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
RI PT
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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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10 11
Biology, Studentski trg 16, University of Belgrade, Belgrade, Serbia 3
Institute for Biological Research “Siniša Stanković”, Despota Stefana 142, University
M AN U
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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
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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
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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
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ppm) and Zostera marina (LC50 – 386 ppm).
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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
69
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.
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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
79
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
87
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
96
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
100
Institute of Marine Biology at the site Dobrota in the Boka Kotorska Bay (Montenegro,
101
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
104
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
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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
112
polychetae. Exposures for genotoxicity assessment and exposures for cardiac activity
113
analyzes were carried out separately.
114
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
117
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
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exposed for 72 h at constant temperature 21±1˚C in a static system and were not fed in
128
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
133
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)
145
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
147
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
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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
163
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
167
electrophoresis, the slides were placed into freshly made cold (4°C) neutralizing buffer
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(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
170
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
181
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
188
(PPG). PPG technology is focused on optical detection of rhythmical changes in blood
189
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
192
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
196
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
204
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
210
test for normality of distribution was used prior to statistical analysis. Considering that
211
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
220
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
226
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
228
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
232
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
239
between the DNA damage in comets and frequency of hedgehogs. Moreover, significant
240
correlation was observed between the value of Tail intensity % and frequency of
241
hedgehogs (rex1 = 0.54, rex2 = 0.95).
242
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
248
determined by HR variability.
249
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
253
SD with stable periods was noted. The washout (the second arrow) caused stable HR
254
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.
258
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
265
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,
267
gradual increase of HR with maximum of 29.8 beats/min occurred. After the washout, the
268
mean HR was stable with low SD.
269
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.
271
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
273
quick HR decrease to 8.3 beats/min. These low HR value followed by unexpectedly low
274
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
280
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,
284
we used maximum concentration of dispersant applicable for aquarium treatment (which
285
did not induce extensive bubbling because of the aeration). Applied concentrations of
286
diesel oil were chosen in correspondence with selected concentrations of dispersant
287
(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.,
291
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
296
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
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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
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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;
587
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
592
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
597
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 +
599
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
601
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 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
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Genotoxic potential detected for S-25 and dispersed diesel oil
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The heart rate disorders recorded for the S-25, diesel oil and dispersed diesel oil
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•