EPA 600/3-81-018
February 1981
SOME EFFECTS OF PETROLEUM ON NEARSHORE MARINE ORGANISMS
b~
D. G. Shaw, L. E. C. tit, D. J. Mcintosh,
and M. S. Stekoll
Institute of Marine Science
University of Alaska
Fairbanks, Alaska 99701
Project R-803922
Project Officer
Barry Reid
Corvallis Environmental Research Laboratory
Corvallis, Oregon 97330
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
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DISCLAIMER
This report has been reviewed by the Corvallis Environmental Research
Laboratory, U.S. Environmental Protection Agency and approved for publica-
tion. Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
ii
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ABSTRACT
The overall objective of this project was to better understand the
effects of chronic, low-level oil pollution on nearshore Alaskan marine or-
ganisms .
The bivalve mollusc Macorna balthioa accumulated hydrocarbons during
180 days of continuous exposure to Prudhoe Bay crude oil in flowing seawater
dispersions with nominal concentrations of 0.03 mg/1, 0.3 mg/1 and 3.0 mg/1.
The animal's ability to concentrate oil from seawater increased with decreas-
ing oil in water concentration. Decreases in M. balthioa's oil burden began
after 30 to 120 days (depending on the oil concentration) and continued for at
least 60 days after oiling ceased. Aliphatic and aromatic hydrocarbons were
fractionated in markedly different ways by the animal. Branched and cyclic
aliphatics in the range dodecane through hexadecane were preferentially
retained over their higher homologs; whereas larger and more substituted
aromatics were selectively concentrated.
Maaoma balthioa showed a number of physical, behavioral, physiological
and biochemical changes during oil exposure. An oil in seawater concentra-
tion of 3.0 mg/1 caused severe dysfunction in the clams including a decreased
burial rate, increased respiration rate, and inhibition of growth leading to
very high mortalities. The lowest concentration of oil tested, 0.03 mg/1,
inhibited growth and caused abnormalities in gonad morphology. One group of
adverse oil effects which was related to sluggishness and disorientation of
the animals appeared after seven days' oiling; another group related to a
negative energy balance was not observed until 60 days. We conclude that
chronic exposure of M. balthioa to oil-in-seawater concentrations as low as
0.03 mg/1 will in time lead to population decreases.
This work was submitted in fulfillment of Grant No. R-803922 by the
Institute of Marine Science, University of Alaska under the sponsorship
of the Environmental Protection Agency.
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Intentionally Blank Page
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TABLE OF CONTENTS
ABSTRACT . . .
LIST OF FIGURES
LIST OF TABLES
CHAPTER 1 1
Introduction 1
CHAPTER 2 3
Accumulation, Fractionation and Release of Oil by the Intertidal
Clam Macoma balthica 3
Introduction 3
Materials and Methods 3
Results 7
Discussion 21
References 24
CHAPTER 3 27
Biochemical and Physiological Effects of Oil on Maoorra balthioa . 2 7
Methods 28
Experimental Set-up 28
Biochemical Assays 29
Protein, Glycogen, and Total Carbohydrates 31
Total Lipid 31
RNA and DNA 32
Preparation of the Crude Homogenate for Enzyme Assays . 32
Na -K -ATPase and Mg -ATPase 32
51-Nucleotidase 33
Phosphodiesterase 33
Physical, Behavioral, and Physiological Assays 33
Wet and Dry Weights 33
Growth and Condition Index 33
Burying Experiment 34
Feeding Rates 34
Respiration 35
Reproduction 35
Electron Microscopy of Gill Tissue 35
Results 36
General 36
Feeding 38
Rates of Burrowing 38
Mortalities 42
Wet and Dry Weights 42
iii
vii
ix
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TABLE OF CONTENTS
CONTINUED
CHAPTER 3 (continued)
Growth 45
Condition Index. 45
Respiration 45
Gill Sections. . 45
Reproduction 49
Protein Content . 49
Glycogen and Total Carbohydrate. ...... 55
Total Lipid 55
RNA and DNA 55
Enzyme Activities 63
Recovery Period 64
Discussion 68
References 78
vi
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LIST OF FIGURES
CHAPTER 2
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Schematic representation of continuous oil exposure
system
Concentration of total oil in seawater in test and
control tanks during the course of the experiment. .
Macoma balthica tissue concentrations of total
hydrocarbons during 180 days of exposure to oil
in seawater dispersions and 60 days of recovery. . .
. 8
Macoma balthica gas chromatograms of oil in water
dispersion and tissue accumulations of hydrocarbons,
Macoma balthica branched, monocyclic, and bicyclic
saturated hydrocarbons with 12 to 16 carbon atoms. .
12
16
CHAPTER 3
Figure 1. Percentage of unburied clams in oil-exposed seawater . . 37
Figure 2. Burying behavior of Macoma in different concentrations
of oiled seawater for day 90 40
Figure 3. Percentage of Macoma which remain unburied after one
hour of being placed on the surface of the sand 41
Figure 4. Cumulative mortalities of Macoma for the various oil
treatments during six months of exposure 43
Figure 5. Percent dry weight for Macoma for points throughout
the six month oiling experiment 46
Figure 6a. Protein (percent of dry weight) for Macoma exposed
to oiled seawater for six months 51
Figure 6b. Total carbohydrate (percent of dry weight) for
Macoma exposed to oiled seawater for six months 52
Figure 6c. Total lipid (percent of dry weight) for Macoma
exposed to oiled seawater for six months 53
Figure 7. RNA and DNA values for Macoma during exposure to
six months of oiling 59
vii
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LIST OF FIGURES
CONTINUED
CHAPTER 3
Figure 8a. Enzyme (ATPases) activities in Maooma as a function
of six months of oiling 65
Figure 8b. Enzyme (5'-Nucleotidase) activities in Maooma as a
function of six months of oiling 66
Figure 8c. Enzyme (Phosphodiesterase) activities in Maooma as
a function of six months of oiling 67
viii
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LIST OF TABLES
CHAPTER 2
Table 1. Macoma balthica - Hydrocarbon Concentrations in Tissues
During Oiling Experiment for Each Column Chromatographic
Fraction 11
Table 2. Macoma. balthica - Percentage of Total Saturated Aliphatic
Hydrocarbons Eluting Between Dodecane and Hexadecane
During Gas Chromatography 13
Table 3. Area and Percent of Total Area for Each of Four Classes
of Alkanes with 12 to 16 Carbon Atoms in Gas Chromatograms
from Samples of the Oil/Water Dispersion and Macoma
balthica Tissue Taken During Chronic Exposure to the
Dispersion 14
Table 4. Percent of Total Area for Each of Four Classes of Alkanes
in Gas Chromatograms from Samples of the Oil/Water
Dispersion and Macoma balthica Tissue Taken During
Chronic Exposure to the Dispersion, Regrouped by Carbon
Number and Normalized within Each Class 15
Table 5. Macoma balthica - Concentration of Aromatic Hydrocarbons
Present in Animals and Exposure Water 18
Table 6. Macoma balthica - Mean Substitution Level of Aromatic
Hydrocarbons Present in Animals Exposure Water 19
Table 7. Macoma balthica Concentration Factor of Aromatic Compounds
in Animals Exposed to 0.03 mg/kg Oil in Water After 60
Days Depuration 20
CHAPTER 3
Table 1. Average Monthly Salinities, Temperature, pH, Dissolved
Oxygen and Phytoplankton Species Densities of Water
Flowing into Exposure Tanks 30
Table 2. A Comparison of the Percentage of Algal Cells Filtered
from the Water by Macoma balthica Subjected to Chronic
Oil Treatment 39
Table 3. Average Values for Wet and Dry Tissue Weights of Clams
Exposed to Continuous Low-Level Oil 44
Table 4. Growth and Condition Index for Macoma balthica After
Exposure to Oiled Seawater 47
ix
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48
50
54
56
57
58
60
61
62
69
76
LIST OF TABLES
CONTINUED
Oxygen Consumption Rates of Macoma balthica After
Exposure to Oiled Seawater
Sex Ratio and Female Reproductive Condition of Maooma
balthica After 120 Days of Exposure to Oiled Seawater . .
Protein Values for Claras Exposed to Oiled Seawater
for Six Months
Glycogen Values for Macoma balthica Exposed to Oiled
Seawater for Six Months
Total Carbohydrate in Macoma balthica During Six Months
of Oiled Seawater . .
Total Lipid in Macoma balthica During Six Months
Exposure to Oiled Seawater. ...... . . . .
RNA Values for Macoma balthica During Exposure to Six
Months of Oiled Seawater
Values for "LOW" DNA in Macoma balthica During
Exposure to Prudhoe Bay Crude Oil in Seawater
Values for "HIGH" DNA in Macoma balthica Exposed
to Six Months of Oiled Seawater
Summary of the Effects of Six Months of Oiled
Seawater on Various Parameters of Macoma balthica . . . .
In Vitro Effect^ ^ of Crude Oil and Components of
Crude Oil on Mg -ATPase from Crude Homogenates
of Macoma balthica
x
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CHAPTER 1
INTRODUCTION
This report describes a research project the goal of which was to better
understand biological changes at the organism and community levels that result
from a defined chronic input of petroleum to an intertidal environment. This
project was undertaken in Alaska in 1975 because at that time several develop-
ment activities were in progress or being contemplated which had the potential
to cause chronic oil pollution of the marine environment through low level
permitted discharges during normal operations. These activities included oil
terminal operations in Port Valdez and outer continental shelf oil exploration
in several regions of the state.
The project focused on the effects of chronic low level inputs since
these, unlike catastrophic spills resulting from shipwrecks or other accidents,
are manageable through the permitting process. Proper setting of permitted dis-
charge levels is a very important matter since levels set too high will not
provide society with the degree of environmental quality which it desires but
levels set too low will lead to cleanup expenses which produce little benefit.
Thus to err in either direction in the setting of discharge levels is likely
to lead to wasting of resources. Clearly no single research project can supply
sufficient scientific information for the evaluation of the effects of various
discharge levels. Yet we believe that the work reported here is an important
step in that direction.
The initial approach adopted to reach this project's overall goal of
better understanding the biological effects of chronic oil pollution was to
examine the effects of natural petroleum seeps on Alaskan intertidal environ-
ments. We hoped that by comparing organisms and communities existing under
the influence of such seeps to carefully selected controls it would be possible
to correlate differences with long term oiling. However, preliminary work at
three oil seep areas in South Central Alaska indicated that the number of
1
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uncontrollable and unquantifiable environmental variables was sufficient to
make the success of this approach doubtful at best.
Consequently, the approach of this project was modified to focus on lab-
oratory experimentation where closer control of variables was possible. This
laboratory approach has two distinct disadvantages in that the practical ex-
posure period is greatly reduced and that there is systematic uncertainty of
extrapolating from the simplified model "environment" of the laboratory back
to the natural environment. However, it was (and is) our opinion that these
disadvantages are out-weighed by the ability to control and manipulate variables
which the laboratory provides. The purpose of this experiment was to examine
the largest possible number and variety of physical, physiological, chemical
and biochemical parameters in a single chronic oiling experiment. The focus
of the experiment was the biological effect of oil pollution. Hence the param-
eters examined concentrated on ones potentially related to oil's mode of
toxicity such as membrane function and respiration and excluded ones related
to oil's fate such as degradative metabolic pathways.
2
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CHAPTER 2
ACCUMULATION, FRACTIONATION AND RELEASE OF OIL
BY THE INTERTIDAL CLAM MACOMA BALTEICA
INTRODUCTION
The rate of hydrocarbon accumulation in marine organisms upon exposure to
sub-lethal concentrations of oil in seawater and the subsequent loss of those
hydrocarbons after termination of oil exposure have been reviewed by Varanasi
and Malins (1977). Some of this information has come from in situ observa-
tions after an oil spill or in a chronically polluted area, and other informa-
tion has come from laboratory studies with static or flow-through systems.
While much work remains to be done, one result of the oil exposure studies is
clear: marine organisms, and in particular bivalves, have the ability to con-
centrate petroleum hydrocarbons from seawater in their tissues.
We have conducted a controlled, laboratory experiment in which the bivalve
mollusc, Maooma balthiaa, was exposed to seawater dispersed crude oil for 180
days. In this chapter we describe the apparatus in which this experiment was
conducted and report the accumulation, fractionation and release of oil by the
test animals.
MATERIALS AND METHODS
In order to provide a realistic simulation of chronic exposure of marine
animals to oil-in-seawater dispersions in the laboratory, we constructed a
continuous-flow system capable of simultaneously delivering dispersions with
nominal oil in water concentrations of 0.03 mg/1, 0.3 mg/1 and 3.0 mg/1. Our
system is based primarily on the design of Hyland et al. (1977). Because we
wished to disperse a moderately viscous crude oil, we also incorporated some
of the features of Roubal et at. (1977).
3
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The components of the system, as shown in Figure 1, are a constant head
pressure tank, a mixing and separation tank, manifold to deliver sea water
and the oil/seawater dispersion to each of eight exposure tanks, and filter
drums to remove the oil. Seawater was maintained at a constant level in the
head tank (A) and flowed into the mixing chamber (B) at 10 £/min. A peri-
staltic pump (D) metered Prudhoe Bay crude oil (C) into the mixing chamber at
0.3 ml/min. Mixing energy was applied by a stainless steel blade rotated at
high speed by a stirring motor (F). The oil/seawater dispersion separated
during its passage through a 340 I tank (G) and was removed from below the
resulting oil slick at 6.66 £/min as it entered the dosing manifold (I). The
tanks used were constructed of marine plywood covered with fiberglass resin.
All plumbing was done with rigid PVC pipe and fittings. The mixing chamber
was constructed of acrylic plastic. The entire system was leached with running
seawater for two weeks prior to use. Evidence of contamination from plastics
was not detected in either water or animals. Overflow from the separation
tank passed through a plastic fibre floss filter (K), two 120 £ separation
drums and one of the two 80 I polyethylene drum filters (L) containing hydro-
phobic plastic strips, plastic floss and activated charcoal.
Different exposure concentrations were achieved by dilution of the oiled
seawater with non-contaminated seawater (H) from the head tank to give a total
flow of 3 S./min through each exposure tank. Seawater was supplemented with
diatom enriched seawater (~ 1:1 mixture) from "upwelling ponds" on the grounds
of the Seward marine facility (Neve et al., 1976) for a period of 4-6 hours
per day beginning 9 July and ending 24 November 1977. The average exposure
temperature was 8.0° (Range = 7.1-9.0°).
Specimens of Macoma balthiaa obtained from Resurrection Bay near Seward,
Alaska (149°27'W; 60°06'N) were maintained in sand filled petri dish bottoms
immersed in flowing seawater. Only clams which buried themselves were used
for experimentation. On 14 June 1977, after two weeks acclimatation, the
180-day oil exposure experiment was begun. Water samples were collected from
all exposure tanks and analyzed for hydrocarbon concentrations at 30-day inter-
vals beginning on day 0 and ending on day 150. Replicate samples of approxi-
mately 50 specimens of Macoma balthiaa were obtained from each treatment
periodically throughout the 180-day exposure period and one set of replicate
4
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SEA WATER
• ft
DRAIN
DRAIN
DRAIN
Figure 1. Schematic representation of continuous oil exposure system: A,
head tank; B, mixing chamber; C, oil supply; D, oil pump; E,
seawater supply; F, stirring motor; G, separation tank; H, non-
contaminated seawater manifold; I, oil/seawater dispersion mani-
fold; J, exposure tanks; K, separation tanks; L, final filter.
5
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samples from the control, low oil, and medium oil treatments after 60 days
recovery in clean seawater. The clams were sieved from the silica sand,
placed in fiberglass mesh buckets and returned to their exposure tanks for
24 h. There was no food available at this time, allowing the clams to clear
their intestinal tract. After 24 h they were removed from the tanks, rinsed
with clean seawater and immediately frozen.
Water samples were liquid-liquid extracted with three 5% (vol.) hexane
washes in a separatory funnel. The hexane extracts were combined, dried over
Na2S0^, and reduced to 2 ml under vacuum in a rotary evaporator. The evapora-
tion parameters were optimized for the recovery of napthalene. Clam tissue
samples were analyzed for hydrocarbons by modification of the method proposed
by Warner (1976). Tissue was removed from the shell while still frozen and
placed in a tared 50 ml centrifuge tube with teflon-lined screw cap. Approxi-
mately 10 g wet weight of tissue was obtained from 50 Macoma balthiea. Samples
were digested with 10 N NaOH at 90° for 3 h, and allowed to cool to room tem-
perature. Hexane was then added, the tube resealed and shaken vigorously for
one minute. The sample was then centrifuged at 2400 rpm for 10 min. The
organic phase was subsequently removed with a 20 ml syringe and the procedure
repeated twice. The hexane extract was dried over Na^SO^ and concentrated to
between 1 and 2 ml on a rotary evaporator, and further concentrated to 0.5 ml
under nitrogen before fractionation on a column of 5 g of 5% deactivated silica
gel. A saturate hydrocarbon fraction was eluted with hexane and an unsaturated
and aromatic hydrocarbon fraction with 40% benzene/hexane or 20% methylene
chloride/hexane.
Quantification and identification of individual compounds was either by
gas chromatography (GC) or by gas chromatography coupled mass spectrometry
(GC-MS). Samples were concentrated to approximately 0.2 ml and an aliquot
injected onto a 30 m x 0.75 mm glass SCOT column coated with OV—101 in either
an Hewlett-Packard (HP) 5710 GC with flame ionization detector, or an HP
5930/5933 GC-MS.
Compounds were identified and quantified on the GC-MS by single ion
monitoring and calibration with an external standard. Aromatic compounds were
quantified by reference to fully deuterated naphthalene and anthracene which
were included in the sample as an internal standard. Alkylated aromatics were
6
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assumed to have the same response as the parent compound which results in an
increasing but unknown underestimation with increasing alkyl substitution.
The percentage recovery after the sample handling process was calculated from
the recovery of an alkane, aromatic standard added to one of the two control
samples from each sampling date before digestion. All hydrocarbon values are
corrected for this handling loss, which averaged 25%.
RESULTS
Total hydrocarbon concentration (mg oil/1 seawater) for each exposure
level measured at 30 day intervals is presented in Figure 2. The greatest
relative variability is encountered at the control and low oil level. The
overall pattern is that of high exposure level or undiluted mixing tank output.
The increased variability of the lower oil levels is due to the widely varying
control levels. Over 150 days, when the water hydrocarbon concentrations were
measured (Fig. 2), the levels were relatively constant and an order of magnitude
apart at nominal values of 3.0 mg/1, 0.3 mg/1, and 0.03 mg/1. Related work
(Shaw and Reidy, 1979) has shown that 86% of the dispersed oil produced in this
experiment was contained in particles larger than 5 pm.
The hydrocarbon concentrations in clam tissue over the 180-day period of
exposure to oil and the 60-day depuration period are presented in Figure 3.
±These results are expressed on a wet weight basis. Those who prefer dry weight
basis will find the information necessary for conversion tabulated in Chapter 9
of this report 1. As one of the two control clam samples for each date had a
hydrocarbon standard added to determine analytical recovery, only the unaltered
control values are presented.
Clams exposed to the highest concentration of oil in water (3.0 mg/1)
rapidly accumulated hydrocarbons to a maximum of nearly 1500 Mg/g wet weight
after 30 days. Their hydrocarbon content then gradually decreased throughout
the rest of the exposure period. Initial uptake decreased with decreasing oil
exposure levels; however, both at medium and low oil concentrations, clams
reached their maximum hydrocarbon content after 120 days of exposure. Hydro-
carbon levels in clams exposed to medium concentrations then began to decline
at a fairly constant rate throughout the rest of the experiment, including the
7
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Medium
.1
.09
.08
.07
.06
.05
.04
.03
.02
.01
Low
Control
60 90
Days of Exposure
120
150
Figure 2. Concentration of total oil in seawater in test and
control tanks during the course of the experiment.
Bars show the range of duplicate determinations;
dots are single determinations.
8
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4.000
2.000
«0
.*
I
s
t
i
i
X
at
m
ISO
90 120
Diyi of Expoturt
Figure 3. Macoma balthica. tissue concentrations of
total hydrocarbons during 180 days of
exposure to oil in seawater dispersions
and 60 days of recovery. "High", "medium",
"low", and "control" correspond to oil in
seawater concentrations shown in Figure 2.
Bars show the range of duplicate determi-
nations; dots are single determinations.
9
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60—day depuration period. At the low oil level, clams showed a decline in
hydrocarbon content during the last 30 days of exposure and a slower rate of
decline after exposure to oil ceased. At the low and medium oil levels, clams
approached the same hydrocarbon level during the 60-day depuration period.
While there was a 100-fold difference in the amount of oil to which the clams
were exposed, after day 60, there was less than a 10-fold difference in hydro-
carbon content among the clams exposed to oil.
The concentration of hydrocarbons found in each column chromatographic
fraction is summarized in Table 1. Fraction 1 contained saturated hydrocarbons
and fraction 2 aromatic hydrocarbons and biogenic olefins. The control clams
contained more fraction 2 material than fraction 1 material as did the clams
in the low concentration of oil after day 60. At the higher concentrations of
oil, clams had more fraction 1 material than fraction 2 material. Chromato-
grams of a typical oil/water dispersion (Fig. 4 A, B), Maooma balthica tissue
after 120 days of exposure to oil (Fig. 4 C, D), 180 days of exposure to oil
(Fig. 4 E, F), and 60 days of depuration (Fig. 4 G, H) demonstrate that a
considerable change occurred in the composition of the oil accumulated by the
clams. For the aliphatics, the relative amount of lighter compounds, those in
the region bounded by dodecane and hexadecane on the chromatogram, increased
from 25% at 30 days of exposure to 56% after 180 days of exposure (Table 2).
The fractionation of aliphatics within the dodecane to hexadecane range
was studied further. Normal alkanes, branched alkanes, monocyclic alkanes
and bicyclic alkanes were assayed by GC-MS to determine the relative intensity
of parent peaks of each class with 12 to 16 carbon atoms. This was done for
extracts of the oil in water and oil exposed clams collected after 90, 180 and
240 days. For each sample, the ion intensity data of the five carbon numbers
investigated was normalized within each compound class (Table 3). Then the
data were regrouped by carbon number and each compound class was renormalized
over the sampling sequence (Table 4). The double normalization eliminated the
effect of variations in relative abundance of the parent ions in the individual
mass spectra of compounds in the four classes; this procedure demonstrated the
enhancement or diminution for each carbon number of each compound class rela-
tive to the total abundance in that compound class at any given time. These
results for the branched, monocyclic and bicyclic alkanes are shown in Figure 5.
10
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Day
0
7
15
30
60
90
120
150
180
240
MACOMA BALTHICA. HYDROCARBON CONCENTRATIONS (ug/g wet weight basis)
IN TISSUES DURING OILING EXPERIMENT FOR EACH COLUMN
CHROMATOGRAPHIC FRACTION
Treatment
Control 0.03 mg/1 0.3 mg/1 3.0 mg/1
F1 ^2 ^1 ^2 ^1 ^2 ^1 ^2_
7.9 22
7.7 13 30 22 60 63 480 310
8.9 6.4 37 32 80 63 570 310
8.2 9.4 48 33 190 120 1020 430
9.7 14 91 84 390 340 780 600
9.5 13 108 130 350 300 630 570
8.8 16 130 140 540 400 630 510
9.5 13 150 110 390 250 500 360
7.2 8.4 68 81 240 130 160 350
6.9 7.4 64 51 61 52
11
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L
I—»
NJ
Figure 4. Maaoma balthioa gas chroraatograms of oil in water dispersion and tissue accumulations of hydro-
carbons. A, saturate fraction of oil in water dispersion; B, unsaturate fraction of oil in water
dispersion; C, saturate fraction of tissue after 120 days; D, unsaturate fraction of tissue after
120 days; E, saturate fraction of tissue after 180 days; F, unsaturate fraction of tissue after
180 days; G» saturate fraction of tissue after 240 days; H, unsaturate fraction of tissue after
240 days. Retention indices are shown for each chromatogram.
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TABLE 2. MA COMA BALTHICA. PERCENTAGE OF TOTAL SATURATED ALIPHATIC
HYDROCARBONS ELUTING BETWEEN DODECANE AND HEXADECANE
DURING GAS CHROMATOGRAPHY
Day
Treatment
Control
0.03 mg/1
0.3 mg/1
3.0 mg/1
0
10
30
3
25
30
28
60
5
34
33
32
90
2
49
39
36
120
3
43
32
34
150
1
40
33
30
180
4
57
52
44
240
0.5
53
55
13
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TABLE 3. AREA AND PERCENT OF TOTAL AREA FOR EACH OF FOUR CLASSES OF ALKANES WITH 12 TO 16 CARBON
ATOMS IN GAS CHROMATOGRAMS FROM SAMPLES OF THE OIL/WATER DISPERSION AND
MAC014A BALTHICA TISSUE TAKEN DURING CHRONIC EXPOSURE TO THE DISPERSION
Carbon Atoms
Normal Alkanes
area %
Branched Alkanes
area %
Monocyclic Alkanes
area %
Bicyclic Alkanes
area %
Oil/seawater Dispersion (o/w)
12
435
18
394
25
892
37
3216
36
13
587
24
400
25
539
22
2939
27
14
321
13
296
19
306
13
1470
17
15
569
24
255
16
316
13
1063
12
16
492
20
238
15
344
14
724
8
Macoma
balthica 90
day exposure
(90)
12
1381
19
2562
28
8697
38
30748
32
13
1675
24
2218
24
6465
28
28179
30
14
1392
20
1775
20
3766
16
19803
21
15
1295
18
1426
16
2318
10
11185
12
16
1354
19
1098
12
1638
7
5247
6
Macoma balthica
180
day exposure
(180)
12
1775
19
5342
29
19059
34
32858
25
13
1843
19
4703
25
16349
29
38562
29
14
1809
19
3504
19
10377
18
31585
24
15
2052
21
2807
15
6658
12
20757
15
16
2069
22
2377
13
4282
8
10236
8
Macoma balthica
180 day
exposure, 60 day depuration
(240)
12
0
0
5318
37
21145
33
38305
20
13
0
0
3981
28
19372
31
55203
29
14
0
0
2554
18
12165
19
49800
26
15
0
0
1447
10
6701
11
33119
17
16
0
0
1045
7
3754
6
15221
8
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TABLE 4. PERCENT OF TOTAL AREA FOR EACH OF FOUR CLASSES OF ALKANES IN GAS CHROMATOGRAMS FROM SAMPLES
OF THE OIL/WATER DISPERSION AND MACOMA BALTHICA TISSUE TAKEN DURING CHRONIC EXPOSURE TO THE
DISPERSION, REGROUPED BY CARBON NUMBER AND NORMALIZED WITHIN EACH CLASS
Carbon Atoms Normal Alkanes Branched Alkanes Monocyclic Alkanes Bicyclic Alkanes Sample
% Norm % Norm % Norm % Norm
12
12
12
12
18
19
19
0
.94
1.0
1.0
0
25
28
29
37
.68
.76
.78
1.0
37
38
34
33
.97
1.0
.89
.86
36
32
25
20
1.0
.89
.69
.56
o/w
90
180
240
13
13
13
13
24
24
19
0
1.0
1.0
.79
0
25
24
25
28
.89
.86
.89
1.0
22
28
29
31
.71
.90
.94
1.0
27
30
29
29
.90
1.0
.96
.96
o/w
90
180
240
14
14
14
14
13
20
19
0
.65
1.0
.95
0
19
20
19
18
.95
1.0
.95
.90
13
16
18
19
.68
.84
.95
1.0
17
21
24
26
.65
.81
.92
1.0
o/w
90
180
240
15
15
15
15
24
18
21
0
1.0
.75
.88
0
16
16
15
10
1.0
1.0
.94
.63
13
10
12
11
1.0
.76
.92
.85
12
12
15
17
.71
.71
.88
1.0
o/w
90
180
240
16
16
16
16
20
19
22
0
.91
.86
1.0
0
15
12
13
7
1.0
.80
.87
.47
14
7
8
6
.0
.50
.57
.43
8
6
8
8
1.0
.75
1.0
1.0
o/w
90
180
240
-------
Branched
1.0
12
0.5
Monocyclic
O °
Bicyclic
Figure 5. Macoma balthica branched, monocyclic, and bicyclic
saturated hydrocarbons with 12 to 16 carbon atoms.
Each graph in the matrix shows the amounts of the
compounds with the indicated structure and carbon
number in oiled seawater (o), and in tissue at day
90 (°), 180 (a), and 240 (•) relative to the total
abundance of the structural class at the time. The
normalization procedure used to derive these results
is described in the text.
16
-------
The results for normal alkanes are not shown since this compound class showed
diminution through time for all chain lengths. The branched alkanes showed
relative enhancement of 12 and 13 carbon atom compounds through time and rela-
tive diminution of larger compounds. In the monocyclic alkanes relative
enhancement was observed for compounds with 13 and 14 carbon atoms. While
the bicyclics showed relative enhancement only for compounds of 14, 15 and
16 carbon atoms.
Aromatic compounds identified and quantified by single ion MS, and their
mean alkyl substitution level are presented in Tables 5 and 6 respectively,
for low oil level clams and the oil/seawater dispersion. Relatively high
levels of these compounds were found in clams before the start of the exper-
iment, suggesting that Maooma balthioa in Resurrection Bay are exposed to low
levels of aromatic hydrocarbons. The control clams, after 180 days in clean
seawater had lost most of the aromatic compounds, but still retained signifi-
cant amounts of the anthracenes, fluoranthenes, and dibenzothiophenes. (Here
and throughout this paper we use the name of one aromatic ring system to indi-
cate that system and its isomers: thus "anthracene" means "anthracene and
phenanthrene"; and by "anthracenes" we mean "anthracene, phenanthrene and
their alkyl homologs".) The mean substitution level for most of the parent
compounds generally increased with continued oil exposure and even upon depu-
ration. There was a considerable difference between the mean substitution
level of the oil-exposed clams after 60 days depuration and that of the oil/
seawater dispersion for all the aromatic systems except the fluoranthenes.
In general, there was selective retention of more substituted compounds
(Table 7), except for the chrysenes for which the mean substitution level was
lower after depuration than in the oil/seawater dispersion.
The concentration of aromatic compounds determined by GC-MS and the total
concentration of hydrocarbons in the second fraction of column chromatography
determined by GC were not the same and the concentration found by GC-MS as a
percentage of the total varied from 25% in clams before oil exposure to 6%
after depuration. The concentration of hydrocarbons determined by GC-MS is an
underestimate, as only the parent compounds were calibrated against standards
and increased alkyl substitution causes decreasing response in single ion
monitoring. The change in the percentage of the total accounted for by MS may
-------
TABLE 5. 14AC0MA BALTHICA. CONCENTRATION OF AROMATIC HYDROCARBONS PRESENT
IN ANIMALS AND EXPOSURE WATER
Macoma balthiaa
(ng hydrocarbons/g wet weight)
Oil/water
Treatment
Day
Compounds
Naphthalenes
Biphenyls
Tetralins
Fluorenes
Anthracenes
Fluoranthenes
Chrysenes
Benzothiophenes
Dibenzothiophenes
Control
0
0.03 mg/kg
60
0.03 mg/kg
120
0.03 mg/kg
180
0.03 mg/kg
240
120
160
5.5
470
2200
220
73
0
1800
Benzonaphthenothiophenes 150
1600
930
640
1400
3000
920
310
140
2300
570
2400
1500
1000
2100
3200
960
510
280
3000
780
980
780
440
960
1900
540
240
120
1300
470
340
330
130
260
740
200
68
44
570
210
Control
180
21
31
0
93
380
130
26
0
130
0
0.03 mg/1
350
86
43
110
200
30
15
14
140
24
-------
TABLE 6. mCOMA BALTHICA. MEAN SUBSTITUTION LEVEL OF AROMATIC HYDROCARBONS PRESENT
IN ANIMALS EXPOSURE WATER
Maaoma balthiaa
Treatment
Day
Control
0
0.03 mg/kg
60
0.03 mg/kg
120
0.03 mg/kg
180
0.03 mg/kg
240
Control
180
Oil/water
0.03 mg/kg
Compounds
Naphthalenes 5.0
Biphenyls 4.5
Tetralins 6.1
Fluorenes 3.3
Anthracenes 2.6
Fluoranthenes 1.7
Chrysenes 0.5
Benzothiophenes 0
Dibenzothiophenes 2.7
Benzonaphthenothiophenes 1.4
5.2
5.0
4.8
4.7
3.3
2.4
1.1
6.1
3.2
1.9
5.3
5.0
4.9
4.5
3.3
2.5
1.2
5.6
3.2
2.0
6.3
5.4
5.4
4.8
3.5
2.5
1.1
6.1
3.2
2.4
7.2
5.6
5.7
5.6
3.8
2.4
0.9
7.0
3.3
2.5
4.3
5.6
0
2.7
3.5
2.2
0.7
0
6.0
0
3.6
3.9
3.4
3.8
3.1
2.4
1.1
4.6
3.0
1.9
-------
TABLE 7. MACOMA BALTHICA. CONCENTRATION FACTOR3 OF AROMATIC COMPOUNDS
IN ANIMALS EXPOSED TO 0.03 mg/kg OIL IN WATER AFTER
60 DAYS DEPURATION. (MOLECULAR WEIGHT OF COMPOUND)
Alkyl Substitution
(No. of carbon atoms) Naphthalenes Dibenzothiophenes Anthracenes Chrysenes
0
0.05(128)
0 (184)
0.26(178)
4.44(228)
1
0 (142)
0.89(198)
0.55(192)
4.93(242)
2
0.01(156)
2.98(212)
2.82(206)
5.61(256)
3
0 (170)
7.12(226)
5.91(220)
4
0.63(184)
7.96(240)
5.84(234)
5
3.01(198)
8.73(254)
10.56(248)
6
5.40(212)
8.77(268)
10.54(262)
7
9.30(226)
a„ . r ug compound/g wet weight of M. halthvoa ,_-3
Concentration factor = —* c ti x 10
pg compound/g seawater
20
-------
be the result of the different levels of substitution encountered, the pre-
sence of hydrocarbon or hetero-aroraatic systems not investigated by GC-MS or
possibly by the formation of oxygenated metabolites whose column chromato-
graphic behavior is similar to the aromatics.
DISCUSSION
The continuous flow system produced reasonably uniform petroleum expo-
sures at the three test concentrations (Fig. 2). The fluctuations of the
measured oil concentrations in the high, medium and low exposures tended to
occur together {e.g., all were lowest on day 90). This is to be expected
since the two lower concentration solutions were produced by dilution of the
high concentration.
Previous work has indicated that non-polar lipids partition between
animal tissues and the aquatic environment in an equilibrium fashion (Hamelink
et at. , 1971) and that the initial uptake rate of hydrocarbons is directly
proportional to the aqueous hydrocarbon concentration (Stegeman and Teal, 1973).
Expressed symbolically these relationships are:
k=[0a]/[0w] (1)
and d[0A]/dt = [0^] (2)
where K is the equilibrium constant for the partition of oil between animals
and water, [0, ] is the concentration of oil in water, [0 ] is the concentration
w A
of oil in the animal and k^ is the rate constant for the initial uptake of oil
by the animal. Using our data from Figure 3, we have evaluated (1) at 120 days
3
and (2) at seven days for the three values of [0 1. K varied from 9.2 x 10
2
at 0.03 mg/1 down to 3.8 x 10 as the oil concentration rose to 3.0 mg/1 and
k^ ranged from 95/day at the high oil concentration to 34/day at the low con-
centration of oil. Both "constants" had intermediate values at 0.3 mg/1 oil.
Both k and K increased as [0IT] decreased; that is M. balthica is more effi-
i w
cient at extracting and retaining oil from water at low oil-in water concen-
trations .
One explanation for this observation is that the concentration of hydro-
carbons in the clams is related not only to the hydrocarbon concentration in
21
-------
the water, but also to the amount and efficiency of water filtration accom-
plished by the clams at the various oil exposure levels. Stainken (1975) has
observed the mode of accumulation of an oil/water dispersion in a filter-
feeding bivalve (Mya arenaria). Oil droplets are processed as food and are
accumulated in the gut and digestive diverticula and from there are trans-
ferred to the rest of the body, probably as individual molecules. No food
was administered to the Macoma balthica in the present study for 24 h prior
to sampling for hydrocarbon analysis, however the animals were left in sea-
water containing oil droplets. No attempt was made to segregate the gut from
the other tissues, thus the hydrocarbon data are for entire clams, including
gut contents without food. At higher oil concentrations in this experiment,
the rate of uptake declined probably as a result of the greatly reduced siphon
activity of the animals exposed to the high oil concentration (Chapter 3).
The uptake rate response of M. balthica over the large range of concentration
of this experiment demonstrated that other behavioral patterns in addition to
shell closure at very high exposure levels may influence the rate at which
hydrocarbons are taken up by organisms.
Bivalves exposed to oil usually fail to return rapidly to pre-exposure
hydrocarbon levels during depuration (Lee et at., 1972; Stegeman and Teal,
1973; Clark and Finley, 1975; Fossato, 1975; Boehm and Quinn, 1977). Stegeman
and Teal (1973) hypothesized a stable compartment in an organism, which after
saturation with hydrocarbons, released those hydrocarbons very slowly. In our
experiment, the low and medium exposure clams returned to approximately the
same hydrocarbon level after 60 days of depuration. It may be that this final
oil concentration 110 mg/g (wet weight) represents the size of this "stable
compartment" for Macoma balthica. However, in the absence of information from
even longer depuration periods, this conclusion is quite tentative.
As illustrated in Figure 4, our experimental work shows that fractionation
of Prudhoe Bay crude oil occurred during both accumulation and depuration by
Macoma balthica. Because of the extreme molecular complexity of the crude oil,
we have not examined the fractionation process for individual compounds but
have investigated the behavior of several classes of compounds present. Table
1, which records the concentrations of aliphatic and aromatic hydrocarbons in
M. balthica shows that aromatics were enhanced in the animals' tissues throughout
22
-------
the experiment. The ratio of aliphatics to aromatics (f^/f2> in the oil in
water dispersion was 3.7. However this ratio in the tissues was generally
less than 1.5 and always less than 2.5. Clearly M. ba.lth.ica selected in favor
of aromatics both in initial uptake rate and in equilibrium partition.
Figure 5 indicates that within the aliphatic fraction cyclic compounds
were generally retained to a greater extent than branched chains which in turn
were preferentially held over linear alkanes. Similar fractionation has been
observed by other workers (e.g. Blumer et at., 1970; Stegeman and Teal, 1973).
Figure 5 and Table 2 also show that molecular size (carbon number) influenced
retention. Although these results are too complex for detailed interpretation,
it seems reasonable that selective metabolism, tissue transport, membrane
transport or final deposition site may be related to the size, solubility or
configuration of the compound (Tanford, 1978). The apparent preference for
accumulation and retention of lighter aliphatic compounds (Table 2), was un-
expected in view of decreasing aqueous solubility with increasing molecular
weight. This may indicate that uptake of aliphatics occurs via the dissolved
phase with attendant fractionation, but that release involves some other
process.
While the relative percentage of the aromatic ring systems remained fairly
constant during the course of our experiment, the mean alkyl substitution level
increased, most noticeably in the lighter aromatic compounds. Given the
inherent change in sensitivity of MS with increased levels of alkyl substitution,
it is difficult to demonstrate a change in relative amount of the sum of each
parent compound and its alkyl substituants. Changes in the relative amount of
alkyl substituted aromatic compounds have been observed in a benthic anemone
following an oil spill (Grahl-Nielsen et al., 1978). Only aromatic compounds
with up to 3 carbon alkyl substitution were examined, but there appeared to be
a pattern favoring higher alkyl substitution with time. A field study of
Modiolus demissus chronically exposed to oil (Lake and Hershner, 1977) demon-
strated a loss of lighter aromatic compounds upon depuration and an apparent
relative increase in 2,3,6-trimethylnaphthalene, fluoranthene, and pyrene.
These authors concluded that most diaromatics are lost upon depuration and tri-
and tetra-cyclic aromatics are retained. When a larger range of alkyl sub-
stituted compounds is available, as in the present study, a pattern of increasing
23
-------
retention with increasing molecular weight emerges, both among unsubstituted
aromatic compounds and among various alkyl substitutions of any given aromatic
ring system. However, for compounds of a given molecular weight, the more
substituted one has a higher concentration factor (Table 7). Thus, in contrast
to the behavior of aliphatic hydrocarbons, retention of aromatic compounds
appears to decrease with increasing aqueous solubility, and the long period of
slow depuration observed in many bivalves may result from relatively high con-
centrations of less water soluble aromatic compounds.
In studies of aromatic sulfur compounds in organisms upon recovery from
oil exposure, Grahl-Nielsen et at. (1978) found an increase in the relative
amount of dibenzothiophene and its alkyl derivatives, whereas Lake and Hershner
(1977) found a decrease in aromatic sulfur compounds that paralleled the de-
crease in aromatic compounds as a whole. In the present study there was little
or no enhancement of the aromatic sulfur compounds relative to other aromatic
compounds. The concentration factor for C, substituted dibenzothiophenes
b
(Table 7) fell between C, naphthalenes and C, anthracenes, about where one
o D
might expect it on the basis of molecular weight.
It thus appears from our laboratory data, that when Maaoma balthioa is
exposed to chronic low levels of an oil in water dispersion, the character-
istics of the oil are extensively modified within the clam. The aliphatic
compounds are handled quite differently than the aromatics. We have suggested
selective metabolism as one of the possible causes of fractionation of petro-
leum by M. balthioa. Although much work under short-term acute conditions
(Varanasi and Malins, 1977) indicates that molluscs have little or no ability
to metabolize hydrocarbons, we believe that the presently available information
does not exclude the possibility of selective metabolism during longer accumu-
lation and release periods.
REFERENCES
Blumer, M., G. Souza, and J. Sass. 1970. Hydrocarbon pollution of edible
shellfish by an oil spill. Mar. Biol. 5:195-202.
Boehm, P. D. and J. G. Quinn. 1977. The persistence of chronically accumu-
lated hydrocarbons in the hard shell clam Mercenaria mercenaria. Mar.
Biol. 44:227-233.
24
-------
Clark, R. C., Jr. and J. S. Finley. 1975. Uptake and loss of petroleum hydro-
carbons by the mussel, Mytilus edulis, In laboratory experiments. Fish.
Bull. 73:508-15.
Fossato, V. U. 1975. Elimination of hydrocarbons by mussels. Mar. Pollut.
Bull. 6:7-10.
Grahl—Nielsen, 0., J. T. Staveland, and S. Wilhelmsen. 1978. Aromatic hydro-
carbons in benthic organisms from coastal areas polluted by Iranian crude
oil. J. Fish. Res. Bd. Can. 35:615-623.
Hamelink, J. L., R. C. Waybrant and R. C. Ball. 1971. A proposal: exchange
equilibria control the degree chlorinated hydrocarbons are biologically
magnified in lentic environments. Trans. Am. Fish. Soc. 100:207-214.
Hyland, J. L., P. F. Rogerson and G. R. Gardner. 1977. A continuous flow
bioassay system for the exposure of marine organisms to oil. pp. 547-
550. In: Proceedings of the 1977 Oil Spill Conference, Washington, D.C.,
American Petroleum Institute.
Lake, J. L., and C. Hershner. 1977. Petroleum sulphur containing components
and aromatic hydrocarbons in marine molluscs, Modiolus derrrissus and
Cvassostvea virginiaa. pp. 627-633. In: Proceedings of the 1977 Oil
Spill Conference, Washington, D.C., American Petroleum Institute.
Lee, R. F., R. Sauerheber, and A. A. Benson. 1972. Petroleum hydrocarbons:
Uptake and discharge by the marine mussel, Mytilus edulis. Science, N.Y.
177:344-346.
Neve, R. A., R. C. Clasby, J. J. Goering and D. W. Hood. 1976. Enhancement
of primary productivity by artificial upwelling. Mar. Sci. Commun.
2:109-124.
Roubal, W. T., D. H. Bovee, T. K. Collier, S. D. Stranahan. 1977. Flow through
system for chronic exposure of aquatic organisms to seawater-soluble
hydrocarbons from crude oil. pp. 551-556. In: Proceedings of the 1977
Oil Spill Conference, Washington, D.C., American Petroleum Institute.
Shaw, D. G. and S. K. Reidy. 1979. Chemical and size fractionation of aqueous
petroleum dispersion. Environ. Sci. Technol. 13:1259-1263.
Stainken, D. M. 1975. Preliminary observations on the mode of accumulation of
No, 2 fuel oil by the soft-shell clam, Mya arenaria. pp. 463-468.
In: Proceedings of the 1975 Conference on the Prevention and Control of
Oil Pollution, Washington, D.C., American Petroleum Institute.
Stegeman, J. J. and J. M. Teal. 1973. Accumulation, release, and retention
of petroleum hydrocarbons by the oyster Cvassostvea vivginica. Mar. Biol.
22:37-44.
25
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Tanford, C. 1978. The hydrophobic effect and the organization of living
matter. Science, N.Y. 200:1012-1018.
Varanasi, V. and D. C. Malins. 1977. Metabolism of petroleum hydrocarbons:
Accumulation and biotransformation in marine organisms. In: Effects of
Petroleum on Arctic and Subarctic Marine Environments and Organisms.
Vol. II Biological Effects, pp. 175-270, Academic Press, New York.
Warner, J. S. 1976. Determination of aliphatic and aromatic hydrocarbons in
marine organisms. Anal. Chem. 48:578-583.
26
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CHAPTER 3
BIOCHEMICAL AND PHYSIOLOGICAL EFFECTS OF
OIL ON MACOMA BALTHICA
One of the more noticeable events that follows severe oil pollution
in a marine system is mass mortality in number of species of plants
and animals (Burk, 1977; Krebs and Burns, 1977; American Petroleum Insti-
tute, 1977). However, mortality, although easy to measure, is the gros-
sest of all effects of oil pollution. Numerous workers have investigated
a variety of more subtle sub-lethal effects of oil pollution including
respiration (Eldridge et al., 1977; Hargreave and Newcombe, 1973), cellular
morphology (Gardner et al., 1975), reproductive development (Berdugo et al.,
1977; Byrne and Calder, 1977), feeding (Atema and Stein, 1974; Morton and Wu,
1977) growth and maturation (Griffin and Calder, 1977; Keck et al., 1978),
behavior (Donahue et al., 1977; Hargreave and Newcombe, 1973), and even some
biochemical effects (Davavin et al., 1975; Heitz et al., 1974; Manwell and
Baker, 1967). To date only a few parameters have been measured for any single
species. It has been difficult to extrapolate what effects will show up in
one particular organism exposed to a particular pollutant.
Many oil pollution studies performed in recent years have been con-
cerned primarily with either uptake and retention of hydrocarbons (Boehm
and Quinn, 1977; Clark and Finley, 1975; Corner, 1975; Fossato and Canozonier,
1976; Harris et al., 1977) and with short term, effects of crude or refined
oils on various parameters of marine organisms (e.g., Atema and Stein, 1974;
Anderson et al., 1974; Gilfillan, 1975; Griffin and Calder, 1977; Hargreave
and Newcombe, 1973; Lee et al., 1978; Lindin, 1978; Malins, 1977). The major
rationale for these studies is to attempt to understand what effects an acute
oil spill will have on various marine ecosystems.
There is, however, need for studies which involve the effects of
low levels of oil pollution over an extended period of time. Such a
27
-------
chronic pollution situation is now in existence in Port Valdez, Alaska,
the terminus of the Trans Alaskan pipeline. The ballast water treatment
plant at Valdez is currently adding a low level of oil into that harbor at
a more or less continuous rate. The effect of this kind of oil input on
the biological populations in the harbor is largely unknown. Since large
populations of the intertidal clam Maeoma balthioa reside in the harbor
this organism was chosen as an experimental animal in these studies.
Previous experiments have shown that M. balthioa is sensitive to oil
treatment. Shaw et at., (1976) have shown that this species will burrow to
the surface of the sediment after the mud flats have been subjected to an
2
oiling regime. High concentrations of oil (5yl/cm ) caused significant
mortalities. Further studies conducted in the laboratory have confirmed
these results (Shaw et at., 1977). Taylor and Karinen (1977) have exposed
M. balthioa to oiled sea water by three different methods. They reported
that oiling caused increased mortality, inhibited the burrowing response
of the clams, and also caused the clams to burrow to the surface of the mud.
Maooma balthioa is therefore a good candidate for studying the effects of oil
on intertidal, benthic marine organisms. Further the clam is circumpolar
in distribution (Gilbert, 1973), and its biology has been studied and
reported by several researchers (Brafield and Newell, 1961; Bubnova, 1972;
Chambers and Milne, 1975; Gilbert, 1973, 1977, 1978).
It was our hope that by measuring several different parameters for an
organism we would be able to arrive at a better understanding of how oil
pollution can affect that organism and possibly its entire population. We
subjected Maooma balthioa to continuous levels of Prudhoe Bay crude oil
suspended in sea water from June to December 1977. Various behavioral,
physiological, and biochemical parameters were assayed in an attempt to
define the potential effects of long-term oil pollution on this marine
organism.
METHODS
Experimental Set-up
Approximately 12,000 Maeoma balthioa were obtained from the mud flats
of Resurrection Bay near Seward, Alaska at low tides during May 3-5, 1977.
28
-------
The clams were placed in fresh running sea water for about two weeks at 6°C.
Approximately two weeks before the commencement of the experiment the clams
were screened through hardware cloth into various size classes. Clams 11-13
mm in length were selected for feeding rate studies. Clams 6-11 mm long were
selected for physiological, behavioral and biochemical studies. The clams
were placed in plastic petri dish bottoms (90 x 25 mm) filled with 170 grams
of pre-washed silica sand (0.1 to 0.7 mm). Fifteen clams of 6-11 nun in
length were placed in each dish. Only ten clams of the 11-13 mm size were
used per dish.
The clams were placed in the exposure tanks which measured 110 cm by
50 cm by 36.5 cm deep (about 200 1 total volume of water). Two tanks were
used for each exposure level. Fresh sea water flowed into and through the
tanks at approximately 3.0 1/min. After one week clams which remained on
the surface were replaced until all individuals had buried themselves.
On 14 June 1977 oil was introduced at various rates by a continuous-
dosing apparatus as described in Chapter 2. Three different oiling levels
plus a control were used in the experiment. The average levels of hydrocarbons
in each treatment were approximately control, 0.0 mg/1; low oil, 0.03 mg/l;
medium oil, 0.3 mg/1; high oil 3 mg/l. Water temperatures, pH, salinity
and dissolved oxygen were essentially the same for all treatments (Table 1).
Tanks were cleaned periodically and dead animals were counted and removed.
The experimental clams were fed an average of 4-6 hours a day by pumping
diatom-rich water into the tanks from a nearby "upwelling pond" (Nevi
et al., 1976). Feeding began on 9 July and was stopped on 21 November 1977.
Biochemical Assays
Clams were sampled for various biochemical assays by removing randomly
selected petri dishes and washing the clams onto a screen. The clams were
then placed back into the tanks from which they had been removed to depurate
for 24 hours (except clams which were to be assayed for enzymes which were
not allowed to depurate but were homogenized at once). After 24 hours
depuration the clams were shucked, and weighed. They were then lyophilized
for 12-24 hours and weighed again to obtain dry weights. The dried clams were
stored in dessicators at -20°C until assayed.
29
-------
TABLE 1. AVERAGE MONTHLY SALINITIES, TEMPERATURE, pH, DISSOLVED OXYGEN AND
PHYTOPLANKTON SPECIES DENSITIES OF WATER FLOWING INTO EXPOSURE TANKS
Salinity Temp. d©2 Phytoplankton Densities Dominant
Day °/oo °C pH ppm cells/ma Species %
0
32.6
7.0
20,500
Sc3
76
UPD
22
30
31.6
7.7
7,900
Sc
84
UPD
10
60
34.0
7.7
1,300
Sc
43
UPD
49
90
33.7
7.4
900
Ls
57
Cc
26
Sc
13
120
33.0
9.0
3,400
Bf
71
Rs
18
150
33.0
9.1
8.1 8.8 9,700
Bf
91
UPD
6
180
—
8.0
_ - —
-
-
3phytoplankton abbreviations: Sc = Skeletonema costatum; UPD = Unidentified
Pennate Diatoms; Ls - Leptocylindriaus spp.; Cc = Cylintrotaeaa closterium;
Bf = Bacieriosira fvagilis; Rs = Rhizosolenia sp.
bphytoplankton densities of upwelling (feeding) water.
30
-------
Protein, Glycogen, and Total Carbohydrates
All assays were performed separately on individual clams. The lyophilized
clams from each treatment were each soaked in 1.0 ml of cold distilled water
for 15 to 30 minutes and then homogenized at 0° in a 5.0 ml glass-Teflon tissue
homogenizer at 2250 rpm for 25-30 seconds. The homogenate was made up to 4.0
ml with distilled water and stored frozen until assayed.
Protein was assayed by the method of Lowry et al. (1951) directly on the
crude homogenate. Bovine serum albumin (BSA, Sigma Chemical Co.) was used as
the standard.
Glycogen was measured on the same crude homogenate by a modification of
the method of Seifter et at. (1950) using 40% KOH digestion for three hours
at 90-100°. The glycogen was precipitated with KCl-saturated 95% ethanol
according to the procedure recommended by Giese (1967). The resuspended
glycogen was assayed by the anthrone procedure (Spiro, 1966) and expressed as
milligrams of glucose equivalents. Total carbohydrate in the crude homogenate
was assayed by the anthrone procedure using glucose as a standard (Spiro, 1966).
Total Lipid
Total lipid was also determined on individual clams. To the lyophilized
clam was added 100 ul of distilled water on ice. After 15-20 minutes 2.0 ml
of benzene:menthanol (2:1 v/v) were added and the clam was homogenized in a
5.0 ml glass-Teflon tissue homogenizer at 2000 rpm for 30 seconds at 3-4°. The
homogenizer was rinsed with an additional 1.0 ml of benzene:menthanol. The ex-
tract was washed with 1.0 ml of 0.9% NaCl in water and centrifuged to separate
the layers. The upper lipid layer was transferred, and the water phase was
re-extracted with 1.0 ml of benzene. The benzene fraction was added to the
lipid fraction. The lipid extract was washed once more with 1.0 ml 0.9% NaCl
and the water phase was again back-extracted with 1.0 ml of benzene. All
lipid phases were combined in a tared vial. The vials were reweighed to obtain
the total lipid weight.
31
-------
RNA and DNA
RNA was separated from DNA by a modified Schmidt-Thannhauser procedure
suggested by Munro and Fleck (1966). RNA was determined by its absorbance
at 260 nm using calf liver RNA (Sigma Chemical Co.) as a standard. DNA was
extracted in hot (70°) 1.2 N HCIO^ and estimated using diphenylamine according
to the procedure of Giles and Meyers (1965). It was found unnecessary to
correct for turbidity by measuring the absorbance at 700nm. Calf thymus DNA
(Sigma Chemical Co.) served as a standard.
Preparation of the Crude Homogenate for Enzyme Assays
At each time point ten clams from each treatment were screened from the
sand and placed in cold (2-4°), fresh sea water. The clams were shucked and
placed into a 15 ml conical centrifuge tube. Three milliliters of cold
"homogenization buffer" (0.25 M sucrose, lmM EDTA, pH 7.0) were added and the
contents mixed and centrifuged. The supernatant was decanted which removed
most of the unabsorbed oil droplets from the clams. The clams from such
treatment were then homogenized in 10.0 ml of the homogenization buffer on
an ice bath at top speed for two, ten second bursts in a Virtis '45' homogeni-
zer. The homogenate was passed through nylon gauze into a 15.0 ml glass-Teflon
tissue homogenizer. The solution was further homogenized at 1000 rpm for three
strokes. This homogenate was stored frozen at -20° in 2.0 ml aliquots for
enzyme assays.
Na+-K+-ATPase and Mg^"*-ATPase (EL No. 3.6.1.4)
For total ATPase the assay mixture contained in a final volume of 1.0 ml:
50 mM imidazole-HC1 (pH 7.5), 4 mM MgCl 0.5 mM Na EDTA, 20 mM NaCl, 20 mM
\ ~t
KC1, 3 mM ATP, and 0.100 ml of the crude homogenate. To measure the Mg
dependent ATPase, KC1 and NaCl were omitted and ouabain was added at a final
concentration of 2 mM. Na+-K+-ATPase activity was taken as the difference
| |
between the total ATPase and the Mg -ATPase activities. Controls included
a blank without crude homogenate, a phosphate standard, and a blank with
crude homogenate added after the reactions were stopped with trichloroacetic
acid (TCA), Incubations were carried out at 20° for 10 minutes and stopped
32
-------
with 2.0 ml of cold ascorbate:TCA (2.5%: 12.5% w/v). The precipitated protein
was pelleted by centrifugation. Inorganic phosphate was measured in the super-
natant by the method of Baginski et al. (1974). Results were expressed as
nanomoles Pi released/minute/rag protein.
5'-Nucleotidase (EL No. 3.1.3.5)
This assay was run essentially as described by Aronson and Touster (1974)
with the following modifications. A volume of 0.100 ml of crude homogenate
was assayed in 0.500 ml total. The reaction was run at 30° for 15 minutes
and stopped with 1.0 ml of ascorbate:TCA (2.5%:12.5% w/v). Inorganic phos-
phate was measured by the method of Baginski et al. (1974). Results were
expressed as nanomoles Pi released/minute/rag protein.
Phosphodiesterase (EL No. 3.1.4.1)
The substrate used in this assay was Thymidine 5' monophospho-p-nitrophenol
ester (Sigma Chemical Co.) . The enzyme was assayed according to the method of
Aronson and Touster (1974). A total volume of 0.5 ml was used, including 50
pi of the crude homogenate. Assays were run at 30° for 15 minutes. Results
were expressed as changes in absorbance at 400nm/rainute/rag protein (AA^^/min/
mg protein). Protein concentrations in the crude homogenates were assayed by
the method of Lowry et al. (1951). The assays of the enzymes were linear with
respect to protein concentration under the conditions employed.
Physical, Behavioral, and Physiological Assays
Wet and Dry Weights
Wet and dry weights were determined on approximately 30 Macoma balthiaa
from each treatment. Wet weights were obtained from depurated and shucked
clams. Dry tissue weights were measured after lyophilization for 12-24 hours.
Growth and Condition Index
One hundred clams from each treatment (10 clams per petri dish) were
selected to monitor growth and condition. These clams (10.5 mm long, SD = 0.15
mm) were left undisturbed from 14 June to 12 October 1977. The measurements
33
-------
taken at the beginning and the end of the experiment were shell length and dry
tissue weight. Dry weight was obtained by drying the tissue in an oven at
80°C until a constant weight was reached. A condition index relating dry
weight to shell length was calculated by:
Condition Index - D^y Weight |g)—x 100
Shell Length (nun)
Burying Experiment
One hundred clams (6-11 mm in length) from each treatment were used in ex-
periments to determine the relative burrowing rates. The clams were screened
from the sand prior to each experiment and replaced on top of the sand in a
pyrex baking dish (25.3 cm x 15.2 cm x 4.4 cm deep) set 4 cm below the surface
of the water. At timed intervals over the next 24 hours the numbers of unburied
clams were recorded. The same clams were utilized for each burying experiment.
Results were expressed as the percent of the clams which remained unburied after
one hour.
Feeding Rates
For feeding experiments one hundred clams for each treatment ranging in
size from 11-13 mm were divided into groups of ten and put into 9 cm diameter
plastic petri dishes containing 170 grams of silica sand and placed into the
treatment tanks. Each month the clams were starved for 24 hours after which
three dishes were selected from each treatment and placed on racks in 2.5 £
plastic cylinders. The cylinders were filled with 1400 ml of water from the
treatment tanks originally containing the clams. One hundred milliliters of
5 6
fresh sea water containing phytoplankton densities of 1.4 x 10 to 7.5 x 10
cells were then added. The species compositions of the phytoplankton were
similar to those listed in Table 1. Water temperature was maintained at
8-9"C. During the first two monthly observations the water in the cylinders
was mixed by aeration for five minutes per hour. At the end of six hours a
phytoplankton sample was withdrawn for counting. During the last three monthly
observations phytoplankton collection occurred after one hour. Phytoplankton
were counted and identified at 600x utilizing the Utermohl technique (Lund,
et al,, 1958).
34
-------
Respiration
Oxygen uptake by Maaoma balthica was measured in a smaller version of the
modified Scholander respirometer described by Steen and Iverson (1965). The air
volume was 15 ml and the water volume 100 ml which allowed changes in air volume
of 1 |al to be detected. The respirometer readings were stable to ± 2 pi O^/hr
after equilibration.
Two or three Maaoma balthica were used for each respiration rate measure-
ment. Clams were removed from the exposure tanks before feeding, screened from
the sand, returned to the exposure tanks for two hours, and then placed in the
respirometer chambers. Respirometers were allowed to equilibrate for one hour
and then oxygen uptake was measured at half-hourly intervals for three or four
hours. Clam respiration was measured at 10°, which was close to the average
daily exposure temperature. After the final measurement, clams were removed
from their shells, weighed, lyophilized, and reweighed. Respiration rate is
expressed as yl 0^ consumed (STP)/rag dry weight-hour.
Reproduction
Maaoma balthica at day 120 of the experiment were depurated and then
placed in formalin. Ten clams were used from each treatment. The gonads were
cut out and dehydrated in ethanol (Davenport, 1960). The tissues were cleared
in xylene and embedded in paraffin. Sections of 20ym thickness were cut on a
microtome, stained with Ehrlich's acid alum hematoxylin, and examined under a
microscope for any anomalies in the gonad tissue.
Electron Microscopy of Gill Tissue
Gill tissue was removed from two Maaoma balthica from each treatment
after 120 days of oil exposure. Tissues were fixed in 5% gluteraldehyde
buffered in 0.1 M cacodylate, pH 7.3, rinsed in buffer and sent to the Electron
Microscope Laboratory at the University of Alaska, Fairbanks, for further
processing. Post-fixation was in 1% osmium tetroxide in cacodylate. All fix-
itive and buffer solutions were adjusted to 980 railliosmols with sucrose to
approximate as closely as possible the osmoality of the tissues in vivo. Tissues
were dehydrated in alcohol and acetone, embedded in Epon-Araldite, and sectioned
35
-------
with glass or diamond knives. These sections were post-stained with uranyl
acetate and lead citrate and viewed in a JEM 6AS electron microscope.
RESULTS
General
When oiling first began, all of the clams were buried in the petri dishes.
During the first week of exposure, clams in both the high (3.0 mg/1) and the
medium (0.3 mg/1) oiled treatments burrowed to the surface. Approximately 10%
surfaced in each treatment. By day 16 almost all of the clams in the medium
exposure had reburied themselves. The clams in the high oiled treatment,
however, remained at the surface of the sand with more clams surfacing through-
out the experiment. The numbers of unburied clams were monitored for each
treatment at monthly intervals. The results are shown in Figure 1. It can
be seen that 3.0 mg/1 oil causes over 90% of the surviving clams to burrow to
the surface after six months (180 days) of exposure. It was not until day 90
that the 0.3 mg/1 oiled clams began to burrow to the surface in significant
numbers (p < 0.01).
We also noted that clams which were unburied at 3.0 mg/1 oil seemed unable
to orient themselves with respect to the surface of the sand. Clams would
extrude their feet which would probe in various directions, but were unable
to penetrate the sand. Thus the clams were unable to rebury themselves once
they had come to the surface.
During feeding periods the clams in the control and the low oiled (0,03
mg/1) tanks would extend their siphons and begin very active feeding behavior,
both suspension-type and deposit feeding (Brafield and Newell, 1961). The
clams in the medium oil treatment displayed less feeding activity, and those
in the high oil treatment were very sluggish in any type of feeding behavior.
It was rare that any extended siphons were seen. In general, it appeared that
oiling caused a degree of narcosis and/or disorientation in the clams at 3.0
mg/1. These clams remained in this condition throughout the six months of oil
exposure.
36
-------
UJ
O
UJ
EE
3
m
z
D
S
<
-I
o
u.
O
I-
Z
UJ
o
cc
UJ
a
100
50
. 1
y
¦ft sfc
yP" —
]
-/ * a $ 3
% .
30
60
90 120 ISO
DAY OF EXPOSURE TO OIL
180
240
Figure 1» Percentage of unburied clams in oil-exposed seawater. Points marked with an
asterisk. (*) are significantly different from the control values at p < 0.01
(arcsin t-test). (•) control; (o) 0.03 mg/1; (a) 0.3 mg/1; (a) 3.0 mg/1; (+)
end of oiling regime.
-------
Feeding
Experiments performed to determine relative feeding rates of the clams
verified the apparent inactivity of the oiled clams. The results of the
feeding experiments are shown in Table 2. The values for days 30 and 60 were
taken after six hours of feeding. Obviously, this time period was too long
since the clams cleared almost all the algal cells in each treatment. There-
fore, we used one hour feeding periods subsequent to day 60. There is a general
trend of lowered feeding activity with increasing oil dosage. It is likely that
even the one hour time period is too long for an accurate estimate of the rate
of feeding. The differences between treatments are, therefore, probably under-
estimates of the actual situation.
Rates of Burrowing
When the clams were measured for their ability to rebury themselves, it was
found that oil exposure has almost immediate effects on this behavior pattern.
Figure 2 shows the results of a typical experiment at day 90. We picked a time
period of one hour to measure the percentage left unburied. This was based on
experiments such as the one shown in Figure 2. Figure 3 summarizes the results
for all the burying experiments throughout the exposure period. At day 1, the
first day of oil exposure the high oiled clams showed a significant decrease in
their burying rate with respect to the control (p < 0.02, arcsin t-test). This
decrease became even greater as the experiment progressed (p < 0.001 at days
5-180). Claras at 0.3 mg/1 also began to show significant effects by day 5 of
the experiment (p < 0.001), and they also remained consistently slower than the
controls except for the point at day 60. (The point at day 60 was high for the
controls due to a power outage and subsequent cessation of the water flow for
12 hours. Many clams in the control tanks burrowed to the surface at this time,
probably due to decreased dissolved oxygen levels.) Clams at 0.03 mg/1 oil
were also slightly affected with respect to the controls, showing significant
difference in burrowing rates at days 5, 30 and 90 (p < 0.05) (Figure 3).
38
-------
TABLE 2. A COMPARISON OF THE PERCENTAGE OF ALGAL CELLS FILTERED FROM
THE WATER BY MAC014A BALTHICA SUBJECTED TO CHRONIC OIL TREATMENT
Days of
Exposure
to Oil
Initial
Cell Count
(in 500 ml)
%
of Cells Filtered by 30 Macoma
Control
Low
(0.03 mg/1)
Med.
(0.3 mg/1)
(3,
High
. 0 mg/1)
30a
5.2 x
10
98
79
81
67
60a
7.3 x
10
99
99
99
99
90b
0.14 x
10
99
50
14
0
114b
1.6 x
10
86
86
69
69
150b
1.0 x
10
36
44
6.6
2.7
See Table 1 for algal compositions: experiment details are given in Methods.
asix hour feeding study
''one hour feeding study
39
-------
2 4 6
8 1 0 12 14 16 18 20 22 24
HOURS
Burying behavior of Macoma in different
concentrations of oiled seawater for day
90. The number of clams for each treat-
ment were: control (•) 98; 0.03 mg/1 (o)
98; 0.30 mg/1 (*) 99; 3.0 mg/1 (a) 65.
Time at 0 hours denotes the beginning of
the experiment with all the clams lying
flat on the surface of the sand.
40
-------
90
80
70
60
SO
40
30
20
10
0
re
60 90 120 150
DAYS OF EXPOSURE TO Oi L
180
240
Percentage of Macoma which remain unburied after one hour of being placed on the
surface of the sand. Asterisk (*) denotes values which are significantly differc
from the control values at p < 0.01 (except for 3.0 mg/1 at day 0, p < 0.02 and (
rag/1 for day 90, p < 0.05). Arcsin t-test was used to evaluate the data. Contrc
(•); 0.03 mg/1 (o); 0.3 rag/1 (<*); 3.0 mg/1 (~); end of oiling regime (!).
-------
Mortalities
Although the major concerns in this experiment were with sublethal effects,
there were significant mortalities in the oiled treatments with respect to the
controls. The total cumulative mortality in the control tanks after six months
was about 3.1% (Figure 4). The low oiled (0.03 mg/1) clams had a similar mor-
tality of 3.2% after the six months of treatment. In the 0.3 mg/1 treatment
mortalities reached 8.0% by day 180 and were significantly different from the
controls by day 120 (p < 0.01, arcsin t-test). The 3.0 mg/1 exposed clams
began to die by day 30 (2.9%, p < 0.001). By day 180 the mortality had reached
81%. In fact there were not enough clams left after day 180 in the 3.0 mg/1
treatment to take a recovery point two months later. The trend in mortality
is approximately in linear proportion to dosage. That is, ten times the oil
concentration gave about a ten-fold mortality rate. ^^59 ^or days = 1.8
mg/1).
Wet and Dry Weights
The initial wet tissue weight for the clams was about 153 mg per clam
(Table 3). Over the course of the next six months this value decreased for
all four treatments. The control values dropped to 118 mg/clam by day 180.
The clams at 0.03 mg/1 showed little change in wet weight until day 150 when
a large decrease took place. The clams at 0.3 mg/1 oil had values similar to
the controls up to day 120 after which the treated clams showed a greater loss
in wet weight. The most marked decrease was in the 3.0 mg/1 treatment. The
wet weight began to decrease immediately. By day 180 the clams averaged only
99 mg wet weight each.
When the dry weights for the lyophilized tissues are compared, again
decreases are shown over the six month period (Table 3). Here again the 3.0
mg/1 and the 0.3 mg/1 treated clams show a greater decrease in weight compared
to the controls and the 0.03 mg/1 treated clams. When the dry weights are
divided by the wet weights to correct for size differences, a clear pattern
emerges. The controls and 0.03 mg/1 clams maintain their dry/wet ratio at
around 17-18% throughout the exposure period. The clams at 0.3 mg/1 initially
have higher values, but by day 120 these values are significantly lower than
42
-------
80
60
to
iu
h-
cc
o
s
»-
z
UJ
o
oc
yj
30
a.
20
0*
90 120 ISO
DAYS OF EXPOSURE TO OIL
240
180
Figure 4.
Cumulative mortalities of Macoma for the various oil treatments during six
months of exposure. Control (•); 0.03 mg/1 (o); 0.3 mg/1 (a); 3.0 mg/1 (~);
end of oiling regime ( + ) .
-------
TABLE 3. AVERAGE VALUES (± S.D.) FOR WET AND DRY TISSUE WEIGHTS OF
CLAMS EXPOSED TO CONTINUOUS LOW-LEVEL OIL
Day of Exposure to Oil
240
Treatment 0 5 16 30 60 90 120 150 180 (recovery)
0.0 ppm wet 153±32 153±46 151±41 151±40 148±39 131±36 145+39 129+34 118±25 120±37
dry 27.1±6.1 28.8±9.5 28.1±8.0 26.9±7.3 26.2±7.4 22.5±6.5 25.9±7.3 22.1±4.7 20.0±5.7 20.9±6.1
dry (%) 17.7±1.1 18.6+1.2 18.2±1.0 17.8±1.0 17.7±1.1 17.2±1.3 17.9±1.6 17.4±1.9 16.9±1.9 17.6±1.6
N 30 30 19 30 30 30 30 30 30 30
0.03 ppm wet - 148±36 147±38 147±37 146±36 145±48 150±40 145±33 128±42 116±35
dry - 27.2±7.0 27.6±7.0 27.016.9 25.9±6.5 24.9±7.9 25.7±7.3 24.9±6.2 21.0±6.6 20.3±5.8
dry (%) - 18.4±1.2 18.9±1.2 18.311.1 17.8±1.1 17.3+1.2 17.1±1.7 17.1±1.9 16.4±1.6 17.6±1.7
N 30 30 30 30 30 30 30 30 30
c
0.30 ppm wet - 153±40 155±44 147±47 149±40 137±42 136±51 112±34 105±28 96±30
dry - 28.8+8.0 30.2±8.2 27.4+8.6 26.0±6.9 23.117.0 23.2+8.8 18.0+4.4° 16.7±4.0 16.7±4.4C
dry (%) - 18.7±1.1 19.1±l.lc 18.4H.3 17.411.1 16.9±1.3 16.9±1.7 16.2+1.9 16.0+1.5 17.7+1.9
N 30 30 30 30 30 30 29 28 30
3.0 ppm wet - 150129 140138 139+32 112+36° 108+29b 112+40° 97+38° 99145
dry - 27.916.9 26.317.3 24.315.8 18.0l6.0c 17.5+5.6° 15.415.8° 13.6+6.3° 13.416.7°
dry (%) - 18.6H.5 18.9+1.3 17.5H.0 16.1+1.4° 16.1+1.7 14.0+2.8° 14.2±3.6C 13.3ll.5C
N 30 30 30 27 18 22 15 12
day 0 values were assayed before the oil was added
percent of the wet weight.
^significant difference with respect to the control
c
significant difference with respect to the control
to the tanks. The values for dry (%) are expressed as
at p < 0.05.
at p < 0.01.
-------
in the control (Table 3). The clams in the 3.0 mg/1 treatment have signifi-
cantly lower values for dry to wet weight ratios than the controls for most
of the points measured. These trends can be visualized by referring to Figure
5.
Growth
Lengths of Maooma balthica shells were measured at the beginning of the
experiment and after exposure to four months of oiling (Table 4). Although the
experiment took place during a time of probable slow growth (Beukema and deBruin,
1977; Gilbert, 1973), the control group of clams showed an average increase of
0.50 mm in shell length during this period. Clams from the oiled treatments
showed significantly less growth with increases of 0.10 mm, 0.00 mm and 0.01 mm
from the 0.03 mg/1, 0.30 mg/1, and 3.0 mg/1 treatments respectively. Initially
all of the clams for this experiment measured 9.8-11.2 mm in length.
Condition Index
The condition index was calculated for the clams after four months of ex-
posure to oil. This time period was chosen rather than after six months due
to the decrease in algal numbers in the upwelling pond used for feeding the
clams (Table 1). The values in Table 4 show that the condition index for the
control clams was lower than that of the clams exposed to 0.03 mg/1, but was
significantly higher than those for the 0.3 mg/1 and the 3.0 mg/1 clams.
Respiration
Respiration rates of Maooma balthica after 120 and 150 days of oil exposure
are summarized in Table 5. Significant increases in respiration rate were ob-
served in the clams at the 0.3 mg/1 and 3 mg/1 exposure level. The slight
increases in rate observed in clams at the lower exposure level, although not
statistically significant may in fact be real as there were too few determinations
to detect small changes in respiration data.
Gill Sections
Examination of Macoma balthica gill tissue electron micrographs did not
reveal any gross morphological differences between oil treatments and controls.
45
-------
¦C*
X
a
LU
s
>
oc
o
DAYS OF EXPOSURE TO OIL
Figure 5. Percent dry weight for Maaoma for points throughout the six month oiling
experiment. Asterisks (*) indicate values significantly different from the
control (see Table 5). Controls (•); 0.03 mg/1 (o); 0.3 mg/1 (a); 3.0 mg/1
(~); end of oiling regime (+).
-------
TABLE 4. GROWTH AND CONDITION INDEX FOR MACOMA BALTHICA
AFTER EXPOSURE TO OILED SEA WATER
Treatment
Day
Mean Length
of Shell
(mm ± SE)
N
Condition
Index
(± SE)
N
Control 0 10.50 ± 0.015 100
Control 120 11.00 ± 0.028 99
0.03 mg/1 120 10.60 ± 0.017b 95
0.30 mg/1 120 10.46 ± 0.094b 98
3.0 mg/1 120 10.51 ± 0.054b 47
0.165 ± 0.0007 100
0.142 ± 0.0012 99
0.149 ± 0.0007b 90
0.138 ± 0.0008b 80
0.109 + 0.0010b 47
acondition index = (§) x iqo
shell length (mm)
bvalues significantly different from controls (day 120) at p < 0.01
(student t-test)
47
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TABLE 5. OXYGEN CONSUMPTION RATES OF MACOMA BALTHICA
AFTER EXPOSURE TO OILED SEA WATER3
Oxygen Consumption
plO^/mg dry wt. hr + SE
Mean Dry Weight/clam
mg ± SE
N
Control
0.406
+
0.027
30.7
+
2.4
6
0.03 mg/1
0.477
±
0.036
30.1
+
2.8
5
0.3 mg/1
0.486
+
0.034b
24.2
+
1.6
5
3.0 mg/1
0.578
±
0.039°
17.1
+
3.9C
6
2 determinations of control and 3.0 mg/1 clams after 120 days oil exposure
4 determinations of control and 3.0 mg/1 clams after 150 days oil exposure
5 determinations of 0.03 mg/1 and 0.3 mg/1 clams after 150 days oil exposure
^values significantly different from controls p < .05
Cvalues significantly different from controls p < .01 (student t-test)
48
-------
Sample size was too small to assess subtle changes that may have taken place.
There may have been some destruction or disorganization of the latero-frontal
cilia cell cytoplasm in animals from the highest exposure level, but it was not
possible to delineate the range of variation in control clams, Maooma balthioa1s
method of feeding (Gilbert, 1977) may have removed most of the oil from the
water before exposure of the gill. If this did indeed occur, then the palps, not
the gills might have been adversely affected. Unfortunately the palps were not
microscopically examined.
Reproduction
Three different stages in the female reproductive cycle of Maooma balthioa
could be recognized from histological examination of gonads removed from indi-
viduals after 120 days of oiling. Females were categorized as mature if the
follicles were completely full of mature ova, spawning or spent if the follicles
were only partially full or completely empty, and resorbing gametes if the ova
were being digested by amoebocytes. The number of females in each category and
the sex ratio are summarized for each treatment in Table 6. Although this was a
small sample, there appears to be some resorption of gametes in all of the oil
treatments, but none in the controls. There were no obvious differences,
between treated males and controls and the sex ratio was close to unity for all
groups.
Protein Content
There was no definite trend for values of protein/dry weight during the ex-
posure period. Control values ranged from 44.4% to 51.3% of the dry weight
(Figure 6a). At day 30 (and day 60 for the 0.3 mg/ml treatment) all the oil-
treated clams had significantly lower values for percent protein compared to
the control, but the clams appeared to recover this deficiency during the re-
mainder of the experiment. The absolute values of the protein for all the
treatments, in mg protein per clam, did decrease in the same fashion as the
corresponding dry weight (Table 7).
49
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TABLE 6. SEX RATIO AND FEMALE REPRODUCTIVE CONDITION OF MACOMA BALTHICA
AFTER 120 DAYS OF EXPOSURE TO OILED SEA WATER
Treatment
Sex Ratio
66
99
Female
Mature
Reproductive
Spawning
Condition
Egg
(# of 99)
Resorption
Total
66 + 99
Control
.86
6
1
0
13
0.03 mg/1
.86
1
4
2
13
0.3 mg/1
1.17
2
2
2
13
3.0 mg/1
.75
3
1
7
50
-------
70
60
S 30
K
o
QC
Q.
20
10
0
30
60 90 1 20 150
DAY OF EXPOSURE TO OIL
180
240
Figure 6a. Protein (percent of dry weight) for Maooma exposed to oiled seawater for six
months. Asterisks (*) indicate values significantly different from the control
(see Tables 7, 9 and 10). Control (*); 0.03 mg/1 (o); 0.3 mg/1 (£>); 3.0 nig/1.
(~); end of oiling regime (+).
-------
U1
ho
90 120 150
DAYS OF EXPOSURE TO OIL
240
Figure 6b. Total carbohydrate (percent of dry weight) for Maaoma exposed to oiled sea-
water for six months. Asterisks (*) indicate values significantly different
from the control (see Tables 7, 9 and 10). Control (•); 0.03 mg/1 (o);
0.3 mg/1 (a); 3.0 mg/1 (~); end of oiling regime (+).
-------
I L
o 30 60 90 120 150 180 240
DAYS OF EXPOSURE TO OIL
6c. Total lipid (percent of dry weight) for Macoma exposed to oiled seawater for
six months. Asterisks (*) indicate values significantly different from the
control (see Tables 7, 9 and 10). Control (•); 0.03 mg/1 (o); 0.3 rag/1 (a);
3.0 rag/1 (u); end of oiling regime (+).
-------
TABLE 7. PROTEIN VALUES (mg/clam and % of dry weight, d.w.) FOR CLAMS EXPOSED TO
OILED SEAWATER FOR SIX MONTHS
Day of exposure to
oil
240
Treatment
0
5
16
30
60
90
120
150
180
(recovery)
Control
mg/clam
11.0±1.8
14.1±3.8
11.9+3.6
12.713.1
11.013.2
12.1+3.0
14.1+4.1
11.6+1.7
10.6+2.9
11.2+3.5
% of d.w.
44.4+7.4
54.0±5.7
43.7±6.9
43.9+2.9
48.7+2.8
50.214.7
50.2+3.0
46.5+3.5
47.513.9
51.313.7
N
10
10
7
10
10
10
10
10
10
10
0.03 mg/1
mg/clam
-
13.4±3.6
12.4±3.4
11.212.3
11.8+2.8
13.4+4.4
12.2+2.7
13.3+2.5
11.7+2.8
10.412.1
% of d.w.
-
51.4±5.1
47.7±3.4
39.0+6.3a
46.1+4.4
48.1+3.3
49.4+1.5
47.112.8
47.2+2.5
47.0+4.2a
N
-
10
10
10
10
10
10
10
10
10
0.30 mg/1
mg/clam
-
13.3±4.2
15.513.5
11.213.5
12.3+4.1
11.9+3.2
13.2+2.9
9.6+2.6
7.7+2.1
8.5+2.1
% of d.w.
-
51. 1±6. 2
48.6+3.1
41.4+1.3a
43.8+4.6a
52.8+2.9
50.3+4.0
48.6+5.3
46.6+2.9
47.2l4.4a
N
-
10
10
10
10
10
10
9
10
10
3.0 mg/1
mg/clam
-
15. 4±2.8
11.0±2.0
10.1+3.2
8.8+2.0
11.0+3.6
9.5+3.6
7.915.5
6.7+2.0
-
% of d.w.
-
52.4±8.2
47.9±5.3
39.8+3.I3
46.713.9
51.214.5
52.4+4.1
49.5+4.2
43.1+1.5
-
N
10
10
10
9
6
6
4
3
All values
are means ±
S.D.
significantly different from the control
at p < 0.05 (student
t-test)
-------
Glycogen and Total Carbohydrate
The values obtained for glycogen showed much scatter. There was no dis-
cernible pattern in the data, and the values appeared atypicallv low compared
to those reported in the literature (Beukeraa and deBruin, 1977). It was deter-
mined that the initial procedure used to extract the glycogen did not suffi-
ciently inactivate breakdown of glycogen to glucose. The homogenate lost 90%
of the reactive glycogen when it was stored at -20° for one week. These values
are reported in Table 8. We relied on the values obtained for total carbohy-
drate as estimates of energy reserves.
The values for total carbohydrate per milligram of dry weight in Figure
6b indicate that the oil-stressed clams were depleting their carbohydrate
reserves more rapidly than either the protein or lipid (Figure 6a,b,c). By day
60 the clams exposed to 3.0 mg/1 had lost half of their carbohydrates as indi-
cated in Table 9. The clams exposed to 0.3 mg/1 also showed a slower, but still
significant, decrease in total carbohydrate content. Again the low-oiled clams
(0.03 mg/1) showed essentially an identical pattern to the control group.
Total Lipid
The third major biochemical constituent of Maaoma balthica the lipid con-
tent, showed considerable scatter (Table 10). The lipid content of the control
clams started out at about 9.2% of the dry weight, rose to a peak at day 90
(12.0%), then fell to 7.0% by day 180 (Figure 6c). The clams from 0.03 mg/1
and 0.3 mg/1 showed essentially the same patterns with significant differences
from the controls at day 30 for both, and at day 90 for the 0.3 mg/1 treatment
(p < 0.05). The lipid pattern for the highest oiled treatment (3.0 mg/1)
generally showed an increased lipid content with respect to the control. There
were highly significant differences between the 3.0 mg/1 oiled clams and the
controls at day 120 (p < 0.005, 15 d.f.) and day 180 (p < 0.01, 12 d.f., student
t-test).
RNA and DNA
RNA values were measured periodically as an estimate of relative ongoing
protein synthesis in the clams. Figure 7 and Table 11 show that the RNA values
55
-------
TABLE 8, GLYCOGEN VALUES FOR MACOMA BALTHICA EXPOSED TO OILED SEA WATER FOR
SIX MONTHS
Day of exposure to oil
240
Treatment 0 5 16 30 60 90 120 150 180 (recovery)
Control
— ^ / _ I _ _
mg/clam
% of d.w.
0.31+0.27 0.35+0.20 0.36±0.20 0.53±0.37 0.35±0.22 0.17+0.09 0.17+0.10 0.16+0.09
1.2+1.2 1.4+0.9 1.4±0.7 1.9+1.4 1.6±0.9 0.67 + 0.26 0.66 + 0.43 0.66±0.38
10
10
10
10
10
10
10
0.21±0.09 0.33+0.29
1.1+0.5 1.4+1.1
9 10
0.03 mg/1
mg/clam
% of d.w.
N
0.39±0.20 0.44±0.50 0.52±0.15 0.24x0.14 0.18±0.09 0.17+0.08 0.24±0.21 0.15+0.11 0.36+0.29
1.5+0.7 1.5±1.1 1.9±0.7 0.94±0,46 0.71±0.50 0.71±0.38 0.80+0.59 0.56±0.32a 1.611.2
, 5 + 0,
10
1.5±1.1
10
1.910.7
10
10
,71±0.50 0.71±0.38 0.80+0.59 0.56+0.32
10 10 10 10
10
0.3 mg/1
mg/clam
% of d.w.
0.34±0.34 0.7210.48 0.44+0.27 0.3310.13 0.10+0.02 0.0810.05 0.13±0.07 0.17+0.09 0.1910.14
1.3±1.4 2.2+1.5 1.6+1.0 1.310.6 0.44+0.07a0.33+0.2ia0.70±0.42 1.0+0.4 1.010.7
10
10
10
10
10
10
10
3.0 mg/1
mg/clam
% of d.w.
N
0.3910.25 0.42+0.26 0.32+0.27 0.14+0.09 0.09+0.03 0.0310.02 0.14+0.15 0.11+0.08
1.3+0.8 1.8+1.0 1.210.9 0.7510.40a0.43+0.08 0.19+0.09a 1.0+1.0 0.76+0.52
10
10
10
All values are means ± S.D.
avalues significantly different from control at p < 0.05 (student t-test).
-------
TABLE 9. TOTAL CARBOHYDRATE IN MACOMA BALTHICA DURING SIX MONTHS OF
OILED SEA WATER
Day of exposure to oil
240
Treatment 0 5 16 30 60 90 120 150 180 (recovery)
Control
rag/clam
% of d.w.
N
3.07±0.99 2.54+0.62 2.56±0.93 2.70±0.84 2.27+0.62 2.60+1.61 2.81+0.74 2.22±0.35 2.19+0.78 1.8411.05
12.113.2 9.93+1.90 9.29+1.44 9.81+3.36 9.75+1.74 10.6±2.23 9.88+2.01 8.94H.36 9.74±2.62 7.92±3.03
10 10 7 10 9 10 9 10 10 10
0.03 mg/1
mg/clam
% of d.w.
N
2.53+0.87 2.71+1.45 2.80+0.35 2.21±0.71 2.77+0.81 2.36±0.61 2.71+0.67 2.1811.12 1.89+0.64
9.66+2.07 10.012.31 9.86+1.73 8.5811.53 10.3+3.25 9.69+1.95 9.64+1.55 8.44+2.78 8.44+2.39
10 10 10 10 10 10 10 10 10
0.3 mg/1
mg/clam
% of d.w.
N
2.54+0.78 3.58+0.75 2.68+1.25 2.47+0.80 1.68±0.56 2.01+0.59 1.45+0.40 1.15+0.62 1.13+0.50
9.78+2.26 10.8+2.15 9.60±2.83 8.98+2.76 7.49+1.83a7.60+1.14a7.33+1.26a6.67±2.2la5.13+2.30a
10
10
10
10
10
10
10
10
3.0 mg/1
mg/clam
% of d.w.
N
2.86+0.89 2.3310.95 2.10+1.32 1.16+0.40 1.39+0.52 1.04+0.52 0.85+0.57 1.01+0.45
9.56±2.35 9.8312.83 7.7912.65 6.1611.62a6.49+1.59a6.07±1.97a6.64+5.25 6.23+1.63
10 10 10 9 6 7 5 3
All values are means + S.D.
3significantly different from the control at p < 0.05 (student t-test)
-------
TABLE 10. TOTAL LIPID IN MACOMA BALTHICA DURING SIX MONTHS EXPOSURE TO
OILED SEA WATER
Day of exposure to oil
240
Treatment
0
5
16
30
60
90
120
150
180 (recovery)
Control
mg/clam
2 .61±0.65
2.51H.27
2.41+0.83
2.5210.85
2.9110.71
2.42+0.74
2.44+0.37
1.78+0.63
1.34+0.46
2.04+0.74
% of d.w.
9.1911.28
8.8912.06
9.31+1.50
9.65+2.04
11.2+2.30
12.112.31
10.211.82
8.81+1.34
6.92+1.75
9.87+1.45
N
10
10
6
10
10
10
10
9
10
10
0.03 mg/1
mg/clam
-
2.65+0.86
3.03+0.71
2.1010.56
2.37+0.81
2.72+1.20
2.7411.00
2.0810.71
1.7110.85
1.6110.49
% of d.w.
-
10.5+1.17
10.911.55
8.1310.93 9.50+1.25
11.011.76
9.3411.66
8.53+1.81
7.7511.76
8.3511.21a
N
-
9
10
10
10
10
10
10
10
10
0.30 mg/1
mg/clam
-
3.08+0.97
2.7410.93
2.3111.41
2.87+0.97
2.5211.47
2.7610.97
1.8710.68
1.16+0.19
1.4210.26
% of d.w.
-
10.2+1.38
10.411.61
7.49+1.74 11.1+1.77
9.7912.15 11.612.14
9.2311.96
6.9811.35
8.9011.73
N
-
10
10
10
10
10
10
10
9
10
3.0 mg/1
mg/clam
-
2.7610.42
2.59+0.83
2.7510.62
2.7010.72
1.91+0.60
1.9610.63 1.13+0.47
1.8510.96
% of d.w.
-
9.6211.33
10.912.03
10.9ll.62
12.612.56
11.511.56
15.4l2.77a9.14+1.44
10.9+2.11a
N
-
10
9
10
9
6
7
5
4
-
All values are expressed as means ± S.D.
values significantly different from control at p < 0.05 (student t-test)
-------
180
160
140
120
ioo'
80
20
10
30
20
10
Hi#)' DNA
Low' DNA
240
DAY OF EXPOSURE TO OIL
. RNA and DNA values for Maaoma during exposure to six months of oiling. Asterisks
(*) indicate values which are significantly different from the controls at p < 0.05
(see Tables 11-13). Note change in scale on y-axis (yg/mg). Control (•); 0.03 rag/1
(o); 0.3 mg/1 (a); 3,0 mg/1 (o); end of oiling regime (+).
-------
TABLE 11. RNA VALUES FOR MACOMA BALTHICA DURING EXPOSURE TO SIX MONTHS OF
OILED SEA WATER
Day of exposure to oil
240
Treatment 0 30 60 120 180 (recovery)
Control
mg RNA/clam 0.96±0.37 0.62±0.29 0.76±0.22 0.52±0.18 0.40±0.10 0.44±0.09
Mg/mg d.w. 33.9±8.0 23.8±5.0 26.7±7.4 21.213.6 23.0±7.2 22.4±5.2
N 10 10 10 10 10 10
0.03 mg/1
mg RNA/clam - 0.67±0.31 0.61±0.23 0.52+0.25 0.35±0.65 0.44+0.20
yg/mg d.w. - 25.6±6.2 23.5±7.0 22.0±6.6 21.4±3.7 22.5±4.3
N 10 10 10 10 10
0.3 mg/1
mg RNA/clam - 0.61±0.25 0.66±0.36 0.39±0.12 0.34±0.11 0.30±0.12
Mg/mg d.w. - 23.717.7 25.3+7.6 22.2+5.2 20.814.2 18.7+4.0
N 10 10 10 9 10
3.0 mg/1
mg RNA/clam - 0.57+0.27 0.3310.09 0.34+0.07 0.18+0.03
yg/mg d.w. - 25.3+7.7 24.815.4 22.716.7 24.6+3.6
N - 10 9 7 4 -
All values are means ± S.D.; d.w. = dry tissue weight of the clams
-------
TABLE 12. VALUES FOR "LOW" DNA IN MCOMA BALTHICA DURING EXPOSURE TO
PRUDHOE BAY CRUDE OIL IN SEA WATER
Day of
exposure to oil
240
Treatment
0
30
60
120
180
(recovery)
Control
yg/clam
381±120
289±80
3971107
293+163
264133
380+92
yg/mg d.w.
12.4+2.2
11.4+2.1
14.6+3.5
13.6+2.8
16.5+2.0
20.213.1
N
6
3
8
3
6
8
0.05 rag/1
Mg/clam
-
277+91
344+35
371+101
271+53
271+122
yg/mg d.w.
-
11.Oil.6
13.1+3.0
16.3+1.7
17.3+4.9
16.1+2.9a
N
-
8
3
4
5
6
0.30 mg/1
yg/clam
-
2971100
371+42
184+116
234154
249+75
Mg/mg d.w.
-
11.511.8
13.7+3.2
10.8+2.2
13.1+l.la
20.412.4
N
-
4
4
4
5
2
3.0 mg/1
yg/clara
-
311±76
225+43
164+74
120+4
-
yg/mg d.w.
-
14.5+3.6
16.4+2.0
10.412.2
16.2+2.4
-
N
6
5
5
4
All values are
means ± S.D.; d.w.
= dry weight
of clam
significantly different from the
control at p
< 0.01 (student
t-test)
-------
TABLE 13. VALUES FOR "HIGH" DNA IN MACOMA BALTHICA EXPOSED TO SIX MONTHS OF
OILED SEA WATER
Day of
exposure to oil
240
Treatment
0
30
60
120
180
(recovery)
Control
Mg/clam
2340±440
2130+650
6570±4500
3570±840
2570+920
4520+140
Mg/mg d.w.
105+34
88+19
164±4 9
142134
123+40
174+17
N
4
6
2
6
4
2
0.03 tng/1
Mg/clam
-
3060±680
2920+710
3290H400
2600+680
4500+1100
Mg/mg d.w.
-
110±5
128±33
136127
151+32
197 + 9
N
-
2
6
6
5
4
0.3 mg/1
Mg/clam
-
2640+1200
30201660
2870+220
233011000
3430+1600
Mg/mg d.w.
-
100±32
136±30
147+18
133+56
204+76
N
-
6
6
5
3
8
3.0 mg/1
a
yg/clam
-
2490±600
2170±260
273012100
a
-
yg/mg d.w.
-
111±14
177±17
153+68
-
-
N
4
4
2
All values are
means ± S.D.; d.w.
= dry tissue
weight of clams
a ,
there were no
'higher' out clams
in the treatment at day 180.
-------
for all the treatments are virtually the same throughout the entire experiment.
There was an initial drop of RNA from 34 yg RNA per mg dry weight to about 24
yg/mg by day 30. After this point the values remained essentially constant.
The relative amount of DNA per mg of dry tissue weight can give an esti-
mate of the relative size and numbers of cells in the clams. If the dry weight
is decreasing, but the DNA content remains the same or increases, the ratio of
DNA to dry weight will increase, indicating that the average cell size is
shrinking. If the total DNA content drops as well as the dry weight, then the
clams are probably reducing the numbers of their cells. The results we ob-
tained were complicated by a bimodal distribution of DNA in the clams (Tables
12 & 13). One group of values ('low' DNA) fell within 10-30 ug DNA/mg and the
other group ('high' DNA) had much higher values ranging from 90 to 120 yg DNA/mg.
Very few values fell in between these two groups. Based on histological studies
made on the clams at day 120, we concluded that the low value DNA clams represent
females and the high values represent males, since we found no evidence of clams
with immature gonads.
Figure 7 shows the plot of 'low' DNA values for each of the three treatments
and the control. Overall there were no differences between the control values
and the values for any of the oiled treatments, indicating relatively little
effect of oil on DNA content in the clams. However, there appears to be a
general trend in all of the treatments to higher values of DNA from day 0 to
day 180. Since all the clams had decreased dry weights by day 180, this trend
indicates that the average cell size of the animals was getting smaller.
Figure 7 also shows the plot for the 'high' values of DNA. The pattern
is similar to that of the 'low' DNA clams with no significant differences between
the controls and any of the oiled treatments.
Enzyme Activities
It has become evident in recent years that many membrane-bound enzymes
require an intact membrane in order to function normally (White et al., 1973).
There is evidence also that hydrophobic molecules which are soluble in the
membrane matrix may disturb the lipid structure (Roubal, 1974). Such distur-
bances could have profound effects on the associated membrane-bound enzymes.
63
-------
We picked four enzymes to study which are known to be associated with the plasma
membrane. If oil did in fact penetrate the plasma membrane of the clams, we ex-
pected to see alterations in the specific activities of some of these enzymes.
The four enzymes we assayed were the Na -K -ATPase, Mg -AXPase, 5'- nucleoti-
dase, and phosphodiesterase.
For the Na+-K+-ATPase we saw no effect of oil treatment on the specific
activities (Figure 8a). Two enzymes were noticeably affected by at least one
++
of the oil treatments. The Mg -ATPase in the control clams (Figure 8a) showed
a gradual increase in activity throughout the duration of the experiment. This
increase was inhibited by the oil treatment. Linear regressions were fitted to
the data points and an analysis of covariance was calculated for the treated
clams with respect to the controls. The 0.03 mg/1 treatment showed no signifi-
cant effect of oil treatment, but the 0.3 mg/1 and the 3.0 mg/1 treatments had
slopes significantly different from the control with p < 0.05 and p < 0.01 respec-
tively. This corresponds to an inhibition by day 180 of about 24% at 0.3 rag/1
and 41% at 3.0 mg/1 oil.
The level of activity of phosphodiesterase in the control clams remained
more or less constant during the experiment (Figure 8b). Oiling the clams
caused an increase in specific activity that was significantly different from
the control for the 3.0 mg/1 treatment (p < 0.01). The activity of the phos-
phodiesterase was increased about 55% with exposure to 3.0 mg/1 oil.
Because the specific activities of the 51 nucleotidase showed no correla-
tion with respect to day of exposure (Figure 8c), the values for each treatment
were pooled, averaged, and the means tested for significance against the controls
using a two-tailed t-test. For days 5 through 90 the 3.0 mg/1 treatment showed
significantly higher values at p < 0.02 (d.f. = 8). For days 5-180 the 0.03 and
0.3 mg/1 treatments had significantly higher values than from the controls at p
< 0.05 (d.f. = 15). By day 180 all of the values from all of the treatments were
identical.
Recovery Period
After 180 days of oiling, the clams were allowed to remain in the tanks with
fresh flowing sea water. The same parameters were measured after two months of
64
-------
70
Na+ - K * - ATPase
DAYS OF EXPOSURE TO OIL
Figure 8a. Enzyme (ATPases) activities in Macoma as a function of six months of oiling.
Assays were performed on pooled samples of ten clams each. For experimental
details see Methods section. Control (•); 0.03 mg/1 (o); 0.3 mg/1 (a);
3.0 mg/1 (D); end of oiling regime (+).
-------
o>
90 120 150
DAYS OF EXPOSURE TO OIL
240
Figure 8b. Enzyme (5'-Nucleotidase) activities in Maaoma as a function of six months of
oiling. Assays were performed on pooled samples of ten clams each. For
experimental details see Methods section. Control (•); 0.03 mg/1 (o); 0.3
rag/1 (£»); 3.0 mg/l. ("); end of oiling regime ( + ) .
-------
*-4
cn
b
i
c
I
<3
90 120 150
DAYS OF EXPOSURE TO OIL
240
Figure 8c. Enzyme (Phosphodiesterase) activities in Maooma as a function of six months
of oiling. Assays were performed on pooled samples of ten clams each. For
experimental details see Methods section. Control (•); 0.03 mg/1 (o); 0.3
wg/1 (A); 3.0 mg/1 (~); end of oiling regime (+).
-------
recovery. This is noted as day 240 in the figures and tables. There were
not enough clams remaining in the 3.0 mg/1 treatment after day 180 to assay,
and so the recovery point was taken only for the control and the two lower
oil concentrations with the exception of the burying experiment.
In general, the clams showed only partial recovery after two months in
fresh sea water. The rate of burrowing of the 3.0 mg/1 treated clams increased
significantly, but the clams at 0.3 mg/1 remained at the day 180 level (Figure
3). Mortalities also continued to increase in the 0.3 mg/1 treatment (Figure
4), Wet weights continued to decline for the oil-treated clams (Table 3). The
dry weights, however, leveled off which caused the dry to wet weight ratios to
regain the same value as for the controls (Table 3, Figure 5). The number of
clams unburied, lying on top of the sand also continued to increase for the 0.3
mg/1 treatment (Figure 1).
Protein values dropped significantly from the controls at day 240 for the
oil-treated clams (Figure 6a). Total carbohydrate also continued to decline in
the 0.3 mg/1 oiled clams, remaining significantly lower than the values for the
controls (Figure 6b). Lipid levels showed a significant difference between the
0.03 mg/1 and the control clams (p < 0.05), but no differences between the 0.3
mg/1 and the controls (Figure 6c). KNA and DNA values were not significantly
different (Figure 7), except for the 0,03 mg/1 treatment where there is a sig-
nificant decrease in the relative amount of 'low' DNA (p < 0.05).
The phosophodiesterase activity of the 0.3 mg/1 oiled clams appeared to
| |
remain higher than the control value (Figure 8b). Likewise the Hg -ATPaae
activities, although increased, were still below the value for the controls
(Figure 8a).
DISCUSSION
The results of exposing Maaoma halthioa to six months of Prudhoe Bay crude
oil in seawater are summarized in Table 14. Before we can compare the effects
of the oiling treatment on the clams, it is important to establish that the
clams in the control treatment were not in turn subjected to an unusual stress
in the laboratory. If one uses mortalities as an index of clam stress, it
would seem that with only 3% mortalities over six months, the clams were not
68
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TABLE 14. SUMMARY OF THE EFFECTS OF SIX MONTHS OF OILED SEAWATER ON
VARIOUS PARAMETERS OF MACOMA BALTHICA
Level of
oil in exposure
tanks
Parameters measured
0.03 mg/1
0.30 mg/1
3.0 mg/1
1.
Behavior
general activity
normal3
decreased
decreased
burying rate
normal
decreased
decreased
% unburied
normal
increased
increased
feeding rate
normal
decreased
decreased
2.
Mortalities
normal
increased
increased
3.
Respiration rate
normal
normal?
Increased
4.
Physical
wet weight
normal
normal
decreased
dry weight
normal
decreased
decreased
dry/wet ratio
normal
decreased
decreased
growth
decreased
decreased
decreased
condition index
increased
decreased
decreased
5.
Morphology
gonads
reabsorbed
abnormal
abnormal
gills
6.
Biochemical
protein
normal
normal
normal
carbohydrate
normal
decreased
decreased
lipid
normal
normal
increased
DNA
normal
normal
normal
RNA
normal
normal
normal
Mg^-ATPase
normal
normal
decreased
Na+-K+-ATPase b
normal
normal
normal
5'-nucleotidase
increased?
increased?
increased?
phosphodiesterase
normal
normal
increased
anormal, decreased, increased - are levels relative to those in the controls
^see Results section for discussion of the effects on these parameters
Csee Table 10 and Figure 6C
69
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highly stressed. However, changes in wet and dry weights, condition factor,
and carbohydrate content (in mg/clam) indicate a lack of sufficient energy in-
put for the control clams requiring a reliance on energy reserves.
There are two factors that might have made significant differences between
the controls in the laboratory and normal clams in the field. One is the ab-
sence of tides in our experimental system and this is related to the second
factor: the type of sediment in which the clams were buried. In natural
conditions around the coast of Alaska, Macoma balthioa is found primarily in
intertidal mud flats. The species also seems to be adapted to deposit feeding
as opposed to suspension feeding (Gilbert, 1977). The movement of the tides
brings fresh sediment to the M. balthioa in the mud flats on a periodic basis.
In our experiment the clams were placed in sand, which for the most part, had
a larger grain size than the sediment from the mud flats from which the animals
were taken. Most of the grains in the experiment were larger than those M.
halthica is reported to ingest (Gilbert, 1977). We, therefore, were forcing
the experimental animals to suspension feed on the incoming algae from the up-
welling pond. If M. balthioa is truly adapted primarily for deposit feeding,
and if it therefore takes more energy to feed on suspended material, it is to be
expected that the control clams were under more stress than they would have been
under natural conditions.
The histological sections done on control M. balthioa at day 120 revealed
full guts in most of the animals. Thus they were apparently eating well even
while their dry weights were declining. Beukema and deBruin (1977) described
seasonal variations in physical and chemical composition of Macoma balthioa in
the Dutch Wadden Sea. They discovered that growth takes place mainly during
April through June. For the rest of the year the rate of growth is nearly zero
and the ash-free dry weight falls to 55% of the June value by the end of Decem-
ber. The condition index, therefore, was also found to drop a similar amount
during the same period. The experiment presented here was performed from the
middle of June to the middle of December in 1977. This coincides with the
period of low growth and decreased weight found in normal clams in the Dutch
Wadden Sea. Resurrection Bay at 60°N latitude is obviously a different habitat
than Dutch Wadden Sea at 53°N, but it seems not unlikely to us that the control
clams in our experiment were acting in an essentially normal seasonal pattern.
70
-------
It is obvious from Table 14 that higher oil concentrations cause many
more disturbances than the lower oil concentrations. The clams exposed to low
oil (0.03 mg/1) showed significant differences from the controls in growth and
condition index. They also showed greater anomalies in the histology of the
gonads. But, otherwise, the low oiled clams appeared to be little different
from the controls during the six months of oiling.
The condition index at day 120 for the low oiled clams was significantly
greater than that for the controls, but the condition indices for medium (0.3
mg/1) and high (3.0 mg/1) oiled clams were lower than controls (Table 4). The
increased condition index at the low oil concentration was due to a decrease in
the rate of shell growth during oil exposure and the same weight as controls
at the end of the exposure period. Shell deposition in M. balthica begins with
the formation of the periostracum composed of tanned protein (Bubel, 1973). The
quinone tanning process hypothesized for periostracum formation is similar to
the process of byssal thread formation in mussels (Hillman, 1961) which has been
shown to be adversely affected by oil exposure (Swedmark et at, , 1973; Dunning
and Major, 1974; and Eisler, 1975). This process appears to be extremely sensi-
tive to petroleum exposure, as out of the many parameters examined in M. balthica,
it was one of only two which showed a significant difference from controls at the
lowest level of oil exposure.
Clams exposed to the higher oiling levels (0.3 mg/1 and 3.0 mg/1) showed
many signs of stress resulting from a negative energy balance (Table 14).
Losses in dry weight, decreases in carbohydrate content, resorption of gametes,
and decreases in shell growth are all indicative of a state of energy utiliza-
tion rather than storage. This condition arose primarily from a decreased food
intake (Table 2), but was compounded by the increased oxygen demand of a higher
metabolic rate (Table 5).
The amount and efficiency of water filtration need to be examined when there
is a decrease in food intake. A noted reduction in apparent feeding behavior of
Macoma balthica upon oil exposure suggests that the volume of water processed
for food was probably reduced. A reduction in filtering efficiency cannot be
ruled out however, as petroleum products have been observed to affect cilia
behavior (Johnson, 1977).
71
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Increased oxygen consumption has been observed in many organisms due to
petroleum exposure, but decreases in oxygen consumption have also been observed
(Johnson, 1977; Stainken, 1978). It is well known that total oxygen consump-
tion is the sum of a basal metabolic rate and a rate due to activity such as
feeding or locomotion. Each of these processes may be affected differently by
oil exposure. Percy (1977) has hypothesized that decreases in oxygen consump-
tion are the result of activity reduction which may mask a general increase in
basal metabolic rate. To support this hypothesis, he has shown that whereas
intact oiled araphipods showed considerable variation in magnitude and direction
of respiration rate change from controls, cell-free homogenates of oil exposed
individuals all had a higher metabolic rate than the corresponding control cell-
free homogenates. The Macoma balthica from the present set of experiments were
not being fed at the time of oxygen consumption measurements, thus the increase
in metabolism after oil exposure probably represents a change in the basal meta-
bolic rate.
Increased oxygen demand could result from enzyme degradation of petroleum
constituents, however Lee et al. (1972) could detect no metabolic products of any
of a number of hydrocarbons accumulated by Mytilus edulis over short periods of
time and, in general, bivalves seem to be lacking in aryl hydrocarbon hydroxylase
(Varanasi and Malins, 1977). As Stainken (1978) has suggested, increased meta-
bolic rate may be the result of oxidative phosphorylation uncoupling. In such a
situation, metabolic rate is dependent only on the amount of substrate available
for oxidation and not on the concentration of ATP, as no ATP is produced. Thus
the animals receive little benefit from energy utilization. If some constituent
or group of constituents of the ingested or absorbed petroleum were acting as
an uncoupler of oxidative phosphorylation, this would be an extremely deleterious
effect of oil exposure and certainly worthy of further investigation.
All of the Macoma balthica experienced a decline in biomass, those exposed
to 0.3 mg oil/1 showing a greater decline than controls on day 150 and those
exposed to 3 mg oil/1 on day 60 (Table 3). The average feeding rate over 120
days of oiling was depressed only slightly at 0.03 mg oil/1, but 51% at 0.3 rag
oil/1 and 61% at 3 mg oil/1 (Table 2). This was coupled with a 20% increase in
metabolic rate at 0.3 mg oil/1 and a 42% increase at 3 mg oil/1 (Table 5).
72
-------
With little energy available for growth or reproduction and a greater expendi-
ture than energy intake, resorption of gametes as an energy reserve can occur,
which appears to have happened in Macoma balthioa from all oil treatments by
day 120 (Table 6). The exhaustion of energy reserves was the probable cause of
the increased mortality of M. balthioa from the 0.3 mg oil/1 and 3 mg oil/1
treatments.
Starvation cannot explain all of the results in the experiment. That expla
nation will not clarify why the oiled clams had reduced burying rates within 24
hours after exposure to the oiled seawater. Nor does it explain why the clams
came to the surface of the sand in the presence of oiled seawater.
The slow burying rate appears to be the result of either a general narcoti-
zation as described by others (Morton and Wu, 1977; Linden, 1976; Percy and
Mullin, 1977), or a disorientation mechanism. The clams in the high oiled (3.0
mg/1) treatment would make efforts to bury themselves by protruding their feet,
but they seemed to have an impaired sense of orientation. It is possible that
along with being narcotisized, causing sluggish movements of the foot, the
statocysts in the clams were affected by the oiling, causing them to be dis-
oriented with respect to the gravitational pull of the earth, thus retarding
the burying rate of the clams. The ability to burrow is of critical importance
to the clams. Macoma balthioa have been reported to surface periodically and
move about over the mud and then rebury themselves. This may be a behavior
pattern performed to find a better site in the mud for feeding (Brafield and
Newell, 1961) or as a result of infection by trematodes (Swennen, 1969; Hulscher
1973). In the course of our studies, we observed trematode infection in some
clams. However, we did not investigate the frequency of this infection or its
relation to other parameters. Whatever the reason, if the clams are inhibited
in their reburial rate, they expose themselves longer to the dangers of preda-
tion plus the chance of being transferred to an area of unsuitable substrate by
the action of the tides.
The same hazards caused by being slow to rebury are also presented to clams
which spend more of their time on the surface. Feder et al. (1976) reported
that oiling M. balthioa caused them to come to the surface of the sediment.
Shaw et al. (1977) found that their tendency to come to the surface after three
73
-------
days of exposure to an oil slick seemed to be a function of sediment depth.
Animals in 3.5 cm of sediment did not surface after the oiling treatment, but
those clams in only 1.0 cm thick sediment did come to the surface in signifi-
cant numbers. The sediment in the petri dishes in the present experiment was
2.5 cm deep and the clams came to the surface again in significant numbers as
shown in Figure 1. However, in order to verify the results of Shaw et al.
(1977), we placed groups of ten clams in large beakers filled with sand to a
depth of about 9 cm. These beakers were placed in the tanks along with the
petri dishes. Although the clams were slower to surface in the beakers, after
150 days of oiling eight of the ten clams in the beaker in the high oiled
treatment had surfaced. This indicates that under conditions of chronic oil
pollution the Maeoma balthiaa will eventually burrow up and out of the sediment.
Further, they will also be slower to rebury themselves again. This behavior
pattern will certainly cause a serious decrease in the population in an oil
polluted area.
It was part of our hypothesis that oiling marine organisms could have
serious consequences on membrane structures due to the fact that both membranes
and oil are hydrophobic. It was our belief that if the oil was able to dissolve
into the membranes of the clam, then enzyme functions which depended on an in-
tact membrane would be impaired. Enzymes such as the Na+-K+-ATPase (sodium
pump) are well known to require associated lipid for proper function (White et
al., 1973). It has also been shown that compounds found in crude oil are able
to enter the membranes of fish (Roubal, 1974).
The work done to date on enzyme systems with respect to oil pollution has
been concentrated in two areas. One area is concerned with aryl hydrocarbon
hydroxylase (AHH) activity in animals after exposure of various marine organisms
to aromatics found in crude oil (e.g. Gruger et al., 1977; Kurelec et al.,
1977). The emphasis in these experiments is on the exposed organism's ability
to degrade aromatic hydrocarbons by mixed function oxidases. To date there have
been no reports for such AHH activity in bivalve molluscs. The other area of
interest has been in screening a large number of enzyme activities from a number
of organisms as a function of either short term exposure to oil (Heitz et al.,
1974) or in vitro exposure of tissue samples (Manwell and Baker, 1967).
74
-------
In this experiment, we have specifically looked at enzyme activities
that are associated with the plasma membranes. Our results, however, do not
support a model of general membrane disruption caused by oiling. Only the
| |
Mg -ATPase activity was significantly reduced by exposure to both 3.0 mg/1
and 0.3 mg/1 oil in seawater. The Na+-K+-ATPase appeared to be unaffected by
oiling, and the 5'-nucleotidase and phosphodiesterase appeared to be activated
slightly by the oiling regime. It is likely that these enzymes occupy differ-
ent sorts of environments in the membranes which are differentially affected by
oiling (Roubal, 1974).
A decrease in the activity of an enzyme caused by oiling could be a result
of direct action on the enzyme or a result of action at another level such as
on synthesis or degradation of the enzyme or even effects on the relative levels
of modifiers of the enzyme. We performed a series of experiments in vitro on
| |
crude homogenates from Macoma balthica which showed that the Mg -ATPase acti-
vity is decreased after crude oil and other hydrocarbons are mixed with the
homogenates (Table 15). This lends support to the idea that the oil can
directly interfere with membrane-bound enzyme activity. More work needs to be
done in this area in order to understand the effect of oiling on membranes in
living systems.
| |
It is unclear to us exactly what the Mg -ATPase activity represents.
Most of the activity (90%) was found to be associated with the membrane fraction
as determined by differential centrifugation. About 50% of this membrane acti-
vity was associated with the mitochondrial pellet (15,000 xg pellet of 1,600
xg supernatant). It is likely that half of the ATPase activity is from mito-
chondrial electron transport, but the rest is associated with the plasma mem-
brane fraction. Since we did not differentiate between membrane fractions in
this experiment, it is unknown whether the decrease in Mg -ATPase activity is
due to mitochondrial or plasma membrane interference by the oil or both. If
the ATPase in the mitochondria were affected, this might be a result of the
effect of oil on membrane permeability causing dysfunction in coupling ionic and
pH membrane potentials to ATP synthesis (Hinkle and McCarty, 1978). It is
possible that such interference would cause uncoupling of oxidative phosphory-
lation leading in turn to an increase in the respiration rate. In fact, the
75
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TABLE 15. IN VITRO EFFECTS OF CRUDE OIL AND COMPONENTS OF CRUDE OIL ON
Mg -ATPase FROM CRUDE HOMOGENATES OF MACOMA EALTHICA
Amount added per ml
Hydrocarbon
of crude homogenate
% inhibition
with
treatment
(in yl/ml)
respect to the
control
h2o
100
(0)
Prudhoe Bay Crude oil
10
20
20
22
Benzene
1
0
10
31
100
50
Toluene
1
0
10
23
100
60
Tetradecane
1
13
10
16
100
38
Crude homogenates from Macoma were prepared arid assayed according to the
procedure in Methods. Test hydrocarbons were added after preparation of
the crude homogenate in the amounts specified in the table. Water was
added to obtain a final volume of 1.1 ml. These solutions were then
vortexed to mix the hydrocarbons with the crude homogenate. From this
100 pi were assayed for Mg -ATPase activity. Controls contained 100 yl
of water per ml of crude homogenate.
76
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oiled clams in this experiment did have an increased rate of oxygen consumption
| j
(Table 5). On the other hand, the Mg -ATPase in the plasma membrane may be
instrumental in supplying the energy needed to generate the ciliary movement
necessary for pumping water through the clam over the labial palps and the
gills. If this ATPase activity were decreased by the oiling, we would expect
a decrease in the filtering rate of the clams. The observed decrease in the
feeding rate of the clams may reflect such a decrease in the pumping rate, al-
though it should be pointed out that there could be a difference between the
rate that water is pumped through the clams and the rate at which particles are
cleared from the incoming water.
Another question raised by this experiment is why there was a decrease in
dry weight relative to wet tissue weight in the oiled clams. It is reasonable
to conclude that as the clams use up their energy reserves, their dry weight
will decrease. The controls displayed such a decrease in dry weight. Yet
their dry to wet weight ratio remained at around 17.5% (Figure 5). The high
oiled (3.0 mg/1) clams, however, lost dry weight faster than wet weight, showing
a ratio of under 14% by day 180. It seems possible that a relative increase in
wet weight to dry weight would indicate that the clams are becoming osmotically
unbalanced. That is, they are becoming hypotonic with respect to the seawater
and by day 180 are acting as though they are in lower salinity than they in fact
are. Thus they have the problem of regaining ionic balance with the surrounding
high salinity seawater. Such response to altered salinities is usually accom-
plished in bivalves by changing the levels of free amino acids in their tissues
(Schoffeniels, 1976; Gainey, 1978). Jeffries (1972) has reported that the hard
clam, Meraenaria mercenaria shows significant changes in free amino acid composi
tion in clams that have come from a chronic hydrocarbon polluted area. Clams
from the polluted area had less free amino acids (on a ymole/dry weight basis)
and different ratios of taurine to glycine than the unpolluted controls. We did
not measure free amino acids in this experiment, but would like to suggest this
as another area for future investigation.
The results reported here have shown the potential deleterious effects of
crude oil on Macoma balthica which had remained in a fairly constant, uniform
environment. The clams were not subjected to any of the sorts of stresses that
normally occur in their natural environment in the mud flats, such as changes
77
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in salinity, temperature, oxygen availability, and wave action. If oiling the
clams under constant conditions causes a general slow-down of activity and
weakening of the clams, then they may not be able to respond to a change which
normally occurs on the mud flats. For example, when the tides recede from the
mud flats in Resurrection Bay, the salinity of the water remaining on the flats
is lowered due to the freshwater run-off. As mentioned above, to compensate
for a change in salinity, bivalves alter the composition and amounts of their
free amino acids in their tissues. If the clams are already in a weakened
state induced by oiling, such natural changes in their environment would prob-
ably lead to a much higher mortality rate than one would have projected based
on the laboratory data reported in this paper. Experiments of this nature, an
artificial stress imposed with natural stresses on biological systems, are just
beginning.
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