Effect of dispersed crude oil on cardiac function in seabass Dicentrarchus labrax

Chemosphere 134 (2015) 192–198

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Effect of dispersed crude oil on cardiac function in seabass Dicentrarchuslabrax

http://dx.doi.org/10.1016/j.chemosphere.2015.04.0260045-6535/� 2015 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: EA 4324 ORPHY (Optimisation des RégulationsPHYsiologiques), UFR Sciences et Techniques, Université Européenne de Bretagne,Université de Brest, 6 avenue LE GORGEU, CS 93837, 29238 BREST cedex 3, France.

E-mail address: [email protected] (M. Dussauze).

Florine Tissier a, Matthieu Dussauze a,b,⇑, Nina Lefloch b, Michael Theron a, Philippe Lemaire c,Stéphane Le Floch b, Karine Pichavant-Rafini a

a EA 4324 ORPHY (Optimisation des Régulations PHYsiologiques), UFR Sciences et Techniques, Université Européenne de Bretagne, Université de Brest,6 avenue LE GORGEU, CS 93837, 29238 BREST cedex 3, Franceb Cedre: Centre de Documentation, de Recherche et d’Expérimentations sur les pollutions accidentelles des eaux, 15 rue Alain Colas, CS 41836,29218 BREST Cedex 2, Francec TOTAL Fluides S.A., 92907 Paris La défense, France

h i g h l i g h t s

� Dicentrarchus labrax were used in this ecotoxicological study.� Fish were exposed to dispersant, mechanical and chemical oil dispersion.� Cardiac contraction parameters were impacted in the presence of oil.� Cardiac energy metabolism was impacted by dispersant alone.

a r t i c l e i n f o

Article history:Received 27 October 2014Received in revised form 7 April 2015Accepted 9 April 2015

Handling Editor: Caroline Gaus

Keywords:Dispersed oilSeabassCardiac functionTissue respiration

a b s t r a c t

In this study, the impact of dispersed oil was assessed in Dicentrarchus labrax, a fish frequently used as anoil contamination indicator species. Fish were exposed for 48 h to (mechanically and chemically) dis-persed oil and dispersant alone. The impact of these exposure conditions was assessed on cardiac func-tion by measuring (i) the contraction strength, the contraction and the relaxation speeds (ii) the cardiacenergy metabolism using respirometry on permeabilized cardiac fibers. Compared to control, the increaseof polycyclic aromatic metabolites observed in the bile indicated oil contamination in our fish. Following48 h of oil exposure at realistic oil concentrations, alterations of cardiac performances were observed. Adecrease in contraction strength, contraction and relaxation speeds was observed in the presence of oilwithout effect of dispersant on these three parameters. Looking at cardiac energy metabolism, dispersantalone decreases all the activity of the respiratory chain and increases the proton leak. From these results,it appears that the observed decrease in cardiac performance in fish exposed to oil was not linked to adecrease in energy availability.

� 2015 Elsevier Ltd. All rights reserved.

1. Introduction by microorganisms (Cerniglia, 1992). To limit oil spill damages,

Global industrial development has resulted in a constantdemand for oil, the most common world source of energy. As oilextraction areas are not distributed uniformly, large amounts mustbe transported across the seas leading to oil spills (Percebois,2001). After the evaporation of volatile components, the oil is par-tially dissolved in sea water and broken up into microdropletsthroughout the water column by physical mixing and degradation

dispersants have been used, mainly in offshore areas, since the1970s. However by increasing oil compounds’ bioavailability, theymay enhance pollution damages (Chapman et al., 2007). Thus, theiruse near the coast is still debated, due in part to the lack of knowl-edge of their effects on organisms, and the question of the effects ofboth fuel oil and dispersed oil on coastal organisms should beraised.

Petroleum compounds have been shown to affect numerousphysiological functions such as respiration (Duarte et al., 2010),immunity (Fabiani et al., 1999; Reynaud et al., 2002), cell differen-tiation (Perez et al., 2003), development (Incardona et al., 2005),growth, reproduction and gene expression (Zhang et al., 2013).Alterations of fish metabolism are also reported (Davoodi and

F. Tissier et al. / Chemosphere 134 (2015) 192–198 193

Claireaux, 2007). Amongst the functions studied, cardiac functionseemed of great interest: (i) it could be altered by petroleum com-pounds (Hicken et al., 2011; Milinkovitch et al., 2012, 2013) and (ii)changes or alterations to the heart may have consequences onother physiological functions (Heideman et al., 2005). To allowheart contraction, energy production by cardiac mitochondriashould be maintained. Petroleum compounds have been shownto alter mitochondrial oxidative phosphorylation and membranepotential, and to induce an increase in superoxide production lead-ing to a potential decrease in ATP production (Salazar et al., 2004;Xia et al., 2004; Stabenau et al., 2008; Westman et al., 2013).

Thus, this work aims at assessing the effects of a dispersed fueloil on the cardiac function of seabass, Dicentrarchus labrax. Thisfish, often used as a biological model, is highly represented intemperate coastal areas. Moreover, this species is exposed toxenobiotics via the ingestion of bioaccumulated molecules throughthe trophic network (Wolfe et al., 1998).

In this study, fish were exposed for 48 h to mechanically dis-persed oil, to one commercial formulation of dispersant and tothe corresponding chemically dispersed oil. Cardiac function wasestimated by measuring contraction strength, contraction andrelaxation speeds. Cardiac energy metabolism was evaluated byrespirometry on permeabilized cardiac fibers. The exposure condi-tions were assessed by measuring the total petroleum hydrocarbonconcentration and the oil droplet size in the water.

2. Material and methods

2.1. Animals

Experiments were conducted on seabass, Dicentrarchus labrax(n = 32; weight: 492 ± 41 g; length: 33 ± 1 cm; mean ± SD) pur-chased from Gravelines hatchery (Gravelines, France). Two weeksbefore the experiments, the fish were acclimatized to their 2500 Lseawater tanks. The photoperiod was according to the season(February to March: 10 h light/14 h dark). The pH (8.0 ± 0.2), oxy-gen saturation (greater than 90%) and temperature (13.9 ± 0.4 �C)were measured daily. Fish were fed daily ad libitum (until they donot catch food anymore) with dried pellets (Neo Grower ExtraMarin Col. 5� from Le Gouessant aquaculture). Around 400 g of foodper kg of fish were distributed per day. The diet composition was43% protein, 20% lipids, 3% cellulose, 5.6% ash, 10% moisture, 0.9%and 18.4% nitrogen free extract.

2.2. Chemicals

The petroleum used in this study was a Crude Arabian Light(CAL) composed of 54% saturated hydrocarbons, 10% polar com-pounds and 36% aromatic hydrocarbons. CAL was evaporated (withair bubbling) until a weight loss of 7%. This process caused thelighter compounds to evaporate mimicking the weathering of anoil slick at sea (Milinkovitch et al., 2011). This weathered CALwas used in other ecotoxicological studies (Milinkovitch et al.,2012; Claireaux et al., 2013; Theron et al., 2014).

Finasol OSR 52, a commercial formulation from TOTAL Fluides(Puteaux, France), was used as dispersant in this study. It is a thirdgeneration oil-based dispersant combining surfactants (amphiphi-lic molecules) and solvents. Components of Finasol OSR52 were:docusate sodium (20–25%); Hydrocarbons, C1–C14, n-alkanes,ioalkanes and cylics (<2%); aromatics (15–20%); 2-methoxymethy-lethoxy propanol (15–20%); Carboxylic acids, diC6-12 compounds,with ethanolamine and boric acid compound with ethanolamine(0–2%); Ethanolamine (0–1%). According to the safety data sheet,physicochemical parameters of Finasol OSR 52 were: viscosity30.1–36.7 m2 s�1 at 40 �C; pH 9–10.5; density 990–1015 kg m�3

at 20 �C; boiling point higher than 150 �C; flash point higher orequal than 65 �C.

2.3. Experimental design

The fish were allocated to four experimental conditions: a con-trol group (C), a group exposed to mechanically dispersed CAL(MD), a group exposed to chemically dispersed CAL (Finasol OSR52: CD) and a group exposed to the dispersant alone (D). In thecase of the MD and CD conditions, 25 g of oil was poured into300 L seawater tanks (concentration around 80 mg L�1), in the CDcondition 1.25 g of dispersant was also added (dispersant oil ratio:1/20) in accordance with the manufacturers terms of use. For the Dcondition, 1.25 g of dispersant was added to the 300 L seawatertank.

Feeding was stopped 24 h before the experiment; the fish werethen randomly assigned to their experimental condition (8 fish pergroup) and placed in the 300 L exposure tanks for 48 h withoutwater renewal. The total petroleum hydrocarbon (TPH) concentra-tion was measured at the beginning, after 24 h of exposure and atthe end of the 48 h fish exposure period for the four experimentalconditions. The obtained results allowed us to adjust the TPH con-centration to around 80 mg L�1 by adding the appropriate CALquantity when necessary. Each of these tanks was equipped witha pumping system allowing continuous water homogenization(see Milinkovitch et al., 2011 for details).

At the end of the exposure, the fish were sized, weighed andkilled with a cerebral dislocation. The heart was sampled andplaced in ice cold medium isoosmotic solution (in mM: NaCl 152,KCl 3.4, MgSO4 0.8, Na2HPO4 0.44, KH2PO4 0.44, NaHCO3 5, Hepes10, Glucose 10, CaCl2 2.5, pH 7.8, 320 mosmol�1). Gall-bladderswere also sampled and stored at �80 �C.

2.4. Measurements of Total Petroleum Hydrocarbon (TPH) seawaterconcentrations

The total petroleum hydrocarbon concentration was measuredin triplicate. One hundred mL samples of water were extractedthree times with 10 mL of dichloromethane (Carlo Erba Reactifs,SDS). The combined organic phases were dried on anhydrous sul-phate and the absorbance was measured at 390 nm (UVeVis spec-trophometer, Unicam, France) as described by Fusey and Oudot(1976). The results are expressed in mg L�1, and the linearity ofthe response was checked between 5 and 100 mg L�1.

2.5. Measurements of droplet size

Droplet size (diameter in microns) in the CD and MD conditionswere measured 6 h after the beginning of fish exposure. The mea-surements were performed by laser granulometry (MalvernMastersizer 2000, Malvern Instruments Ltd, Worcestershire,United Kingdom) based on the principle of Fraunhofer accordingto the intensity of diffracted radiation, where the diffraction angledepends on the particle size. It is completed in our case with theMie theory taking into account the refractive indices of the sampleand the carrier medium (ISO 13320-1, 1999). A flow rate of1200 mL min�1 and an obscuration of 10 ± 0.01% were the condi-tions used during the measurements.

2.6. Fixed wavelength fluorescence analysis of bile

Bile contained in gall-bladder was used to perform semi-quan-titative analysis of PAH biliary metabolites (Vuorinen et al., 2006).The bile was diluted in absolute ethanol (1/2000). Fluorescencemeasurements were performed with a Jasco FP-6200 (Tokyo,Japan). The measurements were made at wavelengths of excitation

Table 1Droplet sizes (lm) in CD and MD (n = 4). CD (Chemical Dispersion) and MD(Mechanical Dispersion). Values not sharing common letters (a or b) in the lineindicate a significant difference (P < 0.05). In this table, d(0.5), d(0.1) and d(0.9)correspond respectively to the median and the two deciles of a Normal distribution.

CD MD

d(0.1) 3.0 ± 0.0a 20.0 ± 0.0b

d(0.5) 10.7 ± 0.2a 62.4 ± 0.3b

d(0.9) 88.3 ± 2.2a 147.8 ± 2.2b

194 F. Tissier et al. / Chemosphere 134 (2015) 192–198

and emission, 380:430 characteristics of benzo[a]pyrene typemetabolites and 343:383 characteristics of 1-hydroxy-pyrene typemetabolites (Lin et al., 1996; Aas et al., 2000). Values are expressedas Arbitrary Units (AU).

2.7. Cardiac contraction measurements

The method used was adapted from Shiels et al. (1997).Ventricular strips were introduced in a system to measure theforce developed during contraction. Ventricular strips are stan-dardized with dimensions of 0.2 ⁄ 0.7 cm. Fresh weight was mea-sured in order to calculate the average cross section of thesample according to the method of Layland et al. (1995).

Strips were placed in a saline solution at 17 �C with air bubblingbetween two clamps connected to a strain gauge (FORT10, WorldPrecision Instruments, Sarasota, USA) connected to an amplifier(TBM4M, World Precision Instruments, Sarasota, USA) and theBiopac interface MP35. Two stimulation electrodes were placedon either side of the strip and connected to a stimulator (6012 stim-ulator, Harvard). The Acquisition software used was BSL pro 3.7.

20 min were necessary for the establishment of the strip understimulation at a frequency of 0.1 Hz, a voltage of 4 V and a stimu-lation time of 10 ms. The amplitude of contraction was initially sta-bilized at 1 g. The force–frequency relationship was recorded witha progressive stimulation of 0.1 Hz to 1 Hz in increments of 0.1 Hz.Each frequency is applied for 4 min. Two strips were used for eachfish. The amplitude of contraction, its duration and the timerequired for half relaxation were measured on the last 5 contrac-tions of each tested frequency.

2.8. Tissue respiration

Measurement of the O2 consumption of permeabilized cardiacfiber was performed using four thermostated respiration chambers(volume 1.4 mL) and an oxymeter Oxy-4 (Presens, Regensburg,Germany). Permeabilization of cardiac fibers was performed fol-lowing a method adapted from Veksler et al. (1987) andKuznetsov et al. (2008) at 4 �C using saponin and collagenase.Briefly, cardiac fibers were cut in solution A (in mM: EDTA 5.5;MgCL2 2.5; Imidazol 10; HEPES 20; KCl 70; ATP 3.3; PCr 2;Dithiothreitol 0.5; pH 7.4 at 4 �C) and were placed in incubationfor 30 min in solution B (Solution A plus 0.05 mg mL�1 of Saponinand 2 mg mL�1 of Collagenase). The fibers were then washed for10 min in solution A and 10 min in respiration medium (in mM:EDTA 0.08; MgCl2 7.5; KCl 150; Tris 20; NaH2PO4 10; pH 7.4).

After permeabilization, the fibers were placed in respirationchambers filled with respiration medium. All measurements wereperformed in excess ADP. Successive addition of substrates(Pyruvate/Malate, Succinate and Ascorbate/TMPD), cytochrome Cand oligomycin were performed in the respiration chamber.Cytochrome C was used to check the integrity of mitochondrialexternal membranes (Kuznetsov et al., 2008). Pyruvate/Malate,Succinate and Ascorbate/TMPD allowed the analysis of differentinputs in the mitochondrial respiratory chain. Oligomycine blocksthe fifth complex of the respiratory chain and was used to measurethe proton leak through the inner mitochondrial membrane. Protonleak was calculated as the O2 consumption with Oligomycinedivided by the O2 consumption with Ascorbate/TMPD. At the endof the experimentation, the fibers were dried and weighed to deter-mine the O2 consumption in lmol O2 min�1 mg�1 of dry tissues.

2.9. Statistical analysis

All analyses were performed using STATGRAPHICS software.Firstly all the data were tested with a Levene test to assess the vari-ance homogeneity and a Kolmogorov–Smirnov and Shapiro–Wilk

test were used to ensure that data followed a normal distribution.The samples with a normal distribution were tested with a one-way ANOVA followed by a Fisher test. Samples that did not followa normal distribution were tested with a Kruskal–Wallis analysis.All data are expressed as the mean ± standard error of the mean.

3. Results

3.1. Measurements of Total Petroleum Hydrocarbon (TPH) seawaterconcentrations

No hydrocarbon was detected in the C and D conditions. In theCD and MD conditions, the mean of the TPH concentrations mea-sured during the 48 h of exposure were 75 ± 15 and 82 ± 6 mg L�1

for the CD and MD conditions respectively corresponding to a nom-inal concentration of 83 mg L�1 for both conditions. No statisticaldifference was observed between CD and MD TPH concentrations.

3.2. Measurements of droplet size

Droplet sizes (lm) in CD and MD obtained with laser granulom-etry are reported in Table 1. For the median, d(0.5), and the twodeciles, d(0.1) and d(0.9), of droplet distribution, droplets were sig-nificantly smaller in the CD condition compared to the MDcondition.

3.3. Fixed wavelength fluorescence analysis of bile

The fluorescence intensities of bile samples at characteristicwavelengths of OH-BaP (380:430 nm) and OH-pyrene(343:383 nm) metabolites are presented in Fig. 1. After the 48 hof exposure, for the two couples of wavelengths, no significant dif-ference was observed between C and D groups. A significantincrease in bile fluorescence was observed in CD and MD comparedto the C and D groups with significantly higher values in the MDcondition compared to the CD condition.

3.4. Cardiac contraction parameters

As shown in Fig. 2, ventricular strips of control fish exhibited asignificant decrease in contraction strength (2.15 times), contrac-tion (1.2 times) and relaxation (1.8 times) speeds when stimula-tion frequency increase from 0.1 to 1 Hz. Exposure to dispersantalone has no effect on these three parameters compared to the con-trol condition. Except at 0.1 Hz, at each frequency used (from 0.2 to1 Hz), contraction strength, contraction and relaxation speeds ofcardiac tissue were lower in the CD and MD conditions comparedto control. This result is true at each frequency except for 0.1 Hz.

3.5. Tissue respiration

3.5.1. Mitochondrial integrityFor each condition, no significant difference was observed

between O2 consumption with Ascorbate/TMPD and after additionof Cytochrome C (Table 2).

C D CD MDFluo

resc

ence

inte

nsity

at 3

43/3

83 (A

rbitr

ary

Uni

t)

0

20

40

60

80

100

120

140

160

180

200 (a)

C D CD MDFluo

resc

ence

inte

nsity

at 3

80:4

30 n

m (A

rbitr

ary

Uni

t)

0

5

10

15

20

25

30

a a

b

c(b)

a a

b

c

Fig. 1. Fluorescence intensities of bile samples at characteristic wavelengths of OH-pyrene (343:383 nm) and OH BaP (380:430 nm) metabolites (a) and (b) respec-tively. Results expressed as arbitrary fluorescence units (mean ± standard error ofthe mean; n = 8). C (Control), D (Dispersant alone), CD (Chemical Dispersion of oil),MD (Mechanical Dispersion of oil). Values not sharing common letters indicates asignificant difference (P < 0,05).

Frequency (Hz)0,0 0,2 0,4 0,6 0,8 1,0

Con

tract

ion

stre

ngth

(mN

.mm

2 )

0

1

2

3

4

*

**

**

**

**

#

##

##

##

##

(a)

Frequency (Hz)

0,0 0,2 0,4 0,6 0,8 1,0

Con

tract

ion

spee

d (m

N.m

m-2

.s-1

)

0

2

4

6

8

* * **

* ** *

*

# # ##

##

##

#

(b)

Frequency (Hz)0,0 0,2 0,4 0,6 0,8 1,0

Rel

axat

ion

spee

d (m

N.m

m-2

.s-1

)

0

2

4

6

8

10

**

** *

* **

*

##

## #

# ## #

(c)

Fig. 2. Contraction strength (2a), contraction speed (2b) and relaxation speed (2c)on cardiac tissue for the four experimental conditions: control, C (d), dispersant

F. Tissier et al. / Chemosphere 134 (2015) 192–198 195

3.5.2. O2 consumption of permeabilized cardiac muscle fibersThe results are reported in Fig. 3. Compared to the control,

exposure to the dispersant alone induced in a significant decreasein O2 consumption of permeabilized cardiac muscle fibers of sea-bass, after the addition of the three substrates tested (pyru-vate/malate, 2.7 times; Succinate, 3.4 times; Ascorbate/TPMD,2.4 times). Previous exposure to oil chemically dispersed inducedalso a decrease in O2 consumption of the cardiac fibers after addi-tion of pyruvate/malate (2.7 times) whereas no significant changewas observed after addition of the other substrates (succinateand ascorbate/TMPD). Compared to the control, previous exposureto oil dispersed mechanically has no significant effect on O2 con-sumption whatever the tested substrate was. Moreover, O2 con-sumption values measured in CD and MD conditions was similarfor the three substrates tested.

alone, D (j), mechanical dispersion, MD (�), chemical dispersion, CD (N). Resultsare expressed as arbitrary fluorescence units (mean ± standard error of the mean;n = 8). ⁄ indicates a significant difference between MD and C. # indicates asignificant difference between CD and C. No difference was observed between CDand MD.

3.5.3. Proton leakThe results of proton leak are reported in Fig. 4. A significant

increase in proton leak was observed in the D condition comparedto the three other conditions.

4. Discussion

This study aimed at evaluate the impact of dispersed oil on amajor physiological function, the cardiac function, in seabassDicentrarchus labrax. Two approaches were associated: the first

one focused on cardiac contraction parameters and the secondone focused on energy metabolism.

In this experimentation, the duration of exposure (48 h) andhydrocarbon concentrations used correspond to the upper rangeof what is observable during an oil spill (Blackman et al., 1978).Thus, our experimental conditions can be considered as realistic.

Table 2Measurements of O2 consumption after addition of Ascorbate/TMPD and CytochromeC for the 4 experimental conditions.

O2 consumption (lmol O2 min�1 mg�1 of drytissues)

Ascorbate/TMPD Cytochrome C

C 1.15 ± 0.11 1.19 ± 0.11D 0.47 ± 0.04 0.51 ± 0.06CD 0.89 ± 0.14 0.94 ± 0.13MD 1.25 ± 0.32 1.35 ± 0.30

Results expressed as lmol O2 min�1 mg�1 of dry tissues (mean ± standard error ofthe mean; n = 8). C (Control), D (Dispersant alone), CD (Chemical Dispersion of oil)and MD (Mechanical Dispersion of oil).

aab

bc bc bc

c

a

ab

abab

abb

pyruvate/malate Succinate Ascorbate/TMPD

O2 c

onsu

mpt

ion

(µm

olO 2/m

in/m

g of

dry

tiss

ue)

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

ab b ab

a

b

aab

a

b

ab

a

Fig. 3. O2 consumption of permeabilized cardiac muscle fibers for the fourexperimental conditions after addition of substrates. Results expressed aslmol O2 min�1 mg�1 of dry tissues (mean ± standard error of the mean; n = 8). C(Control, ), D (Dispersant alone, ), CD (Chemical Dispersion of oil,

), and MD (Mechanical Dispersion of oil, ). Values not sharing commonletters in the column indicate a significant difference (P < 0.05).

C D CD MD

Prot

on le

ak (R

atio

)

0,0

0,2

0,4

0,6

0,8

1,0

1,2

a aa

b

Fig. 4. Measurement of proton leak of permeabilized cardiac muscle fibers for thesix experimental conditions. Results expressed as (mean ± standard error of themean; n = 8). C (Control, ), D (Dispersant alone, ), CD (ChemicalDispersion of oil, ), and MD (Mechanical Dispersion of oil, ). Valuesnot sharing common letters in the column indicate a significant difference(P < 0.05).

196 F. Tissier et al. / Chemosphere 134 (2015) 192–198

TPH measurements performed daily were used to monitor expo-sure conditions while fish contamination was assessed by measur-ing bile fluorescence, one efficient biomarker of exposure topetroleum compounds in fish (Lin et al., 1996; Aas et al., 2000).The results of TPH measurements show no detectable levels ofhydrocarbons in the conditions C and D. In the case of the mechan-ical and chemical dispersion, the measured concentrations were

similar allowing comparisons between experimental conditions.Measurements of bile fluorescence indicate that fish exposed tooil chemically and mechanically dispersed were contaminatedwhereas no contamination occurred in the two other experimentalconditions (control and dispersant alone). The highest bile fluores-cence measured in fish of MD group compared to this of CD groupcould be linked to a higher contamination level due to difference inthe size of droplets measured in these two conditions. First, fishcould have actively ingested large droplets observed in MD condi-tion. Second, in MD condition oil droplets and gills could haveremained in contact for a longer time than for CD condition.

To assess the impact of dispersed oil on cardiac function, severalparameters (strength, contraction and relaxation speeds) weremeasured on fresh ventricular strips at different frequencies. Theprogressive increase in imposed frequencies makes it possible toconsider the adaptability of organ functioning to the body’s needs.Compared to the control group, a significant decrease in the mea-sured parameters was observed in the MD and CD groups whereasCD and MD were not different whatever the frequency. As in thisstudy, the dispersant used had no effect on these parameters,observed changes in cardiac function are mainly due to effects ofpetroleum compounds rather than effects of dispersant. Theseresults are in accordance with previous studies. For example, car-diac malformations have been observed on zebrafish embryos(Incardona et al., 2004) or on Pacific herring Clupea pallasi(Middaugh et al., 1998; Incardona et al., 2009) exposed to variousPAHs. PAHs are lipophilic compounds and therefore could be inte-grated into cellular membranes (Sikkema et al., 1995). They alsohave been shown to potentially interact with some carriers:Channels L and T for example (Richard and Nargeot, 1998). Sothe presence of oil could disturb calcium exchange and especiallycalcium transport across the cytoplasmic and the sarcoplasmicreticulum membranes, thus altering the kinetics of calcium releaseand uptake needed for muscle contraction (Bláha et al., 2002). Thishypothesis is supported by works of Brette et al. (2014). Theyreported a decrease in calcium current and calcium cycling con-ducting to a disruption of excitation–contraction coupling in car-diomyocytes on tuna exposed to crude oil which could alsooccurred in seabass.

The decrease in heart contraction capacity observed in the CDand MD conditions could also be linked to a decrease in ATP avail-ability. Studies have shown that after contamination, PAHs arepreferentially localized in the mitochondria (Zhu et al., 1995).Moreover, a decrease in mitochondrial O2 consumption rate hasbeen observed in zebrafish after exposure to a cocktail of PAHs(Knecht et al., 2013). In this study, the permeabilization of cellsusing saponin made possible to analyze functional mitochondriain situ, in their normal intracellular position, and the use of selectedsubstrates and inhibitors allowed the analysis of different elementsof the respiratory chain. First, mitochondrial integrity was verifiedafter the permeabilization process by the addition of cytochrome Cunder maximal oxygen consumption conditions h with ascorbateand TMPD as substrates (Kuznetsov et al., 2008). In our case, theaddition of the exogenous cytochrome C had no significant effecton O2 consumption, showing that the integrity of external mito-chondrial membranes was preserved.

Pyruvate/malate, succinate and ascorbate/TMPD are respec-tively substrates of the first, second and fourth complexes of therespiratory chain. The measurement of maximal ADP-Stimulated(state 3) respiration with these substrates made it possible to ana-lyze different parts of the respiratory chain. Our results showedthat compared to the control, exposure to dispersant alone andto dispersed oil induced a reduction in the maximal O2 consump-tion when the respiratory chain was fed with pyruvate/malate.When succinate and Ascorbate/TMPD were used, a decrease inmaximal O2 consumption was observed in group D, whereas

F. Tissier et al. / Chemosphere 134 (2015) 192–198 197

groups CD and MD were not different from the control group.Taken together, these results seemed to indicate that dispersantcould alter the activity of the complexes one, two and four (cyto-chrome oxidase).

When F0–F1 ATPase (the fifth complex of the respiratory chain)is inhibited by oligomycin, the remaining oxygen consumption ismainly due to proton leak through the inner mitochondrial mem-brane (Kuznetsov et al., 2008). In this work, an increase in protonleak was observed in the D group compared to the control whereasno change was observed for CD and MD groups. Taken togetherwith the fact that a decrease in O2 consumption was observed inthe presence of succinate for the D group, it could be hypothesizedthat the dispersant used could alter the internal membrane ofmitochondria. This could lead to a modification of the microenvi-ronment of the respiratory chain complexes and of the permeabil-ity of the inner membrane to ions.

Furthermore, production of reactive oxygen species (ROS) was apossible mechanism of alteration of the respiratory chain. In fact,these highly reactive products are known to have a negative impacton complexes I, III and IV of the respiratory chain (Musatov andRobinson, 2012). If, petroleum compounds are known to inducemitochondrial alteration (Zhu et al., 1995; Knecht et al., 2013), dis-persants could also have the same mechanism of action. Theyappear to stimulate ROS production: a study of Milinkovitch et al.(2013) has shown increased antioxidant defenses in the hearts ofLiza aurata exposed to Finasol OSR 62. It is interesting to note thatmechanical dispersion of oil has no effect on tissue respiration com-pared to the control, whereas the chemically dispersed groupexhibited intermediate results between the MD and D conditions.Thus, this dispersant of the third generation could be potentiallymore effective in disrupt the respiratory chain in heart musclefibers of fish than oil after chemical and mechanical dispersion.

In conclusion, this work clearly demonstrates that dispersedcrude oil has an impact on seabass cardiac contraction parametersand that the dispersant Finasol OSR 52 has an effect on maximalmitochondrial energy production and proton leak. Thus, the disper-sant oil mixture could lead to a decrease of fish metabolic capacity.This metabolic reduction could have an impact on the swimmingperformance or capabilities of the circulatory system. This couldalso lead to a decrease in fitness of fish in case of episodes of varia-tion of environmental conditions (oxygen, temperature).

Acknowledgments

This study was supported by a Ph.D grant from Total fluids. Theauthors thank Sally Fergusson (Alba Traduction) for reading thisdocument.

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