ilable at ScienceDirect

Toxicon 55 (2010) 396–406

Contents lists ava

Toxicon

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

Analyses of paralytic shellfish toxins and biomarkers in a southernBrazilian reservoir

Zaira Clemente a, Raquel H. Busato a, Ciro A. Oliveira Ribeiro b, Marta M. Cestari c,Wanessa A. Ramsdorf c, Valeria F. Magalhaes d, Ana C. Wosiack e, Helena C. Silva de Assis a,*

a Departamento de Farmacologia, Universidade Federal do Parana, Setor de Ciencias Biologicas CEP 81531-990, Caixa Postal 19031, CEP 81531-970,Curitiba-Parana, Brazilb Departamento de Biologia Celular, Universidade Federal do Parana, Setor de Ciencias Biologicas CEP 81531-990, Caixa Postal 19031, CEP 81531-970,Curitiba-Parana, Brazilc Departamento de Genetica, Universidade Federal do Parana, Setor de Ciencias Biologicas CEP 81531-990, Caixa Postal 19031, CEP 81531-970,Curitiba-Parana, Brazild Instituto de Biofısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundao, Bloco G, CEP 21941-902, Rio de Janeiro, Brazile Instituto Ambiental do Parana, CEP 80215-100 Curitiba, Brazil

a r t i c l e i n f o

Article history:Received 27 May 2009Received in revised form 1 September 2009Accepted 15 September 2009Available online 22 September 2009

Keywords:Paralytic shellfish toxinsBiomarkersFish

* Corresponding author. Tel.: þ55 41 33611743; faE-mail address: helassis@ufpr.br (H.C. Silva de A

0041-0101/$ – see front matter � 2009 Elsevier Ltddoi:10.1016/j.toxicon.2009.09.003

a b s t r a c t

The Alagados Reservoir (Brazil) is an important source for the supply of water, recreationand fishery. Since 2002, the occurrence of cyanobacterial blooms (paralytic shellfish toxins –PST producers) have been noted. This study was aimed at the monitoring of PST occurrencein the Reservoir’s water and fish. Biomarkers such as ethoxyresorufin-O-deethylase (EROD),glutathione S-transferase (GST), catalase (CAT), and acetylcholinesterase (AchE) activities,lipoperoxidation (LPO), histopathology, and comet assay were analyzed in fish. Water andfish were sampled in spring, summer and autumn. The PST concentrations in water were5.15, 43.84, and 50.78 ng equiv Saxitoxin/L in the spring, summer and autumn, respectively.The PST muscle concentration was below the limit for shellfish. Gonyautoxins (GTX) werefound in water samples and fish muscle, and GTX 5 was the major analogous found inmuscle. In the summer samples, the LPO, genetic damage, and the GST and AchE activitiesincreased while in the autumn an increase in EROD activity and genetic damage wereobserved. In all samplings, histopathological alterations in the fish gills and liver werefound. The results showed a seasonal variation in the fishes health, which could be relatedalso to farming activities and to the contaminants bioavailability during the year.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The alteration of landscapes and the pollution of waterresources by both natural and human-made processes haveserious ambient, economic, and public health conse-quences. Thus, the use of water, either for regular supply orfor generation of energy, must be managed in a responsiblemanner.

x: þ55 41 3266 2042.ssis).

. All rights reserved.

The enrichment of nutrients in aquatic ecosystems,especially those containing phosphorus and nitrogen, is animportant factor that leads to the eutrophication of thesesystems and to an accelerated growth of algae (Briand et al.,2003). Cyanobacterial proliferations, also known as blooms,cause negative impacts on the ecosystem, on the health ofanimals living in these systems, and on human populationsthat use these water bodies for water supply and/orrecreational purposes (Chorus and Bartram, 1999; Wiegandand Pflumacher, 2005). Cyanobacteria that producepoisonous toxins (cyanotoxins) have been reported fromaround the world over the past few decades (Carmichael,

Z. Clemente et al. / Toxicon 55 (2010) 396–406 397

1994; Codd et al., 2005). Cyanotoxins are classified on thebasis of their chemical (cyclic peptides, alkaloids, or lipo-polysaccharides) or toxicological (hepatotoxins, neuro-toxins, or dermatotoxins) properties (Patocka, 2001).

In 1999, Lagos et al. published the first report describingthe production of saxitoxin analogs by the freshwatercyanobacterium Cylindrospermopsis raciborskii in SouthAmerica. Because the occurrence of blooms due to theproliferation of this species was observed in different reser-voirs in Brazil (Chellappa and Medeiros Costa, 2003; Tucciand Sant’Anna, 2003; Yunes et al., 2003; Sperling et al., 2008),saxitoxin analysis of water assumed great importance.

The Alagados Reservoir supplies water to three cities inthe State of Parana in southern Brazil. Electrical energygeneration, recreation, and fishing are the other importantactivities associated with the reservoir. Farming activitiesand disordered occupancy near the reservoir are the maincauses of water eutrophication. Consequently, frequentcyanobacterial blooms, with concomitant production ofsaxitoxins, have occurred since 2002 (Yunes et al., 2003).Analyses conducted over the past several years have showncyanobacterial levels as high as 8� 105 cells/ml (InstitutoAmbiental do Parana, 2007).

Saxitoxins comprise a group of more than 20 moleculeswith a tetrahydropurine structure, which are also known asparalytic shellfish toxins (PST) due to their occurrence andassociation with seafood. They are produced by some speciesof dinoflagellates and cyanobacteria and can be classifiedinto four groups based on their chemical structure asfollows: saxitoxins (STX, decarbamoyl saxitoxin-dcSTX,neosaxitoxin-neoSTX, decarbamoyl neosaxitoxin-dcneoSTX,and nonsulfated STX); gonyautoxins (GTX 1 to 6, dcGTX 1 to4, and single-sulfated GTX); C-toxins (C 1 to 4, doublysulfated C-toxins); and other variants identified in Lyngbyawollei (LWTX 1 to 6).

All these toxins block neuronal transmission by bindingto site 1 of the voltage-gated Naþ channels in nerve cells,causing neurotoxic effects (Patocka, 2001; Briand et al.,2003; Wiegand and Pflumacher, 2005). Moreover, there areevidences of STX being a gating modifier of hERG K, andthat can block L-type Caþ2 currents in myocytes (Wanget al., 2003; Su et al., 2004).

The aim of this study was to analyze the presence ofPSTs in both water and muscle tissue of a fish species fromthe Alagados Reservoir and to assess the toxic impact of thiswater on a selected group of biomarkers during differentseasons of the year. In this study, the activities of ethoxyr-esorufin-O-deethylase (EROD) and glutathione S-trans-ferase (GST) were estimated to identify the changes inhepatic biotransformation; hepatic lipoperoxidation (LPO)and activity of catalase (CAT) were evaluated to investigatethe disturbances in cellular oxidative stress. Brain andmuscle tissues were used to study the acetylcholinesterase(AchE) activity. Histopathology studies of the gill and liverwere used as physiological and morphological biomarkersdue to the constant contact of the gill with water and theimportance of the liver as the major center of xenobioticmetabolism.

Finally, the genotoxic effect was applied as an indicatorof general damage and its assessment was carried out usingthe comet assay.

2. Material and methods

2.1. Fish

Geophagus brasiliensis (Perciformes, Cichlidae) isa native fish widely distributed in Brazil. It is the mostcommon species in the Alagados Reservoir and was henceused in this study to analyze the effect of pollutants presentin the Alagados waters.

Specimens of G. brasiliensis and samples of water werecollected from the Alagados Reservoir in November 2007(corresponding to spring in the southern hemisphere),February 2008 (summer), and May 2008 (autumn). Fish wereanesthetized with 2% benzocaine and then killed by medullarsection. The total length of each fish was measured, and bloodwas collected for the comet assay. The third-left gill arch anda liver fragment were sampled for histopathological studies.Samples of the brain, liver, and axial muscle were frozen at�70 �C for biochemical analyses. The muscle was alsocollected and stored in the dark at�20 �C for PST analysis.

2.2. Water analysis

The water was harvested always at two different points:at the deepest point of the Reservoir (near the dike, P1) andat the edge (P2), a shallow point at a distance of 720 m fromP1. The water was harvested always at 11 a.m. at a depth of50 cm under the water surface (euphotic zone). One samplewas preserved with a Lugol solution for studying the cya-nobacterial count (Utermohl, 1958), and another was storedat 4 �C for 24 h until used for analysis of PSTs by high-performance liquid chromatography (HPLC). The corre-sponding results were expressed as the mean values of thetwo points (P1 and P2).

The pH, temperature, and levels of dissolved oxygen inthe water were measured during the samplings.

2.3. Determination of PSTs in water and fish-muscle tissue

Water (500 ml) was first lyophilized, then resuspendedin acetic acid (0.5 M), and stored at �20 �C for subsequentanalysis. The water samples were filtered with celluloseacetate filters (0.45 mm) before HPLC analysis.

Pools of approximately 10 g of fish muscle were used todetermine the PST content in each group. The pools werehomogenized in HCl (0.1 N) and then centrifuged at10,000� g at 19 �C for 10 min. The supernatants were filteredwith cellulose acetate filters (0.45 mm) before HPLC analysis.

Analyses of the two classes of PST (GTX and STX) inwater and fish-muscle tissue were carried out by HPLC withpostcolumn oxidation using a fluorescence detector, asdescribed by Oshima (1995). PST standards, from theNational Research Council, Canada, were also run before andafter sample analyses. The detection limits were deter-mined as follows: STX, 0.89 ng/L; NeoSTX, 2.33 ng/L; GTX 1,1.03 ng/L; GTX 2, 0.72 ng/L; GTX 3, 0.21 ng/L; GTX 4,0.24 ng/L, and GTX 5, 0.01 ng/L.

The concentrations of each toxin analog, as determinedby chromatographic analyses, were converted to equiva-lents of STX (STX eq.) for comparing the toxicity of eachvariant with that of STX (Hall et al., 1990).

Z. Clemente et al. / Toxicon 55 (2010) 396–406398

2.4. Biomarkers

Samples of liver were homogenized (10% w/v) in cold(4 �C) phosphate buffer (0.1 M, pH 6.5). Homogenates werecentrifuged at 10,000� g for 20 min at 4 �C. Aliquots of thesupernatant (S9 fraction) of each sample were collected foranalyses of LPO and activities of EROD, GST, and CAT.

Samples of brain (10% w/v) and muscle (5% w/v) werehomogenized in cold (4 �C) potassium phosphate buffer,0.1 M, pH 7.5. Homogenates were centrifuged at 10,000� gfor 20 min at 4 �C. The supernatant (S9) was used forassessing AchE activity.

EROD activity was measured according to the method ofBurke and Mayer (1971), as modified by Silva de Assis(1998). The measurement was carried out with a Shimadzuspectrofluorimeter using 530 and 590 nm as the excitationand extinction wavelengths, respectively. Enzyme activitywas calculated as pmol of resorufin formed/min/mg ofprotein, using a standard curve of resorufin.

GST activity was measured at 340 nm using the methoddescribed by Keen et al. (1976). The enzyme activity wascalculated as mmol 1-chloro-2,4-dinitrobenzene conjugateformed/min/mg protein.

Analysis of LPO was carried out using the ferrousoxidation-xylenol orange assay at 570 nm (Jiang et al.,1992). LPO levels were expressed in nmol of hydroperox-ides/mg protein.

The CAT activity was measured at 240 nm based on themethod described by Aebi (1984). Enzyme activity wasexpressed in mmol H2O2 consumed/min/mg protein.

Measurements of AchE activity were recorded using themethod of Ellman et al. (1961), modified for microplates bySilva de Assis (1998). The absorbance was recorded at405 nm, and the enzyme activity was expressed as nmol ofthiocholine formed/min/mg protein.

Protein concentration was determined using the Brad-ford’s (1976) method, with bovine serum albumin as thestandard.

2.5. Histopathology

Samples of gill and liver were preserved in ALFAC fixa-tive solution (every 100 ml of solution contained 85 ml of80% ethanol, 10 ml of 40% formaldehyde, and 5 ml of 100%acetic acid) for 16 h, dehydrated in a graded series ofethanol baths, diaphanized in xylol, and embedded inParaplast-Plus resin (Sigma). The sections (5 mm) werestained with 1% Harris hematoxylin and eosin/floxin andobserved under a Zeiss-Axiophot photomicroscope.

Histopathological alterations were evaluated accordingto Bernet et al. (1999) by considering the proposed alter-ations and their respective factors of importance. Scorevalues, ranging from 0 (unchanged) to 6 (severe occur-rence), were attributed to each alteration depending on thedegree and extent of the alteration. The liver- and gill-injury indexes (IL) were calculated as IL¼

Prpþ

Palt (aw),

where rp¼ reaction pattern, alt¼ alteration, a¼ scorevalue, and w¼ importance factor. Melano-macrophagecenters (MMCs/mm2) were determined microscopicallyusing an eyepiece grade. The structures were quantified in15 fields (sorted randomly) on each slide.

2.6. Comet assay

A blood sample was diluted in fetal bovine serum,stored in ice in the dark for 24 h, and then prepared for thecomet assay (Ramsdorf et al., 2009), which was carried outas described by Singh et al. (1988), with some modificationsproposed by Ferraro et al. (2004).

The slides for microscopy were prepared with 10 ml ofthe cell suspension in low melting-point agarose (120 mL)at 37 �C, followed by incubation in a lysis solution at 4 �C for7 days. After lysis, the slides were placed in a solution ofNaOH (10 N) and EDTA (200 mM),buffer (pH> 13) for30 min to unravel the DNA. Electrophoresis was carried outat 25 V and 300 mA for 25 min at 4 �C, and the slides wereneutralized for 15 min with 0.4 M Tris, pH 7.5, fixed in 95%ethanol for 5 min, and stained with 0.02 mg/ml ethidiumbromide. DNA strand breaks were scored using a Leicaepifluorescence microscope at a magnification of 400�. Foreach fish, 100 cells were visually analyzed according to themethod of Collins et al. (1997).

2.7. Statistical analysis

The Kolmogorov–Smirnov normality test preceded dataanalysis. One-way analysis of variance (ANOVA) was used,followed by the Bonferroni test, to analyze biochemicaldata, Melano-macrophage centers, and lengths of the fish.The correlation between the AchE activities in the muscleand brain and the total lengths of the fish was analyzed bythe Pearson correlation. The data from the comet assaywere analyzed using the Kruskal–Wallis test. The dataregarding the histopathological injury indexes and the PSTconcentrations in muscle were analyzed using the Kruskal–Wallis test, followed by the Dunn’s test. All data werestatistically analyzed by the GraphPad Prism v5.00.288(GraphPad Software, Inc.). All tests were regarded asstatistically significant when p< 0.05.

3. Results

3.1. Analysis of PST levels in water and fish muscle

The percentage of cyanobacteria in relation to the totalphytoplankton was about 40% in spring, whereas it was 88%in summer and autumn. Only gonyautoxins (GTXs) weredetected in the water (Tables 1 and 2).

In autumn, the weather made the sampling of G. brasi-liensis difficult; consequently, the number of fish sampledwas smaller than in other seasons of the year.

The toxin profile of fish muscle from all samplingsshowed the presence of GTXs (Table 3). GTX5 was thedominant compound in this profile, representing almost100% of total PST concentration in all samplings. There wasno significant difference in the PST concentrations inmuscle among the three samplings.

3.2. Biomarkers

EROD activity (Fig. 1A) was significantly higher inautumn than in spring and summer. GST activity in the liver(Fig. 1B) was significantly higher in summer than in spring

Table 1Physical-, phytoplankton-, and PST analyses of the water from AlagadosReservoir in spring of 2007, and summer and autumn of 2008 (mean of P1– near the dike; and P2 – at the edge).

Spring Summer Autumn

Temperature (�C) 23.5 26.9 16.8Dissolved O2 (mg/l) 8.5 9.5 6.2pH 8.2 8.6 7.4Cyanobacteria (Cells/ml) 28,237 119,743 116,911Cylindrospermopsis

raciborskii (Cells/ml)27,180 110,758 107,398

Total phytoplankton(Cells/ml)

63,269 124,927 123,178

STX eq. (ng/L) 5.15 43.84 50.78PST GTX 2, 3

and 5GTX 2, 3, 4and 5

GTX 2, 3, 4and 5

Table 3Total PST concentration, concentration in STX eq. and toxins in muscle ofGeophagus brasiliensis from Alagados Reservoir.

Spring(7 pools,n¼ 24)

Summer(5 pools,n¼ 25)

Autumn(2 pools,n¼ 15)

Total PST concentration(mg/100 g)

2.21� 1.56 3.06� 1.45 2.40� 0.09

STX eq. (mg/100 g) 1.22� 0.87 1.97� 0.93 1.54� 0.05PST GTX 3, 4 and 5 GTX 3 and 5 GTX 3 and 5

Z. Clemente et al. / Toxicon 55 (2010) 396–406 399

and autumn. LPO (Fig. 1C) was significantly higher insummer than in autumn, but it was not different comparedto that of spring. No differences were observed among thethree samplings for CAT activity (Fig. 1D) in the liver andAchE activity in the brain (Fig. 1E). AchE activity in muscle(Fig. 1F) was significantly higher in summer than in springand autumn.

In all the seasons, no significant correlation wasobserved between the brain AchE activity and total lengthof fish. Only in the spring, a negative correlation(r¼�0.6497) was observed between AchE activity inmuscle and the total length of fish. The total length of fish(mean value� SEM) showed significant differences amongthe seasons and were recorded as follows: 14.05� 0.67 cmin spring, 11.93� 0.29 cm in summer, and 9.92� 0.49 cm inautumn.

Several alterations in the gills and liver (Figs. 2 and 3) offish from the Alagados Reservoir were observed, witha notable histopathological injury index in all the seasons;nevertheless, these indexes were not significantly differentamong the various seasons for both tissues. Hyperplasia ofthe epithelium (with partial or total lamellar fusion) wasthe most frequent lesion in gills in all the seasons. Parasiteswere observed in all groups. In both spring and summer,aneurysms were observed, in addition to agglomerations ofcells that remain unidentified until date. Inflammation and

Table 2Total PST concentration, concentration in STX eq. and PST profiles in % ng/Lin water from Alagados Reservoir, in spring of 2007 and summer andautumn of 2008, in P1 (near the dike) and P2 (at the edge).

STX eq.(ng/L)

Total PSTconcentration(ng/L)

Percentage of total PST (% ng/L)

GTX 2 GTX 3 GTX 4 GTX 5

SpringP1 4.60 38.25 13.69 7.68 bdl* 78.61P2 5.69 9.71 58.7 41.29 bdl* bdl*

SummerP1 48.81 82.76 27.45 12.04 56.74 3.75P2 38.87 63.50 27.85 13.32 58.81 bdl*

AutumnP1 18.11 37.39 54.15 27.89 15.19 2.75P2 83.44 117.21 bdl* 5.40 93.17 1.41

* bdl¼ below detection limit

preneoplastic lesions were observed only in summer. In theliver, necrosis was the most frequent lesion in all groups. Inthe spring and summer samplings, vacuolar degeneration,cytoplasmatic deposits, and tissue alterations wereobserved, whereas in autumn, preneoplastic lesions werefound to occur. The frequency of the alterations is shown inTable 4.

The Melano-macrophage centers count was signifi-cantly higher in the spring than in both summer andautumn (Fig. 4).

The genetic damage in summer and autumn was higherthan that in spring (Fig. 5).

4. Discussion

4.1. Paralytic shellfish toxins

The cyanobacterial bloom observed during this studyfollowed the same profile reported years ago (InstitutoAmbiental do Parana, 2007). The bloom increased insummer in comparison to spring and remained at the samelevel up to the end of autumn, with a predominance ofCylindrospermopsis raciborskii (Table 1). The cyanobacterialcount in the water in the three studied seasons of the yearexceeded the limit proposed by the World Health Organi-zation (WHO) (20,000 cells/ml) for protection of healthwith mild and/or low probabilities of adverse effects relatedto recreational water (Chorus and Bartram, 1999). PSToccurrence in the water was found in all samples (Table 2).The PST (in STX eq./L) and the C. raciborskii count in thewater were enhanced eight and four times, respectively, inthe summer compared to the values in spring. Although thePST concentration in the water during the three samplingswas below the limit of 3 mg STX eq./L suggested by Fitzgeraldet al. (1999), which was adopted by Brazilian legislation asthe standard for water meant for human consumption, theWHO considers that there is not enough knowledge toestablish a safe limit for PST ingestion.

The PST concentration (in STX eq./100 g) in fish musclein this study was found to be similar in all the three seasonsof the year, in spite of the differences in C. raciborskii countsand in the levels of STX eq. in the water. Nassem (1996)stated that the level of saxitoxinol (a synthetic analogous ofSTX) in the muscle was not reduced significantly 48 h afterexposure, suggesting that the binding of this toxin tomuscle is irreversible; this finding may explain the absenceof differences among the PST concentrations in muscleobserved in this study.

Fig. 1. Biochemical biomarkers evaluated in Geophagus brasiliensis from Alagados Reservoir in spring 2007, summer and autumn 2008. Results are expressed asmean values � SEM. Different letters indicate significant differences among the groups (p< 0.05.), One-Way Anova, followed by Bonferroni test. (A) Specificactivity of EROD in the liver (pmol of resorufin/min/mg protein). (B) Specific activity of GST (nmol of conjugated CDNB/min/mg protein) in the liver. (C) LPO (nmolof hydroperoxides/mg protein) in the liver. (D) Specific activity of CAT (mmol of degradated H2O2/min/mg protein) in the liver. (E) Specific activity of AchE (nmol ofthiocholine/min/mg protein) in the brain. (F) Specific activity of AchE (nmol of thiocholine/min/mg protein) in the muscles.

Z. Clemente et al. / Toxicon 55 (2010) 396–406400

Cases of human toxicity caused by consumption of fishcontaining PSTs were reported in 1976 in Brunei and in1983 in Indonesia (Deeds et al., 2008). However, most of thereports were related to toxicity caused by ingestion ofshellfish (Chorus and Bartram, 1999; Garcia et al., 2005;Deeds et al., 2008). Internationally, a limit of 80 mg STX eq./100 g has been established for shellfish (Chorus and Bar-tram, 1999). The current results showed that the species G.brasiliensis from the Alagados Reservoir accumulated PST inthe muscle during C. raciborskii blooms, with concentra-tions remaining below the limit established for shellfish.

In spite of the apparent low concentration of PST in boththe water and the fish muscle analyzed, the hypothesis oftrophic transference and biomagnification along the food

chain was not refuted. This phenomenon has been studied inseveral xenobiotics, such as dichloro-diphenyl-trichloro-ethane or DDT, polychlorinated biphenyls, toxaphene,methyl mercury, and arsenic (Gray, 2002; DeForest et al.,2007), whose effects can be more dangerous in organisms atthe highest trophic levels. No data was found concerning PSTbiomagnifications, regardless of some recent studies thatshowed PST being transferred through predation and accu-mulating in carnivorous organisms (Jiang et al., 2007).

Further studies are necessary for a comprehensivepicture, considering that the cyanobacterial count in thepresent work was not as high as that found in other years.In December 2006, the cyanobacterial count in Alagadoswas 867,721 cells/ml, with a predominance of C. raciborskii

Fig. 2. Cross-sections of gills of Geophagus brasiliensis from Alagados Reservoir: (A) gills without lesions showing primary- and secondary lamellae (white- andblack arrows, respectively), (B) preneoplastic lesion (C) agglomeration of unidentified cells (arrow), (D) partial epithelial hyperplasia (arrow), (E) epithelialhyperplasia with lamellar fusion. Bar scale¼ 100 mm

Z. Clemente et al. / Toxicon 55 (2010) 396–406 401

(Instituto Ambiental do Parana, 2007). Thus, contaminationprofiles can differ, and consumption of water and fish canrepresent a higher risk for public health. Laboratory studiesof both the biomagnification and the interaction of cyano-toxins with other contaminants are essential for a betterunderstanding of the results obtained in biomonitoringstudies.

In this study, GTX 5 was the predominant toxin presentin the muscles, representing almost 100% of the total PST inall samplings. Nevertheless, it was not the predominanttoxin present in water, except at P1 in spring. The greaterpart of the PST found in water is formed by GTX 2, 3, and 4,whose relative toxicities are higher than that of GTX5 (Hallet al., 1990). In summer, more than 50% of PST concentra-tion in the water was constituted by GTX4, which is themore toxic subtype among the detected toxins, beingapproximately 11 times more toxic than GTX5 (Hall et al.,1990). Other studies from around the world also showedsubstantial differences in (i) the toxin profiles amongaquatic organisms sampled from the same area and (ii) thecomposition of PST producers (Sagou et al., 2005; Choiet al., 2006; Samsur et al., 2006; Sephton et al., 2007; Deedset al., 2008). Our results are in agreement with the data

reported in literature and suggest that aquatic organismsare capable of metabolizing these toxins into a less toxicform.

Kwong et al. (2006) reported differences in the tox-icokinetics of individual PST in mussels (Perna viridis) andshowed that the fish Acanthopagrus schlegeli can convertone analogous type to another. Differences among PST iningestion, distribution, metabolism (chemical and enzy-matic transformations), accumulation, and excretion in fishor even metabolism by gastrointestinal flora could explainthe differences in PST profiles between the water and thefish. The characteristics of the enzymes involved in themetabolism of PSTs are poorly understood, but some recentworks (Naseem, 1996; Andrinolo et al., 2002; Fast et al.,2006; Donovan et al., 2008; Garcıa et al., 2008) have pre-sented some clarifications regarding this issue.

4.2. Biomarkers

A few morphological, biochemical, and genetic alter-ations were observed in G. brasiliensis collected from theAlagados Reservoir in the three seasons of the year. Thesesuggest that there is a seasonal difference in the health of

Fig. 3. Cross-sections of liver of Geophagus brasiliensis from AlagadosReservoir. (A) Liver without lesions, (B) Preneoplastic lesion, (C) Area ofinflammatory response (arrow), (D) Necrosis area (black arrow) andpancreas tissue (white arrow). (E) Areas of cellular alteration (arrow)enlarged in the inset (e), (F) Melano-macrophage center (white arrow) andarea of cellular alteration (black arrow) enlarged in the inset (f). Barscale¼ 100 mm.

Table 4Prevalence (%) of the alterations in liver and gills observed in Geophagusbrasiliensis from Alagados Reservoir in spring of 2007, summer andautumn of 2008.

Spring Summer Autumn

Liver lesions n¼ 23 n¼ 20 n¼ 15Necrosis 69.56 80 73.33Leucocytic infiltration 8.69 30 40Melano-macrophage centers 91.30 85.00 86.66Vacuolar degeneration 4.34 5 0Cytoplasmatic Deposits 60.86 35 0Preneoplastic lesion 0 0 13.33Parasites 17.39 35 13.33Cellular alterations 30.43 10 0

Gill lesions n¼ 20 n¼ 22 n¼ 9Partial epithelial hyperplasia 85 68.18 33.33Epithelial hyperplasia

with Lamellar fusion30 50 44.44

Preneoplastic lesion 0 9.09 0Aneurysm 10 4.54 0Unidentified cells agglomeration 35 13 0Leucocytic infiltration 0 9.09 0Parasites 30 36.36 33.33

Z. Clemente et al. / Toxicon 55 (2010) 396–406402

fish. There was no difference in the CAT activity among allthe samplings, but no reference values were found for thespecies studied. It is, therefore, difficult to know whetherthe values were normal or not. Compared to the values ofother control fish species, the LPO of G. brasiliensisappeared to be high in all the samplings, indicatingoxidative stress.

In all the samplings, the prevalence of epithelial hyper-plasia in the gills, in addition to necrosis and Melano-macrophage centers in the liver, was high. Gill lesions can beinterpreted as the result of the acute effects of xenobiotics,depending on the type of lesion (Zodrow et al., 2004).Hyperplasia plays a role in the defense mechanism but alsoimpedes the occurrence of gaseous interchanges (McDonaldand Wood, 1993; Garcia-Santos et al., 2007). Necrosis isconsidered an irreversible lesion, associated with chronicexposure to pollutants (Bernet et al., 1999), and can arisefrom different mechanisms, such as lysosomal breakage,tissue hypoxia, disturbance in protein synthesis, and

Fig. 4. Melano-macrophages centers (MMC/mm2) in liver of Geophagusbrasiliensis from Alagados Reservoir in spring of 2007, summer and autumnof 2008. Results are expressed as mean values� SEM. Different lettersindicate significant differences among the groups (p< 0.05.). One-WayAnova, followed by Bonferroni test.

Fig. 5. Score of genetic damage in blood cells of Geophagus brasiliensis fromAlagados Reservoir in spring of 2007, summer and autumn of 2008. Resultsare expressed as mean and 10–90 percentiles. Different letters indicatesignificant differences among the groups (p< 0.05.), Kruskal–Wallis test.

Z. Clemente et al. / Toxicon 55 (2010) 396–406 403

carbohydrate metabolism (Oliveira Ribeiro et al., 2002;Hinton et al., 2008). Melano-macrophage centers aredistinctive groupings of pigment-containing cells (melanin,lipofuscin, or hemosiderin are the most common pigments)in the tissues of heterothermic vertebrates. The function ofthe Melano-macrophage centers in the liver of fish remainsuncertain, but some studies have suggested that it is relatedto the destruction, detoxification, or recycling of endo- andexogenous compounds. Several authors have suggested thathistopathological analysis of Melano-macrophage centerscould provide sensitive indicators of fish health or stressfulenvironmental conditions (Agius and Robert, 2003; Leknes,2007; Hinton et al., 2008).

In spring, the genetic damage and the activities of EROD,GST, and muscular AchE were low compared to the otherseasons of the year. In this season, however, the Melano-macrophage centers concentration was two times as muchas in the other seasons.

In spring and summer, tissue differentiation, which hasbeen suggested as an early stage in the stepwise formationof hepatic neoplasia (Hinton et al., 1992), was observed. Theprevalences of cytoplasmic deposits and tissue differentia-tion were 60 and 30%, respectively, in spring, as against 35and 10%, respectively, in the summer. It may be related to thestorage of lipids and glycogen and may additionally beaffected by other factors, such as availability of food, thereproductive cycle, season, or alteration in the metabolismof these cells (Stentiford et al., 2003; Arellano et al., 1999).The reproductive period of G. brasiliensis in Brazil is fromSeptember to January (Magalhaes, 1931; Suzuki andAgostinho, 1997). Thus, the vacuolization and cytoplasmicdeposits observed in this study can be related with thereproductive cycle of these organisms. However, a possiblerelation with pollutants cannot be rejected because OliveiraRibeiro et al. (2005) described the association of this type oflesion with exposure to polycyclic aromatic hydrocarbons.

There was no difference in the CAT activity among all thesamplings, and the activity was lower than that reported byWilhelm Filho et al. (2001) in G. brasiliensis sampled fromreference and polluted areas. Previous studies have shownthat some xenobiotics are able to inhibit CAT activity (Vander Oost et al., 2003; Bagnyokova et al., 2005) and that GSTactivity can increase to compensate for the CAT activity. The

GST group of enzymes engages in reactive conjugation withepoxide species and other electrophiles, and hence induc-tion of these enzymes must be considered beneficial underchemical stress (Van der Oost et al., 2003). In this study, theGST activity of G. brasiliensis was higher than that deter-mined by Wilhelm Filho et al. (2001).

In summer, an additional increase in the GST activitywas observed, along with increases in the genetic damage,LPO levels, and AchE activities. The EROD activity wassimilar to that in spring, and the results were similar to thevalues of EROD activity observed in G. brasiliensis ofa reference area (Wilhelm Filho et al., 2001; Parente et al.,2008). In the summer, there was a decrease in Melano-macrophage centers concentration, and preneoplasticlesions were observed in the gills of 9% of the individuals.Exposure of aquatic species to genotoxic substances andprocesses (such as oxidative stress) can produce effectssuch as neoplasia (Nacci et al., 1996; Wester et al., 2002).Moreover, because genotoxins may induce changes in DNA,which are passed on to future generations, the biomarkersof genetic damage can be used in a predictive manner,thereby avoiding irreversible ecological consequences(Valavanidis et al., 2006; Montserrat et al., 2007).

The increase in GST and EROD activities, in addition tothe increases in genetic damage and LPO, coincided withthe periods of highest concentrations of both PST andcyanobacteria in the water from the Reservoir. Therefore,further studies on the effects of PSTs in these biomarkersare necessary. Some authors have previously correlated PSTwith an increase or a decrease in GST and EROD activities(Hong et al., 2003; Choi et al., 2006), whereas no suchcorrelation has been found by others (Hong et al., 2003;Kozlowsky-Suzuki et al., 2009). Moreover, PST exposure hasbeen reported to cause an increase in LPO in clams (Gali-many et al., 2008; Choi et al., 2006).

PST concentrations found in G. brasiliensis muscle weresimilar among the three samplings. In spite of the similarityof profiles of cyanobacteria and PSTs in water in bothsummer and autumn, the behavior of biomarkers variedamong the three seasons of the year. The current resultssuggested that the PST concentration in water was not themain cause for the changes in biomarkers. As othercontaminants were not investigated, the seasonal alter-ations found herein could also be probably associated withtheir presence. The EROD activities found in autumn weresimilar to those observed in a polluted area by WilhelmFilho et al. (2001).

The major part of the Alagados hydrographic basin isused for farming activities, and the main crops grown aresoy and corn. Studies by Miranda et al. (2008) on Hopliasmalabaricus from the Ponta Grossa Lake (located in the sameregion as the Alagados Reservoir) also showed an impact bythe activities described above, with the bioaccumulation of27 different pesticides and metabolites in the muscles andliver. Pesticides, such as glyphosate, imazethapyr, anddiflubenzuron; organophosphorus compounds, such asmonocrotophos, methamidophos, and clorpiriphos, pyre-throids, such as deltamethrin; and triazoles are the mostfrequently used in the region (NUCLEAM, 2002). Therefore,the mixture of xenobiotics transported to the AlagadosReservoir must be complex, and their bioavailability can

Z. Clemente et al. / Toxicon 55 (2010) 396–406404

vary throughout the year. Thus, exposure of fish to thecomplex mixture can alter their metabolism by differentenzymatic pathways (GST and EROD), as reported in thepresent work. These pesticides have been correlated withalterations in the biomarkers studied (Almli et al., 2002; Leeand Steinert, 2003; Sayed et al., 2003; Bagnyukova et al.,2005; Valavanidis et al., 2006; Glusczak et al., 2007; Yinget al., 2007; Ansari et al., 2009; Huculeci et al., in press; Luet al., 2008; Contardo-Jara et al., 2009).

The AchE activity may be used as a biomarker of expo-sure to pollutants that are able to inhibit it, such asorganophosphorus and carbamates compounds, delta-methrin, and/or glyphosate (Van der Oost et al., 2003;Glusczak et al., 2007; Velisek et al., 2007). There is noreference value for AchE activity in the species studied,making the interpretation of the results difficult. The AchEactivity of the brain did not vary among the seasons of theyear, and the AchE activities in the brain and muscle weresimilar to the values in other fish species (Sturm et al., 1999;Oliveira Ribeiro et al., 2002; Ferrari et al., 2007; Guimaraeset al., 2007; Torre et al., 2007). Beninca (2006) reported anactivity of 33.31�8.44 in G. brasiliensis captured ina reference area in summer, a value below that found in thisstudy. Thus, it does not appear likely that an inhibition ofAchE occurred in the fish analyzed in this study.

In summary, the present work showed that the Alaga-dos Reservoir is impacted by anthropogenic activities;consequently, the occurrence of cyanobacterial blooms hasbeen more frequent in recent years. The neurotoxinsproduced by the blooms are bioavailable to Geophagusbrasiliensis, and the present findings indicate severe effectsto biota that are not only exclusively due to cyanotoxinexposure, but also due to exposure to other pollutants.Thus, a biomonitoring program is necessary becauseunsuitable water quality can compromise the health ofhumans and wildlife alike.

Acknowledgments

This research was supported by the CNPq, Brazil. Theauthors acknowledge the help rendered by the Environ-mental Military Police (Força Verde) in the sampling effortsand the Parana Environmental Institute for support in theanalysis of water and for making available previous datarelating to the Alagados Reservoir.

Conflict of interest

The authors declare that there are no conflicts ofinterest.

References

Aebi, H., 1984. Catalase in vitro, 105. Academic Press. 121–126.Agius, C., Roberts, R.J., 2003. Melano-macrophage centres and their role in

fish pathology. Journal of Fish Diseases 26, 499–509.Almli, B., Egaas, E., Christiansen, A., Eklo, O.M., Lode, O., Kallqvist, T., 2002.

Effects of three fungicides alone and in combination on glutathione S-transferase activity (GST) and cytochrome P-450 (CYP 1A1) in theliver and gill of brown trout (Salmo trutta). Marine EnvironmentalResearch 54 (3-5), 237–240.

Andrinolo, D., Iglesias, V., Garcıa, C., Lagos, N., 2002. Toxicokinetics andtoxicodynamics of gonyautoxins after an oral toxin dose in cats.Toxicon 40, 699–709.

Ansari, R.A., Kaur, M., Ahmad, F., Rahman, S., Rashid, H., Islam, F.,Raisuddin, S., 2009. Genotoxic and oxidative stress-inducing effects ofdeltamethrin in the erythrocytes of a freshwater biomarker fishspecies, Channa punctata Bloch. Environmental Toxicology 24 (5),429–436.

Arellano, M.J., Storch, V., Sarasquete, C., 1999. Histological changes andcooper accumulation in liver and gills of the Senegales Sole, Soleasenegalensis. Ecotoxicology and Environmental Safety 44, 62–72.

Bagnyukova, T.V., Storey, K.B., Lushchak, V.I., 2005. Adaptive response ofantioxidant enzymes to catalase inhibition by aminotriazole ingoldfish liver and kidney. Comparative Biochemistry and PhysiologyPart B 142 (3), 335–341.

Beninca, C., 2006. Biomonitoramento das lagoas estuarinas do Camacho –Jaguaruna (SC) e Santa Marta- Laguna (SC), utilizando Geophagusbrasiliensis (Cichlidae). Master Dissertation, Federal University ofParana, Curitiba, Brazil. p.112.

Bernet, D., Schimidt, H., Meier, W., Burkhardt-Holm, P., Wahli, T., 1999.Histopathology in fish: proposal for a protocol to assess aquaticpollution. Journal of Fish Diseases 22, 25–34.

Bradford, M., 1976. A rapid and sensitive method for the quantification ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Analytical Biochemistry 72, 248–254.

Briand, J.F., Jacquet, S., Bernard, C., Humbert, J.F., 2003. Health hazard forterrestrial vertebrates from toxic cyanobacteria in surface waterecosystems. Veterinary Research 34, 361–377.

Burke, M.D., Mayer, R.T., 1971. 7-Ethoxyresorufin: direct fluorimetricassay of a microsomal O-dealkylation which is preferentially induc-ible by 3-methylcholanthrene. Drug Metabolism and Disposition 2,583–585.

Carmichael, W.W., 1994. The toxins of cyanobacteria. Scientific American270 (1), 78–86.

Chellappa, N.T., Medeiros Costa, M.A., 2003. Dominant and co-existingspecies of cyanobacteria from a eutrophicated reservoir of Rio Grandedo Norte State, Brazil. Acta Oecologica 24, 3–10.

Choi, N.M.C., Yeung, L.W.Y., Siu, W.H.L., So, I.M.K., Jack, R.W., Hsieh, D.P.H.,Wu, R.S.S., Lam, P.K.S., 2006. Relationships between tissue concen-trations of paralytic shellfish toxins and antioxidative responses ofclams, Ruditapes philippinarum. Marine Pollution Bulletin 52, 572–597.

Chorus, I., Bartram, J., 1999. Toxic Cyanobacteria in Water: a Guide to theirPublic Health Consequences, Monitoring and Management. E & FNSpon, London.

Codd, G.A., Lindasay, J., Young, F.M., Morrison, L.F., Metcalf, J.S., 2005.Harmful cyanobacteria. In: Huisman, J., Matthhijs, H.C.P., Visser, P.M.(Eds.), Harmful cyanobacteria. Springer, Netherlands, pp. 1–23.

Collins, A., Dusinska, M., Franklin, M., Somorovska, M., Petrovska, H.,Duthie, S., Fillion, L., Panayiotidis, M., Raslova, K., Vaughan, N., 1997.Comet assay in human biomonitoring studies: reliability, validationand applications. Environmental Molecular Mutagenicity 30 (2), 139–146.

Contardo-Jara, V., Klingelmann, E., Wiegand, C., 2009. Bioaccumulation ofglyphosate and its formulation Roundup Ultra in Lumbriculus varie-gatus and its effects on biotransformation and antioxidant enzymes.Environmental Pollution 157, 57–63.

Deeds, J.R., Landsberg, J.H., Etheridge, S.M., Pitcher, G.C., Longan, S.W.,2008. Non-traditional vectors for paralytic shellfish poisoning. MarineDrugs 6, 308–348.

DeForest, D.K., Brix, K.V., Adams, W.J., 2007. Assessing metal bio-accumulation in aquatic environments: the inverse relationshipbetween bioaccumulation factors, trophic transfer factors and expo-sure concentration. Aquatic Toxicology 84 (2), 236–246.

Donovan, C.J., Ku, J.C., Quilliam, M.A., Gill, T.A., 2008. Bacterial degradationof paralytic shellfish toxins. Toxicon 52 (1), 91–100.

Ellman, G.L., Courtney, K.D., Andres Jr., V., Featherstone, R.M., 1961. A newand rapid colorimetric determination of acetylcholinesterase activity.Biochemical Pharmacology 7, 88–95.

Fast, M.D., Cembella, A.D., Ros, N.W., 2006. In vitro transformation ofparalytic shellfish toxins in the clams Mya arenaria and Protothacastaminea. Harmful Algae 5, 79–90.

Ferrari, A., Venturino, A., D’Angelo, A.M.P., 2007. Muscular and braincholinesterase sensitivities to azinphos methyl and carbaryl in thejuvenile rainbow trout Oncorhynchus mykiss. Comparative Biochem-istry and Physiology Part C 146, 308–313.

Ferraro, M.V.M., Fenocchio, A.S., Cestari, M.M., Mantovani, M.S., Lemos, P.M.M., 2004. Genetic damage induced by trophic doses of lead in theneotropical fish Hoplias malabaricus (Characiformes, Erythrinidae) as

Z. Clemente et al. / Toxicon 55 (2010) 396–406 405

revealed by the comet assay and chromosomal aberrations. Geneticsand Molecular Biology 27 (2), 270–274.

Fitzgerald, D.J., Cunliffe, D.A., Burch, M.D., 1999. Development of healthalerts for cyanobacteria and related toxins in drinking water in SouthAustralia. Environmental Toxicology 14 (1), 203–207.

Galimany, E., Sunila, I., Hegaret, H., Ramon, M., Wikfors, G.H., 2008.Experimental exposure of the blue mussel (Mytilus edulis, L.) to thetoxic dinoflagellate Alexandrium fundyense: Histopathology, immuneresponses, and recovery. Harmful Algae 7, 702–711.

Garcia, C., Lagos, M.L., Truan, D., Lattes, K., Vejar, O., Chomorro, B.,Iglesias, V., Andrinolo, D., Lagos, N., 2005. Human intoxication withparalytic shellfish toxins: clinical parameters and toxin analysis inplasma and Urine. Biological Research 38, 197–205.

Garcıa, C., Rodriguez Navarro, A., Dıaz, J.C., Torres, R., Lagos, N., 2008.Evidence of in vitro glucuronidation and enzymatic transformation ofparalytic shellfish toxins by healthy human liver microsomes fraction.Toxicon 53 (2), 206–231.

Garcia-Santos, S., Monteiro, S.M., Carrola, J., Fontainhas-Fernandes, J.,2007. Alteraçoes histologicas em branquias de tilapia nilotica Oreo-chromis niloticus causadas pelo cadmio. Arquivo Brasileiro deMedicina Veterinaria e Zootecnia 59 (2), 376–381.

Glusczak, L., Miron Ddos, S., Moraes, B.S., Simoes, R.R., Schetinger, M.R.,Morsch, V.M., Loro, V.L., 2007. Acute effects of glyphosate herbicide onmetabolic and enzymatic parameters of silver catfish (Rhamdia quelen).Comparative Biochemistry and Physiology Part C 146 (4), 519–524.

Gray, J.S., 2002. Biomagnification in marine systems: the perspective of anecologist. Marine Pollutants Bulletin 45, 46–52.

Guimaraes, A.T.B., Silva de Assis, H.C., Boeger, W., 2007. The effect oftrichlorfon on acetylcholinesterase activity and histopathology ofcultivated fish Oreochromis niloticus. Ecotoxicology and Environ-mental Safety 68, 57–62.

Hall, S., Stricharz, G., Moczydlowski, E., Ravindran, A., Reichardt, P.B., 1990.The saxitoxins: sources, chemistry and pharmacology. In: Hall, S.,Stricharz, G. (Eds.), Marine Toxins: Origin, Structure and MolecularPharmacology, 418. American Chemical Society Symposium Series,Washington, DC, pp. 29–65.

Hinton, D.E., Baumen, P.C., Gardener, G.C., Hawkins, W.E., Hendricks, J.D.,Murchelano, R.A., Okhiro, M.S., 1992. Histopathological biomarkers.In: Huggett, R.J., Kimerle, R.A., Mehrle, P.M., Bergman, H.L. (Eds.),Biomarkers: biochemical, physiological and histological markers ofanthropogenic stress. Lewis Publishers, Boca Raton, pp. 155–210.

Hinton, D.E., Segner, H., Au, D.W.T., Kullman, S.W., Hardman, R.C., 2008.Liver toxicity. In: Di Giulio, R.T., Hinton (Eds.), Toxicology of Fish.Taylor and Francis, Boca Raton, pp. 327–400.

Hong, H.-Z., Lam, P.K.S., Hsieh, D.P.H., 2003. Interactions of paralyticshellfish toxins with xenobiotic-metabolizing and antioxidantenzymes in rodents. Toxicon 42, 425–431.

Huculeci, R., Dinu, D., Saticu, A.C., Munteanu, M.C., Costache, M., Dini-schiotu, A. Malathion-induced alteration of the antioxidant defencesystem in kidney, gill, and intestine of Carassius auratus gibelio.Environmental Toxicology, in press.

Instituto Ambiental do Parana (IAP), 2007. Laudos tecnicos de analise daagua do Reservatorio Alagados de 2003 a 2007. Private Communication.

Jiang, Z.Y., Hunt, J.V., Wolf, S.P., 1992. Ferrous ion oxidation in the presenceof xylenol orange for detection of lipid hydroperoxide in low densitylipoprotein. Analytic Biochemistry 202, 384–389.

Jiang, T.J., Niu, D.W.T., Xu, Y., 2007. Trophic transfer of paralytic shellfishtoxins from the cladoceran (Moina mongolica) to larvae of the fish(Sciaenops ocellatus). Toxicon 50, 639–645.

Keen, J.H., Habig, W.H., Jakoby, W.B., 1976. Mechanism for several activi-ties of the glutathione S-transferase. Journal of Biological Chemistry251, 6183–6188.

Kozlowsky-Suzuki, B., Koski, M., Hallberg, E., Walle, R., Carlsson, P.,2009. Glutathione transferase activity and oocyte development incopepods exposed to toxic phytoplankton. Harmful Algae 8 (3),395–406.

Kwong, R.W.M., Wang, W.X., Lam, P.K.S., Yu, P.K.N., 2006. The uptake,distribution and elimination of paralytic shellfish toxins in mussels andfish exposed to toxic dinoflagellates. Aquatic Toxicology 80, 82–91.

Lagos, N., Onodera, H., Zagatto, P.A., Andrinolo, D., Azevedo, S.M.F.Q.,Oshima, Y., 1999. The first evidence of paralytic shellfish toxins in thefreshwater cyanobacterium Cylindrospermopsis raciborskii, isolatedfrom Brazil. Toxicon 37, 1359–1373.

Lee, R.F., Steinert, S., 2003. Use of the single cell gel electrophoresis/cometassay for detecting DNA damage in aquatic (marine and freshwater)animals. Mutation Research 411, 43–64.

Leknes, I.L., 2007. Melano-macrophage centres and endocytic cells inkidney and spleen of pearl gouramy and platyfish (Anabantidae,Poeciliidae: Teleostei). Acta Histochemica 109, 164–168.

Lu, G.H., Wang, C., Zhu, Z., 2008. The dose-response relationships forEROD and GST induced by polyaromatic hydrocarbons in Carassiusauratus. Bulletin of Environmental Contamination and Toxicology 82(2), 194–199.

Magalhaes, A.C., 1931. Monografia brasileira de peixes fluviais. Graphicars,Sao Paulo.

McDonald, D.G., Wood, C.M., 1993. Branchial mechanisms of acclimationto metals in freshwater fish. In: Fish Ecophysiology. Chapman & Hall,London, pp. 297–321.

Miranda, A.L., Roche, H., Randi, M.A.F., Menezes, M.L., Oliveira Ribeiro, C.A.,2008. Bioaccumulation of chlorinated pesticides and PCBs in thetropical freshwater fish Hoplias malabaricus: histopathological, physi-ological and immunological findings. Environment International 34,939–949.

Montserrat, J.M., Martinez, P.E., Geracitano, L.A., Amado, L.L., Martins, C.M.G., Pinho, G.L.L., Chaves, I.S., Ferreira-Cravo, M., Ventura-Lima, J.,Biancini, A., 2007. Pollution biomarkers in estuarine animals: criticalreview and new perspectives. Comparative Biochemistry and Physi-ology Part C 146, 221–234.

Nacci, D.E., Cayulab, S., Jackim, E., 1996. Detection of DNA damage inindividual cells from marine organisms using the single cell gel assay.Aquatic Toxicology 35, 197–210.

Nassem, S.M., 1996. Toxicokinetics of [3H]saxitoxinol in peripheral andcentral nervous system of rats. Toxicology and Applied Pharmacology141, 49–58.

Nucleo de Estudos em Meio Ambiente (NUCLEAM), 2002. Bacia hidrog-rafica do manancial Alagados. UEPG, Ponta Grossa.

Oliveira Ribeiro, C.A., Schatzmann, M., Silva de Assis, H.C., Silva, P.H.,Pelletier, E., Akaishi, F.M., 2002. Evaluation of tributyltin subchronicefects in tropical freshwater fish (Astyanax bimaculatus, Linnaeus,1758). Ecotoxicology and Environmental Safety 51, 161–167.

Oliveira Ribeiro, C.A., Vollaire, Y., Sanchez-Chardi, A., Roche, H., 2005.Bioaccumulation and the effects of organochlorine pesticides, PAHand heavy metals in the eel (Anguilla anguilla) at the CamargueNature Reserve, France. Aquatic Toxicology 74, 53–69.

Oshima, Y., 1995. Post-Column derivatization HPLC methods for paralyticshellfish poisons. In: Hallegraeff, G.M., Anderson, D.M., Cembella, A.D.(Eds.), Manual on Harmful Marine Microalgae. UNESCO, Paris, pp. 81–94.

Parente, T.E., De-Oliveira, A.C., Paumgartten, F.J., 2008. Induced cyto-chrome P450 1A activity in cichlid fishes from Guandu River andJacarepagua Lake, Rio de Janeiro, Brazil. Environmental Pollution 152,233–238.

Patocka, J., 2001. The toxins of cianobacteria. Acta Medica 44 (2),69–75.

Ramsdorf, W.A., Guimaraes, F.S.F., Ferraro, M.V.M., Gabardo, J., Trindade, E.S., Cestari, M.M., 2009. Establishment od experimental conditions forpreserving samples of fish blood for analysis with both comet assayand flor cytometry. Mutation Research 673, 78–81.

Sagou, R., Amanhir, R., Taleb, H., Vale, P., Blaghen, M., Loutfi, M., 2005.Comparative study on differential accumulation of PSP toxinsbetween cockle (Acanthocardia tuberculatum) and sweet clam (Callistachione). Toxicon 46, 612–618.

Samsur, M., Yamaguchi, Y., Sagara, T., Takatani, T., Arakawa, O., Noguchi, T.,2006. Accumulation and depuration profiles of PSP toxins in theshort-necked clam Tapes japonica fed with the toxic dinoflagellateAlexandrium catenella. Toxicon 48, 323–330.

Sayed, I., Parvez, S., Pandey, S., Bin-Hafeez, B., Haque, R., Raisuddin, S.,2003. Oxidative stress biomarkers of exposure to deltamethrin infreshwater fish, Channa punctatus Bloch. Ecotoxicology Environ-mental Safety 56 (2), 295–301.

Sephton, D.H., Haya, K., Martın, J.L., LeGresley, M.M., Page, F.H., 2007.Paralytic shellfish toxins in zooplankton, mussels, lobsters and cagedAtlantic salmon, Salmo salar, during a bloom of Alexandrium fundyenseoff Grand Manan Island, in Bay of Fundy. Harmful Algae 6 (5), 745–758.

Silva de Assis, H.C., 1998. Der Einsatz von Biomarkern zur SummarischenErfassung von Gewasserverschmutzungen. Thesis, Berlin TechnicalUniversity, Germany, p. 99.

Singh, N.P., McCoy, M.T., Tice, R.R., Sch, E.L., 1988. A simple technique forquantitation of low levels of DNA damage in individual cells. Exper-imental Cell Research 175 (1), 184–191.

Sperling, E., Silva Ferreira, A.C., Ludolf Gomes, L.N., 2008. Comparativeeutrophication development in two Brazilian water supply reservoirswith respect to nutrient concentrations and bacteria growth. Desali-nation 226, 169–174.

Stentiford, G.D., Longshaw, M., Lyons, B.P., Jones, G., Green, M., Feist, S.W.,2003. Histopathological biomarkers in estuarine fish species for theassessment of biological effects of contaminants. Marine Environ-mental Research 55, 137–159.

Z. Clemente et al. / Toxicon 55 (2010) 396–406406

Sturm, A., Silva de Assis, H.C., Hansen, D., 1999. Cholinesterases of marineteleost fish: enzymological characterization and potential use in themonitoring of neurotoxic contamination. Marine EnvironmentalResearch 47, 389–398.

Su, Z., Sheets, M., Ishida, H., Li, F., Barry, W.H., 2004. Saxitoxin blocks L-Type Ica. The Journal of Pharmacology and Experimental Therapeutics308 (1), 324–329.

Suzuki, H.I., Agostinho, A.A., 1997. Reproduçao de peixes do reservatoriode Segredo. In: Agostinho, A.A., Gomes, L.C. Reservatorio de Segredo –bases ecologicas para o manejo. EDUEM, Maringa, pp. 163–182.

Torre, F.R., de la; Salibian, S., Ferrari, L., 2007. Assessment of the pollutionimpact on biomarkers of effect of a freshwater fish. Chemosphere 68,1582–1590.

Tucci, A., Sant’Anna, C.L., 2003. Cylindrospermopsis raciborskii (Wolos-zynska) Seenayya & Subba Raju (Cyanobacteria): variaçao semanal erelaçoes com fatores ambientais em um reservatorio eutrofico.Revista Brasileira de Botanica 26 (1), 97–112.

Utermohl, H., 1958. Zur Vervollkommnung der quantitative Phyto-plankton-Methodik. Limnology 5, 567–596.

Valavanidis, A., Vlahogianni, T., Dassenakis, M., Scoullos, M., 2006.Molecular biomarkers of oxidative stress in aquatic organisms inrelation to toxic environmental pollutants. Ecotoxicology and Envi-ronmental Safety 64, 178–189.

Van der Oost, R., Beyer, J., Vermeulen, N.P.E., 2003. Fish bioaccumulationand biomarkers in environmental risk assessment: a review. Envi-ronmental Toxicology and Pharmacology 13, 57–149.

Velısek, J., Jurcıkova, J., Dobsıkova, R., Svobodova, R., Piackova, V.,Machova, J., Novotny, L., 2007. Effects of deltamethrin on rainbowtrout (Oncorhynchus mykiss). Environmental Toxicology and Phar-macology 23, 297–301.

Wang, J., Salata, J.J., Bennett, P.B., 2003. Saxitoxin is a gating modifier ofhERG K channels. The Journal of General Physiology 121, 583–598.

Wester, P.W., Van der Ven, L.T.M., Vethaak, A.D., Grinwis, G.C.M., Vos, J.G.,2002. Aquatic toxicology: opportunities for enhancement throughhistopathology. Environmental Toxicology and Pharmacology 11,289–295.

Wiegand, C., Pflumacher, S., 2005. Ecotoxicologycal effects of selectedcyanobacterial secondary metabolites a short review. Toxicology andApplied Pharmacology 203, 201–218.

Wilhelm Filho, D., Torres, M.A., Tribess, T.B., Pedrosa, R.C., Soares, C.H.L.,2001. Influence of season and pollution on the antioxidant defenses ofthe cichlid fish acara (Geophagus brasiliensis). Brazilian Journal ofMedical and Biological Research 34, 719–726.

Ying, Y., Jia, H., Sun, Y., Yu, H., Wang, X., Wu, J., Xue, Y., 2007. Bio-accumulation and ROS generation in liver of Carassius auratus,exposed to phenanthrene. Comparative Biochemistry and PhysiologyPart C 145, 288–293.

Yunes, J.S., Cunha, N.T., Barros, L.P., 2003. Cyanobacterial neurotoxins fromsouthern Brazilian freshwaters. Comments on Toxicology 9, 103–105.

Zodrow, J.M., Stegeman, J.J., Tanguay, R.L., 2004. Histological analysis ofacute toxicity of 2,3,7,8-tetrachlorobibenzeno-p-dioxin (TCDD) inzebrafish. Aquatic Toxicology 66, 25–38.