Ecotoxicology and Environmental Safety 114 (2015) 164–170

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Toxicological assessment and management options for boat pressurewashing wastewater Marko Gerić a, Goran Gajski a, Višnja Oreščanin b, Robert Kollar c, Jasna Franekić d, Vera Garaj-Vrhovac a,n a

Institute for Medical Research and Occupational Health, Mutagenesis Unit, 10000 Zagreb, Croatia ORESCANIN Ltd., 10000 Zagreb, Croatia c Advanced Energy Ltd., 10000 Zagreb, Croatia d Faculty of Food Technology and Biotechnology, Laboratory for Biology and Microbial Genetics, 10000 Zagreb, Croatia b

art ic l e i nf o

a b s t r a c t

Article history: Received 27 October 2014 Received in revised form 14 January 2015 Accepted 19 January 2015

Boats are washed periodically for maintenance in order to remove biofoulants from hulls, which results in the generation of wastewater. This study aimed at evaluating the cyto/genotoxic and mutagenic properties of wastewater produced by pressure washing of boats. The chemical characterisation of this wastewater showed that Cu, Zn, V, Cr, Fe, Pb, and select organic contaminants exceeded the maximum allowable values from 1.7 up to 96 times. The wastewater produced negative effects on human lymphocytes resulting in decreased cell viability after 4 and 24 h of exposure. Chromosome aberration, micronucleus, and comet assay parameters were significantly higher after 24 h of exposure. At the same time, the Salmonella typhimurium test showed negative for both TA98 and TA100 strains at all of the concentrations tested. After the treatment of wastewater using electrochemical methods/ozonation during real scale treatment plant, removal rates of colour, turbidity and heavy metals ranged from 99.4% to 99.9%, while the removal of total organic carbon (TOC) and chemical oxygen demand (COD) was above 85%. This was reflected in the removal of the wastewater's cyto/genotoxicity, which was comparable to negative controls in all of the conducted tests, suggesting that such plants could be implemented in marinas to minimise human impact on marine systems. & 2015 Elsevier Inc. All rights reserved.

Keywords: Wastewater Mutagenicity Cytotoxicity Genotoxicity Electrocoagulation Electrooxidation ozonation

1. Introduction The fouling of biological material in vessel hulls is a major issue in modern ship transport. If not specially protected, a boat bottom can gather up to 150 kg of fouling per 1 m2 within six months time, which subsequently increases fuel consumption by 40–50% and shipping costs by up to 70% (Yebra et al., 2004). In the last several years, the development of ship hull protection has improved the management of biofouling. This includes: slippery surface coating, enzymes that prevent bacteria from attaching to the hull, electricity, more frequent hull cleaning, and antifouling paints based on heavy metals such as copper (Oreščanin et al., 2012; Yebra et. al, 2004). At the same time, copper-based antifouling paints are considered an important anthropogenic source of copper in the aquatic environment, where approximately 2 kg of copper per n Correspondence to: Institute for Medical Research and Occupational Health, Ksaverska cesta 2, 10000 Zagreb, Croatia. Fax: þ 385 1 46 73 303. E-mail address: [email protected] (V. Garaj-Vrhovac).

http://dx.doi.org/10.1016/j.ecoenv.2015.01.018 0147-6513/& 2015 Elsevier Inc. All rights reserved.

boat is released annually (Boxall et al., 2000; Jones and Bolam, 2007). Once a year, each boat is dry docked and washed as part of regular maintenance, which generates roughly 100 L of boat pressure-washing wastewater (BPWW) containing antifouling paint (Oreščanin et al., 2012). Therefore, this study set out to evaluate the toxic potential of such wastewater from the aspects of mutagenicity and cyto/genotoxicity. First, a chemical characterisation of wastewater produced from high pressure washing of boat paints was conducted in order to detect the amount of heavy metals released into the environment. Using two different bacterial strains, we tried to determine whether such BPWW poses a threat from the aspect of mutagenicity. The chromosome aberrations, comet, and micronucleus tests on human and animal cells have been used in a large number of environmental studies (Au et al., 2001; Almeida et al., 2013; Frenzilli et al., 2009; Gajski et al., 2012; Gerić et al., 2012) and the present study highlights their possible application in the detection of cyto/genotoxicity induced by BPWW. Finally, we will demonstrate toxicological evidence of a possible cost-effective solution for purifying BPWW using electrochemical treatment with simultaneous ozonation. According to the UN Water Development

M. Gerić et al. / Ecotoxicology and Environmental Safety 114 (2015) 164–170

Report (2012), as much as 80% of wastewater is released into the environment without any treatment therefore similar purification methods could be implemented in marinas around the world.

2. Materials and methods 2.1. Wastewater treatment and analysis Wastewater was treated in a full-scale plant (Fig. 1) developed according to the patented technology invented by Oreščanin et al. (2013) (patent no. WO 2013/144664). BPWW was collected in the Kaštela Marina, Kaštel Gomilica, Croatia. One thousand litre of wastewater were pumped into one of the three settlement tanks of the treatment plant. After 1 h, the coarse particles settled and 200 L of wastewater was pumped into the reaction vessel equipped with two sets of electrode plates (iron and aluminium). Each set consisted of nine plates separated by an electro insulator (Oreščanin et al., 2011). The surface of each plate was 450 cm2, the distance among the plates was 10 mm, the applied current and voltage were 45 A and 12 V, respectively. Treatment with the iron electrode set lasted 15 min followed by 20 min of treatment with aluminium electrode plates. Simultaneously with electrochemical treatment, wastewater was ozonated with an ozone generator at a rate of 3500 mg/h. Finally, the suspension of electrochemically generated flocks and water was mixed by ozone bubbles for an additional 20 min, while the settlement time lasted 30 min. The analysis of heavy metals in original and treated wastewater was done by energy dispersive X-ray spectrometry as previously described (Oreščanin et al., 2011). pH value was determined by a PHT-027 water quality multiparameter monitor (Kelilong Electron, China). The colour, turbidity, chemical oxygen demand (COD) and total organic carbon (TOC) were determined by HACH DR890 colorimeter (Hach Company, USA). For sample digestion, a DRB 200 reactor (Hach Company) was used. All measurements were done in pentaplicates. 2.2. Ames mutagenicity test Salmonella typhimurium TA98 and TA100 strains were kindly provided by Maron and Ames (University of California, CA, USA). The modified standard plate incorporation procedure–preincubation assay was used (Maron and Ames, 1983) where 100 μL of Salmonella typimurium strains (TA98 and TA100) was incubated with different concentrations of BPWW (1%, 5%, 25%, 50%, and original) for 20 min at 37 °C. Subsequently, 2 mL of top agar was added to minimal Vogel-Bonner plates and incubated at 37 °C for

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72 h. Methyl methane sulphonate (MMS, Kodak, NY, USA) was used as positive control without metabolic activator and 2-aminoantracene (2-AA, Sigma, Chemical Company, MO, USA) with S9 activation. The induction of mutation was obtained after dividing the number of induced revertants with the number of spontaneous revertants (Durgo et al., 2009). Each experiment was repeated twice and three plates were taken for each concentration. A positive result was considered to be achieved when the number of revertants exceeded the corresponding control value 2.5 times. 2.3. Cell treatment Peripheral blood was taken from a healthy, female, nonsmoking donor. The study was a part of a project approved by the Institutional Ethics Committee and observed the ethical principles of the Declaration of Helsinki. A volume of 100 μL of untreated and purified boat washing wastewater was then added to a blood volume of 900 μL, whereas distilled water was used for negative control. A total of 1 mL of each sample was placed into the incubator at 37 °C in an atmosphere with 5% CO2 (Heraeus Heracell 240 incubator, Germany) and incubated for 4 and 24 h. 2.4. Cell viability test Cell viability test was performed according to Duke and Cohen (1992). The HPBLs were isolated using modified Ficoll-Histopaque centrifugation technique (Singh, 2000), stained with 2 μL of acridine orange/ethidium bromide (Sigma) and analysed using an Olympus BX51 microscope (Japan). Depending on the staining, 100 cells per repetition were classified either viable (uniform green) or non-viable (uniform orange) and four repetitions were done per sample. 2.5. Chromosome aberrations test The chromosome aberration test was performed in agreement with IPCH guidelines (Albertini et al., 2000) with minor modifications (Gajski et al., 2014). Whole blood cultures were established, where 0.5 mL of each sample was added to 6 mL of RPMI 1640 medium (Gibco, Life Technologies, USA) containing antibiotics, 1 mL foetal bovine serum (Gibco), and 0.1 mL phytohaemagglutinin (Remel Europe, UK). The cultures were placed at 37 °C and 5% CO2 and incubated for 48 h. Colchicine (Sigma, 0.004%) was added 4 h prior to the harvest which arrested lymphocytes division in metaphase. When the incubation period expired, the cultures were fixed in freshly prepared ice cold methanol/acetic acid (3:1 v/v) dropped onto slides and air-dried. After staining with 5% Giemsa solution (Merck, Germany), a total of 200 metaphases per

Fig. 1. A full-scale plant for boat pressure washing wastewater treatment installed in Kaštela Marina, Kaštel Gomilica, Croatia (capacity 5 m3/day). (A) exterior view describing the location of the plant in the Kaštela Marina. (B) interior view of the plant showing: two receiving horizontal tanks for coarse particle separation (1000 L each) (1), reaction vessel with iron and aluminium electrode plates (200 L) (2), corona discharge based ozone generator (3), two sedimentation tanks for the separation of purified water from the sludge (300 L each) (4), and untreated and purified water samples (5).

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slide were analysed and the total number of chromosome aberrations was evaluated. 2.6. Comet assay The alkaline comet assay was performed according to Singh et al. (1988) with minor modifications (Gerić et al., 2012), during which 3 layer-agarose-samples were placed in freshly prepared lysis solution and left overnight. The slides were kept in denaturation for 20 min in freshly prepared electrophoresis buffer followed by another 20 min of electrophoresis in horizontal electrophoresis tank at 25 V (300 mA). The slides were then washed with PBS, stained with 10 μg/mL ethidium bromide, and stored at 4 °C prior to analysis. A total of 100 randomly captured nuclei per slide was analysed in duplicate using the Comet Assay II (Perceptive Instruments Ltd., UK) image analysis software. For data presentation, we chose the tail intensity and the number of atypically sized tails (AST) representing the number of comets per each slide with tail intensity exceeding the 95th percentile of their distribution in the control. 2.7. Micronucleus test The micronucleus test was performed following guidelines by Fenech and Morley (1985) with minor modifications (Gerić et al., 2012) where 0.5 mL of blood was added to Euroclone medium (Chromosome kit P, Euroclone, Italy) and incubated at 37 °C and 5% CO2 for 72 h. Then, 28 h before the end of the incubation period, Cytochalasin-B (Sigma) was added at a final concentration of 6 μg/mL in order to prevent cytokinesis. Fixed HBPLs were stained with 5% Giemsa solution (Merck). A total of 1000 binucleated lymphocytes were analysed per slide in duplicate. The frequency of micronuclei (MNi), nuclear buds (NBs), nucleoplasmic bridges (NPBs), and the cytokinesis-block proliferation index (CBPI) was scored according to Fenech (2007). 2.8. Statistical analysis For statistical evaluation, the STATISTICA 7.0 software package was used. The level of statistical significance was set at p o0.05. Differences among the groups were assessed by one-way analysis of variance (ANOVA) and Newman–Keuls test.

3. Results 3.1. Chemical characterisation of BPWW prior/after the treatment The BPWW was characterized (Table 1) by high values of colour (7943 PtCo units) and turbidity (1220 NTU). Among the measured heavy metals, copper exceeded the limit value 96 times, vanadium 18.3 times, zinc 15.7 times, iron 5.2 times, chromium 2.9 times, and lead 1.7 times. Both TOC and COD also exceeded limit values by 5 and 3.3 times, respectively. The combined electrochemical treatment/ozonation resulted in a clear, colourless, and odourless effluent with the values of all measured parameters in accordance with regulated values. Removal of colour, turbidity, and heavy metals ranged from 99.4% to 99.9%, while the removal of TOC and COD was higher than 85%.

Table 1 Mean values 7standard deviations of the parameters measured in the wastewater originated from boat pressure-washing prior/after the treatment from five independent analyses, Limit values (LV) and average removal efficiency of each parameter. Parameter

Wastewater before treatment

LVa

Treated water Measured value Average removal efficiency (%)

Colour (PtCo) Turbidity (NTU) V (mg/L) Cr (mg/L) Fe (mg/L) Ni (mg/L) Cu (mg/L) Zn (mg/L) Pb (mg/L) pH TOC (mg/L) CODCr (mg/ L)

7943 7127

–

28 74

99.7

1220 780

–

4 72

99.7

0.917 70.037 1.43 70.071 10.3 70.33 0.121 70.002 48.0 70.912 31.2 70.481 0.872 70.021 7.34 70.038 149 74.4 412 77.9

0.05 0.5 2 0.5 0.5 2 0.5 6.5–9 30 125

0.006 70.0001 0.008 70.0002 0.011 70.0008 0.002 70.0001 0.014 70.0002 0.054 70.0008 0.003 70.0001 7.43 70.06 21 70.9 51 72.1

99.4 99.4 99.9 99.9 99.9 99.8 99.7 – 85.9 87.6

a Ordinance on the limit values for the emission of wastewaters (Off. Gazzette 80/13).

Table 2 Mutagenicity effects of different concentrations of wastewater produced by boat pressure-washing in Salmonella typhimurium strains TA98 and TA100 in the absence and presence of S9 fraction (Values are presented as mean values7 standard deviations of triplicate plates.). Dose

Number of revertants per plate TA98

NC 1% 5% 25% 50% ORG PUR PC

TA100

 S9

þS9

– S9

þ S9

12.5 7 2.2 12.0 7 1.0 11.0 7 1.4 10.5 7 0.7 11.5 7 0.7 10.0 7 2.2 11.0 7 1.4 212.0 7 12.7a

18.5 7 0.7 15.5 7 0.7 16.5 7 0.7 15.0 7 1.4 17.5 7 0.7 17.0 7 14.0 16.5 7 2.1 293.0 7 11.3a

197.0 7 7.1 185.5 7 9.2 199.5 7 0.7 186.5 7 4.9 187.5 7 12.2 174.0 7 6.3 195.5 7 1.0 487.0 7 7.8a

241.5 7 3.5 240.0 7 9.9 221.0 7 4.2 230.0 7 4.9 226.0 7 18.3 200.0 7 5.6 253.0 7 1.1 670.0 7 16.9a

a ( Z 2.5-times control sample value); ORG, original wastewater sample; NC, negative control; PUR, purified wastewater samples; PC, positive controls: methyl methane sulphonate (MMS) was positive control without metabolic activator and 2-aminoanthracene (2-AA) with S9 activation.

presence and absence of the S9 mix, respectively, for both of the tested strains (Table 2). 3.3. Cell viability A significantly lower number of viable cells was observed in samples exposed to untreated BPWW in both the 4 h (31.6% decrease) and 24 h (35.2% decrease) exposure period. At the same time, cells exposed to purified wastewater did not show lower cell viability for both exposure periods (Fig. 2A). 3.4. Chromosome aberrations

3.2. Ames mutagenicity assay BPWW failed to induce point mutations at all of the concentrations tested (1%, 5%, 25%, 50%, and 100%) in the presence or absence of the S9 mix. Meanwhile, 2-AA and MMS-treated bacterial strains showed an increased number of revertants in the

After 4 h of exposure, there were no differences between the tested samples, although a slightly higher total number of aberrations was detected in the untreated BPWW sample (1.572.1) compared to 0.5 7 0.7 in both the control and purified samples. After 24 h of exposure, the sample treated with BPWW had a

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Fig. 2. The effects of untreated and purified wastewater produced by boat pressure-washing on the viability and genome integrity of human peripheral blood lymphocytes after exposure periods of 4 and 24 h. The percentage of viable cells (A), total number of CAs (B), percentage of DNA in comet tails (C) and number of ASTs (D), total number of: MNi (E), NBs (F), NPBs (G) and CBPI (F) are presented. Values in A, B, D–F are given as mean values 7standard deviation; whereas C is given as mean values7 standard error. *Statistically significant compared to corresponding control (p o 0.05). AST, atypical sized tail; CA, chromosome aberration; CBPI, cytokinesis-block proliferation index; MN, micronucleus; NB, nuclear bud; NPB, nucleoplasmic bridge.

significantly (p o0.05) higher total number of aberrations (9.07 4.2) compared to the control sample (0.5 70.7). The purified sample again did not differ significantly (2.0 71.4) from the negative control (Fig. 2B).

3.5. Comet assay Significantly (po 0.05) higher values of tail intensity and AST for samples treated with original wastewater after exposure periods of 4 and 24 h were observed. The purified samples did not differ from the corresponding negative control after the same exposure periods (Fig. 2C and D).

3.6. Micronucleus test HPBLs exposed to untreated BPWW resulted in a significantly higher total number of MNi (19.5 73.5 and 36.5 70.7) compared to control (4.5 70.7 and 9.0 71.4) after 4 and 24 h, respectively. At the same exposure period the total number of NBs was also higher after being exposed to untreated BPWW (13.5 7 2.1) compared to control sample (5.0 71.4), while there was no difference in the number of NPBs after treatment. On the contrary, purified wastewater did not show any significant differences in MNi, NBs, and NBPs compared to the control sample. After both 4 and 24 h of exposure, CBPI results did not show any significant differences compared to the corresponding controls (Fig. 2E–H).

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4. Discussion Finding an effective way of preventing aquatic organisms to attach to the submerged surfaces of vessels is one of the main issues in ship transport, primarily due to fuel consumption and shipping costs. Heavy metal-based anti-fouling paints are commonly used and the greatest problem that arises is their leaching into aquatic environments (Jessop and Turner, 2011; Karlsson et al., 2010; Ytreberg et al., 2010). Usually, once a year each vessel is dry docked and washed under high pressure, which generates wastewater-containing antifouling paint. The chemical characterisation of wastewater tested in our study showed large amounts of copper, zinc, vanadium, chromium, and iron as well as increased values of organic contaminants, all of which were discharged into the environment. Similarly, high concentrations of heavy metals originating from antifouling paints have been found in marinas and areas with heavy boat traffic (Boxall et al., 2000; Matthiessen et al., 1999). Due to the nature of anti-fouling paint, which prevents organisms such as bacteria, diatoms, spores of macroalgae, protozoa, or larvae of macrofoulers to attach to submerged surfaces (Yebra et. al, 2004), the wastewater tested in our study proved to be cytotoxic to HPBLs. Studies by Karlsson et al. (2010) and Ytreberg et al. (2010) tested the effects of copper and zinc leached from antifouling paints in natural and artificial brackish sea water. Growth inhibition was observed in three trophic levels: bacteria Vibrio fischeri, algae Ceramium tenuicorne, and crustacean Nitocra spinipes larvae. Additionally, CuCl as a source of copper was also lethal to ascidian Botryllus schlosseri with an LC50 of 17.8 mg/L, whereas concentrations lower than LC50 showed immunosuppressive effects on phagocytes and morula cells (Cima and Ballarin, 2012). Short-term exposures to high concentrations managed to decrease phagocytosis by preventing adhesion to the substrate rather than affecting the cytoskeleton. The mechanism of altering cell adhesion could originate from the increase of cellular calcium by affecting Ca2 þ channels, release of Ca2 þ from intracellular stores, or binding to sulfhydryl groups on the membrane proteins involved with Ca2 þ transport, but also from the damage of the systems of integrins (Ballarin et al., 2002; Gonzàlez et al., 2010; Viarengo et al., 1994). The increase of calcium alongside the disruption of mitochondrial crest enzyme activity resulted in a higher percentage of apoptosis and, whereas the neutral red test showed possible alterations in the cell membrane. Taken together, copper exposure demonstrated cytotoxic effects on marine organisms (Cima and Ballarin, 2012). Another endpoint observed in our study was genotoxicity. The results of the chromosome aberration test, comet assay, and micronucleus test demonstrate that BPWW can cause DNA damage to HPBLs. Chemical characterisation showed high concentrations of heavy metals, particularly copper which exceeded the limit values (LV) 96 times. This could have caused the increase of parameters in the genotoxicity assays. Similar negative effects were observed when aquatic organisms (bivalves and fish) were treated with copper sources. The higher comet parameters (longtailed nuclei (LTN) for bivalve Scapharca inaequivalvis and both AST and LTN for fish Sparus aurata) represent an induction of DNA damage in animals’ erythrocytes (Gabbinelli et al., 2003), which is in accordance with our results. A similar effect was observed in a study by Trevisan et al. (2011) on mussel Mytilus edulis haemolymph cells, where the tail intensity increased 70% compared to the untreated control group. Subchronic exposure (25 and 50 μg Cu/L) to copper affected juvenile Centropomus parallelus, in which a higher number of MNi was detected. It should also be noted that the MNi number failed to recover to control values even after 30 days of post-exposure (Oss et al., 2013). In addition, the bioacumulation of copper in fish tissues represents another of its

negative effects, as it has the ability to appear in concentrations higher than found in the environment (Ates et al., 2015). The genotoxicity of chromium and lead has been studied many times before and their negative effects have been recorded in different species and cells (Ahmed et al., 2013; Ciacci et al., 2012; García-Lestón, 2010). As for BPWW, the effects of vanadium and zinc could have had a larger influence on the observed effects as they exceeded the LV 18.3 and 15.7 times, respectively. Vanadium can be considered a slightly genotoxic compound due to its poor absorption; still, it can impair enzymes involved in DNA damage response and become involved in ROS production in animals and humans (Leopardi et al., 2005; Rodríguez-Mercado et al., 2003). On the other hand, zinc plays an essential role in many physiological functions (for instance in superoxide dismutase which is important in antioxidant defence), but certain metal complexes can generate ROS through the catalysis of Fenton-type reactions and cause DNA damage (Linder, 2001). As demonstrated in a paper by Sharif et al. (2012), zinc concentrations within the range of 4– 16 mM provided better viability of human oral keratinocytes cells as well as lower DNA damage compared to cells that were zincdeficient (r0.4 mM) or zinc-replete ( Z32 mM), suggesting that high Zn concentrations can induce DNA damage. The genotoxic potential of BPWW could emanate from multiple additive or synergistic metal interactions as well as ROS production generated by heavy metals. Gabbinelli et al. (2003) and Trevisan et al. (2011) also reported changes in the activity of enzymes or scavenging molecules involved in oxidative stress defence in S. inaequivalvis, S. aurata, and M. edulis, which evidence the formation of oxidative stress. When cells are exposed to ROS generating agents combined with decreased antioxidant capacity, damage in membranes, proteins, and DNA can be expected (Camhi et al., 1995). In the case of the wastewater tested in our study, it is difficult to distinguish whether the antifouling paint was genotoxic thus generating cytotoxicity or if the cytotoxicity of the paint induced cell death which in turn increased the genotoxicity assay parameters. Nevertheless, using a battery of tests, we were able to detect several possible origins of primary DNA damage, where MNi served as reliable biomarkers of chromosome/chromatide breakage or error in chromosome segregation caused by a dysfunctional mitotic spindle. Other alternations in the genome include NPBs, which indicate dicentric chromosomes that arise on the grounds of DNA miss-repair, chromosome rearrangement, or telomere end-fusion. Finally, NBs are formed during the S-phase, representing an extrusion of either amplified DNA or chromatin whose replication has failed, and are considered to be a biomarker of gene amplification and/or DNA-repair complexes (Fenech et al., 2011). On the other hand, using a standard alkaline comet assay, we detected the consequences of DNA strand breaks, DNA–DNA or DNA–protein cross linkage, and alkali-labile sites, i.e. AP (apurinic/ apyrimidinic) sites or baseless sugars (Collins et al., 2008; Kumaravel et al., 2009). From the aspect of mutagenicity, none of the samples tested induced histidine revertants in the presence or absence of the S9 metabolic activator. Similar negative results were observed in leachates produced from the bottom ash of a municipal solid waste in two out of tree sampling sites (Chen et al., 2014), as well as in leachates from river sediments (Magdaleno et al., 2008). The chemical composition of these leachates comprised heavy metals (Cu, Zn, Cr, Pb) similar to those found in the BPWW tested in the present study, but their combinations were different. On the other hand, in a study by Tabrez and Ahmad, (2011) the mutagenicity of wastewater with higher heavy metals concentrations was evaluated. The Ames fluctuation assay with TA102 strains demonstrated that the tested wastewater possesses mutagenic properties. The higher concentrations of heavy metals in wastewater needed to induce mutagenicity may have been result of the

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differences in cellular mechanisms and mechanism of defence between prokaryotic and eukaryotic cells. Furthermore, because of the difference in response to oxidative damage, prokaryotic cells could have been more resistant to the genotoxic effects of certain compounds including heavy metals (Durgo et al., 2009), which were also present in the wastewater tested in our study. Even furthermore, the purification methods used in this study were confirmed as an efficient way of removing toxic compounds from such wastewater. According to the UN World Water Report (2012), groundwater abstraction has risen three times during the last 50 years and by 2050, water demand will increase for another 70%. Taking into account that only 1/3 of wastewater in Central and Eastern Europe is treated, there is room for the implementation of this method in wastewater treatment plants. In a previous study by Oreščanin et al. (2011), it was demonstrated that combining electrochemical methods and ozonation for the removal of inorganic as well as organic contaminants yielded a high efficiency rate. After purifying the BPWW in the present study, neither of the toxicological parameters differed significantly from the values of the corresponding negative controls, indicating that this method could be considered for implementation in marinas. There are several other studies that detected the problem of wastewater discharge and each offered a different solution to the same problem. A similar method for the removal of tin, zinc, and copper was presented by Vreysen et al. (2008), but the removal efficiencies were somewhat lower than in our study. At the same time, using adsorption/flocculation only, the costs of water purification are economically more acceptable. Song et al. (2005) offered a possible way of removing organotin compounds from sandblast waste and wastewater produced during ship washing using heat treatment and solvent extraction, respectively. On the other hand, Mazue et al. (2011) and Pelletier et al. (2009) presented ideas that could be used in the future to avoid the use of paints with biocidal properties. Cleaning hulls using 20 kHz ultrasound is one way how boats can be cleaned effectively and fast, thus generating neither wastewater nor leachate during a ship's voyage (Mazue et al., 2011). Another interesting solution was presented by Pelletier et al. (2009) according to which polymer chitosan, derived from the natural molecule chitin, was used to prevent fouling in cold waters where fouling is slower in general. Their results went in favour of this polymer because it proved to be no worse than paints containing biocides (copper) where they proved to be rather inefficient. At the same time, chitosan is a biodegradable, non-toxic, and non-allergenic green-product.

5. Conclusion The present study proved that BPWW could be cyto/genotoxic to HPBLs. Copper and zinc were detected in the highest concentrations, while vanadium, chromium, iron, lead, COD, and TOC were present in concentrations higher than permissible. After applying electrochemical treatment/ozonation, excellent removal results of over 99% for all heavy metals and over 85% for organic constituents were achieved. Accordingly, the parameters of cytoand genotoxicity assays were statistically higher in untreated wastewater samples compared to control and purified wastewater samples. In order to maintain balance in marine ecosystems and protect the environment from anthropogenic influences, it would be reasonable to use at least one water purifying method before wastewater is discharged into the environment. New environment-friendly ways to minimise fouling or combinations of presently available methods that do not affect non-target organisms will need to be developed.

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Conflicts of interest None declared.

Acknowledgments Supported by the Ministry of Science, Education and Sports of the Republic of Croatia (Grant no. 0022-0222148-2125).

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