C .-Y. Horng, G.L. Taghon J. Exp. Mar. Biol. Ecol. 242 1999 41 –57
43
There have been numerous studies of the effects of sediment chemical composition on the feeding and growth rates and reproductive output of Capitella spp. Tenore, 1977;
´ Gremare et al., 1988; Marsh et al., 1989; Forbes and Lopez, 1990; Tsutsumi et al., 1990;
Bridges et al., 1994. Only one previous study has quantitatively evaluated particle selectivity by Capitella spp., and that study used uncontaminated sediments Self and
Jumars, 1988. In this study, we used two different approaches to examine the influence of contaminants on particle selection by Capitella sp. I. The first approach tested
whether particle selection on natural sediments varied with the degree of contamination. Organically enriched sediments from four different collection sites were used. Particle
selection was examined by comparing the particle size distributions in fecal pellets with those in the sediments. This approach provided information about the sizes of mineral
particles being ingested from natural sediments. From the perspective of testing particle selection, however, this method is relatively insensitive with respect to small particles,
due to the very great abundance of small particles in natural sediments. The second approach was to determine the effect of a single PAH on particle selection. Phenan-
threne-spiked sediments were used for this selection experiment. Glass beads were used as a tracer to evaluate size selection, because they offer the advantage of testing
preference for different particle sizes with equal sensitivity Self and Jumars, 1988.
2. Materials and methods
2.1. Particle selection on natural sediments 2.1.1. Sediment sources and properties
Relatively uncontaminated sediments were obtained from the intertidal zones of two Spartina-dominated salt marshes. Sediment from Sippewissett Marsh, on Cape Cod,
Massachusetts, is used for laboratory culturing of Capitella sp. I Grassle et al., 1992 and was a generous gift from Dr J.P. Grassle. Sediment was collected from Schooner
Creek, a tidal creek in an extensive salt marsh surrounding the Rutgers University Marine Field Station in Tuckerton, New Jersey. Moderately contaminated sediment was
obtained from a subtidal site in Barnegat Bay, New Jersey. The sampling spot was in a bottom depression depth 6.7 m where fine-grain sediments and organic matter
accumulated. Anoxic conditions existed in the summer and no benthic infauna were present. During the winter, the water was oxygenated and Capitella spp. were the only
macrofauna identified in the sediments. Since the site is bordered on two sides by marinas serving commercial fishing boats, sail, and power pleasure boats, the major
contaminant inputs are probably contributed by boating activities. The most heavily contaminated sediments were obtained from Piles Creek 408 36.539 N, 748 13.69 W, a
tidal creek emptying into the Arthur Kill, the navigational channel between New Jersey and Staten Island, New York. Piles Creek is in the center of an industrial park,
surrounded by petroleum refineries and oil storage tanks.
All sediments were wet-sieved through a 90-mm mesh screen using filtered 1 mm
32‰ seawater. The , 90 mm fraction was used for all subsequent experiments. Total carbon and nitrogen contents of freeze-dried samples were measured using a Carlo Erba
44 C
.-Y. Horng, G.L. Taghon J. Exp. Mar. Biol. Ecol. 242 1999 41 –57
NA-1500 elemental analyzer. Sediment protein content was estimated using the method of Mayer et al. 1995, a biomimetic assay of digestible protein based on direct
incubation of sediments with a proteolytic enzyme. The single-point method with a 6-h incubation was used. PAHs were analyzed by placing an aliquot of approximately 1 g
dry weight sediment into a 50-ml Teflon centrifuge tube. Methylene chloride 15 ml, Burdick Jackson, HPLC GC grade was added and the tube was placed into a 408C
ultrasonic bath for 30 min. The extract was separated from sediment by centrifugation at 8820 3 g for 15 min and decanted into a 50-ml evaporation flask. These steps were
repeated two more times and the three successive extracts were combined. Activated copper turnings pre-oxidized with 1 N HCl and cleaned with methylene chloride were
added to the evaporation flask to remove elemental sulfur. This ultrasonic extraction technique had an average efficiency of 87 compared with a 60-cycle Soxhlet extraction
Horng, 1998. The extract was reduced in volume to | 2 ml using a rotary evaporator under a slight vacuum 250 kPa at 358C. The extract was quantitatively transferred to
an amber vial and further reduced in volume to | 0.5 ml under a stream of high purity nitrogen. The extract was then solvent-exchanged to acetonitrile by adding 2 ml of
acetonitrile. The mixture was reduced again to | 0.5 ml with a nitrogen stream and moderate heating 608C. After volumetric quantification with a 1-ml microsyringe, the
acetonitrile extract was transferred to an autosampler vial for HPLC analysis.
PAHs were separated and quantified with a Hewlett-Packard Rockville, MD 1050 series HPLC equipped with a binary gradient elution control, a diode array detector set
at 254 nm, a C reversed phase column Vydac, Hesperia, CA, USA; 250 3 4.6 mm, 5
18
mm, and a guard column Vydac, 20 3 4.6 mm, 5 mm. The column was initially equilibrated with a 50 v v acetonitrile water mixture for at least 15 min. PAHs were
separated using a gradient elution in which the solvent linearly increased to 100 acetonitrile after 50 min and remained isocratic for another 5 min. The flow rate was 1
21
ml min . Peaks were identified by comparing retention times and spectra of samples
with those of 16 PAH standards TCL PAH mix standard, Supelco, Bellefonte, PA. The PAHs were then quantified by calibration curves developed in advance using known
concentrations of standards. Although a PAH standard mixture with 16 EPA priority compounds was used as the reference, only ten PAHs from the sediment samples are
reported here. These ten phenanthrene, fluoranthene, pyrene, benz[a]anthracene, chrysene,
benzo[b]fluoranthene, benzo[k]fluoranthene,
benzo[a]pyrene, ben-
zo[ ghi]perylene, and indeno[1,2,3-cd]pyrene were selected because their quantification could be reasonably achieved within 30 variation upon analyzing a standard marine
sediment NIST SRM [1941a.
2.1.2. Selection experiments Cultures of Capitella sp. I Grassle and Grassle, 1976 were established with worms
provided by Dr J.P. Grassle. Stock cultures were maintained in aerated pans 20 3 30 cm with 32‰ seawater and a food source consisting of sediment from Schooner Creek
that had been sieved through a 1-mm mesh, frozen, then thawed. Three-week-old juvenile worms were used. The thoracic width of each worm was measured and
converted to body volume using a previously established regression equation Horng, 1998. Worms were placed individually into glass dishes 5 cm in diameter containing
C .-Y. Horng, G.L. Taghon J. Exp. Mar. Biol. Ecol. 242 1999 41 –57
45
test sediment five replicates each and allowed to feed for 7 days in a 158C environmental chamber. Fecal pellets were separated from residual, uneaten sediment by
sieving with a 90-mm mesh. Pellets were completely disaggregated by treatment with 30 w v hydrogen peroxide in an ultrasonic bath. Samples were taken from the
sediment stocks used to feed the worms and treated similarly.
Size–frequency distributions of particles in pellets and ambient sediment were determined using a laser-based particle counter Spectrex, Redwood City, CA, which
analyzes the forward scattering interference patterns created by illuminating suspended particles with a laser beam. Particles were kept in suspension by a magnetic stirring bar
in a 250-ml beaker. Calgon Benckiser, Danbury, CT, a commercial detergent, was added to disperse particles in the suspension. Potential interference from background
particles was prevented by the use of 0.2-mm filtered, ultra-pure deionized water and by background subtraction. The system was configured to detect particles between 1 and
100 mm in diameter, and a maximum count of 800 particles was set to reduce the probability of more than one particle simultaneously passing through the laser beam.
Particle counts and sizes were within 5 of actual values, based on analysis of NIST calibration standards.
2.1.3. Data analysis Data were analyzed following the methods of Petch 1986. The particle counter
categorized particles into 33 size classes. Particle size–frequency distributions of fecal pellets and ambient sediment were compared by G-test. Size categories causing
significant differences between size–frequency distributions were identified by compar- ing values of the adjusted residuals for each size category to 1.96, the value of the 5
standard normal deviate Petch, 1986. The adjusted residual also provides information about the direction and strength of selection, because it is symmetrically distributed
about zero.
2.2. Effect of phenanthrene on particle selection 2.2.1. Sediments and glass bead tracers
Schooner Creek sediment was used for this study. This sediment was relatively clean, in terms of PAHs, but was high in organic matter. Glass beads were used as a particle
tracer. Glass beads from four stocks 1–20, 28–53, 53–74, and 88–105 mm, specific
23
gravity 5 2.42 g cm , Cataphote Corporation, Jackson, MS were sieved and re-
combined to give roughly equal numbers of beads in consecutive 15-mm size intervals: , 15, 15–30, 30–45, 45–60, 60–75, 75–90, and 90–105 mm. The bead mixture was
soaked in laboratory glassware detergent, rinsed thoroughly, boiled in one change of deionized water, rinsed twice with acetone, and finally rinsed with deionized water.
Approximately 1:1 w w of sediment and beads were placed into six 250 ml glass flasks. The mixtures were equilibrated on an orbital shaker 100 rpm with filtered
seawater 1.2 mm glass microfibre filter, Whatman grade GF C for 24 h. Different concentrations of phenanthrene Sigma, St. Louis, MO, . 96 HPLC were prepared in
acetone to achieve nominal PAH concentrations of 0, 1, 5, 10, 50, and 100 mg g
21
sediment . The phenanthrene solution was added to the sediment slurry in each flask
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.-Y. Horng, G.L. Taghon J. Exp. Mar. Biol. Ecol. 242 1999 41 –57
and shaken 100 rpm for 24 h. To avoid interference of dissolved and particulate phenanthrene, sediment–bead mixtures were rinsed twice with filtered seawater follow-
ing the equilibration. Samples were taken from each mixture for quantifying phenan- threne concentration and for estimating the size distribution of particles.
2.2.2. Selection experiment Juvenile worms 4–5 weeks old were used in this experiment. After measuring its
size, each worm was randomly assigned to a glass vial 20 ml, 2.5 cm in diameter containing 2 g wet wt test sediment and 10 ml seawater 32‰, 1-mm filtered. Five
vials were prepared for each treatment. Vials were kept in a 158C environmental chamber for 7 days. Worms, pellets, and residual sediments were subsequently separated by
sieving on a 90-mm mesh.
Particle sizes in ambient sediment and fecal pellets were measured using different procedures. Ambient sediment samples | 0.5 g were placed into small test tubes with 2
ml 30 w v hydrogen peroxide, then placed into an ultrasonic bath. Following disaggregation, each tube was vigorously mixed on a vortex mixer. A 50-ml aliquot was
immediately withdrawn from the middle of the tube. The particle sample was then placed on a micro-slide with a few drops of 50 glycerol solution. Particles were spread
evenly by camel-hair brush. Three slides were prepared for each sediment sample. For fecal pellet samples, ten pellets were haphazardly picked from the group of pellets
produced by each individual worm total of 50 pellets per treatment. Each pellet was transferred to a depression micro slide. After measuring the major and minor axes of the
pellet under a dissecting microscope, the pellet was disaggregated by adding a few drops of 30 hydrogen peroxide and letting the reaction proceed for at least 24 h.
Glass beads were counted and measured using a compound microscope Zeiss Axioplan interfaced with a computer-based image analysis system Sony DXC-750
image controller, Sony 3CCD camera, Macintosh IICi computer. On the microscope, a blue light filter, 250 3 magnification, and phase-contrast optics were used. For the
ambient sediment samples, at least ten fields and a minimum of 100 glass beads were measured on each slide. These ten fields were arbitrarily spread out to cover more area
on the slide. For the pellet samples, the whole slide was scanned. The maximum and minimum diameters of glass beads were measured ColorImage v1.36, National
Institutes of Health and used to calculate the mean diameters.
2.2.3. Data analysis Particles were classified into seven size classes from , 15 to 90–105 mm, each
spanning a 15-mm interval. In addition to analyzing data with the G-test and adjusted residuals, the log of the odds ratio LOR was used as the selectivity index for
estimating the strength of selection Cock, 1978 and testing the treatment effect with a multiple regression of LOR against particle sizes Self and Jumars, 1988. The LOR is a
symmetric index, with positive values indicating preference for the particle and negative values indicating avoidance. A value of 1 indicates that a particle is taken ten times more
frequently than would occur with no selection.
Multiple regression and analysis of variance procedures were used to examine the
C .-Y. Horng, G.L. Taghon J. Exp. Mar. Biol. Ecol. 242 1999 41 –57
47
effect of phenanthrene concentration using a particle preference model modified after Self and Jumars 1988:
2
LOR 5 a 1 b log D 1 c log
D 1 d C 1
e s
d
s d
10 10
phenanthrene
where D is the mean test particle diameter, C is the concentration of
phenanthrene
phenanthrene in sediment, and e is the residual error. Self and Jumars 1988 used
geometric mean diameter within a size interval to represent the mean size of the particles within that size range. The use of the geometric mean presumes a lognormal size
distribution, which is largely true for natural sediments. For this selectivity experiment, the glass bead mixture was made from four types of bead stocks, each distributed in a
rather narrow range about 20 mm. The size distributions within every 15-mm wide size category may not be continuous and lognormal. Therefore, the mean size of particles for
each size interval was estimated by pooling all size data collected from ambient sediments and fecal pellets, and then averaging every single particle within the size
range for the mean. The mean diameters for ,15, 15–30, 30–45, 45–60, 60–75, 75–90, and 90–105-mm intervals were 12.7, 20.7, 39.1, 51.3, 64.1, 86.6, and 94.2 mm,
respectively. The method of least-squares was used to determine the values of the coefficients a, b, c, and d in the multiple curvilinear model. The most preferred particle
size and its variance were estimated as in Self and Jumars 1988.
3. Results
