10.1007_s00244-008-9223-5

7/28/2019 10.1007_s00244-008-9223-5

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Diseases of the Chilean Flounder, Paralichthys adspersus(Steindachner, 1867), as a Biomarker of Marine Coastal Pollution

Near the Itata River (Chile): Part II. Histopathological Lesions

Maritza Leonardi Æ Eduardo Tarifen ˜ o ÆJeanett Vera

Received: 15 April 2008 / Accepted: 11 August 2008 / Published online: 4 September 2008

Ó Springer Science+Business Media, LLC 2008

Abstract This study evaluated the histopathological

lesions of the Chilean flounder, Paralichthys adspersus,inhabiting the marine coastal area influenced by the Itata

River (central Chile) in order to provide an environmental

baseline given the plans to discharge effluents from a cel-

lulose plant through a submarine pipe in the area. Flounder

were also sampled at two reference sites over the course of

1 year. Pathological examinations and descriptions of his-

topathological lesions follow the ICES suggested protocol:

internal and histopathological lesions, condition factor, and

hepatosomatic and spleen indexes. The prevalence of fish

with histopathological lesions differed significantly among

sites. The flounder sampled in the Itata area were the most

affected. Evaluation of histopathological lesions observed

on the flounder caught in the Itata area revealed (i) 16

different types of histopathological lesions, (ii) a high

prevalence of lesions in gills and epidermal and hepatic

tissue, and (iii) a normal K factor and hepatosomatic index.

Significant differences were found in epidermal hyperpla-

sia (EH), chronic dermatitis, telangiectasis (TEL),

localized edema in the base of lamellae (LE), fusion of

secondary lamellae (FSL), foci of cellular alteration (FCA),

melanomacrophagic centers (MMCs), and hemosiderin

(HEM) lesions among sites and among sites by season of

the year. Winter 2006 was the most affected. A seasonal

analysis of histopathological lesions on flounder caught in

the Itata area showed significant differences for EH, pro-gressive focal invasion of muscle fibers (PFIMF), TEL, LE,

FSL, lamellar bifurcation, hepatitis, FCA, MMCs, and

HEM lesions between the sampled seasons of the year;

flounder caught in winter 2006 had the highest prevalence

of these lesions except for PFIMF, which was higher in

winter 2007. These results are discussed in relation to the

contents of inorganic and organic compounds in the water

column and the contents of organic compounds in sedi-

ments of the subtidal environment in the Itata area.

Coastal marine areas are important spawning and nursery

grounds for marine fish, given their greater availability of

food material in comparison with the open ocean. In some

cases, these are principally estuarine areas that should be

free of contamination and pollution (Vethaak 1993).

However, in the last few decades, coastal areas have been

increasingly used for dumping domestic and industrial

effluents, which, in some cases, produce dead zones.

In Chile, the need to deal with health improvement

issues in large urban areas has recently triggered the use of

the ocean for dumping untreated wastewater. The justifi-

cation of this health engineering solution is based on theassumption that the immensity of the marine medium and

the current systems in coastal areas will assure adequate

diffusion and dilution of potentially contaminating ele-

ments to levels that are innocuous for the functioning of

local ecosystems and human health.

In particular, the Bio-Bio Region’s coastline off central

Chile, a pole of commercial, industrial, and military

activities, provides habitats for several commercial fish

species. Nevertheless, the large amount of industrial

M. Leonardi (&) Á E. Tarifeno Á J. Vera

PIMEX-ARAUCO Program, Faculty of Natural Sciences

and Oceanography, University of Concepcion, Concepcion,

Chile

e-mail: [email protected]

E. Tarifeno

Department of Zoology, Faculty of Natural Sciences

and Oceanography, University of Concepcion, Concepcion,

Chile

123

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DOI 10.1007/s00244-008-9223-5

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activity and the resident and floating populations result in

the discharge of several untreated industrial and municipal

contaminants along the coastline (Ahumada and Martinez

1983, Ahumada 1992, Carrasco and Gallardo 1994). This,

along with the area’s peculiar oceanographic conditions

(Ahumada and Arcos 1976, Ahumada and Chuecas 1979,

Ahumada and Martinez 1983), constitutes a very fragile

ecosystem that tends to experience alterations in the naturalprocesses determining its load capacity.

An experimental study conducted with domestic efflu-

ents originating from a submarine pipe in Concepcion Bay

revealed macroscopic and histopathological lesions in the

flounders Paralichthys microps and Paralichthys adspersus

(Leonardi and Tarifeno 1996). Field studies done by Leo-

nardi (1998) in Concepcion Bay, a repository for effluents

originating from two municipalities, fish meal plants, and

unknown origins (petroleum hydrocarbons) (Ahumada

1992), confirmed the anterior lesions and cited the presence

of several hepatic lesions as well. Macroscopic and histo-

pathological lesions were also determined in flounder(Paralichthys sp.) during a field study conducted in San

Vicente Bay, a site that receives steel mill effluents con-

taining several heavy metals and petroleum hydrocarbons

from an oil refinery (Ahumada 1992, 1994, Larrain et al.

1998) and a study in the Gulf of Arauco, a site contami-

nated with untreated pulp and paper mill effluents (George-

Nascimento et al. 2000).

Given these and other reports of pollutants along the

coastline of Chile’s Bio-Bio Region, and the fact that the

Chilean maritime environmental authorities approved the

installation of a submarine pipe for discharging secondary

effluents from a cellulose plant near Nueva Aldea, north of

the Itata River (368160S, 728490W) at a pristine (contami-

nation-free) site, a study was done to evaluate histo-

pathogical lesions in the skin, gills, spleen, and liver of

Chilean flounder (Paralichthys adspersus) under natural

seawater conditions at the mouth of the Itata River before the

submarine pipe began operations. In September 2006, the

Nueva Aldea cellulose plant began operations, discharging

its effluents into the Itata River as a provisionary measure

until the submarine pipe could be built and installed.

Material and Methods

Sampling Sites and Fishing Procedures

Three hundred eighty flounders (P. adspersus) were sam-

pled seasonally from three coastal marine areas between

June 2006 and August 2007 (Table 1, Fig. 1): (i) 107

flounder were caught off Cobquecura (3681602400 S,

7284902000 W), a site located 11 km north of the Itata River;

(ii) 133 fish were sampled from the area directly influenced

by the Itata River runoff (3682301800 S, 7285300800 W); and

(iii) 140 fish were taken from Coliumo Bay (368310S,

728570W), a site located 17 km south of the Itata River

(Fig. 1). The Cobquecura and Coliumo Bay sites were used

to determine the extent of the coastal area affected by the

river plume, which is known to head southward in winter

and northward in summer (Sobarzo 1999). Tables 2 and 3

show the organic compounds and metals in the water col-

umn and the sediments from the Itata site between autumn

2006 and winter 2007 (Monitoring Program for the Marine

Environment, Nueva Aldea CFI; CONAMA 2008). Fishing

procedures were done according to Leonardi and Tarifeno

(1996). As fishing gear, a 10 9 5-m midwater trawling net

was used; it was operated for less than 10 min to avoid

physical injuries to the fish caused by the trawl procedures.

Once collected, the flounder were kept in 50-L thermo-

boxes with constant running seawater and taken alive to the

laboratory facility for further processing within 8 h.

Table 1 Number of fish caught and number of trawls done off

Cobquecura (nearby reference site), at Itata (a site influenced by the

Itata River), and in Coliumo Bay (far-off reference site), between

winter 2006 and winter 2007

Site No. of fish caught (No. of trawls)

Winter

2006

Spring

2006

Summer

2007

Autumn

2007

Winter

2007

Cobquecura 20 (3) 30 (4) 20 (2) 20 (3) 17 (3)

Itata 47 (10) 26 (3) 20 (3) 20 (2) 20 (2)

Coliumo Bay 47 (19) 30 (7) 20 (8) 20 (6) 23 (7)

Fig. 1 Coastal locations where flounder were sampled: Cobquecura

coast (j; 36°1602400 S, 72°4902000 W), Itata (d; 36°2301800S,

72°5300800 W), and Coliumo Bay (d; 36°3105200 S, 72°5708500 W)

Arch Environ Contam Toxicol (2009) 56:546–556 547

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Table 2 Hydrography, inorganic compounds, nutrients, organic compounds, and microbiological variables detected in the water column of the

Itata area subtidal environment

Variable Season (X ± SD)

Autumn 2006 Winter 2006 Spring 2006 Summer 2007 Autumn 2007 Winter 2007

Hydrographic

Dissolved O2 (mg/L) 77.18 ± 8.08 6.60 ± 0.50 7.83 ± 1.07 4.97 ± 1.03 5.43 ± 0.97 5.93 ± 0.69

Dissolved O2

(% saturation)4.64 ± 0.54 77.47 ± 4.73 55.47 ± 5.33 60.00 ± 13.07 65.67 ± 11.4 71.00 ± 8.33

Salinity (psu) 33.54 ± 0.68 31.13 ± 1.93 32.48 ± 1.39 33.87 ± 0.30 34.07 ± 0.6 33.48 ± 1.87

Temperature (8C) 12.09 ± 0.12 12.50 ± 0.23 12.82 ± 0.47 13.44 ± 0.47 12.37 ± 0.27 12.25 ± 0.18

Inorganic compounds

Aluminum (lg/L) 0.41 ± 0.45 1.06 ± 0.44 1.47 ± 0.60 1.15 ± 0.40 1.82 ± 0.86 3.19 ± 2.41

Cadmium (lg/L) 0.00 ± 0.01 0.02 ± 0.03 0.13 ± 0.07 0.01 ± 0.02 0.21 ± 0.36 0.08 ± 0.06

Chromium VI

(lg/L)

0.37 ± 0.25 0.60 ± 0.37 1.13 ± 0.30 0.77 ± 0.21 0.33 ± 0.17 0.38 ± 0.09

Total chromium

(lg/L)

0.38 ± 0.26 0.63 ± 0.38 1.17 ± 0.30 1.12 ± 0.31 3.10 ± 0.19 3.70 ± 0.85

Copper (lg/L) 0.30 ± 0.48 0.48 ± 0.22 1.00 ± 0.83 0.96 ± 0.63 1.68 ± 1.82 4.66 ± 2.24

Dissolved iron(lg/L)

2.45 ± 2.96 1.30 ± 0.78 2.47 ± 3.53 1.07 ± 0.64 1.35 ± 0.68 1.69 ± 1.00

Manganese (lg/L) 3.55 ± 1.24 2.79 ± 1.61 1.67 ± 0.63 1.73 ± 0.52 1.23 ± 0.43 2.78 ± 1.95

Nickel (lg/L) 0.13 ± 0.13 0.39 ± 0.14 0.20 ± 0.10 0.19 ± 0.08 0.15 ± 0.07 0.32 ± 0.20

Zinc (lg/L) 1.43 ± 1.65 0.62 ± 0.29 1.23 ± 1.13 1.12 ± 0.94 0.71 ± 0.41 1.12 ± 1.03

Nutrients and others

Total nitrogen

(mg/L)

2.92 ± 0.96 2.97 ± 0.70 5.20 ± 1.03 2.83 ± 2.27 10.10 ± 10.53 110.90 ± 62.10

Nitrite (lM) 1.10 ± 0.96 0.47 ± 0.13 0.33 ± 0.40 0.60 ± 0.10 0.70 ± 0.10 0.50 ± 0.17

Nitrate (lM) 9.55 ± 4.44 7.5 ± 1.60 4.53 ± 2.97 14.63 ± 2.30 16.50 ± 5.50 4.67 ± 2.30

Ammonium (lM) 1.43 ± 0.65 1.07 ± 0.43 1.00 ± 0.67 2.97 ± 0.63 2.20 ± 0.70 0.50 ± 0.13

Total phosphorus

(lM)

2.83 ± 0.55 1.97 ± 1.13 0.77 ± 0.33 1.70 ± 0.33 2.67 ± 0.63 3.87 ± 1.53

Sulfates (mg/L) 6396.54 ± 403.75 6050.83 ± 747.03 2628.67 ± 186.00 2768.07 ± 215.70 2788.63 ± 161.93 2496.00 ± 152.00

Chlorates (mg/L) * N.D. N.D. 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Sulfides (lM) 0.08 ± 0.08 0.37 ± 0.17 0.07 ± 0.10 0.10 ± 0.00 0.20 ± 0.10 0.20 ± 0.13

Organic compounds

AOX (mg/L) 0.08 ± 0.03 0.09 ± 0.04 0.20 ± 0.07 0.14 ± 0.06 0.17 ± 0.13 0.03 ± 0.03

Dioxins (ng/L) 0.00 ± 0.00 0.01 ± 0.01 0.00 ± 0.00 0.01 ± 0.02 0.01 ± 0.01 0.01 ± 0.01

Furans (ng/L) 0.00 ± 0.00 0.00 ± 0.01 0.00 ± 0.00 0.01 ± 0.01 0.02 ± 0.02 0.00 ± 0.01

Resin acids (ppb) 0.00 ± 0.00 0.00 ± 0.00 N.D. 0.00 ± 0.00 N.D. N.D.

Fatty acids (ppb) 14.01 ± 17.93 19.63 ± 19.11 109.57 ± 123.43 28.43 ± 13.13 10.30 ± 9.87 24.41 ± 13.91

Oils and fats (mg/L) 3.89 ± 3.26 3.79 ± 2.32 32.17 ± 26.80 3.07 ± 2.37 9.91 ± 3.88 7.33 ± 2.16

Pentachlorophenol

(lg/L)

0.01 ± 0.02 0.00 ± 0.00 N.D. N.D. N.D. N.D.

Chlorophenols

(lg/L)

1.12 ± 1.14 1.37 ± 1.09 N.D. N.D. N.D. N.D.

Volatile

hydrocarbons

(lg/L)

0.00 ± 0.00 0.00 ± 0.00 N.D. N.D. N.D. N.D.

Total hydrocarbons

(lg/L)

0.00 ± 0.00 0.00 ± 0.00 1.73 ± 5.77 N.D. N.D. N.D.

Microbiological

Fecal coliforms

(NMP/100 mL)

0.30 ± 0.00 45.00 ± 59.00 8.00 ± 14.00 2.00 ± 2.00 5.00 ± 9.00 239.00 ± 391.00

548 Arch Environ Contam Toxicol (2009) 56:546–556

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Table 4 Comparison of

biometric data and

histopathological lesions in

flounder sampled off the coast

of Cobquecura (a site with no

urban or industrial discharges),

at Itata (a site with urban and

industrial discharges, locatednear a cellulose plant), and in

Coliumo Bay (a site with only

urban discharges)

Note: Significant difference

( p\ 0.05): a Between Itata and

northern reference area;b Between Itata and southern

reference area;c

Between

northern and southern areas

Variable Site (X ± SD)

Cobquecura

coast (n = 107)

Itata

(n = 133)

Coliumo Bay

(n = 140)

Biometric data

Length (cm) 18 ± 5 20 ± 5a 25 ± 8 bc

Weight (g) 60 ± 75 83 ± 62 212 ± 164bc

Condition factor 0.86 ± 0.15 0.90 ± 0.10 1.02 ± 0.32bc

Hepatic somatic index 1.42 ± 0.55 1.54 ± 0.44 1.96 ± 0.86bc

Spleen somatic index 0.12 ± 0.06 0.12 ± 0.66a

0.13 ± 0.11

Histopathological lesions, no.

Epidermal hyperplasia 23 (21%) 60 (45%)ab

29 (21%)

Chronic dermatitis 12 (11%) 42 (32%)ab

18 (13%)

Progressive focal invasion of muscle fibers 15 (14%) 18 (14%) 9 (6%)

Telangiectasis 5 (5%) 35 (26%)ab

20 (14%)

Localized edema 38 (36%) 73 (55%)ab 38 (27%)

Generalized edema 10 (9%) 26 (20%)a 19 (4%)

Fusion of secondary lamellae 10 (9%) 48 (36%)

ab

24 (17%)Lamellar bifurcation 3 (3%) 8 (6%)a 0 (0%)

X cells 0 (0%) 9 (7%)ab

0 (0%)

Hepatitis 2 (2%) 11 (8%) 13 (9%)

Foci of cellular alteration 6 (6%) 37 (28%)ab 7 (5%)

Focal necrosis 0 (0%) 1 (1%) 0 (0%)

Progressive focal invasion of hepatic cells 2 (2%) 7 (5%)b 0 (0%)

Hydropic vacuolization of bile duct 0 (0%) 3 (2%) 2 (1%)

Center of melanomacrophages 0 (0%) 45 (34%)ab

7 (5%)

Hemosiderin 24 (22%) 49 (37%)ab 19 (14%)

Table 3 Organic compounds detected in sediments of the subtidal environment in the Itata area

Organic compound Season (X ± SD)


TOM (%) 2.96 ± 2.12 2.4 ± 1.2 3.4 ± 3.3 1.8 ± 0.7 2.0 ± 0.5 2.0 ± 0.7

AOX (mg/kg) 57.80 ± 13.09 127.6 ± 18.2 175.1 ± 53.6 217.3 ± 17.1 247.5 ± 22.1 79.9 ± 16.6

Total hydrocarbons (lg/g) 289.58 ± 613.88 8.4 ± 27.9 5.4 ± 4.1 N.D. N.D. N.D.

Pentachlorophenol (lg/g) 0.01 ± 0.01 0.0 ± 0.0 N.D. N.D. 0.6 ± 1.2 N.D.

Source: Monitoring Program for the Marine Environment, Nueva Aldea CFI (CONAMA 2008)

Note: N.D., Not detected; * not analyzed

Table 2 continued



Total coliforms

(NMP/100 mL)

353.71 ± 512.06 1351.00 ± 1066.00 41.00 ± 74.00 23.00 ± 62.00 94.00 ± 143.00 298.00 ± 400.00

Source: Monitoring Program for the Marine Environment, Nueva Aldea CFI (CONAMA 2008)

Note: N.D., Not detected; * not analyzed


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Biometric Data and Organ Sampling

At the laboratory, a pathological assessment was conducted

following ICES (1989). First, the biometric data (weight

[g], total length [cm], condition factor [K = g/

cm39 100]) were recorded. Then the fish were inspected

externally and internally for macroscopic lesions. To

minimize handling-induced stress and to optimize tissuequality, the following sampling sequence was always used:

(i) gills, (ii) skin, (iii) spleen, (iv) liver. This sequence was

carried out in less than 10 min. Once the body cavity was

opened and the viscera exposed, the spleen and liver were

examined in situ and their size and color were noted

carefully. Next, the presence of any macroscopically visi-

ble nodules or other lesions was recorded. The liver was

dissected away from the remaining viscera and removed

from the fish for examination.

Histopathological Diagnosis

To carry out the histopathological sampling routine, 50%

of the flounder caught at each site were removed at random

from the thermoboxes and placed individually in dissection

trays. In order to prevent the appearance of postmortem

artifacts, flounder were handled with extreme care and all

tissue was placed in individual prelabeled containers of fixative (10% neutral buffered formalin; NBF). The diag-

nostic features of toxicopathic lesions were based on the

conventional method of paraffin wax-embedded material

stained with hematoxylin-eosin dye according to Human-

son (1962). For conventional histology, sections were cut at

4–5 lm using a rotary microtome. Perls Prussian Blue for

ferric iron, a special histopathological staining technique

that detects resistance to iron uptake, was used to diagnosis

spleen histopathology.

Table 5 Comparison of biometric data and histopathological lesions in flounder sampled off the Itata river (a site with urban and industrial

discharges, located near a cellulose plant) during each season of the year


Winter 2006

(n = 47)

Spring 2006

(n = 26)

Summer 2007

(n = 20)

Autumn 2007

(n = 20)

Winter 2007

(n = 20)

Biometric data

Length (cm) 20.42 ± 0.65 18.00 ± 0.88 19.51 ± 1.05 22.00 ± 1.00 18.02 ± 1.00Weight (g) 87.12 ± 8.71 71.13 ± 11.70 89.05 ± 13.36 113.35 ± 13.3

a50.55 ± 13.36

Condition factor 0.88 ± 0.01 0.91 ± 0.02 0.93 ± 0.02 0.98 ± 0.02a 0.83 ± 0.02

Hepatic somatic index 1.45 ± 0.05 1.67 ± 0.07 1.73 ± 0.08 1.88 ± 0.08a 1.06 ± 1.08

Spleen somatic index 0.13 ± 0.00 0.13 ± 0.01 0.08 ± 0.01 0.12 ± 0.01 0.10 ± 0.01

Histopathological lesions, no.

Epidermal hyperplasia 33 (70%)a

2 (8%) 9 (45%) 8 (40%) 8 (40%)

Chronic dermatitis 20 (43%) 1 (4%) 8 (40%) 7 (35%) 6 (30%)

Progressive focal invasion

of muscle fibers

0 (0%) 0 (0%) 5 (25%) 4 (20%) 9 (45%)a

Telangiectasis 24 (51%)a 3 (12%) 5 (25%) 3 (15%) 0 (0%)

Localized edema 40 (85%)a

4 (15%) 9 (45%) 10 (50%) 10 (50%)

Generalized edema 18 (38%) 0 (0%) 1 (5%) 4 (20%) 3 (15%)

Fusion of secondary lamellae 39 (83%)a 2 (8%) 1 (5%) 3 (15%) 3 (15%)

Lamellar bifurcation 8 (17%)a

0 (0%) 0 (0%) 0 (0%) 0 (0%)

X cells 9 (19%)a 0 (0%) 0 (0%) 0 (0%) 0 (0%)

Hepatitis 11 (23%)a

0 (0%) 0 (0%) 0 (0%) 0 (0%)

Foci of cellular alteration 31 (66%)a 0 (0%) 5 (25%) 0 (0%) 1 (5%)

Focal necrosis 0 (0%) 0 (0%) 1 (5%) 0 (0%) 0 (0%)

Progressive focal invasion

of hepatic cells

0 (0%) 0 (0%) 4 (20%) 1 (5%) 2 (10%)

Hydropic vacuolization of bile

duct

0 (0%) 0 (0%) 2 (10%) 1 (5%) 0 (0%)

Centers of melanomacrophages 45 (96%)a

0 (0%) 0 (0%) 0 (0%) 0 (0%)

Hemosiderin 33 (70%)a

3 (12%) 4 (20%) 3 (15%) 6 (30%)

Note:a

Significant differences between seasons ( p\0.05)


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Histopathological lesions were described according to

standardized ICES (1989) procedures and those described

in the literature (Robert 1981, Kohler 1990, MacLean

1993, Vethaak 1993, Vethaak and Wester 1996, Lindesjoo

and Thulin 1994, Leonardi and Tarifeno 1996, Vethaak and

Jol 1996, Leonardi 1998, Au 2004, Kohler 2004) and

correspond to (i) telangiectasia lamellae, (ii) hyperplasia in

gills or ‘‘X cells’’ (cellular growth accelerated between

secondary lamellae), (iii) fusion of secondary lamellae, (iv)

bifurcation of secondary lamellae, (v) localized edema in

the base of lamellae, (vi) generalized edema, (vii) chronic

dermatitis, (viii) epidermal hyperplasia, (ix) hemosiderin in

spleen, (x) hepatitis, (xi) abundance of melanomacrophagic

centers, (xii) foci of cellular alteration, (xiii) hepatic

necrosis, and (xiv) hydropic vacuolization of the bile duct.

Statistical Analysis

The variables weight, total length, condition (K) factor

[(body mass/length3) 9 100], hepatosomatic index [(liver

0

20

40

60

80

100

P r e v a l e n c e

( % )

E H

C D

P F I M F

T E L

L E

G E

F S L

L B

X C E L L

H E P A

F C A

F N

P F I H C

H V B D

M M C s

H E M

Lesions

Histopathological lesions in Winter of 2006

Coliumo Bay

Itata

Cobquecura coast

ab

ab

ab

ab

abab

ab

ab ab

bb

a

Fig. 2 The observed prevalence of histopathological lesions in

flounder captured at the Cobquecura coast, Itata, and Coliumo Bay

sites in winter 2006. EH, epidermal hyperplasia; CD, chronic

dermatitis; PFIMF, progressive focal invasion of muscle fibers;

TEL, telangiectasis; LE, localized edema; GE, generalized edema;

FSL, fusion of secondary lamella; LB, lamellar bifurcation; X-CELL,

X cells; HEPA, hepatitis; FCA, foci of cellular alteration; FN, focal

necrosis; PFIHC, progressive focal invasion of hepatic cells; HVBD,

hydropic vacuolization of bile duct; MMCs, centers of melanomac-

rophage; HEM, hemosiderin. (a) Significant differences between Itata

and northern area ( p\0.05); (b) significant differences between Itata

and southern area ( p\ 0.05)

0

20

40

60

80

100

P r e v a l e n c e ( % )

E H C

D

P F I M F

T E L

L E

G E

F S L

L B

X C E L L

H E P A

F C A

F N

P F I H C

H V B D

M M C s

H E M

Lesions

Histopathological lesions in Spring of 2006

Coliumo Bay

Itata

Cobquecura coast



sites in spring 2006. Abbreviations as in the legend to Fig. 2

0

20

40

60

80

100


E H

C D

P F I M F

T E L

L E

G E

F S L

L B

X C E L L

H E P A

F C A

F N

P F I H C

H V B D

M M C s

H E M

Lesions

Histopathological lesions in Summer 2007

Coliumo Bay

Itata

Cobquecura coast



sites in summer 2007. Abbreviations as in the legend to Fig. 2

0

20

40

60

80

100


E H

C D

P

F I M F

T E L

L E

G E

F S L

L B

X C E L L

H E P A

F C A

F N

P

F I H C

H

V B D

M

M C s

H E M

Lesions

Histopathological lesions in Autumn of 2007

Coliumo Bay

Itata

Cobquecura coast

c



sites in autumn 2007. Abbreviations as in the legend to Fig. 2. (c)

Significant differences between northern and southern areas ( p\0.05)


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mass/body mass) 9 100], and spleen index [(spleen mass/

body mass) 9 100] were compared between sites using

one-way ANOVA and Tukey’s a posteriori test (Zar 1984).

Differences were considered to be significant at p\ 0.05.

Significant differences in the occurrence of different his-

topathological lesions were compared between sites and

between sites by season; data were analyzed using the

(nonparametric) chi-square independence test, with 2 9 2

contingency tables and a significance level of p\ 0.05.

Results

A number of significant differences were observed between

flounder caught in the Itata area near the Nueva Aldea

cellulose plant and those from the northern (Cobquecura)

and southern (Coliumo Bay) reference sites. Significant

differences were found in the prevalence of histopatholo-

gical lesions ( p\ 0.05) among the three areas; flounder in

the Itata area ( p\ 0.05) had the highest prevalence of

histopathological lesions (Table 4). Comparisons showed

that total length, weight, condition (K) factor, and he-

patosomatic index were significantly lower for floundercaught in the Itata area than in Coliumo Bay. Histopa-

thological lesions were more frequent in the flounder

collected in the Itata area than in those caught off Cob-

quecura and in Coliumo Bay. Sixteen different types of

histopathological lesions were recorded on the flounder in

the Itata area: (1) epidermal hyperplasia, (2) chronic der-

matitis, (3) progressive focal invasion of muscle fibers, (4)

telangiectasis, (5) localized edema, (6) generalized edema,

(7) fusion of secondary lamellae, (8) lamellar bifurcation,

(9) X cells, (10) hepatitis, (11) foci of cellular alteration,

(12) focal necrosis, (13) progressive focal invasion of

hepatic cells, (14) hydropic vacuolization of the bile duct,

(15) melanomacrophagic centers, and (16) hemosiderin

(Table 5).

Epidermal hyperplasia, chronic dermatitis, telangiecta-

sis, localized edema, fusion of secondary lamellae, X cells,

foci of cellular alteration, centers of melanomacrophages,

and hemosiderin lesions were significantly more frequent

in flounder from the Itata site than in those caught off

Cobquecura and in Coliumo Bay. On the other hand, only

generalized edema lesions were significantly more frequent

in flounder caught at the Itata site than in those caught off

the coast of Cobquecura; and lamellar bifurcation and

progressive focal invasion of hepatic cell lesions were

significantly more frequent in flounder caught at the Itata

site than in those caught in Coliumo Bay (Table 4).

An assessment of lesion prevalence in the study areas

during different seasons of the year indicated that, in winter

2006, epidermal hyperplasia, chronic dermatitis, telangi-

ectasis, localized edema, generalized edema, fusion of

secondary lamellae, foci of cellular alteration, centers of

melanomacrophages, and hemosiderina lesions were sig-

nificantly more frequent in flounder from the Itata site than

0

20

40

60

80

100


E H C

D

P F I M F

T E L

L E

G E

F S L

L B

X - C E L L

H E P A

F C A

F N

P F I H C

H V B D

M M C s

H E M

Lesions

Histopathological lesions in Winter of 2007

Coliumo Bay

Itata

Cobquecura coast

c



sites in winter 2007. Abbreviations as in the legend to Fig. 2. (c)

Significant differences between northern and southern areas ( p\0.05)

Fig. 7 Histological section of a P. adspersus skin showing (a)

epidermal hyperplasia (bar = 20 lm) and (b) chronic dermatitis

(bar = 450 lm)


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in those sampled at the Cobquecura and Coliumo Bay sites

( p\ 0.05) (Fig. 2). Hepatitis lesions were significantly

more frequent in the Itata area than off Cobquecura,

whereas lamellar bifurcation and X cell lesions were sig-nificant more frequent in flounder caught in the Itata area

than in those from Coliumo Bay.

In spring 2006 and summer 2007, no significant differ-

ences between sites were found in the lesions (Figs. 3 and

4). However, in autumn and winter 2007, significant dif-

ferences were found in histopathological lesions ( p\ 0.05)

between the reference sites; flounder at the Coliumo Bay

site ( p\ 0.05) had the highest prevalence of telangiectasis

lesions in autumn (Fig. 5), and flounder caught off

Cobquecura ( p\ 0.05) had the highest prevalence of pro-

gressive focal invasion of hepatic cell lesions in winter

2007 (Fig. 6).

The seasonal analysis of the variables recorded forflounder caught in the Itata area show significant differ-

ences between the seasons of the year monitored (Table 5).

Epidermal hyperplasia, telangiectasis, localized edema,

fusion of secondary lamellae, lamellar bifurcation, X cells,

hepatitis, foci of cellular alteration, centers of melano-

macrophages, and hemosiderin lesions were significantly

more frequent in winter 2006 than in the other seasons of

the year. Body weight, condition (K) factor, and hepato-

somatic index of flounder caught in autumn 2007 were

Fig. 8 Histological section of a

P. adspersus gill showing (a)

laminar telangiectasis, (b)

localized edema in the base of

lamellae, (c) generalized edema

in the secondary lamellae, and

(d) fusion of secondary

lamellae. Bar = 100 lm


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significantly more frequent than in those caught in other

seasons of the year. No significant differences were found

in length and spleen indexes across the seasons. The main

lesions for each organ are shown in Figs. 7–10.

Discussion

The occurrence of histopathological lesions in the skin,

gills, liver, and spleen of fish considered to be exposed to

anthropogenic and chemical contaminants has already been

recorded for flounder from several polluted coastal areas

(Murchelano and Wolke 1991, Johnson et al. 1993, Myers

et al. 1993, Vethaak 1993, Lindesjoo and Thulin 1994,

Khan 1995, ICES 1997, Wood 2001, Khan 2006) as well as

from the Chilean marine coast (Leonardi and Tarifeno

1996, Leonardi 1998, George-Nascimento et al. 2000). For

Chile, however, the present study is the first, to our

knowledge, to focus not only on macroscopic lesions

(Leonardi et al. 2008) but also on other types of histopa-

thological liver changes (e.g., focal necrosis, hydropic

vacuolization of the bile duct, progressive focal invasion of

hepatic cells). Although most of the histopathologies

reported in this study have also been described by other

authors (see above), it is interesting to note that the focalnecrosis and progressive focal invasion of hepatic cell

lesions were observed herein for the first time in the

Chilean marine environment.

The differences observed in histopathological lesions

among the three coastal sites included in this study suggest

that the Itata area was already affected by discharges that

were contaminating the river. The Itata area is far from

heavy urban and industrial centers; nevertheless, the

observed predominance of the different histopathological

lesions in flounder found in the area during the year of

sampling indicates that the fish were being exposed to some

type of pollution. The high prevalence of histopathologicallesions recorded in flounder caught in the Itata area could be

explained mainly by the mix of chemical compounds present

in the water column and concentrations of AOX and total

hydrocarbons in sediments, which are contributed by Itata

River runoff. Diverse studies show the occurrence of lesions

in flounder collected in winter from areas heavily contami-

nated with organic xenobiotics (i.e., PAHs, its derivatives,

and chlorinates), which correlate significantly with pollutant

concentrations in sediments (Johnson et al. 1992).

Fig. 9 Histological section of a P. adspersus liver showing (a) foci of

cellular alteration (bar = 450 lm) and (b) melanomacrophage cen-

ters (bar = 100 lm)

Fig. 10 Histological section of a P. adspersus spleen showing the

presence of hemosiderin. Bar = 100 lm


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Furthermore, the major prevalence of epidermal hyperpla-

sia, chronic dermatitis, telangiectasia, localized edema,

fusion of secondary lamellae, foci of cellular alteration,

centers of melanomacrophages, and hemosiderin lesions has

already been related to chemical contamination (Malins

et al. 1988, Myers et al. 1993, Lindesjoo and Thulin 1994,

Khan 1995, Vethaak and Wester 1996, Wood 2001). These

results indicate that this coastal marine ecosystem is alreadyaffected by Itata River runoff.

The high prevalence of epidermal hyperplasia, telangi-

ectasis, localized edema, fusion of secondary lamellae,

lamellar bifurcation, X cells, hepatitis, foci of cellular

alteration, centers of melanomacrophages, and hemosiderin

lesions recorded in winter 2006 in flounder caught in the

Itata area suggests that sulfates (6396.54 mg/L) in the local

water column and total hydrocarbons (289.59 lg/g) in

local sediments in autumn 2006 (CONAMA 2008) were

the underlying cause of these lesions in later months

(winter 2006). On the other hand, hepatic lesions (foci of

cellular alteration) are manifested only after a lag of morethan 6 months (Nagazawa et al. 1985, Vethaak and Jol

1996). Therefore, the field situation observed in winter

2006 had developed months before, when river runoff was

at its lowest level. Furthermore, the better fish health

conditions observed in spring 2006 and in summer,

autumn, and winter 2007 (compared to winter 2006) sug-

gest that the effluents dumped into the Itata River from

September 2006 until August 2007 were diluted by the

river runoff, reducing their impact on the studied flounder.

That is, the lesions were present but were less prevalent

than in winter 2006. However, the high prevalence of

epithelial hyperplasia, progressive focal invasion of muscle

fibers, localized edema, and hemosiderin in flounder from

the Itata area during the time in which the cellulose plant

was discharging its effluent into the Itata River should be

noted. Nevertheless, it cannot be assumed that this seasonal

dilution of the plant’s effluents by river runoff will also

occur with the discharges from the submarine pipe, since

the effluent will be dumped independently of the river

runoff. Instead, the effluent discharges could aggravate the

fish pathologies already present in the environment due to

its sublethal effect after a long period of time.

Since histopathological lesions, localized edema, and

hemosiderin have been associated with cellulose effluent

discharges (Lindesjoo and Thulin 1994, George-Nasci-

mento et al. 2000, Khan 2000, 2006) and the increased

frequency of the histopathologic lesion ‘‘progressive focal

invasion of muscle fibers’’ is commonly associated with

steroid-resistant forms of inflammatory myopathy (De

Bleecker et al. 2002), the impact of effluent discharges

from the cellulose plant must be evaluated by carrying out

controlled bioassays that expose the fish to different mix-

tures of seawater plus the effluent.

Acknowledgments We thank the crew of the Kay-Kay scientific

research vessel for their assistance during field sampling, students

from the University of Concepcion Marine Biology Undergraduate

Program for their collaboration, and the PIMEX-ARAUCO Program

for funding.

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