Q2

lable at ScienceDirect

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55

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Contents lists avai

Marine Environmental Research

journal homepage: www.elsevier .com/locate/marenvrev

56575859606162636465666768697071727374757677787980818283

Ecological relevance of Sentinels’ biomarker responses: A multi-levelapproach

Camilo D. Seabra Pereira a,b,*, Denis M.S. Abessa c, Rodrigo B. Choueri a,Victor Almagro-Pastor d, Augusto Cesar a,b, Luciane A. Maranho d,María Laura Martín-Díaz d, Ronaldo J. Torres a, Paloma K. Gusso-Choueri c,e,João E. Almeida a, Fernando S. Cortez b, Antonio A. Mozeto f, Helcy L.N. Silbiger g,Eduinetty C.P.M. Sousa g, Tommas Angel Del Valls d, Afonso C.D. Bainy h

aDepartamento de Ciências do Mar, Universidade Federal de São Paulo, Av. Almirante Saldanha da Gama 89, 11030-490 Santos, SP, BrazilbDepartamento de Ecotoxicologia, Universidade Santa Cecília, Av. Oswaldo Cruz 266, 11045-907 Santos, SP, BrazilcNEPEA, Campus do Litoral Paulista, Universidade Estadual Paulista Júlio de Mesquita Filho, Praça Infante Dom Henrique, s/n,11330-900 Saão Vicente, SP, BrazildUniversidad de Cádiz, Polígono Río San Pedro s/n, 11510 Puerto Real, Cádiz, Spaine Laboratório de Toxicologia Celular, Departamento de Biologia Celular, Universidade Federal do Paraná, CP19031, 81531-990 Curitiba, PR, Brazilf Laboratório de Biogeoquímica Ambiental, Departamento de Química, UFSCar, Rod. Washington Luiz, 13565-905 São Carlos, SP, Brazilg Instituto Oceanográfico da Universidade de São Paulo, Praça do Oceanográfico, 191, 05508-900 São Paulo, SP, BrazilhDepartamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil

8485

8687888990919293949596

a r t i c l e i n f o

Article history:Received 31 July 2013Received in revised form10 November 2013Accepted 14 November 2013

Keywords:Caged bivalvesBiomarkersMacrobenthic communityEcological status

* Corresponding author. Departamento de Ciênciasde São Paulo, Av. Almirante Saldanha da Gama 89, 110fax: þ55 1335235094.

E-mail addresses: camilo.seabra@unifesp.br, camPereira).

0141-1136/$ e see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.marenvres.2013.11.002

979899

100101102103104

Please cite this article in press as: Seabra PeMarine Environmental Research (2013), http

a b s t r a c t

In response to the need for more sensitive and rapid indicators of environmental quality, sublethal effectson the lowest levels of biological organization have been investigated. The ecological relevance of theseresponses assumes a prevailing role to assure effectiveness as indicator of ecological status. This studyaimed to investigate the linkages between biomarker responses of caged bivalves and descriptive pa-rameters of macrobenthic community structure. For this purpose a multi-level environmental assess-ment of marine and estuarine zones was performed in São Paulo coast, Brazil. Multivariate analysis wasapplied to identify linkages between biological responses and ecological indices, as well as to charac-terizing the studied stations. Individuals of the marine mussel Perna perna caged along Santos Bayshowed signs of oxidative stress, lysosomal membrane destabilization, histological alterations andreduced embryonic development. The estuarine oyster Crassostrea rhizophorae caged along Santos PortChannel showed alterations on biotransformation enzymes and antioxidant system, DNA damage andlysosomal membrane destabilization. The benthic community analysis showed reduced richness anddiversity in the same areas of the Santos bay and estuary where biomarker responses were altered. Ourresults revealed that xenobiotics are inducing physiological stress, which may lead to changes of thebenthic community structure and deterioration of the ecological status over time. Integrating biomarkerresponses and ecological indexes improved certainty that alterations found at community level could berelated to xenobiotic as stressors, which was very useful to improve the discriminatory power of theenvironmental assessment.

� 2013 Elsevier Ltd. All rights reserved.

105106 107 108109110111

1. Introduction

Benthic macroinvertebrates have been investigated to assess theecological status of aquatic ecosystems. Different approaches

do Mar, Universidade Federal30-400 Santos, SP, Brazil. Tel./

ilo@unisanta.br (C.D. Seabra

ll rights reserved.

112113114115116

reira, C.D., et al., Ecological r://dx.doi.org/10.1016/j.maren

ranging from population-community level to sublethal responsesof sentinel species are the current basis for most biomonitoringprograms (WFD, 2000/60/EC; MSFD, 2008/56/EC).

Community based indices can detect ecologically relevant ef-fects that usually involve the eradication of one or several speciesfrom a particular site. Thus, such indices may diagnose distur-bances possibly caused by long-term exposure to environmentalpollutants. On the other hand, short-term responses on lower levelsof biological organization have been used to detect first signs of

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impairment caused by xenobiotics. These responses have beengenerally indicated as “biomarkers”, here defined as a biochemical,cellular, physiological or behavioral variation that can be measuredin tissue or body fluid samples, or at the level of whole organismthat provide evidence of exposure to and/or effects of, one or morechemical pollutants and/or radiations (Depledge, 1993).

Biomarkers have received substantial efforts to be developedand finally applied in environmental risk assessments. These effortsare justified by the need of early indicators appearing beforemeasurable effects on individual performance and population/community dynamics occur, and also by the need to identify thecauses of population- and community-level effects induced bypollutants (Forbes et al., 2006). The application of these tools isusually intended to provide an early warning of disturbances athigher levels of biological organization. Nonetheless, one of themajor criticisms on the use of biomarkers has been the link be-tween molecular and cellular effects and impacts at or abovepopulation level (Hagger et al., 2006).

Ecotoxicological studies have sought to fill the gap between sub-individual and higher level biological/ecological responses inaquatic ecosystems exposed to contaminants (Moore et al., 2006a;Tlili et al., 2010). The use of sublethal responses to complementbiological indices based on benthic macroinvertebrate taxa isespecially interesting in moderately polluted sites, where stressorsare already affecting communities but not too strongly to bedetected by biotic indices (Damásio et al., 2011).

Although composed by a mosaic of Protected Areas (PAs), theSão Paulo coast is surrounded by industries, harbors, marines andcities, which continuously affects the coastal zone. This regioncomprises the largest South American commercial harbor andBrazilian oil terminal (DTCS). Several studies have identified por-tions of this coast as chronically contaminated by polycyclic aro-matic hydrocarbons (PAHs), polychlorinated-biphenyls (PCBs),linear alkylbenzenes (LABs) and metals (Abessa et al., 2008; Cesaret al., 2007; Lamparelli et al., 2001; Martins et al., 2008). Ecolog-ical assessments have also reported signs of disturbances onbenthic communities (Abessa et al., 2008; Choueri et al., 2010; Jerezet al., 2001).

Our study presents a multi-level environmental assessment ofmarine and estuarine zones in São Paulo coast (Brazil). Consideringorganic compounds as major pollutants in this coastal zone, CYP450 like e proteins, glutathione S-transferase, glutathione peroxi-dase and catalase were assessed as biomarkers of defense. DNAstrand breaks, lipid peroxidation, lysosomal membrane destabili-zation, histological damages and embryo-larval anomalous devel-opment were assessed as biomarkers of effects. Simultaneously,benthic community structure (in terms of n�. species, n�. in-dividuals, richness, Shannon’s diversity, Margalef’s richness andPielou’s Equitability) was assessed in the same areaswhere bivalveswere transplanted. The linkages between biomarker responses anddescriptive parameters of macrobenthic community were investi-gated in order to provide a better understanding on the mecha-nisms underlying effects on higher levels of biological organizationas well as the ecological relevance of a suite of biomarkers.

2. Material and methods

2.1. Study areas

The study area comprises 200 km along São Paulo coast,extending from São Sebastião channel to the Santos EstuarineSystem, in the Southeast Brazilian coast. It was established tocomprise zones influenced by different contamination sources.Brown mussels Perna perna were transplanted to three marinecoastal areas with different degrees of contamination: 1) Ilhabela

Please cite this article in press as: Seabra Pereira, C.D., et al., Ecological rMarine Environmental Research (2013), http://dx.doi.org/10.1016/j.maren

(M1), a clean site belonging to a Protected Area on the São PauloNorth Shore; 2) Palmas Island (M2) in the mouth of the Santosestuary, where the Port of Santos is located; 3) Itaipu (M3), in themouth of São Vicente estuary, where two domestic sewage sub-marine outfalls are located. Mussels were acquired from a farm atCocanha beach, selected based on the results of the environmentalmonitoring performed by the São Paulo Environmental Agency,which has detected aquatic contaminants below threshold levels inthis area (CETESB, 2006).

Mangrove oysters Crassostrea rhizophorae were caged alongSantos estuarine system; this system is surrounded by the Cubatãoindustrial park, the Port of Santos and also by densely urbanizedareas of Baixada Santista Metropolitan Region. Four sampling sta-tions were chosen: O1 located near a steel industry, in the innerestuary; O2 and O3 in the estuarine channel under influence of portactivities, solid waste disposal (industrial and domestic) and in-dustrial effluents; and O4 at the mouth of estuary (Fig. 1). Oysterswere acquired in a farm located at Paranaguá Estuarine System, anestuary composed by 16 PAs and defined as “Biosphere reserve” byUNESCO (Choueri et al., 2009a).

2.2. Biological material and study design

The marine survey employed about 600 individuals of thebrown mussel P. perna, divided between three ropes containingeach 200 organisms. The mussels were transplanted to the studiedareas, where they remained for three months. At the end of theexposure period, they were recovered, and organisms of approxi-mately 60 mm in length were analyzed. These individuals wereused for the biomarker analyses in gill tissues (n ¼ 10), to developNeutral Red Retention Assay (NRRT) in haemolymph (n ¼ 30) andfor the embryo-larval assay (n ¼ 60).

The estuarine survey employed individuals of C. rhizophorae. Toexclude variability, all animals used in this study were of similarsizes (length of 74.6 mm � 6.8). One caged system was placed ineach study site for 28-days and contained 10 oysters each. Cagesinstallation and retrieval were performed by a scuba diver since theselected sites had depths ranging between 3 and 7 m. After theexposure period, oysters’ gills (n ¼ 10) were used for thebiochemical analyses, whereas haemolymph was employed todevelop the Neutral Red Retention Assay (NRRT).

2.3. Biological responses

Gills of ten mussels or ten oysters of each stationwere dissectedimmediately after the collection. Thereafter, the samples werehomogenized separately, following the procedure developed byLafontaine et al. (2000). Once samples were homogenized in buffer(NaCl 100 mM, Hepes-NaOH 25mM, EDTA 0.1 mM, DTT 0.1 mM, pH7.5), those for enzymatic activity determinationwere centrifuged at15,000 g for 20min at 4 �C, and the supernatant (S15) was extractedand frozen for posterior determination of enzyme activities. Sam-ples obtained to determine DNA damage and lipid peroxidationwere not centrifuged, and the homogenized fraction was analyzed.Total protein contents in S15 and homogenized fraction wereanalyzed according the dye-binding principle (Bradford, 1976). Todevelop the NRRT assay, haemolymph was withdrawn from theadductor muscle of living organisms. For this assay, mussels werekept for a few hours (6e12 h) in 300 L aquariums with controlledsalinity (34e36 ppt) and temperature (22 � � 2 �C), whereas oysterswere analyzed immediately after collection.

2.3.1. Ethoxyresorufin O-deethylase (EROD) activityThe mixed-function oxidase activity (MFO) for the 7-

Hydroxyresorufin substrate was measured using the adapted

elevance of Sentinels’ biomarker responses: A multi-level approach,vres.2013.11.002

Fig. 1. Study area with the location of the mussels (M) and oysters (O) caging stations (Extracted from NOAA/NGDC).

C.D. Seabra Pereira et al. / Marine Environmental Research xxx (2013) 1e9 3

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EROD assay (Gagné and Blaise, 1993). According to this study, ERODwas measured as a more specific substrate for CYP1 like proteinsactivity. 50 ml of S15 were added to 10 mM 7-ethoxyresorufin and1 mM reduced NADPH in 100 mM KH2PO4 buffer (pH 7.4). Thereaction was initiated by the addition of NADPH, allowed to pro-ceed for 60 min at 30 �C, and stopped by the addition of 100 ml of0.1 M NaOH. 7-hydroxyresorufin was determined fluorometricallyusing 485 nm (excitation) and 580 nm (emission) filters. Resultswere expressed as nmol. min�1. mg�1 total protein.

2.3.2. Dibenzylfluorescein (DBF) activityThe mixed-function oxidase activity (MFO) for the dibenzyl-

fluorescein dealkylase substrate was determined as previouslydescribed by Stresser et al. (2000) and Gagné et al. (2007). Ac-cording to these studies, DBF was measured as a more specificsubstrate for CYP3 like proteins activity. The S15 of the gills wasincubated with 10 mM of substrate in 100 mM NaCl containing10 mM HepeseNaOH (pH 7.4). The reaction was started by theaddition of 50 mMNADPH (blanks consisted of the reaction mixturewithout the substrate) and incubated for 0, 15, 30, 45 and 60 min at30 �C. Fluorescence was measured at 485 nm excitation/516 nmemission. Results were expressed as nmol. min�1. mg�1 totalprotein.

2.3.3. Catalase (CAT) activityCatalase activity was determined by using themethod described

by Beutler (1975). This method quantifies the rate of decompositionof hydrogen peroxide (H2O2) by the enzyme through decrease ofabsorbance at 240 nm, using the molar extinction coefficient (ε) of

Please cite this article in press as: Seabra Pereira, C.D., et al., Ecological rMarine Environmental Research (2013), http://dx.doi.org/10.1016/j.maren

0.04 M. The values of CAT activity were expressed as U/mg protein.One unit of CAT is the amount of enzyme that hydrolyzes 1 mmol ofH2O2 min�1 mg�1 total protein.

2.3.4. Glutathione peroxidase (GPx) activityGlutathione peroxidase activity was determined using the

method described by McFarland et al. (1999). GPx activities weremeasured spectrophotometrically at 340 nm every 2 min for10 min, using 1 mM cumene hydroperoxide as the substrate. Thedecrease in NADPH absorbance measured at 340 nm during theoxidation of NADPH to NADP was indicative of GPx activity. Resultswere expressed as nmol min�1 mg�1 total protein.

2.3.5. Glutathione S-transferase (GST) activityThe procedure utilized for the determination of GST activity was

adapted from McFarland et al. (1999). The activity was analyzedusing 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 1 mMGSH assubstrates and measured spectrophotometrically at 340 nm every30 s for 3 min. Results were expressed as nmol min�1 mg�1 totalprotein.

2.3.6. DNA damageDNA damage was assessed by the alkaline precipitation assay

(Olive, 1988) based on the K-SDS precipitation of DNA-proteincrosslink, followed by fluorometric detection of DNA strands(Gagné et al., 1995). DNA quantitation was achieved using Hoeschtdye at a concentration of 100 nM in 200 mM TriseHCl, pH 8.5,containing 300 mM NaCl and 4 mM sodium cholate. Salmon spermDNA standards were used for calibration and fluorescence readings

elevance of Sentinels’ biomarker responses: A multi-level approach,vres.2013.11.002

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were taken at 360 nm excitation and 460 nm emission. The resultswere expressed as mg of DNA mg�1 total protein.

2.3.7. Lipid peroxidation (LPO)Lipid peroxidationwas measured according toWills (1987). LPO

was determined in gill homogenates by the thiobarbituric acid.Thiobarbituric acid reactants (TBARS) were determined by fluo-rescence at 530 nm for excitation and 630 nm for emission using afluorescence microplate reader. Because the reagent could reactwith other aldehydes, the results were expressed as mg ofTBARS mg�1 total protein.

2.3.8. Neutral red retention time assay (NRRT)NRRT assay is based on the principle that only lysosomes in

healthy cells take up and retain the vital dye neutral red. Lysosomalmembranes’ damage caused by the impact of xenobiotics candecrease the retention time of the dye by inducing the leaking oflysosomal components (Dailianis et al., 2003). NRRT assay wascarried out following the method described by Lowe et al. (1995).This non-destructive method employed haemolymph withdrawnfrom the posterior adductor muscle of living bivalves. The mussels’haemolymph was mixed to physiological saline solution (pH 7.3containing 4.77 g/l HEPES, 25.48 g/l NaCl, 13.06 g/l MgSO4, 0.75 g/lKCl, 1.47 g/l CaCl2), whereas oysters’ haemolymph was mixed tophysiological saline solution adjusted to the equivalent ionicstrength of the low salinity environments where they werecollected. The haemolymph was spread on slides and transferred toa lightproof chamber, where it remained 15 min to allow cellsattachment. Excess liquid was removed and 40 mL of the NeutralRed (NR) dye were added to the cell monolayer. A cover slip wasadded. After a 15-min incubation period, slides were examinedevery 15 min by optical microscopy (400�) for both structuralabnormalities and NR dye loss from the lysosomes to the cytosol.The same analyst carried out the assessment for all slides duringthe study. The endpoint was considered the time when at least 50%of the examined cells exhibited these characteristics and a NRRTmean value was calculated for each group.

2.3.9. Histological analysis in P. perna (HF)The gill tissue was excised from the inner and outer demibranch

on the left-hand side of each mussel. The tissue were fixed in 10%neutral formalin and prepared for histology. Tissue sections werecut with a rotary microtome at 5 mm thicknesses, and stained witheosin and haematoxylin. Histological sections were examined un-der light microscopy, and with a Zeiss LSM 510 confocal microscopefor the presence of morphological alterations, such as epithelialhyperplasia and fusion of the gill filaments. The prevalence (thepercentage of mussels affected) was calculated as the fraction ofindividuals with alterations detected.

2.3.10. Embryo-larval development assay with P. pernaIn order to assess the zygote viability to develop to normal

larvae after previous parental exposure in each station, experi-ments were performed according to the protocol recommended byASTM (1992) for mussels, with minor adaptations proposed byZaroni et al. (2005). Sixty adult individuals were induced to spawnby thermal stimulation. The gametes from males and females werecollected separately from each sampling site and transferred toglass beakers. The fertilization was attained by adding 2 ml ofsperm solution to the 200 mL of ovules solution.

With the aid of a Sedgwick-Rafter chamber, the density offertilized eggs was estimated, an about 500 embryos were trans-ferred to glass tubes containing filtered seawater, for a period of48 h at a temperature of 25 �C and salinity of 35 ppt. Four replicateswere used for each group. After 48 h, the assay was finished and the

Please cite this article in press as: Seabra Pereira, C.D., et al., Ecological rMarine Environmental Research (2013), http://dx.doi.org/10.1016/j.maren

first 100 larvae from each replicate were analyzed. Larvae devel-oped to D-phase were considered normal, whereas those present-ing delay and/or morphological anomalies in their developmentwere considered abnormal. A mean percentage of normal devel-opment was obtained for each tested group.

2.3.11. Benthic community structureBenthic community sampling was conducted from each sam-

pling station using a 0.04 m2 van Veen metal hand grab. Collectedsediments (3 replicates per station) were sieved through a 500 mmmesh. The macroinvertebrates were fixed with 4% bufferedformalin, and subsequently transferred to 70% isopropyl alcohol.Then, they were sorted and identified to the lowest possible taxonlevel. The benthic community structure was assessed according toN� individuals, N� species, Richness, ShannoneWiener’s diversity,Margalef’s richness, Pielou’s Equitability. These classical descriptorsare well explained in the literature (Odum and Barrett, 2005).

2.4. Multivariate analysis

Multivariate analysis was applied to the complete data set toidentify linkages between biological responses as well as to char-acterize the studied stations. Initially, the data were submitted toChi-square and Hartley’s tests to verify normality and homogeneityof variance, respectively. Thereafter, Factor Analysis, employingPrincipal Components Analysis as the extraction procedure,analyzed the original data set. Datawere rearranged in a correlationmatrix and two factors (or new variables) were extracted consid-ering eigenvalues higher than 1.0 (Kaiser’s criteria). For the FactorAnalysis, the variables were auto scaled (Varimax normalized) to betreated with equal importance. Following Tabachnic and Fidell(1996), variables having loadings �0.40 to a particular factorwere considered associated to the respective factor. Beside theanalysis of the variables aggregated by PCA, the factor scores fromeach studied area were employed in order to confirm the factordescriptions and also to characterize the studied stations. All ana-lyses were performed using the PCA option of the multivariateexploratory techniques procedure, followed by the basic set-up forFactor Analysis procedure from the STATISTICA software tool (StatSoft, Inc., 2001; version 6.0).

3. Results

3.1. Marine survey employing mussels

As result of the marine survey (Table 1), mussels transplanted toM3 showed increased CYP 450 like proteins and GST activitiesindicating exposure to organic compounds. CAT and GPx activitieswere also increased in mussels transplanted to M2 and M3, indi-cating the activation of defencemechanism against oxidative stress.Mussels transplanted to M3 showed signs of impaired health, sincereduced NRRT, histological damage in gills and reduced embryo-larval development were observed after the exposure period.Moreover, community descriptors tended to show a worse condi-tion in M3, with the lowest richness, diversity and abundance(Table 1). In turn, the descriptors for M1 had the best conditionsamong studied sites, while those for M2 exhibited intermediaryvalues.

The application of Principal Components Analysis indicated thatthe original set of variables could be narrowed down to two newfactors, those explained 100% of the total variance (Table 2).

The first principal factor (F1) accounted for 70.44% of the vari-ance. Factor 1 negative loadings account for the relationship be-tween NRRT, embryo-larval development and all macrobenthicparameters (excepting Pielou Equitability). It was representative for

elevance of Sentinels’ biomarker responses: A multi-level approach,vres.2013.11.002

Table 2Sorted rotated factor loadings of the original variables on the two principal factors.

Variance % Factor 1 Factor 2

70.44 29.56

EROD e �1.00DBF 0.51 �0.86GST e �1.00GPx e �0.96CAT 0.75 0.66DNA e �0.98NRRT �0.55 0.84HF 0.89 �0.45Embryolarval develop. �0.40 0.93N� species �0.96 e

N� individuals �1.00 e

Richness �0.99 e

Diversity Shannon �0.86 0.51Pielou Equitability (J0) 1.00 e

Margalef Specific Richness (d) �0.90 0.44

Table

1Biomarke

rresp

onses(m

eansan

dstan

darddev

iation

s)an

decolog

ical

param

etersof

themarinesu

rvey

.

EROD

(nmol/m

in/

mgprot)

DBF

(nmol/m

in/

mgprot)

CAT

(U/m

gprot)

GPx

(nmol/m

in/

mgprot)

GST

(nmol/m

in/

mgprot)

DNA

( mg/mgprot)

NRRT

(min.)

HF(%)

Embryo

-larva

ldev

elop

.(%)

n�sp

s.n�

ind.

Richness

Shan

non

eW

iener

diversity

(H0 )

Pielou

Equitab

ility

(J0 )

Marga

lefsp

ecific

rich

ness(d)

M1

0.19

�0.05

0.28

�0.09

17.9�3

.391

2.1�

395.5

1073

.3�1

37.7

147.55

�24.96

35.2�1

7.3

070

3519

2359

3.55

0.77

6.78

M2

0.07

�0.02

0.25

�0.08

26.1�3

.43

577.1�

307.7

669.8�

248.2

135.06

�45.38

35.2�1

3.1

5080

1432

014

2.43

0.92

4.03

M3

0.29

�0.06

0.97

�0.34

20.95�

3.24

1762

.7�3

86.2

1458

.9�4

85.7

168.66

�84.09

27.3�1

3.2

100

313

513

1.04

0.94

1.44

C.D. Seabra Pereira et al. / Marine Environmental Research xxx (2013) 1e9 5

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66676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130

MERE3823_proof ■ 3 December 2013 ■ 5/9

Please cite this article in press as: Seabra Pereira, C.D., et al., Ecological rMarine Environmental Research (2013), http://dx.doi.org/10.1016/j.maren

M1, which showed a negative factor score. With positive loadings,this factor has associated DBF, CAT and histological alterations. Itwas representative for stations M2 and M3, which showed positivefactor scores.

The second factor (F2) accounts for 29.56% of the variance. Itrepresents the relationship between Phase I CYP 450 (EROD andDBF), Phase II enzyme GST, antioxidant enzyme GPx, DNA damageand histological alterations (negative values). This association wasrepresentative to mussels caged in M3, which showed a negativefactor score. With positive loadings, this factor has associated CATactivity, NRRT and embryo-larval development with Shannon’sdiversity and Margalef’s Specific Richness. Station M2 showed apositive factor score.

Biomarkers loadings were plotted in a 2D representation (Fig. 2),where it could be noted the association of NRRT and embryo-larvaldevelopment with descriptive parameters of macrobenthic com-munity structure, specially diversity and specific richness. Bio-markers of defense, DNA and histological damages exhibited anegative relationship with macrobenthic community structure.

3.2. Estuarine survey employing oysters

As result of the estuarine survey (Table 3), DBF showed higheractivity in organisms caged in O1, as well as DNA damage, lipidperoxidation and lysosomal membrane destabilization. Oysterscaged in O2 and O3 showed higher EROD and GST activities, inaddition to reduced Neutral Red retention time. Community de-scriptors tended to show a worse condition in O2 and O3, with thelowest richness, diversity and abundance. In turn, the descriptorsfor O4 presented the best conditions among studied sites, whilethose for O1 exhibited intermediary values.

The application of Principal Components Analysis indicated thatthe original set of variables could be narrowed down to two newfactors, which explained 94.49% of total variance (Table 4).

The first principal factor (F1) accounted for 53.13% of the vari-ance. Considering the positive loadings, GPx activity and NRRTappeared to be associated with the most of parameters of macro-benthic community structure (excepting Pielou Equitability). It wasrepresentative for oysters caged in O4, which showed a positivefactor score. F1 negative loadings associated EROD and GST activ-ities. It was representative for O1, O2 and O3 stations (negativefactor score).

The second factor (F2) accounts for 41.36% of the variance. Itrepresents the relationship between EROD, GST and GPx activitieswith NRRTand Equitability. Stations O2, O3 and O4 showed positivefactor scores. With negative loadings, this factor has associated DBF,

elevance of Sentinels’ biomarker responses: A multi-level approach,vres.2013.11.002

Fig. 2. 2D representation of factors 1 and 2 variable loadings e Marine survey.

Table

3Biomarke

rresp

onses(m

eansan

dstan

darddev

iation

s)an

decolog

ical

param

etersof

estuarinesu

rvey

.

EROD

(nmol/m

in/

mgprot)

DBF

(nmol/m

in/

mgprot)

GPx

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DNA damage and LPO. It was representative for oysters caged inO1 (negative factor score).

Fig. 3 exhibits the 2D representation of factors 1 and 2 variableloadings. It is possible to observe the association of NRRT and GPxwith descriptive parameters of macrobenthic community struc-ture, with positive loadings in both factors. Biomarkers of defense(EROD and GST) exhibited a negative relationship with parame-ters of macrobenthic community structure.

4. Discussion

Characterizing the responses of stress indices in target tissues,such as haemolymph and gills of bivalves, represents a reliableapproach for assessing the potential harmful impact of xenobi-otics into aquatic environment. Such characterizations are usefulto provide information on pre-pathological alterations in aquaticorganisms, before diseases, mortality or population changes occur(Tsarpali and Dailianis, 2012).

The mechanism of detoxification of pollutants involving a se-ries of enzymes is divided into Phase I (biotransformation) andPhase II (conjugation). Themetabolic process of Phase I is basicallydescribed as oxidative reactions catalyzed by the mixed-functionoxygenase (MFO) system dependent on a group of proteins (Cy-tochrome P450), while Phase II is characterized by the activity ofthe enzyme Glutathione e S-transferase (Stegeman andLivingstone, 1998; Dauterman, 1994). In the present study, mus-sels caged in M3 showed increased activities of EROD, DBF andGST indicating exposure to organic pollutants. Exposure to organicxenobiotics is supposed to product Reactive Oxygen Species (ROS),which must be counteracted by the activity of antioxidant en-zymes. Catalase and Glutathione Peroxidase are known to protectthe cell by reducing H2O2 to H2O. Thus, GPx increased activity inmussels caged in M3 revealed that they could be suffering pro-oxidant challenges.

Xenobiotics, its metabolites or ROS may lead to impairment ofcells and tissues. DNA alterations promoted by chemical agentsinclude single and double strand breaks, which may also beinduced by an interaction with oxygen radicals or as a conse-quence of apoptosis or necrosis processes (Viarengo et al., 2007).Our study revealed slightly increased of DNA strand breaks inmussels caged in M3, as well as impairments in lysosomalmembranes. Alterations in lysosomal system are well-known as amechanism of action for xenobiotics, leading to tissue damagesand dysfunction (Moore et al., 2006b). Furthermore, 100% of

Please cite this article in press as: Seabra Pereira, C.D., et al., Ecological relevance of Sentinels’ biomarker responses: A multi-level approach,Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.11.002

Table 4Sorted rotated factor loadings of the original variables on the two principal factors.

Variance % Factor 1 Factor 2

53.13 41.36

EROD �0.43 0.91DBF e �0.94GST �0.42 0.70GPx 0.69 0.50DNA e �0.97LPO e �0.98NRRT 0.88 0.48N� species 0.99 e

N� individuals 0.91 e

Richness 0.97 e

ShannoneWiener Diversity (H0) 0.98 e

Pielou Equitability (J0) e 0.97Margalef Specific Richness (d) 0.99 e

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mussels examined in M3 had several gill changes, which includedepithelial hyperplasia leading to variable fusion of the gill filament.No damage was observed in specimens caged in M1.

The integrated analysis of the marine survey data pointed outthe association between biomarkers and population-communityresponses. M3 seems to be the most threatened marine site tak-ing into account the overall biological responses (Factor 1 positiveand Factor 2 negative scores). Exposure to organic pollutants couldbe resulting in increased radical generation leading to cellulardamage, tissue injury and physiological dysfunction. The distur-bances reported formacrobenthic community from M3 could beresult of diseases and mortality promoted by such exposure. Thisinterpretation is supported by previous studies, which detected theoccurrence and bioavailability of PAHs, PCBs, surfactants andmetals in the Santos bay, especially in M3 zone (Abessa et al., 2008;Cesar et al., 2007; Lampareli et al., 2001; Pereira et al., 2012). Thisarea is surrounded by two submarine domestic sewage outfalls,which have been considered significant sources of contaminationto the local ecosystems (Abessa et al., 2005). In fact, untreateddomestic sewage has been considered the main cause of marineand estuarine pollution in Brazilian coastal zones. Bainy et al.(2000) and Pereira et al. (2007) have already observed alteredbiomarker responses in mussels P. perna transplanted to othercoastal areas contaminated by wastewater.

As result of the estuarine survey, EROD and GST showed higheractivities in organisms caged in O2 and O3, whereas DBF activitywas increased in oyster caged in O1, denoting a physiological

Fig. 3. 2D representation of factors 1 and 2 variable loadings e Estuarine survey.

111112113114115116117118119120121122123124125126127128129130

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adaptation to environmental contamination. DNA damage, lipidperoxidation and lysosomal destabilization were also increasedafter oyster exposure in O1, O2 and O3. Kirschbaun et al. (2009) andMaranho et al. (2012) reported similar genotoxic and oxidativeeffects in native fishes and oysters from Santos estuarine system.

Factor analysis revealed the association of NRRT and GPx withdescriptive parameters of benthic structure, which were negativelycorrelated to EROD, GST, DNA damage and LPO. This result indicatesthe remaining risk to benthic populations living in the O1, O2 andO3 estuarine areas, where the ecological indices showed signs ofdegradation. Hence, the stress indices observed can be regarded asa toxicity indication, increasing benthic organism susceptibility topathologies and mortality. This information was useful for assess-ing the ecological status of the inner estuary, where reduced rich-ness and diversity of themacrobenthic communities were expectedas result of the variations on salinity, dissolved oxygen and grainsize, which can alter benthic structure as much as anthropogenicpollution.

This interpretation is supported by previous studies thatdetected environmental concentrations of PAHs and PCBs in sedi-ments above the Threshold Effect Level in the industrial and portareas (Abessa et al., 2008; Cesar et al., 2007; Choueri et al., 2009b;Torres et al., 2012). Beside the intense marine traffic and industrialdischarges, the Santos estuarine channel receives input of un-treated domestic sewages from illegal occupations situated in themangrove zone. Moreover, former domestic landfills were consid-ered contamination sources by the Environmental Agency, sincesuperficial drainage and leachates flow toward the estuary(Lamparelli et al., 2001).

In addition to providing reliable information of the differentialimpact of human-derived activities on the investigated stations, themulti-level approach was able to identify relationships betweeneffects on different levels of biological organization. This integratedapproach shed light on the possible mechanisms underlying im-pacts at population and community levels.

It is worth noting the association of lysosomal membraneintegrity (NRRT) and embryo-larval development in mussels (Fac-tor 1 e Table 2; Fig. 2). Ringwood et al. (2004) observed thatadverse effects on embryo development were consistentlyobserved when parental lysosomal destabilization rates were>35%. Lysosomal catabolism and the subsequent recycling andutilization of metabolites are critical for many biological processes,as highlighted by the serious consequences to human and animalhealth when these functions are disrupted (Futerman and vanMeer, 2004). Moore et al. (2006b) observed that the PAH 3-methylcholanthrene (3-MC) was taken up by yolk granules inmussel oocytes, which are pro-lysosomes. The accumulation of 3-MC in lysosomally related organelles in the ovarian oocytes in-dicates that these cells are target for lipophilic xenobiotics. Thepresence of such compounds in the yolk granules is likely to havedetrimental consequences for the development of gametes fromsuch contaminated oocytes due to functional impairment of thelysosomal-yolk granule system.

Furthermore, degradation of macromolecules by lysosomal hy-drolases is essential for yolk utilization during embryogenesis.Embryos rely on the contents of their yolk cell as the primarysource of amino acid, carbohydrate, and lipid building blocksthroughout their early development (Fan et al., 2010). According toBayne (1976), in bivalves, the early stages of larval developmentrepresent a period of intense morphogenetic activity when there iscomplete dependence on the stored energy reserves acquired fromthe parental adult. Thus, destabilization of lysosomal membranecould promote nutrient unbalance during embryogenesis leadingto disturbances on larval development, here evaluated as delayedgrowth and abnormal D-shape. Ultimately, effects on larval

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development would produce decrease in bivalve populations.Casarini and Henriques (2011), after a long-term study, found thatnatural stocks of the mussel P. perna are significantly decreasing inSantos bay.

Our results also revealed linkages between biochemical, cellularand physiological alterations and ecological indexes. Macrobenthiccommunity structure revealed signs of degradation in M4, O1, O2and O3, where altered biomarkers responses were also observed.Caged bivalves showed CYP 450 and GST increased activities,indicating bioavailability of organic xenobiotics. Increased CAT andGPx were associated to lysosomal membrane destabilization andDNA strand breaks, suggesting ROS generation inflicting damage onmembrane lipids and DNA. Lysosomesmembrane impairments andDNA damages can be linked with many aspects of pathology andtoxicity (Danellakis et al., 2011; Toufexi et al., 2013). Moore et al.(2006b) proposed a conceptual model linking the cellular uptakeof pollutants such PAHs with damage to lysosomes, radical gener-ation and oxidative injury, through to tissue pathology and harmfulphysiological consequences. Tsarpali and Dailianis (2012) showedthe ability of leachate to induce cytotoxic and genotoxic effects intissues of mussels before mortality occurs. As reported in the pre-sent study, Damásio et al. (2011) found that biochemical responsesof Hydropsyche exocellatawere able to differentiate samples withincommunities having a good and deteriorated ecological stage,providing complementary information on factors impairing mac-roinvertebrate communities.

5. Conclusion

Biomarker responses of caged bivalves showed linkages withpopulation-community alterations in both marine and estuarineenvironments, indicating that xenobiotics are inducing physiolog-ical stress, which may lead to changes of the macrobenthic struc-tures and deterioration of the ecological status over time. Thismulti-level approach increased certainty that disturbances foundat community level could be related to xenobiotic as stressors,which was very useful to improve the discriminatory power of theenvironmental assessment.

Acknowledgments

This project has been developed as part of UNESCO/UNITWIN/WiCoP e Europe, funded by Spanish Ministry of Science and Edu-cation and by Brazilian Ministry of Science and Education (CAPES/MECD-DGU # 099/06). Rodrigo B. Choueri and Camilo D. S. Pereirawere supported by CAPES scholarships numbers (BEX2492/08-03and BEX2300/08-7, respectively) and Luciane A. Maranho wassupported by Erasmus Mundus scholarship. Afonso C.D. Bainy,Antonio A. Mozeto, Augusto Cesar, Denis M.S. Abessa and EduinettyC.P.M. Sousa thank CNPq for productivity fellowships awarded.Denis Abessa would like to acknowledge to São Paulo ResearchFoundation (FAPESP) grant #98/00808-6 and CNPq (Process308649/2011-7). Camilo D. Seabra Pereira thanks CAPES-PAEX bysupporting his participation in the 17th PRIMO Conference (PAEX1155-13-0).

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