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The impact of an oil spill on organs of bream Abramis brama in the Po River

L. Giari, B.S. Dezfuli n, M. Lanzoni, G. Castaldelli

Department of Biology & Evolution, University of Ferrara, Borsari St. 46, 44121 Ferrara, Italy

a r t i c l e i n f o

Article history:

Received 2 August 2011

Received in revised form

6 October 2011

Accepted 9 October 2011Available online 24 October 2011

Keywords:

Aquatic pollution

Hydrocarbon

Histopathology

Rodlet cells

Biomarker

a b s t r a c t

An oil spill into the River Lambro occurred on 23 February 2010 and reached the Po River the following

day. Breams captured here on 1 March 2010, along with a sample from a control site, were examined by

light and electron microscopy. The main affected organs were skin and gill with slight or no damage to

liver, kidney, and intestine. The gills exhibited lamellar aneurisms, fusion of secondary lamellae, edema

with epithelial lifting, mucous cell hypertrophy, and mucus hypersecretion. Significantly higher

mucous cell density was observed in the skin of exposed fish. Histochemical staining revealed that

acid glycoconjugates were prevalent in epidermal mucous cells in the exposed Abramis brama, whereas

neutral and mixed glycoconjugates were dominant in the control fish. Rodlet cells were significantly

more abundant in the kidney of exposed fish and showed ultrastructural differences compared to

controls. These histopathologic effects were indicators of chemical stress due to exposure to oil. The

present study is one of the first which explores the acute effects of this incident and makes part of a few

reports focused on freshwater oil spill.

& 2011 Elsevier Inc. All rights reserved.

1. Introduction

The Po is the most important Italian river, originating in theAlps and flowing from west to east for 653 km across the entirewidth of northern Italy to the Adriatic Sea. On February 2010hydrocarbons were spilled into the Lambro River, a small lefttributary to the Po. As part of this study, fish were sampled from alower stretch of the Po River near Ferrara, for the purpose ofdetecting the effects of oil contamination on ichthyofauna. Atten-tion was focused on Abramis brama (L.) (bream) a benthopelagicfreshwater species belonging to the Cyprinidae. Breams have arelatively short life cycle, wide trophic and spatial niches, andwide geographical distribution in Europe and Asia (see www.fishbase.org). In northern Italy, including in the Po where it wasintroduced in the 1980s, bream is rapidly becoming the mostabundant species (Castaldelli and Rossi, 2008).

Oil and its refined products consists of seventyfive percentshort and long hydrocarbon chains and represent the mostcomplex and variable mixtures to evaluate toxicologically (Neff,1979). Several techniques have been traditionally used to estimatethe uptake by fish of aromatic hydrocarbons from petroleum, butthe results have not been satisfactory (Krahn et al., 1986).Chemical analysis allows determination of the extent of pollutionbut not evaluation of its damage to organisms. Histological andultrastructural studies may provide useful information on the

effects of pollutants on fauna (Alazemi et al., 1996; Bernet et al.,1999) especially in acute exposures, as reported by Hinton et al.(1992). Extensive studies in the USA and Europe have establisheda causal relationship between levels of pollution in the aquaticenvironment and fish pathology (Au, 2004).

The surface of the skin, gill, and intestine is the primaryinterface between the fish and its environment. The outer con-stituent of this barrier is a layer of mucus covering the epitheliumwhich is secreted from mucous or goblet cells (Shephard, 1994).The presence of xenobiotics and alterations of ion concentrationsand of pH in water can lead to changes in the density of themucous cells (Paul and Banerjee, 1997). These cells respond to avariety of toxicants and/or irritants by undergoing hyperplasia orhypertrophy and, if the noxious stimulus persists, cellular deple-tion may occur (Mallat, 1985).

Rodlet cells (RCs) are found exclusively in fish, primarily in theepithelia, and represent a component of a generalized hostresponse to a variety of stress conditions (Manera and Dezfuli,2004; Reite and Evensen, 2006), including exposure to xenobio-tics (Iger and Abraham, 1997; Dezfuli et al., 2003, 2006; Giariet al., 2007, 2008). Macrophage aggregates (MAs), groupings ofpigmented macrophages characteristic of heterothermic verte-brate tissues, in fish are normally located in the liver, kidney, andspleen (Agius and Roberts, 2003). Macrophage aggregate hyper-plasia was reported in fish species inhabiting degraded environ-ments and those experimentally treated with high levels ofchemicals (Fournie et al., 2001). Macrophage aggregates, andmore recently, RCs have been suggested as reliable biomarkersof exposure to toxicants (Rice, 2001; Agius and Roberts, 2003;Dezfuli et al., 2006; Giari et al., 2007, 2008).

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

Ecotoxicology and Environmental Safety

0147-6513/$ - see front matter & 2011 Elsevier Inc. All rights reserved.

doi:10.1016/j.ecoenv.2011.10.014

n Corresponding author. Fax: þ39 0532 455715.

E-mail address: dzb@unife.it (B.S. Dezfuli).

Ecotoxicology and Environmental Safety 77 (2012) 18–27

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The first aim of this study was to investigate histologically andultrastructurally the impact of the Lambro oil discharge on themain organs of A. brama inhabiting the lower Po River and to getinformation about the threat imposed by this incident. Thesecond objective was, using bream as an environmental indicator,to confirm histopathological lesions and cellular parameters (i.e.density, content, and/or ultrastructure of some cell types) assuitable biomarkers for monitoring the status of, and trackingchanges in, the ecosystem.

2. Materials and methods

2.1. Oil spill event

On 23 February 2010, 2600 t of hydrocarbons (1800 t of diesel fuel and 800 t of

fuel oil) were discharged from tanks at the disused Lombarda Petroli refinery near

Monza, about 30 km north-east of Milan (Po River Basin Authority, 2010). The oil

mass ran into the Lambro river, a small tributary of the middle reaches of the Po

River (Fig. 1a). The hydrocarbon spread downstream, reaching the Po the following

day. On 1 March the oil was present in the delta and the Adriatic Sea.

2.2. Study sites

Breams were sampled from the Po River near Ferrara (44155055.76*N,

11132017.26*E), 300 km downstream of the oil spill site, and in the Cavo

Napoleonico (control site; 44155033.96*N, 11126027.01*E), an irrigation canal

deriving its water only from the Po but not exposed to any discharge (Fig. 1a).

The inlet had been closed before the polluting wave, and the Cavo Napoleonico

was not reached by the oil spill (neither total nor dissolved hydrocarbons were

detectable). The physico-chemical characterization of the two sampling sites is

reported in Table 1.

2.3. Fish sampling

Sampling was conducted on 1 March, in the last phase of oil slick, to maximize

the exposure time. Fish were captured using sinking trammel nets (30 mm mesh,

50 m long, and 2 m high) in collaboration with professional fishermen. Electro-

fishing was not used due to high conductivity (Table 1) and to avoid the risk of

causing tissue lesions, invalidating histological analysis. Fish were transported live

to the laboratory within an hour of capture in two aerated tanks, measured for

total length and wet weight (Table 2), and a standard necropsy was performed

after killing by percussive stunning and pithing. The captive maintenance

procedures and research protocols were approved by the University of Ferrara

Institutional Animal Care and Use Committee and by the Italian Ministry of Health

Q2 (license no. 116 of 27 January 1992) and the European Union (Directive 86/

609/EEC of the EU Parliament and of the Council of 24 November 1986).

2.4. Tissue processing and histopathologic analyses

For light microscopy, pieces of skin, gill, liver, intestine, and kidney measuring

about 15�15 mm2 were fixed by immersion in ten percent neutral buffered

formalin at 4 1C for 24 h, dehydrated in a graded series of ethanols, and paraffin

embedded. Tissue section (5 mm) were stained with hematoxylin & eosin (H&E)

and/or alcian blue 8 GX pH 2.5 /Periodic acid Schiff (AB/PAS) to visualize the

glycoconjugates. Alcian blue stains acid glycoconjugates, the PAS reaction stains

the neutral glycoconjugates, and both AB and PAS stain mixed glycoconjugates.

Light photomicrographs were taken using a Nikon microscope ECLIPSE 80i (Nikon,

Tokyo, Japan). For electron microscopy the samples were processed with routine

methods as detailed in Giari et al. (2008).

The numbers of mucous cells, RCs, and MAs, as well as their dimensions, were

evaluated through a blind count, using light microscope and computerized image

analyzer software (Nis Elements AR 3.0; Nikon, Tokyo, Japan). The density of cells

(number of cells in 10 000 mm2 of tissue) was counted in ten fields from two

sections per each fish.

The data obtained from cell counting were checked for normal distribution

using the Kolmogorov–Smirnov test. Differences in the density of cells between

the fish exposed to polluted water and the reference group were tested using

ANOVA repeated measure. The size (area occupied) of mucous cells and MAs in the

bream from the two sites was compared with Student’s two tailed t-test. All

statistical analyses were performed using the statistics package Statistica 7

(StatSoft, Tulsa, OK, USA) with the level of significance set at p¼0.05.

3. Results

The concentration of hydrocarbons at the impacted site,measured by the Ferrara Environmental Protection Agencybetween 25 February and 3 March, are presented graphically inFig. 1b. At depths of 1 and 2 m hydrocarbon concentrations werebelow 1 mg/L, the threshold for total hydrocarbons in waterpotabilization (Po River Basin Authority, 2010). No bream deathswere observed in the sampling site, and no fish mortalities in thePo River were reported (Po River Basin Authority, 2010).

Total length (TL), body weight (W) and condition factor K

(calculated K¼W in g�100/TL3 in cm) of the A. brama examinedare reported in Table 2. These parameters were not significantlydifferent in the two fish groups (t-test, p40.05). In both groupsthe sex ratio was 1:1. No macroscopic changes were found in theorgans of any fish, whereas the histopathological examinationwith light and transmission electron microscopy demonstratedthe presence of some anomalies in the exposed group, especiallyin the skin and gills. No parasites neither neoplastic lesions weredetected in the tissues examined from either group.

The epidermis of control fish was principally composed oflayers of epithelial cells interspersed with mucous cells, especiallyat the surface, and mast cells located more deeply. The skinepithelium of exposed A. brama contained the same cell types,and the main feature was mucous cell hyperplasia (Table 3). Thenumber of mucous cells as well as their content differed betweenthe two groups of fish. The histochemistry of complex carbohy-drates revealed the presence of neutral and mixed glycoconjugates(PAS positive and AB/PAS positive, respectively) in the epidermalmucous cells of the control A. brama, and the occurrence of acidglycoconjugates (AB positive) in those of the exposed specimens

Fig. 1. (a) Map of the Po River basin showing the spillage point and the sampling

sites (exposed site and control site). (b) Total (T) and dissolved (D) hydrocarbon

concentrations measured in the lower Po River in the superficial layer (0–0.2 m)

on the left axis, and in the water column (1 m and 2 m depths) on the right axis.

Each point is the mean of two water samples.

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Table 1Physicochemical parameters of the two sampling sites: Po River near Ferrara (exposed site) and Cavo Napoleonico (control site). Values are minimum, maximum, and

mean of monthly analyses conducted by local Environmental Protection Agency from 2002 to 2010.

Parameters Control site Exposed site

min mean max min mean max

Temperature (1C) 5.2 16.66 29.6 6 16.38 30

Conductivity (mS/cm) 307 414.11 535 269 413.75 512

pH 7.05 8.06 8.65 7.29 7.88 8.3

O2 (mg/L) 5.1 8.02 13.20 5.8 8.84 14.1

O2 sat (%) 49 79.25 125 52 90.33 166

BOD5 (mg O2/L) 2 4.09 18 2 2.70 5

Ammonia (N mg/L) 0.02 0.08 0.26 0.02 0.11 0.40

Nitrate (N mg/L) 0.451 2.12 4 0.79 2.40 4.52

Nitrite (N mg/L) 0.01 0.03 0.06 0.01 0.04 0.13

Total nitrogen (N mg/L) 2.22 4.23 8.2 1.55 4.88 12.32

Orthophosphate (P mg/L) 0.01 0.06 0.12 0.02 0.07 0.17

1.1 dichloroethane (mg/L) o0.5 o0.5 o0.5 o0.5

1.1 dichloroethylene (mg/L) o0.5 o0.5 o0.5 o0.5

1.1,2 trichloroethane (mg/L) o3 o3 o3 o3

1.1.2.2 tetrachloroethane (mg/L) o0.5 o0.5 o1 o1

1.2 dibromoethane (mg/L) o3 o3 o3 o3

1.2 trans-dichloroethylene (mg/L) o1 o1 o1 o1

1.2 dichloropropane (mg/L) o1.5 o1.5 o1.5 o1.5

1.2,3 trichloropropane (mg/L) o4 o4 o4 o4

1-1-1 trichloroethane (mg/L) o0.1 o0.1 o0.1 o0.1 o0.5

1–2 dichloroethane (mg/L) o2.5 o2.5 o2.5 o2.5

3.4 dichloroaniline (mg/L) o0.01 o0.01 o0.01 o0.01

Acenaphthene (mg/L) o0.01 o0.01 o0.01 o0.01

Acenaftilene (mg/L) o0.01 o0.01 o0.01 o0.01

Alachlor (mg/L) o0.01 o0.01 o0.01 o0.01

Aluminum (mg/L) o100 o100 o100 o100

Antimony (mg/L) o5 o5 o5 o5

Anthracene (mg/L) o0.01 o0.01 o0.01 o0.01

Arsenic (mg/L) o2 o2 o1 o2

Atrazine (mg/L) o0.05 o0.05 o0.01 0.03 0.05

Azinphos-methyl (mg/L) o0.05 o0.05 o0.01 o0.05

Barium (mg/L) o20 41 120 o20 33 141

Benzene (mg/L) o0.5 o0.5 o0.5 o0.5

Benzo a anthracene (mg/L) o0.01 o0.01 o0.01 o0.01

Benzo a pyrene (mg/L) o0.01 o0.01 o0.01 o0.01

Benzo ghi perylene (mg/L) o0.01 o0.01 o0.01 o0.01

Benzo(b)þbenzo(k)fluoranthene (mg/L) o0.01 o0.01 o0.01 o0.01

Beryllium (mg/L) o1 o1 o1 o1

Boron (mg/L) o50 55 128 o50 53 110

Bromoform (mg/L) o0.5 o0.5 o0.5 o0.5

Cadmium (mg/L) o0.5 o0.5 o0.5 o0.5

Chlorpiryphos (mg/L) o0.01 o0.01 o0.01 o0.01

Cloridazon (mg/L) o0.01 o0.01 o0.01 o0.01

Clorotalonil (mg/L) o0.05 o0.05 o0.05 o0.05

Vinyl chloride (mg/L) o0.5 o0.5 o0.5 o0.5

Cobalt (mg/L) o5 o5 o5 o5

Crisene (mg/L) o0.01 o0.01 o0.01 o0.01

Total chrome (mg/L) o5 o5 o5 o5

Desethyl atrazine (mg/L) o0.01 o0.01 o0.01 o0.01

Dibenzo ah anthracene (mg/L) o0.01 o0.01 o0.01 o0.01

Dibromochloromethane (mg/L) o0.5 o0.5 o0.5 o0.5

Dichlorobromomethane (mg/L) o0.1 o0.1 o0.1 o0.1

Dichloromethane (mg/L) o1.5 o1.5 o1.5 o1.5

Dimethoate (mg/L) o0.01 o0.01 o0.01 o0.01

Ethylbenzene (mg/L) o0.5 o0.5 o0.5 o0.5

Ethofumesate (mg/L) o0.01 o0.01 o0.01 o0.01

Phenanthrene (mg/L) o0.01 o0.01 o0.01 o0.01

Fluoranthene (mg/L) o0.01 o0.01 o0.01 o0.01

Fluorene (mg/L) o0.01 o0.01 o0.01 o0.01

Indeno 123 cd pyrene (mg/L) o0.01 o0.01 o0.01 o0.01

Isoproturon (mg/L) o0.01 o0.01 o0.01 o0.01

Lenacil (mg/L) o0.01 o0.01 o0.01 o0.01

Linuron (mg/L) o0.01 o0.01 o0.01 o0.01

Manganese (mg/L) o5 7 28 o5 8.7 103

Mercury (mg/L) o0.5 o0.5 o0.5 o0.5

Metamitron (mg/L) o0.01 o0.01 o0.01 o0.01

Metobromuron (mg/L) o0.05 o0.05 o0.05 o0.05

Metolaclor (mg/L) o0.01 o0.01 o0.01 o0.01

Molinate (mg/L) o0.01 o0.01 o0.01 o0.01

Naftalene (mg/L) o0.01 o0.01 o0.01 o0.01

Nickel (mg/L) o2 4 8 o2 2.3 7

Oxadiazon (mg/L) o0.05 0.06 0.26 o0.05 o0.05

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(Fig. 2a, b). There was no significant difference between epidermalmucous cells size in A. brama from the exposed site and those ofthe control group (see Table 3). The gills of control fish conformedto the normal cyprinid pattern. On the upper and lower surface ofeach filament (primary lamella) there was a row of distinct andregular secondary lamellae (Fig. 2c). The epithelial cells coveredthe primary and secondary lamellae. Numerous mucous cells anda few RCs were distributed in both types of lamellae, whilechloride cells and mast cells occurred chiefly in the primarylamella (Fig. 2c). There was no evidence of damage in controlspecimens, except for a single case of lamellar aneurysm andepithelial lifting limited to a few secondary lamellae. All fish fromthe exposed site exhibited lamellar capillary aneurysms (Fig. 2d)from five to twenty percent of the secondary lamellae in gillsections. The gills of many exposed A. brama also showedepithelial proliferation associated with thickening of the primarylamella and the fusion of two or more neighboring secondarylamellae (Fig. 2e). More rarely, mild edema with detachment ofthe epithelial layer from the basement membrane was seen withinthe secondary lamellae (Figs. 2f, 3a). No significant difference wasfound in mucous cell abundance in branchial tissue of exposed fish(either in primary and secondary lamellae) compared to the gillsof control specimens (Table 3). In bream from the exposed sitemucous cells were hypertrophic (Table 3). Mucus accumulation onthe gill surface was pronounced in the exposed fish. In theinterlamellar spaces, intensely AB positive mucus and cellulardebris were frequently observed (Fig. 2g). TEM examination ofboth primary and secondary lamellae confirmed the presence ofmature mucous cells filled with mucus granules (Fig. 3b, c) and theevidence of intense secretory activity (Fig. 3d). In the gill epithe-lium of bream from both sites, the majority of the mucous cellsstained positively for acid glycoconjugates with AB, followed bymucous cells containing mixed glycoconjugates (AB/PAS positive)and, to a lesser degree, those staining positively for the presence ofneutral glycoconjugates (PAS positive).

Compared with the control group, minor histological andultrastructure differences in internal organs (kidney, intestine,and liver) of exposed fish were found. The renal tubules ofA. brama were arranged as is typical (Fig. 4a, b), and theinterstitial tissue contained numerous granulocytes (both neu-trophils and mast cells) and a few pigmented MAs. Density(Table 3) and surface area (p40.05) of these MAs did not differbetween the two fish groups. The collecting ducts (especially thelargest ones) were particularly rich in RCs, which were locatedsuperficially in the epithelium, parallel to the other cell types (i.e.epithelial cells and mucous cells) (Figs. 4c, d and 5a, b). In exposedfish, the number of RCs was significantly higher (Table 3, Figs. 4d,5b) with about thirty percent displaying signs of degenerationsuch as capsule deformation, cytoplasmic vacuolization, anom-alous mitochondria (thin and elongated in shape), and absence orpaucity of other organelles (Fig. 5c). Moreover, their inclusions(called rodlets) often appeared fused and without the typicalelectron-dense core (Fig. 5c). The occurrence of mature RCs readyto discharge contents, along with rodlet expulsion into the ductlumen, were common in kidney of exposed fish (Fig. 5d). Aboutfive–ten percent of the epithelial tubular cells of almost all thebreams from the impacted site were affected by ‘‘blebbing’’(apical expansion of the cell) and contained dilated endoplasmicreticulum cisternae (Fig. 5e, f); however, these pathologic phe-nomena were of moderate extension and severity.

In sections of posterior intestine of fish from both control andexposed sites, intact and regular folds were observed (Fig. 6a, b).The submucosal layer was rich in mast cells, and the liningepithelium was composed of normal enterocytes, numerous ABpositive mucous cells, and rare RCs (Fig. 6a, b). The liver of all fishshowed the typical morphological aspect for this species (Fig. 6c,d), and the hepatocytes examined with TEM did not reveal severeabnormalities (Fig. 7a–c). In the liver parenchyma no MAs wereencountered.

4. Discussion

The majority of petroleum spills worldwide have occurred inthe marine environment as a consequence of the transport ofcrude oil and refined petroleum products by sea (Law and Hellou,1999). Generally, field studies following pollution incidentsinvolve animals from contaminated and reference sites locatedin the same area and are based on the assumption that differencesin microscopic lesions between impacted and reference sites are a

Table 1 (continued )

Parameters Control site Exposed site

min mean max min mean max

Lead (mg/L) o2 o2 o2 o2

Pyrene (mg/L) o0.01 o0.01 o0.01 o0.01

Propanyl (mg/L) o0.01 o0.01 o0.01 o0.01

Copper (mg/L) o5 6 11 o5 6 11

Simazine (mg/L) o0.05 o0.05 o0.05 o0.05

Total tin (mg/L) o5 o5 o5 o5

Styrene (mg/L) o0.5 o0.5 o0.5 o0.5

Terbuthylazine (mg/L) o0.01 0.05 0.55 o0.01 0.04 0.33

Carbon tetrachloride (mg/L) o0.1 o0.1 o0.1 o0.1

Tiobencarb (mg/L) o0.05 o0.05 o0.05 o0.05

Toluene (mg/L) o0.5 o0.5 o0.5 o0.5

Trichlorobenzene (mg/L) o1 o1 o1 o1

Trichloroethylene (mg/L) o0.1 o0.1 o0.1 o0.1

Trichloromethane (mg/L) o0.2 o0.2 o0.2 o0.2

Triphenylene (mg/L) o0.01 o0.01 o0.01 o0.01

Trifluralin (mg/L) o0.01 o0.01 o0.01 o0.01

Zinc (mg/L) o10 o10 o10 o10

Table 2Biometrical data of Abramis brama specimens used in this study. Values are

means7standard deviation (SD).

Parameters Control fish Fish exposed

Fish sample n 6 6

Body weight (g) 350.507143.21 283.17790.77

Total length (cm) 30.8772.58 27.6772.50

Condition factor 1.1470.23 1.3170.08

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Table 3Mucous cells, rodlet cells (RCs) and macrophage aggregates (MAs) densities and mucous cells sizes in organs of Abramis brama exposed to hydrocarbons and those from

control site. Cell density is expressed as mean number of cells7SD in 10 000 mm2 of tissue. The size of mucous cells is expressed as mean area 7SD (n¼250 per each fish

group). Different superscript letters in the same line indicate significant differences (ANOVA repeated measures, po0.05).

Organ Cell parameter Control fish Fish exposed

Gill (primary lamellae)n Mucous cells density 9.1578.45 12.9975.16

Gill (secondary lamellae)n Mucous cells density 9.9476.82 7.8874.27

Gilln Mucous cells size 63.10717.47 mm2a 70.42717.05 mm2b

Skiny Mucous cells density 5.9374.93 a 13.3877.04 b

Skiny Mucous cells size 177.34745.44 mm2 173.63750.30 mm2

Intestiney Mucous cells density 15.4574.98 15.2475.09

Kidney (collecting ducts)y RCs density 1.0871.53 a 14.86712.01b

Kidney (interstitial tissue)y MAs density 0.4070.67 0.3070.46

n Sagittal sections.y Transverse sections.

Fig. 2. (a) Transverse section of skin of A. brama of fish from control site stained with AB/PAS: epidermis had mucous cells fuxia (PAS positive) (arrowheads) or purple

(AB/PAS positive) (curved arrows) containing neutral and mixed glycoconjugates, respectively. BM¼basement membrane. Scale bar¼20 mm. (b) Transverse section of

epidermis of fish from exposed site stained with AB/PAS: the mucous cells were blue (AB positive) (arrowheads) due to the presence of acid glycoconjugates.

BM¼basement membrane. Scale bar¼20 mm. (c) An AB/PAS stained saggittal section of gill filament of a control fish; distinct regular secondary lamellae (arrows) project

from both sides of the filament (primary lamella). In the epithelium of both primary and secondary lamellae mucous cells (arrowheads) occur, and in the filament several

mast cells (curved arrows) are seen. Scale bar¼50 mm. (d) A row of secondary lamellae with aneurisms in gill section of an exposed bream. Some secondary lamellae are

completely dilated (arrowheads), others only at the tip (arrows). Scale bar¼100 mm. (e) Gill filament of an exposed fish showing epithelial proliferation and fusion of

neighboring secondary lamellae (arrows). Scale bar¼50 mm. (f) Edematous secondary lamellae with the epithelium lifted from the basement membrane (arrows) in bream

from the exposed site. Scale bar¼20 mm. (g) Abundant mucus alcian blue positive (arrows) between the secondary lamellae and the gill filaments of an exposed A. brama.

Scale bar¼50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

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result of oil exposure (Marty et al., 1998). Most of the faunamonitoring programmes are conducted from one to several yearsafter the spill and focus on the organs most likely to exhibit

chronic lesions (liver, kidney, spleen). The current study wasdesigned to assess acute effects and rely on histopathologicalexamination of fish sampled a few days after the oil release.

Fig. 3. TEM micrographs of A. brama gill. (a) Two adjacent secondary lamellae of an exposed fish gill, note the presence of abundant empty spaces inside the secondary

lamellae and the epithelial lifting (arrow). Scale bar¼7.4 mm. (b) Sagittal section of control fish primary lamella: arrows indicate mucous cells very close to lamella upper

edge. Scale bar¼5.0 mm. (c) High magnification of an entire mucous cell (arrow) filled with mucus granules within secondary lamella of A. brama from exposed site. Scale

bar¼0.7 mm. (d) Gill of bream from exposed site showing two mucous cells (arrows) at the base of the secondary lamellae discharging contents into the interlamellar

space. Scale bar¼5.5 mm.

Fig. 4. Sections of kidneys of breams from control and exposed sites stained with toluidine blue (a, b) and AB/PAS (c, d). (a, b) Kidney semithin sections of a control fish

(a) and of an exposed fish (b): normal renal tubules (arrows) are interspersed in interstitial tissue. Scale bars¼20 mm. (c) Kidney of control bream showing several rodlet

cells (RCs) (arrows) in the epithelium of a collecting duct. Scale bar¼20 mm. (d) Transverse renal collecting duct section of an exposed fish. Note the abundance of RCs

(arrows). Scale bar¼20 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

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The results presented here are among the first on this incidentand the record makes part of a few studies available for fresh-water oil spill (see Krahn et al., 1986; Caldwell, 1997; Boegeret al., 2003).

Fish have been used successfully as indicators of change inenvironmental quality of a wide variety of aquatic habitats (Soto-Galera et al., 1998), offering numerous advantages for monitoringprogrammes. Data on occurrence of diseases and histo-cytologicalalterations in fish organs are increasingly used as biomarkers forstudying aquatic toxicology and oil spill effects (Boeger et al.,2003; Stentiford et al., 2003; Au, 2004; Marigomez et al., 2006).

In Oreochromis niloticus exposed to petroleum refinery effluentthe main target organs were skin and gills. Hemorrhaging of finsand edematous fused lamellae congested with blood wereobserved (Onwumere and Oladimeji, 1990). Seven days after therelease of a mixture of aromatic hydrocarbons into the NemadjiRiver (Wisconsin, USA), fish, collected approximately 25 km

downstream from the spill site, showed histological abnormalitiesin gill, such as basal hyperplasia, fusion of lamellar epithelia, excessmucous production, and swollen lamellae (Caldwell, 1997). Accord-ingly, the status of several organs were examined microscopically,revealing that the skin and gills, which are in continuous contactwith water and directly exposed to toxicants, were the most affectedin A. brama sampled from the impacted site.

The skin of fish is a dynamic tissue with cellular make-upknown to be influenced by factors including stress and environ-mental conditions (Shephard, 1994). Our results on mucous cells inthe skin are in agreement with data obtained by other researchersworking on fish experimentally exposed to various pollutants.Increases in mucous cell abundance and in mucus secretion inthe skin were reported for catfish (Saccobranchus fossilis) followingsublethal exposures to copper or chromium (Khangarot andTripathi, 1991, 1992). The cellular response in the skin of rainbowtrout exposed to Rhine water includes intense mucus secretion and

Fig. 5. TEM micrographs of collecting ducts in the trunk kidney of A. brama. (a) Portion of a collecting duct in the kidney of a control fish showing the occurrence of two

normal rodlet cells (RCs, arrows) parallel to a mucous cell (asterisk). Scale bar¼4 mm. (b) Area of renal duct epithelium in an exposed fish. Numerous RCs (arrows) were

present near a tubular epithelial cell (arrowhead) and a mucous cell (asterisk). Curved arrow indicates an RC with highly vacuolated cytoplasm. Scale bar¼4 mm.

(c) Magnification of a degenerated rodlet cell in kidney tissue of an exposed bream. Note the deformation of the capsule (arrow), the fusion of the rodlets (arrowhead), and

the presence of elongated and thin mitochondria (curved arrows). Scale bar¼0.8 mm. (d) Micrograph showing the presence of an exhausted RC (arrow) and the expulsion

of rodlets (arrowheads) into the collecting duct lumen in exposed bream. Scale bar¼1.7 mm. (e) Epithelial tubular cell (arrow) in the kidney of bream from exposed site

exhibiting ‘‘bleb’’ (arrowhead) at the plasmalemmar surface. Scale bar¼2 mm. (f) Kidney tissue of A. brama exposed to oil spill showing the presence of endoplasmic

reticulum dilated cisternae (arrows) within an epithelial tubular cell. N¼nucleus. Scale bar¼0.3 mm.

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the stimulation of mucous cell differentiation; moreover, some ofthe mucous cells synthesized mucus of high electron density,probably of serous composition (Iger et al., 1994). Zaccone et al.(1985), studying the effect of an anionic detergent on the epider-mis of catfish, found a marked increase in the number of mucouscells that produce acid glycoproteins and suggest that this changein mucus chemical composition may be induced by a pollutant. Thealtered mucus secretion (both quantitative and qualitative)observed here may be an adaptive defense mechanism, even at alow toxic pressure.

Most of the gill lesions observed in this work have been foundin fish naturally (Spies et al., 1996; Caldwell, 1997; Khan, 1998,2003) or experimentally (Brand et al., 2001; Rudolph et al., 2001;Akaishi et al., 2004; Simonato et al., 2008) exposed to petroleumcompounds and also in fish exposed to other contaminants(including pesticides, heavy metals) and hypoxia (Alazemi et al.,1996; Dezfuli et al., 2006; Giari et al., 2007, 2008; Koca et al.,2008). The reviews of Mallat (1985) and Wood (2001) haveprovided comprehensive information on structural changes infish gill in response to toxicant exposure. Gill histopathologicchanges are, in general, responsive but non-specific to pollutantexposure. Aneurisms, cellular proliferation, and disorganization ofthe secondary lamellae are the most frequent alterations in gillsof adult Astyanax sp. and juveniles of Prochilodus lineatus acutelytreated with water-soluble fraction of oil (Akaishi et al., 2004;Simonato et al., 2008) and of embiotocid fishes from a naturalpetroleum seep (Spies et al., 1996). Khan (1998) documentshyperplasia of the lamellar epithelium and epithelial lifting ingills of flounder (Pleuronectes americanus) collected near an oilrefinery, relating the occurrence of these lesions to the oil spills.Some alterations, like aneurisms, are believed to be directdeleterious effects of toxic compounds while others, such asepithelial lifting, epithelial hyperplasia and enhanced mucussecretion can be considered adaptive response, since they try to

reduce the entrance of xenobiotics; however, these defenseresponses can decrease the exchange of respiratory gases andions in gills (Mallat, 1985). The histological (Rudolph et al. 2001;Akaishi et al., 2004) and physiological (Brauner et al., 1999)effects described in gills of freshwater fish exposed to crude oilindicate a relationship between hydrocarbon exposure andimpairment of respiratory system.

Liver is particularly sensitive to chemical injury, but itshistopathologic lesions are not specific to pollutants (Au, 2004).In the present study, no structural abnormalities were recorded inhepatic tissue, while some minor alterations were documented inthe renal epithelium. These results tally with those by Caldwell(1997) who found that liver, spleen, and head kidney of fishesexamined a few days after the hydrocarbon release in theNemadji River were histopathologically similar to those of thesame species from a reference site. Also Onwumere and Oladimeji(1990) did not find damage in the liver and kidney of Nile tilapiaexposed to petroleum refinery effluent. On the contrary, severalstudies reported the occurrence of severe hepatic modifications infish in response to oil and derivatives exposures with hepatocytestypically showing nuclear and cellular degeneration (such asatrophy, pyknosis, pleomorphism, and hypertrophy) (Brandet al., 2001; Simonato et al., 2008; Rodrigues et al., 2010) and/ornecrosis (Spies et al., 1996; Brand et al., 2001; Pietrapiana et al.,2002; Akaishi et al., 2004). A histopathologic approach was usedto evaluate the effect of an oil spill occurring in July, 2000 in theArroio Saldanha and Rio Iguacu (Brazil) on gill and liver ofCorydoras paleatus and Astyanax spp. The data from histopathol-ogy of gill yielded more information than that produced by liveranalysis, which did not show significant variation betweensampling sites (Boeger et al., 2003). Gill tissue apparentlyresponds more rapidly to the presence of toxic agents, whilehepatic tissue effects seems to be more related to chronicexposure (Mallat, 1985; Boeger et al., 2003).

Fig. 6. Sections of internal organs of breams from control and exposed sites stained with AB/PAS (a, b) or H&E (c, d). (a, b) Intestinal transverse sections of A. brama from

control (a) and exposed site (b): observe the intact folds (arrows) with numerous mucous cells AB positive (arrowheads) in the epithelium and mast cells PAS positive

(curved arrows) at the base of epithelium and in the submucosal layer. Scale bars¼50 mm. (c) Liver of control A. brama: hepatocytes and their nuclei (arrowheads) appear

normal and with regular shape. Scale bar¼10 mm. (d) Cords of hepatocytes with central spherical nuclei (arrowheads), a sinusoid (arrow) and a small bile duct (curved

arrow) were visible. Scale bar¼10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

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Macrophage aggregates and RCs belong to the nonspecificpiscine immune system, and their presence has been related tomany physiological and pathological factors and also to pollution(Agius and Roberts, 2003; Manera and Dezfuli, 2004). Theincrease in MAs (number and/or size) and RCs in organs has beendocumented by numerous authors in both experimentally andnatural chemical exposures, and there is a growing interest inusing these aggregates and cells as biomarkers of stress (Couillardand Hodson, 1996; Iger and Abraham, 1997; Meinelt et al., 1997;Stentiford et al., 2003; Tkatcheva et al., 2004; Dezfuli et al., 2006;Giari et al., 2007, 2008). In this survey, only RCs showed increasednumbers in A. brama immediately following exposure to the oilspill compared to control fish. Moreover, the renal RCs of exposedspecimens often showed ultrastructural modifications and highdischarge activity, phenomena which could be symptoms of

stress and consequence of tissue injury (Leino, 1996; Ghadially,1997). Similar findings were reported for Leuciscus cephalus andDicentrarchus labrax experimentally exposed to herbicides(Dezfuli et al., 2003, 2006) or metals (Giari et al., 2007, 2008)and for natural fish populations inhabiting polluted water(Tkatcheva et al., 2004). This is the first report on the involvementof RCs in response to oil contamination, while recent data withregard to MAs are available from the monitoring campaign 2003following the Prestige oil spill (Marigomez et al., 2006). Stentifordet al. (2003) associated the richness of MAs in flounder(Plathichthys flesus) with high levels of polycyclic aromatic hydro-carbon pollution.

5. Conclusion

Although the concentration of hydrocarbons in the watercolumn of the Po River at the study site was low, the histopatho-logic approach revealed alterations in bream immediately afterthe event, providing evidence for the usefulness of A. brama assentinel species. The histological changes documented (especiallyin gill and skin) seem to indicate that this discharge was astressful experience for bream, as confirmed by the response ofRCs in the kidney of fish from the exposed site. It might also besupposed that RCs react more sensitively (with both proliferationand ultrastructural modifications) and rapidly than MAs underthese environmental conditions. With regard to the absence ofmortalities and of macroscopic lesions in A. brama from the PoRiver, it is possible that the long distance between the spill siteand the sampling area and the high flow of the Po River may havecontributed, together with the cleanup operations, to a decreasein the concentration of hydrocarbons.

Acknowledgments

We thank the Environmental Protection Agency of Ferrara(ARPA) for kindly providing chemical data. We are grateful toA. Lui, S. Squerzanti, and E. Salemi of the University of Ferrara fortechnical assistance and to The Lucidus Consultancy for Englishcorrection of the manuscript. This investigation was supported bygrants from the Italian Ministry of the University and ScientificResearch and Technology.

References

Agius, C., Roberts, R.J., 2003. Melano-macrophage centres and their role in fishpathology. J. Fish Dis 26, 499–509.

Akaishi, F.M., Silva de Assis, H.C., Jakobi, C.G., Eiras-Stoffela, D.R., St-Jean, S.D.,Courtenay, S.C., Lima, E.F., Wagener, A.L.R., Scofield, A.L., Oliveira Riberio, C.A.,2004. Morphological and neurotoxicological findings in tropical freshwaterfish (Astyanax sp.) after water waterborne and acute exposure to water solublefraction (WSF) of crude oil. Arch. Environ. Contam. Toxicol. 46, 244–253.

Alazemi, B.M., Lewis, J.W., Andrews, E.B., 1996. Gill damage in the freshwater fishGnathonemus petersii (Family: Mormyridae) exposed to selected pollutants: anultrastructural study. Environ. Technol. 17, 225–238.

Au, D.W.T., 2004. The application of histocytopathological biomarkers in marinepollution monitoring: a review. Mar. Pollut. Bull. 48, 817–834.

Bernet, D., Schmidt, H., Meier, W., Burkhardt-Hol, P., Wahli, T., 1999. Histopathol-ogy in fish: proposal for a protocol to assess aquatic pollution. J. Fish Dis 22,25–34.

Boeger, W.A., Bittencourt Guimar~aes, A.T., Rom~ao, S., Ostrensky, A., Zamberlan, E.,Falkiewicz, F.H., 2003. Histopathology as an approach to evaluate the effect ofan oil spill on fishes of the Arroio Saldanha and Rio Iguacu (Brazil). In:International Oil Spill Conference, 2003, Vancouver, Canada, April 6–11, 2003,pp. 1–7.

Brand, D.G., Fink, R., Bengeyfield, W., Birtwell, I.K., Mcallister, C.D., 2001.Salt water-acclimated pink salmon fry (Oncorhynchus gorbuscha) developstress-related visceral lesions after 10-day exposure to sublethal concentra-tions of the water-soluble fraction of North Slope crude oil. Toxicol. Pathol. 29,574–584.

Fig. 7. TEM micrograph of liver of A. brama. (a) Some hepatocytes (arrows) of

control fish. Note the clear limit between the cells and the rounded nuclei (N).

Scale bar¼1.6 mm. (b) A hepatocyte (arrow) of fish from exposed site showing the

typical polyhedral format and the regular shape of the nucleus (N). Scale

bar¼1.6 mm. (c) High magnification of a hepatocyte of an exposed bream: the

nucleus (N) with normal distribution of eu- and etero-chromatin and few intact

organelles. The arrow indicates well-developed rough endoplasmic reticulum and

arrowheads show mitochondria. Scale bar¼0.7 mm.

L. Giari et al. / Ecotoxicology and Environmental Safety 77 (2012) 18–2726

Author's personal copy

Brauner, C.J., Ballantyne, C.L., Vijayan, M.M., Val, A.L., 1999. Crude oil exposureaffects air-breathing frequency, blood phosphate levels and ion regulation inan air-breathing teleost fish. Hoplosternum littorale. Comp. Biochem. Physiol123, 127–134.

Caldwell, C.A., 1997. Aromatic hydrocarbon pathology in fish following a large spillinto the Nemadji River, Wisconsin, USA. Bull. Environ. Contam. Toxicol. 58,574–581.

Castaldelli, G., Rossi, R., 2008. Emilia-Romagna Fish Inventory, A and B zones.Emilia-Romagna Region, Greentime, Bologna.

Couillard, C.M., Hodson, P.V., 1996. Pigmented macrophage aggregates: a toxicresponse in fish exposed to bleached-Kraft mill effluent? Environ. Toxicol.Chem 15, 1844–1854.

Dezfuli, B.S., Giari, L., Simoni, E., Palazzi, D., Manera, M., 2003. Alteration of rodletcells in chub caused by the herbicide Stam M-4 (Propanil). J. Fish Biol. 63,232–239.

Dezfuli, B.S., Simoni, E., Giari, L., Manera, M., 2006. Effects of experimentalterbuthylazine exposure on the cells of Dicentrarchus labrax (L.). Chemosphere64, 1684–1694.

Fournie, J.W., Summers, J.K., Courtney, L.A., Engle, V.D., 2001. Utility of splenicmacrophage aggregates as an indicator of fish exposure to degraded environ-ments. J. Aquat. Anim. Health 13, 105–116.

Ghadially, F.N., 1997. Ultrastructural Pathology of the Cell and Matrix. Butter-worths, London.

Giari, L., Manera, M., Simoni, E., Dezfuli, B.S., 2007. Cellular alterations in differentorgans of European sea bass Dicentrarchus labrax (L.) exposed to cadmium.Chemosphere 67, 1171–1181.

Giari, L., Simoni, E., Manera, M., Dezfuli, B.S., 2008. Histo-cytological responses ofDicentrarchus labrax (L.) following mercury exposure. Ecotoxicol. Environ. Safe70, 400–410.

Hinton, D.E., Baumann, P.C., Gardner, G.R., Hawkins, W.E., Hendricks, J.D., Murch-elano, R.A., Okihiro, M.S., 1992. Histopathologic biomarkers. In: War, C.H.,Walton, B.T., LaPoint, T.W. (Eds.), Biomarkers: Biochemical, Physiological, andHistological Markers of Anthropogenic Stress, Lewis Publishers, Boca Raton,pp. 155–209.

Iger, Y., Abraham, M., 1997. Rodlet cells in the epidermis of fish exposed tostressors. Tissue Cell 29, 431–438.

Iger, Y., Lock, R.A., van der Meij, J.C., Wendelaar Bonga, S.E., 1994. Effects of water-borne cadmium on the skin of the common carp (Cyprinus carpio). Arch.Environ. Contam. Toxicol. 26, 342–350.

Khan, R.A., 1998. Influence of petroleum at a refinery terminal on winter flounder,Pleuronectes americanus. Bull. Environ. Contam. Toxicol. 61, 770–777.

Khan, R.A., 2003. Health of flatfish from localities in Placentia Bay, Newfoundland,contaminated with petroleum and PCBs. Arch. Environ. Contam. Toxicol. 44,485–492.

Khangarot, B.S., Tripathi, D.M., 1991. Changes in humoral and cell-mediatedimmune responses in skin and respiratory surfaces of catfish, Saccobranchusfossilis, following copper exposure. Ecotoxicol. Environ. Safe 22, 291–308.

Khangarot, B.S., Tripathi, D.M., 1992. The stereoscan observations of the skin ofcatfish, Saccobranchus fossilis, following chromium exposure. J. Environ. Sci.Health Part A 27, 1141–1148.

Koca, S., Koca, Y.B., Yildiz, S., Gurcu, B., 2008. Genotoxic and histopathologicaleffects of water pollution on two fish species, Barbus capito pectoralis andChondrostoma nasus in the Buyuk Menderes River, Turkey. Biol. Trace Elem.Res. 122, 276–291.

Krahn, M.M., Kittle, L.J., MacLeod, W.D., 1986. Evidence for exposure of fish to oilspilled into the Columbia River. Mar. Environ. Res. 20, 291–298.

Law, R.J., Hellou, J., 1999. Contamination of fish and shellfish following oil spillincidents. Environ. Geosci. 6, 90–98.

Leino, R.L., 1996. Reaction of rodlet cells to a myxosporean infection in kidney ofthe bluegill, Lepomis macrochirus. Can. J. Zool 74, 217–225.

Mallat, J., 1985. Fish gill structural changes induced by toxicants and otherirritants: a statistical review. Can. J. Fish. Aquat. Sci. 42, 630–648.

Manera, M., Dezfuli, B.S., 2004. Rodlet cells in teleosts: a new insight into theirnature and functions. J. Fish Biol. 65, 597–619.

Marigomez, I., Soto, M., Cancio, I., Orbea, A., Garmendia, L., Cajaraville, M.P., 2006.Cell and tissue biomarkers in mussel, and histopathology in hake and anchovyfrom Bay of Biscay after the Prestige oil spill (Monitoring Campaign 2003).Mar. Pollut. Bull. 53, 287–304.

Marty, G.D., Okihiro, M.S., Hinton, D.E., 1998. Fish Histopathology DamageAssessment after the Exxon Valdez Oil Spill. Technical Services Study Number2, Final Report.

Meinelt, T., Kruger, R., Pietrock, M., Osten, R., Steinberg, C., 1997. Mercurypollution and macrophage centres in pike (Esox lucius) tissues. Environ. Sci.Pollut. Res. 4, 32–36.

Neff, H.M., 1979. Polycyclic Aromatic Hydrocarbons in the Aquatic EnvironmentSources, Fates and Biological Effects. Applied Science Publishers Ltd, Essex.

Onwumere, B.G., Oladimeji, A.A., 1990. Accumulation of metals and histopathol-ogy in Oreochromis niloticus exposed to treated NNPC Kaduna (Nigeria)petroleum refinery effluent. Ecotoxicol. Environ. Safe 19, 123–134.

Paul, V.I., Banerjee, T.K., 1997. Histopathological changes induced by ambientammonia (ammonium sulphate) on the opercular linings of the livefishHeteropneustes fossilis (Bloch). Dis. Aquat. Organ 28, 151–161.

Pietrapiana, D., Modena, M., Guidetti, P., Falugi, C., Vacchi, M., 2002. Evaluating thegenotoxic damage and hepatic tissue alterations in demersal fish species: a casestudy in the Ligurian Sea (NW-Mediterranean). Mar. Pollut. Bull. 44, 238–243.

Po River Basin Authority, 2010. Emergenza e post-emergenza ambientale deri-vante dallo sversamento di idrocarburi da raffineria nei fiumi Lambro – Po.Rapporto sintetico. Po River Basin Authority, Italy.

Reite, O.B., Evensen, Ø., 2006. Inflammatory cells of teleostean fish: a reviewfocusing on mast cells/eosinophilic granule cells and rodlet cells. Fish ShellfishImmunol 20, 192–208.

Rice, C.D., 2001. Fish immunotoxicology: understanding mechanisms of action. In:Schlenk, D., Bensen, W.H. (Eds.), Target Organ Toxicity in Marine and Fresh-water Teleosts, Taylor & Francis, London, pp. 96–138.

Rodrigues, R.V., Miranda-Filho, K.C., Gusm~ao, E.P., Moreira, C.B., Romano, L.A.,Sampaio, L.A., 2010. Deleterious effects of water-soluble fraction of petroleum,diesel and gasoline on marine pejerrey Odontesthes argentinensis larvae. Sci.Tot. Environ 408, 2054–2059.

Rudolph, A., Yanez, R., Troncoso, L., 2001. Effects of exposure of Oncorhynchusmykiss to the water accommodated fraction of petroleum hydrocarbons. Bull.Environ. Contam. Toxicol. 66, 400–406.

Shephard, K.L., 1994. Functions for fish mucus. Rev. Fish Biol. Fish 4, 401–429.Simonato, J.D., Guedes, C.L.B., Martinez, C.B.R., 2008. Biochemical, physiological,

and histological changes in the neotropical fish Prochilodus lineatus exposed todiesel oil. Ecotoxicol. Environ. Safe 69, 112–120.

Soto-Galera, E., Diaz-Pardo, E., Lopez-Lopez, E., Lyons, J., 1998. Fish as indicators ofthe environmental quality in the Rio Lerma Basin, Mexico. Aquat. Ecosys.Health Manage 1, 267–276.

Spies, R.B., Stegeman, J.J., Hinton, D.E., Woodin, B., Smolowitz, R., Okihiro, M., Shea,D., 1996. Biomarkers of hydrocarbon exposure and sublethal effects inembiotocid fishes from a natural petroleum seep in the Santa Barbara channel.Aquat. Toxicol. 34, 195–219.

Stentiford, G.D., Longshaw, M., Lyons, B.P., Jones, G., Green, M., Feist, S.W., 2003.Histopathological biomarkers in estuarine fish species for the assessment ofbiological effects of contaminants. Mar. Environ. Res. 55, 137–159.

Tkatcheva, V., Hyvarinen, H., Kukkonen, J., Ryzhkov, L.P., Holopainen, I.J., 2004.Toxic effects of mining effluents on fish gills in a subarctic lake system in NWRussia. Ecotoxicol. Environ. Safe 57, 278–289.

Wood, C.M., 2001. Toxic responses of the gill. In: Schlenk, D., Bensen, W.H. (Eds.),Target Organ Toxicity in Marine and Freshwater Teleosts, Taylor & Francis,London, pp. 1–89.

Zaccone, G., Lo Cascio, P., Fasulo, S., Licata, A., 1985. The effect of an anionicdetergent on complex carbohydrates and enzyme activities in the epidermis ofthe catfish Heteropneustes fossilis (Bloch). Histochem. J 17, 453–466.

L. Giari et al. / Ecotoxicology and Environmental Safety 77 (2012) 18–27 27