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Chemical and Physiological Measures on Oysters (Crassostrea virginica) from Oil-Exposed Sites in LouisianaAuthor(s): Thomas M. Soniat, Sarah M. King, Matthew A. Tarr and Megan A. ThorneSource: Journal of Shellfish Research, 30(3):713-717. 2011.Published By: National Shellfisheries AssociationDOI: http://dx.doi.org/10.2983/035.030.0311URL: http://www.bioone.org/doi/full/10.2983/035.030.0311

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CHEMICAL AND PHYSIOLOGICAL MEASURES ON OYSTERS (CRASSOSTREA

VIRGINICA) FROM OIL-EXPOSED SITES IN LOUISIANA

THOMASM. SONIAT,1,2* SARAHM. KING,

3MATTHEWA. TARR

3ANDMEGANA. THORNE

1

1Department of Biological Sciences; 2Pontchartrain Institute for Environmental Sciences; 3Department ofChemistry, University of New Orleans, New Orleans, LA 70148

ABSTRACT Potential lethal and sublethal effects of oil from the Deepwater Horizon spill to oysters (Crassostrea virginica)

in Louisiana east of the Mississippi River were examined along a biophysical gradient of oil pollution, salinity, and disease.

Approximately 6 mo after the capping of the Deepwater Horizon wellhead, no polycyclic aromatic hydrocarbons were detected in

oysters from oil-exposed sites. Variations in oyster condition and reproductive state, and infection with the oyster parasite

Perkinsus marinus are consistent with natural differences along the salinity gradient and not with impacts of polycyclic aromatic

hydrocarbon contamination.

KEY WORDS: oil, polycyclic aromatic hydrocarbons, Crassostrea virginica, oysters, condition, reproduction, disease, Perkinsus

marinus, Louisiana

INTRODUCTION

OnApril 20, 2010, an explosion on theDeepwater Horizon oil

well, located 80 km off the coast of Louisiana, triggered a human,economic, and environmental disaster.When the well was cappedon July 15, 2010, nearly 5 million barrels of oil had been released

into the offshore waters (National Commission on the BPDeepwater Horizon Oil Spill and Offshore Drilling 2011), anddemonstrated the risk of offshore oil activities to inshore oysterpopulations.

Oysters are a priority species for study because of potentialimpacts to an economic resource used for human consumption,and because they are biomonitors that can in turn be used to

access biological and environmental impacts. The utility ofoysters as environmental sentinels is associated with their sessileand filter-feeding habit. Because of their immobility, they are

more likely to be in constant contact with pollutants, thusmaking them more contaminated than fish and other mobilespecies (Milan & Whelan 1978). As filter feeders, they integrateenvironmental conditions over time and, because they have

a limited capability of metabolizing contaminants, can increasethe concentration of contaminants in their tissues abovebackground levels found in the ambient water (Capuzzo 1996).

Biological responses to hydrocarbon contaminants havebeen observed from the cellular through the community levelsof organization (McIntyre & Pearce 1980, Moore et al. 1989,

Capuzzo 1996). Organismal effects include enhanced suscepti-bility to disease and diminished reproductive effort, whereaspopulation effects include altered age–size structure, decreased

recruitment, and increased mortality (Capuzzo 1996). Poly-cyclic aromatic hydrocarbons (PAHs) are hydrocarbon con-taminants of special concern because of their ability tobioaccumulate in lipid-rich cells and tissues of bivalves (Stegeman

& Teal 1973, Capuzzo 1996), and potentially harm human con-sumers (Obana et al. 1981).

In April 1979, the Ixtoc I exploratory well in the Bay of

Campeche suffered a blowout. As a result, oil from the relativelyshallow well was released at the seafloor for almost 10 mo.

Coastal lagoons along the Bay of Campeche were partiallyimpacted by the Ixtoc spill, and oysters were shown to be a usefulindicator of oil contamination because of their effective bio-accumulation of these analytes (Botello et al. 1983). During the

Ixtoc spill, the presence of oil hydrocarbons, including PAHs,were observed in oysters in a lagoon impacted by the spill;however, the total hydrocarbon content of the oysters declined

substantially after the exposure ceased.The purpose of this study was to determine lingering impacts,

if any, of oil from theDeepwaterHorizon spill to oysters at sites in

Louisiana east of the Mississippi River. Special emphasis wasplaced on responses at the organismal level (gonad and egg de-velopment, susceptibility to disease), and at the population level

(age–size structure, mortality). These potential lethal and sub-lethal effects of PAHs on oysters along a biophysical gradient ofoil pollution, salinity, and disease were examined.

MATERIALS AND METHODS

Sampling was conducted from Lake Borgne and Mississippi

Sound in Louisiana on January 4, 2011. Position (GPS) and depth(SONAR) were determined using a Garmin 440SChartplotter/Fishfinder (Salem, OR). Because of concerns that the de-

composition of accumulated oil on the reef might promotehypoxia (Shelton & Hunter 1974), especially under conditions ofthermohaline stratification, top and bottom temperatures,salinities, and oxygen concentrations were measured (Yellow

Springs Instrument model 85 T/S/Ometer, Yellow Springs, OH).Two areas were sampled (Fig. 1A): an area in Lake Borgne

comprising stations 1–3 that was unaffected by oil (designated

area 1) and an area in Mississippi Sound comprising stations 4–6(designated area 2) that was exposed to oil (Fig. 1B). A minimumof 3 dredge samples were taken at each station. If insufficient

numbers of oysters were obtained, additional dredges were takenuntil at least 15 adult oysters (shell height, >75mm)were collected.Live oysters and dead oysters (boxes) were enumerated as spat

(0–24 mm), seed (25–74 mm), and adults. A subsample of 10oysters (>75 mm) from each station was used to determine oystercondition, Perkinsus marinus (Dermo) infection, percent female,and gonadal condition. The height of each oyster was measured.

All length measures (oyster height, adductor muscle diameter,*Corresponding author. E-mail: tsoniat@uno.edu

DOI: 10.2983/035.030.0311

Journal of Shellfish Research, Vol. 30, No. 3, 713–717, 2011.

713

gonadal width) were made using digital Vernier calipers. Oysters

were shucked and the adductor muscle diameter (AMD) wasmeasured. Shell weight (SW) and meat wet weight (MWW) weredetermined for each oyster and used to calculate a condition

index (CI), where

CI ¼ MWW=SWð Þ 3 100

(Baird & Drinnan 1957).

Gonadal width (GW) was measured on the left side of theoyster froma cross-sectionmade at the intersection of the gills andlabial palps. Gonadal index (GI) was calculated as

GI ¼ GW=AMDð Þ 3 100

(Soniat & Ray 1985).Each oyster was designated as female, male, hermaphrodite,

or unknown by blotting gonadal material onto a slide and ob-

serving it at 1003magnification. Percent female was calculatedfor each site. A piece of mantle tissue (�4 mm2) on the right sideof the oyster near the intersection of the gills and labial palps was

taken for Dermo analysis. After incubation in fluid thioglycolate

for about 7 days, the tissue was stained with Lugol’s iodine andobserved at 403 and 1003magnification (Ray 1966). Infectionof individual oysters was rated as a disease code number from

0 (uninfected) to 5 (heavily infected), using the scale of Craig et al.(1989). Disease metrics include percent infection, weightedprevalence (WP), and infection intensity (II), where WP is the

sum of disease code numbers divided by the number of oysters inthe sample, and II is the sum of disease code numbers divided bythe number of infected oysters.

For the PAH analysis, oysters were homogenized and the

PAHs were extracted using the Agilent QuEChERS method(Gratz et al. 2010). The extracts were filtered and diluted 1:1 withwater, and were analyzed using an Agilent 1100 high-performance

liquid chromatograph coupled to a HP 1046A fluorescence de-tector.Fluorescence experimentswere conductedonaPerkinElmerLS 55 luminescence spectrometer. To verify the extraction

procedure, purchased oysters were spiked with a 16-componentmixture of PAHs. Calibration curves were constructed and thepercent recoveries for each of the PAHs monitored were

Figure 1. (A) Map of collection sites. Area 1 contains sites 1, 2, and 3 (uncontaminated sites); area 2 contains sites 4, 5, and 6 (contaminated sites). (B)

The May 11, 2008, SCAT oiling ground observations. Oil contamination is reported as follows: heavy ( ), moderate ( ), light ( ), very light ( ), no oil

found ( ), trace < 1% (Environmental Response Management Application 2011).

SONIAT ET AL.714

calculated. Detection limits for the PAHswere 15 ng/g wet oysterweight. For the 16 PAHs, 13 had a recovery similar to that seen

in the study by Gratz et al. (2010). In addition, 11 of the 13 PAHrecoveries were 75% or more, which represents an acceptablerecovery for biological samples. The 2 PAHs with less than 75%

recovery were the larger PAHs—benzo[a]pyrene (74.3%) andbenzo[g,h,i]perylene (57.6%)—which are known to be difficult torecover.

RESULTS AND DISCUSSION

A comparison of top and bottom temperatures, salinities,and oxygen concentrations showed no evidence of stratifica-tion of the water column or depletion of oxygen (Table 1).

The mean salinity (top and bottom) of the oiled area (23.56ppt) was higher than the salinity of the unoiled area (11.25ppt). Mean top and bottom oxygen concentrations were

similar in the oiled and unoiled areas (9.34 ppm and 9.89ppm, respectively). Mean water depths at sites in the oiled andunoiled areas were both 3.0 m. No oil was visible in any of the

samples.The dredge survey was designed to evaluate wholesale oyster

mortalities and the presence or absence of visible oil; this was notdesigned as a quantitative survey. However, the presence and

relative abundance of spat, seed, and adult oysters in oiled andunoiled areas was noted. More oyster spat, and seed werecollected from the oiled area than from the unoiled area, whereas

more adult oysters were collected from the unoiled area than inthe oiled area. No boxes (articulated shells) of spat or seed were

found in either area, and only one adult box was found in eacharea, indicating little recent mortality. Furthermore, mortalitywas similarly low in the two areas. No oil was observed on the

water surface, in the water column, or on or in oysters.Oysters from the oiled area had a higher gonadal index, a

similar condition index, and higher percent females than oystersfrom the unoiled area (Table 2). Because the expectations of

the effects of contamination are diminished reproductivecapacity and condition, these results from sublethal measuresof stress can be explained as a response to salinity differences

and not pollution.No Dermo infection was found in oysters in the unoiled

area, in contrast to mean 43% infection, a 0.31 WP, and a 0.47

II of oysters in the oiled area. Although there is evidence that

TABLE 1.

Environmental conditions at oyster collection sites.

Area Site

Bottom Temperature

(�C)Bottom Salinity

(ppt)

Bottom Oxygen

(ppm)

Top Temperature

(�C)Top Salinity

(ppt)

Top Oxygen

(ppm) Depth (m)

1 1 10.5 9.0 10.2 10.5 8.9 9.9 2.9

1 2 10.4 12.8 9.5 10.5 9.7 10.15 2.9

1 3 10.5 15.0 9.4 10.4 12.1 10.21 3.2

2 4 10.9 17.8 9.64 11.3 17.5 9.84 4.3

2 5 10.6 25.3 9.23 10.6 24.8 9.27 2.3

2 6 11.2 28.4 9.08 11.1 27.6 8.98 2.8

Area 1 includes the unoiled sites (1–3), whereas area 2 includes the oiled sites (4–6).

TABLE 2.

Reproductive, condition, and disease metrics based on

composites of 10 oysters.

Area Site GI CI PF WP II PI

1 1 6.38 8.58 60 0 0 0

1 2 5.35 10.24 50 0 0 0

1 3 5.15 9.43 60 0 0 0

2 4 7.17 11.05 60 0 0 0

2 5 7.61 8.46 80 0.33 0.56 60

2 6 8.75 8.86 80 0.6 0.86 70

Area 1 includes the unoiled sites (1–3), whereas area 2 includes the oiled

sites (4–6). CI, condition index; GI, gonadal index; II, infection

intensity; PF, percent female; PI, percent infection ofPerkinsus marinus;

WP, weighted prevalence.

Figure 2. (A) LC-FLD chromatogram of the PAH standard (- - -),

uncontaminated (---), and contaminated samples (���). (B) Florescencescan of the 12-min peak: benzo[a]pyrene (---), purchased oyster (���),unoiled oyster (�-�), and oiled oyster (- - -).

CHEMICAL AND PHYSIOLOGICAL MEASURES ON OYSTERS 715

Dermo is exacerbated by pollutants such as hydrocarbons(Chu&Hale 1994, Bushek et al. 2007), in the absence of PAHs,

the elevation in disease in the oiled area is likely simply theresult of the well-known pattern of elevated disease at salinitiesmore than 20 ppt (Mackin 1962, Powell et al. 1996, Soniat1996).

Oiled and unoiled samples were analyzed for PAH content(Fig. 2A). It is evident from the chromatogram that 12 of the13 PAHs monitored were not found in either of the oyster

samples. A peak at 12 min coincided with the retention time ofbenzo[a]pyrene; however, this peak was also observed in theuncontaminated and reference oyster samples. To study this peak

further, the compound was collected and analyzed using fluores-cence spectroscopy (Fig. 2b). From the spectrait is obvious thatthe 12-min peak observed in the oyster extracts had a differentemission spectrum then the benzo[a]pyrene. The difference in the

spectra confirmed that the peak was not benzo[a]pyrene, but anextracted compound that was normally present in the oysters.Because the peak was observed in all the oyster samples, even

those with no history of oil contaminations, it was concluded tobe a component of the oysters.

The persistence of oil and its degradation products in the

marine environment is extremely variable, depending on thenature of the oil, exposure to sunlight, ambient water tempera-ture, and other factors (Blumer& Sass 1972, Corredor et al. 1990,

SenGupta et al. 1993, Owens et al. 2008, Short et al. 2007). Oil oflighter molecular weight fractions in lower latitudes and warmer

waters generally exhibits rapid degradation (Corredor et al.1990). In the current study, the emphasis was placed on the

persistence of PAHs in oysters. Neff et al. (1985) found PAHcontamination in oysters (Crassostrea gigas) 27 mo after theAmoco Cadiz oil spill. In contrast, Michel and Henry (1997)studied oyster PAH concentrations over 10 mo after an El

Salvador oil spill in which dispersants were also used. In thatstudy, PAHs were detected after 1 mo of exposure, but no PAHswere detected in oyster samples after 10 mo of exposure.

Approximately 6 mo after the capping of the DeepwaterHorizon wellhead, no PAHs were detected in oysters fromoil-exposed sites. Variations in oyster infection, condition,

and reproductive state are consistent with natural variationalong the salinity gradient, not impacts of PAH contamina-tion. Although no impact was observed in this study, wecaution the overapplication of the results of this spatially and

temporally limited study to other areas and other timeswhere impacts from the Deepwater Horizon spill may haveoccurred.

ACKNOWLEDGMENTS

This research was made possible by grant 10-BP GRI-UNO-01 from BP/The Gulf of Mexico Research Initiative. Field orlab assistance was provided by Brian Lezina, Chris Schieble,

Patrick Slattery, Ellen Isbell, Janice Jacobi, Alicia Wylie, andElisabeth Trinh.

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