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Ecotoxicology and Environmental Safety 62 (2005) 376–382

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Effects of salinity on biomarker responses in Crassostrea rhizophorae(Mollusca, Bivalvia) exposed to diesel oil

Angela Zaccaron da Silvaa, Juliano Zanettea, Jaime Fernando Ferreirab, Joao Guzenskic,Maria Risoleta Freire Marquesa, Afonso Celso Dias Bainya,�

aLaboratorio de Biomarcadores de Contaminac- ao Aquatica e Imunoquımica, Departamento de Bioquımica, CCB, Universidade Federal de Santa

Catarina, 88040-900, Florianopolis, SC, BrazilbLaboratorio de Moluscos Marinhos, Departamento de Aquicultura, CCA, Universidade Federal de Santa Catarina, 88040-900,

Florianopolis, SC, BrazilcEmpresa de Pesquisa Agropecuaria e Extensao Rural de Santa Catarina, 88034-901, Florianopolis, SC, Brazil

Received 8 October 2004; received in revised form 10 December 2004; accepted 10 December 2004

Available online 25 January 2005

Abstract

Crassostrea rhizophorae is a euryhaline oyster that inhabits mangrove areas, which are widely distributed along the Brazilian

coast. The aim of this study was to investigate the effects of salinity (9, 15, 25, and 35 ppt) on the activities of glutathione S-

transferase (GST), glucose 6-phosphate dehydrogenase (G6PDH), catalase (CAT), and acetylcholinesterase (AChE) in the digestive

gland of this species after exposure to diesel oil for 7 days at nominal concentrations of 0.01, 0.1, and 1mlL�1 and after depuration

for 24 h and 7 days. GST activity increased in a diesel oil concentration-dependent manner at salinities 25 and 15 ppt and remained

slightly elevated even after depuration periods of 24 h and 7 days. No changes were observed in the activities of G6PDH, CAT, and

AChE in the oysters exposed to diesel and depurated. Based on these results, GST activity in the digestive gland of C. rhizophorae

might be used as a biomarker of exposure to diesel oil in sites where the salinity is between 15 and 25 ppt, values usually observed in

mangrove ecosystems.

r 2005 Elsevier Inc. All rights reserved.

Keywords: Mangrove oyster; Crassostrea rhizophorae; Diesel; Salinity; Glutathione S-transferase; Biomarkers

1. Introduction

Coastal regions are frequently impacted by humanexploration of natural resources. Many foreign organicand inorganic compounds may enter the marineenvironment through discharge of domestic sewageand industrial effluents, harbor activities, and applica-tion of biocides, deteriorating the quality of the aquaticenvironment. Polycyclic aromatic hydrocarbons(PAHs) are present in the marine environment due totheir widespread occurrence in petroleum, coal, soot,air pollutants, and oil spillages (Walker et al., 1996), and

e front matter r 2005 Elsevier Inc. All rights reserved.

oenv.2004.12.008

ing author. Fax: +5548 331 9672.

ess: bainy@mbox1.ufsc.br (A.C.D. Bainy).

carcinogenic properties have been associated withthe exposure to these compounds (Reynaud et al.,2002).

The impact of these contaminants in biologicalsystems can be evaluated through biomarkers relatedto xenobiotic biotransformation and excretion mechan-isms. Biotransformation of xenobiotics may enhance theproduction of reactive oxygen species (ROS) andelectrophilic intermediates derived from the parentchemical. These intermediates can be conjugated withendogenous molecules such as reduced glutathione(GSH) through the action of glutatione S-transferase(GST) (Livingstone, 1985). This conjugation mechanismproduces more hydrophilic xenobiotics yielding expel-lable metabolites (Mannervik and Danielson, 1988).

ARTICLE IN PRESSA. Zaccaron da Silva et al. / Ecotoxicology and Environmental Safety 62 (2005) 376–382 377

Cellular antioxidant defenses, such as the enzymessuperoxide dismutase (SOD), catalase (CAT), andglutathione peroxidase, and free radical scavengers,such as vitamins C and E, carotenoids, and GSHamong others (Stegeman et al., 1992), protect thecell against oxidative stress, inactivating the producedROS and/or repairing oxidized biomolecules.Glucose-6-phosphate dehydrogenase (G6PDH) is aregulatory enzyme of the pentose phosphateshunt, which produces NADPH for xenobiotic bio-transformation and to recycle oxidized glutathione(GSSG) to its reduced form (GSH) through a reactioncatalyzed by glutathione reductase (GR). Acetylcholi-nesterase (AChE) is a serine hydrolase found inneuromuscular junctions that decomposes acetylcholinein the synaptic cleft (Galloway et al., 2002). AChEactivity has been widely used as a biomarker of exposureto and effects of organophosphates and carbamates inaquatic organisms. Payne et al. (1996) showed thatAChE is inhibited by aromatic hydrocarbons andsuggested the use of the activity of this enzyme tomonitor the exposure of aquatic organisms to thesecompounds.

The International Council for the Exploration of theSea (ICES) has proposed the use of antioxidant andbiotransformation enzymes and the inhibition of AChEas biomarkers of exposure to xenobiotics. Theseparameters have been studied in mussels (Livingstoneet al., 1985; Le Pennec and Le Pennec, 2003) and oysters(Alves et al., 2002; Niyogi et al., 2001a). Bivalves aresessile, filter-feeding organisms found in coastal andestuarine zones, which bioaccumulate chemical com-pounds present in the surrounding seawater; this is thereason that they are used as sentinel organisms inbiomonitoring programs (Viarengo and Canesi, 1991;Bainy et al., 2000; Cheung et al., 2001; Oliver et al.,2001; Gowland et al., 2002).

The mangrove oyster Crassostrea rhizophorae is aeuryhaline osmo-conformer bivalve widely distributedalong the Brazilian coast that has been proposed as arelevant biomonitor of environmental contamination intropical systems (Nascimento et al., 1998; Wallner-Kersanach et al., 2000; Monserrat et al., 2002; Rebelo etal., 2003). They are found attached to mangrove rootsand coastal rocks (Queiroz and Junior, 1990) wheredrastic changes of salinity may occur, determining theirdistribution and affecting their structural and functionalproperties (Dame, 1996). Therefore, examining theeffects of salinity on the toxicity of water-bornecontaminants is essential in assessing the risk ofexposure of estuarine organisms to these compounds(Wang et al., 2001).

The aim of this study was to assess the effects ofsalinity on the activities of GST, G6PDH, CAT, andAChE in digestive glands of C. rhizophorae exposed todiesel oil.

2. Material and methods

2.1. Animals

Mangrove oysters, C. rhizophorae (average lengthbetween 60 and 80mm), were sampled at an oysterfarming area at Sambaqui beach (Laboratorio deMoluscos Marinhos, Departamento de Aquicultura,CCA, UFSC) in Florianopolis, Santa Catarina State,Brazil. The animals were collected, cleaned, andtransported to the laboratory where the experimentswere carried out. No sex identification was performedfor these organisms.

2.2. Experimental exposure of the oysters

The oysters were divided into 16 20-L aquaria,containing filtered seawater disinfected through UVtreatment, with constant aeration and temperature at2372 1C (mean7SD). The animals were divided intofour groups of 4 aquaria. Each aquarium containedinitially 20 oysters. Each group was gradually accli-mated to the experimental salinities of 35, 25, 15, and9 ppt during a period of 16 days. After the acclimationperiod, 5 oysters from each salinity group were killed foranalysis. The remaining oysters, kept in salinities of 35,25, 15, and 9 ppt, were exposed to diesel oil at nominalconcentrations of 0.01, 0.1, and 1ml L�1 for 7 days. Thediesel oil was bought at a commercial gas station fromPetroleo Brasileiro S.A., with a certified quality control.The diesel oil was directly added to water and mixedthroughout for 5min. The oysters were immediatelyplaced in the aquarium and remained immersed duringthe whole period of exposure. Control groups, withoutdiesel addition, were set up in all salinity groups. Afterthe 7-day exposure period, the oysters exposed toincreasing diesel oil concentrations, at different sali-nities, were killed for biochemical analysis. Other groupsof oysters that were exposed to diesel were transferred toaquaria containing clean water at different salinities fora 24-h and a 7-day depuration period, when theorganisms were subsequently killed for analysis.

During the acclimation, exposure, and depurationperiods, the oysters were fed with Isochrysis sp. andChaetoceros muelleri at a density of 1� 105 cells peroyster twice a day and the water was renewed daily.

2.3. Sample preparation and biochemical assays

At all sampling periods, the digestive gland from eachorganism was dissected out and frozen individually inliquid nitrogen. The tissues were homogenized (1:4 v/v)with a tissue tearor (Biospec Prod. Inc.) in buffer(20mM Tris–HCl, pH 7.6, containing 1mM EDTA,0.5M sucrose, 1mM dithiothrectol, 0.15M KCl, and0.1M phenylmethylsulfonyl fluoride). The homogenate

ARTICLE IN PRESSA. Zaccaron da Silva et al. / Ecotoxicology and Environmental Safety 62 (2005) 376–382378

was centrifuged at 9000g at 4 1C for 30min. Thesupernatant fractions were stored at �80 1C.

Glutathione S-transferase (EC 2.5.1.18) activity wasestimated as described by Habig and Jakoby (1981),adapted to a 96-well microplate reader (Tecan Sunrise).The assay was performed using 1-chloro-2,4-dinitroben-zene (1mM) and reduced glutathione (1mM) inpotassium phosphate buffer (0.1M), pH 7.0. Theactivity was monitored for 2min at 340 nm. Glucose 6-phosphate dehydrogenase (EC 1.11.49) activity wasmeasured according to Glock and McLean (1953) withadaptations to a 96-well microplate reader. The methodis based on the transformation of glucose 6-phosphateby the enzyme through the reduction of NADP+ intoNADPH, which is monitored for 2min at 340 nm.Catalase (EC 1.11.1.6) activity was measured asdescribed by Beutler (1975), but the hydrogen peroxidedecomposition was monitored at 240 nm for 1min.Acetylcholinesterase (EC 3.1.1.7) activity was deter-mined according to Ellman et al. (1961), using 5,50-dithiobis-2-nitrobenzoate (0.01M, pH 8.0) and acet-ylthiocholine (0.3mM) as previously adapted by Mon-serrat et al. (2002). The reaction was monitored at412 nm for 2min at 25 1C. The protein content wasdetermined according to Peterson (1977), using bovineserum albumin as standard.

2.4. Statistical analysis

A normal probability test was used to test the datanormality. The influences of salinity and diesel oil

Fig. 1. Specific activity of GST at salinities 35, 25, 15, and 9 ppt. Each bar i

and numbers of individuals are given between brackets. Data are presented a

between the groups. #Indicates statistical difference between the control group

15, after the exposure period.

exposure on the biochemical parameters were evaluatedusing two-way analyses of variance (Po0:05), followedby multiple average comparisons according to the Tukeytest, for unequal sample sizes (Zar, 1984). StatSoftStatistica 5.1 was used to perform the tests.

3. Results

Oyster mortality was observed only in the groupexposed to the highest diesel oil concentration(1ml L�1). Four oysters kept at salinity 35 ppt diedduring the oil exposure, two died during the 24-hdepuration period, and three died during the 7-daydepuration period. At salinity 15 ppt, one oyster diedduring the exposure period. No mortality was observedin the oysters exposed to diesel oil and further depuratedin salinities 9 and 25 ppt.

Fig. 1 shows the GST activity in the digestive gland ofoysters sampled after the acclimation, exposure, anddepuration periods, at different salinities. The controloysters from the diesel exposure groups at salinities 25and 15 ppt that were killed after 7 days showedsignificantly lower GST activity than the oystersanalyzed after the acclimation period of 16 days. Thecontrol oysters kept at salinity 9 ppt and killed after theexposure period showed higher GST activity than theoysters kept at higher salinities (Fig. 1). The oystersexposed to increasing diesel oil concentrations that werekept at salinities 15 and 25 ppt showed significantincreases in GST activity compared to that of the

ndicates nominal diesel concentration used during the exposure period

s mean7standard deviation. *Indicates statistical difference (Po0:05)kept at alinity 9 ppt and the control groups kept at salinity 35, 25, and

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Fig. 2. Specific activity of G6PDH at salinities 35, 25, 15, and 9 ppt. Each bar indicates nominal diesel concentration used during the exposure period

and numbers of individuals are given between brackets. Data are presented as mean7standard deviation.

Fig. 3. Specific activity of CAT at salinities 35, 25, 15, and 9 ppt. Each bar indicates nominal diesel concentration used during the exposure period

and numbers of individuals are given between brackets. Data are presented as mean7standard deviation.

A. Zaccaron da Silva et al. / Ecotoxicology and Environmental Safety 62 (2005) 376–382 379

controls (Fig. 1). After both 24-h and 7-day depurationperiods, the GST activity in the oysters kept at salinities15 and 25 ppt were still elevated but not statisticallydifferent from that of the controls (Fig. 1). GST activityof oysters exposed to increasing diesel oil concentra-tions, which were kept at salinities 35 and 9 ppt, was notstatistically different from that of the controls (Fig. 1).

Activities of G6PDH, CAT, and AChE in thedigestive glands of oysters exposed to the various dieselconcentrations (0.01, 0.1, and 1ml L�1) in all testedsalinities were not different from those of the controls(P40:05) (Figs. 2–4). Likewise, no changes in the

activities of G6PDH, CAT, and AChE were observedin the organisms analyzed after both periods ofdepuration at different salinities (Figs. 2–4).

4. Discussion

The influence of salinity in biomarker responses isespecially relevant in euryhaline mollusks, such asC. rhizophorae, which has been proposed as sentinelorganism in biomonitoring programs in mangroveand estuarine zones (Nascimento et al., 1998;

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Fig. 4. Specific activity of AChE at salinities 35, 25, 15. and 9 ppt. Each bar indicates nominal diesel concentration used during the exposure period

and numbers of individuals are given between brackets. Data are presented as mean7standard deviation.

A. Zaccaron da Silva et al. / Ecotoxicology and Environmental Safety 62 (2005) 376–382380

Wallner-Kersanach et al., 2000; Monserrat et al., 2002;Rebelo et al., 2003). Frequently these areas receivecontaminant inputs associated to domestic and indus-trial effluent discharges and elevated stormwater runoff.In addition, these factors under low tides can signifi-cantly contribute to decrease the salinity where theorganisms live.

Field studies have shown that the concentrations ofhigh-molecular-weight PAHs were related to the in-crease in GST activity in the digestive gland of themussel Perna viridis (Cheung et al., 2001) and in Mytilus

edulis (Gowland et al., 2002). In an in vitro model, LePennec and Le Pennec (2003) observed that cells fromthe digestive gland of Pecten maximus, incubated withPAHs for 96 h, showed significant increases in GSTactivity up to 48 h upon exposure. Longer periods ofincubation were followed by lower enzymatic activityand elevated cell death compared to the nontreated cells.In the present study, oysters acclimated to salinities of25 and 15 ppt and exposed to different concentrations ofdiesel for 7 days showed concentration-dependentincreases in GST activity. This effect was not seen inthe oysters acclimated and kept at salinities of 35 and 9ppt. According to Castro et al. (1985), C. rhizophorae

shows higher filtration rates at salinities 20 and 25 ppt,with values of 1.33 and 1.43L/h, respectively, attemperature of 24 1C. Lower filtration rates of 0.85and 0.4 L/h were observed at salinities 10 and 35 ppt,respectively. Based on this study, we could suggest thatthe oysters acclimated to salinities 25 and 15 pptdisplayed higher filtration rates. An increase in theenergy availability supplied by feeding could provideadequate conditions for efficient xenobiotic conjugation

reactions. It is worth pointing out that the oysters werefed with microalgae daily throughout the experiment.On the other hand, we cannot discard the possibilitythat higher filtration rates, observed at salinity 25 ppt,imply higher PAH exposure, possibly contributing tothe observed elevation in the GST activity. PAHanalysis of the tissues of the exposed oysters could testthis hypothesis; however, due to technical problems wewere unable to perform such an analysis.

The elevation of GST activity in the diesel-oil-exposedoysters could also be related to its function in thedefense against oxidative stress (Fiander and Schneider,1999; Amicarelli et al., 2004), reducing organic hydro-peroxides possibly formed during the biotransformationof diesel oil compounds. This is consistent with the factthat no changes in the levels of lipid peroxidation wereobserved in the oysters acclimated to 25 ppt and exposedto diesel oil, but higher levels of this parameter wereobserved in the organisms exposed at salinities 9, 15,and 35 ppt (J. Zanette, unpublished data).

The lack of GST response in the digestive gland ofoysters exposed to diesel oil that were kept at salinitiesof 35 and 9 ppt might be attributed to a greater energeticcost for these organisms to adapt to hyper- andhypoosmotic conditions of the external environment,respectively. Another possibility would be lower con-taminant uptake, since in these salinities the filtrationrates might have been decreased. According to Dame(1996) and Gosling (2003), bivalves may immediatelyclose their valves when the surrounding salinity ischanged as a strategy of protection against osmoticstress. This behavior has been observed by differentauthors who demonstrated that at low salinities animals

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such as the brown mussel Perna perna (Salomao et al.,1980) and the tropical clam Anomalocardia brasiliana

(Leonel et al., 1983) are able to isolate themselves fromthe surrounding media using this mechanism. Based onthese data, we could suggest that PAHs availability tothe oysters could be affected by valve closure. Moreover,it is plausible to consider that the absence of a significanteffect in the GST response in diesel-oil-exposed oystersthat were maintained at 9 ppt could be also associated tothe elevated levels of GST activity observed in thecontrol oysters kept in this salinity. The etiology of thisresponse remains to be clarified.

During the experiment, death was observed in theoysters exposed to 1ml L�1 and kept at salinity 35 ppt.This observation could be associated with a decreasedcapacity to conjugate xenobiotics in these animals,which would lead to a rise in the levels of intracellulartoxic metabolites and disrupt the homeostasis of theorganisms.

No significant changes in the G6PDH activity in thedigestive gland of the oysters exposed to diesel oil for 7days at different salinities were seen. G6PDH is the mainregulatory enzyme in the pentose phosphate shunt whichproduces NAPDH, required for the maintenance of thecellular antioxidant capacity (Sies, 1993), fatty acidbiosynthesis (Bayne, 1976), and biotransformation ofxenobiotics (Timbrel, 1991). NADPH donates electronsto reduce GSSG through the enzyme GR, recyclingGSH, required for the GST activity. Considering thatno changes in the G6PDH activity in the digestive glandwere seen, we could hypothesize that the levels ofcellular NAPDH were not affected during the exposureperiod at different salinities. In fact, the increases in theGST activity at salinities 25 and 15 ppt suggest that theGSH availability was not limited during the exposure.However, when the oysters were exposed to diesel atsalinities 35 and 9 ppt, a lack of GST response wasobserved. This result could be related to lower GRactivity, which could decrease GSH recycling capacity.However, this enzyme was not analyzed in the presentstudy. Previous studies carried out in the liver of tilapia(Oreochromis niloticus) caught at a site contaminated bydomestic and industrial effluents showed lower activitiesof both G6PDH and GR together with a smaller GSHcontent compared to those of noncontaminated refer-ence fish (Bainy et al., 1996).

CAT activity in the digestive gland of the oysters wasnot significantly different in any diesel-exposed groupcompared to that of controls. Considering that CAT isan important antioxidant enzyme that decomposeshydrogen peroxide that is produced in larger amountsduring the biotransformation process, we could suggestthat this antioxidant system was not affected byexposure to diesel, at least in the tested concentrations.However, this assumption must be made with cautionsince some studies have shown positive relationships

between CAT activity and PAH levels in the digestivegland of the oyster Saccostrea cucullata (Niyogi et al.,2001a), in the digestive tissue of the barnacle Balanus

balanoides (Niyogi et al., 2001b), and in the gills of themussel P. viridis (Cheung et al., 2001). Cheung et al.(2001) observed an inverse correlation between CATand hydrocarbons in the digestive glands of the sameanimals. Krishnakumar et al. (1997) did not observecorrelations between CAT activity and PAHs indigestive glands of M. edulis exposed to microencapsu-lated PAHs for periods of 6 and 30 days. These apparentconflicting results could be partially explained by eitherspecies- or tissue-specific factors or by abiotic factors,such as the period of exposure, the method of analysis,and the PAH composition.

AChE inhibition has been extensively used as abiomarker of exposure to and effects caused bypesticides, especially organophosphates and carbamates(Bocquene et al., 1997). Some authors have observedAChE inhibition in mussels caused by exposure to heavymetals (Najimi et al., 1997), and domestic effluents andpetroleum (Payne et al., 1996). El-Alfy and Schlenk(1998) observed the inhibition of AChE activity in theeuryhaline fish Oryzias latipes exposed to aldicarb andkept at higher salinities. In the present study, neither asignificant inhibitory response nor an elevation in theAChE activity was seen upon exposure to diesel atdifferent salinities. This observation indicates that thecholinergic transmission system in the digestive gland ofthis species was not affected by the diesel exposure andthat AChE does not seem to be an adequate biomarkerfor this purpose.

After the 24-h and 7-day depuration periods, the 1-ml L�1 diesel-exposed oysters kept at salinities 25 and15 ppt showed decreases in GST activity compared tothe groups exposed for 7 days to 1ml L�1 diesel at thesame salinities (Fig. 1). This decrease might beassociated with lower levels of intracellular toxicmetabolites due to an efficient excretion of conjugatesduring depuration.

In addition to the conjugation systems, it is possiblethat the monoxygenases were also activated in theoysters during the diesel exposure and depurationperiods. A similar study showed elevated cytochromeP450 content in the mussel M. edulis exposed to dieselfor 4 months, but after 8 days of depuration theexpression of these proteins was not different from thatof the controls (Livingstone et al., 1985). Wallner-Kersanach et al. (2000) showed that the oyster C.

rhizophorae, exposed for 60 days to a site contaminatedby trace metals and depurated for 30 days, eliminated35% of absorbed lead.

In conclusion, the observed dose-dependent increasesin GST activity in oysters exposed to diesel at salinities15 and 25 ppt, followed by decreases in GST activityafter depuration, suggest that GST activity can be used

ARTICLE IN PRESSA. Zaccaron da Silva et al. / Ecotoxicology and Environmental Safety 62 (2005) 376–382382

as a biomarker of exposure to diesel in mangrove andestuarine areas.

The lack of changes in the activities of G6PDH, CAT,and AChE in this study support the idea that theactivities of these enzymes in the digestive gland of theoyster C. rhizophorae are not suitable biomarkers of anacute exposure to diesel at the concentrations tested. Itis possible that additional studies using differentexposure periods and diesel oil concentrations wouldproduce different results.

Acknowledgments

This work was supported by grants from BrazilianAgency CNPq-CTPetro No. 474513/01-7 and Plano Sulde Pesquisa e Pos-Graduac- ao No. 520741/99-4. A.B. is aresearch fellow from CNPq. The authors thank Dr.Paulo S.M. Carvalho for revising the manuscript andthe two anonymous referees for the excellent suggestionsto improve the quality of the manuscript.

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