U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
PB-252 671
AN ECOLOGICAL STUDY OF HEXACHLOROBUTADIENE (HCBD)
NEW ORLEANS UNIVERSITY
PREPARED FOR
ENVIRONMENTAL PROTECTION AGENCY
9 APRIL 1976
-------
-------
EPA 560/6-76-010
AN ECOLOGICAL STUDY OF HEXACHLOROBUTADIENE (HCBD)
ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF TOXIC SUBSTANCES
WASHINGTON, D.C. 20460
April 1976
-------
TECHNICAL REPORT DATA
(Please read Imtnutiont on the nvtne before completing/
i. REPORT NO.
EPA-560/6-76-010
3. RECIPIENT'S ACCESSION NO.
4. TITLS AND SUBTITLE
An Ecological Study of Hexachlorobutadlene (HCBD)
8. REPORT DATE Date Of
April 9, 1976Printing
6. PERFORMING ORGANIZATION CODE
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/GRANT NO.
EPA 68-01-2689
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. SPONSORING AGENCY CODE
IB. SUPPLEMENTARY NOTES
16. ABSTRACT . •
Hexachlorobutadiene (HCBO). has been found in the environment in southeastern Loui-
siana 1n addition to other parts of the world. In this region it is a byproduct of the
petrochemical industry. HCBD has been used as an insecticide in central European
orchards and vineyards where excessive contact has caused illness in humans. In this
study, soil, water and organism samples were collected periodically 1n 1974 and 1975
from sites in southeastern Louisiana, with emphasis along the Mississippi River and an
Industrial region of known contamination of HCBD near Geismar, Louisiana. Maximum HCBD
concentrations in water from the two areas were 1.9 and 4.7 vig/£ (ppb). Maximum HCBD
concentrations in soil from the two areas were 790 and 1,080 yg/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.
Accumulation in fish was erratic and fairly low, with concentrations normally well
below SOX. In algae and sediment* concentration factors were between 200X and 300X. In
water, LCso's were found above approximately 2mg/£ (ppm) for fish and crayfish. Expo-
sure to HCBD in ambient water caused histological damage to hepatopancreases of cray-
fish and liver and kidney of bass. It also elevated serum cortisol level in bass.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
8. DISTRIBUTION STATEMENT
19. SECURITY CLASS (ThisReport) 121. NO. OF PAGES
20. SECURITY CLASS (Thispage)
EPA form 2120-1 (»-7J)
-------
EPA 560/6-76-010
AN ECOLOGICAL STUDY OF HEXACHLOROBUTADIENE (HCBD)
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
10,
-------
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 Boyer, 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.
^^
-------
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 commercial products constitute
endorsement or recommendation for use.
-------
TABLE OF CONTENTS
Page
List of Tables vi
List of Figures vli
I. Summary and Conclusions 1
II. Introduction . . . 2
III. Field Studies,Methodology 4
A. Overview 4
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. . . . .12
D. Gas Chromatography • 16
E. Mass Spectrometry. • • 17
F. Corticosteroid Analysis. ........... 17
G. Fish Blood Hematocrit • 18
H. Respirometry 18
I. Histology. 19
J. Photochemistry . 19
V. Results 20
A. Field Studies- ......... 20
i • <
1. Overview • • • 20
2. Area of Higher Concentration ....... 20
B. Acute Toxicity 24
1. Crayfish Injections. . 24
2. Fundulus Injections- ........... 24
3. Crayfish LCso's- .......... , . . 25
iv ' . ' .
-------
Page
4. Fish LC50's 25
5. General Pathological Effects . 27
C. Chronic Toxicity '.<.... 71
\
1. Crayfish Tissue Morphology - Normal and Pathological. . 27
2. Bass Tissue Morphology - Normal and Pathological. ... 31
3. Retention and Distribution of HCBD in Crayfish 31
4. Retention and Distribution of HCBD in Bass 32
5. Fish Blood Hematocrit 35
6. Fish Corticosteroid Level 35
D. Accumulation and Clearance 36
1. Crayfish 36
2. Mollies 38
3. Bass 40
4. Algae 43
5. Bottom Sediment 43
6. Effect of Food Chain . 43
7. Crayfish Uptake of HCBD in Field Environment. .... . 44
E. Respirometry 47
1. Crayfish. . . .' 47
2. Mollies 48
F. Photochemistry 5°
VI. Discussion of Results 51
Literature Cited 58
-------
LIST OF TABLES
No. Page
1 Field localities for overview collections . . 6
2 Concentrations of HCBD in water and soil samples from sites
removed from Mississippi River transect 22
3 Mean concentrations of HCBD in Mississippi River mosquitofish
(Gambusia affinis) in comparison with levels measured in water
and soil 22
4 Mean concentrations of HCBD in crayfish (Procambarus sp.) from
ditches in comparison with levels measured in soil 22
5 HCBD residues in water, mud and organism samples from area of
higher concentration 23
6 Injections of HCBD in oil into crayfish (Procambarus clarki) . . 24
7 Distribution of HCBD residues in tissues of crayfish
(Procambarus clarki)exposed to 2.97 ppb for 10 days. . 32
8 HCBD content of tissue samples from bass exposed to HCBD in
diluter for 10 days 33
9 Hematocrit of. bass (Micropterus salmoides) exposed to HCBD ... 35
10 Whole-body HCBD residue concentrations in male and female
crayfish (Procambarus clarki) during uptake and depuration ... 37
11 Whole-body HCBD residue concentrations in sailfin mollies
(Poecilia latipinna) during uptake and depuration. . 39
'
12 Whole-body HCBD residue concentrations in largemouth bass
(Micropterus salmoides) during uptake and depuration 41
13 Concentration of HCBD by a green alga, Oedogoniurn cardiacum
exposed to a flowing solution of 16.9 ppb HCBD in water 43
14 HCBD concentrations in largemouth bass (Micropterus salmoides)
feeding on contaminated sailfin mollies (Poeci 1 iaTatipinna) . . 44
15 HCBD content of crayfish exposed to 4.3 ppb HCBD in a field
locality 44
16 Short and long-term exposure of crayfish to a contaminated
field site 45
17 Effect of time of exposure to HCBD on respiration rate of
juvenile crayfish (y£02/mg/hr) 47
v^
-------
No. Page
18 Effect of time of exposure to HCBD (0.5 ppm) on respiration rate
of juvenile mollies (y£ C^/mg/hr) ................. 49
19 Summary of acute toxicity experiments with HCBD ......... %
20 Minimum concentrations of HCBD tested that resulted in an
observed response in organisms .................. 57
LIST OF FIGURES
1 Localities sampled for presence of HCBD 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 HCBD .................. 9
5 Gas chromatographic traces of HCB and HCBD (chromatographic
conditions as described in Section IV. D.) ............ . 10
6 Water Treatment system ............ ......... ..... 11
7 Modified proportional diluter ................... 13
8 Major test tank .................... ...... 14
9 Model ecosystem .......................... 14
10 Distribution of HCBD in soil along the Mississippi River,
Louisiana ................... ...... .... 21
11 Photomicrographs of bass tissue sections1 ..... ........ 29
12 Average HCBD concentrations of tissue samples from bass exposed to
HCBD for 10 days .................... ..... 34
13 Mean plasma cortisol levels (pg/100 ml) in bass (Micropterus
salmoides) exposed to HCBD for ten days.. . , • ...... ..... 35
14 Uptake and depuration of HCBD in crayfish (Procambarus clarki)
exposed to a mean concentration of 3 ppb (ug/£) .......... 38
15 Uptake and depuration of HCBD in sail fin mollies (Poecilia
latipinna) exposed to a mean concentration of 11.3 ppb (vg/t). . . 40
16 Uptake and depuration of HCBD in bass (Micropterus salmoides). . . 42
17 Levels of HCBD in crayfish during depuration following 10 days
exposure to a contaminated environment (4.7 ppb HCBD) ....... 46
18 The effect of time of exposure to HCBD on respiration rate of
juvenile crayfish (Procambarus clarki) ......... ..... 48
vii
-------
No. Pa_ge
19 The effect of time of exposure to HCBD (0.5 ppm) on respiration
rate of juvenile mollies (y£ 02/mg/hr). . 49
20 Gas chromatographic separation of photoproducts resulting from
ultraviolet irradiation of HCBD 50
-------
I. SUMMARY AND CONCLUSIONS
The purpose of the study summarized in this report was to determine
the distribution and toxic effect of hexachlorobutadiene (HCBD) in selected
aquatic systems and organisms. Field collections of soil and water were
made in southeastern Louisiana, and were supplemented by samples of aquatic
organisms from sites where available. In laboratory experiments, the
principal animals used were the red swamp crayfish, Prpcambarus clarki , the
sailfin molly, Poecilia lati'pinna. and the largemouth black bass, Micropterus
Both acute and chronic effects of HCBD were studied.
's were established for organisms in flow- through experiments which
utilized a modified proportional diluter. Twenty-five to 30-hour LCgn's
resulted when sailfin mollies were challenged with 4.2 to 4.5 ppm (yg/£)HCBD.
Concentrations of 1.2 to 1.7 ppm HCBD resulted in LCso's of 77 to 138 hours
for bass. HCBD concentrations of 2.7 and 2.9 ppm resulted in 57-hour and
80-hour LCso's, respectively, for adult crayfish. When dissolved in oil,
HCBD produced lethal effects following administration by injection. Twenty-
four hour LDso's resulted from injections of 3.4 mg/g body weight in crayfish
and 1.7 mrj/g body weight in Gulf killifish.
The compound did not accumulate to high levels in test animals, and
rates of uptake as well as distribution in organs were irregular. 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 liver and kidney of bass exposed to 32.8 ppb HCBD
for 10 days. Hepatopancreas in crayfish was affected by 10 days' exposure
to 3.7 ppb HCBD.
Analysis of blood samples taken from freshly-sacrificed fish revealed
no differences in hematocrit following chronic exposure to HCBD; however,
serum cortisol levels were observed as indicators of stress. HCBD signifi-
cantly elevated cortisol level in bass exposed to 3.43 ppb HCBD for ten days.
Oxygen consumption rates were not significantly altered by HCBD 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 compounds by algae (Oedogonium cardiacum) and sediment. Concentration
factors remained below 300X for both during the relatively short-term
experiments. A food-chain study compared the relative accumulation effects
on a predator feeding upon HCBD-contaminated food fish and a similar predator
taking up the compound both through its food and through contaminated water.
Uptake of HCBD was erratic and bass accumulated more from water than from
food when taken as an average.
Laboratory experiments were compared with patterns of accumulation under
field conditions. HCBD-free crayfish were caged, placed at a contaminated
field site and removed periodically for GC analysis, or for laboratory
depuration and subsequent GC analysis. HCBD was not concentrated to a great
extents and accumulated irregularly. Concentration factors for crayfish left
in the field site for 17 days ranged from 7.8 to 300X (33.7 to 1,290.3 yg/Kg
HCBD). The great majority of HCBD accumulated in specimens during a 10-day
exposure was lost following 12 days' depuration.
1
-------
.Since HCBD 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. Irradia-
tion of HCBD yielded several products of higher molecular weight which
decrease in number and concentration with exposure time.
II. INTRODUCTION
4
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. HCBD was found to be
a co-contaminant of the processes causing these problems. The field portion
of this study was undertaken to measure HCBD levels in the environment in
southeastern Louisiana. Laboratory experiments were aimed at observing
acute and chronic effects of the compound under controlled conditions.
Hexachlorobutadiene (HCBD), a chlorinated compound, is a volatile dense
liquid often found in association with HCB as an industrial by-product.
Many studies of its toxicity are oriented toward its use as a controlling
agent for insect pests, e.g., the vine louse (Khokhryakova e_t al., 1963),
the housefly larva (Levinson, 1955), and the apple tree borer "[Vashchinskaya,
1971). Subsequent changes in the microflora following application have been
noted (Khokhryakova ejt aK, 1963, Mirzonova and Perov, 1968, Mukasheva, 1974).
Toxic effects of HCBD have been observed in metabolic processes of rats
(Gudumak, 1968) following ingestion. Jacobs £t a_l_. (1974) noted accumulation
of the compound in fatty tissue of rats, and Dmitrienko (1972) observed kidney
damage in rats fed HCBD. Murzakaev (1963a) provided LD5Q values for HCBD in
mice, rats and guinea pigs and reported effects upon the central nervous
system of rats subjected to prolonged exposures of low doses (Murzakaev, 1967).
In the aquatic environment, effects of HCBD upon fish behavior were, reported
by Hiatt e_t al_. (1953), and Stroganov (1968) presented toxicity data for fish
(Leucaspius delineatus) exposed to HCBD.
Russian farm workers were found to suffer several maladies as a result of
exposure to HCBD fumigation (Krasnyuk e_t aK, 1969). These, and other conse-
quences of agricultural uses of HCBD have prompted studies to determine
maximum permissible limits in water supplies (Murzakaev, 1963b, 1964) and air
near industrial sites in the USSR (Poteryaeva, 1972).
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 HCBD in the southern central portion of
the state of Louisiana, extending eastward to Mississippi and following the
Mississippi 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 immediate vicinity of the Vulcan Materials Company at
Geismar, La. Here a peribdic monitoring of HCBD concentrations in soil, water
and selected aquatic organisms at various trophic levels was conducted. During
the course of field worki localities in this latter 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 HCBD upon local
fauna more precisely, laboratory experiments were designed to expose aquatic
biota to the test compound. Histological preparations, behavioral observa-
tions and gas chromatographic analyses were used to obtain data and gain a
greater understanding of the significance of the presence of HCBD in the
environment.
Tests of acute toxicity have been central to studies of contaminants
introduced into environmental systems. Tarzwell (1966, 1971) has discussed
the use of acute toxicity levels and of application factors in 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 HCBD. The
physiological and morphological indications of long term effects of sublethal
concentration of HCBD are 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 commonly-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 HCBD in aquatic systems. Condition of animals
was observed regularly, and any abnormal behavior or appearance was noted for
inclusion as possible pathological effects of HCBD.
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 HCBD 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 HCBD. Samples
of blood taken from fish were used in determinations 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 Porthe'-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 HCBD in ambient water of fish and
crayfish, respirometry experiments were carried out in the laboratory.
Accumulation and clearance of HCBD 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 HCBD 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 HCBD was determined
in animals, initially free of the compound, which were placed in an HCBD-
contaminated field locality. ,
Experiments were designed to observe possible breakdown products of HCBD.
The 'substance was subjected to UV light and the resultant photoproducts
determined.
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
were made 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 HCBD in soil and water.
r,uir or MUI co
SCALE OF MILES
ttt : « • SITE SAMPLED
Legend to Figure 1.
Sites
A - T
B^E^ U-Z
i, i i, i i i, v
iv
vi
vi i
East bank of Mississippi River
West bank of Mississippi River
Inland, east of Mississippi River
Pass Manchac, inland tidal lake
Spillway of Mississippi River, connected to Lake Pontchartrain
Bay
5 ' •- ••••'• '
-------
Table 1. Field localities for overview collections.
Type of Sample Type of
Location Description Water* Soil* Biota**0rganism
A Hwy. 90, North of Baton Rouge (E)
B Intersect, River Rd., La. Hwy 327 (E)
BI Addis at La. Hwy. 990 & La. 998 (W)
82 PTaquemine at La. Hwy, 988 & La. 1148 (w)
C 5 Mi. South of B (E)
D Sunshine (E)
E Carville (E)
E! La. 405, 4 mi. South of White Castle (W)
F Ashland Plantation (E)
G Darrow (E)
H Intersect, La. 44 & La. 942 (E)
I Romeville (E)
J Convent (E)
K Lutcher (E)
L Garyville (E)
M Reserve (E)
N Laplace (E)
0 South of Bonnet Carre Spillway (E)
P Destrehan (E)
Q St. Rose (E)
R River Ridge (E)
S New Orleans, River Rd. at Causeway (E)
T New Orleans, Audubon Park (E)
U Lower Algiers (W)
V Belle Chase (W)
W Myrtle Grove (W)
X Mile Marker 42 (W)
Y .8 Mi. South of X (W)
Z Port Sulphur (W)
i Walker (I)
ii Hammond (I)
Hi Covington (I)
iv Pass Manchac (T)
v Sorrento (I)
vi Spillway (S)
vii Lake Grand Ecaille (B)
t
Key
E: East bank of Mississippi River
W: West bank of Mississippi River
I: Inland, east of Mississippi River
T: Tidal lake
S: Spillway
B: Bay
X
X
X
X.
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
.X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
F
F
C, F
C, S
C, F
C, F
C, F
F & C
C
C, F
F & C
C
C
F
Sh
C, F, S
C, F
C, Sh
C
F, Sh, S
C, F
C, F
C
C
C
F, Sh, Cl
C
C
C, F
Key_
C: Crayfish
Cl: Clam
F: Fish
S: Snail
Sh: Shrimp
* : GC analyses performed on soil and water samples from all locations.
**: GC analyses oerformed on biota from selected locations (Ref. Tables 3 & 4
-------
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 in 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 m.iuliirly,
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 common green alga provided food and substrate for
microcrustacea. 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 s.a 1 mo ides") which
were stocked as sport fish. At the Recreation Pond, samples of soil, water,
aquatic plants, invertebrates and fishes were collected and analyzed to
determine HCBD 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 HCBD 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 sp.) were taken occasionally, along with regular samples
of water, mud and fish.
Organisms were collected on several occasions, and their HCBD content
determined and tabulated. Concentration factors were computed as a function
of HCBD 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 HCBD 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 for algae samples in the Methodology Section (IV.D.) of this report.
Concentration is expressed in terms of wet weight of solid material.
-------
Figure 3. Location of Geismar field sites (area of higher concentration)
POND ADJACENT TO
HEX LANDFILL
-------
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 1,3-hexachloro-
butadiene (HCBD) was obtained from Chem Service, West Chester, Pa. 19380.
A purity check vi^ gas chromatography was made using a standard of 0.1 ppm
of the pesticide in 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 90° magnetic sector mass spectrometer (MS) equipped with
a Bell and Howell Datagraph 5-134 galvanometer driver recording oscillograph,
Hexachlorobutadiene was introduced via the batch inlet system. 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 HCBD
starting material is given in Figure 4. Figure 5 represents gas chromato-
graphic traces of the two compounds, HCB and HCBD.
Figure 4. Mass spectrometry plot of HCBD.
60
60
60
147
ISO
140
260
m/e
-------
Figure 5. Gas chromatographic traces of HCB and HCBD (chromatographic
conditions as described in Section IV.D.).
LU
2
S3
cr
o
a:
§
a:
STANDARD
HCBD
HCB
10
TIME (MNUTES)
20
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, Oedogom'um cardiacurn
(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 cTarki) 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 'HCBD-free chicken meat and small
fish.
Small estuarine fishes, including saiTfin 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 sp.) 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.
10
-------
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 HCBD 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 the 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,
diagrammed 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.
Figure 6. Water Treatment system.
V
/
//
'
float
switch
float
switch
-
water // submersible ' \ jj
in ^J pump •-•^^*l \
i! ^partition
II B
-
*
water
out
CM
c
to
(U
c
O
$_
IB
O
TJ
(U
-------
C. Static and Flow-Through Assay Systems
Aqueous experiments were of two basic types, static and flow-through.
Static tests with mollies (Pbec11ia 1 a t ipinna) were carried out in five-
gallon glass jugs filled approximately half full with 10£ 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 wi.th crayfish (Procambarus 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 propor-
tional diluter. The design for this apparatus was based upon developments
by DeFoe (1975) and preceding workers (Benoit and Puglisi, 1973, 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.
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 wi.thin each flow-splitting chamber deliver the water
to respective test tanks thr.ough glass tubing.
Each of the six major test tanks (Fig.8 ) have a filled capacity of
70£, but were usually filled to a level of 30£ as controlled by an adjustable
*: 4528 Pitt St., Duluth' Minn. 55804.
12
-------
Figure 7 . Modified proportional diluter.*
solenoid
flow-
splitting
chambers
water metering
cells
venturi vacuum system
microswitch
teflon toxicant
tube from injector
* one of six similar units schematically represented.
13
-------
Figure 8. Major test tank.
•70 cm-
36 cm
.overflow standpipt;
Figure 9. Model ecosystem.
water supply tube
14
-------
overflow standpipe. The smaller tanks (Fig. 9) are model ecosystems in which
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 120£ 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 determined by the number
and type of samples needed for analysis. Availability of animals and their
tolerance to conditions were other factors contributing to this figure.
HCBD 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 HCBD in the test animals' aquatic medium. Some injection experiments, however,
were carried out with HCBD dissolved in peanut oil. A dosage of .01 cc/g 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 HCBD were used.
In an effort to achieve a more uniform dispersion of HCBD to be injected into the
hemocoel of crayfish, we prepared an emulsion as the carrier of HCBD. HCBD was
first dissolved in peanut oil. The oil was then sonicated with lecithin as an
emulsifier in 0.38 M sucrose solution.
Fish were also used in injection experiments. In the absence of sufficient
numbers of bass, the Gulf killifish (Fundulus grandis) was selected for use in acute
injection experiments. A stock solution of 1.2 ml HCBD was dissolved in 6 ml of
peanut oil. Dosages administered intraperitoneally were 3.4, 1.7, .8 and .2 mg/g
body weight, and an oil control, injected at the rate of .01 ml/g body wt. Each
injected group consisted of 10 fish. Following injection, fish were held in HCBD-
free water in diluter tanks until they succumbed. Some survivors, including the
controls, were kept for more than six weeks.
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 HCBD by a common filamentous green alga, a culture of
Oedogonium cardiacum (I.U. #39) was prepared. Original stock was grown in 4£ flasks
in 31 of 1:1 3N BBM; H£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 HCB experiment.
Protocol for diluter tanks consisted of acetone and water controls and 16.9
ppb HCBD solution. Illumination to the tanks was provided by three Sylvania lifeline
15
-------
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 refriger-
ated at approximately 3°C. Preparation of samples for GC analysis is discussed
in section III.D. of this report.
An experiment was designed to measure uptake of HCBD 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 HCBD, were screened through 1.6 mm 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 HCBD 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 fn Section III.B. HCB and HCBD content of the
water was monitored. Periodically, crayfish were removed from the cages 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 Eberbach
reciprocating action shaker. Following passage through a separatory funnel an
aliquot from the benzene layer .was ready for injection into the GC. In pre-
paring 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 HCBD were expressed tn terms of wet weight of the algae.
16
-------
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
thre6 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 571QA gas chromatograph (GC) equipped with an electron capture
detector utilizing °^Ni 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-1 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 ml/min.
The injection port temperature was held at 250°C and the detector was set at
300°C.
Quantification was accomplished using external standards of 1,3-hexachloro-
butadiene at concentrations of 1 ppm and 0.1 ppm in a benzene solvent. Concentra-
tions of HCBD in water were computed in terms of yg/£ of sample, and expressed in
parts per billion (ppb). Other samples reported in yg/g are expressed in parts
per million (ppm).
E. Mass Spectrometry
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 4 mm I.D. glass
column packed with 10% OV-1 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 (lights on at 0730)
for the entire duration of exposure to HCBD. 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 Porthe-Nibelle,
1974, Meier and Srivastava, 1975). A 10 yl 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
17
-------
corticosteroid 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 N2.
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
uCi of 1 jZH^-cortisol (obtained from New England Nuclear Corp.) and 5 ml of pooled
male horse serum (obtained from the LSD Veterinary School) with distilled water to
volume. Horse serum was used as a source of corticosteroid binding globulin
(CBG) since Ficher ejb aJL (1973) have found that it gives greater specificity
for cortisol.
After incubation, the samples were transfered to an ice bath for 30 min and
then 40 ml 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.
H. Respirometry
the effect of exposure of juvenile crayfish and mollies to HCBD was measured
using a Gilson Respirometer (Unbreit, et_ al., 1972). Each set of tests using
juvenile crayfish (average weight = 10 mgTemployed animals from one hatching of
one female. ..Juvenile mollies (average weight = .3 g) were collected from uncontami-
nated environments and used immediately after collection.
Five crayfish were placed in each respirometer vessel in 3 mis of water. In
the experiments using mollies a single fish was placed in each vessel in 7 mis 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
daily. Mollies were kept in deionized water reconstituted to 2.5 ppt with artificia]
sea salt. Crayfish were kept in deionized water reconstituted to 1 ppt. The experi
mental groups were exposed to water that had been stirred with appropriate amounts
of HCBD for at least 24 hours before the animals were placed in the solutions.
18
-------
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 were determined for
HCBD 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 animals.
I. Hj stology
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 HCBD utilized for GC analysis. Animals were sacrificed on the day of termination
of toxicant accumulation. Brain, green gland, hepatopancreas, one gill and a 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 dissecting 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 Harris1 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.
J. Photochemistry
Solutions of HCBD in both hexane and benzene (1 yg/10 ml) were irradiated at
varying time intervals. HCBD was irradiated for 15 minutes, 30 minutes and 60
minutes. Irradiations at 253.7 nm were conducted in serum capped quartz test tubes
employing a Rayonet RPR-100 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.
19
-------
V. Results
A. Field Studies
Field work consisted on two distinct phases. The first, a regional
overview, dealt with HCBD 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
i
Figure 1 shows localities in the immediate vicinity of the Mississippi
River sampled for HCBD residues in collections of water, mud, crayfish and
fish made during the first phase of field work. Concentrations of HCBD in
mud and soil are presented in Figure 10. Supplementary sites are outlined
in Table 1 in the Field Methodology section. Comparisons of HCBD residues
in soil, water and organisms from various sites can be seen in Tables 2
through 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 HCBD 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 s'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.
20
-------
Figure 10. Distribution of HCBD in soil along the Mississippi River,
Louisiana. Baton Rouge - Port Sulphur transect.
900.
800
I700
e eoo
|.
" 100
£ 500
s
°- 200
100
LOCALITY
CQDI_
DITCH HUD
11
800
700
3
73 eoo
a
g 500
S 100
oe
£ 500
co
I 200
100
LEVEE SOIL .
,
1 HCBD
1
1 .
B C D E E, F G II I
MMOPQRSTUVKXYZ
LOCALITY
PIPE
LOCALITY
CflDi_
LOCALITY
A HMY. 90, NORTH OF BATON ROUGE
B INTERSECT, RIVER RD.. LA. HHY, 527
B, Aoois AT LA. HNY. 990 t LA. 998
B, PLAOUEMINE AT L». HMY. 988 I LA. 1118
C 5 MI. SOUTH OF B
D SUNSHINE
E CARVILLE
E, LA. 105. 1 MI. SOUTH OF WHITE CASTLE
F ASHLAND PLANTATION
G DARROM
H INTERSECT. LA. 11 I U. 912
I ROHEVILLE
J CONVENT
K LUTCHER
L GARYVILLE
H RESERVE .
N LAPLACE
0 SOUTH OF BONNET CARRE SPILLNAY
P DESTREHAN
0 ST. ROSE
R RIVER RIDGE
S NEH ORLEANS. RIVER RD. AT CAUSEMAY
T MEN ORLEANS, AUDUBON PARK
U LONER ALGIERS
V BELLE CHASSE
W MYRTLE GROVE
X KILE MARKER 12
Y .8 MI. SOUTH OF X
Z PORT SULPHUR
21
-------
Table 2 . Concentrations of HCBD in water and soil samples from sites
removed from Mississippi River transect.
Location
Walker
Hammond
Covington
Pass Manchac
Sorrento,
SpiVlway
Lake Grand
Ecaille
Code
(i)
(11)
(iii).
(iv)
(v)
(vl)
(vll)
Water
HCBD in pg/1 (ppb)
.7
1-2
*
1.5
.7
1.5
1.0
Soil
HCBD in pg/Kg (ppb)**
f *
*
*
*
*
321.5 (433.0)
21.6 (62.6)
*: < .7 ppb
**: Figures in parentheses are corrected for dry weight of sample.
Table 3 . Mean concentrations of HCBD in Mississippi River mosquitofish
(Gambusia affinis) in comparison with levels measured in water
and soil.
Water Soil Fish
Location Code HCBD in yg/1 (ppb) HCBD in yg/Kg (ppb)**HCBD in yg/Kg (ppb)
Garyville (L)
Romeville (I)
Baton Rouge (A)
.9
1.4
1.9
793.4 (1001.3)
112.8
197.4
827.3
*: < .7 ppb
**: Figures in parentheses are corrected for dry weight of sample.
Table 4 . Mean concentrations of HCBD in crayfish (Procambarus sp.) from
ditches in comparison with levels measured in soil.
Location
Walker
Romeville
Ashland
Da r row
Code
(i)
(I)
(F)
(.6)
Soil
HCBD in yg/Kg (ppb)**
. •*
489.9 (665.3)
204.9 (410.1)
219.4 (420.2)
Crayfish
HCBD in yg/Kg (ppb)
10.6
24.1
70.1
22.9
*: < .7 ppb
**: Figures in parentheses are corrected for dry weight of sample,
-------
Table 5. HCBD residues in water, mud and organism samples from area of higher concentration.
(Number of organism samples in brackets; Figures in parentheses corrected for dry weight of mud samples)
ro
CO
Organism # of
Mean HCBD
concentration Concentration Abiotic Dec. April May Aug. Oct.
analyses in yg/Kg(ppb) factor Component 1974 1975 1975 1975 1975
Recreation Pond
Plankton
Chara
Najas
Eleocharis
Physa [3]
Gambusia [27]
Lepomi s [45]
Micropterus [1]
1
1
1
1
1
3
15
1
1016. 2,701x
12- 120x
9- 13x
20. 29x
345 • 3,446x
89°- 2,225x
600 • l,546x
liver: 2405. - l ,093x
muscle: 72. 33X
Water (ppb) * !-2 -7 -1 .38
Mud (nob) * 19°- 58°- 55°-
Mud tppb> * (27Q } (67Q } (73Q }
South Effluent
Anisoptera [1]
Procambarus [9]
Gambusia [24]
1
3
8
315. 7,507x
997. l,404x
817. l,151x
;
Water (ppb) ** ** i.o .04 .65t
Mud (ppb) ** 250. ** 700. 1080. t
(430.) (1010.M2370.)
Landfill Pond
Gambusia [27]
9 16,205. 3,525x
* no sample taken.
** no value determinate from GC analysis.
t site changed due to construction.
Water (ppb) ** 4^5 4.7 * 4.49
Mud (ppb) * 380. ** * 650.
(500.) (920.)
-------
B. .Acute Toxicity
1. Crayfish Injections
Several experiments were carried out in which HCBD, dissolved in
peanut oil alone, or prepared as an emulsion, was injected into crayfish.
Although acute toxicity was observed in the emulsion injection experiments,
GC analysis of the emulsions later indicated large variations in the
concentration of HCBD in the Injected material. These results from GC
indicated an unstable emulsion and rendered the results of six injection
experiments useless.
Because of the above difficulties a more direct method was adopted.
HCBD was dissolved in peanut oil at three different concentrations. Each
animal was injected with .01 cc/g body weight. As in earlier experiments,
oil was injected directly into the hemocoel of each animal, at the base
of its second walking leg on the left side. Results of this work are given
in Table 6 . HCBD was obviously toxic to crayfish at doses in excess of
336 yg/g (ppm) body weight, when injected in an oil medium.
Table 6. Injections of HCBD in oil into crayfish (Procambarus clarki).
Concentration
of solution
336 ppt
33.6 "
3.4 "
Oil control
N
injected
10
10
10
10
yg/g
body wt.
3,360
336
34
Number of crayfish
LD5Q surviving 6 days
<1 days
5 days
0
o
7
10
2. Fundulus Injections
Dosages of HCBD administered to Gulf killifish through intraperitoneal
injection were 3.4, 1.7, .8 and .2 mg/g body weight, and an oil control, all
injected at the rate .of .01 ml/g body weight. The first three of these
concentrations were .four-hour, 20-hour and 18-day LDso's, respectively. No
fish died during a three-week period at the lowest dosage, 200 ppm.
24
-------
3. Crayfish
In static experiments, four groups of eight animals each were distributed
as follows: water controls, water with acetone controls and experimental at
5 and 10 ppm, respectively (nominal concentrations of HCBD). Two water control
crayfish molted and died during the test. Two acetone control animals died;
one of these had also molted first. Two crayfish in 10 ppm jars died; one
of these had also molted first. These results do not suggest that HCBD caused
death under these particular experimental conditions.
In the diluter, 64 full -size adult crayfish were tested in two tanks
(nominal: 10 ppb; GC J: 2.97 ppb HCBD), 32 animals were in water with acetone
and 32 others were water controls. Uptake lasted 10 days and depuration
another 10 days. Of a total of 64 crayfish, two experimental animals in a
single HCBD tank and two controls in the water control tank died during the
test. Their deaths were apparently the result of cannibalism following
molting. No deaths, therefore, could be attributed to exposure to this
concentration of HCBD.
'In another diluter experiment, nine crayfish were held in each of two
experimental tanks with GC-measured HCBD levels of 2.7 ppm and 2.9 ppm. Half
of the animals were dead within 57 hours in the first tank, while half of the
crayfish in the second tank had died within 80 hours. Controls in uncontamin-
ated water and water with acetone carrier survived the experiment.
A static experiment yielded acute toxicity data for juvenile crayfish in
HCBD. In a series of animals, each of which weighed approximately 12 to 14 mg,
the 70-hour LC50 was 5 ppm (nominal concentration).
4. Fish LC5Q's
Tests with both bass and mollies were carried out to provide LC$Q data.
The first test with bass was a static experiment in which tanks were kept in
an environmental chamber. Temperature was maintained at 25°C and a 12:12
light:dark photoperiod was maintained. Tanks were filled to 10 liters with
deionized water reconstituted to 1 ppt salinity with Rila marine mix. HCBD
was inoculated directly into the experimental tanks. Droplets remained dispersed
1n the tank but not dissolved, indicating that actual concentration in water
was lower than calculated doses. In this preliminary experiment, three bass
each were placed in tanks with nominal concentrations of 40, 20 and 5 ppm yg/£)
and a water control .
In these static experiments, a nominal calculated concentration of 40 ppm
HCBD killed all three test animals in less than 45 hours. The average body
HCBD concentration of two fish analyzed was 2.06 yg/g (ppm). A concentration
of 20 ppm killed all three within 70 hours. The HCBD content of one fish
analyzed was 2.83 yg/g. All three fish in the tank with 5 ppm HCBD died in
less than 96 hours.
25
-------
Findings' from the static experiment were^ applied to tests in the diluter.
The first flow-through experiment used six bass per tank. In one tank, all
six animals died within 20 hours of being subjected to 2.5 ppm HCBD, as
determined by GC. In another tank, half of the six animals tested were dead
within 43 hours and all had died within 48 hours at a measured level of 4.3
ppm HCBD. At the GC-measured level of 2.2 ppm HCBD, all six fish died in
less than 114 hours. At a measured level of .9 ppm HCBD, all specimens were
distressed within 24 hours, immobilized and inverted in 114 hours, but revived
with depuration at the termination of the experiment.
Two bass experiments were carried out at substantially lower concentrations
of HCBD. In the first of these, tanks of twelve fish each were subjected to
the following conditions: 3.43 ppb HCBD, 31.95 ppb HCBD (both measured on GC;
mean of 3 separate analyses), water control and acetone control. All specimens
survived 10 days of exposure in thediluter, at which time they were sacrificed
and frozen for subsequent GC analysis.
In the second experiment, bass were tested in the following format:
Tank A B C D E F
Number 11 11 10 10 11 11
of Fish
Nominal 1C 10 H20 Acetone 100 100
Concentration Control Control
in ppb (yg/£)
GC
Concentration 5.65 7.80 _ _ 59.10 57.12
in ppb(X of 3)
Fish were exposed to toxicant for 10 days and were depurated for 10 days.
Groups of bass were removed and sacrificed on days 1, 5, 10, 11, 15 and 20.
No fish died due to toxicant levels during the entire test.
The seasonal ity of young bass made it necessary to continue LC§0
tests with a substitute species of fish. Two diluter experiments with
mollies were directed toward levels known to have lethal effects on bass.
Test conditions were the same as for bass, with two notable exceptions.
Salinity was increased to 2.5 ppt and tank volume reduced by 50%. The
latter change was possible due to decreased biomass of specimens and served
as one means to increase turnover rate of aquarium water.
In the first series of experiments a 77-hour LC50 of 1.9 ppm (mg/£)
HCBD was found for a series of 11 mollies. A 138-hour LCsg of 1.2 ppm
HCBD was found for a second series of 11 fish. Sixteen specimens had a
77-hour LC.5Q in a tank containing 1.4 ppm HCBD, and another 14 had a 115-
hour LCso at 1 .7 ppm.
A diluter experiment at higher concentrations of HCBD resulted in
of less than 48 hours. Twenty mollies were placed in each experimental tank
and subjected to a GC-measured level of 4.5 ppm and 4.2 ppm respectively. The
LCso ln tne flrM: tan'( was attained after 30.5 hours of exposure. In the second
tank, half of the animals had died within 26.5 hours.
26
-------
5. General Pathological Effects
HCBD had acutely lethal effects upon organisms tested during our work.
A number of behavioral changes were noted in exposed organisms. Crayfish
held in 2.7 and 2.9 ppm HCBD in the diluter appeared sluggish within 16 hours.
Some were lying on their sides in the tanks. Following 24 hours, one specimen
was lying on its back and died within two more hours. In 1.2 to 1.4 ppm HCBD,
mollies were affected in less than 24 hours. Darker pigmentation appeared
almost immediately. Within 19 hours loss of equilibrium was observed with
some animals lying on their sides and fibriHating the pectoral fins. Others
swam in circles and ultimately assumed inverted positions, usually with the
trunk curved stiffly to one side. These fish lived another two days in this
condition. Some of those affected after 24 hours lived for 10 more days. In
2.5 ppm HCBD, bass were swimming erratically within 18 hours and all had died
two hours Tater. These assumed an inverted position and were gasping during
several hours before death. In a lower concentration, .9 ppm, bass were
distressed within 18 hours, swimming in circles. They all were inverted
within two days and remained alive, gasping, for three more days. The toxicant
.was removed at this point and fish recovered within 4 days in HCBD-free water.
These animals were fed and behaved normally during the week that followed.
In summary, fish showed a consistent pattern of behavior in response to
HCBD poisoning. Pigmentation increased, even at sublethal doses. Loss of
equilibrium and muscle tetany resulted in a rigid flexing of the body. Fins
were held widely spread, and pectorals vibrated rapidly.
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
glycogenjipid and mineral storage (Fingerman e_t «]_. 1967). These facts .
coupled with reports of liver damage in other species following HCBD
exposure led to priority being given to histological analysis of this organ.
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 eipthelium are derived. These are surrounded by a "basket" of myoepithe-
lial 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
27
-------
the epithelium lies in the distal-most region of each diverticulum where the
epithelium consists of relatively small, slender columnar cells with basophilie
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 ejt ajL, 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 tfie 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 HCBD-exposed crayfish.
Grossly the majority of hepatopancreases from HCBD-exposed crayfish did
not differ from H20 and acetone controls except in occasional instances of
necrosis as noted also for HCB.
Macroscopic examination of the best controlled laboratory environmental
groups shows remarkably healthy tissues, for the most part. HCBD distribution
in these tissue samples may be considered in conjunction with histological
observations. These data are included in the account of an HCBD distribution
experiment 1n section V C3. Tissue appeared healthy upon gross examination,
however, there were areas of epithelial exfoliation more distal than normal
within the canals and, most importantly, a heightened rate of mitosis in the
germinative tips which also contained small areas of seemingly disorganized
growth. Such histological findings were seen in animals from »even*day:static
experiments with a nominal concentration of 5 ppm HCBD in water, and animals
from a 10-day diluter experiment with a concentration of 3.7 ppb HCBD in water
and a whole-body tissue concentration of 0.173 ug/g HCBD.
These findings would indicate that the crayfish hepatopancreas is a very
promising system for further study of longer-term chronic exposure to see the
direction that these early indications of abnormal growth may take. Furthermore,
the hepatopancreas with its diversified cell population, where function varies
with morphological type, may prove to be an excellent cell system for answering
more specific questions as to the nature of toxicant damage.
28
-------
Figure 11, A-E. Photomicrographs of bass tissue sections.
t&^&^y '*':'J*'*
W£X»:£##t'fy
^*''&*?T%J'-£,
ipf^
^V-"rt"- it •
29
-------
Explanation of Figures
(All figures are photomicrographs at the same mangification, 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. (^0 Control Bass)
B. This section illustrates the existence of normal liver lobulation
but cytological change with marked eccentricity of pyknotic nuclei
and loss of stainability (H & E) of the cytoplasm. The arrow
indicates cells interpreted as pancreatic acinar cells in the
connective tissue stroma of the portal canal. (Bass exposed to
32.8 ppb HCBD for 10 days in diluter; 10.8 is mean HCBD concentration
in tissue. See section VII C4.)
C. A section of fingerling bass kidney illustrates the normal histology.
Note the size of the glomeruli and the tubules. (H?0 Control Bass)
D. A section from the kidney of a fingerling bass showing some intact
tubules and a portion of a rather large lesion containing extra-
vasated blood. (Bass exposed to 32.8 ppb HCBD for 10 days in diluter;
3.7 ug/g is mean HCBD concentration in tissue. See section VII C4.)
E. 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. (H20 Control Bass)
30
-------
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. 11A).
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.HE).
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. 11A, arrow). Exocrine pancreas also is situated
between the muscularis and serosa of the gall bladder (F1g. HE, arrow).
Exocrine and endocrine cells are intermixed to form small nodules of pancreas
1n the gastro-hepatic ligament. Variability among fish species has been noted
previously in the location of endocrine and exocrine pancreas (Bengelsdorf
and Ellas, 1950, Bertolini, 1965, Falkmer and Wlnbladh, 1964, FujHa, 1964,
Weis, 1972).
The kidney, an opisthonephros, shows relatively large renal corpuscles and
short proximal and distal tubules. A thin loop of Henle is not present (Fig. 11C)
Other bass tissues (gill, brain, and muscle) have been excised, examined grossly
and scanned histologically. Approximately two hundred blocked specimens are
available for further detailed study.
Histopathological change has been noted in one bass (specimen El in Tx 37)
following HCBD exposure (See Section V C4). It differed from that seen following
HCB exposure. Macroscopically, the liver was noted to be pale. On microscopic
examination, the parenchyma showed an accentuated lobulation with hepatocytes
that were small, with pale eccentric nuclei and an empty-looking cytoplasm
(Fig. 11B). The kidneys from TX37-E1 were abnormal showing areas of leucocytic
infiltration (Fig. 11D). They were not as damaged as those 1n HCB tanks. In
this same bass, the gall bladder was observed as opaque white, an Indication
that HCBD may cause damage to this organ similar to the damage found in HCB
exposure.
3. Retention and Distribution of HCBD in Crayfish
Distribution of HCBD in crayfish tissue was observed in a experiment which
exposed a series of .crayfish to 2.97 ppb (yg/£) of HCBD for TO days. At the
end of the exposure period, four animals were sacrificed and their tissue samples
grouped and frozen together. Subsequent GC analysis gave the results seen in
Table 7.
31
-------
Table 7 . Distribution of HCBD residues in tissues of crayfish (Procambarus
clarki) exposed to 2.97 ppb for 10 days.
Tissue HCBD in yg/g (ppm) Concentration factor
Brain 35.27 11.875X
Green gland 2.188 737X
Hepatopancreas .173 58X
Gills .165 56X
Muscle .046 15X
4. Retention and Distribution of HCBD in Bass
Distribution of HCBD in various organs of bass was determined in an
experiment in which the fish were exposed to two concentrations of HCBD in
the diluter in addition to the appropriate controls. Exposure lasted 10 days,
at the end of which nine specimens in each tank were dissected and the tissues
frozen in groups of three. The groups were extracted and analyzed separately.
Resulting data are given in Table 8 and the average values in Figure 12.
Highest levels were found in gut, liver,kidney and brain of bass exposed to
approximately 33 ppb HCBD. The highest concentration, in the gut, was 52.73 yg/g
(ppm), a concentration factor of 1610X.
32
-------
Table 8 . HCBD content of tissue samples from bass exposed to HCBD in diluter for 10 days. Values in yg/g (ppm
Tank
B
3.37 ppb
(yg/£)HCBD
E
32.76 ppb
(ygAe)HCBD
SO
" . c
H20 Control
.74. ppb (yg/£)
D
Acetone Control
1.0 ppb (ug/£)
Sample
Numbers
4-6
7-9
10-12
X
4-6
7-9
10-12
7
4-6
7-9
10-12
X
4-6
7-9
10-12
7
Gut
.410
.203
22.531
7.715
122.452
15.937
19.803
52.731
.560
.565 -
.181
.435
.510
.035
.257
.267
Gills
.050
.184
4.795
1.676
.296
.099
.250
.215
.124
.278
.024
.142
.012
.035
.056
.034
Organ
Ki dney
2.575
1.574
.203
1.451
6.700
1.012
3.236
3.650
16.810
49.384
2.621
22.938
.989
1.139
3.752
1.96
Type
Brain
.970
*
.107
.539
.809
.298
6.106
2.404
.851
2.401
.270
1.174
1.510
*
.841
.784
Liver
.417
.114
.492
.341
**
15.208
6.282
10.745
.371
1.880
.125
.792
.010
1.513
.207
.577
Muscle
.172
*
.013
.093
.033
.070
.089
.064
.035
.296
.045
.125
*
.076
.004
.04
Body
.008
.044
.001
.018
.003
.142
.023
.056
.004
.020
.020
.0147
.064
.001
.008
.024
* ;
**.
below detectable levels.
lost during processing.
-------
Figure 12. Average HCBD concentrations of tissue samples from bass
exposed to HCBD for 10 days. Concentration factors in parentheses.
3
03
1/1
11 ppm-
-------
5. Fish Blood Hematocrit
Twelve bass were maintained in test conditions given 1n Table 9 for
a period of 10 days. The Individual variation and mean values do not Indicate
an effect upon hematocrU by HCBD at the concentrations tested.
Table 9. Hematocrit of bass (Micropterus salmoides) exposed to HCBD
TANK
Conditions
Water
Control
Acetone
Control
X" Hematocrit (%) 27.85 24.83
* range (%) 17-33.8 14.3-30.9
B
4ppb nominal
3.43ppb (GC)
28.0
20-33.8
40ppb nominal
31.95ppb (GC)
23.23
12-34
6. Fish Corticosteroid Level
Figure 13 shows the results of an assay of plasma cortisol levels (yg/100 ml)
in bass exposed to HCBD for ten days. A t-test shows that the mean values for the
control groups are significantly lower (5% level) than those of the experimental
(exposed to 3.43 ppb and 31.95 ppb) indicating that the experimentals were under
more stress than the controls. Since experimental conditions were exactly the
same for all groups except for the concentration of toxicant these results
indicate a significant stressful effect of HCBD.
Figure 13. Mean plasma cortisol levels (yg/100 ml) in bass (Micropterus salmoides)
exposed to HCBD for ten days.
55 T
1 water
50-
- 45-
E
1 40-
O)
a 35-
30-
25-
20-
1 1 control
L- «N=12
•
- | m
M
^
*%>
\m\sStt
^
•
o!
X
x$
-------
D. Accumulation and Clearance
It 1s 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 HCBD 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 fijr such grazers as snails
(Physa), crayfish (Procambarus). mollies (Pqecilia latipinna), and mosquitofish
(Gambusia affinis).These invertebrates and fishes are common food items of
larger predators, including sunfish (Leppmis) and bass (Micropterus). Several
organisms named above were used in a number of laboratory experiments discussed
here, in an effort to increase available knowledge of accumulation of HCBD in
the food chain.
1. Crayfish
_ In the diluter, 64 crayfish were tested in two tanks (nominal: lOppb; GC
x: 2.97 ppb HCBD). Thirty-two animals were acetone controls and 32 others were
water controls. Uptake lasted 10 days and depuration another 10 days.
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 10 and Figure 14.
Concentration factors are relatively low under these experimental conditions.
Following 5 days1 exposure, males had a mean concentration factor of 11.3 x.
The comparable concentration factor for females is 59.3 x. The mean concentration
factors for both males and, females were lower following 10 days'exposure,
suggesting that a critical maximum level of' toxicant had been reached. Organisms
do not accumulate and retain the substance to the same extent as they do a more
stable compound.
36
-------
Table 10. Whole-body HCBD residue concentrations in male and female
crayfish (Procambarus clarki)during uptake and depuration. Concentrations in
ug/Kg (ppb). ^
Days Exposure
o
CD
0
«C X
^ JD
Z O.
CM
HCBD (ug/Kg) in tissue
Males
Females
CM
0
CO
o
co o=
^ jQ
Z Q.
c
i£ O
z o
^ 0
CM
o:
"o
c
Q q
^ OJ
-------
Figure 14. Uptake and depuration of HCBD in crayfish (Procambarus clarki)
exposed to a mean concentration of 3 ppb (yg/£). Average values*
from Table 10.
JD
Q.
Q.
O>
0)
3
I/)
•f
+•>
Q
CO
O
200i
100-
A
Females / \
Days exposure
Days depuration
* four animals per data point
2. Mollies
Mollies were subjected to nominal concentrations of 10 ppb HCBD and control
conditions in the diluter, to provide uptake and depuration information. As
shown in Table 11, uptake does not proceed steadily, but rather it reaches a
maximum in less than one week. Further exposure does not appear to increase
the level of HCBD in test animals, and concentration factors remain relatively
low. A low level of HCBD contamination was found in control tanks, probably
resulting from diffusion of the volatile substance through the air. The same
low concentration of HCBD is also seen in controls listed in Table 11 .
Figure 15 shows the erratic pattern of uptake and depuration of HCBD
in mollies. Each data point represents two separate GC analyses of an aggregate
of four males and two females. ' '
38
-------
Table 11. Whole-body HCBD residue concentrations in sail fin
mollies (Poecilia latipinna) during uptake and
depuration. Concentrations in yg/Kg (ppb).
Days exposure
Days depuration
C
(O
-------
Figure 15. Uptake and depuration of HCBD in sailfin mollies (Poecilia latipinna)
exposed to a mean concentration of 11.3 ppb (ug/f): Average values
from Table 11.
a
a.
CT
Ol
o
03
CJ
!,500-
1,000-
500-J
Days exposure
Days depuration
3. Bass
Table 12 gives concentrations of HCBD in bass tissue during both uptake
and depuration. As has been observed with other organisms, uptake rate is not
consistent in whole body samples. Concentration factors remain relatively low,
reaching a maximum of 112X in this experiment, and do not correlate with size
(weight) of the test organism. The levels accumulated, however, do reflect the
concentration of HCBD in the environment, within limits. Figure 16 shows this
and illustrates the irregularity with which specimens responded to the compound.
Increases in levels during depuration are not readily explained.
40
-------
Table 12 . Whole-body HCBD residue concentrations in largemouth bass
(Micropterus salmoides) during uptake and depuration.
Days exposure
1 5 10
Days depuration
1 5 10
< a.
a.
i
U_ O.
Q
i
-------
Figure 16 . Uptake and depuration of HCBD in bass (Micropterus
salmoides). Average values from Table 12.
15,000-
DL
Q.
o>
3.
10,000-
Q
00
O
5,000-
Bass in tanks E and F;
X HCBD cone. 58.1 ppb
300-
200-
100-
Bass in tanks A and B; X HCBD cone. 6.7 ppb.
10 1
10
Days exposure
Days depuration
-------
4. Algae
In a flow-through experiment with green filamentous algae (Oedogom'um
cardlacum), higher concentration factors were observed than in laboratory
tests with other organisms. Results of an accumulation experiment are given
in Table 13.
Table 13. Concentration of HCBD by a green alga, Oedogom'um cardiacum
exposed to a flowing solution of 16.9 ppb HCBD in water.
Days exposure HCBD in ug/Kg (ppb) Concentration factor
(wet weight)
1
3
7
965.8
2,547.2
2,700.6
57. Ix
150.7x
159. 8x
5. Bottom Sediment
In a brief laboratory experiment, soil was found to accumulate HCBD more
readily than did experimental animals. Following one day of exposure to a regular
flow of 3.6 ppb HCBD, a sediment sample contained a concentration of 725 yg/Kg
HCBD (725 ppb), a concentration factor of 201.4x. Four days after initiation
of the test, soil contained 938 ppb HCBD, a concentration factor of 260.5x.
Depuration began on the fourth day. After four days of depuration 632 ppb of
HCBD were measured by GC in the sediment.
6. Effect of Food Chain
Experiments with HCB show a significant difference between amount of the
compound taken in by bass from water sources' in comparison with HCB included
in their diet alone. A similar test was made with HCBD to determine if that
compound showed a similar pattern in bass. Five bass were placed in each of
two test tanks. Tank 1 received an average concentration of 3.0 ppb HCBD.
Tank 2 was a water control. Bass being tested were fed ad libitum HCBD-
contaminated mollies containing approximately .02 pg/g (20 ppb) HCBD for seven1
of the nine days of the experiment. All bass were fasted the last two days
to permit clearance of all solid food material from the gut. Results of this
experiment are given in Table 14.
43
-------
Table 14. HCBD concentrations in largemouth bass (Micropterus salmoides)
feeding on contaminated
Bass held in 3 ppb HCBD
Bass spec.
f
1
2
3
4
5
wt in
24.5
26.0
26.3
34.0
40.6
HCBD in
yg/Kg (ppb)
802.4
5.0
2,227.4
3.3
1.2
sail fin mollies (Poecilia lati pinna).
Bass held in HCBD-free water
Bass spec.
f
1
2
3
4
5
wt in
15.2
18.2
20.3
36.9
42.4
HCBD in
yg/Kg (PPP)
4.1
3.8
50.0
11.8
2.7
These data draw attention to the great variability in the uptake of
HCBD by individual organisms. No useful trends are apparent in a sample of
this size, except that, on the average, bass in contaminated tanks accumulated
more HCBD than their counterparts which were fed contaminated mollies in control
water.
7. Crayfish Uptake of HCBD in Field Environment
To provide broad information concerning* uptake of HCBD (and HCB) in a
field situation, a group of crayfish was caged and placed in a waste disposal
pond at the site of the Vulcan Materials Company at a time when HCBD concentra-
tions were 4.3 to 4.6 ppb in water and 381.7yg/Kg (381.7 ppb) in mud. At the
end of 17 days' exposure, the mean concentration of HCBD in eight mature crayfish
was 438 ppb (range 33.7 - 1,290.3 ppb). Distribution of results is given in
Table ,15.
Table 15. HCBD content of crayfish exposed to 4.3 ppb HCBD in a field locality.
Number of
Specimens
4
1
1
1
1
Mean HCBD concentration
in yg/Kg (ppb)
93
357
443
1 ,042
1,290
Concentration
factor
22X
83X
103X
242X
300X
44
-------
Three other crayfish exposed to similar conditions for five days in the
same field site contained 9, 33, and 240 ppb HCBD which represent concentration
factors of 2.OX, 7.2X, and 55.8X.
Six crayfish were exposed for different periods and depurated either not
at all or for two months. Data resulting from this part of the study are
presented in Table 16. HCBD concentration in water was 4.6 ppb and in mud was
381.7 ppb. The variation both in uptake and depuration rates is readily seen
in this table.
Table 16 . Short and long-term exposure of crayfish to a contaminated
field site (4.6 ppb HCBD).
Weight (in g) HCBD Concentration
Days exposure Days depuration of specimen in yig/Kg (ppb)
4 63 19.32 8
4 63 13.93 132
15 63 17.39 186
51 0 12.30 501
51 0 13.55 320
51 0 24.61 394
Thirty-one crayfish were placed at the field site where they were exposed
to 4.7 ppb HCBD for 10 days. A sample was sacrificed on removal from the field.
Remaining animals were depurated in environmental chambers in the laboratory,
and samples were sacrificed for GC analysis on days 3, 7, 12, 25 and 48 of
depuration. The results, given in Figure 17, show a higher level of uptake by
females and a rapid loss of the substance by both sexes. The samples having
highest levels of HCBD, sacrificed on day three of depuration yielded concen-
tration factors of 151X (712 ppb) in males and 289X (1,360 ppb) in females.
45
-------
Figure 17 . Levels of HCBD in crayfish during depuration following
10 days exposure to a contaminated environment (4.7 ppb HCBD).
1 ,300 .-,
a
a.
at
Ol
3.
C
O
00
o
1,100-
M
900-
-
700-
500-
300 -
100 -
MALES N = 19
712.4
/\
/ \418.7
418.2 \
\
iQ.av — -1—- : — . _j2.5
III! 1 1
0 3 7 12 25 48
DAYS DEPURATION
1,300^
CL
a.
01
a>
c
•r*
O
CO
o
1,100-
—
900-
_
700-
500-
™
300-
-
100-
FEMALES N = 12
• 1 ,00 1 .£
l\
1 \
/ \
I \
754.9 \
\
\
\
i
\
VH5.6
^22^ &*_„.
1 1 1 1 , 1 1
03712 25 48
DAYS DEPURATION
46
-------
E. Respirometry
1. Crayfish
Experiments were designed to measure possible effects of HCB upon
respiration in crayfish. The average oxygen consumption rates (y£ 02/mg wt/hr)
for HCBD-exposed and for control animals are shown in Table 17. Each value
is based upon an average of 20 animals whose oxygen consumption rates were
measured for a total time period of two hours. In Figure 18 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.
Exposure of juvenile crayfish to 5 ppb HCBD shows no significant effect
upon oxygen consumption rate during the first two hours of exposure. Chronic
exposures to 5 and 0.5 ppm and 50 ppb for periods as long as eight days
produced complex patterns which are difficult to interpret until more extensive
studies are completed. A preliminary pattern which is emerging (Fig. 18)
indicates that respiration rate may be initially depressed and then accelerated
after several days of exposure.
An HCBD concentration of 5 ppm was selected for the first experiment
because it was known to be an acute lethal dose in crayfish. At 5 ppm half of
the juveniles in our experiment died within 72 hours. The HCBD concentration
was reduced to 0.5 ppm, and fifty percent of the animals died within 120 hours.
At 50 ppb the juvenile crayfish survived indefinitely.
Table 17. Effect of time of exposure to HCBD on respiration rate of juvenile
crayfish (y£ 02/mg/hr).
Experiment^Group Duration of exposure (in days)
01 2468
1
2
3
4
Control
Exp.
(5 ppm)
Control
Exp.
(0.5 ppm)
Control
Exp.
(50 ppb)
Control
Exp.
(50 ppb)
0.3866 0.3439
0.3434 0.4550
0.4613
0.2488
0.5725
0.3680
0.2746 0.1835 0.3817
0.2608 0.1749 0.2871
0.3116
0.4419
0.4532
0.3852
0.5614
0.4856
0.3171
0.3111
0.3265
0.3872
0.2562 0.1931
0.1793 0.2143
47
-------
Figure 18. The effect of time of exposure to H.CBD on respiration rate of
juvenile crayfish (Procambarus clarki).
Ol
Q.
X
0)
-------
Table 18. Effect of time of exposure to HCBD (0.5 ppm) on respiration rate
of juvenile mollies (\it 02/mg/hr).
Experiment # Group
Control
1
Exp.
Control
2
Exp.
Duration of exposure (in days)
0 2
0.4750
0.5240
0.3022. 0.4277
0.2707 0.4856
4
0.4685
0.4404
0.2932
0.2960
8
0.3778
0.3692
0.3905
0.6212
Figure 19. The effect of time of exposure to HCBD (0.5 ppm) on respiration
rate of juvenile mollies (Poecilia latipinna).
0)
O.
X
•o
c
(O
I/I
'o
$_
01
4J
O)
.a
a;
o
c
QJ
-------
F. Photochemistry
HCBD in benzene shows a dramatic change upon irradiation. After only
15 minutes of irradiating at a wavelength of 2735 A , numerous products
having a molecular weight higher than HCBD are evident in large quantities
relative to the initial amount of HCBD. The number of these products decreases
with the time of exposure. GC traces of resultant products taken at intervals
are shown in Figure 20.
Figure 20. Gas chromatographic separation of photoproducts resulting
from ultraviolet irradiation of HCBD.
K> 20
TME (MINUTES)
50
-------
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 extracts9 while the
remaining analyses included samples collected during more than 40 field trips.
During the field phase which encompassed a transect along the Mississippi
River from Baton Rouge to Port Sulphur, Louisiana, HCBD residues were found in
several soil and water samples. The level in water did not reach 2 yg/£ (ppb)8
but concentrations of HCBD in mud or soil samples exceeded 200 yg/Kg (ppb)
between Ashland Plantation and Romeville and at New Orleans. All these sites
were along the east bank of the Mississippi River. Significant levels of HCBD
were also found at Addis, on the west bank and at Myrtle Grove, below New
Orleans, also on the west bank.
Substantially greater loads of HCBD were found in mud samples when compared
with HCBD concentrations in most organisms (mosquitofish, Gambusia affinis, and
crayfish, Procambarus sp.) taken from field sites along the Mississippi River.
At Romeville, for instance, river water had a level of 1.4 ppb, soil had 793.4
ppb and fish had 197.4 ppb HCBD. Concentrations of HCBD in river samples tended
to be more variable than those from ditch sites.
Variable patterns of generally low-level contamination of HCBD were seen
as a result of seasonal collections from localities at the area of higher concentra-
tion in Geismar, Louisiana. Most water samples contained less than 1 ppb HCBD and
none had more than 5 ppb. Levels in mud were noticeably higher, reaching a
maximum of 2,370 ppb. Organism samples contained less than 1000 ppb HCBD
with few exceptions. Gambusia, the mosquitofish, had an average of 16,000 ppb
HCBD. These specimens came from a pond of highest contaminations where
concentrations in water ranged from 4 to 5 ppb. Observations of HCBD levels
in the Mississippi River survey and the more limited Geismar area corroborate
one another as much as might be expected with the variability seen in HCBD.
There is a general association of HCBD with the Mississippi River. The principal
source of contamination is very likely to be petrochemical industries that border
the river.
Studies of HCBD acute toxicity in test animals have emphasized uptake by
oral administration, injection, and topical application. Gul'ko et al. (1965)
reported LDso's of 41 mg/Kg (ppm) in mice, taken orally. Topical applications
of 500 mg of HCBD to the shaven skin of guinea piys caused intoxication in some,
as well as some deaths (Murzakaev, 1966).
Murzakaev (1963a) also performed injection experiments with mammals. He
reported LD5Q*s resulting from single intraventricular injections of 87,90 and
350 mg/Kg (ppm) in mice, guinea pigs and rats, respectively. Poteryaeva (1966)
reported mortalities in offspring of white rats born up to three months after
the mothers had been injected subcutaneously with 20 mg HCBD/Kg body wt.
Injection experiments in the present study used crayfish (Procambarus clarki)
and Gulf killifish (Fundulus grandis). Injection of 3.4 mg HCBD per gram body
weight (ppt), ir. peanut pil as a carrier, was a 24-hour LD5Q for crayfish. A
51
-------
five-day LDKQ was 336 yg/g. In killifish, intraperitoneal injection of 3.4 mg
HCBD/g (ppt; was a four-hour LDso. A 1.7 ppt injection was a 20-hour
An LD50 of 18 days was 800 yg/g (ppm), but no fish deaths were noted during
a three-week period at a dosage of 200 ppm. The greater toxicity of HCBD to
crayfish may be in part due to the site of injection. Crayfish were Injected
directly into the hemocoel, permitting more rapid circulation throughout the
body. Killifish were injected into the peritoneal cavity, and some of the
oil was observed to have been lost from the fish when they were released into
tanks. Therefore, killifish might not have retained the full dosage administered
and the distribution in the body may have been slow.
Very little work has been published on toxicity of HCBD to aquatic animals.
Hiatt et al. (1953) noted a behavioral response in schooling fish to applications
of 20 ppm~Tmg/£). HCBD had been dispersed but was not necessarily dissolved
in water. Stroganov and Kolosova (1968) exposed cyprinid fish, Leucaspius
delineatus to toxic levels of HCBD in water and reported total mortality even
when, after a brief exposure to the toxicant, the fish were transferred to
clean- water and showed some recovery. They stated that concentrations below
15.6 ppm had no toxic effects, even after 11 days of exposure. Experiments
carried out during the present study found HCBD to be acutely toxic at lower
concentrations than previously reported. A 54-hour LC$Q for crayfish held in
the dlluter was 2.9 ppm.
Bass were not overtly affected by 10 days' exposure to 3.43, 31.95, or
59.10 ppb (yg/£) HCBD in the diluter. At a level of 900 ppb (.9 ppm) bass
were distressed in 24 hours but survived more than four days under these
conditions, although they were inverted and immobilized. Some variability
was noted in tests with bass. Concentrations of 2.5 ppm and 4.3 ppm were
24- and 48-hour LCso's, respectively. Larger numbers of mollies were available
for acute experiments. I^Q'S for 26 and 30.5 hours were 4.2 ppm and 4.5 ppm
HCBD, respectively. Little difference was1 apparent in the response of the two
species to HCBD. It should be noted, however, that molly experiments took place
in 2.5 ppt salinity, while 1.0 ppt salinity water was used for bass. Furthermore,
bass were substantially larger than mollies.
More subtle effects of chronic doses of a substance are expected to be seen
at the tissue and cellular level. Reports of damage at the histological level
to aquatic animals exposed to chronic doses of HCBD have not been published.
However, microscopic observations of morphological changes following HCBD
exposure are reported for the rat in liver (Gul'ko e_t aJL , 1965 and Murzakaev,
1967) and in kidney tissues (Dmitrienko et aJL , 1972; Gage, 1970; and Murzakaev,
1967). In histological sections of crayfish tissue prepared during the course
of the present study, abnormalities in site and degree of hepatopancreas epithelial
exfoliation were noted. This change and the increased mitotic rate and indications
of disorganization in growth in the germi native tips suggest possible chronic
effects of an exposure to 2.97 ppb (yg/£) HCBD for 10 days. Analyses of crayfish
tissue extracts on GC showed the brain as having the highest concentration of
HCBD, (35 yg/g) with the hepatopancreas having 200 times less HCBD residue,
(.173 yg/g). The green gland has a concentration an order of magnitude higher
than the hepatopancreas; 2.188 yg/g (ppm).
Histological slides of tissue from bass exposed to 32 ppb for 10 days
showed damage tc liver parenchyma, gall bladder, and kidney. In GC analyses of
extracts from these tissues, highest HCBD residue concentration was found in
gut, followed by liver and kidney.
-------
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 histological sampling to determine the extent of
tissue recovery would lend further significance to the findings.
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 had been 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 microscopic examination
of the accumulated material. It seems particularly appropriate to continue to
explore the liver and gall bladder response to HCBD noted in the bass and molly
in light of the findings of cholecystitis in vineyard workers having contact
with HCBD as a fumigant (Krasnyuk ejt al_., 1969).
The extent to which a substance is accumulated by an organism during prolonged
exposure to it may have an effect upon health and survival. Concentration and
period of exposure may influence both the maximum level attained and the time
required for depuration. Laboratory experiments included in this report exposed
organisms to HCBD in both static and flow-through systems. In general, concen-
trations in whole organisms increased with time of exposure and decreased with
time of depuration. Rates of accumulation and depuration varied widely between
organisms.
In laboratory experiments, crayfish accumulated relatively small amounts
of HCBD. Concentration factors in females were higher than in males, but did
not exceed SOX during 10-day exposures to a mean of 2.97 ppb (yg/£). In mollies
exposed to a mean of 11.29 ppb, concentration factors reached 110X during a 10-
day test period. Bass held in tanks with 6.73 ppb HCBD had a maximum concentra-
tion factor of 29X during a 10-day exposure. The lower concentration factors
of bass in comparison with mosquitofish may in part be explained by
Murphy's (1971) observation, that smaller organisms in aquatic systems accumulate
proportionately greater amounts of a toxin than larger ones do. Bass were, on
the average, 2 to 4 times larger than mollies at the time of their respective
experiments.
Crayfish in laboratory experiments accumulated proportionately less HCBD
than mollies did. In the field, crayfish accumulated proportionately less than
mosquitofish, a member of the same fish family, Poeciliidae, as mollies, it
appears that either the fishes are more efficient at taking in and concentrating
the substance or the crayfish have more effective means of altering the structure
of accumulated HCBD. A combination of these.factors may be at work. Mosquitofish
at the Geismar site accumulated higher levels and had higher concentration factors
than mollies tested in the laboratory. This may be a result of extended periods
of exposure time.
Algae samples in the laboratory accumulated HCBD at higher levels than
animals did. After 7 days of exposure to 16.9 yg/£ (ppb), the level attained
was 2,700 yg/Kg (ppb); a concentration factor of 160x. Sediment was subjected
to 3.6 yg/£ (ppb) HCBD. The highest level attained was 938 yg/Kg after four
days; a concentration factor of 261x. Depuration was gradual, since 632 ppb of
HCBD remained in a sample taken after four days. The highest level of HCBD
found in soil at Geismar was 2,370 yg/Kg (ppb).
53
-------
A food chain experiment was designed to compare the effect of HCBD uptake
by ingestion with the combination of uptake by ingestion and through a contaminated
environment. A considerable variability was observed among individual organisms
and results were inconclusive. Only when considered on the average did bass in
HCBD-contaminated tanks accumulate more HCBD than those which were fed contaminated,
mollies in clean water.
Experiments in which HCBD-free crayfish were caged and placed in a field
site with known HCB and HCBD contamination provided a more natural situation for
observing uptake rates. Mean concentration factors and individual variability
were similar to those for crayfish tested in the laboratory. Crayfish exposed
to 4.6 ppb HCBD in pond water for 17 days accumulated a wide range of residue
concentrations. The mean level was 438 yg/Kg (ppb), but the range was 33 to 1,290
ppb. The concentration factors for these were from 7X to 300X. Females accumu-
lated more of the compound than did males, and HCBD levels in both sexes decreased
at a rapid rate between days 3 and 12 of depuration. In animals held in the field
site for 10 days, approximately 95% of the HCBD measured at the onset of depuration
was lost within 12 days.
Laboratory experiments revealed physiological effects upon test animals
subjected to HCBD. Stress by HCBD in water was indicated as a result of analysis
of corticosteroid level in blood. Plasma cortisol levels in bass exposed to 3.43
and 31.95 ppb (ug/£) HCBD for 10 days were significantly higher than corresponding
levels in controls (5% level).
As a further test for parameters indicating stress due to a chronic
exposure, several experiments were carried out in which juvenile mollies and
crayfish were subjected to HCBD in water and their oxygen consumption rate
monitored 1n a Gil son respirometer. HCBD seemed to have no significant effect
upon oxygen consumption rate of crayfish during a two-hour exposure to 5 ppb HCBD.
Complex patterns of response were seen in crayfish exposed to 50 ppb and .5 and 5
ppm HCBD for extended periods of time as long as 8 days. Tentative results show
an initial depression in rate followed by an accelerated rate after several days.
Experimental mollies in these studies had extremely variable response patterns.
A great deal more data is essential before any definitive statements can be
made on this subject.
Oxygen consumption studies utilized the Gilson Differential Respirometer
instead of a flow-through respirometer 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 a flow-through respirometer. 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. 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 HCBD, 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 18 and 19 often indicated high percentage differences. HCBD
54
-------
is a hydrophobia substance. Its distribution may be through passive partitioning
through lipid phases in the organism. Any effects on respiration may be a
result of the accumulation of HCBD in membranes. Many enzymes depend upon the
integrity of the membrane 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
phosphorylation, 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 hydro-
phobic substances. Establishment of detailed 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.
Pertinent information from acute toxicity experiments is summarized
in Table 19. Data from injection and diluter exposures are included.
Table 20 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.
55
-------
Table 19. Summary of acute toxicity experiments with HCBD.
'**«"- SosSre
Injection Experiments
Procambarus . .
rlarkS Single
CiariC1 iniortinn
(Crayfish) injection
Fundulus
qrandis "
(Gulf Killifish)
Concentration
34 yg/g body
wt
336 "
3,360 "
200 yg/g body
wt
800 "
1 ,700 "
3,400 "
Total # of
organisms
exposed
10
10
10
10
10
10
Duration
of
exposure
single
injection
"
Duration
of
observation
6 days
6 "
2 "
21 "
18 "
22.5 hr
4 hr
Mortality
30
100
100
60
100
100
Diluter Experiments
Procambarus aqueous
clarki solution
(Crayfish) 1n d1luter
Poecilia
lati pinna "
(Sail fin molly)
M 'n
Micropterus
salmoides „
(Largemouth
black bass)
2.7 ppm
2.9 "
1.2 ."
1.4 "
1.7 "
1.9 "
4.2 "
4.5 "
.9 "
2.2 "
2.5 "
4.3 "
9
9
11
16
14
11
20
20
6
6
6
6 -
12 days
12 "
6 "
6 "
6 "
5 "
5.5 " 5
5 "
4 "
4 "
1 "
1.5 " 1
12 days
12 "
9 days
5 "
.5 "
5 "
8 "
4 "
1 "
.5 "
100
100
100
100
100
100
100
100
100
100
100
56
-------
Table 20. Minimum concentrations of HCBD tested that resulted in an observed
response in organisms.
a)
b)
c)
Method Lowest tested level (in
ppb) at which changes
were observed
Direct injection 34,000. yg/Kg body wt
in oil medium
800,000. ug/Kg body wt
Diluter 2,700.
900.
1,200.
4,200.
Histology 3.
Observations Section
containing
discussion
30% mortality to
crayfish in 6 days
following injection
18-day 1050 for
injected Gulf killifish
57-hour LCso
for crayfish
Distress within
24 hours for bass
138-hour LCso
for bass
26.5-hour LC50
for mollies
Heightened rate of
V Bl
V B2
V B3
V B4
V B4
V B4
V Cl
d) Cortisol level
in blood plasma
33.
mitosis in germinative
tips of hepatopancreas;
Distal epithelial
exfoliation in crayfish.
Accented lobulation of
liver parenchyma
Leukocytic infiltration
of kidney in bass
Cortisol level in blood
elevated (Significant
at 5% level) in bass
V C2
V C6
Maximum HCBD concentrations found in environmental survey
Water
Mud
Area of Higher
Concentration
Overview
4.7 ppb (ygAe) 1,080. (2,370.) ppb (ug/Kg)
790. (1,088.) ppb (ug/Kg)
(Romeville)
1.9 ppb
(Baton Rouge)
*: Figures corrected for dry weight in parentheses.
57
-------
Literature Cited
Beams, H. W., E. Anderson, and N. Press. Light and electron microscope
studies on the cells of the distal portion of the crayfish nephron
tubule, Cytologia. 21(1): 50-57 (1956).
Bengelsdorf, H. and H. Elias, The structure of the liver of Cyclostomata.
Chicago Med. Sch. Quart. (1950).
Benoit, D. A. and F. A. Puglisi. A simplified flow-splitting chamber and
siphon for proportional diluters. Water Res. 7: 1915-1916 (1973).
Bertolini, B. The structure of the liver cells during the life cycle of a
brook-lamprey (Lampetra zanadreai). Z_. Zellforsch. 67: 297-318 (1965).
Coleman, Roger. Membrane-bound enzymes and membrane ultrastructure.
Biochemica et Biophysica Acta 300: 1-30 (1973).
Defoe, D. L. Multichannel toxicant injection system for flowthrough
bioassays. J_. Fish. Res. Board Can. 32: 544-546 (1975).
Dmitrienko, V. D. and A. F. Vasilos. Kidney damage during the experimental
acute poisoning with hexachlorobutadiene. Zdravookhranenir (Kishinev)
15 (1): 11-12 (1972). Abstracted.
Falkmer, S. and L. Winbladh. An investigation of the pancreatic islet
tissue of the Hagfish (Myxine glutinosa) by light and electron microscopy.
In; The Structure and Metabolism of the Pancreatic Islets, S. E. Brolin,
B. Hellman, and H. Knutson (eds.): 17-32. Pergamon Press. Oxford (1964).
Fingerman, M., T. Dominiczak, M. Miyawaki, C. Oguro and Y. Yamamoto,
Neuroendocrine control of the hepatopancreas in the crayfish. Procambarus
clarki. Physio!. Zoo!. 40: 23-30 (1967).
Fujita, T. Cytologic studies on the pancreatic islets of Chimaera monstrosa.
In: The Structure & Metabolism of the Pancreatic Islets, S. E. Brolin,
B. Hellman, and H. Knutson (eds): 11-15, Pergamon Press. Oxford (1964).
Gage, J. C. The subacute inhalation toxicity of 109 industrial chemicals.
Brit. Jf. Industr. Med. 27(1):1-18 (1970).
Gudamak, V. S. [The change in activity of the oxidative enzymes in the organs
of white rats given hexachlorobutadiene in acute experiments.] Zdravookhr
(Kishinev) 3: 47-50 (1968). Abstracted in Ref. Zh.. 0_td_. Vy^. Farmakol.
Khimtoter. Sredstva. Toksikol 4.54.828 (1969).
Gul'ko, A. G., N. I. Zimina and I. G. Shroit. [A toxicological study of the
insecticide hexachlorobutadiene.] Ref. Zh. Otd. Vypusk. Farmakol. Toksikol
3.54.377 (1965) Abstract. ^
Hargreaves, G. and Porthe-Nibelle, H. Plasma cortisol concentrations in two
teleost fishes: Anguilla anguilla L. and Carassius auratus L.: A
comparison of two assay systems. Steroids 24(2): 112-124 (1974).
58
-------
Hiatt, R. W., J. J. Naughton and D. C. Matthews. Effects of chemicals on a
schooling fish, Kuhlia sandvicensis. Biol. Bull. 104: 28-44 (1953).
Huxley, T. H. The Crayfish, An Introduction to the Study of Zoology. C.
Kegan Paul & Co. London (1880T
Jacobs, A., M. Blangetti and F. Hellmond. Accumulation of noxious chlorinated
substances from Rhine River water in the fatty tissue of rats. Vom. Nasser
43: 259-274 (1974). Abstracted in Chem. Abst. 82: 165547.
Krasnyuk, E. P., L. A. Zaritskaya, V. G. Bioko, G. A. Voitenko and L. A.
Matokhnyuk. Health of vineyard workers having contact with the
fumigants hexachlorobutadiene and polychlorobutane - 80. Vrach. Delo
7: 111-115 (1969). Abstracted.
Khrokhryakova, V. S., G. Ya. Robas, and G. P. Deniskina. [The problem of the
phytotoxicity of hexachlorobutadiene in the first year following the
fumigation of vineyards.] Aspirantskie Raboty Nauch Inst. Udobr.
Mnsektofung 124-128 (1963). Abstracted in Ref. Zh.. Biol. 10E85 (1964).
Levinson, Z. H. Chemicals affecting the preimaginal stages of housefly.
V. Vapor toxicity of dichlorobenes to housefly pupae. Riv. Parasitol.
16, 253-256 (1955). Abstracted.
Loizzi, R. F., Interpretation of crayfish hepatopancreatic function based
on fine structural analysis of epithelial cell lines and muscle network.
Z. Zallforsch. 113: 420-440 (1971).
Meier, A. H., and A. K. Srivastava. Alteration by drugs of the daily rhythm
of plasma corticosteroid concentration in the gulf killifish (Fundulus
grandis). Gen. Comp: Endoc. 26; 253-258 (1975).
Mirzonova, M. N. and N. N. Perov. Effect of hexachlorobutadiene and
polychlorobutane - 80 on the microflora of a carbonate chernozem. Khim.
Sel. Khoz. 6(5): 345-348 (1968). Abstracted.
Miyawaki, M., M. Matsuzaki, and N. Sasaki. Histochemical studies on the
hepatopancreas of the crayfish, Procambarus clarkii. Kumamoto J. of Scj. Ser.
. B. 5(2) (1961).
Mount, D. I., and W. A. Brungs. A simplified dosing apparatus for fish
toxicology studies. Water Res. 1: 21-29 (1967).
Mikasheva, G. M. Effect of hexachlorobutadiene on mycoflora of grape vine
roots. Mikrobiol. Zh. (Kiev) 36(1): 105-110 (1974). Abstracted in
Chem. Abstr. 81: 34352.
Murphy, B. E. P. Some studies of the protein-binding of steroids and their
application to the routine micro and ultamicro measurement of various
steroids in body fluids by competitive'protein-binding radioassay. J_. Clin.
Endo. Metab. 27: 973-990 (1967).
59
-------
Murphy, B. E. P. "Hormone assay using binding proteins in blood" in
Principles of Competitive Protein-Binding Assays VI. D. Odell and Wm.
H. Daughaday (eds.) Lipincott: Phila. (1971).
Murzakaev. F. G. [Some data on the toxicity of a new insecticide, hexachloro-
butadiene, and its intermediate metabolic products]. Farmakol. J_.
Toksikol. 26(6): 750-753 (1963). Abstracted.
Murzakaev, F. G. Maximum permissible concentrations of hexachlorobutadiene
and polychlorobutanes in the water of reservoirs. Gigiena i Sanit.
28(2): 9-14 (1963). Abstracted.
Murzakaev, F. G. Experimental determination of the permissible limit concen-
trations of hexachlorobutadiene and of polychlorobutanes in cistern water.
Sanit. Okhrana Vodoemov crt Zagryazneniya Prom. Stochnymi Vodami 6: 179-
20TT1964). Abstracted.
Murzakaev, F. G. Toxicological and hygienic characteristics of hexachloro-
.butadiene and its intermediate products. Dokl. Neftekhim. Sekts. Bashkir.
Rasp. Pravl. Vses. Khim. Obs. 2: 245-250 (1966). Abstracted in Chem.
Kbstr."6771392807
Murzakaev. F. G. Action exerted by low hexachlorobutadiene doses on the
activity of the central nervous system and morphological changes in
animals so poisoned. Gj£. Tr_. Prof. Zabol. 11(3) 23-28 (1967) English
summary.
Ogura, K. Midgut gland cells accumulating iron or copper in the crayfish,
Procambarus clarkii. Annotationes Zoologicae Japonenses 32(3): 133-
142 (1959).
Pease, D. C. Fine structure of the kidney seen by electron microscopy.
4. Histochem. Cytochem., 3(4): 295-308., (1955).
Peterson, D. R., and R. F. Loizzi. Ultrastructure of the crayfish kidney-
coelomosac, labyrinth, nephridial canal. *L Morph. 142(3): 241-264 (1974).
Poteryaeva, G. E. Effect of hexachlorobutadiene on the offspring of albino
rats. Gig, i Sanit. 31: 33-35 (1966). Abstracted.
Poteryaeva, G. E. [Information substantiating the maximum permissible
concentration of hexachlorobutadiene in the air of industrial premises].
Gig. Sanit. 37(2): 32-36 (1972). Abstracted.
Stroganov, N. S. and L. V. Kolosova. Effect of small concentrations of
hexachlorobutadiene on small organisms. Tr. Mosk. Obshenest. Ispyt.
Prir. 30: 126-138 (1968). Abstracted in ChemTIBstr. 72: 97852(1970).
Tarzwell, C. M. Water quality requirements for aquatic life. National
Symposium on Quality Standards for Natural Water Proceedings. 185-197.
Ann Arbor, Mich. (1966).
60
-------
Tarzwell, C. M. I. Measurements of pollution effects on living organisms.
Bioassays to determine allowable waste concentrations in the aquatic
environment. Proc. Roy. Soc. Lond. ]J. 177: 279-285 (1971).
Unbreit, W. W., R. H. Burn's and J. F. Stanffer. Manometric and Biochemical
Techniques. (5th edition): 100-110. Burgess Pub. Co., Minn. (1972).~
Vashchinskaya, N. V. Apple-tree borers and measures for combatting them.
Izv. Sel'skokhoz. Na.uk 14(8): 51-57 (1971), Abstracted in Chem. Abstr.
77: 57S3T
Warren, C. E. Biology and Mater Pollution Control. W. B. Saunders Co.,
Philadelphia (1971).
Weis, P. Hepatic ultrastructure in two species of normal fasted and
gravid teleost fishes. Am. ,h Anatomy 133(3): 317-332 (1972).
61
------- |