Figure 15 . Uptake and depuration of HCB in crayfish ( Procambarus
clarki ) exposed to a mean concentration of 31.7 ppb
(pg/ ). *
6,000 -
5,000 -
4,000
3,000 -
2,000
1,000
1
I
I
I
I
I
5
10
1
5
10
Days exposure Days depuration
*: Average values from Table 7 ; Four specimens per data point.
Accumulated levels reached a high point after five days’ exposure and
tended to show no further increase. Males had an average HCB level of 4460
ug/Kg(ppb), a maximum concentration factor of 141X. For females, which were
measured at 5,140 iig,kg, a maximum concentration factor was 162X. After 10 days
of depuration the factors had been reduced to 60X for males and 53X for
females.
C,
-‘
C
a,
In
In
9-
4- )
C
•1
C-,
38
-------
2. Mollies
Two static experiments and three diluter experiments were carried out
with mollies challenged with HCB. Results from two flow-through experiments
are reported here. In each protocol, a set number of male and female
sailfin mollies were placed in each test tank. Duplicate tanks received the
same concentration of HCB. Groups of three fish per tank were removed at
intervals given in the figures below. GC analyses of these groups from
individual tanks were done separately and the results combined to form a mean
used in tabulation. Data available In this work did not suggest that one
sex accumulated a greater proportion of compound than the other. The
experiments were carried out well after the season’s young had been born.
Results of these two experiments are given in Figures 16 and 17.
A steady uptake and subsequent loss of 1KB are indicated in most of these
figures, with the highest concentration factor occurring after eight days’
exposure to 62.2 ppb ( ‘g/ ), at which point the HCB residue concentration
reached 149.1 pg/g; a level 2,397 times that measured in water. The
concentration factor for mollies exposed to 7.9 ppb for the same period of
time was very close to this, 2241X. The concentration measured in this
group was 17.7 pg/g.
Figure 16. Uptake and depuration of HCB in sailfin mollies*
( Poecilia latipinna ) exposed to 55 ppb (pg/e) HCB.
0’
C
1
Ln
U,
U,
•1
4.,
C
70
60
50
40
30
20
10
Days exposure
Days depuration
two separate GC analyses
1
110 j
-U-
5 10
*: Four males and two females per data point;
39
-------
Figure l7.Uptake and depuration of HCB in sailfin mollies*
( Poecilia latipinna ) exposed to 7.9 and 62.2 ppb
( pg/ ) HCB .
140
/
120
I
a
a
; . 100
I —
I —
•1
62.2 ppb
80 1 —
U) /
I
I
60
U
. An
•1
U) I
U
S.-
20 7.9ppb
Days exposure Days depuration
* Three males and three females per data point; Two separate GC
analyses
** One GC analysis of three fenales
3. Bass
Rate of HCB uptake and depuration was studied in an experiment with finger-
ling bass (6-10cm, total length). HCB concentrations in the four experimental
diluter tanks were 9, 10, 2 and 2 ppb (pg/i). Twenty fish were placed in each
tank at the outset. Three randomly selected animals per aquarium were removed
and frozen individually following 5, 9 and 15 days’ exposure and after 4, 9
and 13 days’ depuration.
40
-------
Results of this experiment are shown in Table 8. There was a direct
correlation between the length of the period of exposure to HCB and the
concentration of HCB in tissue. Highest levels attained in whole body extracts
reflect accumulation of more than 44,000 times the concentration measured by
GC in test water. These maximum levels were found in fish from all groups,
sacrificed on the fifteenth and last day of exposure. No apparent correlations
between size of fish and concentration factors were noted in this experiment.
Levels of HCB in whole body extracts decreased fairly regularly during
depuration, with 8.6 to 26.9% of the residue remaining following 13 days’
exposure to HCB-free water (Fig. 18 ). During this time period, fish
initially exposed to lower levels of HCB had succeeded in eliminating a greater
proportion of the substance than had those exposed to higher levels.
41
-------
Table 8. Whole-body HCB residue concentrations in largemouth bass
( Micropterus salmoides ) during uptake and depuration.
Number of fish analyzed in parentheses.
Days exposure Days depuration
5 9 15 4 9 13
HCB (pg/g)
in tissue 6.12 160.96 163.92 124.14 54.61 42.42
U 4
ean w in
g 2.6(4) 2.9(3) 3.9(3) 3.3(2) 4.8(2) 3.3(3)
Concentration
factors 680x 17,885x 18,214x 75 • 7 %a 33.3% 25.8%
HCB (pg/g)
in tissue 17.49 171.49 325.13 116.09 177.14 87.54
. _u
Mean wt in
g .9(4) .9(3) 1.2(3) 1.4(2) 1.8(2) 2.5(1)
Concentration
factors 1,749x 17,149x 32,513x 35.7% 54.5% 26.9%
HCB (pg/g)
in tissue 2.72 14.40 88.88 20.14 8.46 7.62
L)
c Mean wt in
g 2.4(4) 2.9(3) 4.2(3) 3.2(2) 3.9(2) 3.2(2)
Concentration
factors 1,043x 7,198x 44,437x 22.7% 9.5% 8.6%
EICB ( ig/g)
in tissue 1.00 16.34 67.65 41.00 20.61 13.90
. .o Mean wt in
g .9(4) .9(3) 1.3(3) 1.4(2) 1.6(2) 1.5(2)
Concentrati on
factors 498x 8,170x 33,824x 60.6% 30.5% 20.5%
a Percent remaining of maximum measured level.
42
-------
Figure 18 . Uptake and depuration of HCB in bass ( Micropterus
salmoides ) exposed to 2 ppb and 9.5 ppb (pg/e).
Average values from Table 8.
250•
200.
150•
100•
50
1
9
1
9
Days exposure
Days depuration
In flow-through experiments with green filamentous algae ( Oedogonium
cardiacum) , a lower rate of accumulation was observed in comparison with
that of animals. Results of one such experiment are given in Table 9.
Table 9 - Concentration and depuration of HCB by green alga, Oedogonium
cardiacum exposed to a flowing solution of 11.5 ppb (1ig/ )
HCB in water
Days
exposure
HCB in g/
(ppb) wet
Kcj
wt
Concentration
factor
1,973.
172x
1
3
3,472.
302x
7
7,161 .
623x
Days
depuration
4
8
6,314.
682.
E
C.
C-
C ..
0
a)
U)
U,
•.-
4-)
C
•I-
a)
-r
U,
a)
I-
9.5
—-I
2p
S
— —
—
——4——
— — —
I I I
4 13
4. Algae
43
-------
5. Bottom sediment
Results of a brief experiment with sediment showed soil accumulated
proportionately less HCB than did organisms, but retained it longer. Following
one day of exposure to a regular flow of 8.3 ppb (pg/i) HCB, a soil sample
contained a concentration of 332 pg/Xg(332 ppb) FICB, a concentration factor
of 40x. Four days after initiation of the test, soil contained 269 pg (g HCB,
a concentration factor of 32x. Depuration began on the fourth day. After
four days of depuration the sample of sediment removed and analyzed on GC
still contained 303 pg/KgHCB, a level nearly as high as that of the first
day’s sample.
6. Effect of Food Chain
An experiment was carried out to determine the difference in HCB uptake
between bass fed HCB-contaminated mollies in a clean environment and others
fed similar mollies in an HCB-contaminated environment. Mollies were exposed
to HCB until a sample tested contained a substantial amount of the substance;
in this instance, 64 pg/g (ppm). The entire test with bass lasted 8 days,
during which time numerous attempts to feed the bass were made under constant
observation. Results of the test are included in Table 10.
Table 10, HCB concentrations in largemouth bass ( Micropterus salmoides)*
feeding on contaminated sailfin mollies ( Poecilia latipinna).
Bass held in 37.6 ppb HCB Bass held in HCB-free water
Bass wt 1/ mollies HCB in Bass wt # mollies HCB in
spec. # in g eaten pg/g spec. # in g eaten pg/g
1 10.24 4 95.24 1 8.58 5 3.33
2 13.56 5 78.53 2 10.59 0 0.43*
3 18.28 4 56.96 3 16.33 5 3.42
*The diet initially fed these test animals contained .08 pg/g HCB.
These data illustrate the effect size has upon concentration factors of
fish in a contaminated medium. The smallest bass in the HCB-contaminated
water of Table 10 had an HCB concentration factor of 2,533x, while the largest
had a comparable value of l,515x. Bass accumulated a much greater proportion
of the compound from the ambient water than from ingestion of HCB included
in mollies.
Since a body is not likely to retain all of the compound ingested,
calculations were made to determine the approximate proportion excreted or other-
wise eliminated by the bass.
44
-------
Of the approximately 106 pg of HCB estimated to have been contained in
five mollies and taken in by bass #1 in clean water, approximately 25 pg
remaining in the whole body of the bass can be attributed to this source,
indicating that one-fourth of the HCB ingested was retained. A longer period
of ad libitum feeding would be expected to have given a greater concentration
of HCB through ingested contaminated food fish.
The seasonality of bass juveniles made it impossible to repeat this test
with a larger number of the same species. However, two groups of sunfish,
Lepomis macrochirus , also in the family Centrarchidae, were tested in a similar
way. In this experiment, HCB concentration in the contaminated tank was
lowered to a mean level of 2.7 ppb, and the six sunfish in each tank were fed
contaminated mollies twice daily ad libitum . Mollies contained approximately
16 jJg/g HCB. It was ascertained that all fish had eaten daily for the seven
days of feeding. All fish were fasted for one day prior to sacrifice to
eliminate remains of solid food from the gut. Results of this experiment are
given in Table 11.
Table 11 HCB concentrations in sunfish ( Lepomis macrochirus ) feeding
on contaminated mollies ( Poecilia latipinna).
Sunfish held in 2.7 ppb (pg/e) HCB Sunfish held in HCB-free water
Sunfish wt in HCB in Sunfish wt in HCB in
spec. # _____ ______ spec. # _____ ______
1 21.0 4,248. 1 17.0 576.
2 24.6 3,965. 2 26.2 665.
3 27.2 4,782. 3 26.3 799.
4 32.2 3,186. 4 28.7 504.
5 38.1 3,084. 5 31.6 780.
6 42.4 2,205. 6 51.4 258.
The mean concentration of HCB in the two groups is 3,578. and 594. pg/Kg
respectively. For an experiment of this duration, it appears that HCB in water
contributes a greater proportion of HCB to sunfish than does HCB taken in with
contaminated food, since sunfish in control water accumulated less than 800 pg/Kg
HCB through contaminated food while those in HCB-contaminated water had levels of
2,200 to 4,700 pg/Kg HCB. Sunfish ate approximately 7 grams of mollies which
would have contributed a maximum of 112 pq of HCB to each of them. Assumina
ingestion of ll2pg of HCB, a 30 g sunfish would have contained 3,733 pg HCB per Kg
body weight, if all HCB were retained. Since HCB content in sunfish of this size
was approximately 640 pg/Kg, it appears that sunfish assimilated approximately
6% of the HCB from food taken in. Specimens in the contaminated tank show an
inverse relationship between fish size (weight) and concentration of HCB.
45
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7. Crayfish uptake of HCB in Field Environment
A series of field studies was carried out to demonstrate uptake of HCB
in clean crayfish ( Procambarus clarki ) under contaminated field conditions
at the Geismar site. In the first experiment, crayfish were placed in cages
and submerged in the pond adjacent to the hex landfill. HCB levels in water
measured 74.9 ppb and in mud were 29.08 ppm. Animals had access to the mud
bottom.
Following 10 days’ exposure all specimens were removed from the site.
Six were frozen immediately for analysis and the remainder were transferred
to trays of clean water, which was changed regularly. These latter specimens
were sacrificed periodically. Figure 19 shows mean levels of HCB measured in
whole body extracts of crayfish, before and during depuration. Males usually
weighed more than females, but females tended to accumulate and retain greater
proportions of HCB.
At the end of the initial ten days’ exposure whole bodies of males contained
an average of 90 pg/g (ppm) HCB, which is a concentration factor of l,200x
the amount measured in water. Females contained 129.5 pg/g HCB; a concentration
factor of 1,729x. Following 3 days of depuration, males retained 82% and
females held 87% of initial HCB. At the end of 25 days, this figure was
reduced to 53% in males and 36% in females.
A short-term experiment in the same waste pond consisted of introducing
clean crayfish on one day and removing a sample at daily intervals for seven
days. The increase in HCB levels of whole-body extracts is given in
Figure 20.
Depuration rates following shorter-term exposures appear to be more rapid.
Table 12 gives the mean HCB concentrations attained after one to six days’
exoosure, and the subsequent level reached at the end of one to nine days’
depuration. In this abbreviated experiment, more than half of the samples
lost more than 50% of their HCB content if depurated for a period of five
to nine days.
Table 12. Short-term exposure and depuration of crayfish introduced into
a contaminated environment (43.5 ppb HCB).
Mean Days Mean
Days exposed HCB concentration depuration HCB concentration
______________ in pg/g (ppm ) ____________ in yg/g (ppm )
1 14.04 9 3.47
2 21.74 5 8.79
6 5.60
8 13.22
3 44.28 7 15.15
4 39.37 1 53.34
4 33.13
7 16.21
5 46.76 7 59.38
6 50.64 7 24.70
46
-------
Figure 19. Levels of HCB in crayfish during depuration following 10 days’
exposure to a contaminated environment (74.9 ppb HCB).
100
L)
— 50
0 ,
I
48
150
100
0,
0,
50
Figui
50
40
=
0,
0,
10
1 2 3 4 5 6 7
Days exposure
47
MALES N=19
0 3 7 12
Days 25 depuration
FEMALES N=12
0 3 7 12 Days 25 depuration 48
20.HCB concentrations in crayfish exposed to a contaminated environment (43.5 ppb)
-------
E. Respirometry
1. Crayfish
Exposure of juvenile crayfish to 1903 ppb HCB resulted in variable oxygen
consumption patterns. The average oxygen consumption rates (pe 0 /rng wt/ hr)
for HCB-exposed and for control animals are shown in Table 13. Each value
is based upon an average of 20 animals whose oxygen consumption rates were
measured for a total time period of two hours. For Figure 21 the percentage
deviations of the experimental groups from control groups are plotted against
time (in days). Time “zero” represents the initial exposure period. By
establishing the control values as a base line, any trends in the experimental
values should become evident through this method of plotting the results.
There were no significant differences between experimentals and controls
during the first three hours of exposure. Determinations on days 2, 4 and 10
gave variable results among repeated experiments. The trend, which was not
consistent, indicated an initial decline in rate for the first four days
followed by an increase to control levels after a period of ten days. These
observations must be considered tentative.
Table 13 Effect of time of exposure to HCB on respiration of
juvenile crayfish (p.c 02/ mg/ hr).
Experiment II Group
0
Duration of
exposure
(days)
2
5
10
Control
0.4425
0.2844
0.7078
0.5243
1
Exp.
(1903 ppb)
0.3874
0.2996
0.4470
0.4495
Control
0.4080
0.4984
0.5233
2
Exp.
p437 ppb)
0.2600
0.3942
0.4812
48
-------
Figure 21.
I-
(0
4-.’
4) ....
US-
.0 U
UX
ow
a v
4-
4- V.,
.r- —
00
5-
4J
0
U
10
0
-10
-20
-30
The effect of time of exposure to HCB (1903 ppb, •
2437 ppb, o) on respiration rate of juvenile crayfish
( Procambarus clarki) .
2. Mollies
Three sets of experiments were carried out to determine the possible
effect of HCB upon oxygen consumption in mollies ( Poecilia latipinna) . The
average oxygen consumption rates (p 0 2 /mg wt/ hr) for HCB-exposed and for
control animals are shown in Table 14. Each value is based upon an average
of 4 animals whose oxygen consumption rates were measured for a total time
period of one hour. In Figure 22 the percentage deviations of the experimental
groups from control groups are plotted against time (in days). Time “zero’
represents the initial exposure period. By establishing the control values
as a base line, any trends in the experimental values should become evident
through this method of plotting the results.
Juvenile mollies exposed to 2,783 opb HCB showed no significant change in
respiration rate initially (first 3 hours) during exposure. Rates were
compared in three sets of experiments at 2, 4, 8 and 12 days of exposure.
Differences as great as 50% increase of experimentals over controls were
observed. The patterns resulting from each experiment were not clearly re-
peatable and therefore definitive conclusions based upon the present data are
not possible. Both increased and decreased rates in experimentals were observed.
As a preliminary pattern there was a tendency for rates in experimentals to
rise between the first and fourth days and to decline to control levels
around days ten to twelve.
Days exposure
49
-------
Table 14. Effect of time of exposure to HCB on respiration rate of juvenile
mollies (p O /mg/ hr).
a’
Experiment #
Group
0
2
4
Duration of exposure (in
days)
12
14
5
6
8
10
Control
0.5397
0.4704
0.3577
1
Exp.
(2783 ppb)
0.4919
0.4789
0.2935
Control
0.3308
0.3314
0.3568
0.3568
0.3685
2
Exp.
(2783ppb)0.3130
0.4282
0.3619
0.4404
0.3383
Control
0.3244
0.2654
0.2272
0.4088
0.2859
0.2596
0.2391
3
Exp.
(1587 pib)
0.3578
0.3742
0.2548
0.4524
0.2915
0.2622
0.2429
-------
Figure 22
The effect of time of exposure to HCB (2783 ppb, • and
o; 1587 ppb, a ) on respiration rate in juvenile mollies
( Poecilia latipinna) .
In
I-
C
a,
E
•1
a,
0
x
a,
V
C
In
0
C
0
0
C
U
a)
4- )
a)
a)
U
C
U
5.-
C)
14
‘4-
•1
Days exposure
51
-------
F. Metabolic Fate
Autoradiograms of HC 14 B extracts run on thin layer plates were developed
and showed only one labelled component in the following samples: feces,
stomach and intestines, liver, and remaining body. Since the label from each
of these extrafjs migrated on the chromatograms with the same relative
mobility as HC’ ’B standards, there was no indication that HCB had been
metabolized. Had metabolites been produced, thin layer chromatography/auto-
radiography may have shown more than one labelled spot. The relative mobility
of HCB in this experiment was 0.91.
Extracts of kidney, the only other organ studied in this experiment,
contained low radioactivity of only twice the background level as measured by
liquid scintiflation counting. Autoradiographs of chromatograms of kidney
extract did not develop spots even after two months of exposure because of
the low level of radioactivity applied to the origin.
The relative distribution of labelled HCB in the extracts as determined
by liquid scintillation counting is shown in Table 15.
Table 15 . Activj y (cpm/g) of extracts of bass ( Micropterus salmoides )
fed C -HCB
Sample
BASS #1
BASS #2
feces
10,677
5,951
gut
1.494
1,840
liver
253
165
kidney
160
135
remaining
body
44
*
* sample lost in processing
52
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G. Photochemistry
Irradiation at a wavelength of 2735 causes the disappearance of HCB.
After 60 minutes of irradiation in benzene, less than 10% of the original
HCB remained. Lower molecular weight compounds appear after 30 minutes of
irradiation and continue to increase proportionately with time of exposure.
GC traces of resultant products taken at intervals are shown in Figure 23.
Figure 23. Gas chromatographic separation of photoproducts resulting
from ultraviolet irradiation of HCB.
L i i
U)
z
0
a.
U)
L i i
w
0
C.)
L i i
5 10
TIME (MINUTES)
O mm.
65 mm.
120 mm.
15
53
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VI. DISCUSSION OF RESULTS
Analytical support for the studies contained in this report included GC
analyses of more than 1,300 separate water, soil and organism samples.
Laboratory specimens accounted for more than 1,000 of these prepared extracts,
while the remaining analyses included samples collected during more than 40
field trips.
Results of GC analysis of HCB extracts from samples taken from the
environment in southeastern Louisiana revealed a consistent association of
HCB with the Mississippi River. The principal source of contamination is very
likely to be petrochemical industries that border the river. Three major areas
having considereble HCB concentrations are immediately north of Baton Rouge,
Addis and Plaquemine on the west bank and a stretch of river between Ashland
Plantation and Darrow along the east bank.
Samples taken at the most contaminated sites on property of the Vulcan
Materials Company in Geismar had substantially more HCB residues than any
taken in the regional survey. Mud samples at Geismar had as much as 45 times
the highest level found elsewhere during the MississipDi River survey. In
water samples from the river survey, HCB contamination rarely exceeded 2 pg/i
(ppb), but at Geismar, levels up to 30 times this concentration were found
at a site where wastes were being properly disposed. A pond, less than 500
meters from the disposal site, called the Recreation Pond, had very low levels
of HCB, exceeding 2 ppb on only one occasion. The highest concentration factor
was found in a naturally-occurring organisms at this site, however. The snail,
Physa , was found to have a concentration of 561 pg/Kg (ppb); a concentration
factor of nearly 7000X at the time of water sampling. The mosquitofish,
Gambusia , had a mean of 3,291 pg/Kg HCB residues, which in terms of HCB-content
in water at the sampling times, was a concentration factor of 2,789X. These
results are consistent with data from a model ecosystem study by Metcalf
et al. (1973) in which Physa had accumulated 4,099 pg/Kg HCB, a level pro-
portionately higher than the 3,154 pg/Kg found in mosquitofish at the same
exposure of 6.44 pg/i (ppb). A comparison between mosquitofish from the
Baton Rouge site and the Recreation Pond at Geismar reveals an order of magnitude
higher HCB level in fish from the latter site, while HCB levels in water were
close to the same for the two sites. This is probably a function of the static
nature of the Geismar pond in comparison with the dynamic situation in the
Mississippi River. Fish in the river are probably not exposed to a constant
dosage of HCB.
Injections of HCB in oil were not lethal to animals in this study.
Dosages were given to a maximum of 125 pg/g body weight (125 ppm). Since
the injected solution was saturated with HCB, the only means to increase
dosage would have been to increase volume injected, which, in the case of our
specimens might have resulted in physical damage due to the volume entering
the organisms. HCB did not produce acutely toxic results in experiments
reported by Davis etal. (1959). In those tests, rats were injected
intraperitoneally with 500 pg/g (ppm) HCB which was a fourfold multiple of
concentration used in the experiments reported here. Parke and Williams (1960)
noted that much of the HCB injected subcutaneously remained near the injection
site. We did not analyze the distribution in our organisms following injection.
54
-------
No HCB toxicity studies have been published to date using bass as the
experimental animal. However, in an experiment with pinfish ( Lagodon
rhomboides ) in a sea-water system, Parrish etal. (1975) reported a maximum
concentration factor of HCB in muscle of 34,000 times the tank level (maintained
at 5.2 pg HCB/I for 42 days). Following 14 days’ exposure to a mean
concentration of 1.87 pg/I , they measured 63.5 pg/g (ppm) of HCB in liver
tissue, 36.9 pg/g in muscle and 67.4 jig/g in the entire remaining bodies. At
the end of 15 days’ exposure to 2 jig/I HCB in our experiments, the average
concentrations for each tissue were: liver, 535; muscle, 11.8; and remaining
body, 39.5 pg/g.
In general, the highest concentrations were found in the gut, kidney and
gills. Each of these organs has an abundant supply of blood and relatively
high metabolic rate. Since HCB is lipophyllic, it would be expected to
partition from the blood into the hydrophobic lipid-rich cell membranes of these
organs. This transfer would result in the passive accumulation by the tissues.
The higher concentrations in the brain as compared with muscle tissue may be
related to the relatively high lipid content of the brain. The accumulation
of HCB in these essential organs, the gut, kidney, gills, liver and brain
would suggest that if HCB is a toxic substance, these tissues may be the ones
most adversely affected.
Reference to examination of crayfish or largemouth bass tissues following
exposure to HCB has not been seen in the literature. The present findings of
tissue damage to liver of fingerling bass exposed to 3.5 ppb HCB add to those
findings thus far noted in HCB-exposed rats (Ockner and Schmid, 1961 and
Medline etal. 1973) and Japanese quail (Vos etal., 1971). Hepatomegaly reported
for man and rat (Ockner and Schmid, 1961), and the quail (Vos etal., 1971),
was not observed in the bass in which the liver weight relative to body weight
remained within normal range in those animals utilized for histological
examination. Kidney damage was noted in bass following HCB exposure (3.5 ppb)
and has been reported also in quail (Vos etal., 1971). Gall bladder necrosis,
severe in the fingerling bass after 10 days’ exposure to 25.8 ppb HCB, has not
been mentioned in the literature relative to this or other species exposed to
this pesticide.
Although to date there are relatively few animals in each category that
have been submitted to a thorough histological scanning, the findings are so
consistent that continued examination of the accumulated tissue blocks is
warranted. Furthermore, repetition of diluter exposure experiments and
subsequent depuration with attention to obtaining additional histological
sampling of those organs now known to be affected would lend further significance
to the findings. Also pertinent in such a repeat run would be histological
examination of liver, gall bladder and kidney after depuration to determine the
extent of organ recovery.
In addition to histological preparations of bass tissue, a considerable
volume of material has been prepared from a second fish species, the sailfin
molly ( Poecilia latipinna) . Mollies were used in several experiments during
the present study, and substantial information on their response to the test
compounds has been included in this report. Observations of gross pathology
of the liver and gall bladder have been noted and warrant histological examina-
tion of the accumulated material.
55
-------
Useful correlations can be made between histological observations of
tissue changes and the concentration of a substance in the various tissues
in chronically-exposed bass. As a part of a metabolic fate experiment,
another means of determining distribution used radiolabeled HCB. xtracts
made from tissues of bass exposed to a single acute oral dose of C 4 HCB
were analyzed with a scintillation counter.
The results of feeding experiments with C’ 4 HCB (See Table 15 , Section V F)
provide a means of following the distribution of ingested HCB in bass living in
clean water but given only one feeding with contaminated food. Our other
studies (section V C4) were done either by exposing the animals to a contaminated
environment for varying periods and then analyzing tissues for HCB content by
gas chromatography or by feeding contaminated food in a clean environment.
Under these latter conditions, bass showed very high concentrations of HCB in
gut and kidney. These two tissues have abundant interphases which would allow
the passive accumulation of HCB because of its lipid:water partition
coefficient.
The C 14 HCB study has slightly different results. Kidney tissue in both
cases had one of the lowest activities measured, only the remaining body being
less active in proportion to weight. It must be remembered, however, that the
previous studies involved much longer term exposure and feeding (15 days) which
would allow for much greater accumulation of HCB in an excretory organ. In
this experiment the exposure was acute (only one feeding) rather than chronic
and this may account for the difference observed in kidney tissue.
Values for feces were not obtained in previous studies. The results here
show the highest concentration in feces after acute exposure indicate that
much of the HCB is defecated soon after ingestion. This is in agreement with
inferences made from data from food chain experiments, reported in Section V 06.
Total HCB content of fish used as food for bass was compared with the final
whole-body HCB content in bass sacrificed after feeding and digestion. It was
concluded that three-fourths of the HCB ingested was lost through defecation
by the predator. Mehendale (1975) noted an elimination of only 16% of a single
oral dose of HCB in the feces of rats and 1% in urine. He reported that the
majority of HCB was retained in fat tissue.
Parke and Williams (1960) stated that much of the ingestedHCB remained in the
gut of rabbits. Work with bass in the present study agrees with this observation
(Section V F, Table 15). Since any solid waste remaining in the gut was
removed prior to extraction, the high concentration of radiolabelled HCB in the
gut indicates a high retention of HCB by the digestive system. Passive
accumulation by partitioning of hydrophobic HCB into lipid phases seems the most
likely mechanism. Liver showed more accumulation than kidney by weight but is
still 5 to 10 times lower than the gut.
Tissue extracts from C 14 HCB-fed bass discussed above were part of an
experiment to determine whether or not HCB was being metabolized. Results of
autoradiography of thin-layer chromatograms were negative, with only a single
spot migrating at the same rate as HCB. Metcalf etal. (1973) reported a
biodegradability index (BI) of .46 for HCB in mosquitofish in a model ecosystem
study. They found little indication of products of degradation, but noted the
presence of “highly polar materials and conjugates.” They noted no metabolites
of HCB in the saltmarsh caterpillar, Estigmene acrea .
56
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In uptake and depuration experiments with bass during the present study,
levels of HCB in whole body extracts decreased fairly linearly during depuration,
with 8.6 to 26.9% of the residue remaining following 13 days’ exposure to HCB-
free water. This is a substantially higher rate of loss than that observed by
Parrish etal. (1975), who reported maximum loss of 50% from tissue following
28 days’ depuratton. Their period of exposure (42 days) however, was nearly
3 times that of the present study. Time of exposure may be very important
in relationship to time of depuration. Since HCB probably partitions passively
in the organism, it may gradually accumulate in lipid deposits with slow
turnover rates. Depuration of that HCB component might be much less rapid than
the HCB in gut, liver and kidney.
The significance of contamination along the Mississippi River might be
put in better perspective if FICB levels in fish from this area are compared
with HCB levels in fish reported by authors working in other locations. Zitko
(1971) reported .002 to .006 1.Ig/g (ppm) HCB in herring from Nova Scotia and
.006 to .019 pg/g in American eel from freshwater sites in New Brunswick.
Johnson etal. (1974) reported .016 to .34 i’g/g in white perch from New York
and New Jersey. A high point of 62 pg/g was found in carp from near a source
of industrial pollution, but most other fish samples from North America sources
had less than .1 pg/g HCB in whole-body extracts (Johnson etal. 1974). In
Australia, Best (1973) reported .01 pg/g HCB from fatty tissue of freshwater
catfish. She noted 102 .ig/g HCB in blacktip shark. In our laboratory, an
analysis of ocean perch intended for feeding yielded .064 to .262 pg/g HCB
(unpublished). The perch had been purchased locally, already cleaned and
frozen.
Mosquitofish collected during our Mississippi River transect had a maximum
level of .379 pg/g HCB. These specimens were from a site near Baton Rouge,
immediately downstream from a heavily industrialized area. While this
concentration is higher than most others reported above, the effect of size
of specimens must not be overlooked. Mosquitofish weighed less than 3 grams
each, while individuals of most species analyzed by other authors weighed more
than 30g and usually exceeded 100 g. Observations in the present study supported
Murphy’s (1971) statement of an inverse correlation between fish size and capacity
for concentration of test compound.
Experiments with crayfish in field and laboratory situations provided useful
comparative information. Females accumulated more FICB than did males in both
field and laboratory. Net concentration of HCB and concentration factors were
much higher in the field than the laboratory.
HCB exposure appears to exert some physiological stress upon bass in the
form of elevated plasma cortisol levels. Experimental groups differed
significantly from controls at the 6.5% level (F-test). Further experimentation
must be done to clearly establish the relationship between HCB exposure and
corticosteriod level in the fish.
Oxygen consumption studies utilized the Gilson Differential Respirometer
instead of a flow-through respironieter which we designed, because of several
advantages which became evident during our experiments. During the season of
the year when the flow-through system was completed we had available only small
juvenile animals which could not be used in the flow-through resoirometer. The
Gilson Respirometer was more sensitive. Significant differences between control
and experimental animals did not appear within 24 hours. The flow-through
system was designed to measure oxygen consumption during periods up to 12 hours.
57
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Bass did not survive in the confined chambers of the flow-through system.
These circumstances, among others, made the Gilson unit a more useful
instrument.
The results of respiration measurements both in mollies and crayfish did
not produce repeatable patterns from one experiment to another. It must be
concluded that, in the case of HCB, oxygen consumption data from whole
animals was not particularly useful as an indication of chronic stress.
The data, presented as percentage variation of the experimental from
control rates, in Figures 21 and 22, often indicated high percentage dif-
ferences. HCB is a hydrophobic substance which is probably not metabolized
or actively transported in the system; the distribution may be through passive
partitioning through lipid phases in the organism. Any effects on respiration
may be a result of the accumulation of HCB in membranes. Many enzymes depend
upon the integrity ofthemembrane elements in the immediate vicinity of the
enzyme (Coleman, 1973). Steric distortion of membranes by hydrophobic substances
which partition into membranes may be an important and chronically cumulative
problem in toxicology. The effects on a central metabolic process, such as
oxidative phosyphorylation, may be complex. For example, if steric alteration
of membranes in the vicinity of the electron transport system in mitochondria
occurs, the transport system may be inhibited. Oxygen utilization would be
decreased. If, on the other hand, oxidative phosphorylation were uncoupled
from electron transport by the steric alteration, oxygen utilization would
increase. The absence of rate changes upon initial exposure may reflect the
time required for transfer into the organism. Variations in exposure time
and concentrations of hydrophobic substances such as HCB for longer exposure
periods may result in highly complex effects when the whole organism is studied.
Respiration studies utilizing mitochondria and specific experiments utilizing
membrane-bound enzymes may produce much more useful results. Such experiments
are particularly recommended in view of the tissue alterations which we have
reported in the present study.
An intensive investigation of cellular and subcellular damage in critical
organs utilizing electron microscopy in combination with subcellular physiological
and enzymatic studies would be an important direction in the toxicology of
hydrophobic substances. Establishment of detail syndromes of toxicological
effects which are legally defensible are much more likely at the subcellular
level, than at the organismic level. It is expected that early warnings of
toxic effects would first be observed at the levels of enzymatic activity and
subcellular structure. We recommend this direction as potentially important.
Table 16 presents an overview of the critical results from this project.
The table includes only those experimental results which clearly showed some
alteration in experimental animals. From each of those experiments only the
minimum concentration resulting in the effect is included in the table.
Maximum observed concentration in the environment at Geismar and along the
Mississippi River are included for comparison purposes.
58
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Table 16. Minimum concentrations of HCB tested that resulted in an observed
response in organisms
Method Lowest tested
level
(in Observations Section
containing
ppb) at
were
which changes
observed
discussion
a) Histology 5. Heightened exfoliation V Cl
of hepatoparicreas
epithelium in crayfish
3.5 Liver and kidney necrosis, V C2
and gall bladder
epithelium damage in bass
b) Cortisol level 6. Cortisol level in blood V C6
in blood plasma elevated (significant at
6.5% level) in bass
Maximum HCB concentrations found in environmental survey
Water Mud *
Area of High 74.9 ppb (pg/i) 53,130 (74,970) ppb (pg/Kg)
Concentration
(Gei smar)
Overview 90.3 ppb (pg/e) 870. (1,950) ppb (pg/Kg)
(Plaquemine) (Darrow)
*: Figures corrected for dry weight in parentheses.
59
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1 REPORT NO 2.
EPA-560/6-76-009 I
4 TITLE AND SUBTITLE
An Ecological Study of Hexachlorobenzene (HCB)
3. RECIPIENTS ACCESSIO NO.
5. REPORT DATE Date of
April 9, l976Printing
6.PERFORMINGORGANIZATIONCODE
7 AUTHOR(S)
J. L. Laseter; C. K. Bartell; A. L. Laska; D. G.
Holmquist; D. B. Condie; J. W. Brown and R. L. Evans
8. PERFORMING ORGANIZATION REPORT NO
9 PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Biological Sciences
University of New Orleans
Lakefront
New Orleans, Louisiana 70122
10. PROGRAM ELEMENT NO.
2LA328
11 CONTRACT/GRANTNO
PA 1 2689
- - -
12 SPONSORING AGENCY NAME AND ADDRESS
Environmental Protection Agency
Office of Toxic Substances
4th and M Streets, S.W.
Washington, D.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final Report
14.SPONSORINGAGENCYCODE
15. SUPPLEMENTARY NOTES
15 noo u nn .
Hexachlorobenzene (HCB) has been found in the environment in southeastern Louisiana
in addition to other parts of the world. In this region it is a byproduct of the petro
chemical industry. HCB is a fungicide and has been found to accumulate in fatty tissue
of wild and domestic animals. It has had toxic effects upon humans. A number of cases
of porphyria cutanea tarda were traced to ingestion of treated grain. In this study,
soil, water and organism samples were collected periodically in 1974 and 1975 from
sites in southeastern Louisiana, with emphasis along the MississIpni River and an in-
dustrial region of known contamination of HCB near Geismar, Louisiana, Maximum HCB
concentrations in water from the two areas were 90.3 and 74.9 pg/L (ppb). Maximum HCB
concentrations in soil from the two areas were 874 and 53,130 jig/Kg (ppb). Laboratory
experiments with the compound included acute toxicity studies in aquatic systems and
through injection in fish and crayfish. Accumulation and depuration rates were deter-
mined and observations made with histological slides of tissue. Other potential mea-
sures of stress were made, including blood cortisol levels and oxygen uptake rate. HCB
was not lethally toxic during our experiments, but its chronic effects were noted in
histology and cortisol levels. Damage to kidney, liver and gall bladder in fish was
observed and serum cortisol levels were elevated in response to exposure to HCB in
ambient water. Highest levels of uptake were measured in gut, kidney and gills, attain-
ing a maximum concentration factor of 181,000X in gut of bass.
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