A . Bennett et al. J. Exp. Mar. Biol. Ecol. 242 1999 1 –20
3
diversity of primary producers i.e., vascular plants, microphytobenthos, phytoplankton, and macroalgae, resulting in a heterogeneous mixture of organic matter sources in
coastal sediments. Plant pigment biomarkers have been widely used to identify different sources of algal organic matter in aquatic systems and to differentiate trophic dynamics
in aquatic food webs Mantoura and Llewellyn, 1983; Bianchi et al., 1993, 1996. Chlorophyll-a is used to determine general algal biomass, while carotenoids are more
class specific Wright et al. 1991. For example, the carotenoid fucoxanthin is a biomarker for the presence of diatoms, while zeaxanthin is a biomarker for cyano-
bacteria Wright et al., 1991; Bianchi et al., 1996. Phaeopigments, such as phaeophytins and phaeophorbides, are the decay products of chlorophylls; phaeophorbides are
typically used as markers for metazoan grazing Schuman and Lorenzen, 1975; Welschmeyer and Lorenzen, 1985; Bianchi et al., 1988, 1995; Bennett et al., 1999.
Thus, plant pigments can be used to determine the composition and heterotrophic consumption of benthic algal sources in wetland ecosystems.
Louisiana wetlands represent 41 of the coastal wetlands in the US Turner and Gosselink, 1975; Turner, 1997; many of these wetlands have been chronically exposed
to petroleum-hydrocarbon pollution from oil exploration in the Gulf of Mexico Fang, 1990. In particular, coastal salt marshes of Pass Fourchon, LA, were subjected to
produced water discharge from the petrochemical pumping processes of Chevron until 1994, creating a dramatic PAH gradient over a very short distance ca. 1 km Rabalais
et al., 1991; Means and McMillin, 1993; Means, 1995. Carman et al. 1997 suggested that there was an enhancement of microphytobenthic abundance due to reduced
meiofaunal grazing in PAH-contaminated sediments from Pass Fourchon. However, to fully understand the complexity of parameters that control the abundance and com-
position of microphytobenthos in Pass Fourchon sediments, further work is needed on the interactive effects of contaminants, nutrients, and macrofaunal grazing.
In this study, the effects of PAH contamination and macrofaunal grazing on the abundance and composition of microphytobenthos in sediments were investigated using
a laboratory microcosm experiment. This laboratory experiment was conducted in conjunction with a 2-year field study in an effort to understand the effects of PAH
contamination on the salt marsh at Pass Fourchon, LA Bennett et al., 1999. Our overall hypothesis was that PAH concentrations in sediments influence the classes and relative
abundances of microphytobenthos, which affect the trophic and population dynamics of the epibenthic gastropod herbivore Littorina irrorata. Thus, the objectives for the
microcosm portion of the study were to determine the effects of PAH concentration and grazing by L
. irrorata on the abundance and composition of microphytobenthos in sediments from Pass Fourchon, LA, and to determine the effects of PAH concentration
on the somatic growth and food resources of L . irrorata.
2. Materials and methods
2.1. Site description Sediments and organisms were collected at a field site located in a Spartina
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. Bennett et al. J. Exp. Mar. Biol. Ecol. 242 1999 1 –20
alterniflora salt marsh located between Bayou LaFourche and Bay Champagne, near Pass Fourchon, LA Fig. 1. This location is in close proximity to a field station owned
and operated by the Louisiana Universities Marine Consortium LUMCON. The tidal range at Port Fourchon is ca. 0.3 m and the salinity ranges from 4 to 26 ppt. Stations 1
and 3 represented the High- and Low-PAH contamination stations, respectively, that were described in a field study by Bennett et al. 1999. The range of total PAH
concentrations in sediments at Stations 1 and 3 between 1996 and 1997 were ca. 2 to 18 ppm and 0.01 to 0.1 ppm, respectively Bennett et al., 1999. Sediments at the
High-PAH site were more coarse 32 silt and clay than at the Low-PAH site 80 silt and clay Bachman, 1997. The marsh periwinkle L
. irrorata was used as a model macrofaunal species in this S
. alterniflora-dominated marsh due to its dominance as a macrofaunal epibenthic grazer in this region. A macroinfaunal species was not used in
Fig. 1. Map of study area at Pass Fourchon, LA, showing the three sampling stations from the field study.
A . Bennett et al. J. Exp. Mar. Biol. Ecol. 242 1999 1 –20
5
this study because macroinfauna do not represent a significant fraction of the benthic community in northern Gulf coastal systems.
2.2. Microcosm experiment A microcosm experiment was performed using a 2 3 3 3 9 factorial design, with two
sediment conditions, three snail conditions, and nine exposure times, resulting in the following treatments: Low- and High-PAH sediments without snails; Low- and High-
PAH sediments with snails collected from High-PAH sediments in the field Station 1; Low- and High-PAH sediments with snails collected from Low-PAH sediments Station
3. Five replicates of each treatment 30 total microcosms were maintained for a period of 60 days and nondestructively sampled on Days 0, 4, 12, 20, 28, 36, 44, 52, and 60.
Snails and sediments were collected from both the High-PAH site Stations 1a for sediments and 1b for snails and the Low-PAH site Stations 3a for sediments, and 3b for
snails. The experiment consisted of Low-Exposure snails from Station 3b in both High- and Low-PAH sediments Stations 1a and 3a, respectively and High-Exposure
snails from Station 1b in both sediment treatments. Controls for both sediment treatments were also used to examine microphytobenthic communities in the absence of
snails.
Sediments and snails were collected on July 1996 from field Stations 1 and 3 at Pass Fourchon, LA. Sediments were sieved 500 mm mesh to remove macrofauna and then
added to Petri dish microcosms of 14 cm diameter to a depth of approximately 1 cm. Meiobenthos, which represented a significant fraction of total benthic community
Chandler and Fleeger, 1983; Carman et al., 1997 in Pass Fourchon sediments, were not removed for the microcosm experiment. To prevent the escape of snails, each Petri dish
microcosm was wrapped with plastic screening and secured with a rubber band, with the screening extended from the bottom of the dish to a height of 30.5 cm, and was capped
with Petri dish lids. A portion of the screening was above the water line and provided ample space for snails to move in and out of the water.
Thirty Petri dishes were placed randomly in a flow-through water table. Ambient
marsh water of 13‰ salinity was filtered 5 mm to remove particles and continuously circulated through a wet table. Approximately 70 of the water was replaced every 8
days. Water temperature was maintained at approximately 288C, light penetration was 20
22 21
mmol m s
, pH was approximately 7.8, and the dissolved oxygen concentration was
21
8 mg l . Lighting from banks of 40-W fluorescent lights was cycled on an approximate
12:12 light dark cycle. Although light intensity was lower than that found in the field, an algal film was observed on microcosms surface sediments in the early stages of the
experiment, suggesting that light was not a limiting factor. Snails were depurated for 1 day prior to their addition to microcosms in order to
remove any residual gut contents remaining from the field, then added in groups of three to each of 20 microcosms; the remaining ten microcosms represented treatments with no
22
snails grazing controls. Snail density in the microcosms | 190 snails m was
22
slightly higher than the highest observed field population densities 5–140 snails m ,
Bennett et al., 1999. Sediments and snails were collected on Day 0 for PAH and
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. Bennett et al. J. Exp. Mar. Biol. Ecol. 242 1999 1 –20
pigment analysis, after which sediments were sampled on Day 4 and approximately every 8 days following for a period of 60 days. Snail wet-weights were measured at
every sampling period in order to monitor snail biomass changes in the four different L .
irrorata treatments. Sediment PAHs, snail gut pigments, and total sedimentary carbon and nitrogen were measured at the beginning Day 0, midpoint Day 28, and endpoint
Day 60 of the experiment. Snails removed at Day 28 were replaced by L . irrorata
from a nonstudy site in order to maintain consistent population density within each treatment.
2.3. PAHs Surface sediments 0–1 cm and whole snail tissues were extracted for PAHs
according to the methods of Means 1998. All PAH analyses were performed on a GC MS Hewlett-Packard 5890 gas chromatograph coupled to a Hewlett-Packard 5970B
Mass Selective Detector Carman et al., 1996; Means, 1998.
2.4. Total carbon and nitrogen Lyophilized sediments were prepared for organic carbon and nitrogen POC and
PON analyses by placing them in a glass desiccator along with a small beaker containing 25 ml of 12 N HCl for 24 h in order to remove inorganic carbon. Following
acid treatment, analyses for POC and PON were performed using a Carlo Erba NA1500 NCS system. Acetanilide 71.09 C and 10.36 N was used as a standard.
Reproducibility between replicate samples always differed , 5 for both C and N.
2.5. Plant pigments Surface sediments and dissected digestive tracts of L
. irrorata were lyophilized, weighed, and extracted for pigments with 100 acetone. Samples were sonicated at 40
W within a beaker of ice for 40 s and allowed to sit overnight at 2 48C Bianchi et al., 1995. Extracts were filtered through a Gelman cartridge filter 25 mm diam., 0.2 mm
pore size prior to analysis by HPLC.
Reversed-phase HPLC analysis was conducted according to the method of Wright et al. 1991 as modified by Bianchi et al. 1996. The Wright et al. 1991 method has
been shown to be the most effective means for separating over 50 carotenoids, chlorophylls and their derivatives, including elution of the isomers lutein and zeaxanthin.
High purity HPLC standards for chlorophylls-a and -b were obtained from Sigma, whereas standards for fucoxanthin and lutein were obtained from Hoffman LaRoche.
Total phaeophytins and phaeophorbides a and b were made in the laboratory according to procedures described by Welschmeyer 1994. Purity of all standards was spectro-
photometrically verified. Pigment identification was based on retention times of standards and confirmed with a Waters 996 spectral diode-array. All peaks were
quantified with calculated response factors using a Waters Millennium software package.
21
Detection limits of pigment concentrations were approximately 2 ng l ; replicate
n 5 3 and standard precision for pigments ranged between 3 and 6.
A . Bennett et al. J. Exp. Mar. Biol. Ecol. 242 1999 1 –20
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2.6. Statistical analyses Analysis of variance ANOVA analyses were conducted using the software STAT-
GRAPHICS Plus 2.0. Values of P , 0.05 were considered to be significant. When ANOVA differences were significant, the Fisher’s Least Significant Difference, LSD, a
posteriori multiple range test was used to detect differences among effects. A 2 3 3 3 9 repeated measures ANOVA design was used to detect significant differences among
sediment type, snail type, PAH concentration, and time effects in the microcosm experiment. Regressions and correlations were determined using both STATGRAPHICS
Plus 2.0 and SigmaPlot 1.02 software.
3. Results
