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RESIDUE DYNAMICS AND AVAILABILITY OF
2,#,5,2',4',5'-HEXACHLOROBIPHENYL IN
AQUATIC MODEL ECOSYSTEMS

BY

Thomas Richard Lynch

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Department of Fisheries and Wildlife

1979

ABSTRACT

RESIDUE DYNAMICS AND AVAILABILITY OF
2,u,5,2',H',5'-HEXACHLOROBIPHENYL
IN AQUATIC MODEL ECOSYSTEMB

By

Thomas Richard Lynch

The polychlorinated biphenyls (PCB's) are organic
compounds of industrial origin which have become ubiquitous
contaminants of aquatic systems. Because of their hydro-
phobic nature, the PCB's are attracted to surfaces which
results in their accumulation on bottom sediments. Sorption
onto bottom sediments creates a potential reservoir of
material which can buffer changes in residue concentrations
in the water via equilibrium exchange reactions. Fine
textured sediments and sediments with high organic contents
are often enriched with hydrophobic compounds. The signifi-
cance of these substrate-bound residues, their availability
to aquatic organisms, and the principal route of uptake by
organisms are largely unknown.

The present study was undertaken to determine the
residue dynamics and metabolism of a representative PCB
isomer in a semi-natural model of a woodland stream ecosystem

and in a series of simplified laboratory experiments. The

laboratory experiments were designed to determine the

Thomas Richard Lynch
the relative importance of residue accumulation by a repre-
sentative benthic invertebrate from water, from contaminated
food, and from direct contact with contaminated substrates.
The bioavailability of substrate-bound residues as a
function of substrate type, organic matter content, and
particle size distribution was also investigated. It was
hoped that the laboratory studies would also support and
clarify the results derived from the more complex model
streams experiment.

Hexachlorobiphenyl was accumulated rapidly by the
substrates and benthic organisms of the model streams and
concentrations increased with duration of exposure. Residues
in substrates after thirty days of exposure ranged from 630-
3200 times the concentrations in the water and were signi-
ficantly correlated with the organic matter content.

The rate of uptake of the isomer by the biota was
biphasic with the most rapid accumulation occurring during
the initial three hours of exPosure. Maximum residue
concentrations in the biota usually occurred after thirty
days of exposure. Bioaccumulation factors ranged from about
300,000 in amphipods to 1200 in clams while fish, crayfish,
and other macroinvertebrates had intermediate values. There
was no evidence that residue concentrations were increased
by trophic transfer.

Residues of hexachlorobiphenyl were present in all

benthic organisms after sixty days of depuration but

Thomas Richard Lynch
repeated exposure to the isomer did not result in stepwise
increases in concentration with each exposure.

Metabolites of hexachlorobiphenyl were not detected in
the substrates or biota of the model streams.

Amphipods accumulated hexachlorobiphenyl residues
directly from water as a function of time and exposure
concentration under both static and continuOus flow conditions.
Residue levels in amphipods under continuous flow exposure
were 3.3-6.7 times higher than under static conditions
although bioconcentration factors were similar under both
exposure conditions.

The accumulation of residues by ingestion of contaminated
food was less important than direct uptake from water
although residues accumulated from each pathway appear to be
additive.

Accumulation of residues by amphipods was enhanced by
direct contact with contaminated substrates although direct
uptake of substrate-desorbed residues directly from the water
was the major route of uptake. The bioavailability of
substrate-bound residues is controlled by the level of organic
matter and to a lesser extent by the particle size of the
substrates.. Residue levels in amphipods were inversely
related to the organic content and directly related to the

particle size of the substrate.

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to all
of those who contributed to the success of my doctoral
program, eSpecially the following individuals:

Dr. William J. Adams for his assistance in the
construction, maintenance and daily operations of the model
streams.

Ms. Linda Koivuniemi and Ms. Terry Aiken for their
dedication to a job well done and for help in sample
preparation and analysis. I am also indebted to Terry for
her help in the preparation of graphic materials.

Drs. Richard Cole and Kenneth Cummins for their
encouragement and suggestions and for serving on my grad-
uate committee during the early part of my program.

Drs. Charles Liston and Richard Merritt for their
willingness to serve as replacements on my graduate
committee and for their time and assistance in reviewing
my dissertation.

Dr. Mathew Zabik for his consultation and for serving
on my committee.

I am eSpecially indebted to Dr. Howard E. Johnson for

the privilege of having worked under his guidance as my major

ii

professor, for the opportunities and responsibilities he
presented me, and for the many hours of effort Spent on

my behalf.

Finally, I would like to thank my wife, Ann, for
her undying patience, endurance and understanding and
without whose love and moral support this dissertation

would not have been possible.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES . . . . . . . . . . . vi
LIST OF FIGURES . . . . . . . . . . x
INTRODUCTION . . . . . . . . . . 1
MATERIALS AND METHODS 9
Model Streams - Description . . . . 9
Model Streams — Experimental Procedure . . . 13
Laboratory Studies - Objectives . 17
Laboratory Studies - Experimental Procedures . 18
Accumulation from Water - Static Exposure . 20
Mechanism of Accumulation . . 20
Accumulation from Water - Continuous Flow

Exposure . . . . . . . . 21
Accumulation from Food . . . . . . 22
Availability from Substrates . . . 23

Intact Substrates . . . . . . 24

Sieved Substrates . . . . . . 28

Sample Extraction and Analysis . . . . . 29
Metabolite Analysis . . . . . . . 32
RESULTS AND DISCUSSION . . . . . . . . 3A
The Model Streams . . . . . . . . 3h
Chemical Fate . . . . . . . . 35

Water . . . . . . . . . 35

Substrate . . . . . . . . 37

Biota . . . . . . . . . 43
Bioaccumulation . . . . . . . 54

Metabolite Analysis . . . . 58
Laboratory Studies . . . . 6O
Accumulation from Water - Static Exposure . 6O
Mechanism . . . . . 6h

Accumulation from Water - Continuous Flow

Exposure . . . . . . 67
Accumulation from Food . . . . . . 78

iv

Page

Residue Availability from Substrates . . 85

Intact Substrates - Trials 1 and 2 . . 85

Intact Substrates - Trial 3 . . . 90

Intact Substrates - Trial 4 . . . . 98

Sieved Substrates . . . . . 102

SUMMARY . . . . . . . . . . . 111
APPENDIX . . . . . . . . . . . 1L4
LIST OF REFERENCES . . . . . . . . . 116

LIST OF TABLES

Table Page

1. The chronological sequence of major events
in the model streams study . . . . . 15

2. Summary of procedural differences for experi-
ments on HCB availability from sieved
substrates . . . . . . . . . 30

3. Mean concentrations of HCB (ug/liter) in
water from treated streams during the treat-
ment phase of each experimental period . . 36

4. Concentrations (ug/g dry wt.) and regression
equations for accumulation and depuration of
H03 in the stream substrates. Each value
represents the mean of four samples . . . 38

5. Mean HCB concentrations (ug/g dry wt.) and
organic matter content (%) in the laboratory
stream substrates and correlation (r) of
residue concentration with organic matter.
Standard deviation in parentheses . . . 42

6. Hexachlorobiphenyl concentrations (ug/g wet
wt.) accumulated by the benthos during the
initial 24 hours of each treatment phase.
Each value is a mean obtained by averaging
values from the individually treated streams.
Except for the fall treatment, the
pre-eXposure concentrations are day 60 values
from the prior depuration period . . . 44

7. Mean HCB concentrations (ug/g wet wt.) in the
benthic flora and fauna during the treatment
and depuration phases of the model streams
study. Each value was obtained by averaging
the mean values for each eXperimental period

(season) . . . . . . . . , 46

vi

Table

8.

10.

11.

12.

13.

14.

15.

Page

Mean HCB concentrations (ug/E) in the
benthos of individual streams on day 30 of
the treatment phase. Except where indicated
the mean values were obtained by averaging
single values for a composite sample across
experimental periods. Values for clams,

~crayfish and fish are the grand means of the

experimental period means of single individ-
uals , O O O O I O O O O O “’7

Rate of accumulation of HCB (ug/g/hour) in

the stream benthos over several intervals of

time for each treatment phase. Values were
calculated as the change in HCB concentra-

tion divided by the corresponding time

interval (hours) . . . . . . . 49

Regression equations calculated for the
accumulation and depuration of HCB in the

stream benthos over time. Correlation
coefficients (r) indicate goodness of fit.

Data points for calculating the equations

were the mean concentrations for a given

sampling day . . . . . . . . 50

Hexachlorobiphenyl concentrations (ug/g wet

wt.) in tissues of watercress plants

(Nasturtium officinale). Each value is the

grand mean of experimental period means . . 53

The range of HCB bioaccumulation factors for
organisms in the laboratory streams. Ranges
reflect seasonal variations . . . . . 55

Rate of accumulation of HCB by amphipods
exposed to three concentrations in water
under static conditions . . . . . . 63

Bioconcentration of HCB by amphipods exposed
to three concentrations in water under
static conditions . . . . . . . 65

Recovery of HCB residues in amphipods and
water during static exPeriments . . . . 66

vii

Table Page

16. Rate of accumulation of HCB residues by amphipods
exposed to 0.200 ug/liter under static conditions
during experiments on the mechanism of uptake . 70

17. Bioconcentration of HCB by amphipods exposed to

three concentrations in water under continuous
flow conditions . . . . . . . . . 74

18. Rate of accumulation of HCB by amphipods exposed
to three concentrations in water under continuous
flow conditions . . . . . . . . . 76

19. Concentrations of HCB in water (ug/liter) of the
upper aquaria two hours after food was added.
Each value represents the mean of two observa-
tions . . . . . . . . 79

20. Ratio of HCB residues in amphipods from upper

and lower aquaria (upper/lower). Accumulation
1n upper tanks was from food and water whereas
accumulatlon 1n lower tank was from water only . 82

21. Water concentrations (ug/liter) which would be
required in upper and lower aquaria if HCB
accumulatlon by amphipods was from water only.

Values were backcalculated using the bioconcen-
tration factors from the continuous flow study . 83

22. Particle size distribution, organic matter
content and HCB concentrations in various

fractions of Cheny Creek and Arboretum Pond
substrates at the end of Trial 2 . . . . 89

23. Hexachlorobiphenyl concentrations in water
(ug/liter) overlying Cheny Creek and Arboretum
Pond substrates during the equilibration and
exposure phases of Trial 3. . . . . . 91

viii

Table
214‘.

25.

26.

A1.

AZ.

Page

Bioaccumulation and bioconcentration factors

for amphipods which were exposed to HCB
contaminated substrates and to contaminated

water respectively. The factors were

computed as the HCB concentration in the

organisms (ng/g)/concentration in water

(ug/liter) . . . . . . . . . 94

Particle size distribution and HCB concen-
trations in various fractions of Cheny Creek

and Arboretum Pond substrates at the end of

Trial 3. Substrates had been combusted

prior to use . . . . . . . . . 96

Hexachlorobiphenyl concentrations (ug/g) in
amphipods and organic matter content (fl) of
substrate particle size fractions on day 8

of each experiment. During Trials 1 and 3

the substrate particle size fractions had
naturally occurring levels of organic matter
Prior to Trials 2 and 4 the substrates were
combusted to remove natural organics . . . 103

The daylength maintained in the laboratory
during the experimental periods of the model
streams experiments . . . . . . . 114

Temperature and water chemistry for the

water used in the model streams study.
Values are means for each month . . . . 115

ix

Figure

LIST OF FIGURES

Diagram of one pair of laboratory streams.
The direction of water flow is indicated
by arrows . . . . . . . . .

Uptake and depuration of hexachlorobiphenyl
in the organic matter of the laboratory
stream substrates. Data points are averages
for each stream which were then averaged
across experimental periods. Vertical bars
indicate standard errors about the mean .

Disappearance of HCB from water at three
nominal concentrations under static condi-
tions. Vertical bars indicate one standard
deviation above and below the mean . .

Accumulation of HCB by amphipods exposed to
three nominal concentrations in water under
static conditions. Vertical bars indicate
one standard deviation above and below the
mean . . . . . . . . .

Residue concentrations of HCB in acetone
extracts of amphipods which had been rinsed
quickly in water or acetone at the time of
sampling. Each data point represents the

mean of three samples . . . . .

Percent of total extracted radioactivity that

was found in the acetone rinse of amphipods
exposed to 0.200 ug HCB/1 . . . .

Concentrations of HCB in water for three

nominal test concentrations during continuous

flow exposure . . . . . . .

Accumulation of HCB by amphipods exposed to
three nominal concentrations in water with
continuous flow conditions . . . .

Page

11

41

61

62

68

69

71

73

Figure
9.

10.

ll.

12.

13.

Page

Accumulation of HCB by amphipods which were

exposed to contaminated food and water and

to contaminated water only. Each data

point represents the mean of two replicate

samples . . . . . . . . . . 81

Accumulation of HCB residues by substrate-

exposed and water-exposed amphipods for two
different intact substrates. Substrates

contained naturally occurring levels of

organic matter . . . . . . . . 86

Accumulation of HCB residues by amphipods

exposed directly to contaminated inorganic
substrates (substrate-exposed) or to residues

in contaminated overflow water only (water-
exposed) . . . . . . . . . . 93

Concentrations of HCB in water (ug/liter)

from aquaria at a water-to-substrate ratio of
11.2521 (wt/wt) and in aquaria receiving the
overflow water from both the high and low

ratio (1:1, wt/wt) of water-to-substrate

aquaria during the period of amphipod

exposure . . . . . . . . . . 99

Accumulation of HCB residues by amphipods

which were exposed to Cheny Creek substrates

at high (11.25:l.00) and low (1.00:1.00) water

to substrate ratios (grams of H Ozgrams of
substrate). Substrate-exposed amphipods were
subject to uptake from water and substrates

while uptake by water-exposed amphipods was

from contaminated water only . . . . . 100

xi

INTRODUCTION

The polychlorinated biphenyls (PCB's) are complex
mixtures of chlorinated organic compounds of industrial
origin and have become wideSpread contaminants in the envi-
ronment. Their structure, chemical properties, commercial
uses, acute and chronic toxicity, environmental residues,
rates and route of transport and metabolism have been
reviewed by Gustafson (1970), Maugh (1972), Hammond (1972),
Peakall and Lincer (1970), Peakall (1972), Risebrough and
Brodine (1970), Ahmed (1976), Nisbet and Sarofim (1972),
and Veith and Lee(1970, 1971a). The toxicity of the PCB's
to fish and aquatic invertebrates has been described by
Stalling and Mayer (1972), Halter and Johnson (1974),
Nebeker et a1. (1974), Mayer et a1. (1977), Defoe et a1.
(1978), and Mauck et a1. (1978).

In general, the PCB's are hydrophobic, lipophilic
and persistent compounds of low water solubility and volatility
which behave in the environment in a manner similar to many
of the chlorinated hydrocarbon pesticides, eSpecially dichloro-
diphenyldichloroethylene (DDE). Because of their hydrophobic
nature, the PCB's and other chlorinated hydrocarbons are

attracted to surfaces which results in their accumulation on

pond and stream bottoms and by inorganic and organic materials

1

2

suSpended in the water column (Cope 1966: Gerakis and Sficas
1974). Thus, almost all of the PCB's released into fresh
water and substantial portions of the chlorinated hydrocarbons
entering the sea are adsorbed on bottom sediments (Nisbet
and Sarofim 1972; Hom et a1. 1974). The bottom sediments of
Escambia Bay below an industrial outfall had PCB residues
as high as 486 ppm (Duke et a1. 1970) while Baltimore Harbor
sediments ranged as high as 84.2 ppm (Morgan and Sommer
1979). The worst case of environmental pollution by PCB's
'is in New York where Hudson River sediments in the Fort
3Edward area contain PCB concentrations ranging from 6.6 -
f %' '6700.0.ppm (Nadeau and Davis 1976). In the Great Lakes there
7V is an estimated 84-100 tons of PCB in the water column and
V bottom sediments have been proposed as the immediate source
\of this material (Nisbet and Sarofim 1972; Neely 1977).
Sorption of neutral hydrophobic compounds onto suspended
material and bottom sediments is usually attributed to an
equilibrium exchange reaction which.oc¢urs rapidly.and is
reversible (Baughman and Lassiter 1978: Paris et a1. 1977:
Paris and Lewis 1976). This reaction can be described by a
simple distribution coefficient which is a function of the
differential solubility of the solute in two different
solvents. Thus the water solubility of the PCB's which is
inversely related to their chlorine content will determine

the distribution of the mixture with the result that the lower
chlorinated chlorohomologues and isomers will be preferentially

dissolved in the water (Hague et a1. 1974; Paris et a1. 1978).

3

Adsorption onto sediments creates a reservoir of material
which can buffer changes in water concentrations (Frank et a1.
1974: Baughman and Lassiter 1978) and may stabilize the
compounds against volatilization and microbial degradation
(Pionke and Chesters 1973; Oloffs et a1. 1972, 1973). Adsorp-
tion may also reduce toxicity to aquatic organisms by making
the material unavailable for uptake (Ferguson et a1. 1965;
Terriere et a1. 1966; Veith and Lee 1971b). However, several
workers have reported that DDT and methoxychlor adsorbed on
suspended particulates are a very effective means of control-
ling filter-feeding simulid larvae for as much as 98 miles
downstream from the point of application (Arnason et a1. 1949;
Fredeen et a1. 1953; Wallace et a1. 1976).

sediment characteristics can affect the extent of
residue accumulation by sediments which may lead to a hetero-
geneous distribution of the compounds in the environment.
Highest concentrations of hydrophobic compounds are often
associated with fine textured sediments (Browman and Chesters
1977; Frank et al. 1974; Hetling et al. 1978; Hargrave and
Phillips 1974; Steen et a1. 1978; Karickhoff and Brown 1978;
Routh 1972; Richardson and Epstein 1971). In addition, organic
content is often inversely related to particle size with the
result that fine textured sediments are often high in organic
matter (Hargrave 1972; Odum and de la Cruz 1967).

Hydrologic conditions affect the distribution of sediments

and thus influence the distribution of PCB residues in the

aquatic environment. Higher concentrations of DDT

4

and PCB have been detected in the sediments of deep basins
of the Great Lakes than in nearshore sands and gravels
(Leland et a1. 1973; Glooschenko et a1. 1976). Impoundments
may also produce conditions conducive to dieldrin buildup

in the benthos as silt settles out (Morris and Johnson 1971).
Significant differences in sediment pesticide levels were
found between sampling points in ponded and fast flowing
waters which were attributed to greater amounts of organic
matter and fine particles in the sediments of the ponded
areas (Dimond et a1. 1974). The bed load (the shifting mass
of detritus and sediment which forms the interface between
water and bottom materials) of Ontario streams contained

3 - 6 times more DDT, dieldrin and endosulfan than did
bottom sediments (Miles 1976).

Residue levels of chlorinated hydrocarbon insecticides
in stream sediments are often correlated with the organic
matter content (Pionke and Chesters 1973: Richardson and
Epstein 1971). Hetling et a1. (1978) found that PCB residues
in Hudson River sediments were higher when associated with
either muck or sand which contained wood chips than in sand
without wood ships. In Lake Michigan sediments, Leland et a1.
(1973) reported that the DDT residue concentrations were
significantly related to the organic carbon content but
Glooschenko et a1. (1976) were not able to establish such a

relationship for either DDT or PCB's.
The association of chlorinated organics with fine

textured sediments has often been attributed to increased

5

surface area available for adsorption. Even among organic
detritus particles, higher concentrations of DDT were

noted in small particles than in larger undecayed pieces
(Odum et a1. 1969). However, Aroclor 1254 was found to

be evenly distributed in particle size fractions of Escambia
Bay sediment (Nimmo et a1. 1971) while PCB concentrations
in Great Lakes sediments were not clearly correlated with
sediment texture (Glooschenko et a1. 1976). More work is
needed to elucidate the role of the various substrate
characteristics in the accumulation of chlorinated organics
by bottom sediments.

A number of workers have speculated that benthic
organisms can accumulate hydrophobic organics from
contaminated substrates and thus provide a means of entry
into aquatic food chains (Holden 1965; Odum et a1. 1969;
Leshniowsky et a1. 1970; Yule and Tomlin 1971; Veith and
Lee 1971b: Reinert.l972; Herbes 1977: Karickhoff and Brown
1978; Weininger 1978). There is little experimental evidence
to substantiate this hypothesis although some observations
from survey data do lend some support to the idea. Wallace
and Brady (1971) observed that most of the organisms with
high dieldrin concentrations in their study were near the
bottom of the food chain and were either detritus or
omnivorous filter feeders. In another study, benthic feeding

fish contained larger amounts of dieldrin than species whose

feeding habits were less benthically oriented (Morris and

6
Johnson 1971). Hindin et a1. (1968) observed that

organisms which ingested sediment had the greatest
concentrations of chlorinated organics in their body
tissues. Accumulation of PCB's by snails has also been
attributed to ingestion of contaminated Hudson River
sediments by Nadeau and Davis (1976). matsumura and
Benezet (1973) observed that the highest residues of TCDD
were accumulated by mosquito larvae which were also the
only benthic feeding organisms in their studies with
contaminated substrates. Thus, the potential for
increased accumulation of residues due to direct exposure
to contaminated substrates appears to exist and should be
examined in greater detail.

Many aquatic organisms exhibit Species specific
preferences for a given range of substrate particle sizes
which is often exhibited in different distribution patterns
for various macro- and microhabitats (Shelford and Eddy
1929; Wene and Wickliff 1940; Pennak and Van Gerpen
1947; Bell 1969; Chutter 1969; Hynes 1970; Cummins and Lauff
1969; Barber and Kevern 1973; Reice 1974; Higler 1975). In
fact, Cummins and Lauff (1969) have proposed that substrate
particle size may represent a common denominator in habitat
selection by benthic stream invertebrates.

Food resources are also partitioned on the basis of
particle size and whether they are active, stationary or in

suspension (Cummins 1973, 1974; Petersen and Cummins 1974;

Cummins et a1. 1973). Community structure may also depend

7

on the distribution of detritus within aquatic ecosystems
as Egglishaw (1969, 1964) has shown a definite correlation
between numbers of organisms present in substrate samples
and the quantity of detritus available for consumption.

Because of the tendency of hydrophobic organics to
accumulate in bottom sediments, benthic organisms are
exposed to higher concentrations than are pelagic species.
In addition, those organisms which are attracted to, or
feed on, fine textured sediments of high organic content
may be exposed to the highest concentrations. Whether
these residues are available to the organism is largely
unknown and needs to be evaluated as does the mechanism
and route of uptake by sediment dwelling organisms. It is
vitally important to our understanding of aquatic toxicology
that we determine whether sediments can be considered sinks
and safely ignored or if they will continue to be long-
term sources of contaminants to the overlying water and
to benthic and pelagic organisms.

The commercial PCB's are very complex mixtures and
are difficult to analyze. In order to simplify analysis
and data interpretation a representative isomer was
chosen for study. The isomer selected was 2,4,5,2',4',5'—
hexachlorobiphenyl which is a major component of the
technical PCB's with chlorine contents greater than 50%

(Jensen and Sundstrom 1974). Hexachlorobiphenyls comprise

8

4%, 34% and 38% of Aroclors 1248, 1254 and 1260 respectively
(Hammond et a1. 1972). Chiou et a1. (1977) reported that
2.4.5.2',4',5'-hexachlorobiphenyl is soluble in water at
0.95 ug/liter (24°C) and has an octanol/water partition
coefficient (log) = 6.72.

The primary objectives of this study were to determine;
1) the residue dynamics and metabolism of the isomer in
the water, substrate and benthos of a laboratory model
of a woodland stream, 2) the respective roles of uptake
from water and from contaminated food, 3) the availability
of residues to the benthos from contaminated substrates,
4) the relative importance of substrate type, organic
matter content and particle size in the availability of
residues from contaminated substrates to the overlying

water and to the benthos.

MATERIALS AND METHODS

The fate and behavior of 2.4.5.2',4',5'-hexachloro-
biphenyl (HCB) in aquatic systems were studied in two
phases; 1) a year-long model streams study and 2) a series
of short-term laboratory studies during which exposure
conditions were systematically altered.

The model streams study was designed to determine the
accumulation, depuration and metabolism of HCB residues in
various abiotic and biotic components of seminatural labor-
atory streams. The laboratory streams represented a complex,
multi-species ecosystem which retained many of the natural
characteristics of a small woodland stream.

The short-term laboratory studies were designed to
determine the relative importance of HCB uptake from
exposure to contaminated water, food, or substrate and the
effect of substrate type, organic content, and particle size
on the availability of substrate-adsorbed HCB to a represent-
ative benthic macroinvertebrate.

Model Streams-—Description
The laboratory streams were constructed in the fish

hatchery building of the Michigan Department of Natural

10

Resources located at Paris, Mecosta County, Michigan.
The site provided a natural stream water supply and an
abundance of aquatic organisms for colonization of the
laboratory streams.

The hatchery was supplied with water from Cheny Creek,
a woodland stream of approximately 5.9 mB/minute discharge
which originates from several springs within a fenced water-
shed adjacent to the hatchery. Water was diverted to the
hatchery through an underground pipe that leads from a
concrete diversion dam and headbox 320 m upstream. Trash
screens within the headbox removed large debris but allowed
the free entry of aquatic organisms and particulate material
drifting in the water supply.

Within the hatchery building, the laboratory streams
were constructed by modification of existing concrete
troughs. An upper tier of six troughs, each 3.96 m x 0.58 m
x 0.27 m. served as constant-head reservoirs for a lower tier
of identical sized troughs that served as the laboratory
stream channels (Figure 1). Water entered each reservior
trough through paired pipes (3.17 cm diameter) and then
drained by gravity through paired 5.08 cm diameter pipes
to the matching stream channel below. Water flow to each
stream was maintained at 60 liters/minute by adjustment'

of a gate valve at the head of each reservoir trough.

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12

A single drain (10.2 cm diameter) at the end of each
stream channel diverted the water to a common filter box
which contained alternate layers of activated carbon and
polyurethane foam. The filtered effluent was discharged
to the Muskegon River.

The stream channels were illuminated by 48 high-
intensity, cool—white fluorescent lamps attached to plywood
sheets suspended above the streams. Light intenSity at the
stream water surface was 900 t 50 foot candles. The lights
were wired through an automatic time switch which was
adjusted at two-week intervals to conform with the natural
day length of the local area (Table A1).

Water temperatures in the laboratory streams followed
the natural daily and seasonal fluctuations of Cheny Creek
which ranged from a maximum of 15.5°C in may to a minimum of
0.5°C in December (Table A2). Data for dissolved oxygen,
pH, alkalinity, and hardness are also presented in Table A2.

In June 1975, natural substrate from Buckhorn Creek,
Mecosta County, Michigan, was transferred to the stream
channels. Equal quantities of substrate which consisted of
silt, sand, gravel, and rubble (maximum 10 cm diameter) were
uniformly distributed in each stream channel to a depth of

5 to 10 cm.

Biological colonization resulted from the organisms

introduced with the substrate and from immigration of

13

organisms drifting in the water supply. A diverse
community of algae, invertebrates, and bryophytes was in
evidence eight weeks after substrates were added.

Larger stream organisms, i.e., fish, clams, and
crayfish, were added to the stream communities prior to
each chemical treatment. These organisms were collected
from nearby streams and acclimated to the Cheny Creek water
for a minimum of two weeks prior to stocking in the streams.
The test animals were not fed either before or during their
residence in the test streams, but were free to forage
within the biological communities existent within the

streams.

Model Streams-Experimental Procedure

The model stream experiments were planned to determine
the fate and residue dynamics of HCB in the seminatural
streams through one annual period. The design of the
streams and the water supply allowed "natural" seasonal
fluctuation in temperature, photoperiod, water chemistry,
and biological community structure, but required control of
stream flow, light intensity, and the stocking and replace-
ment of larger stream organisms, i.e., clams, fish and cray—
fish.

One year was allowed for biological colonization of the
streams and pretreatment measurements. In the second year,

four replicate treatments were timed to coincide with the

14

fall, winter, Spring, and summer seasons. During each
experimental period (season), HCB was metered into the test
streams during a 30-day treatment phase which was followed
by a 60-day depuration phase. This sequence Was repeated
for each season except a depuration phase was not included
in the summer eXperimental period.

Two streams were treated with HCB, and one was
designated as a solvent control. The control stream received
acetone equivalent to the amount which was added to the
test streams with the chemical stock solutions.

The chronological sequence of the major experimental
events is listed in Table l.

Radiolabeled and unlabeled 2,4,5,2',4',5'-hexachloro-
biphenyl were obtained from Pathfinder Laboratories, Inc.,

lac-ring-labeled compound

St. Louis, Missouri. The uniformly
had a specific activity of 14.06 mCi/mM. Gas and thin layer
chromatography indicated that the compounds were:>98% pure.
Stock solutions were prepared by mixing appropriate quan-
tities of labeled and unlabeled HCB in acetone to provide a
concentration of 127.12 mg/liter and a Specific activity of
8.60 dpm/ng.

Syringe pumps (Harvard model 975) which were equipped
with 100 m1 capacity glass syringes and teflon needles

delivered the stock solution to mixing chambers at the

lower end of each reservoir trough. Water turbulence in

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16

the mixing chambers dispersed and mixed the stock solution
with diluent water before discharge to the stream channels.
A 1.27 x 106-fold dilution of the stock solution gave a
nominal stream HCB concentration of 100 ng/liter.

Acetone was metered into the control streams at the same
rate as in the test streams (0.69 ul/liter).

Syringes were filled daily and the quantities of stock
solution used during the previous 24 hours were recorded.
Syringe failure occurred on several occasions but pumping
rates were always reestablished within a 12-hour period.

Concentrations of HCB in water, substrate, and
selected plants and animals were determined from samples
collected during each experiment. Substrate was collected
from all streams on days 5, l5, and 30 of each treatment
phase and days 15, 30, and 60 of each depuration phase.
Crayfish and sculpins were collected on days 15 and 30
and clams were removed on day 30 of the treatment phases.
Water, benthic invertebrate, and plant samples were
collected from the streams at 3, 6, 12, 24, 48, and 96 hours,
and on days 8, l6, and 30 of each treatment phase.

During the depuration phase of each experiment, samples
were collected on days 4, 8, 16, 32, and 60.

Biological samples usually included amphipods, snails,
stoneflies, mayflies, aquatic earthworms, filamentous algae

and bryophytes, but it was occasionally necessary to omit

certain groups and/or to sample others depending upon their

1?

seasonal availability. Samples of watercress, an aquatic
macrophyte, which was abundant in the lower sections of the
streams were subdivided into leaf, stem, root, and seed
pod components when possible.

Small invertebrates were collected by removing indi-
vidual organisms with forceps and small dip nets. A sample
of 10-20 organisms of a Single species was placed in a
glass vial and stored at -20°C until analyzed. Larger
organisms, including fish, clams, and crayfish, were
wrapped in aluminum foil and frozen.

Substrate cores were collected with a 10.2 cm diameter
core sampler which was pushed to the stream bottom. The
entire core was lifted with a stainless steel end-plate,
placed in a plastic bag and stored at -20°C.

Composite 2 liter water samples were collected by
siphoning equal quantities of water from the upper, middle,
and lower portions of each stream. The samples were

extracted within a few hours after collection.

Laboratory Studies - Objectives

Laboratory studies were conducted to determine the
residue dynamics of HCB under simplified exposure conditions.
Organisms were exposed to the chemical in water under static
and continuous flow conditions and to a contaminated food
source in order to determine the contribution of each

exposure condition to the accumulation of body residues.

18

Static tests are often used to Simulate exposure to a Spill
or other single dose introduction whereas continuous flow
conditions would be analogous to a constant input of a
toxicant in a stream, river or well mixed lake. Experi-
ments with contaminated food as a source of residues would
give some indication of the potential for transfer through
food chains and would also indicate whether uptake from food
or from water was the dominant pathway of residue accumu-
lation.

The availability of substrate-bound residues to benthic
organisms was also evaluated. The purpose of these experi-
ments was to determine the potential of contaminated sedi-
ments as long term sources of toxicants in aquatic environ-
ments and the extent to which residue levels in the overlying
water and in benthic organisms are controlled by substrate
characteristics. Of primary interest in this regard were
the respective roles of substrate type, organic matter
content and particle size distribution.

It was hoped that the laboratory studies would also
support and clarify the results and conclusions derived from

the more complex model streams experiments.

Laboratory Studies - Experimental Procedures

Laboratory studies were conducted in the Fisheries
Research Laboratory on the campus of Michigan State Univer-

sity. Well water of the following characteristics was used

19

in all tests; total alkalinity as CaC03, 270-330 mg/liter;
hardness as CaC03, 329 mg/liter; pH, 7.4-7.8; dissolved
oxygen, 9.1-10.0 mg/liter; and temperature, 10-l4°C.

The organism selected for laboratory study was the
amphipod, Gammarus pseudolimnaeus Bousfield, which is
widSSpread and common in unpolluted clear waters including
springs, Spring brooks, and streams. Generally, freshwater
gammarids are cold-stenothermal, photonegative, thigmotactic,
(Holsinger 1976), omnivorous, scavengers.which utilize'
both plant and animal materials (Pennak 1953).

Amphipods were chosen as the test organism because
they were an important component of the model streams
community in terms of abundance and biomass. They are also
important in the diet of stream fish and were observed
to occupy a variety of substrate types within Cheny Creek
and the model streams. In addition, they were; available
in large numbers, easy to maintain in laboratory culture,
and durable when handled.

Amphipods were collected from Cheny Creek and cultured
in the laboratory in a circular stream of the type used by
McIntire et a1. (1964). A paddlewheel circulated the water
and a continuous inflow of well water maintained water tem-
perature at 12-1400. Photoperiod (16 hours light, 8 hours
dark) was provided by natural daylight which was supple-

mented by fluorescent lights suspended over the stream.

Amphipods were fed preleached maple leaves (Acer Sp.) and

20

Purina Trout Chow #2. Amphipods could be maintained

indefinitely when cultured in this manner.

Accumulation from Water — Static Exposure

Amphipods were exposed to HCB under static conditions
in 2 liter glass beakers which contained 1.5 liters of well
water. Known quantities of HCB in 1.0 ml acetone were‘
added to each beaker below the water surface and stirred to
create nominal concentrations of 0.100, 0.200, and 0.400
ug/liter. Amphipods (15-20/beaker) were added to the
beakers which were then placed in water baths (12-16°C).

At 24, 48, 96, and 192 hours of exposure, three
beakers per concentration were removed from the water baths.
Water samples (1 liter) were siphoned from each beaker
into 4 liter amber glass bottles which contained 100 ml of
hexane. The bottles were Shaken for 30 seconds and set
aside at room temperature until extracted. Samples were
usually extracted within one week of collection. Amphipods
were removed by dip net, placed in glass vials, and frozen

until analyzed.

Mechanism of Accumulation

The mechanism of accumulation of HCB residues by
amphipods under static conditions was studied at a nominal
HCB concentration in water of 0.200 ug/liter. During trial
1, water samples (1 liter) and amphipods (14-20) were

collected from each of three replicate beakers at 24, 48,

21

96, and 192 hours of exposure. Water samples were treated

as described. Amphipod samples were divided into two lots;
the individuals of one lot were quickly dipped five times

in a scintillation vial containing 10 m1 of acetone,

the other lot was dipped in distilled water. Both lots

were placed in separate glass vials and frozen. The acetone
was retained for later analysis by liquid scintillation tech-
niques. During trial 2, the entire experiment was repeated
as described but additional samples'were taken after 2, 6,

and 12 hours of exposure.

Accumulation from Water - Continuous Flow Exposure

Accumulation of HCB by amphipods from water under

 

continuous flow conditions was tested in 20 liter glass
aquaria at nominal HCB concentrations of 0.100, 0.200, and
0.400 ug/liter. The aquaria were equipped with standpipes
which maintained the water level at 9 liters. Harvard
syringe pumps (Model 975), which were equipped with 100 m1
glass syringes, delivered known quantities of HCB in acetone
at 1.45 ml/hour into the bottom of 2 liter beakers which
served as mixing chambers. Diluent water (100 ml/minute)
was added to each mixing chamber from which the water over-
flowed through a standpipe into a glass Splitter box which
in turn delivered the water into duplicate test aquaria.
The flow rate of 50 ml/minute per aquarium provided a 90%

replacement time of about 7 hours (Spraque 1969).

22

Hexachlorobiphenyl was injected into the system for 24 hours
to allow the system to equilibrate after which 50-60
amphipods were added to each aquarium.

At 24, 48, 96 and 192 hours of exposure, water samples
(1 liter) were siphoned from each aquarium and treated as
described; 8-10 amphipods were dipnetted from each aquarium

at these times and frozen until analyzed.

Accumulation from Food

Accumulation of HCB residues by ingestion of contaminated
food was determined by placing 120 amphipods into each of six
20 liter aquaria. Each aquarium contained 9 liters of water
which was continuously replaced at 50 ml/minute. Water from
each of the aquaria overflowed via a standpipe into an
identical set of’six aquaria which contained 60 amphipods each.

The food which was used in this study was Purina Trout
Chow #2 pellets. The food was ground with a mortar and
pestle and then contaminated with HCB at concentrations of
1.45, 14.5, and 145 ug/g dry weight by adding known quantities
of stock solution in hexane to l g lots of food. Excess
solvent was removed by evaporation under vacuum and under a
nitrogen stream- Uncontaminated food was treated with the
hexane only.

One particle of the contaminated food was added daily
for each amphipod in the upper set of aquaria. Amphipods

were allowed access to the contaminated food particles for

23

two hours after which any unconsumed food was removed.
Organisms in the lower set of aquaria were fed only uncon-
taminated food which was also removed after two hours. The
number of food particles added to both sets of aquaria was
adjusted for the number of amphipods remaining after previous
sampling activities.

Hexachlorobiphenyl accumulation by amphipods in the upper
set of aquaria was assumed to be from the contaminated food
and/or by direct uptake from water of HCB that had desorbed
from the food. Residues in the amphipods from the lower set
of aquaria were assumed to be from water only.

At 24, 48, 96, and 192 hours of exposure and prior to
feeding, 16-20 amphipods were removed from each of the upper
aquaria. Each amphipod sample was divided into two lots; one
was used for whole body residue analysis and the second for
separate determinations of residues in the body tissues and as
unabsorbed gut contents. Amphipods (8—10) were also collected
at these times from each of the lower aquaria.

Water samples (1 liter) were obtained from each aquarium
on a similar schedule but were taken two hours after the food

was presented.

Availability from Substrates
The availability of HCB to amphipods from artificially

contaminated natural substrates was studied to determine the

role of differences in substrate type, particle Size distri-

bution, individual particle size fractions, and the quantity

24

of organic matter within each soil type or particle Size
fraction. The HCB availability experiments may be separated
into two phases based on whether intact or sieved substrates
were used; each phase will be treated separately because of

procedural differences.

Intact Substrates. The availability of HCB from
unfractionated substrates was studied in 20 liter glass
aquaria which were arranged in two tiers of six aquaria each.
Each of the upper tier aquaria contained 800 g of substrate
and 9 liters of water which was replaced by clean well water
at the rate of 50 ml/minute (90% replacement time = 7 hours,
Spraque 1969). Flow rates were checked daily and adjusted
as necessary. Water levels in the upper aquaria were main-
tained by surface overflow standpipes; excess water from
each upper aquarium flowed through a short length of
glass tubing into one of the lower aquaria which did
not contain substrates. Water levels in the lower aquaria
were maintained at 9 liters by surface overflow standpipes
through which excess water was discharged to floor drains.
Accumulation of HCB by amphipods in the upper aquaria was
assumed to be the result of direct uptake from water of
HCB that had desorbed from the substrate, substrate ingestion,
and/or by direct contact of amphipods with the substrate

while accumulation in the lower tanks was assumed to result

from water uptake only.

25

Two different natural substrates were used in the
availability studies. One was obtained from a riffle in
Cheny Creek, Mecosta County, Michigan while the other was
dredged from a pond in the Fenner Arboretum, Ingham County,
Michigan. Prior to use, both substrates were dry sieved
so that only particles <2.0 mm were utilized. Cheny
Creek Substrate consisted primarily of washed coarse and
medium sand (organic content = 0.67%) while the Arboretum
Pond substrate consisted primarily of medium and fine
sand (organic content = 4.61%).

At the start of each experiment, 800 g (dry wt.) of
Cheny Greek or Arboretum Pond substrate were added to each
of three replicate aquaria. To each substrate, 800 ug of
HCB were added in excess acetone until the substrates were
saturated. The acetone was allowed to evaporate until the
substrates were dry. The nominal HCB concentration in
substrates was thus 1 ug/g; radiolabeled HCB represented
1% of the total HCB. Well water was slowly added so as to
minimize disturbance of the substrates, continuous flow of
clean well water was initiated (50 ml/minute), and the
system allowed to equilibrate for 7 - 15 days prior to the
addition of 50-60 amphipods per aquarium.

Four trials were conducted as described below. During
each trial 7-10 amphipods were dipnetted from each aquarium
after 24, 48, 96, and 192 hours of exposure, placed in glass

vials, and frozen until analyzed.

26

During trial 1, only the upper tier of aquaria was used.
The system was allowed to equilibrate for 12 days prior to
amphipod exposure. Water samples (1 liter) were taken after
24, 48, 96, and 192 hours of equilibration; because residue
levels were below the limit of detection (0.0145 ug/liter)
water sampling was discontinued during the period of amphipod
exposure. .

For trial 2, the lower tier of aquaria was installed
and the substrates from trial 1 were reutilized. No water
samples were taken.

Prior to use in trial 3, Cheny Creek and Arboretum
Pond substrates were combusted at 500°C for eight hours to
remove all organic material. The substrates were then
distributed to the aquaria, contaminated, and allowed to
equilibrate for 15 days. Water samples were taken after
24, 48, 96, and 192 hours of equilibration and after 24, 48,
96, and 192 hours of amphipod exposure.

For trial 4, only Cheny Creek substrates were utilized.
Experimental design was modified in that the two tiers of
aquaria contained only four aquaria each; only the upper
aquaria contained substrate. The standpipes on two of the
upper aquaira were adjusted so that only 0.8 liters of water
covered the substrates (water to substrate ratio = 1:1,
wt./wt.) while the standpipes in the other two aquaria
maintained a water level of 9 liters (water to substrate

ratio = 11.2521, wt./wt.). Upper aquaria overflowed into

27

lower aquaria which contained 9 liters of water. The system
was allowed to equilibrate for seven days. After 24, 48,
96, and 192 hours of amphipod exposure, two water samples
were obtained from each of the 9 liter upper tier aquaria to
determine if a vertical gradient in HCB concentration
existed; one was siphoned from just below the surface and
the other from just above the substrate. No water samples
were obtained from the 0.8 liter aquaria. One middepth
water sample was obtained from each of the four lower tier
aquaria.

Nominal HCB concentration in the substrates was 1 ug/g
for all trials but the percentage of radiolabeled HCB was
increased from 1% in trials l-3 to 2.44% in trial 4 to A
lower the limits of HCB detectability.

At the end of trials 2, 3, and 4 the substrates were
removed from the aquaria, placed in aluminum pans, and frozen
until analyzed for HCB residues. Excess unextracted substrate
from replicate aquaria of trials 2 and 3 which remained
after residue determination were composited by substrate
origin (Cheny Creek or Arboretum Pond) and trial, then frac-
tionated by dry sieving to determine particle size distri-
butions; the organic matter content and HCB residue concen-

trations in each particle size fraction were also determined.

28

Sieved Substrates. The availability of HCB to amphipods
as a function of substrate particle size and organic matter
content was determined in 2 liter glass beakers. The
beakers were equipped with glass standpipes which main-
tained the water level at 1.5 liters. Well water flowed
continuously into each beaker at 30 ml/minute which gave a
90% replacement time of about two hours (Spraque 1969).

Cheny Creek and Arboretum Pond substrates were wet and
dry Sieved (twice each) into five particle size fractions
per substrate. The particle size fractions, in millimeters,
used in the experiment were; 1.0-0.50, 0.50-0.25, 0.25-0.12,
0.12-0.06 and less than 0.06.

Prior to an experiment, four aliquots of known weight
of each Size fraction were placed in individual beakers. A
known quantity of HCB (2.44% lLAC) in acetone (50-100 ml) was
added to each beaker, swirled to insure uniform mixing, and
the beakers set aside for 3-5 days for acetone evaporation.
Water (1400 ml) was slowly added to each beaker so as to
minimize substrate disturbance and allowed to stand for 12-
24 hours. Continuous flow was then initiated and the system
allowed to equilibrate for an additional 24 hours at which
time 15 amphipods were added to each beaker.

At 24, 48, 96, and 192 hours of amphipod exposure, water
samples (1 liter) were siphoned from one beaker of each

particle size fraction, the excess water was discarded, and
amphipods were separated from the substrates. Amphipods and

substrates were frozen until analyzed for HCB residue

29

concentrations.

Five trials were conducted using this design; differ-
ences in experimental conditions are summarized in Table 2.

Prior to trials 3 and 4, the particle size fractions
of each substrate were combusted at 500°C for eight hours
to remove organic materials. During trial 5 preleaChed,
dried maple leaves were chopped in a meat grinder and a
Wylie mill, then dry sieved twice to obtain the five particle
size fractions.

Amphipods were not fed during any of the trials but were

free to graze on organic materials within the substrates.

Sample Extraction and Analysis

Water samples (2 liters) from the model streams study
were extracted with three successive additions of hexane
(200, 100, 100 ml); the combined extracts were evaporated
under vacuum to 20 ml and three 0.5 ml aliquots were counted
in scintillation vials which contained 15 ml scintillation
fluor (3a20 fluor, Research Products International Corp.).
Water samples (1 liter) from laboratory exPeriments were
handled in the same manner but hexane volumes were propor-
tionately reduced. Final HCB concentrations were corrected
for isotope dilution. Recovery of HCB from fortified water
samples averaged 106.2%.

Substrate samples were thawed and air dried, then 50 g

subsamples were extracted in glass chromatographic columns

with acetoneshexane (1:1, v/v), (U.S.E.P.A. 1977), and

Table 2.

Summary of procedural differences for experiments

on HCB availability from sieved substrates.

 

 

Cheny

natural natural
organic organic
levels

levels

Fraction (mm)

1.00-0.50
0.50-0.25
0.25-0.12
0.12-0.06

< 0.06

1.00-0.50
0.50-0.25
0.25-0.12
‘ 0.12-0.06
.4 0.06

100
100
100
15
10

1.0
1.0
1.0
1.0
1.0

2

Substrate Origin and Condition

Arboretum Arboretum Cheny

100
100
100
20
20

1.0
1.0
1.0
1.0
1.0

Trial Number

inorganic
materlals

only

100
100
100
100
100

Nominal HCB

1.0
1.0
1.0
1.0
1.0

only

Substrate Dry Weight (g)

100
100
100
15
10

Maple
leaves

inorganic high
materials organic
content

15
15
15
15
15

Concentrations (ug/g)

1.0
1.0
1.0
1.0
1.0

1.0
1.0
1.0
1.0
1.0

 

31
prepared for counting as described above. Recovery from
fortified samples averaged 82.7%.

Small invertebrate organisms were blotted to remove
excess moisture, then homogenized in glass tissue grinders
containing 2 ml acetone. The homogenates were centrifuged
for 5-10 minutes, the supernatant transferred to centrifuge
tubes, evaporated to 2.0 ml under a nitrogen stream, and
prepared for counting. The extraction efficiency of forti-
fied amphipod samples was 101%; the extraction efficiency was
considered to be the same for other small invertebrates.

Clams, crayfish, and fish were individually weighed,
and then homogenized in a mortar and pestle with anhydrous
sodium sulfate. The homogenate was extracted for four hours
with 200 ml hexane in a Soxhlet apparatus; the extract was
concentrated to 2 ml volume, and cleaned up on a Florisil
column (700 mm x 22 mm glass chromatographic column which
contained 10.2 cm Florisil activated at 130°C.). The
samples were eluted from the columns with 200 ml of ethyl
ether in hexane (6:94, v/v). The eluent was concentrated to
2 ml volume and three 0.5 m1 aliquots prepared for counting.
Recovery from fortified samples of fish, crayfish, and clams
averaged 90.47, 69.94 and 76.80%, rSSpectiyely.

Plant materials, i.e., algae, bryophytes, periphyton,
and watercress, were oven dried (5000) for 24 hours and then

100 mg (dry weight) subsamples were combusted in a Nuclear

Chicago, semiautomated combustion apparatus. The 1["002

32

was captured in a 10 ml solution of 2-methoxyethanol:
ethanolamine (2:1, v/v). A 5 ml aliquot was pipetted into
a vial containing 10 ml scintillation cocktail (3a20 fluor:
2-meth0xyethanol, 2:1, v/v) and counted. Hexachlorobiphenyl
residue recovery from fortified samples averaged 71%,
39.1%, and 50% for algae, bryophytes, and watercress resPec-
tively. The reported concentration values were not
corrected for extraction efficiency.

All samples were counted for 10 minutes on a Nuclear
Chicago Mark I liquid scintillation counter. Counting
efficiency was determined with a series of quenched standards

and the external standard method for quench correction.

Metabolite Analysis
A 0.5 m1 aliquot of the extract of samples collected

after 24 and 48 hours and 30 days of exposure to HOB in
the model streams was concentrated to 0.1 ml from which 10-
30 ul were spotted on a Silica Gel G‘F25O thin layer plate.
The plate was eluted to 10 cm with redistilled hexane and
developed under U.V. light; identified Spots were compared
to co-chromatographed Standards. The developed thin layer
plates were exposed to Medical No Screen X-Ray film (Kodak)
for 30-60 days after which the films were developed. Areas

of the film exposed to radioactivity appeared as dark spots
on the film. Suspected metabolites were then counted by

scraping 1 cm zones from the thin layer plate into scintil-

lation vials to which 15 ml of 3a20 fluor was added. The

33

vials were capped, labeled, refrigerated, and counted as
outlined above. No attempt was made to identify suSpected
metabolites other than comparison to known standards which

were co-chromatographed on the thin layer plate.

RESULTS AND DISCUSSION

The Model Streams

The laboratory streams served as excellent models of a
riffle segment in a small natural stream. In essence, the
model stream channels were modified extensions of Cheny Creek.
The flow-through design of the system permitted the contin-
uous import and eXport of small organisms and of dissolved
and particulate inorganic and organic materials. Water
chemistry, temperature, and the biotic communities in the
laboratory streams were similar to those found in Cheny Creek.

The trophic status of the model stream communities was
_ modified by physical constraints Which were imposed on the
laboratory system. Stream flow-discharge was regulated at
a constant 60 liters/minute which eliminated periodic
spates and in turn permitted the establishment of an auto-
trophic community dominated by filamentous algae and water-
cress throughout much of the year. The important effect of
controlled flow-discharge regimes on the dominance of primary
production in running water has been described by Cummins
(1974)- Additionally, the screened water intake to the

streams prevented entry of large aquatic organisms and coarse

organic materials (e.g., leaves) which are usually associ-

ated with heterotrophic conditions in small first-order streams.

34

35

Chemical Fate

Happy. The mean concentration of hexachlorobiphenyl
(HCB) in the water samples from each treatment phase ranged
between 0.04 and 0.18 ug/liter (Table 31 Eighty-five percent
of the measured values were equal to or less than the nominal
value (0.100 ug/liter) and 15% exceeded this value. The
concentration in stream 3 was usually higher than in stream
2, but the concentrations in both streams followed the same
pattern of variation except for a few isolated samples.

Five individual samples which had concentrations
exceeding 0.20 ug/liter were probably the result of HCB
associated with particulate material suSpended in the stream
water. Excluding these few values, the mean HCB concen-
trations in samples from all treatment phases were 0.04 and
0.05 ug/liter for streams 2 and 3, respectively.

During the depuration phases, the HCB.concentrationS in
the stream water decreased below the limits of detection
(0.006 ug/liter) within 4 to 8 days after treatment ended.

Only 40 to 50% of the nominal concentration of HCB
was found in the water samples; of the remainder, most was
probably adsorbed onto stream sediments and biota, but some
may have accumulated at the air-water interface and was
either discharged in the effluent or volatilized to the
atmosPhere. Oloffs et a1. (1972) reported that significant

losses of chlorinated hydrocarbons occurred by evaporation

36

 

 

 

 

 

Table 3. Mean concentrations of HCB (ug/liter) in water
‘ from treated streams during the treatment phase
of each experimental period.
Experimental Period
Fall Winter Spring Summer
STREAM 2
Number of samples 9 9 9 9
Mean 0.043 0.045 0.149 0.051
Standard deviation 0.044 0.034 0.150 0.107
STREAM_3
Number of samples 10 9 9 9
Mean 0.072 0.069 0.180 0.049
Standard deviation 0.096 0.066 0.376 0.023
Mean 0.057 0.057 0.165 0.050

 

37

from natural waters in the absence of sediments but atmo-
Spheric losses were negligible when natural sediments were
present (Oloffs et a1. 1973). Low solubility compounds like
HCB would be expected to adsorb rapidly onto suSpended parti-
culates and onto bottom sediments (Baughman and Lassiter
1978). Haque et a1. (1974) concluded that the concentra-
tion of PCBS in water will be reduced whenever water comes

in contact with a solid surface and that the type and degree
of surface area, and the organic content of the solid will

be important factors.

Higher concentrations of HCB in stream 3 may have been
caused by undetected leakage of stock solution from the
base of the syringe which supplied stream 2. Both syringes
emptied at equal rates and required equal amounts of replace-
ment stock solution when refilled daily. Small chemical
deposits were observed on both syringes at the point where
the plunger entered the barrel. Because of the very concen-
trated nature of the stock solution, even very Slight loss
differences between the syringes could have affected the
concentration in the water.

Substrate. Hexachlorobiphenyl concentrations in the
stream substrates averaged less than 0.1 ug/g sediment dry
weight (Table 4). Mean HCB concentrations for a particular
sampling day varied between streams and with season of treat-

ment; the concentrations ranged from 0.11 to 0.182 ug/g

during the 30-day treatment phases and from 0.004 to 0.073

38

Table 4. Concentrations (ug/g dry wt.) and regression
equations for accumulation and depuration of
HCB in the stream substrates. Each value represents
the mean of four samples.

 

 

HCB Concentration
ExPerimental Treatment Phase (days)

 

 

Period 5 15 3O Regression Equation
Fall 0.011 0.037 0.182 y=-0.041+0.007x
Winter 0.019 0.037 0.036 y=0.020+0.0006X
Spring 0.049 0.060 0.074 y=0.044+0.001X
Summer 0.029 0.051 0.081 y=0.019+0.002X
Mean 0.027 0.046 0.093

(Std. dev.) (0.024) (0.030) (0.125)

Depuration Phase (days)
15 30 0

 

Fall ' 0.017 0.027 0.027 y=0.l76-0.002X
Winter 0.035 0.004 0.014 y=0.047-0.0004X
Spring 0.073 0.071 0.053 y=0.088-0.0004X
Summer ------------------------------
Mean 0.041 0.034 0.031

(Std. dev.) (0.039) (0.040) (0.026)

 

39

ug/g during the 60-day depuration phases.

The accumulation and depuration of HCB residues in
stream substrates were time dependent as concentrations
increased progressively during the treatment phase and
decreased with time during each depuration phase. Equi-
librium was not approached during the treatment phases
except perhaps in winter when residue concentrations on
days 15 and 30 were nearly equal. Higher concentrations
occurred in stream 3 substrates than in stream 2 which was
consistent with the difference in HCB concentration in the
water of the two streams.

The partition coefficients for HCB residues in the
substrates (HCB concentration in substrates (mg/€1/hean
seasonal HCB concentration in water (ug/liter)) on day 30
of the fall, winter, Spring and summer treatments were 3193,
632, 448, and 1620 reSpectively. These values are in general
agreement with data in the literature for other chlorinated
hydrocarbons. Baughman and Lassiter (1978) reported that
partition coefficients for two tetrachlorobiphenyl isomers
ranged from 420-1270 for four natural sediments tested in the
laboratory. Concentrations of DDT and its metabolites in
the bottom mud of a drainage ditch from an agricultural area
were 820-13,000 times the concentration in the water (Miles
and Harris 1971). The bottom muds of a lake which had been

treated with toxaphene one year previously contained concen-

trations 1000 times those detected in the water (Johnson

40

et a1. 1966) while contemporary Lake Erie sediments accumu-
lated aldrin levels 620 times higher than water concentra-
tions within 20 minutes of exposure in a laboratory test
(Leshniowsky et a1. 1970).
The mean percent organic matter of model stream substrates

varied from 0.88 to 2.71%. When HCB residue calculations
were based on substrate organic matter content, concen-
trations increased linearly with duration of treatment and
decreased in a nearly linear manner during depuration
(Figure 2). Hexachlorobiphenyl residues and organic matter
content were highly correlated in day 30 samples (r= 0.91)
and less so in samples obtained after other durations of
treatment or depuration (Table 5). The importance of sedi-
ment organic matter content in the adsorption.or accumu-
lation of hydrophobic organic compounds has been reported by
a number of investigators (Karickhoff et a1. 1979; Steen
et a1. 1978; Herbes 1977; Dimond et a1. 1974; Leland et al.
1973)-

Regression equations were computed for each phase of
treatment and depuration for HCB residue concentrations in
the substrate as a function of time (Table 4). The maximum
rate of accumulation (Slope) occurred during the fall treat-
ment phase and was slightly higher than the rates occurring
in other treatment phases. This may be due to the previously
uncontaminated nature of the substrates at the beginning of

the fall treatment phase, whereas the substrates at the

41

9.0)-
. STREAM 3 “

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LC)
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an
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IO 20 30 4O 50 60 7O 80 9
DAYS

Figure 2. Uptake and depuration of hexachlorobiphenyl in
the organic matter of the laboratory stream
substrates. Data points are averages for each
stream which were then averaged across

experimental periods. Vertical bars indicate
standard errors about the mean.

 

 

C)

42

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43

beginning of each subsequent treatment phase contained some
residual HCB from the previous treatment. The y intercept
values for the regression equations reflect the carry-over

of residual HCB concentrations from one experimental period
to the next. Hexachlorobiphenyl concentrations in the spring
depuration phase were higher than in the winter phase but

the rates of depuration were essentially the same.

Bippp. All residue data for the biota are presented
on a wet weight basis. As plant materials were analyzed on
a dry weight basis, a dry weight to wet weight conversion
was necessary to facilitate comparison with other data.

Water content of aquatic plant materials was considered to

be 92%; this value is intermediate between those given by
Westlake (1965), Wetzel (1975), and Dr. Clarence McNabb
(Department of Fisheries and Wildlife, Michigan State Univer-
sity, personal communication).

The stream biota rapidly accumulated HCB residues during
the initial 24 hours of the fall treatment phase (Table 6).
Highest residue concentrations were found in Baetis mayfly
nymphs while the lowest concentrations were observed in plant
materials.

During subsequent treatment periods, HCB concentrations
increased after three hours of exposure, but then showed a
slight decrease after six hours. Residue levels in the biota
appeared to stabilize at this time as there was little

difference in HCB concentrations within a given Species in

44'

Table 6. Hexachlorobiphenyl concentrations (ug/g wet wt.) accumulated
by the benthos during the initial 24 hours of each treatment
phase. Each value is a mean obtained by averaging values
from the individually treated streams. Except for the fall
treatment, the pro-exposure concentrations are day 60 values
from the prior depuration period.

 

———-—-
f

Treatment Pre-e osure i n e h S

 

, Organism Phase conc. ug/S) 3 12 2
B o e S 0.41 0.00* 0.12* 0.18* 0.20*
ry phyt 85::25 0.02 0.15 0.28 0.21 0.97
mean ---" 0007 0020 0019 0059
Algae ‘Winter 0.07 0.04* 0.03” 0.14’ 0.20*
Spring 0.03 0.09 0.81 1.10 1.61
summer 0007 0027 1001 0029 ‘ 0017’

Mean ---"’ 00 13 0062 0 051 I 0066

P 1 Winte """'- 0013 0000 0029 0000
at phyton Spring -"""- 0016 0009 0026 0031
Summer ---- 0.07 0.05 0.13 0.27

mean ‘“"-- 0012 0005 0023 0019

Gagggggs Fall 0.00 1.65 2.25 1.95 2.89
pseudolimnaeus Winter 1.89 3.55 2.63 2.27 1.65
Spring 6.97 8.37 6.23 .73 6.90

Summer 3.25 7.94 .17 6.63 6.27

"can -""" 5038 057 #015 40 5

Ph!§ 0 Fall 0000 0077 0091 --""’ 1077
a GP Winter 0.69 1.07 0.93 1.03 0.83
Spring 1.61 2.35 1.51 2.70 1.73

Summer 0.59 0.33 0.65 0.73 0.87

mean "“ 1013 1000 1049 1030

Eggsgbia SPO Fall 0000 0051 Oeua ---" 0033
fiagti 0 Fall 0000 13027 6011 """" 17075,
8 SP Winter 00 83 ---- “005 -""""" 8.1}5

Mean ---- ---- 5.08 ---- 13.10

Nemouridae Spring 4.99 6.77 4.69 5.23 6.03

 

* Stream 2 samples only

45

the 6-, 12-, and 24-hour samples.

Concentrations of HCB residues in the stream biota
increased with time during the treatment phase and decreaSed
during the depuration phase (Table 7). In plant samples,
highest concentrations were generally observednon day 30
of the treatment phase while concentrations in the fauna
were usually the highest on day 4 of the depuration phase.
Very high residue levels were detected in samples of Baetis
mayflies and snails of the genus Hydrobia; residue levels
in the mayflies were highly variable, possibly as a result
of their small sample weights. Samples of bryophytes and
periphyton had consistently low concentrations of HCB when
compared to other benthic samples; concentrations in the
bryophytes were similar to those in periphyton regardless of
exposure duration.

Residue concentrations were similar from season to
season on a given sampling day for any particular component
of the benthos with the possible exception of algae which had
higher concentrations during the winter and Spring treatment
phases than during the summer treatment. Residue concen-
trations in samples from stream 3 were consistently higher
than.Similar samples from stream 2 (Table 8). However, when
the two streams were compared, residues were distributed

among the various components of the benthos in a similar

manner .

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46

 

 

 

 

 

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47

Table 8. Mean HCB concentrations (ug/g) in the benthos of
individual streams on day 30 of the treatment phase.
Except where indicated the mean values were obtained
by averaging single values for a composite sample
across experimental periods. Values for clams,
crayfish and fish are the grand means of the exper-
imental period means of single individuals.

 

 

Stream Number‘

 

 

 

 

 

2 . 3
231000 Pragucgrg I
Algae 7.55 15-78
Bryophytes 0.59 2.33
Periphyton 0.38 1.09
Herbivores
Ehxga sp. 0.85 5.28
.Nemouridae 10.70 30.63
Detritivores
Gammarus 6.92 16.29
pseudolimnaeus
Orconectes 3.03 5.45
virilis+
Lumbriculidae 1.84 3.96
Strophitis rugosus+ 0.31 0.38
Carnivores
Cottus bairdi+ 2.22 3.52

 

+ Grand means

#8

Residual concentrations of HCB were detectable in
all benthic Species after 60 days of depuration and were
higher in invertebrate samples than in plant materials
(Table 7). DesPite the preSence of HCB residues in the
benthos from prior treatment phases, there was no indication
of a consistent stepwise increase in residue concentration
as a result of multiple exposures to the chemical. There
was no evidence of HCB increasing through trophic level
magnification (Table 8).

The rate of accumulation of HCB residues was most
rapid during the initial three hours of treatment and was
slowest during the day l to day 30 interval (Table 9). Good
reproducibility was observed in the rate of accumulation
within a given component of the benthos for the different
seasonal treatment phases. Arthropods had the highest
short-term uptake rates followed by mollusks while plant
materials had the slowest short-term uptake rates. Baetis
mayflies, Hydrobia snails and algae had the highest long-term
(day l - day 30) uptake rates.

Long-term rates of accumulation and depuration of HCB
residues in the various organisms were also compared using
linear regression equations (Table 10)“ The values which
were used in the regression analysis were the mean concen-
trations for a given duration of treatment or depuration
averaged over seasons and did not include samples taken

prior to 20 hours of exposure.

#9

 

 

 

 

 

Table 9. Rate of accumulation of HCB (ug/g/hour) in the stream
benthos over several intervals of time for each
treatment phase. Values were calculated as the
change in HCB concentration divided by the corres-
ponding time interval (hours)

Lilac:
Hours Hours Days

Organism 0-3 3-24 1-30

Gammarus pseudolimnaeus

Fall 0.55 0.06 0.02
Spring 0.47 -0.07 0.01

Nemouridae.

Spring 0.59 -o.03 --——

Baetis Sp.

Fall 4.02 0.21 0.03

Physa Sp.

Fall 0.26 0.05 0.01
Winter 0.13 -0.01 0.00
Spring 0.25 -0.03 0.01
Summer -0.09 0.03 0.01

Hydrgbig Sp.

Fall 0.17 -0.01 0.03

Algae

Winter -0.01 0.01 0.01

Spring 0.02 0.07 0.03

Summer 0.07 -0.01 0.00
Bryophytes

Spring -0.1Ll' 0001 0000

Summer 0.00 0.04 0.00
Periphyton

Spring 0.05 0.01 0.00

Summer 0.02 0.01 0.00

 

50

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51

Hydrobia snails accumulated HCB most rapidly (greatest
slope) followed in decreasing order by algae, Nemouridae,
Baetis, Gammarus, Ehygg. bryophytes, and periphyton. Data
for all organisms showed a reasonably good fit to the
regression lines with the eXception of Baetis. The high
y-intercept values for Baetis, Nemouridae, and Gammarus
reflect rapid short-term accumulation and/or high residues
from previous treatments. Hexachlorobiphenyl was eliminated
by the benthos in a linear manner during each 60-day depuration
phase; Nemouridae had the most rapid rate of depuration.

Based on the data presented, accumulation of HCB appears
to occur in two phases; a rapid short-term uptake followed
by a slower long-term accumulation. matsumura (1975)
suggests that such a biphasic relationship can be eXplained
by quick absorption of the chemical by the insect cuticle
which is then followed by a slower penetration into the body.
Lipid solubility of the chemical will determine the extent
of initial accumulation by cuticular lipids whereas aqueous
solubility will determine the rate and amount of penetration
through cuticular lipids to an aqueous body fluid (matsumura'
1975). Chitin has been shown to be the major factor influ-
encing the uptake of DDT by insects and other arthropods
(Richards and Cutkomp l9h6) and may explain why the arthropods
accumulated more HCB than mollusks, fish, or plant samples.

The elimination of HCB residues from the benthos is
probably the result of slow partitioning of the surface-

bound residues into the surrounding water. Deeper tissues

52

in the organisms would lose residues at a slower rate and
thus remain contaminated for a longer time. If elimination
(occurs in this manner then surfaces of the organism would
be relatively unsaturated at the end of the 60-day depur-
ation phase and would allow a rapid surface sorption of HCB
when the subsequent treatment phase was initiated.

Hexachlorobiphenyl residues were unequally distributed
in the various tissues of the watercress plants (Table 11).
All tissues accumulated HCB residues but for any given
duration of treatment the leaves had the highest concentra-
tions followed in decreasing order by roots, stems, and seed
pods. Concentrations in plant tissues failed to show a
consistent trend with time during the treatment phases but
residue levels decreased on successive sampling days during
the depuration phases.

The distribution of residues in the watercress tissues
may result from differences in surface area available for
sorption. Partitioning of HCB into the plant tissues directly
from water was probably the primary mechanism of accumulation
but translocation from the substrates and storage in the
various tissues may have been an important route for contam-
ination. Meeks (1968) reported that submerged vegetation
usually had higher residues of DDT than emergent plants in
a field study while Witcamp (1960) reported.that the quan-

tity of radionuclides per unit of surface area increased as

the leaf surface area increased.

53

 

 

 

 

 

 

 

 

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moon
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54

Bioaccumulation. The terms bioconcentration and bioac-
cumulation have often been used synonymously in the literature.
For the purpose of clarity. the term bioaccumulation will be
used in this report when uptake of residues by organisms may
have occurred through multiple routes of exposure (i.e.,
food. water and/or direct contact with contaminated substrates)
while the term bioconcentration will be used only when residue
levels are known to have resulted from direct uptake from
water.

Bioaccumulation factors were calculated for each group
of organisms in the model streams as the ratio of the 30-day
HCB concentration in the organisms to the mean HCB concentra-
tion in water for each stream (Table 12). In general, amphi-
pods had the highest bioaccumulation factors which ranged
from 66,000 to 300,000 while clams had the lowest bioaccumu-
lation factors which ranged from 1300 to 16,000. The remainder
of the benthos accumulated HCB residues between 10,000 to
100,000 times the mean.measured water concentrations.

Values for the bioaccumulation factors were similar for
a given organism during the fall, winter and summer treatments
but were lower during the Spring treatment primarily as a
result of higher concentrations detected in the water rather
than a decline in residue levels in the benthos.

The bioaccumulation factors for the benthos of stream 3
were generally higher than those from stream 2: this observa-

tion is consistent with the other data on HCB residues in the

water, substrates, and benthic organisms.

55

Table 12. The range of HCB bioaccumulation factors for

 

 

 

 

 

 

 

(Cottus bairdi)

organisms in the laboratory streams. Ranges
reflect seasonal variations. .
Stream

Organism 2 3
Algae 21,000 - 140,000 41,000 - 253,000
(Rhizoclonium Sp.)
'Bryophyte 6,600 - 8,000 6,000 - 60,000
(Fontinalis Sp.) .
Periphyton 2,000 - 12,000 4,800 - 31,000
Snail 26,000 - 149,000 44,000 - 78,000
(Physa Sp.)
Amphipod 66,000 - 233,000 69,000 - 298,000
(Gammarus pseudolimnaeus)
Crayfish 22,000 - 125,000 18,000 - 134,000
(Orconectes virilis)
Annelid 15,000 - 47,000 18,500 - 104,000
(Lumbriculidae)
Clam 2,000 - 11,000 1,300 - 16,100
(Strophitis rugosa)
Fish 16,500 - 55,000 22,000 - 77,000

 

56

Direct comparison of these results with those of others
is difficult due to the paucity of published studies with
individual PCB isomers. However, some meaningful comparisons
can be made with laboratory and field studies on closely
related compounds. Metcalf et al. (1975) have conducted
model ecosystem studies with tri-, tetra-, and penta-
chlorinated biphenyls. In their results, algae generally
had the lowest "ecological magnification factors" (5,400-
18,000) whereas snails were the highest (5800-59,600) and
fish ranged between 6,400 and 12,100. These authors
concluded that ecological magnification factors increased
with increasing chlorine content of the isomers. The lower
range of the model streams bioaccumulation factors (Table
12) tends to overlap the values presented by Metcalf et a1.
(1975) but the upper range of the model streams data is
approximately one order of magnitude higher. These results
are eXpected because of the higher chlorine content and the
lower water solubility of HCB.

Differences in the design and operation of the model
streams and the Metcalf ecosystems may also account for the
higher concentrations found in the model streams. The
primary differences were: flow-through vs. static design,
direct toxicant introduction vs. trophic transfer, different
Species, and different exposure durations (e.g., fish in
the model streams were exposed for 30 days compared to 3 days

in the model ecosystems).

57

In the laboratory model streams, clams consistently
had the lowest bioaccumulation factors compared to other
benthic organisms. Duke; et. a1. (1970) indicated that
oysters were excellent indicators of Aroclor 1254 contam-
ination in seawater, deSpite the fact that they contained
lower residue concentrations than fish, shrimp, and crabs.
Vreeland (1974) reported that oysters concentrated HCB
48,000 times the level in seawater. This is much higher
than the value calculated for the freshwater clams and is
probably due to differences in exposure conditions.
Freshwater mussels concentrated DDT and dieldrin from lake
water 2,400 and 1,200 times respectively (Bedford and Zabik
1973) which agrees more closely with the values for HCB.

Fish in the model streams had 30-day bioaccumulation
factors which ranged from 16,500 to 77,000; the variation
reflects seasonal and stream differences. These values are
higher than those reported for fish (n212,000) exposed to
tetra- and penta-chlorobiphenyl in model ecosystems
(Metcalf et al. 1975) but are much lower than values reported
for fathead minnows exposed to Aroclor 1248 and 1260
(120,000 and 270,000 reSpectively) (Defoe et al. 1978).
Stalling and Mayer (1972) reported that bluegill sunfish had
bioconcentration factors ranging from 26,300 to 71,400 when
chronically exposed to Aroclors 1248 and 1254 while.Branson
et al. (1975) calculated a bioconcentration factor of 28,700

for rainbow trout exposed to 2,2',4,4'-tetrachlorobipheny1.

58

These laSt two studies agree well with the values for bioac-

cumulation attained by sculpins in the model stream study.

Metabolite Analysis

Unlabeled and Inc-labeled standards of HCB which were
Spotted on thin layer plates and developed with hexane had
a mean Rf value of 0.41 (S.D: = 0.03). Residues in sample
extracts were not visible on the chromatographs when viewed
under U.V. light but were detected on x-ray film after
exposure for 30 to 60 days. Hexachlorobiphenyl residues from
sample extracts had the same Rf value as did co-chromato-
graphed unlabeled and radiolabeled standards.

Positive radioautographs resulted from samples of
Gammarus, Hydrobig, and filamentous algae taken 24 hours
after the initiation of the first treatment and from Baetis
mayflies and ghyga snails within 48 hours of their initial
exposure. Sculpins, crayfish, and substrate samples taken
after 15 days of exposure and samples of all types taken
after 30 days of exposure had positive radioautographs while
sample extracts of control stream benthos and substrates were
not visualized on the x-ray film. No metabolites were
detected on the radioautographs of 342 individual extracts
encompassing samples of water, substrates, and benthic
organisms.

To verify the absence of metabolites, thin layer

chromatographs of samples taken on day 30 of the summer

59

treatment phase were divided into ten 1 cm zones after
development of the radioautograph. The individual zones
were scraped into Scintillation vials and radioactivity
determined by liquid scintillation counting as already
described. Radioactivity above background levels was most
often isolated from zone 4 although, on occasion, activity
was detected in zones 1, 5, 6, 7, and 8. However, similar
results were obtained when radiolabeled HCB standards were

ll‘c-HCB standards

scraped and counted. ~Radioactivity from
which was detected in zones other than 4 was considered to
represent trailing of the compound as the solvent front

1“cs-HCB standard

moved across the plate. Analysis of the
by gas chromatography resulted in only one peak which had
the same retention time aS standards of known purity.

Isensee and Jones (1975) used the same technique for
the analysis of TCDD metabolites and also encountered
trailing in radiolabeled standards which they attributed to
TCDD adsorption to the plate. Based on similar chroma-
tographic behavior of HCB standards and sample extracts it
was concluded that HCB was not metabolized by any of the
components of the model stream ecosystem. Hutzinger et a1.
(1972) found no evidence of reductive dechlorination or hydrox-
1ation of 2, 2',4,4',5,5',-hexachlorobipheny1 in pigeons,
rats, or brook trout, although Jensen and Sundstrom (1974)

found a phenolic metabolite of the same isomer in rat feces.

60

No degradation of Clophen A 50 (a PCB manufactured in
Germany) by Ephemera danica (Ephemeroptera) was observed
after nine days of exposure (Sodergren and Svensson 1973).
Highly chlorinated isomers and mixtures of chlorinated ,
biphenyls can be degraded by acclimated activated sludges or
by isolated bacterial strains but degradation occurs very

slowly (Tucker et a1. 1975; Furukawa and Matsumura 1976).

Laboratory Studies

Accumulation from.Water - Stat;c_Exposug§

Hexachlorobiphenyl residues disappeared rapidly from
the water of the test beakers with 38-52% of the nominal
concentrations recoverable from the water after 24 hours;
only 7-13% of the nominal concentrations were present at 192
hours (Figure 3).

Amphipods accumulated HCB as a function of the concen-
tration in water and duration of eXposure and appeared to be
approaching equilibrium by 96 hours at the lowest concen-
tration (Figure 4). The most rapid uptake (ug/g/hour)
occurred during the initial 24 hours when water concen-
trations were highest; slowest rates were observed between
96 and 192 hours when concentrations in water were lowest
(Table 13). When the uptake rates were adjusted for HCB
concentrations in the water, they were comparable for each

nominal concentration at a given sampling time and less

variable between sampling times at a given concentration

61

 

 

 

 

0.20 -
Nominal Exposure
.. Concentrations (ug / liter)
_ o p ‘OJCKD
A 0.200
0J5? I (1400
’2 - '
.93
z D
a D
3 .
CD 0.I0 -
Q ..
]E
L.
0.05 -
L . , l L
96 l68 I92
TlME (hours)

Figure 3. Disappearance of HCB from water at three
nominal concentrations under static conditions.
Vertical bars indicate one standard deviation
above and below the mean. *

L4

L2

(ug/g)
p
(n

HCB
8

(12

 

Figure 4.

62

Nominal Exposure

 

 

 

 

Concentrations (ug / liter)
a. o O. IOO «-
A. (1200
- I CAKE)
2| u 1 1 1
4 96 |68 l92
TIME ( hours)

Accumulation of HCB by amphipods exposed to
three nominal concentrations in water under
static conditions. Vertical bars indicate
one standard deviation above and below the
mean.

63

 

 

 

Table 13. Rate of accumulation of HCB by amphipods
exposed to three concentrations in water under
static conditions

Adjusted

Nominal HCB Uptake HCB conc. uptake

conc. & conc. rate* in water rate**

time (hour) (ug/g) (ug/g/hour) (ug/liter) (liter/g
hour)
0.100 ug/liter
24 0.105 0.004 0.038 0.11
48 0.028 0.001 0.028 0.04
96 0.163 0.003 0.011 0.31
192 0.059 0.001 0.007 0.09
c. v: =5o.o% c. V.=85.7%

0.200 ug/liter
24 0.290 0.012 0.104 0.12
48 0.080 0.003 0.070 0.05
96 0.330 0.007 0.042 0.16
192 0.150 0.002 0.014 0.11
C. V. =83.3% C. V.=45.5%

0.400 ug/liter
24 0.510 0.021 0.186 0.11
48 0.150 0.006 0.130 0.05
96 0.310 0.006 0.083 0.07
168 0.210 0.003 0.053 0.06
C. V. =88.9% C. V.=42.9%

 

* Uptake rate = HCB concentration (ug/g)/time (hours)

** Adjusted uptake rate

concentration (ug/liter)
+ Coefficient of variation

uptake rate (ug/g/hour)/water

64

(Table 13). Adjusted uptake rates were elevated during the
initial 24 hours, depressed at 48 hours and elevated again
at 96 and 192 hours.

Bioconcentration factors (concentration in amphipods/
concentration in water) were independent of exposure concen-
tration during the initial 48 hours but ranged from 50,000-
60,000 at 192 hours (Table 14). The bioconcentration factors
would be expected to increase until equilibrium was estab—
lished.

Mass balance calculations indicate that there was a
decrease in total recovery of HCB with time (Table 15). 0f
the HCB recovered after 192 hours, 84-90% was associated with
the amphipods.

Declining residues of hydrophobic compounds in the
water of static systems have been reported by a number of
investigators (Weidhaas et al. 1960: Holden 1962: Gakstatter
and Weiss 1967; Wilkes and Weiss 1971; Crosby and Tucker
1971; GilleSpie et al. 1975). Rapid losses of hydrophobic
compounds from water have been attributed to adsorption
onto container walls and accumulation at the air water
interface by Bowman et al. (1959) while longer term losses

are due to evaporative codistillation (Acree et al. 1963;

Bowman et a1. 1959).

Mechanism. The mechanism of accumulation of residues
under static conditions was studied at a nominal concentra-

tion in water of 0.200 ug/liter. Residues disappeared from

65

Table 14. Bioconcentration of HCB by amphipods exposed to
. three concentrations in water under static
conditions. _

 

 

 

Exposure
Time Nominal Exposure Concentration (ug/liter)
(hours) " 0.100 0.200 0.400

24 2763 2788 2763

48 4750 5286 ' 5092

96 26909 16667 11627

192 50714 60714 22245*

 

* 168 hour sample

66

Table 15. Recovery of HCB residues in amphipods and water
A during static experiments.

 

 

 

 

Nominal Total % of Nominal Quantity geggvered
Concentration Recovery HCB in
and Time (hours) (%) Amphipods Water Amphipods
0.100 ug/liter
24 55.1 17.1 38.0 31.0
48 54.3 26.3 28.0 48.4
96 45.1 34.5 10.7 76.4
192 44.3 37.3 7.0 84.2
o . 200 ug/liter
24 80.1 28.2 52.0 35.1
48 71.9 37.1 34.8 51.6
96 75.7 54.7 21.0 72.3
192 69.9 62.9 7.0 90.0
o . L-oo ug/liter
24 61.2 14.6 46.6 23.8
48 53.3 20.8 32.5 39.0
96 49.5 28.9 20.7 58.3
168 58.4 45.1 13.3 77.3

 

67

the water as described for the static tests; concentrations
in water ranged from 68% of nominal at two hours to about
9% after 192 hours.

Amphipod samples which had been rinsed with acetone
prior to freezing had consistently lower concentrations in
body tissue extracts than samples rinsed with water (Figure
5). However, the difference between the acetone-rinsed
and water-rinsed amphipods was Significant (P‘<0.05) only
at 24 hours of Trial 1 and at 12 hours of Trial 2 as
determined by one-tailed t-tests.

When the total radioactivity that was recovered for each
amphipod sample was tabulated the percent that was found in
the rinse acetone ranged from 81% at two hours to about 7%
after 192 hours (Figure 6). The percentage contribution of
the acetone rinse to total recovered radioactivity declined
most rapidly during the initial 24 hours.

Uptake rates were a function of water concentration and
when adjusted for differences in water residue levels they
were elevated at each time interval during the initial 24
hours (Table 16). Rates were depressed between 24 and 96

hours but were again elevated at 192 hours.

Accumulation from Water - Continuous Flow Exposure

Mean concentrations of HCB in water for the eight day
exposure period were 0.031, 0.056, and 0.098 ug/liter
reSpectively at the 0.100, 0.200, and 0.400 ug/liter nominal

test concentrations (Figure 7). Mean values were only

68

LOt
TRIAL l

  
 
 

 

 
 

o————o Water Rinsed
A-.---A Acetone Rinsed

 

 

Loi-
. y TRIAL 2

 
    

n-- ------- -A

 

96
TIME (hours)

   

1
l92

Figure 5. Residue concentrations of HCB in acetone
extracts of amphipods which had been rinsed
quickly in water or acetone at the time of
sampling. Each data point represents the
mean of three samples.

69

- T‘RIAL 2

 

6C)

PERCENT
8

2C)

 

 

96
TIME (hours)

Figure 6. Percent of total extracted radioactivity that
was found in the acetone rinse of amphipods

exposed to 0.200 ug HCB/l.

70

Table 16. Rate of accumulation of HCB residues by amphipods
exposed to 0.200 ug/liter under static conditions
Aduring experiments on the mechanism of uptake.

 

 

 

HCB HCB conc. Uptake Adjusted
Time conc. in H 0 rate uptake rate
(hours) (ug/g) (ug/iiter) (ug/g/hour) (liter/g hour)
2 0.03 0.136 0.0150 ‘ 0.11
0.11 0.128 0.0200 0.16
12 0.20 0.097 0.0150 0.15
24 0.36 0.090 0.0133 0.15
48 0.42 0.056 0.0025 0.04
96 0.49 0.035 0.0015 0.04

192 0.95 0.016 0.0048 0.30

 

71

 

 

 

 

 

L
0"50 Nominal Exposure
Concentrations (ug / liter)
0 O. IOO
_ a 0200
A 0.400
C
.‘2
'-‘- OJOO
\
cm
3
ca
gt) /
ooso - H a}
—-o
l l I g_
2 I92

96
TIME (hours)

Figure 7. Concentrations of HCB in water for three
nominal test concentrations during continuous

flow exposure.

72-

24.5-31.0% of the nominal concentrations: the remainder was
probably adsorbed onto the walls of the mixing chambers,
splitter boxes and test aquaria. Inadequate mixing, which
would result in accumulation at the air-water interface
(Bowman et al. 1959: Oloffs et a1. 1972) and discharge via
the surface overflow standpipes, also cannot be discounted.

A similar loss of material was observed in the model streams.
Lower concentrations in the water at 24 hours were due to
syringe pump failure.

Residue levels in amphipods increased linearly with time
and were a function of exposure concentration (Figure 8)-
Data Showed excellent fit to the regression lines at each
concentration. Residues in amphipods at 192 hours were
3.3 - 6.7 times higher than concentrations in amphipods
after an equivalent time exposure to the same nominal concen-
trations under static conditions.

Bioconcentration factors for HCB accumulation by
amphipods under continuous flow exposure ranged from 5400-
10,200 after 24 hours to 42,600-6l,900 after 192 hours
(Table 17). Bioconcentration factors were generally Similar
for all concentrations at a given sampling time but had a
tendency to increase with increasing exposure concentration.
Calculated values at 96 and 192 hours agree well with values
after Similar intervals in the static tests but both sets of
data underestimate the actual bioconcentration potential which

would continue to increase until the HCB residues in amphipods

_ 8'0 - Nominal Exposure
Concentrations (ug / liter) ~
D
A 0.488
y=‘0.73+0.0439x
5-0 " r=0.98
3 I
\
co
3
”4.0 -
00
cf .. A
y= ‘O.I6+0.0l49x
r=0.99
2.0 -
' y= -o.23+ 0.0lllx
‘ r=0.99
u; - 1 L1
24 96 |92
TIME (hours)
Figure 8. Accumulation of HCB by amphipods exposed to

73

 

 

three nominal concentrations in water with
continuous flow conditions.

74

Table 17. Bioconcentration of HCB by amphipods exposed to
three concentrations in water under continuous flow
conditions.

 

 

 

 

 

Exposure Nominal Expgggre Concentration (uglliter)
(hairs) 0.100 0.200 0.400
24 5,428 8,055 10,189
48 10,740 12,157 13,465
96 17,879 21,458 29,065
192 49,024 42,576 61,811

 

75

were in equilibrium with concentrations in the water.
Bioaccumulation factors for amphipods from the model streams
averaged 98,889, 151,111, 136,666 and 193,333 at 24, 48, 96
and 192 hours reSpectively; these values are substantially
higher than bioconcentration factors determined in the
laboratory and probably reflect multiple routes of accumu-
lation (i.e., from water, food and substrate contact).

As expected, the rate of residue accumulation by amphipods
was concentration dependent but when adjusted for differences
in exposure concentration the data were Similar between test
concentrations and were less variable over time within test
concentrations (Table 18). Adjusted uptake rates were
elevated during the initial 24 hours and again during the
96-192 hour interval.

The data from the static and continuous flow exposures
indicate that accumulation of HCB residues by amphipods is
rapid during the initial 24 hours and is, at least initially,
due to adsorption onto the external surfaces of the organism.
This conclusion is supported by the high uptake rates during
this period for both the static and continuous flow exposures
and by the percentage of radioactivity associated with the
acetone rinse during the early hours of the static exposure.
These results are also consistent with data from the model
streams study.

Johnson and Kennedy (1973) reported that both live and

autoclaved bacteria accumulated 80-90% cf the total residue

levels of DDT and methoxychlor within 30 minutes of exposure

76

 

 

 

Table 18. Rate of accumulation of HCB by amphipods eXposed
to three concentrations in water under continuous
flow conditions.

Adjusted

Nominal uptake

conc. & HCB Uptake HCB conc. rate**

time conc. rate* in water (liter/g
(hour) (ug/g) (ug/g hour) (ug/liter) hour)
0.100 ug/liter
24 ' 0.19 0.008 0.035 0.23
48 0.10 0.004 0.027 0.15
96 0.20 0.004 0.033 0.13
192 1.42 0.015 0.041 0.36
C.V. =62.5% C.V.= 45.5%

o .200 ug/liter
24 0.29 0.012 0.036 0.34
48 0.33 0.014 0.051 0.27
96 0.41 0.009 0.048 0.18
192 1.78 0.019 0.066 0.28
C.V. =30.8% C.V.= 25.9%

0.400 ug/liter
24 0.54 0.023 0.053 0.42
48 0.82 0.034 0.101 0.34
96 1.75 0.037 0.107 0.34
192 4.74 0.049 0.127 0.39
C.V. =30.5% C.V.= 10.8%

 

* Uptake rate
** Adjusted uptake rate

= HCB concentration (ug/g)/time (hours)

= uptake rate (ug/g/hour)/water
concentration (ug/liter)

+ Coefficient of variation

77

g

which would indicate a passive process. No difference was
found in the accumulation of DDE by live and dead chironomid
larvae at various time intervals for up to eight hours (Derr
and Zabik 1974); both live and dead larvae exhibited a time
dependent accumulation which was related to cuticle surface
area. The importance of chitin in the accumulation of DDT
was discussed previously and is further supported by Khan

et al. (1976) who found that unpeeled shrimp had a greater
PCB adsorption potential than peeled Shrimp. Early accumu-
lation is thus a passive process and is based on the physical
and chemical properties of HCB and the external surfaces of
the organism.

Uptake over longer periods of time however appears to
involve an active process. Wilkes and Weiss (1971) reported
that live and dead dragonfly nymphs accumulated DDT at
similar rates at low concentrations but these workers observed
a divergence in accumulated: residues as exposure times and
concentrations increased. They attributed the greater accumu-
lation by live nymphs to biological processes. Crosby and
TuCker (1971) reported that heat killed daphnia accumulated
only about 50% of the residues in live daphnia in a 24 hour
exposure and that uptake occurred principally through the
carapace. Wildish and Zitko (1971) also concluded that
accumulation of Aroclor 1254 by marine amphipods occurred

as an active process across the general integument while

Kenaga (1975) concluded that the first period of residue

78

accumulation is due to adsorption and that later uptake is
by absorption.

Adsorption followed by absorption may also explain
elevations in the uptake rates that were observed during the
first 24 hours and again at intervals beyond 96 hours in the
data presented above. Wilkes and Weiss (1971) observed the
same type of uptake rate fluctuation while Derr and Zabik
(1974) attributed slower uptake after an initial rapid uptake

period to an alteration of the cuticle.

Accumulation from Food

Hexachlorobiphenyl was detected in water two hours after
food was presented to amphipods; concentrations in the water
were a function of HCB concentration in the food and the
quantity of food presented (Table 19). Residues in water
resulted from desorption from the contaminated food and
changed continuously because of two eXperimental design
factors; 1) food was removed after two hours, 2) the quantity
of food presented was adjusted for amphipod removal. These
design features produced a Spike in concentration each day
which was followed by a relatively rapid decline in water
concentrations. Therefore, it was not possible to calculate
a mean daily water concentration over the duration of the
experiment. However, the concentration in water at a given

t

time could be approximated by Ct=Coe-r where Ct and Co were

the concentrations at time t and two hours respectively;

79

Table 19. Concentrations of HCB in water (ug/liter) of
the upper aquaria two hours after food was
added. Each value represents the mean of two
observations.

 

 

Concentration in Food (uglg)

 

Time (hours) ‘ 1.5 14.5 145.0
24 N.D.* 0.0102 0.0729
48 N.D. 0.0049 0.0349
96 N.D. N.D. 0.0313
192 N.D. ' N.D. 0.0083

 

L.0.D. = 0.0029 ug/liter

* None detected 5

8O

r=0.33, the percent of water volume replaced per hour and
e=natural logarithm. 4

Concentrations of HCB in amphipods which were exposed
to the combination of contaminated food and water or to
contaminated water alone increased with duration of exposure
and as a function of HCB concentration in the food (Figure 9).
However, amphipods which were exposed to the combination
had HCB concentrations 4-20 times higher than those exposed
to contaminated water only; uptake from food became less
important as the exposure duration increased (Table 20).
After eight days of exposure, concentrations in amphipods
ranged from 0.09 - 0.13 times the concentrations in the food.

Because of fluctuating water concentrations, it was
impossible to calculate a meaningful bioaccumulation factor.
However, it was possible to back calculate from amphipod
concentrations to water concentrations using the bioconcen-
tration factors which were determined in the continuous flow
exposure. The backcalculated water concentration would be
the mean concentration which would have to be maintained
over the Specified time interval in order to produce the
measured HCB concentration in amphipods.

Water concentrations were backcalculated for 24 hour and
192 hour samples (Table 21). At 24 hours water concentrations
that were measured after food was present for two hours in the

upper tanks (Table 19) were lower than the backcalculated

values; values for the 192 hour sample reveal even more

I 00.0

I U '11I'Ur

I 0.0

' U U r""

(ug/g)

|.0

HCB
I I I I IT'.

0.|

I I IIIIII

 

0.0I-

Figure 9.

81

Type of Exposure

Food and Water
----- Water Only

Nominal Food Concentrations (ug lg)

 

 

    

1
l92

96
TIME (hours)

Accumulation of HCB by amphipods which were
exposed to contaminated food and water and to
contaminated water only. Each data point
represents the mean of two replicate samples.

82

Table 20. Ratio of HCB residues in amphi ads from upper
and lower aquaria (upper/lower . Accumulation
in upper tanks was from food and water whereas
accumulation in lower tank was from water only.

 

 

Concentration in Food (uggg)_

 

Time (hours) 1.5 14.5 145.0
24 - '1 20.0 14.0
48 4.0 29.5 3.5
96 10.0 10.9 10.9

192 5.7 14.4 5-7

 

83

Table 21. Water concentrations (ug/liter) which would be
required in upper and lower aquaria if HCB
accumulation by amphipods was from water only.
Values were backcalculated using the bioconcen-
tration factors from the continuous flow study.

 

 

 

 

 

 

 

 

Time and .
Food Upper Aquaria Lower Aguaria
Conce tration . Bioconcentration Factor
(ug/g Min. Max. M1n. Max.
24 hours 5000; 10,200 5000 10.200
145 0.224 0.110 0.016 0.008
14.5 0.040 0.020 0.002 0.001
1.5 0.006 0.003 - -
192 hours 50,000 80,000 50,000 80,000
145 0.225 0.159 0.044 0.028
14.5 0.037 0.023 0.003 0.002
1.5 0.003 0.002 0.001 0.001

 

84

diSparity between backcalculated and measured concentrations
in the water (compare Tables 19 and 21).

To accumulate their measured HCB body burdens from water
only, amphipods which were exposed to the contaminated over-
flow water from the two upper aquaria treated with 14.5 and
145.0 ug HCB/g of food would have required water concentrations
of 0.001-0.002 and 0.006-0.016 ug/liter respectively during
the initial 24 hour period. These values agree with esti-
mated mean concentrations in the lower tanks of 0.002 and
0.015 ug/liter respectively. The estimated values were calcu-
lated from the previously given equation. However, at 192
hours backcalculated water concentrations in the lower tanks
seem high and could not have been maintained for the duration
of the experiment. Thus, the actual bioconcentration factors
were probably higher than those predicted from the continuous
flow experiment. If, however, the concentrations in the
water-exposed amphipods are assumed to represent the minimum
accumulation from water, then residue accumulation from
contaminated food would represent a maximum of 5.7 to 14.4
times the uptake from water alone.

Relatively few studies have been conducted on aquatic
invertebrates to determine the importance of dietary intake
of hydrophobic organic compounds. Reinert (1972) concluded
that uptake of dieldrin from water was the principal
mechanism of accumulation by Daphnia magna; Crosby and

Tucker (1971) reached the same conclusion with regard to DDT

accumulation by Daphnia. Derr and Zabik (1974) reported

85

that live chironomid larvae eXposed to DDE in food and water
accumulated the same residue levels as dead larvae (uptake
from water only) when tested for up to eight_hours. Kenaga
(1972) probably best summarized the problem when he stated
that the amount of pesticide intake by an organism is
determined by the availability and concentration of the
compound in the diet or in the surrounding environment as
well as by the quantity of the contaminated food consumed
and the quantity of the compound penetrating the organism
through such cellular barriers as the cuticle, lungs, diges-
tive system, gills, etc. In the present study, the role of
ingestion in the accumulation of residues is subordinate to
direct uptake from water although residues accumulated from

each pathway appear to be additive.

Residue Availability_§rgm Substrates

Intact Substpates - Trials 1 and 2. While the system
was equilibrating during Trial 1, only 13 of 24 water samples
had radioactivity above background levels but none were above
the statistically significant limit of detection (0.0145 ug/l,
Pe:0.10, (Seelye 1975)). For this reason, water sampling
was discontinued during the remainder of Trial 1 and all of
Trial 2. .

Amphipods accumulated HCB as a function of exposure time

and there was no difference in accumulation by amphipods as

a function of exposure to the different substrates (Figure

10). Residue levels in amphipods were similar within

86

Figure 10. Accumulation of HCB residues by substrate-

exposed and water-exposed amphipods for two
different intact substrates. Substrates

contained naturally occurring levels of
organic matter.

87

 
  
    

 

 

I00 :-
:Trial I Trial 2
5--.; x—----x Arboretum Pond
' o—o O-—-o Cheny Creek
Substrate-exposed
l.0 _-
3 I
\ I
O) n
3
CD .
O
I
I
0.1 _-
I ‘ I
- x x .’ Water- exposed
\\ 1’
\‘\ I,
O_O| I I x’ I
24 96 I92
TIME (hours)

F igure 10 .

88

substrates of the same origin during Trials 1 and 2 deSpite
the fact that the substrates had depurated for 21 days
between the respective sampling times of each trial.

Residue levels in amphipods exposed only to the
contaminated water, which overflowed from the upper aquaria
(water-exposed), were consistently lower than residues in
amphipods exposed directly to the contaminated substrates
(substrate-exposed). After 192 hours, substrate-exposed
amphipods had accumulated residue concentrations that were
8.8 - 10.5 times higher than in water-exposed organisms.

The extent of bioconcentration from water could not be
calculated because HCB was not detected in water samples.
However, residue levels in substrate-exposed amphipods on
day 8 of Trial 2 were only 5.6 and 3.8 times higher than
concentrations in the Cheny Creek and Arboretum Pond .
substrates respectively. These values wouhibe reduced .
to 5.0 and 3.4 in the respective systems when compensated
for known uptake from water.

Cheny Creek substrate consisted primarily of coarse and
medium sand and had a mean organic matter content of 0.67%
(Table 22). The two Size fractions which were less than
0.125 mm represented less than 0.3% of the total soil dry
weight but had the highest organic matter contents and HCB
concentrations. However, they contributed little to the
total HCB burden of the substrates. Hexachlorobiphenyl

concentrations were significantly correlated with the

89

Table 22. Particle size distribution, organic matter content
and HCB concentrations in various fractions of
Cheny Creek and Arboretum Pondsubstrates at the

end of Trial 2.

___1
-

 

 

 

Substrate category ngcent HCB ug HCB
and particle size total soil organic conc. per
range (mm)* weight matter (ug/g) 800g
Cheny Creek
92:8 coarse 1 - 2 14.61 1.16 1.13 131.4
coarse sand 0.5 - 1 40.21 0.92 0.54 173.0
medium sand 0.25-0.5 42.02 0.54 0.42 145.5
fine sand 0.125-0.25 2.86 5 2.01 1.10 25.5
very fine sand 0.063-0.125 0.16 18.60 8.14 10.4
Silt and clay ( 0.063 0.13 22.87 9.13 9.5
Arboretum Pond
92:8 coarse l - 2 7.03 7.37 1.03 58.0
coarse sand 0.5 - 1 12.50 6.59 1.01 101.0
medium sand 0.25-0.5 28.39 2.67 0.54 122.0
fine sand 0.125-0.25 25.23 3.92 0.69 138.7
very fine sand 0.063-0.125 15.92 5.27 0.96 122.3
Silt and clay < 0.063 10.92 5.32 1.26 110.2

 

* After Cummins

(1962)

90

organic content of the particle size fractions (r=0.99,
P<0.001).

Arboretum Pond substrate had a mean organic matter .
content of 4.61% and consisted mostly of medium and fine
sand with 26% of the soil dry weight represented by particles
less than 0.125 mm. These two fractions contributed 35.6%
of the HCB present in the substrate. Residue levels were
not significantly correlated (P) 0.05) with either the
organic matter content or the particle size of the various
fractions.

At the end of Trial 2, intact Cheny Creek and Arboretum
Pond substrates, which had been depurating for 33 days,
retained mean residue concentrations of 0.55 and 0.86 ug/g
reSpectively. Obviously, once contaminated with hydrophobic
compounds, sediments may remain so for long periods of time
and in so doing provide a reservoir for introduction of toxic
compounds into aquatic organisms. Organisms which have
direct access to contaminated sediments will accumulate
higher residue levels of these compounds than organisms
exposed to the compounds in the overlying water only.

lppact Subsppgtes - Trial 3. Residues of HCB were
readily detected in the water when the Cheny Creek and
Arboretum Pond substrates consisted of inorganic material
only (Table 23). During the equilibration period, concen-
trations in water were higher in the Cheny Creek system than

in the Arboretum Pond system but residue levels in the water

91

 

 

 

 

Table 23. Hexachlorobiphenyl concentrations in water
(ug/liter) overlying Cheny Creek and Arboretum
Pond substrates during the equilibration and
exposure phases of Trial 3.
Running Substpate
Time Time Cheny Arboretum
(days) (hours) Creek Pond
Equilibration Period .
l 24 0.091 0.069
2 48 0.063 0.032
4 96 0.055 0.032
8 192 0.038 0.034
Exposure Period
16 24 0.030 0.022
17 48 0.017 0.017
19 96 0.019 0.020
23 192 N.D.* N.D.

 

L.0.D.= 0.0145 ug/liter

* none detected

92

of the two systems were equivalent during the period of
amphipod exposure.

Residue levels in both substrate-exposed and water-
exposed amphipods were markedly higher than in amphipods from
reSpective exposure conditions during Trials 1 and 2 (Figure
11). This observation is consistent with the higher concen-
trations of residues in the water during the present eXper-
iment. Concentrations in water-exposed amphipods were lower
than in substrate-exposed organisms and there was a slight
difference in residue accumulation by amphipods as a function
of exposure to the different substrates. Differences in
the soil characteristics (e.g., particle size distribution)
may have made HCB less available from the Arboretum Pond
substrates although no differences in HCB concentrations were
observed in the water samples.

Bioaccumulation and bioconcentration factors were calcu-
lated for substrate-exposed and water-exposed amphipods
reSpectively on the assumption that the HCB water concentra-
tions in the two sets of aquaria were equivalent (Table 24).
The factors increased with time for both exposure conditions
and were generally higher in the Cheny Creek system than in
the Arboretum Pond system. After 192 hours, values for
substrate-exposed amphipods were 2.3 and 5.4 times higher
than in water-exposed organisms for the Cheny Creek and

Arboretum Pond systems respectively.

93

 

 

 

I0.0
5.0
’5-
\
OILO :"
£3 .
m I:
(3(15-
I n
l
D I
I
'0
" ,' Substrate- exposed
. c Cheny Creek
.' A Arboretum Pond
,' Water- exposed
OJ _- . o Cheny Creek
' -' A Arboretum Pond
- I
r ,'
005- A
I I I I
I92

4 48 96
2 TIME (hours)

Figure 11. Accumulation of HCB residues by amphipods
exposed directly to contaminated inorganic
substrates (substrate-exposed) or to residues
in contaminated overflow water only (water-
exposed).

94

Table 24. Bioaccumulation and bioconcentration factors for
amphipods which were exposed to HCB contaminated '
substrates and to contaminated water respectively.
The factors were computed as the HCB concentration

in the or anisms (ng/g)/concentration in water
(ug/liter§.

 

 

 

 

Substrate and Time (hours)

Type of Exposure 24' 48 96* 192

Cheny Creek
substrate exposed 27,677 121,176 207,368 1,073,333
water exposed 8,333 42,941 61,053 463,333
ratio* 3.3 2.82 3.40 2.32

Arboretum Pond

substrate exposed 38,182 104,118 156,500 851,111
water exposed 2,273 23,529 28,000 157,778
ratio 16.80 4.43 5.59 5.39

 

* ratio = substrate exposed/water exposed

95

Bioconcentration values for water-exposed organisms were
comparable to bioaccumulation values obtained after 192 hours
in the model streams study. However, substrate-eXposed
amphipods had much higher bioaccumulation values than in
the model streams which may be explained by exposure to the
more highly contaminated substrates in the laboratory study.

The importance of substrate exposure appears to decrease
with time for both substrates as judged by decreases in the
ratio of bioaccumulation to bioconcentration (Table 24).
Direct exposure to the Arboretum Pond substrate appears to
be more significant than exposure to the Cheny Creek
substrate. After 192 hours, HCB concentrations in substrate-
exposed amphipods were 22.5 and 15.0 times the concentrations
in the Cheny Creek and Arboretum Pond soils respectively.

The Cheny Creek and Arboretum Pond substrates differed
slightly in particle size distribution from those used
previously (Table 25). AS in Trial 2 the two fractions of
Cheny Creek substrate which were less than 0.125 mm repre-
sented only a small percentage of the total soil weight and
did not contribute a significant amount to the substrate HCB
burden deSpite elevated concentrations. Hexachlorobiphenyl
concentrations were not significantly correlated (P)r0.05)
with particle size.

Arboretum Pond substrates had a smaller percentage of
particles less than 0.125 mm when compared to substrates used

during Trial 2. Unfortunately, residue data from the

96

Particle Size distribution and HCB concentrations

Table 25.
in various fractions of Cheny Creek and Arboretum

 

 

 

Pond substrates at the end of Trial 3. Substrates
had been combusted prior to use.
Substrate category % of HCB ug HCB
and particle size total soil conc. per
range (mm)* weight (ug/g) 800g
Cheny'Creek
very coarse sand 1 - 2 (16.33 0.78 101.9
coarse sand 0.5 - 1 52.37 0.45 188.5
medium sand 0.25-0.5 28.24 0.26 58.?
fine sand 0.125-0.25 2.85 0.40 9.1
very fine sand 0.063-0.125 0.15 4.42 5.3
Silt and clay < 0.063 0.06 13.45 6.7
Arboretum Pond
very coarse sand 1 - 2 7.12 0.66 37.6
coarse sand 0.5 - l 14.52 0.57 66.2
medium sand 0.25—0.5 46.87 0.48 180.0
fine Sand 0.125-0.25 23.12 0.59 109.1
very fine sand 0.063-0.125 6.32 0.61 30.8
silt and clay -< 0.063 2.05 Lost -

 

* After Cummins (1962)

97

smallest particle Size fraction were lost but it appears
that there was no significant elevation in residue concen-
tration. This assessment is based on the uniform distribu-
tion of residues within the other particle size fractions
and on the fact that the intact substrate had a mean HCB
concentration of 0.51 ug/g. If the HCB quantity in each
particle Size fraction (exclusive of the smallest) is summed
and divided by the total soil weight, the result is a concen-
tration of 0.53 ug/g which agrees well with the measured
concentration. Hence, the smallest particle Size fraction
contributed little to the total HCB burden of the substrate.
The HCB concentrations in each of the particle size
fractions of the oxidized substrates were consistently lower
than when organic matter was present (compare Tables 22 and
25) and this occurred despite the fact that the Trial 2
substrates had depurated for 18 additional days. Evidently,
organic matter, even in small amounts, is important in the
retention of HCB residues by hydrosoils. Removal of organic
materials increases the residue levels in the water and
thereby increases the availability of the residues to aquatic
organisms. V
Substrate partition coefficients (concentration in
the substrate (ng/g)/concentration in the water (ug/liter)
at 192 hours of amphipod exposure were 47,800 and 56,800 for
the Cheny Creek and Arboretum Pond substrates respectively.

These values are much higher than the partition coefficients

 

98

determined for the model stream substrates but are much

lower than the distribution coefficients reported by Halter
and Johnson (1977). The high coefficient values reported

by Halter and Johnson (1977) and in the present study reflect
the fact that the substrates were artifically contaminated
and the contaminant was then allowed to desorb into the
water. In this reSpect the substrates are somewhat analogous
to the columns of inert substrates proposed by Veith and
Comstock (1975) for continuously saturating water with
hydrophobic organic chemicals.

Intact Spbstrates - Trial 4. Hexachlorobiphenyl
concentrations in water decreased with time during the
period of amphipod exposure but were higher than in previous
trials (Figure 12). The higher concentrations were probably
due to the Shorter equilibration period that was used during
this experiment;

Initially, residue levels were slightly lower in the
overflow water than in water overlying the substrates but'
concentrations in all aquaria were Similar by 192 hours.
There was no evidence of a vertical gradient in the HCB
water concentrations of aquaria with the high ratio of water-
to-substrate (data not presented).

Residue levels in substrate-exposed amphipods were
higher than in water-exposed amphipods (Figure 13).
Substrate-exposed amphipods from aquaria with a high ratio

of water-to-substrate (11.25zl) had higher residues at

 

99

 

I
0040'
_ 0 High Ratio - overlying substrates
0 High Ratio - overflow
- A Low RatIo - overflow
(M03Ch-
‘1: ' .
g .
C .
90.020 - '
8 " '
I _ 5- -- -1“ ‘
P “\‘
0.0I0 - ‘~ .
‘A- --------------- a-..
r I I I I
24 48 96 I92
TIME (hours)

F igure 12 .

Concentrations of HCB in water (ug/liter) from
aquaria at a water-to-substrate ratio of
11.2521 (wt/wt) and in aquaria receiving the
overflow water from both the high and low ratio
(1:1, wt/wt) of water-to—substrate aquaria
during the period of amphipod exposure.

100

 

 

 

I6,-
. Substrate-exposed ‘
. A Hugh Ratio /
.. a Low Rat-o X
Water-ex oged I”
l2- A HIgh .otIo ,’
° LOW Ratlo I,
’5. _
\ ’,
OI _ ,
a ,v
Ve- I” '
ca - x'
C) _ "
3E ":
4... x,
.. A.--“ ,r
L -, ‘.oA""------- : --
1 I
24 96 I92
”“5 (hours)

Figure 13. Accumulation of HCB residues by amphipods which
were exposed to Cheny Creek substrates at high
(ll.25:l.00) and low (1.00:100) water to
substrate ratios (grams of H Osgrams of substrate).
Substrate-exposed amphipods acre subject to
uptake from water and substrates while uptake
by water-exposed amphipods was from contaminated
water only.

101

96 and 192 hours than did substrate-exposed organisms from
the low ratio tanks (1:1). There was no difference between
amphipods exposed to the overflow water from either set of
aquaria. The longer residence time and larger volume of
water in the high ratio aquaria may have allowed a greater
quantity of HCB to desorb from the substrates; the HCB would
also remain available for uptake by the amphipods for a
longer period of time.

Bioaccumulation factors at 192 hours ranged from 1.21-

1.28 x 106

for substrate-eXposed amphipods while bioconcen-
tration factors ranged from 2.78 - 3.19 x 105 for water-
exposed organisms. The values for the two exposure conditions
(substrate vs. water) differed by a factor of about four
which could be assumed to represent the maximum contribution
to HCB accumulation from contaminated substrates via direct
contact and ingestion as opposed to uptake from water alone.

Partition coefficients for substrate-exposed amphipods
(concentration in amphipods/concentration in the substrates)
at 192 hours ranged from 19.2 to 22.8 which would drop to
14.4-18.5 when compensated for direct uptake from water.
These values are in general agreement with Similar data on
Cheny Creek substrates from Trial 3.

Substrate partition coefficients (concentration in the
substrate/concentration in the water) ranged from 53,300 at

the high water-to-substrate ratio to 66,700 at the low water-

to—substrate ratio. These values are slightly higher than

102

values obtained in Trial 3 which was not unexpected since the
substrates in the present eXperiment had natural levels of
organic material (0.41%) whereas those in Trial 3 consisted
of inorganic materials only. The presence of organic
material would reduce the availability of the residues to the
water which, in turn, would result in a higher partition
coefficient.

Sigyed_§ubstrates. Of 169 water samples taken during
the experiments, only 11.8% had HCB residues above the limit
of detection (0.0059 ug/liter) and, of these, the mean values
were 0.012 and 0.009 ug/liter for the Cheny Creek and
Arboretum Pond systems respectively.

The residue levels of HCB which were accumulated by
amphipods after 192 hours of exposure to each particle Size
fraction of the different substrates is presented in Table
26.

Amphipods, which were exposed to the contaminated size
fractions of unaltered Cheny Creek substrate (Trial 1),
accumulated the least residues when exposed to particles
less than 0.125 mm. Concentrations in amphipods exposed to
particles less than 0.063 mm were only about 5% of those in
amphipods exposed to the particles larger than 0.25 mm.
Residue levels in amphipods were inversely correlated
(r= -0.94, P-<0.05) with the organic matter content of the

'particle size fractions.

103

 

 

 

 

Table 26. Hexachlorobiphenyl concentrations (ug/g) in
amphipods and organic matter content (%) of
substrate particle size fractions on day 8 of
each experiment. During Trials 1 and 3 the
substrate particle size fractions had naturally
occurring levels of organic matter. Prior to
Trials 2 and 4 the substrates were combusted to
remove natural organics.

Particle Size Range (mm)
1.0- 005- 0025- 00125-
0.5 0.25 0.125 0.063 0.063
Cheny Creek

Trial 1

organic matter 0.51 0.26 2.96 28.39 37.47

HCB conc. 1.91 2.07 1.15 0.24 0.11

Trial 3

HCB conc. 3.34 4.48 7.16 0.69 0.47

Arboretum Pond

Trial 2

organic matter 6.02 2.71 3.83 5.42 5.63

HCB conc. 1.05 3.15 2.07 0.99 0.83

Trial 4

HCB conc. 1.68 2.20 1.97 1.57 1.05

Maple Leaves

Trial 5

organic matter 72.8 74.5 73.2 70.6 66.7

HCB conc. 0.40 0.21 0.16 0.19 0.19

 

104

Amphipods which were exposed to oxidized Cheny Creek
substrates (Trial 3) accumulated higher residue levels from
each particle size fraction compared to residue.uptake
during Trial 1. Organisms which were exposed to particles
less than 0.125 mm accumulated the least HCB.

Accumulation of HCB by amphipods which were exposed to
the various particle Size fractions of unaltered Arboretum
Pond substrate (Trial 2) was inversely correlated with the
level of organic matter in the size fractions (r = -0.97,
P4¢0.01). There was no statistically Significant relation-
ship between HCB uptake by amphipods and the particle size
of the substrates but the lowest residues were found in those
amphipods exposed to the smallest particles.

Compared to the results of Trial 2, a slight increase
in amphipod residue levels was observed at the two smallest
size fractions of oxidized Arboretum Pond substrates (Trial
4). However, lowest residues in organisms were found in
those exposed to the finest particles.

The maple leaf substrate had an organic matter content
which averaged 66.7-74.5% for the different particle Size
fractions. Residue accumulation by amphipods exposed to
particles greater than 0.125 mm was markedly reduced when
compared to the results of Trials 1 - 4. Hexachlorobiphenyl
concentrations in amphipods exposed to particles less than
0.125 mm were comparable to those accumulated by amphipods

from the same particle sizes in Trial 1.' It will be recalled

that these latter two fractions had high organic matter

105

contents. Organic matter in the form of crushed maple
leaves apparently reduced the availability of HCB to the
amphipods exposed to the medium and coarse particle sizes
and behaved in a Similar manner to naturally occurring
organic matter in the fine particle sizes.

The accumulation of HCB by amphipods in the simple
laboratory systems appears to result primarily from direct
uptake from water. Direct contact with, or ingestion of,
contaminated substrates is also significant but of secondary
importance. Furthermore, amphipods are capable of accumu-
lating measurable residue levels in their body tissues from
extremely dilute Solutions within a relatively short time.
The residue levels in water result from desorption from
the substrates according to the properties of adsorption-
desorption equilibrium reactions and may be below the level
of detection in flowing water systems.

Literature data appears to confirm that direct uptake
from water is the dominant mechanism of accumulation of hydro-
phobic organics by aquatic organisms. Direct uptake from
water has been reported to be the dominant uptake mechanism
for Daphnia exposed to DDT (Crosby and Tucker 1971); for
various freshwater invertebrates exposed to DDT and aldrin
(Johnson et a1. 1971), or to polychlorinated biphenyls
(Mayer et al. 1977); for bacteria exposed to DDT and
methoxychlor (Johnson and Kennedy 1973): for marine amphi-

pods exposed to PCB's (Wildish and Zitko 1971); and by a

106

simple food chain exposed to dieldrin (Reinert 1972).

The present study also indicates that body burdens of
hydrophobic contaminants can be enhanced by contact with, or
ingestion of, contaminated substrates. Derr and Zabik (1974)
suggested that exposure of chironomid larvae to DDT contam-
inated substrates may have resulted in the higher bioconcen-
tration factors in their data compared to the results of
Wilkes and Weiss (1971) for dragonfly nymphs exposed to DDT
in water only. Isensee and Jones (1975) discounted the import-
ance of TCDD uptake from contaminated hydrosoils but observed
that concentrations in organisms were within an order of
magnitude of TCDD concentrations in the soils. Residue levels
of PCB's in fish which were exposed to contaminated sediments
were about Six times higher than when fish were denied direct
access to the substrates (Halter and Johnson 1977). These
authors believed that uptake from water, which followed
desorption from the substrates, was the mechanism of accumu--
- lation. Kobylinski and Livingston (1975) attributed a portion
of the mirex burden of the hogchoker (Epipgppgg pgpplgppg, a
carnivorous, bottom dwelling fish) to be from direct contact
with contaminated substrates.

Direct uptake from water of hydrophobic organic compounds
by aquatic organisms and sediments has been most often attri-
buted to chemical partitioning based on exchange equilibria

between two or more media (Hartung and Klingler 1970:

Hamelink et a1. 1971; Lenon 1968; Clayton et al. 1977;

107

Haque et a1. 1974; Branson et al. 1975; Herbes 1977;

Steen et al. 1978; Paris et al. 1978; Karickhoff et al. 1979).
Strong inverse correlations exist between water solubility
and octanol/water partition coefficients (Chiou et al. 1977)
and between aqueous solubility and bioconcentration factors
(Chiou et al. 1977; Metcalf et al. 1973, 1975). A strong
positive correlation between bioconcentration potential and
octanol/water partition coefficients was indicated by Metcalf
et al. (1975) and Branson et al. (1975).

The results of the substrate experiments demonstrate
that substrate characteristics control the availability of
HCB to the overlying water and in turn to aquatic organisms.
The most important control factor appears to be the level of
organic matter while substrate particle size is of less
importance. The data clearly indicate that the uptake of
HCB was increased in both substrate-exposed and water-exposed
organisms when the organic content of the substrates was
reduced by either artificial (combustion) or natural factors
(increase in particle size). Organic materials represent
a medium that enhances partitioning into substrates thereby
reducing HCB equilibrium concentrations in the overlying
water. When organic materials are reduced or eliminated the
exchange equilibria reactions allow greater quantities of
HCB to desorb into the water i.e., organic materials are

simply a better ”solvent” for hydrophobic compounds than

are inert materials.

108

The particle Size of the substrate also affects the
availability of HCB to the water and organisms. Amphipods
which were eXposed to the silt and clay fraction (< 0.063 mm)
had consistently lower levels of HCB than did those exposed
to larger particle sizes. This result was observed in both
the presence and absence of organic materials but was less
influential than organic materials in determining HCB
availability to the amphipods.

The role of substrate characteristics in the sorption
of organic compounds by aquatic sediments has been investi-
gated in detail only recently and most studies have dealt
with the adsorption side of the exchange reaction. However,
any process or characteristic that favors adsorption will
inhibit desorption and thereby lower equilibrium concentra-
tions in the water which would result in reduced uptake by
organisms.

Haque et al. (1974) concluded that reduction of PCB
levels in water depends on the area, organic content,
nature and pH of contacted surfaces. Organic matter is
often the prime determinant of pesticide adsorption by soils
(Browman and Chesters 1977) and organic sediments adsorb many
pesticides more strongly than do mineral sediments (Pionke
and Chesters 1973). Lotse et a1. (1968) found a direct
relationship between the organic content of lake sediments
and.the amount of lindane they adsorbed while Haque et al.

(1974) attributed the high adsOrbing capacity of Woodburn

109
soil to its high organic content. Differences in the Sorption
of hydrophobic pollutants within the Silt and clay fractions
of several natural sediments was also related to differences
in the organic carbon content (Karickhoff et al. 1979).

In a study with aldrin, a high organic matter soil
1 sample adsorbed 44% more of the pesticide than did oxidized
samples of the same soil and after leaching, the organic
soil retained 67% more aldrin than did the oxidized soil
(Yaron et al. 1967). In a similar manner, DDT concentrations
in water increased when organic matter was removed by
oxidation with H202 (Richardson and Epstein 1971) while
ashing reduced the uptake of DDT by substrates in another
study (Hargrave and Phillips 1974).

In a more natural setting, toxaphene detoxification
occurs more rapidly in eutrophic lakes than in oligotrophic
lakes (Terriere et al. 1966) and is due at least partially
to sorption onto sediments and suspended particles (Veith
and Lee 1971b; Johnson et al. 1966).

As Shown in the present study a decrease in the particle
Size of substrates is often associated with higher organic
contents which complicates analysis of partitioning behavior.
Sorption is usually strongest in the fine particle sizes
which often results in toxicant enrichment in these fractions

while sand fractions which often comprise the bulk of

substrate mass usually adsorb only small quantities of

hydrophobic compounds (Baughman and Lassiter 1978;

110

Browman and Chester 1977; Karickhoff and Brown 1978;
Hetling et al. 1978; Haque and Schmedding 1976).

Haque and Schmedding (1976) found that of a number of
compounds tested, adsorption by sand fractions was Signifi-
cant only for hexachlorobiphenyl which may.help to explain
the results obtained in the present study where sand
fractions were observed to contain the bulk of adsorbed
residues.

The interaction of fine particle Size and elevated
organic contents is complex especially if one considers
that in soils with organic carbon contents greater than five
to eight percent only organic surfaces appear to be avail-
able for bonding (Browman and Chesters 1977).. Thus, in
order to interpret and predict partitioning behavior one
must understand the reSpective roles of both organic

matter and particle size (Steen et al. 1978).

SUMMARY

In the present study the residue dynamics and metabolism
of hexachlorobiphenyl (HCB) were determined in a series of
semi-natural laboratory model streams. Laboratory eXperi-
ments were also conducted to determine the relative import-
ance of residue accumulation by amphipods from water, from
contaminated food, and from direct contact with contaminated
substrates. The bioavailability of substrate-bound residues
as a function of substrate type, organic matter content, and
particle Size distribution was also investigated.

V The present study indicates that HCB is rapidly
accumulated by the substrates and biota of laboratory systems
to concentrations that range from about 3-6 orders of magni-
tude higher than concentrations in the water. These
bioaccumulation values would be expected to increase as there
was no evidence that equilibrium was approached even after
thirty days of exposure to the chemical. The compound was
persistent in the substrates and biota of the model streams
and there was no evidence that the compound was metabolized
in either the substrates or the biota of the laboratory streams.
Accumulation of HCB by amphipods was primarily by

direct uptake from water. However, concentrations in the

111

112

organisms after 192 hours of exposure could be increased
from 2-14.4 times by either ingestion of contaminated food
or by direct exposure to contaminated substrates.

Direct uptake from water was most rapid during the first
2-3 hours and was attributed to adsorption onto the substrates
and external surfaces of the organisms; slower rates of uptake
which were observed after the initial rapid accumulation were
attributed to partitioning of the compound into the deeper
tissues of the biota.

Residue levels in amphipods from the laboratory tests
were dependent on exposure concentrations in the water.
The availability of substrate-bound residues to the overlying
water was controlled primarily by the concentration of
organic matter in the substrates and to a lesser extent by
the particle size of the substrate. In the laboratory studies
where each substrate particle size fraction had the same
nominal HCB concentration amphipods had lower concentrations
of HCB when exposed to hydrosoils of high organic content
and fine texture than did organisms exposed to other types
of sediment. However, HCB concentrations and organic matter
contents in unfractionated substrates were highest in the
finest particle sizes when these sediments were sieved and
analyzed. Literature data, previously cited,also indicate
that the highest concentrations of chlorinated organic

compounds in the sediments of natural systems occur in those

of fine texture and high organic content. Thus, in natural

113

systems, organisms which are exposed to these types of
sediment would also be exposed to the highest concentrations
of hydrophobic organic compounds. At some point, exposure
to these higher concentrations will result in increased body
burdens in the benthic organisms. The relationships between
sediment particle size, organic matter content, and

sediment concentrations of hydrophobic organic compounds
need further investigation with regard to accumulation of
these compounds by benthic organisms before the full signi-
ficance of bottom sediments as sinks or reservoirs of toxic

materials is understood.

APPENDIX

114

Table A1. The daylength maintained in the laboratory during
the experimental periods of the model streams

experiment.

 

 

Experimental Period

Daylength (Hours)

 

Fall

Start August 11, 1976
End November 9, 1976

Winter

Start November 10, 1976
End February 7. 1977

Spring

Start February 9, 1977
End May 99 1977

Summer

Start May 11, 1977
End June 8, 1977

14.50
10.75

10.25
11.00

11.00
14.70

14.70
15.30

 

115

 

 

 

Table A2. Temperature and water chemistry for the water used
in the model streams study. Values are means for
each month.

Dissolved Hardness Total
Oxy en (mg/ Alkal-
Month/' Temperature 0C (m pH liter init
Year Mean Range liter) Range CaCoB) (.
liter)
9/75 12.1 10.0-12.8 9.9 7.65-7.8 210 192
10/75 10.0 7.8-10.5 9.4 7.75-7.95 209 191
12/75 4.8 2.8-5.0 10.8 7.85-7.85 203 191
2/76 4.0 2.8—3.9 11.2 7.9 -7.9 204 .190
3/76 6.3 3.3-8.3 10.2 7.5 -7.7 182 177
4/76 9.5 5.5-12.2 9.9 7.7 -8.0 199 191
5/76 -8 7 6.1-12.2 9.8 7.6 -8.0 192 192
6/76 12.3 8.9-12.8 9.0 7.8 -8.0 204 192
7/76 12.0 11.1-12.8 9.3 7.9 -7.9 202 190
8/76 11.4 8.3-13.3 9.8 7.8 -7.9 204 192

9/76 11.1 8.3-13.8 9.7 7.8 -7.9 203 191

10/76 7.8 6.7-9.4 9.8 7.6 -7.7 205 188

11/76 5.7 4.4-6.7 10.6 7.7 -7.8 206 188

12/76 3.7 0.5-5.0 11.3 7.6 -7.7 212 191

1/77 4.0 3.3-5.0 11.2 7.7 -7.8 217 190
2/77 3.3 1.1-7.2 11.4 7.6 -7.9 217 192
3/77 8.3 5.0-11.1 9.4 7.4 -7.7 187 164
4/77 11.0 7.8-13.8 9.7 7.6 -7.8 201 196
5/77 12.0 11.6-15.5 9.6 7.8 -7.8 202 196
6/77 10.5 9.4-11.7 9.7 7.8 -7.8 204 202

 

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