Branchial structure and hydromineral equilibriumin juvenile turbot (Scophthalmus maximus) exposedto heavy fuel oil

Christelle Goanvec • Elisabeth Poirier •

Stephane Le-Floch • Michael Theron

Received: 15 April 2010 / Accepted: 14 September 2010

� Springer Science+Business Media B.V. 2010

Abstract This study is an attempt to go further in the

comprehension of the effects of heavy fuel oil in

the context of an accidental oil spill at sea. It focuses on

the link between morphological and functional impacts

of realistic doses of the dissolved fraction of a heavy

fuel oil on fish gills. Juvenile turbot, Scophthalmus

maximus were exposed to the dissolved fraction of a

heavy fuel oil for 5 days and then placed 30 days in

clean sea water for recovery. During the contamination

period, the concentration of the 16 US EPA priority

poly-aromatic hydrocarbons showed small variations

around a mean value of 321.0 ± 9.1 ng l-1 (mean ±

SEM). The contamination induced a 64% increase in

hepatic cytochrome P 450 1A (Western blot analysis).

Osmolality, [Na?] and [Cl-] rapidly and significantly

increased (by 14, 23 and 28% respectively) and

slowly decreased to normal levels during the recovery

period. At the same time, branchial histology showed

decreases in the number of mucocytes (by 30%) and of

chloride cells (by 95%) in the interlamellar epithelium.

Therefore, it is suggested that the osmotic imbalance

observed after the 5 days of exposure to the dissolved

fraction of the heavy fuel oil is the consequence of the

structural alteration of the gills i.e, the strong reduction

of ionocyte numbers.

Keywords Fish � Heavy fuel oil � Turbot � Chloride

cells � Hydromineral balance � Gill epithelium

Introduction

In teleost fish, the gills are multitasking organs that

play a central role in environmental adaptation. Gills

are the main region for respiratory gas exchange but

they are also involved in osmotic, ionic, acid/base

and nitrogen regulation (Evans 2008; Evans et al.

2005). Due to these interacting functions, the gills

activity results from a compromise between compet-

ing regulatory needs (Sollid and Nilsson 2006;

Gonzalez and McDonald 1992; Claireaux and Dutil

1992). In coastal fish species, osmoregulation is a

gill-based physiological process that plays a central

role in determining individual fitness, and coastal

areas are particularly exposed to changes in water

C. Goanvec � E. Poirier � M. Theron (&)

Laboratoire ORPHY EA4324, Universite de Bretagne

Occidentale, Universite Europeenne de Bretagne,

6 Avenue le Gorgeu, CS 93837, 29238 Brest,

Cedex 3, France

e-mail: Michael.Theron@univ-brest.fr

C. Goanvec

e-mail: Christelle.Goanvec@univ-brest.fr

E. Poirier

e-mail: elisabeth.jaouen@univ-brest.fr

S. Le-Floch

Cedre (Centre de documentation de recherche et

d’experimentations sur les pollutions accidentelles des

eaux), 715 Rue Alain Colas, CS 41836, 29218 Brest,

Cedex 2, France

e-mail: Stephane.Le.Floch@cedre.fr

123

Fish Physiol Biochem

DOI 10.1007/s10695-010-9435-2

salinity. Coastal ecosystems, situated at the interface

between continents and oceans are, moreover, espe-

cially exposed to anthropogenic influence. As a

result, fish living in coast areas heavily utilised by

human activities may be exposed to a wide variety of

pollutants such as heavy metals, nitrogenous com-

pounds and a number of organic substances (Howarth

et al. 2008; Martinez-Gomez et al. 2009; Tin et al.

2009). All these contaminants have been shown to

affect gill functions and in particular osmoregulation

(Mallatt 1985; Evans 1987). A survey of the literature

readily shows that most published works on the effect

of fuel oil exposure upon gill function are histology

based. They revealed histomorphological alterations

such as epithelial lifting, lamellar fusion, aneurysms,

necrosis, clavate lamellae, mucocyte proliferation,

mucus secretion or epithelial hyperplasia (Lopez

et al. 1981; Solangi and Overstreet 1982; Metcalfe

1998). There is, however, a key aspect that has been

seldom tackled by these investigations, which is the

functional implication of these structural impairments

(Simonato et al. 2006; Kennedy and Farrell 2005;

Norton et al. 1985). Moreover, when available, this

information is difficult to extrapolate to field condi-

tion because the contamination level tested can be

much higher than the field concentrations measured

following accidental events (Boehm et al. 2007;

Tronczynski et al. 2004; Readman et al. 2007).

In that context, the present study is an attempt to

investigate the link between the morphological and

functional impacts of hydrocarbon exposure using

realistic levels of a heavy fuel oil dissolved fraction.

This work was conducted on the turbot (Scophthal-

mus maximus), a typical large predator of demersal

coastal ecosystems. Following a 5-day exposure to

the dissolved fraction of heavy fuel oil (HFO), gills’

structural and functional integrity was examined via

histological analysis and hydromineral balance

assessment.

Materials and methods

Chemicals

Dimethylsulphoxide (DMSO), Dithiotreitol, EDTA,

HEPES, lithium heparin and Na phosphate buffer

were purchased from Sigma (Saint Quentin Fallavier,

France).

Experimental heavy fuel oil (HFO) originated

from the North Sea and contained approximately 22%

saturated, 55% aromatic and 22% polar compounds.

Concentrations of the 16 priority PAHs in this fuel

are given in Table 1.

Fishes and rearing conditions

Juvenile turbot, Scophthalmus maximus (n = 160;

age: 15 month; mass: 254 ± 54 g; length: 24 ± 2 cm;

mean ± SD), were purchased from a local fish farm

(France Turbot, 22220 Tredarzec, France). Fishes were

acclimated for 2 weeks to the laboratory conditions in

two, 1,200-l tanks supplied with aerated clean sea water

pumped from the bay of Brest (300 l h-1). Lighting

regime was set according to the season (February to

May). Water salinity (37.0 ± 0.5%), pH (7.92 ± 0.15),

oxygen concentration (4.08 ± 0.24 mg l-1) and tem-

perature (12 ± 2�C) were measured daily. Fish were fed

daily with dried pellets (Le Gouessant�, 4.5 mm

diameter, total protein 54% of dry weight and crude

fat 12% of dry weight).

Sampling schedule and sample preparation

To evaluate the effects of the dissolved fraction

of HFO, fishes were randomly allocated to two

Table 1 Analysis of 16 priority PAHs of the United States

Environmental Protection Agency (US EPA) list in fuel oil

Naphthalene 686 ± 89

Acenaphthylene 51 ± 3

Acenaphthene 272 ± 14

Fluorene 396 ± 20

Phenanthrene 1936 ± 116

Anthracene 213 ± 28

Fluoranthene 125 ± 14

Pyrene 516 ± 57

Benz[a]anthacene 213 ± 13

Chrysene 464 ± 14

Benzo[b ? k]fluoranthene 81 ± 11

Benzo[a]pyrene 167 ± 7

Dibenz[a, h]anthracene 27 ± 5

Benzo[ghi]perylene 48 ± 3

Indenol[1,2,3-cd] pyrene 16 ± 3

PAH detection was performed by gas chromatography/mass

spectroscopy (GC/MS)

Results in lg g-1 of Hydrocarbons (n = 3, means ± SD)

Fish Physiol Biochem

123

experimental groups, a control group (C) or an

exposed group (E). During the 5-day contamination

period, fish were maintained in two identical, close-

circulation tanks (1,200 l). The first tank corre-

sponded to the control condition. The second tank

corresponded to the exposed condition. In this case,

fish were exposed to recirculated water containing

the dissolved fraction of HFO. Water contamination

started 10 days before the introduction of fish into

the tank by flowing sea water through a mixing

device (Goanvec et al. 2008) consisting in a separate

tank allowing contact between sea water and fuel

oil.

Following the exposure period, fish were moved

back into their rearing tanks for a 4-week recovery

period. Experimental groups were sampled at day 1

and 4 during the exposure period and at day 1, 3, 8,

14 and 30 during the recovery period. Each sampling

consisted of 10 fish.

Each was treated as follows. A blood sample was

withdrawn (500 ll) from the caudal vein using a

syringe rinsed with Li-heparin solution (250 U/ml).

The blood sample was immediately centrifuged

(10 min at 600 g) and the plasma frozen in liquid

nitrogen before being stored at -80�C until analysis.

The fish was then killed with a sharp blow on the

head, and the liver was excised, weighted, washed in

KCl solution (0.15 M), frozen in liquid nitrogen and

stored at -80�C for later analysis. The gill (second

arch) was excised and placed in fixative (Bouin) for

24 h before processing.

Sample analyses

Measurement of PAH concentration in the sea water

Five seawater samples (after 1, 3, 4, and 5 days of

fuel oil exposure) were taken to assess PAH concen-

tration in the contamination tank during the exposure

period. These samples (1 l) were collected into

heated Duran glass bottles (500�C). Samples were

extracted using Pestipur grade dichloromethane

(3 9 100 ml per seawater sample). The combined

organic extracts were dried by filtering through

anhydrous sodium sulphate (Na2SO4 Pestipur grade)

and concentrated to 2 ml by means of a Turbo Vap

500 concentrator (Zyman, Hopkinton, MA, USA).

Aromatic compounds were analysed using gas

chromatography coupled with mass spectrometry

(GC–MS, Hewlett Packard HP5890 series II) coupled

with an HP5973 mass selective detector following

procedures previously described (Goanvec et al.

2008). The detection limit of this GC/MS method

was 1 ng l-1.

CyP 1A Western blotting

The protocol of microsome preparation was adapted

from Monod et al. (1994) and Beyer et al. (1997) and

is described in Goanvec et al. (2004). SDS–PAGE

gels (10% polyacrylamide) were run according to the

method of Laemmli (1970). Microsomal suspensions

(30-lg protein loadings, Peters et al. 1997) were

transferred to nitrocellulose membranes for subse-

quent analysis. Immunodetection of Cytochrome

P450 1A was performed using a mouse anti-trout

monoclonal antibody (Cyp 02 Biosense Laborato-

ries� Norway) at a 1/500 dilution and an anti-mouse

goat peroxidase-linked secondary antibody (BA-

9200 Vector Laboratories�) at a 1/2,000 dilution.

The immunoreactive proteins, on nitrocellulose mem-

brane (CriterionTM blotting sandwiches, Bio-Rad

Laboratories), were detected using an Avidin-Biotin-

ylated peroxidase enzyme complex (Vectastain� elite

ABC kit, PK 6100, Vector laboratories) and its

substrate diaminobenzidine (DAB substrate kit for

peroxidase, SK-4100, Vector laboratories�). Mem-

brane was digitalised with a camera (Gel doc 2000,

Bio-rad) and semi-quantified by image analysis using

Quantity One� Software (Quantitation Software, Bio-

rad). Comparative semi-quantitative evaluation was

performed on traces appearing on the same blot, and

results were expressed in arbitrary units from a

reference sample present on all membranes.

Plasma ion concentration and osmolality

All blood parameters were measured (in duplicate) on

plasma samples stored frozen until analysed: osmo-

lality was measured on a VAPRO� vapour pressure

osmometer (model 5520 WESCOR); chloride con-

centration was measured by argentimetric titration

with a chloridometer (CORNING Chloride analyzer

925); plasma concentrations of sodium ([Na?]) and

potassium ([K?]) were obtained with a flame pho-

tometer (IL 243-05 Instrumentation Laboratory).

Fish Physiol Biochem

123

Branchial histology

After 24 h in fixative (Bouin), samples were dehy-

drated in graded concentrations of alcohol and

Neo-clear� (Martoja and Martoja-Pierson 1967).

Paraffin-embedded tissues were sectioned at 5 lm

and stained with H&E according to routine proce-

dures (Martoja and Martoja-Pierson 1967; Stehr et al.

2004). This stained the cytoplasm of cells pink and

the nucleus in brownish-violet.

After 4 day of exposure and 1, 8 and 30 days of

recovery, fish were examined and scored for the

histopathological abnormalities listed by Mallatt

(1985) i.e., epithelial oedema, necrosis, lamellar

fusion, epithelial cell hypertrophy, epithelial hyper-

plasia and rupture.

Particular attention was paid to the structure of gill

filament. Ionocytes are preferentially located on the

afferent edge in primary epithelium between the

secondary lamellae, mucocytes are more present on

the efferent edge. The number of chloride cells and

mucocytes were determined in the region of the

afferent edge of the filament, when the cartilage could

be seen on the slide, over 10 interlamellar regions

(approximately 650 lm), on 5 different gill filaments

from 5 fish in each group. The results are expressed

as number of cells over 100 lm. The thickness of the

filament epithelium was determined from 20 mea-

surements from 5 animals in each group.

Statistical analyses

Statistical tests were performed using Statistica

(Version 8.0, Statsoft). Normality was checked using

Lilliefors test; homoscedasticity was evaluated using

a Bartlett test. When results are not gaussian, they

were expressed as median ± EQ. In this case,

statistical significance was checked with Mann–

Whitney tests performed at the different sampling

times. When the results are Gaussian, they are

expressed as mean ± sem, their statistical signifi-

cance is analysed with a two-way ANOVA and post

hoc tests.

Results

Over the whole experiment, no mortality was

observed.

PAH concentration in the seawater

During the contamination period, no change in the

water concentration of the main PAH was observed

over time. The mean sum of 16 US EPA list PAH was

321.0 ± 9.1 ng l-1 (Fig. 1). The mean concentrations

of the main PAHs were the following: anthracene:

127.5 ± 6.5 ng l-1, benzo[a]pyrene: 30.2 ± 0.7 ng l-1,

fluoranthene: 42.3 ± 4.1 ng l-1, naphthalene: 57.2 ±

9.8 ng l-1, phenanthrene: 36.0 ± 1.9 ng l-1, pyrene:

10.4 ± 1.2 ng l-1. During the recovery period, no

PAH was detected in the water.

CyP-1A

The quantity of CyP-1A (see Fig. 2 and Table 2) rose

quickly following exposure to the contaminant (*1.64

after 4 days into exposure) and remained stable for

the rest of the contamination period. Following

transfer to clean water, CyP-1A level remained high

during the first days (from the first to the third day of

recovery) and then gradually decreased to reach the

control value after 2 weeks.

Plasma ion concentration and osmolality

In the control group, no change in plasma sodium or

chloride concentrations or in plasma osmolality

(Fig. 3) was observed. In the exposed group, on the

other hand, these three parameters were significantly

increased after one and 4 days on exposure. During

the recovery period, these parameters gradually

Days during contamination period

PAH

con

cent

ratio

n (n

g.L-1

)

10

100

1000

Naphtalene Phenanthrene Anthracene Fluoranthene Pyrene Benzo[a]pyrene Sum of the 16 PAH

12C 3C 4C 5C 0C 1C

Fig. 1 GC/MS analysis of PAH in the closed water circulation

system during the experiment. Results in ng l-1 of seawater

(n = 1). Concentrations of PAHs are constant during the whole

period of contamination

Fish Physiol Biochem

123

returned to control level, which was reached after

30 days of recovery.

Plasma potassium concentration was not affected

by exposure to soluble hydrocarbons, and no signif-

icant difference between the control and exposed

groups was observed.

Histology

The histopathological study of the gills did not show

increase in lesion occurrence following fuel oil

exposure. Neither lamellar fusions nor lamellar epi-

thelial ruptures were observed. In both groups (control

and exposed), 30% of fish displayed gill filament

hyperplasia and only one case of aneurism was

observed after 30 days of recovery in the exposed

group.

Epithelial thickness was not affected by exposure

to heavy fuel oil soluble fraction (Fig. 4a). But

exposure to HFO induced a significant reduction of

mucocyte number of 30% (Fig. 4b, two way ANOVA;

P = 0.038). After 8 days of recovery, however, this

parameter was back to the reference level.

Chloride cells’ counts (Figs. 4b and 5) showed a

dramatic fall after transfer to exposed water and only

partial recovery after 30 days in clean water.

Discussion

In fish, the gill is a fundamental site for homeostasis;

its structural and functional integrity will largely

determine individual’s ability to face environmental

contingencies. Following an oil spill at sea, acute

toxical effects are largely due to the penetration of the

pollutant through the gills since uptake rates of most

lipid-soluble toxicants across this organ are very rapid

(Randall et al. 1998). Since most published works on

the effect of fuel oil exposure upon gill function are

histology based, the goal of the present study was to

bring information on the link between morphological

and functional impact of the dissolved fraction of fuel

oil on fish gills, using realistic levels of heavy fuel oil

dissolved fraction. Fish were exposed during 5 days to

realistic contamination conditions (300 ng l-1 of

summed 16 PAH of US EPA). These conditions

78 kDa

57.5 kDa

1 2 3 4 5 6 MW 8

Fig. 2 Densitometry of CYP1A1 expression by Western blot.

Microsomal proteins (30 lg per lane) from fish exposed or not

to fuel oil were separated by SDS–PAGE and transferred to

nitrocellulose filters. The blots were probed with cyp 02 (C10-

7, monoclonal antibodies, Biosense laboratories�, Norway)

antibodies raised against CYP 1A1 from different fish species.

The panel contains the molecular weight markers (MW) in kDa

on lane 7. Lane 1 and 2 are control fish. The exposed fish are

on lanes 3, 4, 5, 8. They were compared to the same sample of

a reference sample (lane 6) identical for and present in all

membranes

Table 2 Evolution of CYP 1A protein evaluated by

Western blot

Experimental Control group Exposed group

Condition Day

Exposure 1 0.86 ± 0.23 0.99 ± 0.07

4 0.75 ± 0.26 1.23** ± 0.14

Recovery 1 0.82 ± 0.13 1.30** ± 0.18

3 0.86 ± 0.02 1.30** ± 0.06

8 0.97 ± 0.11 1.16** ± 0.07

14 1.06 ± 0.20 1.10 ± 0.21

30 0.82 ± 0.10 0.91 ± 0.03

Results (median ± iq, n = 6–7) are expressed in arbitrary

units

** Statistical difference with the corresponding control group

(P \ 0.01)

0 10 20 30

plas

mat

ic io

ns in

mM

ol /

osm

olal

ity in

mO

sm

100

150

200

250

300

350

400

[Cl -] control [Cl -] exposed [Na +] control [Na +] exposed Osmolality control Osmolality exposed

Exposure

Sampling days

Recovery

∗∗∗ ∗∗∗∗∗ ∗∗ ∗∗∗ ∗∗

∗∗∗

∗∗∗∗∗∗

∗∗∗ ∗∗∗

∗

∗∗∗

∗∗∗

∗∗∗

Fig. 3 Plasmatic concentrations in Cl- and Na? and osmo-

lality. Ion results in mMol, osmolality results in mOsm

(median ± iq, n = 9–10). Statistical differences with the

corresponding control group: * P \ 0.05, ** P \ 0.01,

*** P \ 0.001

Fish Physiol Biochem

123

induced a rapid and significant increase in plasmatic

osmolality, [Na?] and [Cl-], concomitant with a 95%

decrease in the number of chloride cells in the inter-

lamellar epithelium.

Following an oil spill, reported water PAH con-

centrations typically range between 20 and

600 ng l-1 (Geffard et al. 2004; Tronczynski et al.

2004; Boehm et al. 2007; Readman et al. 2007). In

the present work, water concentration of the summed

16 PAHs of the US EPA list was in the middle of that

range (300 ng l-1), representing a relevant contam-

ination level.

The significance of the contamination conditions

was assessed using cytochrome P450-1A. A 64%

increase in CYP 1A was observed after 4 days in

exposed water and it took 2 weeks in unexposed

conditions for it to return to reference level. In

comparison with other reports (Aas et al. 2000;

Gagnon and Holdway 2000), this can be considered

as a moderate CYP1A induction. However, this must

A

4 1 8 30

epith

eliu

m th

ickn

ess

(µm

)

0

5

10

15

20

25

30

Control exposed

**

Sampling day

B

4 1 8 30

cell

num

ber

0

1

2

3

4

5

Sampling day

Cell type

∗ ∗

∗∗

∗∗ ∗∗ ∗∗

Exposure Recovery

M I M I M I M I

Exposure Recovery

control exposed

M mucocytesI ionocytes

Fig. 4 Filament epithelium: thickness, number of mucocytes and chloride cells. Results are expressed as mean ± sem (n = 5).

Statistical differences with the corresponding control group: * P \ 0.05, ** P \ 0.01

Fish Physiol Biochem

123

be put in parallel with the relatively low, yet realistic,

level of contamination tested here.

A large number of chemical and physical irritants

can induce intraepithelial oedema, epithelial cell

swelling and epithelial hyperplasia in fish gills

(Mallatt 1985). Exposure to fuel oil is also known

to induce morphological alteration. In the common

sole Solea solea exposed 5 days to a heavy fuel

oil poured at the surface of experimental tanks,

Claireaux et al. (2004) have shown epithelium lifting,

aneurysms and even destruction of the respiratory

epithelium. In the hogchoker Trinectes maculatus, a

38 days exposure to the water-soluble fraction of a

crude mixed oil induces hyperplasia, epithelium

lifting, lamellar fusion (Solangi and Overstreet

1982). Furthermore, field surveys of plaice popula-

tion Pleuronectes platessa after the Amoco cadiz

crude oil spill have demonstrated lamellar fusion,

capillaries telangiectasis and mucous cell hyperplasia

(Haensly et al. 1982). Mucocyte proliferations

have been also shown in pearl dace exposed to diesel

fuel (Khan 1999) or in plaice after oil spills at sea

(Haensly et al 1982). In our own analysis, however,

exposure to dissolved petroleum hydrocarbons does

not lead to important gill histopathological lesions.

For instance, we obtained a 30% decrease in muco-

cyte number, and gill filament hyperplasia was the

only frequent gill alteration that we observed. How-

ever, this last result was independent of heavy fuel oil

exposure since it was also observed in the control

group. The absence of important histopathological

consequences of fuel oil exposure could be explained

either by the short duration of the contamination, by

the absence of suspended particles (since fish were

exposed only to the dissolved fraction of the fuel), or

by the fact that the concentration was low enough that

fish were able to cope with or adapt to the PAHs.

Histomorphological aspects being set aside, func-

tional impairments of the gills have also been

demonstrated in relation to high concentration of

PAH or fuel oil. Norton et al (1985) combining

ionocyte counts and hydromineral balance analysis

gave evidence for the sharp influence of short-term

exposure to PAH (mixture of benzene, toluene and

xylene) on the fat-head minnow osmoregulatory

performance. More recently, Kennedy and Farrell

(2005) have shown that a 96-h exposure to the water-

soluble fraction of crude oil induced alteration of ion

homeostasis with significant increases in plasmatic

chloride, sodium and potassium concentrations.

The histological techniques that we used clearly

prevent us from the precise examination of intracel-

lular structure; hence, we may have underestimated

the chloride cell number reported in Fig. 4. Never-

theless, our observations of cytoplasmic alterations of

chloride cells during the contamination period

strongly support the hypothesis of a reduction of

ionocyte count. This is further supported by the fact

that changes in the hydromineral balance of the

extracellular space were observed shortly after fish

were exposed to fuel. It is most likely that these

changes resulted from structural alteration of the gill

epithelium. During the recovery period, however,

while plasma sodium, chloride and osmolarity slowly

returned to control values, chloride cells’ counts only

partially recovered. It is hypothesised that this partial

restoration allows the fish to extrude sodium and

chloride ions efficiently enough to maintain its

hydromineral balance.

This study confirmed previous works on the effect

of heavy fuel oil exposure upon ion homeostasis in

Fig. 5 Structure of the filament epithelium at day 8. Results

from the control (a) and exposed (b) groups. M mucocytes,

C chloride cells, L lamella, I interlamellar space

Fish Physiol Biochem

123

fish (Kennedy and Farrell 2005; Norton et al. 1985)

but also shows that even short-term exposure to low

concentration of the dissolved fraction of heavy fuel

oil can affect the hydromineral equilibrium. The

observed fall of ionocyte numbers allows us to

propose that this osmotic imbalance is the conse-

quence of the structural alteration of the filament

epithelia. However, further analyses are needed to

precise the cellular effects of the contamination. A

fundamental point will be the determination of the

cellular target of the contaminant in order to explain

its specific action upon chloride cell. Since Norton

et al. (1985) have shown structural alteration of

chloride cell mitochondria, the effects of fuel oil on

the activity of these organelles will be of particular

interest. Finally, since gills play a major role in other

fundamental physiological processes, it would be

interesting to assess the effects of heavy fuel oil on

other homeostatic parameters.

Acknowledgments We thank Guy Claireaux, Myriam Donz-

elot and Adrian Moffat for their valuable comments on the

manuscript and the French ‘‘Ministere de l’Enseignement

Superieur et de la Recherche’’ for financial support.

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