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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).
Contents lists available at SciVerse ScienceDirect
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.
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