Evaluation of the Sub-lethal Toxicity of Bleached Kraft PulpMill Effluent to Carassius auratus and Dicentrarchus labrax

Mário S. Diniz & Ruth Pereira & Ana C. Freitas &

Teresa A. P. Rocha-Santos & Luisa Castro &

Isabel Peres & Armando C. Duarte

Received: 25 February 2010 /Accepted: 20 July 2010# Springer Science+Business Media B.V. 2010

Abstract The effluents from bleached Kraft pulp mill(BKME) and paper industry are toxic to differentaquatic organisms being an important source ofcontamination to aquatic environments due to thepresence of several chemicals produced during theproduction of Kraft pulp. The main objective ofthe present study was to evaluate the exposure effectsof a secondary-treated BKME in two different species of

fish:Carassius auratus and Dicentrarchus labrax. Bothspecies were exposed to different concentrations ofsecondary-treated effluent (1%, 10%, 25%, 50%,100%) in semi-static tests under controlled laboratoryconditions. At the end of the experimental period(21 days), samples of livers were collected for CYP1Adetermination and histopathological evaluation. Theresults show significant changes (p<0.05) of CYP1Ainduction in carp exposed to 50% and in sea bassexposed to 25% of the effluent. Histopathologicalalterations were also observed according to the differentconcentrations of the tested effluent suggesting thattested BKME cause damage to exposed organisms.

Keywords BKME .Carassius auratus .

Dicentrarchus labrax . CYP1A . Toxicity .

Histopathology

1 Introduction

The pulp and paper mill industry is the sixth largestworld polluter after oil, cement, leather, textile, andsteel industries, as it discharges a variety of gaseous,liquid, and solid wastes into the environment (Ali andSreekrishnan 2001). The major concerns are with theecotoxicological impacts in the receiving water bodiesbecause large volumes of wastewater are generated bypulp and paper mills and discharged into freshwater,estuarine, and marine ecosystems affecting aquatic

Water Air Soil PollutDOI 10.1007/s11270-010-0565-z

M. S. Diniz (*)REQUIMTE, Departamento de Química, Faculdade deCiências e Tecnologia, Centro de Química Fina eBiotecnologia, Universidade Nova de Lisboa,Caparica 2829-516, Portugale-mail: mesd@fct.unl.pt

R. PereiraCESAM & Department of Biology, University of Aveiro,3810-193 Aveiro, Portugal

A. C. Freitas : T. A. P. Rocha-SantosISEIT/Viseu, Instituto Piaget,Estrada do Alto do Gaio, Galifonge,3515-776 Lordosa, Viseu, Portugal

L. Castro : I. PeresDepartamento de Ciências e Engenharia do Ambiente,IMAR-Instituto do Mar, Faculdade de Ciências eTecnologia, Universidade Nova de Lisboa,Quinta da Torre,2829-516 Caparica, Portugal

A. C. DuarteCESAM & Department of Chemistry, University of Aveiro,3810-193 Aveiro, Portugal

biota and having a negative impact on human health(Lacorte et al. 2003; Pokhrel and Viraraghavan 2004).

Bleached Kraft pulp mill effluents (BKPME) are acomplex mixture of highly toxic compounds, containingabout 300s of known chemicals, but not all have beenidentified (Nestmann et al. 1980; Mather-Mihaich andDiGiulio 1991). The effluents from Kraft pulpprocesses are mainly composed by filtrates frombleaching, wastewaters from debarking, condensatesfrom cooking and evaporation, black liquor residues,as well as other pills from the different processingstages. In modern pulp mills, most of the water usedin the production process is recycled resulting in adiminished amount of wastewater. Furthermore, beforebeing discharged into the receiving aquatic systems,BKPME always undergo chemical and or/biologicaltreatments in which suspended solids are mechanicallyseparated in a primary wastewater treatment plant andthen dissolved organic matter is mineralized in anactivated sludge secondary treatment plant or, morerarely, in aerated lagoons (Aaltonen et al. 2000). Thetoxic nature of BKPME is attributed to the presence ofseveral naturally occurring compounds extracted fromwood (e.g., tanins and lignins) as well as severalxenobiotic chemicals derived from the whole papermaking process (e.g., chlorinated lignins, resin acids,phenols, dioxins, and furans; Aaltonen et al. 1997).Some of these compounds such as the dibenzodioxinsand polychlorinated dibenzofurans, dibenzodioxins, andfurans are very persistent in the environment and areusually known as persistent organic pollutants (POPs)which have been classified as “priority pollutants” bythe US Environmental Protection Agency (USEPA1998; Ali and Sreekrishnan 2001). Meanwhile, theEuropean Community has signed international instru-ments concerned with POP’s and has ratified the UnitedNations Economic Commission of Europe protocol andthe Stockholm Convention in 2004.1 Among severalother measures, both instruments aimed to reduceemissions of unintentionally produced polychlorinatedbiphenyls (PCBs; e.g., dioxins and furans).

The exposure to BKPME has been associated withseveral adverse effects in the biology and physiologyof fishes, such as impaired reproduction, pathologicalalterations, changes in growth rate, and severalbiochemical responses (Owens 1991; Sodergren

1993; Heuvel et al. 2002; Denslow et al. 2004).Many of these compounds (e.g., dioxins and furans)showed acute and chronic toxicity, since they canpromote genetic alterations, proliferative lesions, andneoplasia in exposed organisms (Nestmann 1985;Couillard et al. 1999).

Molecular biomarkers can be a useful tool tomeasure biochemical responses in organisms,functioning as a useful alarm of exposure in aquaticecosystems, before more deleterious impacts occur(Au 2004). Consequently, several biomarkers havebeen used to evaluate the adverse effects in thephysiology of fish, caused by specific or groups ofchemicals present in BKPME (Woodworth et al.1998). The cytochrome P-450-based mixed functionoxygenase is the primary metabolic system responsiblefor the biotransformation of xenobiotics and endobioticsin vertebrates (Chen et al. 2001). Specific enzymesfrom this system such as CYP1A are induced byseveral components from the BKPME (e.g., PCBs,dioxins, and PAHs) and can be used as indicators orbiomarkers of corresponding exposures (Hodson 1996;Arellano et al. 2001). In fact, as it was demonstrated byJones et al. (2001), the exposure to BKPME has beenfound to give rise to various biochemical responses infish, including induction of hepatic cytochromeP4501A and associated enzymes.

Furthermore, it has been proven that histopatholog-ical alterations in organisms are reliable bioindicatorsof adverse effects caused by exposure to BKPMEcompounds and have been recommended as biomarkersfor monitoring the effects of pollution by this type ofeffluents because they are easily determined and can berelated with the health and fitness of individuals(Mondon et al. 2001; Au 2004).

The aim of the present work was to evaluate thetoxicity of a BKPME, collected after secondarytreatment, to two different fish species from receivingaquatic systems: a marine (Dicentrarchus labrax) anda freshwater (Carassius auratus) one. Thus, to assessthe effects of exposure to this wastewaters, theinduction of CYP1A and histopathological alterationswere assessed on the liver of animals from bothspecies. The somatic indices gonadosomatic index(GSI) and hepatosomatic index (HSI) were used ascomplementary parameters to assess effects at theorgan’s level. Additionally, the present study intendsto contribute for a better comprehension of the effectsof the xenobiotics generated by the Kraft pulp

1 http://www.unece.org/env/lrtap/pops_h1.htm, available on 19July 2009.

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production and subsequently to reinforce the need ofprotection measures.

2 Materials and Methods

2.1 Effluent Source

Effluent samples were collected from a bleached Kraftpulp mill processing Eucalyptus globulus (BKPME),after secondary treatment by activated sludge process,a biological treatment in which wastewaters andactivated sludge are agitated and develop a biologicalfloc that settle to the bottom of the tank which reducesthe organic matter of the wastewater. Samples werecollected in glass bottles, acidified at pH 2, and keptat room temperature before analysis.

2.2 Analytical Procedures for EffluentCharacterization

Sub-samples of the BKPME were taken for physicaland chemical characterization. The absorbance ofBKPME sub-samples was measured at 325, 400, and460 nm using GBC/Cintra 10e spectrophotometer.Before absorbance measurements, the pH was adjustedto 5.5 by addition of sodium hydroxide (0.1–1 M) orhydrochloric acid (0.1–1 M), followed by filtrationthrough grade GF/A glass fibber filters pore 0.45 μm(Whatman, GE Healthcare, Germany). The pHmeasurements were performed with a pH meter Crison.Chemical oxygen demand (COD) was determinedspectrophotometrically following the standard method1252-88D ASTM (1994) and using an aqualytic PCcompact spectrophotometer and an aqualytic AL32COD reactor for digestion of the sub-samples in CODvials.

2.3 Test Organisms

The toxicity of the BKPME, collected after secondarytreatment, was tested for two species: a marine andfreshwater one. D. labrax (sea bass) is a demersaleuryhaline marine species, with a high distribution inthe Black and Mediterranean Seas, as well as in theEastern Atlantic Ocean and with a great ecologicaland economic importance in these regions. Juveniles(<1 year) are usually found in estuaries, toleratingsalinities ranging between 10‰ and 20‰ (Kelley

1988). C. auratus, a freshwater fish species from thefamily Cyprinidae (the largest family of freshwaterfish), has been widely used as a model species inseveral ecotoxicological studies, since they arecommercially available and easy to maintain andhandle in laboratory. Despite their small size, theyprovide enough tissue and blood for histopathologicaland biochemical analysis. Thus, cyprinids andjuvenile sea bass were obtained from nationalcommercial suppliers (Piscicultura de Avis andViveiros Vila-Nova, respectively).

2.4 Experiment Conditions

Previously to the beginning of the assays, the fish wereacclimated for 2 weeks in controlled laboratorial con-ditions (temperature 16±1°C, continuous aeration >6 mgL−1, and photoperiod 16:8 h light/dark (L/D)) inpolystyrene tanks (400 L). D. labrax were acclimatedin clean seawater with a salinity of about 34‰. As faras the cyprinids are considered, the tank was filled withdechlorinated tap water in a closed circuit with filtration.

2.5 Pre-acclimation of D. labrax to DifferentSalinities

In a preliminary assay, D. labrax was exposed to arange of concentrations of seawater during 3 weeks,diluted with dechlorinated tap water to obtainsalinities ranging from 10‰ to 34‰, to test theability of fish to support different water salinitieswithout apparent osmotic stress. As it was expected,because this species tolerates great variations in watersalinity, no mortality was recorded on the differentseawater dilutions during this experiment. Based onthese results, the sea basses were then pre-acclimatedin tanks with clean seawater diluted for the sameconcentrations that will be used in the assay and pre-acclimation conditions, renewed each 48 h. Both fishspecies were daily fed ad libitum with commercialpelleted dry food purchased from Skretting (Burgos,Spain).

2.6 Fish Exposure Assays

For freshwater fish, the assays were carried out to testdifferent concentrations of BKPME, obtained bydilution with dechlorinated tap water and tested (1%,10%, 25%, 50%, and 100% of effluent). Tanks

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containing fish exposed to clean water (dechlorinatedtap water) were used as controls. The experiment hada duration of 28 days. Forty-eight cyprinids (C. auratus)were randomly distributed by 50 L polystyrene tanks,with the different effluent dilutions (two replicates percontrol and treatment).

For sea bass (D. labrax), 36 sea bass juvenileswere, in a similar way, distributed by tanks of 50 L(two replicates by controls and each treatment),containing the different concentrations of BKPME,diluted with seawater (1%, 10%, 25%, 50%, and100% of effluent). Tanks containing fish exposed toseawater (diluted with 1%, 10%, 25%, 50%, and100% freshwater) were used as controls, maintainedfor the same exposure period and in the samelaboratorial conditions described for the cyprinidsassay.

Both fish species were tested under a constanttemperature (16±1°C), pH (7.2±0.3), a 16:8-h L/Dphotoperiod and continuous aeration (>6 mg/L).Throughout the experimental period, the test mediumin each treatment was renewed every 48 h. Tankswere checked daily for mortality and dead animalswere counted and removed from the tanks. Thecumulative mortality was calculated at the end of theexperimental period.

2.7 Somatic Indices

At the end of the experimental period, the fish weremeasured and weighed to the nearest 1 mm and 0.1 g,respectively. The organs (liver and gonads) wereremoved and weighed as well to the nearest 0.1 g.The weight of organs was expressed as percentage ofthe total body mass to provide information on gonadand liver condition, through the GSI and the HSI,according to the formulae:

GSI ¼ total gonad weight=total body weightð Þ � 100

HSI ¼ total liver weight=total body weighð Þ � 100

2.8 Histological Analysis

Sub-samples from each organ were collected alwaysfrom the same portions according to the proceduresdescribed by Blazer et al. (2007). Samples were

then fixed in a solution of Bouin–Hollande during48 h for histological analysis. The samples wereprepared following the procedures described byMartoja and Martoja (1967). Briefly, after thefixation period, the samples were washed in tapwater and then passed through a series of alcohols(70–100%), followed by a bath of xylene (Lab-Scan,Belgium). Then, samples were embedded in paraffinand sliced in sections of 7 μm thickness. Paraffinwas removed from slides using xylene as solvent,followed by a treatment in a graduate series ofalcohols before staining with hematoxylin and eosin(H&E). The histological observations were carriedout using an optical microscope (Leica-ATC 2000,Wetzlar, Germany).

2.9 Preparation of Postmitochondrial Supernatantand Microsomes

Another fraction of liver samples was stored at −80°C forsubsequent analysis of the CYP1A content in the liver asdescribed by Nielsen et al. (1998). Briefly, thepostmitochondrial supernatant (PMS) was preparedusing a buffer solution containing 0.15 M KCl (Merck,Darmstadt, Germany), 1 mM ethylenediaminetetraaceticacid (EDTA; Merck, Darmstadt, Germany), 1 mMdithiothreitol (DTT; Sigma, St. Louis, MO, USA), and10% (v/v) glycerol (pH 7.4). The microsomal (MS)buffer solution was similar to the previous one butwithout glycerol. Additionally, to resuspend the MSfraction, a buffer containing 0.1 M sodium phosphate,0.15 M KCl, 1 mM EDTA, 1 mM DTT, and 20% (v/v)glycerol was used.

The frozen livers were thawed on ice. Afterward,4 mL of PMS buffer was added to each gram oftissue, which was minced with scissors. Sampleswere then homogenized with a rotor (Heidolph-RZR 2100, Heidolph Elektro GmbH & Co., KG,Kelheim, Germany) equipped with a Teflon pestle.The resulting crude homogenate was transferred tocentrifuge tubes and centrifuged at 12,000×g, at4°C, for 20 min, to obtain the PMS fraction. Thesupernatant (PMS fraction) was collected avoidingthe pellet and the floating lipid layer. To obtain theMS fraction, the PMS fraction was transferred toultracentrifuge tubes and centrifuged at 100,000×gfor 60 min, at 4°C. The supernatant was carefullyremoved to leave the microsomal pellet which wasresuspended with the resuspension buffer solution

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(0.5–2 mL) using the homogenizer, on ice, to aprotein concentration of approximately 5–15 mgprotein/mL (normally 1:1 ratio between buffervolume and tissue weight). Afterward, samples werediluted in a coating buffer (50 mM carbonate/bicarbonate at pH 9.6), to a concentration of10–100 μgtotal protein/mL. Then, 100 μL of eachsample and blanks were added to a microplate(96 wells). The microplate was covered and incubatedat 4°C overnight. After the incubation period, themicroplate was washed three times with TPBS (0.05%Tween in phosphate buffered saline (PBS)), andblocking buffer was added (2% bovine serum albumin(BSA) in PBS). The microplate with the blocking bufferwas incubated at room temperature for 45–60 min. Atthe end of this incubation period, the microplate waswashed and 100 μL of diluted monoclonal antibodyagainst CYP1A (Biosense, Bergen, Norway; in 1%BSA in PBS) was added to each well. The microplatewas covered and incubated for 1 h at 37°C. Then, themicroplate was washed three times in TPBS and 100 μLof a diluted (in 1% BSA in PBS) secondary antibody(anti-rabbit IgG, Sigma, St. Louis, MO, USA) wasadded to each well, followed by incubation at roomtemperature for more 60 min. Afterward, microplatewas washed five times in PBS and 100 μL of revealingsolution o-phenylenediamine (Sigma, St. Louis, MO,USA) was added to each well, followed by theaddition of 50 μL of a Stop solution (4 N H2SO4,purity 95–97%, Merck, Darmstadt, Germany), after30 min. The samples were then read using an ELISAmicroplate reader (Bio-Rad, Hercules, CA, USA) at awavelength of 492 nm.

3 Statistical Analysis

Data recorded for each variable measured in thisstudy (HSI, GSI, CPY1A) was analyzed by a moreconservative one-way ANOVA, with type III sum ofsquares, as it is more appropriate for the unbalanceddesign created by the death of the animals (Quinn andKeough 2002). This analysis was used to test the nullhypothesis of no significant differences betweenorganisms exposed to different effluent concentra-tions, in terms of variables measured. Whenever thenull hypothesis was rejected, a Dunnett multiplecomparison test was performed to test each groupagainst the control. Statistical analysis was performed

with a significance level of 5%, using the softwareSPSS® 17.0 Statistics, for Windows.

4 Results

Table 1 describes the general characterization of thebleach Kraft pulp mill effluent after secondarytreatment.

4.1 Exposure Assays

The cumulative mortality, length, and weight oforganisms were registered at the end of the exposureperiod and are shown in Table 2. It is important tonotice that 100% of mortality was recorded in D.labrax exposed for 14 days to 50% and 100% BKPMEconcentrations. Concerning C. auratus, the same levelof mortality was observed only in fish exposed to non-diluted BKPME (100% concentration).

4.2 Somatic Indices

The GSI and the HSI were calculated for bothspecies, and the results are presented in Fig. 2a, b.Significant differences were found among GSIvalues determined in the different effluent concen-trations for both species: D. labrax (F=4.601, d.f.=3, p=0.019) and C. auratus (F=4.583, d.f.=4,p=0.006). An increasing trend of GSI with effluentconcentration was observed for D. labrax (Fig. 1a);however, only animals exposed to the 10% (p=0.023) and 25% (p=0.019) effluent BKPME con-centrations showed a GSI value significant differentfrom the control. In opposition although a decreasingtrend in GSI values was recorded for C. auratus

Table 1 Characteristics of secondary-treated bleached krafteffluent from pulp and paper mill

Parameter Secondary treated

pH 7.0±0.2

COD (mg/L) 392±2

Absorbance(325 nm)

1.373±0.015

Absorbance(400 nm)

0.407±0.003

Absorbance(460 nm)

0.204±0.001

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(Fig. 1a) exposed to different BKPME concentra-tions, significant differences in comparison with thecontrol were recorded only for animals exposed tothe 25% concentration (p=0.007). As far as the HSIwas considered, no significant differences were

recorded for both fish species, exposed to differenteffluent concentrations (Fig. 1b).

4.3 CYP1A

The results for CYP1A induction in the liver ofexposed fish are represented in Fig. 2. Significantdifferences among treatments were recorded for bothspecies: D. labrax (F=10.582; d.f.=3, p=0.001) andC. auratus (F=26.607; d.f.=4; p=0.000). Theinduction of CYP1A was significantly high on fishexposed to the highest BKPME concentrations thatdid not kill the animals, namely 25% for D. labrax(p=0.002) and 25% (p=0.036) and 50% (p=0.000)BKPME concentrations for C. auratus.

4.4 Histopathology

Figure 3 displays representative microscopic imagesof the hepatic tissue of control fish, as well as of fishexposed to particular effluent concentrations. Thecontrols (Fig. 3a, b) show the normal structure ofthe liver tissue, with normal hepatocytes showing a

Fig. 1 a, b GSI and HSI in C. auratus and D. labrax exposedto different concentrations of BKME. Significant differencesfrom controls if *p<0.05

Effluent Control 1% 10% 25% 50% 100%

C. auratus, N 8 8 8 8 8 8

Mortality rate, % 2.50 5 5 2.50 5 100

Length (mm) 42±3 42±5 40±4 41±5 44±3

Weight (g) 3.4±0.5 3.0±0.8 2.8±0.8 3.1±1.4 3.5±0.7

D. labrax, N 6 6 6 6 6 6

Mortality rate, % 12.5 25 25 25 100 100

Length (mm) 77±6 76±9 73±6 77±4

Weight (g) 8.3±2.0 8.6±2.4 7.4±1.4 8.2±1.0

Table 2 Mortality rate,length, and weight (mean ±SD) after exposure tosecondary-treated BKME

C. auratus (total N=48); D.labrax (total N=36)

Fig. 2 Relative intensity (OD) of CYP1A in fish exposed tothe different percentages of BKME. Significant differencesfrom controls if *p<0.05

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polygonal shape, weakly basophilic, with a singlespherical nucleus and containing small lipid droplets,sometimes hardly distinguished. The hepatic paren-chyma is homogeneous, formed by a system ofhepatic cords with hepatocytes supported by asinusoidal net.

With respect to C. auratus, notable histologicalalterations were observed only on fish exposed to25% and 50% BKPME concentrations. These animalshave showed a progressive degeneration of thehepatic tissue with hepatocyte hypertrophy, whichwas more evident in fish exposed to 50% concentra-tion of the secondary-treated effluent, which alsodisplayed tissue necrosis (Fig. 3c).

The morphology of the hepatic parenchyma of D.labrax from the control was similar to that observedfor C. auratus (Fig. 3a, b). However, in this species,several alterations were observed on fish exposed toall the concentrations BKPME tested. The BKPMEcaused severe changes in liver cells, the severity andextension of lesions increased with effluent concen-tration. A progressive degeneration and a decrease ofhepatic tissue integrity were observed as well as theoccurrence of foci of pigmented macrophages(melanomacrophages centers). In some animals, itwas also observed the presence of necrosis foci and/orproliferative lesions (Fig. 3d).

5 Discussion

The results of the present study showed that twodifferent fish species (C. auratus and D. labrax) weresensitive to BKPME after it has undergone secondarytreatment. Pereira et al. (2009) also have recorded theacute and sub-lethal toxicity of this secondary-treatedeffluent for the bacteria Vibrio fischeri, the green algaePseudokirchneriella subcapitata and the cladoceranDaphnia magna.

This effluent has a mean of 0.19 kg/t AOX andalso more 38 organic compounds, including 6% ofphenolic compounds, 53% of carboxylic acids, 16%of saturated and unsaturated alcohols, and 5% ofsterols (Rocha- Santos et al. 2010).

This demonstrates that the biological treatment mayreduce substantially the BKPME toxicity to the aquaticenvironments, but some toxic compounds still persist orare formed during the treatment process, being availableto cause acute and/or chronic toxicity to organisms(Aaltonen et al. 2000). In fact, the main aspect focusedin BKPME studies has been the potential effects ofchlorinated organic compounds, yield as a by-product ofthe bleaching process (Couillard et al. 1999), andMunkittrick et al. (1992) have confirmed that thesecondary treatment did not remove CYP1A-inducingcompounds from the BKPME.

Fig. 3 Control liver from aC. auratus and b D. labrax;c liver from C. auratusexposed to 50% of the finaleffluent, showingdegeneration of the hepaticparenchyma; d liver from D.labrax exposed to 25% ofBKME: tissue degeneration(arrowhead) andmacrovesicular steatosis(asterisk). Bar, 50 μm.Staining H&E

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The liver of teleost fish species is the primaryorgan responsible for the biotransformation of organicxenobiotics and probably also the excretion ofharmful trace metals, the food digestion and storage,and the metabolism of sex hormones (Au 2004).Thus, liver biomarkers are increasingly regarded aspowerful and informative tools in ecotoxicology andenvironmental monitoring. The induction of thecytochrome P450 1A (CYP1A) in fish, for example,has been used as a sensitive “early warning” indicatorof exposures to organic xenobiotics in the aquaticenvironment (Nielsen et al. 1998). In fact, theinduction of CYP1A in the liver of fish is consistentwith the role of this organ in xenobiotic metabolism,detoxification, and excretion (Au 2004). Consequently,to determine exposure and sub-lethal effects ofBKPME, this biomarker as well as histopathologyand/or other health indices have been used as measure-ment endpoints of the responses of organisms (Couillardet al. 1999).

A 100% mortality rate was recorded for C. auratusexposed to non-diluted BKPME (100%) and for D.labrax exposed to 50% and 100% BKPME concen-trations, highlighting the acute toxicity of the effluentto these species and particularly to sea bass. For marinespecies, the pre-acclimation test suggests that aneventual acute osmotic stress is not occurring at lowersalinity causing these mortalities and the resultsindicate an elevated effluent’s toxicity as the maincause of mortality. However, we cannot exclude totallythat adaptation to different salinities may influence theresults even knowing that juvenile sea bass are able tolive in different salinities from freshwater to 60‰salinity (Jensen et al. 1998). On the other hand, wecannot exclude either that the contaminants fromBKPM effluent can impair osmoregulation and conse-quently affect the adaptation to different salinities.

Additionally, the results obtained for the biomarkersevaluated, as well as somatic indices and histopathology,suggest that both species also have different sensitivitiesto BKPME concentrations tested. Furthermore, theCYP1A induction observed for the liver for both speciesexposed to concentrations of BKPME, equal or below50%, suggests once again the presence of toxiccompounds in BKPME. Our data were consistent withobservations made by other authors, when this bio-marker was accessed on other fish species. In fact, Chenet al. (2001) have shown that the BKPME may induceP4501A enzymes in several fish species. Gravato and

Santos (2002) have exposed juvenile sea bass for 72 hto a BKPME produced by a similar Kraft pulp millprocessing, using E. globulus, and demonstrated thepresence of genotoxic compounds in the effluent.Although low ethoxyresorufin-O-deethylase (EROD)levels were measured, a significant P450 induction wasobserved on fish exposed for 8 up to 72 h. The sameauthors carried out an experiment in which eels(Anguilla anguilla) were exposed to BKPME (3.12%,6.25%, and 12.5% (v/v)) for 3, 7, and 30 days and haveshowed that such exposures did not induce significantlythe total EROD activity. In opposition, Andersson et al.(1988) observed that BKPME have increased, inparticular, the EROD activity in perchs living nearthe Swedish coast. Furthermore, Lindstrom-Seppa andOikari (1989, 1990) showed that BKPME affected thecytochrome P450 system of fish captured in Finnishinland waters. According to Aaltonen et al. (2000),BKPME also induced liver EROD activity in roachand impaired the immune responsiveness. Tests usingBKME from the same plant showed that there aresome chemicals having toxicity and able to affect fishphysiology that prevail even after tertiary treatment(Diniz et al. 2009; Freitas et al. 2009).

Changes in the weight of the liver have also beenpointed out as an interesting indicator of contaminantstress (Yang and Baumann 2006). Typically, increases inliver biomass following exposure to Aryl hydrocarbonreceptor agonists are explained by a proliferation of thesmooth endoplasmic reticulum promoted by an increasein the synthesis of CYP1A-related proteins (Larsson etal. 1988). Other authors have shown that fish fromchemically contaminated sites had elevated values ofhepatosomatic index when compared to fish fromrelatively uncontaminated sites (Slooff et al. 1983;Fabacher and Baumann 1985; Everaarts et al. 1993),though a variety of factors other than contaminants mayhave caused changes in the liver size as well (Fabacherand Baumann 1985). However, the present studyshowed no significant differences in HSI levels amongfish of both species exposed to different BKPMEconcentration.

The significant decrease of GSI recorded for C.auratus suggests a negative effect at the reproductivesystem level. This was in agreement with otherstudies, reporting the role of a BKPME sample,resulting from the use of elemental chlorine in thebleaching process, in the inhibition of reproduction infish and in GSI reduction (Larsson et al. 1988;

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Munkittrick et al. 1994; Gagnon et al. 1995; Denslowet al. 2004). According to Sepúlveda et al. (2004),other studies have reported that the decrease in gonadsize may not always occur after exposures of fish toBKME (McMaster et al. 1996a, b). This difference inGSI can be related, for instance, to differences inspecies sensitivity as they can have different metabolicpathways of detoxification or distinct mode of action ofcompounds target sites (Vaal et al. 1997a).

The histological evaluation revealed changes in thehepatic tissue of both species. However, the results ofthe histological evaluation suggest that the BKPMEwasmore toxic for D. labrax, since necrosis foci and/orproliferative lesions were observed in some organisms.

The results of the histological evaluation suggestedonce again a different response of test speciesexposed to a final effluent from Kraft industry (aftersecondary treatment). While the cyprinids revealed tobe more resistant to BKPME, the results obtainedwith the sea bass suggest that this effluent was moretoxic for this marine species, even at low concen-trations. The effluent toxicity was more evident in theeffluent diluted to 25% and 50% as suggested by theresults from CYP1A induction, demonstrating aresponse at the level of hepatic metabolism. Theresults of the histopathological analysis confirm theadverse effects in the hepatic tissue and demonstratedonce again the higher susceptibility of D. labrax toBKPME toxicity.

The interspecific variation found in the results can beattributed to different sensitivities of these species whichare related to fish’s biology, variations in metabolicpathways of detoxification, distinct compound targetsites of toxic action, genetics, and diverse evolutionaryadaptations to habitats (Vaal et al. 1997a, b).

In summary, a significant aspect of the presentwork is a comparative approach regarding BKMEeffects in two different species from different aquaticenvironments, by assessing the induction of a specificbiomarker (CYP1A) and changes at the cellular levelcomplemented with somatic indices evaluation. Inaddition, as few studies have been carried out withmarine species, this work gives a valuable contribu-tion for a better comprehension of the BKME effectsin fish species which can lead to the development ofmore efficient effluent treatment strategies to a betterprotection of the aquatic environments.

Further, this study suggests that the secondarytreatment usually applied to BKPME did not prevent

its potential to yield sub-lethal effects on fish, evenafter a remarkable dilution, which is expected to occurwhen entering into receptor aquatic systems. Sucheffects on fish specimens may give rise to top-downeffects on the trophic chains, compromising thestability of the whole ecosystem.

Acknowledgments This work has been developed under thescope of the FCT (Portugal) funded research project, POCT/CTA/45030/2002: “Decolourisation of effluents from pulp andpaper mills: removal of organic compounds and toxicity(DECORTOX)”.

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