South African Journal of Marine Science

ISSN: 0257-7615 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tams19

Accumulation and depuration of petroleum

hydrocarbons by black mussels. 1. Laboratory
exposure trials

R. P. Mason

To cite this article: R. P. Mason (1988) Accumulation and depuration of petroleum hydrocarbons
by black mussels. 1. Laboratory exposure trials, South African Journal of Marine Science, 6:1,
143-153, DOI: 10.2989/025776188784480582

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S. Afr. J. mar. Sci. 6: 143-153
1988 /43

ACCUMULATION AND DEPURATION OF PETROLEUM HYDROCARBONS

BY BLACK MUSSELS. 1. LABORATORY EXPOSURE TRIALS

R. P. MASON'"

Laboratory experiments were conducted to measure the rates of accumulation and subsequent depuration of
hydrocarbons by black mussels after exposure to various concentrations of water-soluble fractions of crude oil.
The initial rate of uptake was related to the concentration in the water, but a maximum level of accumulation
was found after long-term exposure. The rate of depuration was closely related to the time of exposure prior to
depuration. The depuration profile consists of an initial rapid loss followed by a slower loss of the remaining
hydrocarbons. Increased exposure time decreases the relative quantity of hydrocarbons lost in the initial rapid
depuration. The results indicate that the use of mussels as petroleum hydrocarbon input "monitors" might have
limitations.

Laboratoriumeksperimente is gedoen om die tempo's van ophoping en daaropvolgende suiwering van

koolwaterstowwe deur swartmossels te bepaal na blootstelling aan verskeie konsentrasies wateroplosbare
fraksies van ru-olie. Die aanvanklike opnametempo het verband gehou met die konsentrasie in die water, maar
'n maksimum ophopingsvlak is na langdurige blootstelling bereik. Die suiweringstempo het in noue verband
gestaan met die duur van blootstelling voor suiwering. Die suiweringsprofiel bestaan uit 'n aanvanklike snelle
verlies, gevolg deur stadiger verlies van die oorblywende koolwaterstowwe. Langer blootstelling verminder die
relatiewe hoeveelheid koolwaterstof wat met die aanvanklike snelle suiwering van ontslae geraak word. Die
resultate dui daarop dat die gebruik van mussels as "monitors" van die inset van petroleumkoolwaterstowwe
miskien sy beperkings het.

The presence of petroleum hydrocarbons in tissues
of marine organisms has been used as an indication of
petroleum hydrocarbon input into the marine en-
vironment. Bivalve molluscs are generally accepted
as one of the most suitable organisms for monitoring
such pollutants because of their sedentary nature,
their ability to concentrate pollutants in their tissues,
their world-wide distribution and abundance, their
robustness and their suitability for routine chemical
analysis (Lee 1977, Tripp and Farrington 1984).
Bivalves have been widely used in monitoring pro-
grammes (Goldberg et al. 1978, Farrington et al.
1982b, Law and Andrulewicz 1983, Risebrough et al.
1983, Broman and Ganning 1985, Friocourt et aJ.
1985) - the so-called "mussel watch" programmes.
They have also been used to monitor the effects of oil
spills (Grahl-Nielsen et al. 1978, Boehm et aJ. 1982,
Farrington et aJ. 1982a, Teal and Howarth 1984).
However, in order to interpret successfully the
information obtained in the field during such pro-
grammes, the mechanisms of uptake, accumulation
and loss of petroleum products by bivalves must be
clearly understood. To date, laboratory and field
studies of the rate of uptake and depuration of
petroleum hydrocarbons by bivalves have produced
apparently conflicting results.
Research has indicated that the length and type of
exposure have a marked effect on the ability of
bivalves to depurate accumulated hydrocarbons.
Short-term laboratory trials (Lee et aJ. 1972, Stege-
man and Teal 1973, Lee 1977, Stainken 1977) and
short-term field exposures from oil spills (Grahl-
Nielsen et al. 1978, Boehm et aJ. 1982, Farrington et
ol. 1982a) have shown that, under these conditions,
the majority of the accumulated hydrocarbons are
rapidly lost. In contrast, bivalves exposed to chronic
levels for extended periods do not always readily lose
their accumulated burden (Boehm and Quinn 1977,
Lee op. cit.). In addition, there seems to be some
disagreement about the rate of depuration of hydro-
carbons by mussels after extended laboratory ex-
posures (Lee op. cit.).
The objective of the research reported on here was
first to try to clarify the relationship between the
exposure period prior to depuration and the depura-
tion rate, and second to investigate the relationship
between the water concentration and the rate of
accumulation of hydrocarbons. Long-term and short-
term exposures of the black mussel Mytilus gallo-
.provinciaJis to water-soluble fractions of Qatar light
'Crude oil were undertaken in the laboratory, and
:subsequent depuration rates were compared.

* Sea Fisheries Research Institute, Private Bag X2, Rogge Bay 8012, South Africa
Present Address: University of Connecticut, Dept. of Marine Science, Avery Point, Groton, CT06340, U.S.A.
Manuscript received: June 1986
144 South African Journal of Marine Science 6
Analyses were based on fluorescence. Fluorescence
has been used fairly extensively to monitor oil levels
in various types of marine samples (Keizer and
Gordon 1973, Gordon and Keizer 1974, Law and
Andrulewicz 1983, Friocourt et al. 1985, Kerley et a/.
1985, Jackson and Bidleman in press). The analytical
technique used was an adaptation of that used by
Jackson and Bidleman (op. cit.).
MATERIALS AND METHODS
The laboratory exposure system
Specimens of black mussel Mytilus ga//oprovin-
cialis were collected from a beach in Cape Town.
Mussels were cleaned and placed in glass-fibre tanks
in a flowing seawater aquarium (Chapman et al.
1984).
CONTINUOUS-EXPOSURE EXPERIMENTS
A water-soluble fraction (WSF) of Qatar light
crude oil was continuously added to the glass-fibre
tanks containing the mussels. In a series of experi-
ments, mussels were exposed to WSFs of 0,3-3 ppm
for exposure periods of 3-7 days. Water concen-
trations were measured daily by fluorescence, and the
system was adjusted to compensate for any change.
The variation in concentration during any given
exposure experiment was less than 20 per cent. On
completion of the exposure experiments, mussels
were tranferred to clean flowing seawater and
depurated for up to two weeks. A sample of the
mussels was taken daily during exposure and less
frequently during depuration. Before being frozen,
the mussels were placed in clean flowing seawater for
3 h to enable them to depurate any petroleum
hydrocarbons present in their gut.
BATCH-EXPOSURE EXPERIMENTS
For the long-term experiments of 1-3 months'
exposure, it was not practicable to use the same
system. Instead, mussels were kept in a glass-fibre
tank containing 50 f of seawater. The water was
stirred continuously to simulate the flowing con-
ditions of the short-term experiments. A seawater
solution of the WSF of Qatar light crude oil (5 t),
freshly prepared according to the method of Anderson
et al. (1974), was added every 12 h. In the presence of
mussels, the concentration decreased rapidly, 50 per
cent of the oil components remaining after 4 hand,
1988
for this reason, the tanks were dosed every 12 h rather
than daily. In the absence of mussels the concentra-
tion decreased slowly, less than IO per cent of the
components being lost after 4 h. Every day the
seawater was changed and the tanks were thoroughly
cleaned.
The average water concentration in the tank, after
addition of the WSF, was 0,15 ppm. As the concen-
tration of the WSF was found to vary between
batches by less than 10 per cent, the water concentra-
tion in the tank was measured only every two days.
An "average" exposure concentration of 0,065 ppm
was calculated for each 12-h period, assuming an
exponential decay in concentration.
Mussels were periodically sampled during the 95-
day exposure period. After 46 days' exposure and
after 95 days' exposure, mussels were removed from
the exposure tank and depurated in clean flowing
seawater. Depuration was monitored for 2-3 months
in each case. Prior to freezing, mussels were again
placed in clean flowing seawater for 3 h.
In addition, a series of short-term batch-exposure
experiments was run at "average" exposure concen-
trations of 0,06,0,045 and 0,02 ppm (corresponding
to batch additions of 0,6, 0,3 and 0,1 ppm). The
exposure period was 19 days. During these experi-
ments, depuration was not monitored.
For all experiments, control tanks were maintained
under identical conditions to those used for exposure
and for depuration, but with clean seawater. All
samples were wrapped in aluminium foil and stored
frozen until analysed.
Analytical techniques
WATER SAMPLES
All glassware was pre-rinsed with dichloromethane
'and distilled water, and water samples were collected
in 250-mt glass bottles cleaned in this manner. Water
samples were extracted with two lO-mf aliquots of
dichloromethane within an hour of sampling. The
concentration was measured by fluorescence spec-
troscopy with Qatar crude oil as a standard. The
emis'sion intensity at an excitation wavelength of 280
nm and an emission wavelength of 374 nm was used
for quantification. In some cases, synchronous scans
were also run. The fluorescence techniques used are
discussed in more detail later.
TISSUE DIGESTION
Frozen mussels were shucked and the frozen tissue
1988 Mason: Lab. Exposure of Mussels to Petrol~um Hydrocarbons
removed. For the continuous experiments, samples
consisted of single mussels. Tissue was weighed into a
clean 30-mt centrifuge tube, 15 ml of 2M potassium
hydroxide (KOH) was added, and the samples were
digested overnight at 40-50°C.
Tissue concentrations varied considerably between
single mussels sampled simultaneously during the
short-term exposure experiments. Variance (the stan-
dard deviation as a percentage of the mean) was high
(up to 100 per cent), especially during the initial
stages ofthe accumulation and at the higher exposure
concentrations. There were no discernible trends
according to sex or size, the mussels used in the
experiments varying in size between 5-10 g wet mass
(50-80 mm). For this size range, differences according
to size would be masked by variability between
mussels (Watling 1978). Further, all experiments
were conducted between October and March, when
sex differences are not easily discernible (Orren et al.
1980). Spawning took place before sampling during
the first trial, only within the first day of exposure to 3
ppm and to 1,5 ppm. Therefore, any effects caused by
spawning would not be apparent in the results.
Most studies that have found differences due to
size and sex have used mussels collected from field
sites or mussels that have been exposed for a
reasonable period, e.g. 3 weeks in the experiments of
Watling (1978). In the present experiments, concen-
trations changed so rapidly that differences caused by
different times of "actual" exposure (the time the
mussel is actually filtering) coupled with inherent
variability would mask any of the relationships
discussed above.
It was therefore decided that, for the batch experi-
ments, instead of analysing two or more mussels
singly as was the case in the continuous trials, a
composite sample of 10 mussels would be analysed,
thereby obtaining a more representative average
tissue concentration. The frozen tissue of to mussels
was weighed into a 250-mt flask. An equivalent mass
of 5M KOH was added and the samples were
digested overnight, after which 15 g of digested tissue
was subsampled into a 30-mt centrifuge tube.
The two methods of digestion are comparable. For
the single samples, the ratio of alkali to wet tissue
mass was 3-10 millimoles KOH per gramme wet
tissue (mmol' g-I), whereas the ratio for the composite
samples was 4 mmol·g-1• A review of the literature
showed that ratios of this magnitude are common
(Wise et al. 1980, Awad 1981).
EXTRACTION
Ether (10 mt) was added to the centrifuge tubes,
145
the tubes were vigorously shaken and they were then
centrifuged to obtain an adequate separation of
layers. The ether layer was decanted off and the
samples were re-extracted with a further 10 mt of
ether. The ether extracts were combined.
To enhance the separation of layers, 5 mt of
methanol was added to the composite samples. The
combined ether extracts were washed with clean
seawater to remove any methanol or polar species
co-extracted with the methanol present in the ether
phase. Polar compounds, including methanol, could
seriously affect the column chromatographic step
which follows extraction. Finally, 10 mt of hexane
was added to the combined extracts and the volume
was reduced in a rotary evaporator to approximately
I mt. This replaced the ether as solvent with hexane.
COLUMN CHROMATOGRAPHY
The concentrates were eluted through a chroma-
tographic column to remove biogenic substances. If
desired, separation of the sample into various frac-
tions could also be achieved during this step. The
column used contained 3 g each of alumina and silica
gel, activated overnight at 180°C and then deacti-
vated with 90 p,t of distilled water. Elution was with
15 mt hexane, 10 mt of 30-per-cent dichloromethane
in hexane and finally 10 mt of dichloromethane. The
final fraction was not collected during the short-term
experiments. The three fractions were designated,
respectively, as the aliphatic, aromatic and polar
fractions. Trials with a standard solution containing
alkanes and aromatics revealed 80-100 per cent
recovery of aromatics in the second fraction (fluor-
escence analysis). Qualitative gas chromatography
showed that alkanes eluted in the first fraction and
naphthalene, dimethylnaphthalene, fluorene, phen-
anthrene, anthracene and chrysene in the second.
Benzopyrene was split between the second and third
fractions. Elution of a solution of crude oil revealed
that 84 per cent of the aromatic components eluted in
the second fraction (fluorescence analysis).
The standard calibration curve was constructed
with column-eluted standards, thereby compensating
for any losses during this step of the procedure.
FLUORESCENCE ANALYSIS
Column effluents were collected and the volumes
were noted. All samples were characterized and
quantified by means of fluorescence spectroscopy.
Quantifications were based on the emission intensity
at 374 nm with an excitation wavelength of280 nm,
this being the emission maximum of Qatar crude oil
/46 South African Journal of Marine Science 6
(Jackson and Bidleman in press). Qualitative in-
formation was obtained by running synchronous
scans from an excitation wavelength (EX) of 260-
460 nm and an emission wavelength (EM) of
280-480 nm.
RESULTS AND DISCUSSION
Background levels measured in the control mussels
throughout the experiments were less than 15 fLg' g
wet weighC' (Qatar crude oil equivalents) and averaged
6,7 fLg' g wet weighc' during short-term trials. During
the long-term trials the average value was 11,0 ~g' g
wet weighc'. During the short-term batch exposures,
efforts were made to reduce any background hydro-
carbon input, and the resulting average tissue concen-
tration of the controls was lower (2,8 ppm). These
values are slightly higher than those for mussels taken
from non-urban areas and probably reflect the effects
of small hydrocarbon inputs. Levels measured during
monitoring programmes ranged from 2 to 500 fLg'g
wet weighC for heavily polluted areas such as the
Cape Town harbour (Mason 1987). These values
are similar to those found by others during
monitoring programmes (Farrington et al. 1982b,
Law and Andrulewicz 1983, Broman and Ganning
1985 and others).
Exposure experiments
Exposure of the mussels to 3 ppm WSF resulted in
immediate stress. A large proportion of the mussels
spawned as a result. Mter 6 h of exposure the mussels
were removed and placed in clean seawater. The
concentration in the tissue was found to be 132 fLg' g
wet weight-I. In this instance, mussels were not placed
in clean water prior to sampling and some died after
the exposure.
At an average exposure level of 1,5 ppm, there was
still some spawning within the first six hours of
exposure but to a lesser extent. Hydrocarbons were
rapidly accumulated by the mussels (Table I). Mter
5 h of exposure, levels substantially higher than back-
ground levels were recorded (26,4 ± 31 fLg' g wet
weighcl). At lower levels of exposure, the rate of
accumulation was slower. Levels substantially higher
than background levels (28,2 ± 14 fLg' g wet weighC')
were measured within the first 24 h of exposure. At
the lowest batch-exposure concentration, concen-
trations subtantially higher than the background
were found after seven days (Table I).
1988
The rate of accumulation constant (kJ) was calcu-
lated from a plot of the tissue concentration against
(I-el-k21), where k2 is the rate of depuration and t the
exposure period (in days). The equation used, derived
by Connell and Miller (1984), as cited by Chapman
and Connell (1986), is
Cn = CM.k,/k2 . (I-e-k2/)
where CM is the exposure concentration and CB the
tissue concentration. This equation predicts a rapid
initial increase in concentration and a maximum level
of accumulation (CT) as e-k2/ approaches zero. The
slope of the curve is CM.k,/k2, and from this, kl can
be calculated if k2 is known. From the results of
various experiments made in the laboratory, a value
ofk2 ofO,129'd-1 has been calculated for short-term
exposures. This value was used in the calculations
(Table I, Fig. 1). The values of k, ranged from 42 to
85, d-I for the continuous exposures and from 66 to
90, d-I for the batch exposures. In the latter case, the
equation fits the data remarkably well. The calculated
values of maximum concentration are similar to
those found experimentally at Day 19 of the exposure
trials (Table I). From all the data, an average k, of 68
± 18· d-I was calculated.
Stainken (1977) found, in his exposure trials, that
the concentration in the tissue decreased as the
concentration in the water decreased. Stegeman and
Teal (1973) demonstrated a relationship between
water concentration and the tissue concentration of
oysters after exposure for two days at concentrations
up to 0,45 ppm. However, they found a decrease in
concentration in the tissue after two days of exposure
when the concentration in the water was 0,9 ppm.
The oysters were closed at that level of exposure, thus
accounting for the drop in tissue concentration. In
the present study, mussels remained open even at
exposure levels of 1,5 ppm, although some mussels
closed for extended periods between opening times.
As mentioned previously, such variable actual ex-
posure times could account for the discrepancies in
concentrations in tissue of single mussels calculated
under apparently identical exposure conditions.
There is also evidence of a maximum level of
accumulation (an equilibrium level). After 10 days'
exposure at 0, 15 ppm, mussels had accumulated 52,2
fLg'g wet weighcl. In the following 78 days, tissue
concentrations varied, the overall average value
being 52,8 ± 8,31 fLg' g wet weighcl (Table I).
Stegeman and Teal (1973) also found evidence of a
maximum level of accumulation as well as of a
relationship' between the fat content of oysters and
tissue levels. At an exposure concentration of 0, I
1988 Mason: Lab. Exposure of Mussels to Petroleum Hydrocarbons 147

Table I: Average concentration in mussel tissue after different exposure periods to various concentrations of crude oil and
the corresponding regression data for rate of uptake of hydrocarbons

Exposure time Average concentration

Number of mussels ek2t Regression
time (days) t sampled in tissue 1-
data (*)
(J.'g.g wet weight-I) CB

Continuous exposure 10 /,5 ppm

0 40 6,74 0
0,2 2 26,4 0,025 m = 488 1
1,0 3 79,0 0,132 k, = 42'd-
1,2 2 33,0 0,143 Cor = 488 ppm
2,0 2 165,6 0,227 2p = 0,05
3,0 3 146,0 0,321

Continuous exposure 10 0.7 ppm

0 40 6,74 0
1,0 6 24,5 0,121 m = 296 1
4,1 6 123,5 0,411 k, = 55'd-
5,1 6 144,5 0,482 Cor = 296 ppm
7,1 4 175,8 0,600 2p = 0,001
Continuous exposure 10 0.3 ppm
0 40 6,74 0
0,8 6 16,3 0,098
1,0 6 52,9 0,121
1,9 6 72,5 0,217 m = 198
2,0 7 120,3 0,227 k, = 85'd-1
2,9 7 74,9 0,312 Cor = 198 ppm
3,9 5 70,1 0,395 2p =0,01
4,9 4 130,6 0,469
5,0 5 74,3 0,475
5,8 4 144,3 0,527
Balch exposure 10 0,/ ppm
0 2,79 0
I Composite 3,02 0,121 m = 13,9
4 samples of 9,70 0,403 k, = 90'd l

7 10 mussels 9,29 0,595 CT = 14 ppm

10 in each case 13,62 0,725 2p = 0,001
16 14,21 0,873
19 14,95 0,914
Batch exposure to 0,3 ppm
0 2,79 0
I Composite 4,47 0,121 m = 23,1 1
4 samples of 14,08 0,403 k, = 66'd-
7 IOn mussels 17,92 0,595 CT = 23 ppm
10 in each case 20,37 0,725 2p = 0,001
16 21,01 0,873
19 24,43 0,914

Batch exposure 10 0.6 ppm

0 2,79 0
I Composite 6,67 0,121 m = 33,2 1
4 samples of 19,09 0,403 k, = 71'd-
7 10 mussels 21,57 0,595 CT = 33 ppm
10 in each case 32,99 0,725 2p = 0,001
16 28,17 0,873
19 34,03 0,914

* k2 = rate of depuration (0,129·d-l)

m == the slope of the regression curve (i.e. CM· kd k2)
k, == rate of accumulation
2p == level of significance
CT == maximum accumulated concentration (ppm)
148 South African Journal of Marine Science 6 1988

Table II: Average tissue concentrations found in depurated mussels after short-term exposure to crude oil

Depuration Number of Average concentration Percentage of

period mussels in tissue tn (/lgog-I) accumulated burden
(days) sampled (/lgog wet weight-I) not depurated

Exposure 10 1.5 ppmfor 3 days

o 3 146,0 ± 156,3 4,98 100
3,0 3 56,2 ± 7,9 4,03 38
6,2 2 39,8 ± 25,0 3,68 27
11,0 6 32,8 ± 7,2 3,49 22
Exposure 10 1,5 ppm for 3,8 days
0 6 69,0 ± 60,1 4,23 100
1,1 9 49,4 ± 17,7 3,90 72
2,1 6 42,6 ± 23,5 3,75 62
2,4 6 36,9 ± 25,0 3,61 53
3,0 6 24,7 ± 14,4 3,21 36
3,2 6 18,5 ± 7,0 2,92 27
4,2 5 31,0 ± 21,0 3,43 44
5,2 6 15,6 ± 4,7 2,75 23
8,0 6 15,6 ± 3,6 2,75 23
8,2 6 8,9 ± 2,5 2,19 13
9,2 4 9,5 ± 1,8 2,25 14
10,0 5 20,6 ± 2,2 3,03 30
12,0 4 19,8 ± 4,3 2,99 29

Exposure 10 0,3 ppm for 2,9 days

0 7 74,9 ± 49,5 4,32 100
1,1 2 64,1 ± 2,8 4,16 86
2,1 2 43,7 ± 17,7 3,78 58
2,2 2 51,6 ± 25,5 3,94 69
3,0 2 54,4 ± 18,1 4,00 73
4,2 2 17,0 ± 6,7 2,83 23
6,2 2 31,1 ± 1,5 3,44 42
9,2 2 33,0 ± 28,2 3,52 45
11,0 2 9,75 ± 5,3 2,28 13

Exposure 10 0,3 ppm for 5,8 days

o 4 77 ,2 ± 185 4, 35 I00
2,1 3 66,4 ± 26;5 4,35 86
2,2 2 36,7 ± 6,0 3,60 48
3,0 3 52,6 ± 16,5 3,96 68
3,2 3 45,9 ± 14,5 3,83 59
4,1 2 23,4 ± 11,5 3,15 30
6,2 2 47,1 ± 9,1 3,85 61
7,2 2 24,8 ± 0,6 3,21 32
8,0 2 25,9 ± 2,5 3,21 34

ppm (fuel oil dissolved in ethanol and added con-
tinuously) oysters with a high fat content approached
an equilibrium concentration after 50 days' exposure
(334 Mg'wet weighcl), whereas oysters with low fat
reserves reached a maximum accumulation after 35
days (161 Mg' g wet weighc1). In the present study,
mussels exposed to a batch concentration of 0,15
ppm reached maximum accumulation after 10 days
(53 Mg' g wet weighCI). In the shorter-term experi-
ments, maximum levels of accumulation were not
reached at the higher exposure concentrations. How-
ever, there is evidence that the equilibrium levels were
being approached at the lower concentrations used
during the batch exposures (Table I), because the
concentrations at Day 19 are similar to those expected
from the equation at equilibrium.
The results indicate that the time required to reach
equilibrium depends on the magnitude of the equi-
librium concentration, which depends on the lipid
content and the exposure concentration. The level of
accumulation is maximal after longer-term exposure
and the concentration at this equilibrium is related to
the lipid content of the mussels. Further, an increase
in the exposure concentration leads to an increase in
the initial rate of accumulation and to an increase in
the equilibrium concentration.
1988 Mason: Lab. Exposure of Mussels to Petroleum Hydrocarbons 149

(0)
• (b)
__ 0,6ppm • _ 1.5ppm

0----0 0,3 ppm A----A O.7ppm

30
•....•.• 0,1 ppm 0······0 0,3ppm
•
250

......•
lC
'01
en
:1, •• 150 ,,/0

,/¢/' 0 .. ,... ,..

10
•
o 0

50

0.2 0.4 0,6 0,8 1.0 0,2 0.4 0,6

Fig. 1: Plots of 1-e-k2t against tissue concentration (a) after batch exposure every 12 hours to 0,6, 0,3 and 0,1
ppm and (b) after continuous exposure to 1,5, 0,7 and 0,3 ppm Qatar crude oil water-soluble fraction

Depuration experiments
After exposure for short periods, the mussels
rapidly lost most of their accumulated burden when
placed in clean seawater. After 3,79 days' exposure to
1,5 ppm, mussels lost more than 70 per cent of their
accumulated hydrocarbons within a week of being
placed in clean seawater (Table II). After two weeks,
levels were still substantially higher than background
levels. Depuration after exposure to 1,5 ppm for 3,0
days and to 0,3 ppm for 2,88 and 5,83 days resulted in
similar rates of loss (Table II). Half-lives for depur-
ation were calculated assuming an exponential rate
of depuration (Farrington et af. 1982a and others).
Values ranged from 5 to 6 days (Table Ill) and are
similar to those reported by Lee (1977) for laboratory
exposures of 2-7 days.
After longer exposure, mussels lost their ac-

Table III: Calculated regression data for rates of depuration of hydrocarbons from mussel tissue after various exposure
regimes

Average exposure Exposure Regression Associated Significance

concentration period slope half-life level
(ppm) (days) ( 'd-I) (days) (2p)

Short:term exposures
1,5 3 0,126 5,5 0,1
1,5 3,8 0,121 5,8 0,001
0,3 2,9 0,142 5,0 0,01
0,3 5,8 0,127 5,7 0,025

Long-term exposures
0,15 46 0,0437 15,9 0,001
0,15 95 0,0236 29,4
I 0,02
150 South African Journal of Marine Science 6 1988

80 0,00 I). This finding indicates that exposure period is

& (0) probably a major factor in determining the rate at
60
& which bivalves lose their accumulated hydrocarbons.
& The synchronous scans of the tissue extracts
40
& (Fig. 3) show the changing relative ratios of the low,
&
&
medium and high molecular weight species as indi-
& &
<..? & & cated by the changes in the relative heights of the
Z20
Z && peaks at, respectively, 325, 350 and 410 nm. Initially,
«~ in both the short-term and long-term experiments,
u.J the species of lower molecular weight predominate,
""u.J (b) but as exposure time increases, the relative quantity
<..?
$ of the medium and heavier aromatics increases
Z (Fig, 3). On depuration, the heavier components are
u.J
U
lost more quickly than the lighter compounds. How-
""0..
u.J
6 95-day exposure
'0", ever, the rate of loss decreases as the exposure period
" increases, as found for the overall concentration.
4 46-day''' Different rates of depuration for different compounds
exposure - 0
have been found after a fuel oil spill (Farrington et al.
2 1982b), probably related to differing solubilities in
the tissues and to the molecular weight and con-
figurations of the individual species.
o 4 8 ~ ~ 20 ~ ~ n ~ The results of Farrington et al. (1982a) for short-
DEPURATION TIME (days)
term field exposure and of Boehm and Quinn (1977)
Fig. 2: Rate of depuration of hydrocarbons by mussels after and others reported by Lee (1977) tend to support the
exposure to (a) 1,5 ppm for 3,79 days and (b) 0,15 observations just made. Farrington et al. (l982b)
ppm for 46 and 95 days found an initial rapid release of accumulated hydro-
carbons after a small oil spill, half-lives for individual
compounds varying from 0 to 4 days. In contrast,
cumulated burden much more slowly (Table IV, Boehm and Quinn (op. cit.) found little depuration
Fig. 2). Half-lives for depuration (Table III) show by mussels taken from a chronically polluted field
that the rate of loss is much slower after long-term site. Tissue levels decreased from 42 to 29 J.Lg' g-I in
exposure, even though the exposure concentration 120 days. Similar results were obtained by Di Salvo et
and the accumulated levels in the tissue were sub- al. (1975), who recorded slow release from chroni-
stantially smaller. A significant relationship (Student's cally polluted mussels from San Francisco Bay with
t test) was found between the half-life for depuration depuration half-lives of 48-60 days.
(Table III) and the exposure period (significance level Stegeman and Teal (1973) found an initial rapid

Table IV: Average tissue concentrations found in mussels depurated after long-term exposure

Depuration Number of Average concentration Percentage of

period mussels in tissue in (J.lg.g-I) accumulated
(days) sampled (J.lg'g wet weighc1) burden not depurated

Exposure: 0,/5 ppm/or 46 days

0 52,8 3,97 100
7 Composite 37,4 3,62 71
14 sample of 29,1 3,37 55
28 10 mussels 15,3 2,73 30
49 in each 14,2 2,65 27
84 case 10,9 2,39 21

Exposure: 0,/5 ppm/or 95 days

0 Composite 52,8 3,97 100
6 sample of 32,8 3,49 62
21 10 mussels 29,1 3,37 55
35 in each 24,6 3,20 47
52 case 12,9 2,56 24
1988 Mason: Lab. Exposure of Mussels to Petroleum Hydrocarbons 151

ACCUMULATION tration for at least a month before depuration.

Therefore, it may well be that the period of exposure
Days Days
after bivalves have reached the equilibrium level is a
major factor in determining the rate of release of
10 0,21
hydrocarbons, because the relative quantity of the
heavier components increased even after the mussels
had reached their equilibrium level. Therefore, the
exposure period seems to influence the type of
component accumulated in the tissues, an influence
which, in turn, effects the overall rate of depuration.
Stegeman and Teal (1973), Farrington et al. (1982b)
and others have proposed a "multiple compartment
model" whereby some of the accumulated hydro-
carbons are rapidly released before a much slower
release of those remaining. There seems to be some
evidence from their work and the results of the
present study that the proportion of hydrocarbons
"rapidly lost" to those depurated more slowly is
related to the exposure period. In other words,
chronically polluted bivalves lose their burden more
slowly because the hydrocarbons have been accumu-
DEPURATION lated into "stable compartments" and are not avail-
able for rapid depuration. However, in short-term
Days Days exposure situations, the majority of the hydrocarbons
7 3 are readily depurated because they have not been
strongly accumulated into the tissues.
Most authors, as stated previously, have found a
differential rate of depuration with time. Further
manipulation of the data gathered for depuration
after 46 days' exposure to 0,15 ppm revealed that the
actual depuration curve could be broken down into
two exponential curves superimposed on each other
(Table Y, Fig. 4). The rate of loss was "fast" during
the first 28 days and "slower" thereafter. This finding
further points to two different depuration processes.
Insufficient data were available to obtain a similar set
Control Control of equations for any of the other depuration experi-
ments.
480 380 280 480 380 280 Both the site of accumulation and the rate of
WAVELENGTH (mm) accumulation of a particular hydrocarbon have an
effect on the rate of depuration. Heavier species are
Fig. 3: Synchronous scans of emission intensity of tissue
accumulated more slowly, and the rate of accumula-
extracts made during uptake and depuration. Ex- tion is probably related to the molecular weight,
posure to (a) 0,15 ppm for95 days and (b) 1,5ppm for' configuration and type of components. Further,
3 days these factors will also effect the depuration rate.
Solubility of components in seawater and in the
tissue lipid will effect the rate at which components
decrease followed by a slower loss after 50 days of are taken up and released. Heavy aromatics (e.g.
exposure, the initial rate ofloss with a half-life offive benzopyrene) are relatively insoluble in both seawater
days. However, that result tends to disagree with the and organic solvents, and therefore it would be
results of the present study and the other studies just expected that these compounds would be relatively
mentioned. The oysters in Stegeman and Teal's study insoluble in tissue lipids. Such a theory may account
had not yet reached equilibrium, although the rate of for the quicker loss of the heavier aromatics during
accumulation had decreased. In the present study, depuration, whereas 2-4 ring aromatics, which are
mussels were maintained at their equilibrium concen- most soluble, are less rapidly lost.
152 South African Journal of Marine Science 6 1988

4.0

3.5
....
·e. e Actual data
·· .. pverall role

3.0

UJ
:::> 2.5
- - --
-'
.
..
-- - - - - - -- - -- - - - ---
Vl
Vl
;::
---e
~
Ol 2.0
0>
::l..,
0
9
1.5

24 40 56 72
DEPURATION TIME (days)

Fig. 4: Graphic representation of the two calculated equations which fit the experimental data

The results of these laboratory experiments indicate
that the use of bivalves as pollution monitors may
have limitations. Release of chronically accumulated
hydrocarbons is slow and therefore bivalves exposed
for extensive periods would have significant levels of
accumulation even if the exposure was not con-
tinuous, However, bivalves exposed to relatively high
concentrations for short periods on a relatively
infrequent basis could have low tissue concentrations
generally. Depuration of a portion of each sample

Table V: Data obtaine'd from separation of the depuration curve, obtained after 46 days' exposure to 0,15 ppm, into two
superimposed exponential curves

Time Data used to calculate Values calculated from Actual data Values calculated for the
(days) the "slow" curve "slow" curve equation values "fast" curve

0 2,92 3,97 1,05

7 2,88 3,62 0,74
14 2,84 3,37 0,53
28 2,73 2,75 2,73 -0,Q2
49 2,65 2,62 2,65
84 2,39 2,40 2,39

Regression dara
Slope (·d-I) -0,0062 -0,0437 -0,0371
Correlation
coefficient -0,9883 0,9989 0,9939
Half-life (d) 112 15,9 18,9
1988 Mason: Lab. Exposure of Mussels to Petroleum Hydrocarbons 153

would be a worthwhile test, because the rate of GOLDBERG, E. D., BOWEN, V. T., FARRINGTON, J. W.,
depuration would indicate whether the exposure was HARVEY, G., MARTIN, J. H., PARKER, P. L., RISE-
BROUGH, R. W., ROBERTSON, W., SCHNEIDER, E.
extensive or not. If the possibility of short-term and E. GAMBLE ]978 - The mussel watch. Environ.
exposure intermittently is suspected, sampling at Conserv. 5(2): 101-125.
short intervals over a period of time would give a GORDON, D. C. and P. D. KEIZER 1974 - Estimation of
good indication of the input into the environment at a petroleum hydrocarbons in seawater by fluorescence spec-
troscopy: improved sampling and analytical methods.
particular site. Finally, the quantity of petroleum Tech. Rep. Fish. mar. Servo Can. 481: 28 pp.
hydrocarbons found in the tissue of chronically ex- GRAHL-NIELSEN, 0., STAVELAND, J. T. and S. WIL-
posed bivalves would indicate the overall concentra- HELMSEN 1978 - Aromatic hydrocarbons in benthic
tion present in the water column. organisms from coastal areas polluted by Iranian crude oil.
J. Fish. Res. Bd Can. 35: 615-623.
Consideration of these factors may well permit JACKSON, L. F. and T. F. BIDLEMAN (in press) - Fluor-
more exact and relevant interpretation of monitoring escence spectroscopic studies of differential accumulation
and other field data. of aromatic hydrocarbons by Callianassa kraussi. Mar.
environ. Res.
KEIZER, P. D. and D. C. GORDON 1973 - Detection of trace
amounts of oil in seawater by fluorescence spectroscopy.
LITERATURE CITED J. Fish. Res. Bd Can. 30: 1039-1046.
KERLEY, G. I. H., ERASMUS, T. and R. P. MASON 1985
- Effect of moult on crude oil load in a jackass penguin
A WAD, H. 1981 - Comparative studies on analytical methods Spheniscus demersus. Mar. Pollut. Bull. 16(12): 474-476.
for the assessment of petroleum contamination in the LAW, R. and E. ANDRULEWICZ 1983 - Hydrocarbons in
marine environment. 2. Gas chromatographic analyses. water, sediment and mussels from the southern Baltic Sea.
Mar. Chem. 10: 417-480. Mar. Pollut. Bull. 14(8): 289-293.
ANDERSON, J. W., NEFF, J. M., COX, B. A., TATEM, H. E. LEE, R. F. 1977 - Accumulation and turnover of petroleum
and G. M. HIGHTOWER 1974 - Characteristics of hydrocarbons in marine organisms. In Fate and Effects of
dispersions and water-soluble fractions of crude and Petroleum Hydrocarbons in Marine Organisms and Eco-
refined oils and their toxicity to estuarine crustaceans and systems. Wolfe, D. A. (Ed.). New York; Pergamon: 60-70.
fish. Mar. Bioi. 27: 75-88. LEE, R. F., SAUERHEBER, R. and A. A. BENSON 1972 -
BOEHM, P. D., BARAK,J. E., FIEST, D. L. and A. A. ELSKUS Petroleum hydrocarbons: uptake and discharge by the
1982 - A chemical investigation of the transport and fate marine mussel Myilus edu/is. Science. N. Y. 177: 344-346.
of petroleum hydrocarbons in littoral and benthic environ- MASON, R. P. 1987 - A comparison of fluorescence and GC for
ments: the Tsesis oil spill. Mar. environ. Res. 6: 157-188. the determination of petroleum hydrocarbons in mussels.
BOEHM, P. D. and J. G. QUINN 1977 - The persistence of Mar. Pollut. Bull. 18(10): 528-533.
chronically accumulated hydrocarbons in the hard shell ORREN, M. J., EAGLE, G. A., HENNIG, H. F.-K. O. and A.
clam Mercenaria mercenaria. Mar. Bioi. 44: 227-233. GREEN 1980 - Variations in trace metal content of the
BROMAN, D. and B. GANNING 1985 - Bivalve molluscs mussel Choromytilus meridionalis (Kr.) with season and
(Mytilus edu/is and Macoma baltica) for monitoring sex. Mar. Pollut. Bull. II: 253-257.
diffuse oil pollution in a northern Baltic archipelago. RISEBROUGH, R. W., DE LAPPE, B. W., WALKER, W.,
Ambio 14(1): 23-28. SIMONEIT, B. R. T., GRIMALT, J., ALBAIGES, J.,
CHAPMAN, H. F. and D. W. CONNELL 1986 - Uptake and GARCIA REGUEIRO, J. A., BALLESTER I NOLLA,
clearance of diesel alkanes from sediments by the Great A. and M. M. FERNANDEZ 1983 - Application of the
Barrier Reef gastropod Strombus luhuanus. Mar. BioI. mussel watch concept in studies of the distribution of
92(1): 15-19. hydrocarbons in the coastal zone of the Ebro Delta. Mar.
CHAPMAN, P., BLAMIRE, T. J. and R. T. HARDING 1984 Pollut. Bull. 14(5): 18]-187.
- The marine toxicity aquarium of the Sea Fisheries STAINKEN, D. M. 1977 - The accumulation and depuration of
Research Institute, South Africa. Spec. Rep. Sea Fish. Res. No.2 fuel oil by the soft-shell, Mya arenaria. In Fate and
Inst. S. Afr. I: 8 pp. Effects of Petroleum Hydrocarbons in Marine Organisms
DI SALVO, L. H., GUARD, H. E. and L. HUNTER 1975 - and Ecosystems. Wolfe, D. A. (Ed.). New York; Pergamon:
Tissue hydrocarbon burden of mussels as potential monitor 313-322.
of environmental hydrocarbon insult. Environ. Sci. Tech- STEGEMAN, J. J. and J. M. TEAL 1973 - Accumulation,
nol. 9(3): 247-251. release and retention of petroleum hydrocarbons by the
FARRINGTON, J. W., DAVIS, A. c., FREW, N. M. and K. S. oyster Crassoslrea virginica. Mar. Bioi. 22: 37-44.
RABIN 1982a - NO.2 fuel oil compounds in Mytilus TEAL, J. M. and R. W. HOWARTH 1984 - Oil spill studies: a
edu/is: retention and release after an oil spill. Mar. BioI. 66: review of ecological effects. Environ. Mgml 8(1): 27-44.
15-26. TRIPP, B. W. and J. W. FARRINGTON 1984 - Using sentinel
FARRINGTON, J. W., RISEBROUGH, R. W., PARKER, organisms to monitor chemical changes in the coastal zone:
P. L., DAVIS, A. c., DE LAPPE, B., WINTERS, J. K., progress or paralysis. Report submitted to the Ninth
BOATWRIGHT, D. and N. M. FREW 1982b- Hydro- Annual Conference of the Coastal Society, Atlanlic City.
carbons, polychlorinated biphenyls, and DDE in mussels New Jersey: 12 pp.
and oysters from the U.S. coast, 1976-1978 - The mussel W A TUNG, H. R. 1978 - Selected molluscs as monitors of metal
watch. Tech. Rep. Woods Hole Oceanogr. Instn 82-42: pollution in coastal marine environments. Ph.D. thesis,
106 pp. University of Cape Town: viii + 263 pp.
FRIOCOURT, M. P., BODENNEC, G. and F. BERTHOU 1985 WISE, S. A., CHESLER, S. N., GUENTHER, F. R., HERTZ,
- Determination of polyaromatic hydrocarbons in scallops H. S., HILPERT, L. R., MAY, W. E. and R. M. PARRIS
(Pecten maximus) by UV fluorescence and HPLC com- 1980 - Inter-laboratory comparison of determinations of
bined with UV and fluorescence detectors. Bull. environ. trace-level hydrocarbons in mussels. Analyt. Chem. 52( (2):
Contamin. Toxicol. 34: 228-238. 1828-1833.