Accepted Manuscript Genotoxic potential and heart rate disorders in the Mediterranean mussel Mytilus galloprovincialis exposed to Superdispersant-25 and dispersed diesel oil Rajko Martinović, Stoimir Kolarević, Margareta Kračun-Kolarević, Jovana Kostić, Sandra Marković, Zoran Gačić, Zoran Kljajić, Branka Vuković-Gačić PII:

S0141-1136(15)00068-9

DOI:

10.1016/j.marenvres.2015.05.001

Reference:

MERE 3998

To appear in:

Marine Environmental Research

Received Date: 19 February 2015 Revised Date:

28 April 2015

Accepted Date: 6 May 2015

Please cite this article as: Martinović, R., Kolarević, S., Kračun-Kolarević, M., Kostić, J., Marković, S., Gačić, Z., Kljajić, Z., Vuković-Gačić, B., Genotoxic potential and heart rate disorders in the Mediterranean mussel Mytilus galloprovincialis exposed to Superdispersant-25 and dispersed diesel oil, Marine Environmental Research (2015), doi: 10.1016/j.marenvres.2015.05.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Genotoxic potential and heart rate disorders in the Mediterranean mussel Mytilus

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galloprovincialis exposed to Superdispersant-25 and dispersed diesel oil

3 Rajko Martinović1, Stoimir Kolarević2, Margareta Kračun-Kolarević3,

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Jovana Kostić4, Sandra Marković1, Zoran Gačić4, Zoran Kljajić1, Branka Vuković-Gačić2

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6 1

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Institute of Marine Biology – Kotor, University of Montenegro, Dobrota bb, 85330,

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Center for genotoxicology and ecogenotoxicology, Chair of Microbiology, Faculty of

SC

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Biology, Studentski trg 16, University of Belgrade, Belgrade, Serbia 3

Institute for Biological Research “Siniša Stanković”, Despota Stefana 142, University

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Kotor, Montenegro

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of Belgrade, Belgrade, Serbia 4

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Department of Natural Resources and Environmental Sciences,

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Institute for Multidisciplinary Research, Kneza Višeslava 1,

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University of Belgrade, Belgrade, Serbia

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To whom correspondence should be sent: Tel/fax.: +38 1112637364

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E-mail address: [email protected] (Dr Stoimir Kolarević)

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Abstract

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The effects of ex situ exposure of Mytilus galloprovincialis to Superdispersant-25 (S-25),

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diesel oil and dispersed diesel oil mixtures were studied by the impact on level of DNA

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damage in haemocytes (comet assay) and the cardiac activity patterns of mussels.

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Specimens were exposed for 72h in a static system to diesel oil (100 µL/L and 1 mL/L),

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S-25 (5 and 50 µL/L), and dispersed diesel oil mixtures M1 (diesel oil 100 µL/L + S-25 5

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µL/L) and M2 (diesel oil 1 mL/L + S-25 50 µL/L). For positive control 40 µM CdCl2 was

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used. The comet assay results indicated genotoxic potential of S-25 while the effects of

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diesel oil alone were not observed. The highest response was detected for M1 while the

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effects of M2 were not detected. The heart rate disorders were recorded for the diesel oil

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(1 mL/L), S-25 (50 µL/L) and both dispersed diesel oil mixtures.

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Keywords: Superdispersant-25, dispersed oil, comet assay, cardiac activity pattern,

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Mytilus galloprovincialis

33 Introduction

35 36

Diesel oil, the most commonly used oil derivative, can have a serious biological impact

37

on the marine environment (Fingas and Brown, 2011). The city of Kotor (Montenegro),

38

as one of the leading nautical tourism destinations in the southern Adriatic, contributes to

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intensive maritime transport followed by excessive release of oil related chemicals to the

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sea. Occasionally, reducing the oil levels in the Boka Kotorska Bay were conducted by

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oil slick dispersant, Superdispersant-25 (S-25). Oil dispersants are mixtures of the surface

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active agents (surfactants) and solvents, produced to enhance the rate of oil degradation

43

in the water (GESAMP 1993).

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Toxic properties of dispersants became well known after the 2010 Deepwater Horizon oil

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spill crisis in the Gulf of Mexico. The results of the 38 ecotoxicological studies which

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followed this event are summarized in the review of Wise and Wise (2011). In general, a

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lot of publications considering the toxic effects of dispersants to different sea organisms

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are related to previous chemical formulations with hydrocarbon based solvents with

49

polycyclic aromatic hydrocarbon (PAH) content. S-25 belongs to the third oil dispersant

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generation based on glycol ether solvent intended to be less toxic to the environment

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(European Maritime Safety Agency, 2009). Regardless, five products of the third oil

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dispersants generation, even in low concentrations, were shown to be harmful to early life

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stages of two reef corals, questioning the in situ application of dispersants in the coral

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reefs (Epstein et al., 2000). Moreover, the study of Scarlett et al. (2004) showed toxic

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effect of S-25 in Anemonia viridis (LC50 – 20 ppm), Corophium volutator (LC50 – 260

56

ppm) and Zostera marina (LC50 – 386 ppm).

57

Data in available literature, indicating genotoxic potential of oil dispersants, is scarce.

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The study of De Flora et al. (1985) reported genotoxic potential of 3 commonly used

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dispersants based on bacterial assays. The study of Wise et al. (2014) indicated cytotoxic

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and genotoxic potential of Corexit 9500 and 9527 dispersants in cultures of primary skin

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fibroblast cells of sperm whale. However, data related to genotoxic properties of S-25 is

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currently lacking.

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In this study we have tried to simulate the conditions of a possible environmental

64

accident related to diesel oil pollution. The major goal was to investigate the effects of

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exposure to S-25 and mixtures of S-25 and diesel oil ex situ in haemocytes of

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Mediterranean mussel Mytilus galloprovincialis mainly from the aspect of genotoxicity.

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We decided to use marine mussels M. galloprovincialis as a bioindicator as this

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cosmopolitan species is commonly used as a sentinel organism for the screening of

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pollution and potential environmental harm (Lionetto et al., 2003; Regoli et al., 2004;

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Vlahogianni et al., 2007). For assessment of genotoxicity we have employed the comet

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assay, as this method is considered to be one of the major tools for assessing pollution-

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related genotoxicity in aquatic organisms (Dixon et al., 2002; Chen et al., 2007; Picado,

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2007; Kolarević et al., 2013; Sunjog et al., 2014). The comet assay is a sensitive and

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rapid technique for the detection of DNA damage in individual cells, based on the

75

migration of denatured DNA during electrophoresis.

76

As an indication of exposure of animals to selected compounds we decided to monitor

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additional biomarker – the cardiac activity pattern. Changes in cardiac activity pattern of

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mollusks and crustaceans are reliable physiological biomarker of stress reaction to

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different pollution sources (Bamber and Depledge, 1997; Chelazzi et al., 2004). Infrared

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technology for noninvasively recording the heart rate (HR) of invertebrates was

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introduced by Depledge (1990). Similar methodology, based on fiber-optic sensors,

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developed by Kholodkevich and described by Fedotov et al. (2000), was used in this

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study for recording of cardiac activity of M. galloprovincialis.

84 85

Material and methods

86

2.1. Chemicals

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Diesel oil was purchased from the local Petrol station and S-25 was obtained from

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shipyard Bijela. Cadmium chloride was purchased from E. Merck, Darmstadt, Germany.

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The day before the beginning of treatment, primary solutions were prepared and marked

90

as following: diesel oil in concentrations D1’ (1 mL/L) and D2’ (10 mL/L), S-25 in

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concentrations S1’ (50 µL/L) and S2’ (500 µL/L). Primary mixtures of dispersed diesel

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oil were prepared and marked as following: M1’ (diesel oil 1 mL/L + S-25 50 µL/L) and

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M2’ (diesel oil 10 mL/L + S-25 500 µL/L). Ratio of diesel oil and S-25 used in dispersed

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diesel oil mixtures was prepared based on the instructions given by manufacturer of

95

dispersant (Oil Slick Dispersants ltd, 2014). Primary solutions were prepared in seawater

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collected from the site Dobrota (mussel collection site) in final volume of 800 mL and

97

shaken in plastic containers for 24 h to enable mechanical dispersion.

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2.2. The specimen collection

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The specimens of M. galloprovincialis were collected from the mussel farm of the

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Institute of Marine Biology at the site Dobrota in the Boka Kotorska Bay (Montenegro,

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42°26′ N, 18°45′ E) at a depth of 2-3 m. Temperature, salinity and dissolved oxygen were

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measured in situ by WTW sonda Multi 350i. Detected values were 22ºC for temperature,

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31‰ for salinity and 8.1mg/L for oxygen. Determination of nutrients at sampling site

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such as nitrate, nitrite, ammonium, phosphate, silicate, total nitrogen and total phosphorus

105

were analyzed according to (Parsons et al, 1984; Deggobis et al, 1986). Nutrient

106

concentrations showed the following results: nitrate 1.429 µmol/L, nitrite 0.089 µmol/L,

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ammonia 0.122 µmol/L, phosphates 0.178 µmol/L and silicate 2.012 µmol/L. Values of

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total nitrogen and total phosphorus were 9.012 µmol/L and 0.289 µmol/L. The collection

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site can be considered as relatively unpolluted already as it was used as reference site for

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various biomarkers in the previous studies (Da Ros et al., 2011; Heberger at al., 2014).

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Mussels were taken to the laboratory; shells were cleaned from algae and marine

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polychetae. Exposures for genotoxicity assessment and exposures for cardiac activity

113

analyzes were carried out separately.

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2.3. Exposure for genotoxicity assessment

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Mussels with intact bissus threads were divided in 8 groups per 10 specimens and groups

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were placed in eight 15 L glass aquaria containing 7.2 L of seawater taken from the

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sampling site Dobrota. Mussels were left for 24 h for acclimation to laboratory

118

conditions. Before addition of the test substances, specimens were checked if they are

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attached to aquaria bottom by bissus threads.

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Primary solutions of substances (800 mL per each) prepared as described above were

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added in aquaria with attached animals to obtain following: diesel oil in the final nominal

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concentrations D1 (100 µL/L) and D2 (1 mL/L), S-25 in concentrations S1 (5 µL/L) and

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S2 (50 µL/L), dispersed diesel oil mixtures in concentrations M1 (diesel oil 100 µL/L +

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S-25 5 µL/L) and M2 (diesel oil 1 mL/L + S-25 50 µL/L). For the negative control, 800

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mL of seawater from the sampling site Dobrota was added. For positive, 800 mL of 400

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µM CdCl2 was added to obtain final nominal concentration of 40 µM. Specimens were

127

exposed for 72 h at constant temperature 21±1˚C in a static system and were not fed in

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order to avoid interaction between tested substances and food.

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2.4. Exposure for cardiac activity analyzes

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Cardiac activity analyzes were performed in eight experiments. For each one, eight

131

mussels were placed in an 20 L aquarium with 7.2 L of seawater in constant temperature

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21±1˚C and salinity 25±1 g/L regime. Chemical treatment and exposure period was

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analogous to previously described, used in genotoxicological analysis. With the exception

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of the negative control experiment, in all treatments the chemicals were washed out after

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72 h and replaced with clean seawater.

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2.5. Exposure for genotoxicity assessment

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Over the exposure period, viability of mussels was checked every 12 h by mechanical

138

stimulation of shell closure. If the mussel did not respond to closure stimulation, it was

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considered nonviable and was removed from aquaria. For genotoxicity assessment,

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treatments were performed in two individual experiments.

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2.5.1. Cell viability

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From each specimen, approximately 100 µL of haemolymph was collected from the

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adductor muscle with a 3 mL syringe with a hypodermic 26 G needle. The viability of

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haemocytes was assessed with differential acridine orange/ethidium bromide (AO/EB)

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assay (Squier and Cohen 2001). Briefly, 20 µL of haemolymph was mixed with 2 µL of

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AO/EB (100 µg/mL) and for each specimen 100 cells were examined under fluorescence

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microscope.

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2.5.2. Comet assay

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After exposure, haemolymph was collected from the adductor muscle with a 3 mL

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syringe with a hypodermic 26 G needle. Haemolymph from 2 to 3 specimens was pooled

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in microtubes on ice to obtain 1 mL of sample (total of 4 mL per group of 10 animals).

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Samples were centrifuged for 10 min at 500 x g at 4 °C; the cell suspension was made in

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60 µL of residual supernatant. Prepared cell suspension was subjected to comet assay.

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The alkaline comet assay procedure was performed under yellow light as described by

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Singh et al. (1988). Microscopic slides were pre-coated with 1% NMP agarose and air

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dried for 24 h. To form a second, supportive layer, 80 µL of 1% NMP agarose was gently

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placed on top of the 1% NMP layer and spread over the slide using a coverslip. The slide

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was placed on ice for 5 min to allow complete polymerization of agarose. After the

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coverslips were removed, 30µL of cells suspension (prepared as described earlier) was

160

gently mixed with 70 µL of 1% LMP (37 ºC) agarose, pipetted onto the supportive layer

161

of 1% NMP agarose and covered with a coverslip. After 5 min on 4 °C, the coverslips

162

were removed and the slides were lowered into freshly made cold (4 °C) lysis buffer (2.5

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M NaCl, 100 mM EDTA, 10 mM Tris, 1.5% Triton X-100, pH 10) for 1 h. To allow

164

DNA unwinding, slides were placed in an electrophoresis chamber containing cold (4 °C)

165

alkaline electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH 13) for 20 min.

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Electrophoresis was performed at 0.75 V/cm and 300 mA for 20 min at (4 °C). After

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electrophoresis, the slides were placed into freshly made cold (4°C) neutralizing buffer

168

(0.4 M Tris, pH 7.5) for 15 min. Slides were fixed in ice cold methanol (15 min at 4°C)

169

and air dried in dark. Staining was performed with 20 µl per slide of ethidium bromide (2

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µg/mL). The slides were examined with a fluorescence microscope (Leica, DMLS,

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Austria, under magnification 400 X, excitation filter 510-560 nm, barrier filter 590 nm).

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Microscopic images of comets were scored using Comet IV Computer Software

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(Perceptive Instruments, UK). Fifty nuclei were analyzed per slide, and the TI% (tail

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intensity - the percent of the fluorescence in the comet tail) was scored and served as a

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measure of DNA damage. Based on the level of DNA damage assessed by TI%, two

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additional parameters were monitored: frequency of the highly damaged comets (HDC%)

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and frequency of the atypically sized comets (ASC%). The parameter HDC% represents

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frequency of the comets with the values of TI% higher than 50%, while the parameter

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ASC% represents frequency of comets with value of TI% exceeding the 95% confidence

180

limit of means in reference groups. Highly damaged nuclei in which DNA damage level

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could not be properly assessed were marked as ‟hedgehogs‟ and counted with hedgehog

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tool available in Comet Assay IV software. Frequency of hedgehogs was assessed on 100

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observed nuclei.

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2.6. Cardiac activity analyzes

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Cardiac activity recording of mussels was conducted by non-invasive laser fiber-optic

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method developed in 1999 at the Laboratory of experimental ecology of aquatic systems

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SRCES RAS (Fedotov et al., 2000). The method is based on photoplethysmography

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(PPG). PPG technology is focused on optical detection of rhythmical changes in blood

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vessels derived from contractile activity of the heart (Allen, 2007).

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Experimental unit includes eight PPG devices allowing simultaneous recording of the

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heart rate activity of eight mussels. After removing encrusted organisms, sensor holders

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were glued to shell valves to enable connection made by fiber-optic cables to an IR light

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source and receiver placed in the PPG device. Heart area of mussels was exposed to IR

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light, reflected light containing the information about periodical changes of heart volume

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was detected by the sensor and sent to a personal computer. After appropriate

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amplification and analog to digital conversion, signal is processed by original software

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VarPulse, designed for statistical analyzing of cardiac intervals (Kholodkevich et al.,

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2008).

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Prior to the treatment, it was necessary to determine the initial HR value of mussels in

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clean seawater in order to create baseline as a reference point for the possible changes of

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cardiac activity induced by the application of prepared chemicals. Also, it was necessary

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to obtain stable HR level as a baseline prior and following the exposure to test substance,

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with low standard deviation (SD) for at least of 2-3 hours duration, to be visible in 72h

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treatment displayed on graph. HR was recorded continuously, prior the treatment, under

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the action of test substance and after depuration from the system (following the

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treatment).

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2.7. Statistical analyses

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Statistical analysis of the results obtained in the experiments was carried out using

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Statistica 6.0 Software (StatSoft, Inc.). For the comet assay data, Kolmogorov-Smirnov

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test for normality of distribution was used prior to statistical analysis. Considering that

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the data were not in the line with the requirements for the application of parametric tests,

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differences between each group and corresponding negative control were tested using the

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Mann-Whitney U test. Taking into consideration the size of the groups Students t-test (p

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< 0.05) was used when testing significance for parameters % hedgehogs, HDC % and

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ASC %. Correlation analyses were carried out using Spearman correlation test with

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significance level p < 0.05.

217 3. Results

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3.1. Cell viability

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The results of cell viability assay are summarized in the Table 1. Reduction of

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haemocytes viability was recorded only for the treatments with CdCl2 (29 % reduction).

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3.2. Comet assay

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The comet assay results are summarized on the Fig. 1. Significant increase (p < 0.05) of

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TI% was observed for both concentrations of S-25. The effect of diesel oil was observed

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only for the concentration D1 and only in the experiment 1. M1 induced significant

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increase (p < 0.05) of DNA damage in both experiments. As for the M2 mixture,

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significant increase (p < 0.05) of DNA damage was detected only in the first experiment

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but induction was significantly lower (p < 0.05) comparing individually to S2, D2 and

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M1 treatments. CdCl2 resulted in significant increase (p < 0.05) of DNA damage in both

230

experiments (Fig. 1a).

231

Exposure to selected concentration of S-25 resulted in significant increase (p < 0.05) of

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frequency of ASC in both experiments. Significant increase (p < 0.05) was also observed

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for M1, M2 and CD in the first experiment (Fig. 1b). Considering frequency of HDC,

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significant increase (p < 0.05) was observed for CdCl2 treatments in both experiments

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and M1 mixture in the second experiment (Fig. 1c).

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The highest percentage of hedgehogs in both experiments was detected in groups exposed

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to S2 (Fig. 1d). Increased percentage was also observed for D2 in the first experiment and

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M1 in the second experiment. With exception of D2 similar trends can be observed

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between the DNA damage in comets and frequency of hedgehogs. Moreover, significant

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correlation was observed between the value of Tail intensity % and frequency of

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hedgehogs (rex1 = 0.54, rex2 = 0.95).

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3.3. Cardiac activity analyzes

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The results obtained for controls and exposures to diesel oil are summarized on Fig. 3.

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Obtained results for a negative control were expected. The mean HR value was mainly

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stable during 72 hours with lower SD. Also, cadmium chloride (CD), used as positive

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control, caused increase in mean HR value from 16.5 to 24.4 beats/min, thereafter,

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decline to 10.7 beats/min occurred, as expected (Fig. 2). The rest of the CD exposure was

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determined by HR variability.

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Both concentrations of diesel oil caused changes in cardiac activity pattern of M.

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galloprovincialis. After the HR baseline level of 21.8 beats/min was reached, lower

251

concentration D1, was applied (the first arrow). The mean HR value of eight mussels

252

rapidly increased to 26.3 beats/min. During the next 72h of treatment, HR variability and

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SD with stable periods was noted. The washout (the second arrow) caused stable HR

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pattern with lower SD. The initial effect of D2 was a slight increase of mean HR from

255

18.5 to 22.2 beats/min. Thereafter, the mean HR was decreased significantly to 9.6

256

beats/min followed by variable SD and also retention within these lower values for a 11h

257

and 37min with a maximum of 14.5 beats/min. Then, HR rose sharply to 25.4 beats/min.

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The rest of exposure period was characterized by very high SD and HR variability. The

259

mean HR was very stable on a higher value after washout.

260

The results obtained for exposure to S-25 and dispersed diesel oil are summarized on the

261

Fig. 3. Lower concentration of S-25 (S1), increased the mean HR value from 17.8 to 21.9

262

beats/min. In next 72 hours, high HR variability was observed. Also, on the third day of

263

exposure, very high range of SD value was noted. The washout caused stable HR on 16.5

264

beats/min with low SD. The mean HR baseline level was slightly increased from 17.5 to

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20.2 beats/min, following the application of S2. Afterwards, HR value rose rapidly to 10

266

beats/min and mainly retained on lower values within the first 24 hours. The next day,

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gradual increase of HR with maximum of 29.8 beats/min occurred. After the washout, the

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mean HR was stable with low SD.

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Exposure to lower concentration of dispersed diesel oil (M1) induced increase of mean

270

HR from 19.7 to 24.2 beats/min. In the next 72h, the HR variability was observed.

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Subsequently, the washout caused stable HR with higher SD. M2 application caused

272

significant increase of the mean HR value from 18.7 to 30 beats/min, followed by very

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quick HR decrease to 8.3 beats/min. These low HR value followed by unexpectedly low

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SD retained to the end of exposure. The mean HR value increased rapidly to 18.4

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beats/min followed by higher SD after the washout (Fig. 3).

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4. Discussion

278

In this study we have investigated the effects of short term exposure of mussels M.

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galloprovincialis to dispersant S-25, diesel oil and dispersed diesel oil mixture. In the

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experimental setup, we have tried to simulate the conditions of a possible environmental

281

accident. Therefore, prior to exposure, mixtures were prepared using mechanical shaking

282

to simulate the effect of waves. The diesel oil spill accidents are in most cases of local

283

character in which the aquatic biota is exposed to high amounts of dispersants. Therefore,

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we used maximum concentration of dispersant applicable for aquarium treatment (which

285

did not induce extensive bubbling because of the aeration). Applied concentrations of

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diesel oil were chosen in correspondence with selected concentrations of dispersant

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(respecting 1:20 dispersant: diesel oil v/v ratio). For the exposure period, we have chosen

288

72h considering the local characteristics/endurance of possible accidents.

289

In our study we used the comet assay to assess the level of DNA damage in haemocytes

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of M. galloprovincialis. To avoid the possible interference of cytotoxicity (Tice et al.,

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2000; Collins, 2004; Lovell and Omori, 2008; Frenzilli et al., 2009) we have also

292

assessed the effects of treatments on the viability of haemocytes. The comet assay data

293

were validated as none of the tested concentrations of S-25, diesel oil or tested mixtures

294

had significant impact on the viability of the haemocytes.

295

As a positive control, treatment with cadmium chloride was used based on the findings of

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our previous study on freshwater mussels (Gačić et al., 2014). This mutagen induced

297

significant increase of DNA damage during in vivo exposure in haemocytes of Unio

298

pictorum and U. tumidus in the same concentration used in our current study (40 µM).

299

Genotoxic potential of cadmium chloride was also reported in other aquatic organisms

300

(Dabas et al., 2014; Harabawy and Mosleh, 2014).

301

Our results indicated that S-25 in tested concentrations (5 and 50 µL/L), which are lower

302

then toxic concentrations present in literature, has influence of the level of DNA damage

303

in haemocytes. The study of Scarlett et al. (2004) indicated that the exposure to the S-25

304

in concentration of 250 ppm (250 µL/L) had no toxic effects in Mytilus edulis but did

305

result in valve closure. In our study, the results indicated that exposure to S-25 in higher

306

concentration (50 µL/L) contributes to increase of fragmentation of DNA molecule

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assessed by all studied comet assay parameters indicating genotoxic potential of S-25. S-

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25 is a blend of glycol and glycol ether solvents, combined with ionionic and anionic

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surfactants (AFI, 2003). The study of Elias et al. (1996) indicated that short-term

310

exposure to glycol ethers can result in genotoxic effects in human lymphocytes, while the

311

study of Ave et al. (2010) performed on Chinese hamster ovary cell line showed that

312

propylene glycol can induce DNA damage measured by the comet assay.

313

In general, exposure to diesel oil did not result in the significant increase of DNA damage

314

with exception of D1 in the first experiment. Increase of frequency of hedgehogs was

315

observed only for D2 concentration in the second experiment but without the alterations

316

of other parameters. The studies of Vanzella et al. (2007) and Santos et al. (2010)

317

indicated that water soluble diesel oil fraction can have genotoxic potential but in the

318

concentrations which are several fold higher than concentrations applied in our study.

319

Exposure to mixture M1 resulted in increase of DNA damage comparing to control but

320

also comparing to groups exposed to S1 and groups exposed to D1. Also, increase of

321

frequency of hedgehogs was recorded. The study of Poli et al. (2014) indicated that crude

322

oil dispersed by chemical dispersant had higher potency in inducing apoptosis in germ

323

cells of Cenorabdites elegans comparing to crude oil and to dispersant separately.

324

Enhancement of toxicity of crude oil dispersed by chemical dispersant was also observed

325

in fish rainbow trout (Ramachandran et al., 2004). These studies have also raised the

326

issue of much-increased bioavailability of polycyclic aromatic hydrocarbons (PAHs) in

327

the water fraction caused by the use of dispersants. PAHs are well known for

328

toxic/genotoxic properties and therefore they are recognized as priority hazardous

329

substances in aquatic ecosystems (Cachot et al., 2006; Inzunza et al., 2006; Barbee et al.,

330

2008). Exposure to mixture M2 did not result in any increase of the studied comet assay

331

parameters in comparison to the negative control, suggesting a possible interference of

332

valve closure, which could affect the level of exposure of animals to test substances.

333

Therefore, as an indication of the level of exposure of animals and possible adaptation to

334

stress during exposure to selected compounds we decided to monitor an additional

335

biomarker - the cardiac activity pattern. HR of Mytilid mussels has been widely used in

336

multibiomarker studies as an indicator of stress in response to marine pollution (Astley et

337

al. 1999; Halldorsson et al., 2008; Turja et al., 2014). Decline of HR value followed by

338

shell-closing behavior in toxic exposure to M. galloprovincialis was evidenced by

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Kholodkevich et al. (2009). Also, valve closure of the same species accompanied by

340

either exhalant or inhalant siphon inhibition led to anaerobic metabolism as the

341

adaptation to stress (De Vooys, 1991).

342

All chemicals applied were rapidly detected by the changes of mean HR values, recorded

343

within a group of mussels while in clean seawater as a negative control cardiac activity

344

changes did not occur. Cadmium chloride was used as positive control since we have

345

already obtained decrease in the cardiac activity caused by the same concentration

346

(Martinovic et al., 2013). Recently, we have reported a heart rate disorder in M.

347

galloprovincialis caused by the direct application of S-25 and dispersed diesel oil in 2h

348

exposure (Martinovic et al., 2015). The lower dispersant concentration S1 (5 µL/L)

349

slightly increased the initial mean HR value opposite to a previous study that indicated

350

significant bradycardia caused by 2 ppm (2 µL/L) of S-25. Accordingly, sea waves could

351

reduce the influence of dispersant on M. galloprovincialis. In spite of shaking and

352

dilution in seawater prior to the exposure, S2 caused decrease of the initial mean HR

353

value within a group of mussels. The stress effect of S2 was present during the entire

354

exposure period due to very high HR variability and SD until the washout.

355

The results of diesel oil contamination for two concentrations tested are analogous to

356

effects of dispersant. D1 was detected by a 20% increase of HR level and later occurred

357

HR variability was not high due to low concentration. On the other hand, D2 induced

358

decrease of cardiac activity followed by significant HR oscillations that could be

359

prescribed to the components of diesel oil. HR response to M1, beside the initial increase,

360

caused higher HR variability comparing to separate applications of D1 and S1, probably

361

due to higher accessibility of diesel oil components to mussels after dispersion. HR

362

oscillations in blue mussel Mytilus edulis, occurred under the action of oil pollution were

363

already described by Bakhmet et al. (2009) as an adaptation of mussels to the exposure

364

stress.

365

In our opinion, retention of the HR on lower values during the entire exposure period in

366

M2 treatment, followed by immediate recovery after washout, indicate the lack of

367

physiological mechanism to overcome dispersed oil pollution. In this scenario, mussels

368

could activate other protective mechanisms such as reduction of filtration rate or valve

369

closure which can be explanation of unexpectedly low DNA damage in both M2

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treatments despite of higher concentrations applied. Interference of valve closure with the

371

level of response was considered as an issue in the studies of Siu et al. (2004) performed

372

on Perna viridis exposed to mixtures of PAHs and chlorinated pesticides, and the study

373

of Mersch et al. (1996) performed on Dreissena polymorpha exposed to clastogens.

374

Moreover, to avoid issues related to valve closure, in some genotoxicological studies

375

even mechanical prevention of valve closure is applied (Wilson et al., 1998; Pruski and

376

Dixon, 2002). However, in D2 and S2 exposures, the reversible HR pattern was indicated

377

after the observed retention close to values of 9 beats/min. Later occurred HR variations

378

were considered as adaptive strategy with the consequence of higher DNA damage

379

obtained in genotoxicological treatment.

380

In conclusion, our results demonstrate that exposure to Superdispersant-25 and mixtures

381

of Superdispersant-25 and diesel oil can have harmful effect in marine mussels M.

382

galloprovincialis. Our results also demonstrate that retention of the HR on lower values

383

and subsequent valve closure represent interesting protective mechanism of marine

384

mussels which significantly reduces harmful effects during short period of exposure to

385

tested pollutants.

386

Acknowledgment

387

This paper is part of the project Kotor (Complex research of the ecosystem of the coastal

388

sea of Montenegro), supported by the Ministry of Science, Montenegro. Authors are

389

grateful to Prof. S.V. Kholodkevich from SRCES RAS, St. Petersburg, Russia, and EPA

390

Montenegro for providing experimental equipment. Also, we are grateful to the company

391

‘Adriatic shipyard Bijela’ for supplying oil dispersant. The authors are grateful to Luka

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Gačić who provided improvements to our English.

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Captions for figures and tables

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Fig 1 (a) The level of DNA damage assessed by Tail intensity (mean ± SE), (b) the

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frequency of atypically sized comets ASC% (mean ± SD), (c) the frequency of highly

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damaged comets HDC% (mean ± SD), (d) the frequency of hedgehogs (mean ± SD) in

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haemocytes of M. galloprovincialis after exposure to S-25 in concentrations S1 and S2;

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diesel oil in concentrations D1 and D2 and mixtures M1 and M2; CD – cadmium chloride

588

(40 µM).* p < 0.05.

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Fig. 2 Mean heart rate of the Mediterranean mussel (M. galloprovincialis L.) with

591

standard deviation (SD): a) C in the clean sea water, before the application, under the

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action and after depuration from b) CD (40µM), c) D1 (100 µL/L), d) D2 (1 mL/L);

593

Abbreviations: C – control, CD – cadmium chloride, D – diesel oil, w – washout. The

594

arrows indicate the moment of onset and termination of chemical treatment.

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Fig. 3 Mean heart rate of the Mediterranean mussel (M. galloprovincialis L.) with

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standard deviation (SD) in the clean sea water before the application, under the action

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and after depuration from a) S1 (5 µL/L), b) S2 (50 µL/L), c) M1 (diesel oil 100 µL/L +

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S-25 5 µL/L), d) M2 (diesel oil 1 mL/L + S-25 50 µL/L) Abbreviations: S-25 –

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Superdispersant 25, M – mixture of diesel oil and S-25, w – washout. The arrows indicate

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the moment of onset and termination of chemical treatment.

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Table 1 Percentage of viable haemocytes in comparison with negative control group

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Table 1 Percentage of viable haemocytes in comparison with negative control group S1 S2 D1 D2 M1 M2 CD Experiment1 89 % 96% 99% 100% 100% 100% 85% Experiment2 94 % 93% 93% 100% 92% 100% 58% mean 91% 95% 96% 100% 96% 100% 71%

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Highlights •

Effects of Superdispersant-25 and dispersed diesel oil studied in M. galloprovincialis The DNA damage in haemocytes and the cardiac activity patterns were monitored



Genotoxic potential detected for S-25 and dispersed diesel oil



The heart rate disorders recorded for the S-25, diesel oil and dispersed diesel oil

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Genotoxic potential and heart rate disorders in the Mediterranean mussel Mytilus galloprovincialis exposed to Superdispersant-25 and dispersed diesel oil.

The effects of ex situ exposure of Mytilus galloprovincialis to Superdispersant-25 (S-25), diesel oil and dispersed diesel oil mixtures were studied b...
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