Aquatic Toxicology 44 (1998) 1 – 15

Bioaccumulation of sediment-associated fluoranthene in benthic

copepods: uptake, elimination and biotransformation

Guilherme R. Lotufo *
Department of Zoology and Physiology, Louisiana State Uni6ersity, Baton Rouge, LA 70083, USA

Received 22 May 1997; received in revised form 12 March 1998; accepted 16 April 1998

Abstract

Most polycyclic aromatic hydrocarbons (PAHs) entering aquatic systems reside in sediments and in the storage
lipids of the benthic biota. Massive amounts of PAHs reach estuarine systems and threaten their ecosystems.
Copepods abound in the estuarine benthos, where they are an important component of food webs. The accumulation
of sediment-associated [14C]fluoranthene was examined in adult females of two species of sediment-dwelling copepods,
Schizopera knabeni and Coullana sp., collected from a Louisiana salt marsh. Accumulation was measured throughout
a short- (24 h) and a long-term (10-day) exposure to concentrations in the sediment ranging from 0 to 1652 nmol (g
dry wt.) − 1. Fluoranthene apparent steady state body residue was reached very rapidly (B12 h) at all concentrations
for both species. Lipid and organic-carbon-normalized bioaccumulation factors (BSAFs) calculated at day 1 were
0.57– 0.80 for S. knabeni and 0.35–0.71 for Coullana sp. Fluoranthene body burden in female Coullana sp. changed
dramatically during their reproductive cycle. A significant fraction of the fluoranthene residue was stored in the
lipid-rich maturing eggs which were extruded into paired egg sacs. For S. knabeni, fluoranthene depuration occurred
at similar rates in sediment and water (half life approximately 4.8 h), while it was faster in sediment than in water
for Coullana sp. (half lives =4.2 and 7.4 h). Fluoranthene was biotransformed to polar compounds in both species.
Metabolites accounted for approximately 12% of the 14C activity in the tissues following a 96-h sediment exposure in
both species. Given that fluoranthene was taken up from spiked sediment very efficiently and reached steady-state
levels in the tissues very rapidly, PAH-contaminated sediments may pose a risk for benthic copepods and their
predators. © 1998 Elsevier Science B.V. All rights reserved.

Keywords: PAH; Fluoranthene; Sediment; Bioaccumulation; Copepod; Lipid content

* Present address: AScI Corporation, US Army Corps of
Engineers, Waterways Experiment Station, CEWES-ES-F,
Vicksburg, MS 39180-6199, USA. Tel.: +1 601 6344103; fax:
+1 601 6343120; e-mail: lotufog@mail.wes.army.mil
1. Introduction
Polycyclic aromatic hydrocarbons (PAHs) oc-
cur in most coastal systems of the world, as well

0166-445X/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved.
PII S0166-445X(98)00072-1
2 G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15
as in freshwater and terrestrial systems. Their
elevated concentration in the aquatic environment
is usually associated with human activities, mostly
via discharge of petroleum and its derivatives and
atmospheric deposition of combustion by-prod-
ucts. Because of their high hydrophobicity, ther-
modynamic equilibrium tends to favor
partitioning of the PAH molecules to more non-
polar environments such as the lipids of an organ-
ism or the organic carbon surrounding a sediment
particle. Thus, most PAHs entering aquatic sys-
tems reside in sediments and bioaccumulate in the
benthos.
Organic contaminants, such as PAHs, are taken
up by benthic organisms by direct adsorption of
freely dissolved chemicals from the overlying and
pore water, and also by direct contact and inges-
tion of sediment particles (Landrum et al., 1992a).
Bioaccumulation of sediment-associated PAHs by
benthic invertebrates has been extensively studied,
both in marine (Meador et al., 1995 and refer-
ences therein; Hellou et al., 1995; Forbes et al.,
1996; Kane Driscoll and McElroy, 1996; Mayer et
al., 1996) and freshwater species (Landrum, 1989;
Landrum et al., 1991, 1992b; Lydy and Landrum,
1993; Clements et al., 1994; Harkey et al.,
1994a,b,c; Kukkonen and Landrum, 1994; Lan-
drum et al., 1994; Kane Driscoll et al., 1997;
Wood et al., 1997). Bioaccumulation of organic
compounds in meiobenthic organisms (benthic
metazoans passing through a 0.5-mm sieve), how-
ever, is limited to a single study that examined
PCB uptake by copepods (Wirth et al., 1994).
Most harpacticoid copepods are benthic and
epibenthic. They are typically the second most
abundant group in the meiobenthos, with impor-
tant ecological roles in aquatic ecosystems (Coull,
1988). Meiobenthic copepods have been success-
fully used as test-organisms during hazard assess-
ments of contaminants, including sediment
toxicity testing (Chandler and Green, 1996). Sedi-
ment-associated PAHs were toxic to harpacticoid
copepods, causing both mortality and sublethal
effects such as impaired reproduction and feeding
(Lotufo, 1997; Lotufo and Fleeger, 1997). The
bioaccumulation of PAHs by copepods has been
investigated only in planktonic species (Corner et
al., 1976; Harris et al., 1977a,b).
Assessment of the toxicity and bioaccumulation
of organic contaminants in small invertebrates has
been greatly facilitated by the use of the isotopic
dilution technique, where radioactivity may be
used to estimate total contaminant concentration
in both the exposure media and in test organisms.
This method has been successfully used for mea-
suring uptake from either water (Harding and
Vass, 1977; Pawlisz and Peters, 1993) or sediment
(Landrum, 1989; Forbes et al., 1996). The pur-
pose of the present study was to investigate the
bioaccumulation of sediment-associated
[14C]fluoranthene, a PAH congener, by two spe-
cies of sediment-dwelling harpacticoid copepods,
Coullana sp. and Schizopera knabeni. The tempo-
ral pattern of fluoranthene accumulation over a
short- (24 h) and a long-term (10-day) exposure
was examined under a range of sediment concen-
trations. Fluoranthene elimination in the presence
and absence of sediment, as well as the potential
for biotransformation were investigated. In addi-
tion, the relative fraction of fluoranthene residue
in somatic and reproductive tissue of mature fe-
male Coullana sp. was examined.
2. Material and methods
2.1. Test organisms
S. knabeni Lang is commonly found in salt-
marshes and is about one tenth the size of Coul-
lana sp. S. knabeni is thought to be a selective
deposit feeder, as has been observed in a related
species (Chandler and Green, 1996). Coullana sp.
is the largest and one of the most abundant
benthic copepod in the Gulf of Mexico coastal
waters (J.W. Fleeger, Louisiana State University,
personal communication). Coullana sp. has been
reported to feed primarily from the water column
(Decho, 1986; Pace and Carman, 1996). S. kn-
abeni was obtained from a mono-specific labora-
tory culture based on stock collected in October
1993 from the surface sediment of a Spartina
alterniflora salt-marsh at Port Fourchon, LA. An-
imals were cultured sediment-free in 500-ml Erlen-
meyer flasks at room temperature with 25‰
artificial seawater (Lotufo and Fleeger, 1997).
 G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15
Copepods were fed twice a week with a mixture of
the diatom Chaetocerous muelleri and Microfeast
Plus Larval Diet® (Provesta, Bartlesville, OK).
Coullana sp. was collected in March 1996 from
the same site. Approximately 200 ovigerous fe-
males were sorted into groups of five individuals
and placed in 50×35 mm crystallizing dishes
containing artificial seawater (ASW) at 25‰. Af-
ter 48 h, adults were removed and nauplii were
pooled in a 150×75-mm crystallizing dish. They
were fed daily with a mixture of Isochrysis gal-
bana and Thallassiosira weissflogii, and two thirds
of the water was renewed weekly. Most copepods
had reached the adult stage after 3 weeks and
were used in all experiments.
For wet-weight determination, copepods from
the stock cultures were sorted into groups of
non-ovigerous females consisting of 100 S. kn-
abeni or ten Coullana sp. Each group was trans-
ferred to pre-weighed 2× 2 cm Nytex mesh
squares (100 mm pore size) over tissue paper. Each
square was weighed on a Sartorius MC5 balance
(Goettingen, Germany) as soon as examination
under a dissecting microscope revealed that all the
water in the mesh interstices had been absorbed
by the tissue paper and only a thin film of water
was covering the copepods. Individual wet weight
(mean 9S.D.) was 3.9790.31 mg for S. knabeni
(n=4) and 25.93 95.87 mg for Coullana sp. (n =
4). For dry-weight determination, copepods were
frozen in liquid nitrogen and rinsed in deionized
water. Non-ovigerous adult females, 50 S. knabeni
or ten Coullana sp., and 300 eggs separated from
ovigerous Coullana sp. females were transferred to
pre-weighed, 12-mm diameter aluminum pans
(Cahn, Madison, WI), in four replicates. Samples
were dried at 55°C for 4 days. After drying, pans
were transferred to a desiccator, cooled to room
temperature and weighed using a Sartorius MC5
balance. Mean individual dry weight was 0.969
0.15 mg for S. knabeni, 6.159 1.3 mg for Coullana
sp., and 0.043 9.007 mg per egg for Coullana sp.
Mean individual weights were used to normalize
fluoranthene tissue concentration in copepod sam-
ples from all experiments. Copepods do not molt
after reaching the adult stage. Mean weight values
are, therefore, accurate surrogates for specific
weights.
3
2.2. Test sediment
Sediment was collected by hand-scooping at
low tide from the top 2 cm of a mudflat in a S.
alterniflora salt marsh near Cocodrie, LA. The
typical total PAH concentration in this sediment
was 0.26 mg g − 1 (Carman et al., 1995). Stock
test-sediment was prepared for spiking following
procedures similar to those recommended in
Chandler and Green (1996). Sediment was passed
through 1-, 0.125- and 0.045-mm sieves. Sediment
passing through all the sieves was left to settle
overnight at 4°C. The supernatant was removed
by aspiration, and the sediment was flash auto-
claved (15 min). A sediment slurry (solids content
approximately 12%; particle size B 45 mm, 100%
silt and clay) was created by homogenizing the
autoclaved sediment with the appropriate volume
of 25‰ ASW. Autoclaving sediment slurries has
been recommended for use in spiked-sediment
bioassays with meiobenthic copepods (Chandler
and Green, 1996). Flash autoclaving did not af-
fect the total fraction of organic carbon of a salt
marsh sediment (G.T. Chandler, unpublished
data), the major factor that controls the
bioavailability of hydrophobic organic contami-
nants. Total solids was determined by oven drying
sediment samples at (80°C). The sediment organic
carbon (SOC) of the resultant slurry, measured in
duplicate on a Perkin Elmer 2400 CHN Elemental
Analyzer (Norwalk, CT), was 1.5% after acidifica-
tion with HCl to remove inorganic carbonate.
Radiolabeled [3-14C]fluoranthene (50.4 mCi
mmol − 1) was purchased from Chemsyn Labora-
tories (Lexana, KS), and non-radiolabeled fluo-
ranthene (98% purity) was purchased from
Aldrich (Milwalkee, WI). The 14C purity was de-
termined to be 99% by thin layer chromatography
(TLC) and liquid scintillation counting (LSC).
TLC was performed on K6 silica gel plates
(Whatman, Clifton, NJ) with hexane:benzene (4:1,
v/v) as the solvent (Landrum, 1982). Fluo-
ranthene was amended to the stock sediment to
create five target concentrations: 0, 25, 100, 250,
690 and 2000 nmol (g dry wt.) − 1. Separate dosing
solutions were prepared for each sediment con-
centration by adding 40 mCi of [14C]fluoranthene
in 190 ml of acetone and the appropriate amount
4 G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15
of a 6.7-mg ml − 1 stock solution of non-radiola-
beled fluoranthene to small vials. The final vol-
ume of each stock solution was adjusted to 360 ml
by adding acetone. The 0 nmol (g dry wt.) − 1
(control) dosing solution consisted of 360 ml of
acetone only, and the 25 nmol (g dry wt.) − 1
dosing solution did not receive any non-radiola-
beled fluoranthene. Before spiking, stored sedi-
ment was homogenized with the overlying water
and one aliquot (300 g wet wt., dry-to-wet-wt.
ratio = 0.12) was transferred to each of five 500
ml beakers and vigorously stirred under vortex.
Stock solutions were added dropwise to each sed-
iment slurry and stirred for 4 h. Duplicate sedi-
ment aliquots (approximately 3 g wet wt.) were
taken from each sediment treatment immediately
following dosing to determine total fluoranthene
sediment concentrations via reverse phase HPLC
(Lotufo and Fleeger, 1996). Dosed sediments were
stored in the dark at 4°C. After settling overnight,
the overlying water was removed by aspiration
and replaced with fresh 25‰ ASW and the sedi-
ment was homogenized. This procedure was re-
peated four times to remove excess solvent
(Landrum et al., 1994). Dosed sediments were
stored for 3 weeks at 4°C in the dark. Before use
in the experiments, the overlying water was re-
placed, the sediment was homogenized, and
aliquots were taken in duplicate for chemical
analysis by high pressure liquid chromatography
(HPLC) and solids content determination. Sedi-
ment aliquots (approximately 100 mg wet wt.)
were also taken in duplicate and transferred to
scintillation vials. Biosafe II liquid scintillation
cocktail (Research Products International, Mount
Prospect, IL) was then added and
[14C]fluoranthene activity was measured by LSC
on a Beckman LS 6000IC Liquid Scintillation
Analyzer (Palo Alto, CA). Samples were corrected
for quench using the external standards ratio
method after subtracting background. Mean val-
ues were used to calculate the specific activity for
each sediment treatment (mCi of radiolabeled fluo-
ranthene per nmol of total fluoranthene). Specific
activities were used for estimating total fluo-
ranthene concentrations in sediment and tissue
samples in all experiments.
2.3. Bioaccumulation experiments
Fluoranthene bioaccumulation was examined in
24-h and 10-day experiments using S. knabeni and
Coullana sp. In the 10-day experiment, copepods
were exposed to sediment treatments in 50× 35
mm crystallizing dishes (Kimble, Toledo, OH)
filled with 25 ml of 25‰ ASW. Spiked sediment
treatments stored for 3 weeks at 4°C were pre-
pared for use in the experiments by replacing the
overlying water and homogenizing. Eight
milliliters of sediment slurry were dispensed to the
bottom of each dish creating a 3–4-mm sediment
layer. Dishes were then placed in random order
inside loosely-covered plastic containers and
stored at 4°C in the dark for 7 days. Soaked paper
towels inside the containers created a humid envi-
ronment to retard evaporation from the experi-
mental dishes. Following the storage period,
dishes were kept overnight at 25°C in a tempera-
ture controled incubator. Copepods were then
added and dishes were returned to the incubator
and kept in the dark. In the S. knabeni experi-
ment, 50 non-ovigerous adult females were added
to each dish. Four replicates per treatment were
sampled after 1, 4 and 10 days. Thus, the number
of dishes per sediment treatment was 12. In the
Coullana sp. experiment, 15 non-ovigerous adult
females were added to each dish. Three replicates
per treatment were sampled at days 1, 4 and 10.
Thus, the number of dishes per sediment treat-
ment was nine. Lower replication was used for
Coullana sp. because a limited number of test-or-
ganisms was available. Copepods were fed after
introduction into the experimental dishes and at
days 3, 6 and 9. Each dish with S. knabeni re-
ceived 1 mg of Microfeast Plus Larval Diet® in
0.5 ml of 25‰ ASW, and dishes with Coullana sp.
received approximately 1× 106 T. weissflogii in
0.65 ml of 25‰ ASW.
At each sampling period, the contents of each
dish were sieved through a 45-mm mesh. The
sediment passing through the sieve was collected
in a 50-ml glass beaker and kept in the dark at
4°C. After 24 h, the overlying water was removed
by aspiration. Aliquots of sediment were taken in
duplicate for dry weight and 14C activity determi-
nation. The material retained in the sieve was
 G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15
examined under a stereo-dissecting microscope.
Live copepods were transferred to a plastic cell-
culture dish containing 25‰ ASW, formalin
killed, and transferred to a new dish with 25‰
ASW. All copepods found dead were in an ad-
vanced stage of decomposition, and were dis-
carded. Fifteen non-ovigerous female S. knabeni
per replicate were transferred to a scintillation
vial. Coullana sp. were sorted into ovigerous and
non-ovigerous females. All non-ovigerous females
from each replicate were transferred to a scintilla-
tion vial. Ovigerous females were separated from
their egg sacs and transferred to a scintillation
vial. Eggs were enumerated and transferred to a
scintillation vial. S. knabeni eggs did not contain
enough tissue to be assayed for radioactivity.
Therefore, only non-ovigerous females of this spe-
cies were analyzed. Female copepods or eggs were
solubilized in 200 ml of TS-2 tissue solubilizer
(Research Products International, Mount
Prospect, IL). After 24 h, 100 ml of 1.2 HCl (to
neutralize the tissue solubilizer), 10 ml of Biosafe
II scintillation cocktail were added, and 14C activ-
ity was assayed by LSC for tissue concentration
determination.
The 24-h experiment was conducted in 28 ×45
mm carrier glass vials (Kimble, Toledo, OH) filled
with 10 ml of 25‰ ASW and 1.5 ml of sediment.
Non-ovigerous adult females (15 for S. knabeni or
three for Coullana sp.) were added to each dish.
Three replicates per treatment were sampled after
3, 6, 12 and 24 h. Copepods were not fed. At each
sampling period, copepods were retrieved and as-
sayed for radioactivity as described above.
2.4. Depuration experiment
For the fluoranthene depuration experiment,
non-ovigerous female copepods (300 S. knabeni or
60 Coullana sp.) were exposed to
[14C]fluoranthene-spiked sediment (25 nmol (g dry
wt.) − 1) in 150 ml beakers filled with 50 ml of 25‰
ASW and 15 ml of sediment for 24 h. After
exposure, copepods were retrieved from the sedi-
ment and divided into groups: 18 groups with 15
individuals each for S. knabeni and nine groups
with three individuals each for Coullana sp. Two
groups of S. knabeni and one group of Coullana
5
sp. were immediately used for radioisotopic analy-
sis. For each species, half of the remaining groups
were individually placed in 28× 45 mm vials con-
taining 10 ml 25‰ ASW, while the other half
were placed in vials containing 10 ml 25‰ ASW
and 1.5 ml of uncontaminated sediment for depu-
ration assessment. Vials were sampled at 2, 4, 8
and 12 h. Duplicate vials per sampling period
were used for S. knabeni. Single units were used
for Coullana sp. due to a shortage of test-organ-
isms. At each sampling period, copepods were
retrieved from the sediment or water and assayed
for 14C activity by LSC to determine tissue
concentration.
2.5. Biotransformation experiment
To examine fluoranthene biotransformation,
copepods (approximately 1500 adult S. knabeni or
60 adult female Coullana sp.) were exposed to
[14C]fluoranthene-spiked sediment (25 nmol (g dry
wt.) − 1) in 150 ml beakers filled with 15 ml of 25‰
ASW and 20 ml of sediment for 96 h. After
exposure, copepods were retrieved from the sedi-
ment, frozen in liquid nitrogen, and analyzed for
biotransformation products using procedures
modified from Landrum (1982). Three replicates
with approximately 500 S. knabeni or 30 Coullana
sp. were lyophilized and sonicated for 20 s in 5 ml
of ethylacetate:acetone (1:4, v/v). The extract was
filtered through a sodium-sulfate column and re-
extracted with 2 ml of hexane. All extracts were
combined and the volume reduced under a stream
of nitrogen to approximately 100 ml. The extracts
were analyzed by TLC using hexane:benzene (4:1)
as solvents. Developed plates were divided into
four sections corresponding to the Rf of fluo-
ranthene and three other regions, including the
origin. The silica gel from each section was
scraped from the TLC plate, and 14C activity was
determined using LSC. The amount of total
metabolite was determined to be the sum of all
non-fluoranthene 14C on the TLC plate.
2.6. Lipid content determination
Lipid content of copepods at day 0 in the
10-day experiment was determined in duplicate
6 G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15
from the following: 200 non-ovigerous S. knabeni,
20 non-ovigerous or 20 ovigerous Coullana sp.,
and ca. 500 Coullana sp. eggs. Each sample was
placed in a 50× 6-mm glass test tube in a total
volume of 0.5 ml of water and sonicated for 20 s
with a Branson Sonifier 450 high intensity probe-
sonicator (Danbury, CT) at 50% power.
Methanol (1 ml) and chloroform (2 ml) were
added, and the samples were refrigerated
overnight. Additional water (2 ml) and chloro-
form (2 ml) were added, and the sample was
centrifuged for 5 min at 2000 rpm. Three quarters
of the chloroform fraction (3 out of 4 ml) was
pulled off and transferred to a new tube. All
solvent was evaporated under a stream of nitro-
gen, and the samples were kept frozen. Lipid
classes were quantified using an Iatroscan, which
combines thin layer chromatography and flame
ionization detection (TLC-FID). A known frac-
tion of the lipid extract was spotted onto a silica-
coated rod (Chromarod). Lipid classes were
separated and quantified in a stepwise sequence
by partial scanning between developments (Par-
rish, 1987). Total lipids was determined to be the
sum of the contents of triacylglycerols, fatty acids,
steroids and wax esters. Percent lipid content was
calculated using mean dry weight (see above).
2.7. Calculations and statistical analysis
For both the S. knabeni and Coullana sp. exper-
iments, one-way analysis of variance (ANOVA)
was used to analyze fluoranthene sediment con-
centrations after dosing and at days 0, 1, 4 and 10
in the 10-day experiment and tissue concentra-
tions at days 1, 4 and 10 in the 10-day experiment
and 3, 6, 12 and 24 h in the 24-h experiment.
Treatments were compared using Bonferroni’s
two-tailed t-test (a =0.05). Untransformed data
met the requirement for normal distribution and
equal variance. Accumulation data from the 24 h
experiment were fit to a two-compartment model
(Landrum et al., 1992a; Eq. (1)):
ks · C s
Ca = (1−eket) (1)
ke
where Ca is the concentration in the animal (mmol
(g wet wt.) − 1), ks is the conditional uptake clear-
ance rate coefficient (g dry sediment (g wet tis-
sue) − 1 h − 1), Cs is the concentration in the
sediment (mmol (g dry wt.) − 1), ke is the condi-
tional elimination rate constant (h − 1), and t is
time (h). Depuration rate constants (kd) for both
species was estimated by fitting the data from the
sediment and water depuration experiments to a
first-order decay (Eq. (2)):
C (ta = t) = C (ta = 0) · e − kdt (2)
where C(ta = 0) is the fluoranthene concentration in
the copepods at the beginning of the depuration
experiment. The corresponding half-lives (t1/2)
were determined in terms of kd by the formula
(t1/2)= 0.693 k − d
1
(Meador et al., 1995). Analyses
were performed using Sigma Stat® (Release 3.0,
Jandel Corp, San Rafael, CA).
3. Results
3.1. Sediment
The dry-to-wet ratio of the sediment treatments
added to the experimental dishes ranged from
0.115 to 0.128. The concentrations of fluo-
ranthene parent compound in the sediment imme-
diately after dosing ranged from 0.95 to 1.07 of
nominal concentrations (Table 1), indicating high
incorporation of the dosing compound into the
test sediment. Concentrations of fluoranthene-
parent compound measured prior to use in the
bioaccumulation experiments (day 0) were lower
than those measured immediately after dosing
(Table 1), as determined by HPLC analysis. The
decrease in concentration of parent compound
between the events of spiking and experiment
initiation ranged from 0.5 to 23% across treat-
ments and was likely a consequence of repeatedly
exchanging the overlying water to remove excess
carrier (acetone). Fluoranthene concentrations in
sediments sampled at days 1, 4 and 10 of the
bioaccumulation experiment (Table 1) were calcu-
lated using 14C activity as a surrogate and are,
Table 1
Average sediment concentrations of fluoranthene parent-compound as measured by HPLC after dosing (dosed) and before use in the 10-day experiments (day 0), and
by the isotopic dilution technique at days 1, 4 and 10 of the S. knabeni and Coullana sp. experiments

Nominal Dosed (n= 2) Day 0 (n =2) S. knabeni Coullana sp.

Day 1 (n = 4) Day 4 (n = 4) Day 10 (n = 4) Day 1 (n= 3) Day 4 (n=3) Day 10 (n =3)

25 26 (0.8) 25 [5] (0.4) 23 (1.9) 23 (1.5) 22 (3.0) 22 (1.2) 21 (3.3) 21 (0.84)

100 102 (0.7) 90 [18] (1.1) 88 (7.5) 82 (7.1) 85 (6.4) 75 (2.7) 76 (6.2) 69 (2.4)
250 256 (8.2) 231 [47] (4.0) 217 (14.4) 209 (43.2) 225 (24.4) 178 (25.8) 177 (75.6) 194 (5.6)
690 654 (9.4) 652 [132] (20.8) 544 (99.7) 541 (87.4) 519 (30.6) 489 (44.2) 477 (15.7) 479 (38.3)
2000 2148 (15) 1652 [334] (12.7) 1659 (91.1) 1365 (159.5) 1429 (91.8) 1422 (73.1) 1287 (24.8) 1480 (174.1)

All concentrations in nmol (g dry wt.)−1, except for numbers in square brackets (mg (g dry wt.)−1). Number in parenthesis indicate
1 S.D. of the mean.
G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15
7
8 G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15

therefore, expressed as fluoranthene molar equiva-

lents. Degradation of the parent compound in
sediments, either by light or microorganisms spe-
cialized in PAH breakdown was not determined.
Degradation was not expected, however, because
the sediment was autoclaved prior to use and all
experiments were conducted in the dark. Changes
in sediment concentration during the 10-day expo-
sure period were small in the S. knabeni experi-
ment (3–13.5%) but larger (12 – 25%) in the
Coullana sp. experiment. In the S. knabeni experi-
ment, differences among fluoranthene sediment
concentrations at days 0, 1, 4 and 10 were not
significant for any sediment treatment, except for
the 2000 nmol (g dry wt.) − 1 treatment, where the
concentration at day 4 was significantly lower
than that at day 1 (Table 1). In the Coullana sp.
experiment, differences were detected at the 100
and 690 nmol (g dry wt.) − 1 treatments, where
concentrations at days 1, 2 and 4 were signifi-

Fig. 2. Accumulation of fluoranthene in tissues of S. knabeni

over time in the 24-h experiment (left) and the 10-day experi-
ment (right) in exposures to increasing sediment concentra-
tions as measured at day 0 (indicated in the lower right
corner).

cantly lower than concentrations at day 0, and at

the 2000 nmol treatment, where the concentration
at day 4 was significantly lower than the concen-
tration at day 0 (Table 1). Hereafter, sediment
treatments will be referred to as their concentra-
tion measured at day 0.

3.2. Bioaccumulation

Fluoranthene concentrations in tissue samples

Fig. 1. Fluoranthene tissue concentrations (y) as a function of
sediment concentrations (x) at day 1 of the 10-day S. knabeni
and Coullana sp. experiments. Solid lines are the best fit from
linear regression; dotted lines represent 95% confidence bands.
S. knabeni: y = 0.017x+ 0.076, r 2 = 0.95, PB 0.001; Coullana
sp.: y=0.013x−0.058, r 2 = 0.99, PB 0.001.
were calculated using 14C activity as a surrogate
and are, therefore, expressed as fluoranthene mo-
lar equivalents. For S. knabeni, fluoranthene con-
centrations in the tissues increased linearly with
increasing fluoranthene concentrations in the sedi-
 G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15
ment at day 1 (r 2 =0.95, Fig. 1), day 4 (r 2 =0.91)
and day 10 (r 2 = 0.92) in the 10-day experiment.
A similar pattern was obtained for Coullana sp. at
day 1 (r 2 = 0.99, Fig. 1), day 4 (r 2 =0.97) and day
10 (r 2 = 0.94).
For S. knabeni, mean fluoranthene body residue
was lowest at 3 h, increased significantly at 6 h in
all sediment treatments except 652 nmol (g dry
wt.) − 1, and did not change significatively there-
after (Fig. 2). Body residues in the 10-day experi-
ment tended to decline slightly from day 1
through 10, except at 652 nmol (g dry wt.) − 1
(Fig. 2), but were not significantly different, ex-
cept at the lowest and highest sediment treat-
ments. At 25 nmol (g dry wt.) − 1, tissue concen-
tration at day 1 decreased by 53% to a signifi-
Fig. 3. Accumulation of fluoranthene in tissues of Coullana sp.
over time in the 24-h experiment (left) and the 10-day experi-
ment (right) in exposures to increasing sediment concentra-
tions as measured at day 0 (indicated in the lower right
corner).
9
Table 2
Apparent steady state lipid and sediment organic carbon nor-
malized bioaccumulation factors (BSAF) for non-ovigerous S.
knabeni and Coullana sp. calculated using day 1 sediment and
tissue concentrations, mean lipid content at day 0, and the
organic carbon content of the stock-sediment
Treatment nmol S. knabeni (n =4) Coullana sp. (n = 3)
(g dry wt.)−1 mean 9S.D. mean 9S.D.
25 0.57 9 0.28 0.22 9 0.05
100 0.51 9 0.17 0.27 9 0.10
250 0.61 9 0.16 0.67 9 0.08
690 0.60 90.12 0.50 9 0.17
2000 0.80 9 0.22 0.49 9 0.06
S.D., standard deviation of the mean.
cantly lower mean at day 10. At 1652 nmol (g dry
wt.) − 1, mean tissue concentration decreased sig-
nificantly by 54% at day 4 and was not measured
at day 10 due to a low number of surviving
organisms. Overall, the analysis of the data from
the 24-h and 10-day experiments indicated that S.
knabeni body residues attained apparent steady-
state in less than 12 h in all treatments, and
remained relatively constant through day 10, ex-
cept at 25 and 1652 nmol (g dry wt.) − 1, where it
decreased significatively.
For Coullana sp., body residues remained rela-
tively constant from 3 to 24 h and from day 1 to
10 (Fig. 3) and were not significantly different at
any sediment treatment. Steady state was appar-
ently attained in less than 6 h. Higher variability
in tissue concentration was observed in the 24-h
experiment than in the 10-day experiment, proba-
bly due to the lower number of organisms per
replicate used for 14C activity analysis, three in the
24-h experiment and from 5 to 15 in the 10-day
experiment. Overall, Coullana sp. accumulated
higher concentrations of fluoranthene than S. kn-
abeni in the 24-h experiment, but not in the
10-day experiment.
Accumulation data from both species did not fit
the non-linear model, except for the data from the
25 nmol (g dry wt.) − 1 treatment in the S. knabeni
experiment. At this concentrations, the estimated
conditional uptake clearance rate coefficient (ks)
was 0.51 g dry sediment (g wet tissue) − 1 h − 1
(0.22–0.80, 95% confidence interval (CI)). The
conditional elimination rate constant (ke) was 0.27
10 G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15
h − 1 (0.08–0.45, 95% CI). The time required to
reach 95% steady state body residue can be esti-
mated by the elimination rate constant in sedi-
ment (ke) using the formula (TSS95) =2.99 k − e
1
(Meador et al., 1995). Using the ke value obtained
above, the time for 95% steady state was 11.1 h
for S. knabeni, confirming the visual estimation.
Fig. 4. A. Distribution of the total fluoranthene burden in
ovigerous Coullana sp. recovered at day 1 of the 10-day
bioaccumulation experiment expressed as the percent mea-
sured in the body and in the egg clutch. B. Relative fluo-
ranthene tissue concentration in non-ovigerous females, body
of ovigerous females and in the egg clutch of Coullana sp. C.
Relative lipid normalized fluoranthene tissue concentration in
non-ovigerous females, body of ovigerous females and in the
egg clutch of Coullana sp. Error bars show + 1 S.D. of the
mean. In B and C, the tissue concentration in non-ovigerous
females was arbitrarily normalized to the unit (one) and the
concentration in the body of ovigerous females and in the eggs
is shown as a relative proportion.
3.3. Sur6i6al
Survival was high (\ 90%) in all sediment
treatment during the 24-h experiment and in the
controls during the 10-day experiment for both S.
knabeni and Coullana sp. Significant mortality
occurred at high fluoranthene concentrations dur-
ing the 10-day experiments, at 1652 nmol (g dry
wt.) − 1 for S. knabeni and at 652 and 1652 nmol
(g dry wt.) − 1 for Coullana sp. The relationship
between mortality and fluoranthene body residue
in the 10-day experiments is reported in a com-
panion paper (Lotufo, 1998).
3.4. Lipid content and BSAFs
Mean total lipid content of stock non-ovigerous
female S. knabeni was 22.29 1.0% (S.D.). For
stock Coullana sp., mean lipid content was 31.79
3.1% for non-ovigerous females, 18.29 1.2% for
ovigerous females, and 87.1929.5% for eggs. Ap-
parent steady state lipid and sediment organic
carbon normalized bioaccumulation factors
(BSAF) were calculated for non-ovigerous S. kn-
abeni and Coullana sp. using day 1 sediment and
tissue concentrations, mean lipid content at day 0
and the organic carbon content of the test-sedi-
ment (1.5%). BSAF values ranged from 0.61 to
0.80 for S. knabeni and from 0.22 to 0.67 for
Coullana sp. (Table 2).
3.5. Body residue and reproducti6e cycle
About 30% of the non-ovigerous Coullana sp.
exposed to sediment-associated fluoranthene had
extruded egg sacs at day 1. For each ovigerous
female, the fluoranthene burden was separately
determined for the body and for the extruded egg
clutch. The egg clutch contained approximately
50% of the total fluoranthene accumulated in the
body plus eggs (Fig. 1A), although the biomass of
an egg clutch is approximately only 25% of the
body biomass. The relative concentration of fluo-
ranthene in non-ovigerous females with mature
ovaries, the body of ovigerous females and in the
egg clutch is shown in Fig. 4B and C. The fluo-
ranthene concentration, expressed as mass fluo-
ranthene/mass tissue (Fig. 4B) or mass
 G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15
fluoranthene/mass lipids (Fig. 4C), in non-oviger-
ous females was arbitrarily normalized to the
unit (one) and the concentration in the body of
ovigerous females and in the eggs is shown as a
relative proportion. On a dry-weight basis, fluo-
ranthene tissue concentration in non-ovigerous
females was approximately twice that in oviger-
ous females separated from their eggs, and con-
centrations in the eggs alone were 3 – 4.5 times
higher than in non-ovigerous females and six to
ten times higher than in the body of ovigerous
females (Fig. 4B). Differences were minimized
when fluoranthene tissue concentrations were
lipid normalized (Fig. 4C). Similar trends were
found for days 4 and 10 (data not shown).
3.6. Depuration
The depuration of fluoranthene was evaluated
in both sediment and water in 24-h experiments
following pre-exposure to 25 nmol (g dry wt.) − 1.
For S. knabeni, depuration in water and sedi-
ment occurred at approximately the same rate
constant (kd), 0.15 (90.02) h − 1 and 0.14 (9
0.03) h − 1, respectively. The corresponding half-
lives (t1/2) were 4.7 h in sediment and 4.9 h in
water. For Coullana sp., depuration in sediment
was significantly (P = 0.002) faster (0.179 0.05
h − 1) than in water (0.09 90.05 h − 1). The corre-
sponding half lives were 4.2 h in sediment and
7.4 h in water. After a depuration period of 12
h, virtually no 14C activity was detected in tissues
for S. knabeni in both water and sediment, and
only 10% for Coullana sp. in sediment and 32%
in water.
3.7. Biotransformation
After a 96-h exposure to sediment-associated
fluoranthene, the mean (9S.D.) fraction of 14C
activity in the copepod tissues from non-fluo-
ranthene compounds in TLC plates was deter-
mined to be 10.8 ( 9 8.8)% for S. knabeni and
12.8 (9 4)% for Coullana sp. These fractions are
assumed to correspond to products of fluo-
ranthene biotransformation.
11
4. Discussion
Fluoranthene apparent steady state was
reached very rapidly in the benthic copepods S.
knabeni and Coullana sp., with tissue concentra-
tions increasing over the initial 6 h of the sedi-
ment exposure and remaining at apparent
steady-state thereafter throughout the 10-day ex-
periment. Different temporal patterns of fluo-
ranthene bioaccumulation have been observed
with other invertebrate species in sediment expo-
sures. For fresh water amphipods, body residue
increased continually until reaching apparent
steady state after 10 day in Diporeia spp. and was
maximal after 1 day and declined thereafter in
Hyalella azteca (Kane Driscoll et al., 1997). Fluo-
ranthene body residue in the polychaete Capitella
sp. I attained a maximal level after 1 day and
declined rapidly and dramatically thereafter, be-
ing undetectable after 7 days despite continuous
exposure (Forbes et al., 1996).
Differences in the temporal pattern of fluo-
ranthene bioaccumulation in benthic species are
likely related to differences in their ability to
biotransform PAHs, which may greatly impact
their rate of elimination, and consequently, time
to achieve steady state. Diporeia spp. biotrans-
forms PAHs inefficiently (Landrum, 1988), while
H. azteca and Capitella sp. I biotransform PAHs
at a high rate (Landrum and Scavia, 1983; Forbes
et al., 1996). Invertebrate species that biotrans-
form PAH efficiently also eliminate PAHs very
rapidly, as observed with Chironomus riparius, H.
azteca and Capitella sp. I (Leversee et al., 1982;
Landrum and Scavia, 1983; Forbes et al., 1996).
Although S. knabeni and Coullana sp. have lim-
ited ability to biotransform fluoranthene to polar
metabolites, 14C activity decreased very rapidly in
the depuration experiments, suggesting very effi-
cient elimination of fluoranthene parent com-
pound and metabolites. The high
surface-to-volume ratio in benthic copepods likely
contribute to a fast rate of fluoranthene elimina-
tion. Daphnia magna, a small cladoceran with
small capacity to metabolize PAHs and high sur-
face-to-volume ratio, also eliminates PAHs very
efficiently (Leversee et al., 1981).
12 G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15
Accumulation data from the 24-h experiments
did not fit a two-compartment non-linear general
model, except for S. knabeni exposure to 25 nmol
(g dry wt.) − 1. Likely reasons for the poor fit are
lack of earlier time points (B 3 h) and high
variance. Nevertheless, the conditional uptake
clearance rate coefficient calculated for S. knabeni
(0.51 g dry sediment (g wet tissue) − 1 h − 1) indi-
cate highly efficient fluoranthene uptake from sed-
iment. Uptake clearance rates (ks) estimated for
Diporeia spp. exposed to different concentrations
of sediment-associated fluoranthene ranged from
0.01 to 0.054 g dry sediment (g wet tissue) − 1 h − 1
(Kane Driscoll et al., 1997). High activity level
and high surface to volume ratio are believed to
be associated with high PAH uptake clearance
rate (Landrum, 1988; Meador et al., 1995). These
factors likely explain the higher fluoranthene up-
take rate in S. knabeni than in Diporeia spp. For
S. knabeni exposed to the 25 nmol (g dry wt.) − 1
sediment treatment, the elimination rate (ke) was
estimated by fitting the bioaccumulation data to a
non-linear model and the depuration rate (kd) was
estimated by transferring exposed individuals to
uncontaminated sediment. The 95% CI of the
estimate for elimination rate (0.08 – 0.45 h − 1) in-
cluded the mean estimate for depuration rate
(0.15 h − 1).
For S. knabeni, depuration rates (kd) were very
similar in both sediment and water. For Coullana
sp., however, depuration was significantly faster
in sediment than in water. Faster depuration in
sediment has been previously reported for de-
posit-feeding invertebrates and was related to ac-
tive sediment ingestion as opposed to starvation
in water-only exposures (Landrum and Scavia,
1983; Kukkonen and Landrum, 1994). The faster
depuration in sediment for Coullana sp. was unex-
pected, since it feeds primarily by filter-feeding
from the near-bottom water (Decho, 1986; Pace
and Carman, 1996), and food was not added to
either medium. In addition, there is no compelling
evidence for bulk deposit feeding among harpacti-
coids. It is possible that Coullana sp. engage in
filtering activity in sediment even in the absence of
food, but did not filter feed in water only. The
flow of water over mouth parts could have con-
tributed to a higher depuration rate in sediment.
Apparent steady-state BSAF values obtained
with copepods in this study (from 0.51 to 0.80 for
S. knabeni and from 0.22 to 0.67 for Coullana sp.)
were substantially lower than 1.7, the maximal
value predicted for neutral organic chemicals on
the basis of equilibrium partitioning (McFarland
et al., 1986), or 4, the value recommended for
regulatory screening of dredged materials (US
Environmental Protection Agency, 1991). Values
obtained in the present study were, however,
higher than the mean BSAF for PAHs (0.34)
calculated from selected published values (Tracey
and Hansen, 1996). Overall, the BSAFs for cope-
pods were in the same range as those obtained
from laboratory exposures to fluoranthene with
the amphipods H. azteca (0.23–0.61; Kane
Driscoll et al., 1997) and Diporeia spp. (0.35–
0.82; Kane Driscoll et al., 1997). The BSAFs for
copepods were highest at high or intermediate
sediment concentrations (Table 2), a trend also
observed for freshwater amphipods (Kane
Driscoll et al., 1997). Although mean lipid content
at day 0 was used in the calculations of BSAFs at
day 1, differences in copepod lipid content among
treatments were not expected since treatment-re-
lated changes in lipid content are not expected
after only 1 day.
Lipid content in Coullana sp. was highly associ-
ated with the reproductive status. Non-ovigerous
females with mature ovaries contained approxi-
mately twice as much lipid as ovigerous females
without egg sacs. Consequently, higher concentra-
tions of fluoranthene were measured in non-
ovigerous than in ovigerous females. Because lipid
content was much higher in the eggs than in the
body, so was the fluoranthene concentration on a
dry-weight basis. When lipid-normalized, concen-
trations in the body and eggs were within closer
range (Fig. 4). The contaminant distribution in
eggs and somatic tissues of ovigerous females
indicates that approximately 50% of the body
residue of a female copepod with mature ovaries
resides in the eggs and is released when eggs are
extruded. Similar disposition of lipophilic contam-
inants was observed with copepods exposed to
PCBs (McManus et al., 1983) and in crabs ex-
posed to the pesticide kepone (Roberts and
Leggett, 1980).
 G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15
Little is known about the bioaccumulation of
sediment-associated contaminants in estuarine
meiobenthos. PAH contamination at sublethal
concentrations can pose a significant risk to estuar-
ine systems. Fluoranthene tissue concentration
reaches an apparent steady state very rapidly in S.
knabeni and Coullana sp. Most of the fluoranthene
bioaccumulates as parent compound. PAH bioac-
cumulation in invertebrates has been associated
with adverse effects at the organismal level
(Donkin et al., 1989; Kane Driscoll et al., 1997)
and may also pose a risk to higher trophic levels
in aquatic ecosystems. Benthic copepods are an
important prey item in the diet of numerous
estuarine fish and shellfish species, including some
of commercial importance (Coull, 1990). It is
uncertain, however, whether feeding on PAH-con-
taminated copepods will pose significant risks to
predators. Trophic transfer of PAHs from inverte-
brates to fish is highly inefficient (Niimi and
Dookhran, 1989; Clements et al., 1994). Moreover,
trophic transfer studies suggest that fish feeding on
copepods in sediments contaminated with organic
compounds acquire most of their body burden by
direct contact with the sediment itself rather than
by ingestion of contaminated prey (DiPinto, 1996;
DiPinto and Coull, 1997). High PAH body
residues, in the range observed in this study, are
known to impact exposed organisms at the popula-
tion level, as they were accompanied by high
mortality in amphipods (Kane Driscoll et al.,
1997). Fluoranthene lethal and sublethal effects
were detected in the 10-day experiment with S.
knabeni and Coullana sp. Significant mortality over
10 days was detected at body residues as low as 1.3
mmol (g wet wt.) − 1, and significant reductions in
reproduction and feeding rates were detected at
concentrations as low as 0.2 and 0.1 mmol (g wet
wt.) − 1, respectively (Lotufo, 1998).
5. Conclusions
Fluoranthene apparent steady state body residue
in the benthic copepods S. knabeni and Coullana
sp. exposed to spiked sediment was reached very
rapidly ( B 12 h). Non-linear modeling of bioaccu-
mulation data revealed that S. knabeni uptakes
13
sediment-associated fluoranthene very efficiently
and eliminates at a high rate. Fluoranthene depu-
ration rate was high in both species, and depuration
half-lives in sediment (approximately 4.5 h)
were among the shortest reported for PAHs in
invertebrates. Both S. knabeni and Coullana sp.
have limited ability to biotransform PAHs. Fluo-
ranthene body residue in female Coullana sp. was
strongly associated with their reproductive status.
A significant fraction of the fluoranthene residue
was stored in the lipid-rich maturing eggs and was
disposed of during extrusion of the paired egg sacs.
Lipid and organic-carbon-normalized bioaccumu-
lation factors calculated at steady state (0.35–0.80)
were in the typical range observed reported for
benthic invertebrates. Sediment-associated fluo-
ranthene accumulates rapidly and at high levels in
benthic copepods, posing a risk for population
growth and constituting a potential source for
uptake by higher trophic levels.
Acknowledgements
Thanks are due to Steve Pomarico, Kevin Car-
man and John Fleeger for technical advice and
assistance and for Tom Luong for assistance with
HPLC analysis. The manuscript was improved by
comments by John Fleeger, Kevin Carman, Peter
Landrum and Todd Bridges. Appreciation is also
expressed to the NOAA Great Lakes Environmen-
tal Research Laboratory for providing logistical
support for the elaboration of the final version of
this manuscript. The author received financial sup-
port from CNPq, Brazilian Federal Government.
References
Carman, K.R., Fleeger, J.W., Means, J.C., Pomarico, S.,
McMillin, D.J., 1995. Experimental investigation of the
effects of polynuclear aromatic hydrocarbons on an estuar-
ine sediment food web. Mar. Environ. Res. 40, 289 – 318.
Chandler, G.T., Green, A.S., 1996. A 14-day harpacticoid
copepod reproduction bioassay for laboratory and field
contaminated muddy sediments. In: Ostrander, G.K. (Ed.),
Techniques in Aquatic Toxicology. CRC Press, Boca Ra-
ton, FL, pp. 23 – 39.
14 G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15
Clements, W.H., Oris, J.T., Wissing, T.E., 1994. Accumulation
and food chain transfer of fluoranthene and ben-
zo[a]pyrene in Chironomus riparius and Lepomis
macrochirus. Arch. Environ. Contam. Toxicol. 26, 261–
266.
Corner, E.D.S., Harris, R.P., Kilvington, C.C., O’Hara,
S.C.M., 1976. Petroleum compounds in the marine food
web: short-term experiments on the fate of naphthalene in
Calanus. J. Mar. Biol. Ass. 56, 121–133.
Coull, B.C., 1988. Ecology of the marine meiofauna. In:
Higgins, R.P., Thiel, H. (Eds.), Introduction to the Study
of Meiofauna. Smithsonian Institution Press, Washington
D.C., pp. 18 – 38.
Coull, B.C., 1990. Are members of the meiofauna food for
higher trophic levels? Trans. Am. Microsc. Soc. 109, 233–
246.
Decho, A.W., 1986. Water-cover influence on diatom ingestion
rates by meiobenthic copepods. Mar. Ecol. Prog. Ser. 33,
139 – 146.
DiPinto, L.M., 1996. Trophic transfer of a sediment-associated
organophosphate pesticide from meiobenthos to bottom
feeding fish. Arch. Environ. Contam. Toxicol. 30, 459–
466.
DiPinto, L.M., Coull, B.C., 1997. Trophic transfer of sedi-
ment-associated polychlorinated biphenyls from meioben-
thos to bottom-feeding fish. Environ. Toxicol. Chem. 16,
2568 – 2575.
Donkin, P., Widdows, J., Evans, S.V., Worrall, C.M., Carr,
M., 1989. Quantitative structure-activity relationships for
the effect of hydrophobic organic chemicals on rate of
feeding by mussels (Mytilus edulis). Aquat. Toxicol. 14,
277 – 294.
Forbes, V.E., Forbes, T.L., Holmer, M., 1996. Inducible
metabolism of fluoranthene by the opportunistic poly-
chaete Capitella sp. I. Mar. Ecol. Prog. Ser. 132, 63–70.
Harding, G.C., Vass, W.P., 1977. Uptake from sea water and
clearance of 14C-p,p-DDT by the marine copepod Calanus
finmarchicus. J. Fish. Res. Board Can. 34, 177–182.
Harkey, G.A., Landrum, P.F., Klaine, S.J., 1994a. Preliminary
studies of the effect of feeding during whole sediment
bioassays using Chironomus riparius larvae. Chemosphere
28, 597 – 606.
Harkey, G.A., Landrum, P.F., Klaine, S.J., 1994b. Compari-
son of whole-sediment, elutriate and pore-water exposures
for use in assessing sediment-associated organic contami-
nants in bioassays. Environ. Toxicol. Chem. 13, 1315–
1330.
Harkey, G.A., Lydy, M.J., Kukkonen, J., Landrum, P.F.,
1994c. Feeding selectivity and assimilation of PAH and
PCB in Diporeia spp. Environ. Toxicol. Chem. 13, 1445–
1456.
Harris, R.P., Berdugo, V., O’Hara, S.C.M., Corner, E.D.S.,
1977a. Accumulation of 14C-L-naphthalene by an oceanic
and an estuarine copepod during long-term exposure to
low-level concentrations. Mar. Biol. 42, 187–195.
Harris, R.P., Berdugo, V., Corner, E.D.S., Kilvington, C.C.,
O’Hara, S.C.M., 1977b. Factors affecting the retention of
a petroleum hydrocarbon by marine planktonic copepods.
In: Wolfe, D.A. (Ed.), Fate and Effects of Petroleum
Hydrocarbons in Marine Organisms and Ecosystems. Perg-
amon, New York, pp. 78 – 94.
Hellou, J., Mackay, D., Fowler, B., 1995. Bioconcentration of
polycyclic aromatic compounds from sediments to muscle
of finfish. Environ. Sci. Technol. 29, 2555 – 2560.
Kane Driscoll, S., McElroy, A.E., 1996. Bioaccumulation and
metabolism of benzo[a]pyrene in three species of poly-
chaete worms. Environ. Toxicol. Chem. 15, 1401 – 1410.
Kane Driscoll, S., Harkey, G.A., Landrum, P.F., 1997. Accu-
mulation and toxicokinetics of fluoranthene in sediment
bioassays with freshwater amphipods. Environ. Toxicol.
Chem. 16, 742 – 753.
Kukkonen, J., Landrum, P.F., 1994. Toxicokinetics and toxic-
ity of sediment-associated pyrene to Lumbriculus 6ariegatus
(Oligochaeta). Environ. Toxicol. Chem. 13, 1457 – 1468.
Landrum, P.F., 1982. Uptake, depuration and biotransforma-
tion of anthracene by the scud, Pontoporeia hoyi. Chemo-
sphere 11, 1049 – 1057.
Landrum, P.F., 1988. Toxicokinetics of organic xenobiotics in
the amphipod, Pontoporeia hoyi: role of physiological and
environmental variables. Aquat. Toxicol. 12, 245 – 271.
Landrum, P.F., 1989. Bioavailability and toxicokinetics of
polycyclic aromatic hydrocarbons sorbed to sediment for
the amphipod Pontoporeia hoyi. Environ. Sci. Technol. 23,
588 – 595.
Landrum, P.F., Scavia, D., 1983. Influence of sediment on
anthracene uptake, elimination, and biotransformation by
the amphipod, Hyalella azteca. Can. J. Fish. Aquat. Sci.
40, 731 – 743.
Landrum, P.F., Eadie, B.J., Faust, W.R., 1991. Toxicokinetics
and toxicity of a mixture of sediment-associated polycyclic
aromatic hydrocarbons to the amphipod Diporeia sp. Envi-
ron. Toxicol. Chem. 10, 35 – 46.
Landrum, P.F., Lee, H.I., Lydy, M.J., 1992a. Toxicokinetics
in aquatic systems: model comparisons and use in hazard
assessment. Environ. Toxicol. Chem. 11, 1709 – 1725.
Landrum, P.F., Eadie, B.J., Faust, W.R., 1992b. Variation in
the bioavailability of polycyclic aromatic hydrocarbons to
the amphipod Diporeia (spp.) with sediment aging. Envi-
ron. Toxicol. Chem. 11, 1197 – 1208.
Landrum, P.F., Dupuis, W.S., Kukkonen, J., 1994. Toxicoki-
netics and toxicity of sediment-associated pyrene and
phenanthrene in Diporeia spp.: examination of equi-
librium-partitioning theory and residue-based effects for
assessing hazard. Environ. Toxicol. Chem. 13, 1769 – 1780.
Leversee, G.L., Giesy, J.P., Landrum, P.F., Bartell, S.M.,
Gerould, S., Bruno, M., Spacie, A., Bowling, J., Haddock,
J.D., Fannin, T.E., 1981. Disposition of benzo(a)pyrene on
aquatic systems components: periphyton, chironomids,
Daphnia and fish. In: Cook, M., Dennis, A.J. (Eds.),
Polynuclear Aromatic Hydrocarbons: Chemical Analysis
and Biological Fate. Fifth International Symposium,
Batelle Press, Columbus, OH, pp. 357 – 366.
Leversee, G.L., Giesy, J.P., Landrum, P.F., Gerould, S., Fan-
nin, T.E., Haddock, J.D., Bartell, S.M., 1982. Kinetics and
biotransformation of benzo(a)pyrene in Chironomus ripar-
ius. Arch. Environ. Contam. Toxicol. 11, 25 – 31.
 G.R. Lotufo / Aquatic Toxicology 44 (1998) 1–15
Lotufo, G.R., 1997. Toxicity of sediment-associated PAHs to
an estuarine copepod: effects on survival, feeding, repro-
duction, and behavior. Mar. Environ. Res. 44, 149–166.
Lotufo, G.R., 1998. Lethal and sublethal toxicity of sediment-
associated fluoranthene to benthic copepods: Application
of the critical-body-residue approach. Aquat. Toxicol., 44,
17– 30.
Lotufo, G.R., Fleeger, J.W., 1997. Effects of sediment-associ-
ated phenanthrene on survival, development and reproduc-
tion of two species of meiobenthic copepods. Mar. Ecol.
Prog. Ser. 151, 91 – 102.
Lotufo, G.R., Fleeger, J.W., 1996. Toxicity of sediment-associ-
ated pyrene and phenanthrene to Limnodrilus hoffmeisteri
(Oligochaeta: Tubificidae). Environ. Toxicol. Chem. 15,
1508 – 1516.
Lydy, M.J., Landrum, P.F., 1993. Assimilation efficiency for
sediment-sorbed benzo(a)pyrene by Diporeia spp. Aquat.
Toxicol. 26, 209 – 224.
Mayer, L.M., Chen, Z., Findlay, R.H., Fang, J.S., Sampson,
S., Self, R.F.L., Jumars, P.A., Quetel, C., Donard, O.F.X.,
1996. Bioavailability of sedimentary contaminants subject
to deposit-feeder digestion. Environ. Sci. Technol. 30,
2641 – 2645.
McFarland, V.A., Clarke, J.H., Wiley, R.G., 1986. Testing
bioavailability of polychlorinated biphenyls from sedi-
ments using a two-level approach. In: Willey, R.G. (Ed.),
Water Quality R&D: Successful Bridging Between Theory
and Application. Seminar Proceedings. U.S. Army Corps
of Engineers, Washington, DC.
McManus, G.B., Wyman, K.D., Peterson, W.T., Wurster,
C.F., 1983. Factors affecting the elimination of PCBs in
the marine copepod Acartia tonsa. Estuarine Coastal Shelf
Sci. 17, 421 – 430.
Meador, J.P., Stein, J.E., Reichert, W.L., Varanasi, U., 1995.
Bioaccumulation of polycyclic aromatic hydrocarbons by
marine organisms. Rev. Environ. Contam. Toxicol. 143,
79– 165.
15
Niimi, A.J., Dookhran, G.P., 1989. Dietary absorption effi-
ciencies and elimination rates of polycyclic aromatic hy-
drocarbons (PAHs) in rainbow trout (Salmo gairdneri ).
Environ. Toxicol. Chem. 8, 719 – 722.
Pace, M.C., Carman, K.R., 1996. Interspecific differences
among meiobenthic copepods in the use of microalgal food
resources. Mar. Ecol. Prog. Ser. 143, 77 – 86.
Parrish, C.C., 1987. Separation of aquatic lipid classes by
chromarod thin-layer chromatography with measurement
by Iatroscan flame ionization detection. Can. J. Fish.
Aquat. Sci. 44, 722 – 773.
Pawlisz, A.V., Peters, R.H., 1993. A radioactive tracer tech-
nique for the study of lethal body burdens of narcotic
organic chemicals in Daphnia magna. Environ. Sci. Tech-
nol., 27: 2795 – 2800.
Roberts, M.H.J., Leggett, A.T., 1980. Egg extrusion as a
kepone-clearance route in the blue crab, Callinectes
sapidus. Estuaries 3, 192 – 199.
Tracey, G.A., Hansen, D.J., 1996. Use of biota-sediment
accumulation factors to assess similarity of nonionic or-
ganic chemical exposure to benthically-coupled organisms
of differing trophic mode. Arch. Environ. Contam. Toxi-
col. 30, 467 – 475.
US Environmental Protection Agency, 1991. Evaluation of
Dredged Material Proposed for Ocean Disposal (Testing
Manual). EPA 503/8-91/001. US Environmental Protection
Agency, Washington, DC.
Wirth, E.F., Chandler, G.T., DiPinto, L.M., Bidleman, T.F.,
1994. Assay of polychlorinated biphenyl bioaccumulation
from sediments by marine copepods using a novel microex-
traction technique. Environ. Sci. Technol. 28, 1609 – 1614.
Wood, L.W., O’Keefe, P., Bush, B., 1997. Similarity analysis
of PAH and PCB bioaccumulation patterns in sediment-
exposed Chironomus tentans larvae. Environ. Toxicol.
Chem. 16, 283 – 292.