Potential use of an ultrasound antifouling technology as a ballast water treatment system

Journal of Sea Research xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Journal of Sea Research journal homepage: www.elsevier.com/locate/seares

Potential use of an ultrasound antifouling technology as a ballast water treatment system Noelia Estévez-Calvara,⁎, Chiara Gambardellaa, Francesco Miragliab, Giovanni Pavanelloa,1, Giuliano Grecoa,1, Marco Faimalia, Francesca Garaventaa a b

Istituto di Scienze Marine (ISMAR), Consiglio Nazionale delle Ricerche (CNR), Via de Marini 6, 16149 Genova, Italy Molecolar Energy Systems (M.E.S) S.r.l., Via Val Lerone 9, 16011 Arenzano, Italy

A R T I C L E I N F O

A B S T R A C T

Keywords: Ballast water Efficacy Marine environment Sensitivity Treatment Ultrasound

The aim of this study was to investigate, at a laboratory scale, the potentialities of an ultrasound-based treatment initially designed to eliminate fouling, as a ballast water treatment system. Therefore, early life stages of three different zooplanktonic species (Amphibalanus amphitrite, Brachionus plicatilis and Artemia salina) were exposed to ultrasound waves (20–22 kHz). The experimental set up included static assays with variations of time exposure (30 s, 60 s and 30 s on/60 s off/30 s on), material of tanks (stainless steel, galvanized steel and plastic) and position of the ultrasound source. Results showed that the treatment efficacy increased from 30 to 60 s and no differences were registered between 60 s-continuous exposure and pulse exposure. The highest efficacy was observed in Experiment I (metal-to-metal contact assay) with a mortality value of 93–95% for B. plicatilis and A. salina. It consisted of organisms located inside stainless steel tubes that were located in direct contact with the ultrasound source and treated for 60 s. Further, we found that, generally, A. amphitrite and B. plicatilis were the most resistant species to the ultrasound treatment whereas A. salina was the most sensitive. We further discuss that US may unlikely be used for commercial vessels, but may be used to treat ballast water in smaller ballast tanks as on board of mega yachts.

1. Introduction

rotifers, crustacean, chaetognaths, cnidarian, mollusks, polychaetes, echinoderms and tunicates (Chu et al., 1997; National Research Council, 1996; Williams et al., 1988). Moreover, many aquatic invertebrates are taken aboard as dormant or larval stages but can reproduce during transit (Raikow et al., 2007). Cases of marine invasions of the last years attributable to the ballast tank vector (Cope et al., 2015; Fofonoff et al., 2003; Vanderploeg et al., 2002) have represented a serious threat to ecosystems, economy and human health (Elliot, 2003; Ruiz et al., 2000). Recognition of this problem by governmental and non-governmental bodies, industries and the global scientific community prompted changes in regulations managing BW. In addition, the International Maritime Organization (IMO) adopted in 2004 the International Convention for the Control and Management of Ship's Ballast Water and Sediments that will enter into force on 8 September 2017. The aim of the Convention is to prevent, minimize and ultimately eliminate the transfer of harmful aquatic organisms and pathogens through the control and management of ship's BW and sediments (Globallast IMO, 2015; IMO, 2004). BW management for vessels

Ballast water (BW) transports daily > 10.000 aquatic species across the oceans around the world (Carlton, 1999; Globallast IMO, 2017) representing the most significant source of dispersion of non-indigenous organisms for coastal, estuarine and inland navigable waters (Carlton, 1985, 1996; National Research Council, 1996; Williams et al., 1988). BW is needed to provide stability and maneuverability during a voyage when ships are not carrying cargo, are not carrying heavy enough cargo, or require more stability due to rough seas (EMSA, 2015). However, the biota found in BW are diverse and predicting the presence of a particular unwanted species in a specific vessel or certifying a vessel as free of or safe from these species is extremely difficult (National Research Council, 1996). Organisms travelling in BW comprise mainly viruses, bacteria, algae, plants, invertebrates and vertebrates (Carlton and Geller, 1993; Ruiz et al., 2000; Williams et al., 1988). Zooplanktonic organisms include those that spend all or a part of their life cycle in the water column such as many types of protozoans,

⁎

Corresponding author at: ISMAR-CNR, Via de Marini 6, 16149 Genova, Italy. E-mail addresses: [email protected], [email protected] (N. Estévez-Calvar), [email protected] (C. Gambardella), [email protected] (F. Miraglia), [email protected] (G. Pavanello), [email protected] (M. Faimali), [email protected] (F. Garaventa). 1 Present address. http://dx.doi.org/10.1016/j.seares.2017.04.007 Received 15 September 2016; Received in revised form 10 April 2017; Accepted 19 April 2017 1385-1101/ © 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Estévez-Calvar, N., Journal of Sea Research (2017), http://dx.doi.org/10.1016/j.seares.2017.04.007

Journal of Sea Research xxx (xxxx) xxx–xxx

N. Estévez-Calvar et al.

did not have taken into consideration other different variables such as time exposures, materials of experimental tanks, positions of the transducer according to the tank or even differences on sensitivity among different target species despite all these parameters may influence the efficacy of BWTSs at full scale. In the present work, we analyzed whether an antifouling prototype system based on US-technology may have a potential use as a ballast water treatment. To this aim, a series of laboratory scale experiments was performed exposing early life stages of three marine invertebrates (two crustaceans, Amphibalanus amphitrite and Artemia salina and one rotifer, Brachionus plicatilis; dimension > 50 μm) to the US treatment. Barnacles and rotifers frequently occur in BW whereas the brine shrimp Artemia sp. has been suggested as a surrogate test organism for BWTSs (Holm et al., 2008; Voigt, 2004). The experimental set up included variations in time exposures, tank materials and position of the US source. The efficacy of the US-treatment and the sensitivity of the different species to the treatment were measured.

includes all the measures aimed at preventing aquatic species from being transported with BW between ports (Buck, 2012). Among the different possible options, the physical and chemical treatment of BW is the most widely studied approach and several technologies and methods have been proposed. Different companies around the world offer different ballast water treatment systems (BWTSs) complying with IMO regulations. At least 51 BWTSs have already received the Type Approval Certification by their respective Administrations and can be used on board ships (IMO, 2017). Available water treatment options can be divided into three categories: mechanical, chemical and physical methods but water treatment may combine also several of these categories (David and Gollasch, 2015). Mechanical methods are essential for water pretreatment by capturing organisms and particles during the ballasting operations; this results in a reduction of the organic material in the ballast tanks (Gregg et al., 2009; Werschkun et al., 2012). Filters, hydro cyclones and separation methods by chemical enhancement (i.e. flocculation and coagulation methods) represent examples of mechanical methods (Lloyds, 2012). Chemical options include any method which neutralize aquatic organisms through addition of any active chemical substance; they include oxidants, such as ozone, sodium hypochlorite, chlorine, chlorine dioxide, hydrogen peroxide, hydroxyl radicals and several combinations of them, called multioxidants. Organisms can be neutralized also by addition of other biocides, or any substance that will form a biocide in reaction with seawater, or chemicals that cause de-oxygenation (Dragsund et al., 2003). Physical methods focus on changing the physical properties or hydrodynamic characteristics of the water to remove organisms, and these approaches do not release residuals into the environment (Taylor et al., 2002). These systems make organisms harmless usually damaging their structure or tissues acting through rapid stress variations. Some physical measures are represented by the application of ultrasound (US) and cavitation, which lead to the mechanical destruction of particles and organisms; other include pressure fluctuations, nitrogen/ air super saturation, high energy techniques, such as heating or ultraviolet (UV) irradiation which causes a molecular change in the DNA of microorganisms, killing them or preventing from reproduction. Among physical methods, UV and US represent about 20% of the total treatment units already installed in ships (Lloyds, 2010). Power US is a relatively new technology in BW treatment. It refers to the section of the sound spectrum from 20 kHz through to around 1 MHz (Leong et al., 2011). US are generated through a transducer that converts mechanical or electrical energy into high frequency vibration or acoustic energy producing physical or chemical effects to the liquid being processed (Mahvi, 2005; Sassi et al., 2005). US are not being currently used on board as commonly as other BWTSs based on mechanical, chemical or even physical methods like UV (IMO, 2017; National Research Council, 1996). In fact, only two BWTS that employ US technology have currently received the Type Approval Certification by their respective Administrations (IMO, 2017). In addition, the information regarding the use of US in BWTSs and how these treatments can influence the mortality of marine planktonic stages is still scarce. Only a few works have demonstrated the fatal effect, at laboratory scale, of US to marine organisms belonging to different taxa (Guo et al., 2011a, 2011b, 2012; Holm et al., 2008; Sassi et al., 2005). Gavand et al. (2007) confirmed the effectiveness of US after a 20 mintreatment that induced a 100% of mortality in larval stages and adults of the crustacean Artemia salina. Even, the treatment was approximately 1 and 4 times as effective at inactivating adults of brine shrimp as other treatments such as hydrogen peroxide or ozone. Moreover, Guo et al. (2011a, 2011b, 2012) observed the most effective inhibition of settlement in cyprid larvae of Amphibalanus amphitrite after a 23 kHz-US treatment in comparison with other higher US frequencies (63, 102 kHz). An US-treatment operating at 20 kHz was also able to kill barnacle larvae within 45 s (Seth et al., 2010). Apart from variations on frequency and power, experimental set up of previous research works

2. Material and methods 2.1. Test organisms The barnacle A. amphitrite, the brine shrimp A. salina and the rotifer B. plicatilis were used as test organisms. Naupliar larvae (Stage 2) were obtained directly from laboratory cultures of brood stock of A. amphitrite. Adult barnacles were reared in aerated glass beakers with 700 mL of Filtered Natural Sea Water (FNSW) at 0.45 μm at 37‰ salinity and at 20 °C. Organisms were fed each two days with a mixed diet of Tetraselmis suecica and A. salina. Barnacle spawning was induced by a thermal shock and naupliar larvae (Stage 2) were collected and immediately used in the experiments, as described by Faimali et al. (2006). 24 h post-hatching individuals of the rotifer B. plicatilis (MicroBioTests Inc., Gent, Belgium) were obtained from dehydrated cysts, following the Rotoxkit M protocol, as described in Garaventa et al. (2010). Briefly, dehydrated cysts were transferred into Petri dishes (ø 6 cm diameter), each containing 4 mL of 0.22 μm FNSW at 20‰ salinity. Incubation was performed in a thermostatic room, at 25 °C for 24 h, with a photoperiod of 16 h light: 8 h dark (Lubzens et al., 1985). Newly hatched larvae were obtained and acclimatized for 2 h in FNSW (0.22 μm) at 35‰ salinity before their use in the assays. With regards to A. salina, a volume of 800 mL of FNSW at 0.22 μm (salinity of 37‰) was used to hydrate 0.5 g of dry cysts of brine shrimp (BLUE CO. Soc. Coop Arl, Italy). Brine shrimp culture was maintained with aeration at 28 °C, under 16 h light: 8 h dark conditions. After 24 h, Instar I stage larvae were separated from non-hatched cysts, based on their positive photo taxis. Hatched larvae were transferred with a Pasteur pipette into a beaker and immediately used in the experiments. 2.2. Ultrasound prototype The US prototype used to perform the experimental assays was kindly provided by the Molecolar Energy Systems Company (M.E.S s.r.l, Arenzano, Italy). For each experiment, the prototype was connected to an electrical source power of 220–240 V and 16 A, and the signal was activated. The US signal transmitted from the function generator was fed into the power amplifier and used to drive the connected piezoelectric transducer with a power of 200 W. The transducer included a conical horn made of aluminum and two piezoelectric discs (Fig. 1). A frequency of 20–22 kHz was selected to perform all tests. During the experimental procedure, no temperature elevation was registered as measured with a thermometer (resolution 0.5 °C). 2.3. Experimental set up An ultrasound system created and designed for applications in 2

Journal of Sea Research xxx (xxxx) xxx–xxx

N. Estévez-Calvar et al.

salinity. The Fig. 2 shows the transducer (T) used to perform 1) Metalto-metal contact assays (Experiment I), and 2) Immersion assays in metal and plastic tanks (Experiments II, III and IV). In experiment I, organisms were exposed to US waves in stainless steel tubes filled with 20 mL of FNSW. Tubes were left out of the water and put into direct contact with the surface of the US transducer. This in order to simulate a fouling treatment where the transducer is in direct contact with the pipeline subject to treatment. Assays were performed separately for each species (Fig. 2, Experiment I). Experiment II simulated the treatment of ballast water by means of immersion assays performed in metallic tanks made by galvanized steel (Fig. 2, Experiment II). Organisms were located inside stainless steel tubes as in experiment I. In addition, tubes were placed in the surface of the water at a distance of 5 cm (measured horizontally) from the US source. To analyze the influence of the transducer position according to the metal tank, in assay II.A the transducer was located not in contact with the bottom of the tank, at a distance of 5 cm, whereas in assay II·B the transducer was located in direct contact. Fig. 2, Exp. III shows experiment III which included both plastic (Experiment III.A-B) and metallic (galvanized steel) tanks (Experiment. III·C-D). Four assays were performed and the transducer was located ever in the same position (not in contact with the bottom of the tank at 5 cm distance) this to analyze the effect of the tank material on the final efficacy of the treatment. In addition, the potential effect of a physical barrier was studied. To this aim, organisms were exposed to US inside stainless steel tubes (Experiment III.A, C) and floating net container (Experiment III·B, D). Along the whole experiment III, organisms were placed in the surface of the water at a distance of 5 cm (measured horizontally) from the US source. Last, in experiment IV organisms were located inside floating net containers at different horizontal distances (where x = 5–100) from the

Fig. 1. Picture of US prototype used for the experiments.

fouling scenarios was tested for its efficacy as a potential BWTS. Early developmental stages of test organisms were exposed to US waves through static experiments. A scheme of the ultrasonic experimental set up is given in Fig. 2. The US irradiation system was tested in continuous and intermittently by performing contact assays out of the water and immersion assays. All experiments were performed with FNSW at 37‰

Fig. 2. Diagram of the ultrasonic experimental set up. Four experiments were performed (I, II, III and IV). Organisms were exposed to US waves in stainless steel tubes (Experiments I, II, III. B and III.D) and floating net bags (Experiments III.A, III·C and IV). The transducer (T) was used to perform metal-to-metal contact assay (Exp. I), immersion assay in metal tanks (Exp. II and III·C-D) and in plastic tanks (Exp. III.A-B and IV). Immersion assays were performed with “T” in direct contact (Exp. II·B) and at 5 cm distance from the tank bottom (Exp. II.A, III and IV). Organisms were placed at 5 cm (measured horizontally) from the US source (Exp. II-III) and at different horizontal distances (x cm, where x = 5–100) from the US apparatus (Exp. IV).

3

Journal of Sea Research xxx (xxxx) xxx–xxx

N. Estévez-Calvar et al.

The US transducer was immersed and fixed at a vertical distance of 5 cm from the bottom of the tank. Organisms were placed in floating net containers, fixed in specific points (at horizontal distances ranging from 5 cm to 100 cm from the US source). Mortality rate was evaluated using a stereomicroscope after an US continuous treatment of 60 s. Controls included organisms not exposed to US, immersed at a distance of 5 cm from the US prototype.

Table 1 Factors and corresponding levels involved in the ANOVA statistical analysis. Factors Exp. I Exp. II

Exp. III

Exp. IV

1. 2. 1. 2. 3. 1. 2. 3. 4. 1. 2.

US exposure mode Organisms US exposure mode Transducer position Organisms US exposure mode Container Organism exposition Organisms US source distance Organisms

Levels 60 s, 30 s, pulse mode A. salina, A. amphitrite, B. plicatilis 60 s, 30 s, pulse mode No contact, contact A. salina, A. amphitrite, B. plicatilis 60 s, 30 s, pulse mode Plastic, metal Net container, stainless steel tube A. salina, A. amphitrite, B. plicatilis 5–10–20-30-40-50-60-70-80-90-100 cm A. salina, A. amphitrite, B. plicatilis

2.4. Statistical analysis All experimental assays were carried out in triplicate. All results are expressed as the mean (M) ± standard error (SE). Two-way analysis of variance (ANOVA) tested for differences in the percentage of mortality in Experiments I and IV; for experiment II and III a three-way and fourway one ANOVA, were applied respectively. All the factors involved in the analysis were considered fixed and orthogonal. In detail, variables tested for each of the experiments are included in Table 1. Pair wise comparisons (SNK-tests) were used to test for differences in larval mortality among factors at each level within each one. Prior to analysis, data were tested for homogeneity of variance using Cochran's C-Test (Underwood and Chapman, 1997).

US apparatus (Fig. 2, Exp. IV) in order to study the influence of the transducer position in the final efficacy. All experiments performed here were repeated three times. For all treatments and following exposure to US waves, organisms were transferred into Petri dishes and the percentage of mortality was recorded under a stereomicroscope and compared to the control (organisms not exposed to US waves). Organisms were counted as dead when no movement was observed for 5 s.

3. Results

2.3.1. Experiment I: metal to metal contact assay Cylindrical stainless steel tubes were used as metallic test tanks, as shown in Fig. 2 (Exp. I). 20 organisms belonging to each species were introduced into the tubes, each filled with 20 mL of FNSW. Treatments consisted in exposing larvae to US waves for 30 and 60 s (continuous exposure), separately. A pulse exposure test was also performed by switching US generator 30 s on, 60 s off and 30 s on. The tubes were in contact with the US apparatus. Following the exposure, the percentage of mortality was registered.

3.1. Experiment I: metal to metal contact assay Results of sensitivity obtained for target species exposed to US by direct contact between experimental tank and transducer are shown in Fig. 3. The mode of exposure to US had a significant effect on larval mortality (p < 0,01; F = 332,92) inconsistently with the tested organisms (US exposure mode X Organism p < 0,01; F = 47,48). In particular, mortality increased significantly with exposure time (30 vs 60 s), for the continuous treatment, whereas no significant differences were observed between 60 s exposure and pulse US treatment for any treated species. Considering the more effective mode of US exposure (60s and pulse) the results highlighted that A. salina and B. plicatilis were the more sensitive organisms showing a percentage of mortality very close to 100%.

2.3.2. Experiment II: immersion assay in metal tank A cylindrical metal tank made of galvanized steel was filled with 5 L of FNSW. For each species, 20 organisms were introduced in stainless steel tubes fitted with buoyant rings. Tubes were filled with 20 mL of FNSW and placed inside the metal tank. The US transducer was placed at a vertical distance of 5 cm from the bottom of the metallic tank, in order to avoid the direct contact (Fig. 2, Exp. II.A) and on the bottom of the tank (Fig. 2, Exp. II·B). Tubes were fixed with clamps and a floating support, respectively, to avoid undesirable movements caused by the vibration of the US apparatus. After continuous (30 and 60 s) and pulse, tubes were meticulously rinsed with FNSW, before recording mortality under a stereomicroscope as mentioned above.

3.2. Experiment II: immersion assay in metal tank Results on the percentage of mortality in stainless steel tubes after immersion assay in metallic tank are shown in Fig. 4. Results with the transducer not in direct contact and in contact with the test tank showed overall a low efficacy of the treatment since all mortality values

2.3.3. Experiment III: immersion assay in plastic and metallic tanks A third experiment (Exp. III.A-D) was performed in a plastic (Fig. 2, Exp. III.A, B) and galvanized steel metallic tank (Fig. 2, Exp. III·C, D) introducing about 30–50 organisms in a net container (Experiment III.A, C) and stainless steel tubes (Experiment III·B, D) as described in paragraph 2.3.2. The net container consisted of a nylon mesh of 80 μm. The plastic tank was rectangular and filled with a total volume of 5 L of FNSW. In both assays, the US prototype was located at a vertical distance of 5 cm from the bottom of the plastic tank. Exposure modes included continuous (30 and 60 s, separately) and pulse. Dead organisms were counted as indicated above. Based on the results obtained in experiment II and considering the attenuation of US waves in plastic materials (Raišutis et al., 2008) the immersion assay in direct contact with the plastic tank was not performed. 2.3.4. Experiment IV: ultrasound efficacy vs distance assay To evaluate the effect of the distance between the US source and the target organisms, on the treatment efficacy, a plastic tank containing a volume of 320 dm3 of FNSW was used, as indicated in Fig. 2 (Exp. IV).

Fig. 3. Effect of US exposure mode on A. amphitrite, B. plicatilis and A. salina mortality at 20–22 kHz in metal-to-metal contact assay. Results are reported as M ± SE, n = 3.

4

Journal of Sea Research xxx (xxxx) xxx–xxx

N. Estévez-Calvar et al.

Fig. 4. Effect of US exposure mode and transducer position on A. amphitrite, B. plicatilis and A. salina mortality at 20–22 kHz in metallic tank after immersion assay with US transducer not in contact and in direct contact with the test tank. The US exposure was in continuous (30, 60 s) and pulse mode (30 s on/60 s off/30 s on). Results are reported as M ± SE, n = 3.

ison showed that organisms exposed to US inside the net container are more affected by ultrasound. The significant interaction of the above mentioned factors further sustains the consistency of these results (US exposure mode X Container X Organism Exposition p < 0,01; F = 9,56). The highest mortality values (> 60%) were registered in brine shrimps exposed to 60s US mode in net containers immersed in a plastic tank (Fig. 5) whereas the less sensitive organisms were barnacles and rotifers.

were < 60% for all species. As already observed, three-way ANOVA highlighted that in the experiment I, the mode of exposure to US had a significant effect on larval mortality (p < 0,01; F = 31,16) inconsistently with the tested organisms (US exposure mode X Organism p < 0,01; F = 9,92). On the contrary, the transducer position had an overall not significant effect on larval mortality (p = 0,28; F = 1,17) and the effect of prolonged exposure to ultrasound results to be independent from the transducer position (US exposure mode X Transducer position p = 0,4; F = 0,99). The effect of the different treatment is species specific as indicated by the not significant interaction among the three factors involved in the analysis (US exposure mode X Transducer position X Organisms p = 0,26; F = 1,33). The pair wise comparisons (SNK-tests) highlighted that A. salina showed the highest sensitivity along the whole experiment and for each exposure mode, in comparison with the two other species. Very low percentage of mortality was observed in barnacle nauplii or rotifers for all US exposure times (< 25% mortality).

3.4. Experiment IV: ultrasound efficacy vs distance assay The percentage of mortality after 60 s-US exposure, at a distance from US source that ranged from 5 cm to 100 cm, is shown in Fig. 6. The two-way ANOVA showed that the distance of the US source has a significant effect on invertebrate mortality (p < 0,01; F = 228,75) consistently with the tested organisms has indicated by the positive interaction of the two factors (p < 0,01; F = 25,57). The a posteriori comparison of the means pointed out that the general trend, for all tested species, was a decrease of mortality with increasing distance from the US source and allowed identifying a No Observed Effect Distance (NOED) that corresponded to 90, 80 and 40 cm for A. salina, B. plicatilis and A. amphitrite, respectively. When comparing mortality values for each species at a specific distance from the US source, A. salina resulted to be the most sensitive organism at 5 and 10 cm (mortality percentage equal to 80%, respectively) and at 60 and 70 cm. From the distance of 20 cm to that of 50 cm, B. plicatilis resulted the most affected by US whereas any difference disappeared when the transducer was positioned at a distance higher than 80 cm.

3.3. Experiment III: immersion assay in plastic and metallic tanks The overall not significant effect of the transducer position on invertebrate larval mortality observed in the Experiment II together with attenuation of US waves in plastic materials observed by Raišutis et al., 2008, drove to select the not in contact mode also being that way of exposure more representative of the real use in ballast water tanks. Fig. 5 shows the percentage of mortality registered after the exposure assay with organisms placed in net containers and stainless steel tubes floating in plastic and metallic tanks. The four-way ANOVA confirmed that the 60s and pulse mode were the most effective mode of exposure to US (p < 0,01; F = 336,55) in fact, all species registered mortality values increased with exposure time (30 vs 60 s; 30 s vs pulse) whereas no significant differences were observed between 60 s continuous treatment and the pulse US treatment for all species. The effectiveness of the prolonged or pulse mode of exposure is consistent with the tank material as indicated by the significant interaction of the two factors (US exposure mode X Container p < 0,01; F = 14,00) with an overall higher effect of US in plastic container as indicated by SNK-tests. The effect trend is the same even considering the interaction (US exposure mode X Organism Exposition p < 0,01; F = 26,47). Pairwise compar-

4. Discussion BWTSs currently available used to comply with the IMO Convention for the Control and Management of Ship's Ballast Water and Sediments must be safe in terms of the ship, its equipment and the crew, being environmental acceptable, demonstrate practicability, be effective in terms of costs and last, meet the biological effectiveness requirements stated by the Convention (IMO, 2004). In order to meet the last criteria, BWTSs should remove, or otherwise rendering Harmful Aquatic Organ5

Journal of Sea Research xxx (xxxx) xxx–xxx

N. Estévez-Calvar et al.

Fig. 5. Effect of US exposure mode on A. amphitrite, B. plicatilis and A. salina mortality at 20–22 kHz, with organisms placed in net containers and stainless steel tubes floating in plastic and metal tanks with the transducer not in contact with the tank. The US exposure was in continuous (30, 60 s) and pulse mode (30 s on/60 s off/30 s on). Results are reported as M ± SE, n = 3.

Comb™ BW 250 (Aquaworx ATC GmbH), employ both a mechanical treatment (i.e. filtration) as a pre-treatment step to remove organisms ≥ 20 μm in order to prevent microorganisms and sediments entering ballast tank. Following filtration, OceanGuard® BWMS employs electrocatalysis (chemical treatment) able to produce large numbers of hydroxyl radicals and other highly active oxidizing substances. Then, US clean the surface of the electrocatalysis unit regularly to keep the long-term treatment effectiveness of the electrocatalysis material (Headwaytech, 2017). Besides, AquaTriComb™ BW 250 applies a filtration stage followed by a treatment with UV light to which the water is exposed inside UV reactors and then, US technology cleans the UV Quartz sleeves avoiding accumulations of biofilms and/or inorganic salts. This assures that the produced UV light is able to penetrate the water to achieve the required killing rate of the organisms (MEPC 67/INF.28 8 August, 2014; Veldhuis, 2011). The present study showed that mortality rate of A. amphitrite, A. salina and B. plicatilis increased rapidly with increasing exposure time, from 30 s to 60 s (continuous mode), in all the assays performed in this work. Exposure mode has been defined together with the power level as the most important practical parameter in disinfection technologies. Indeed, these two parameters determine treatment cost and effectiveness (Seth et al., 2010). On the other hand, both continuous exposure (60 s) and pulse treatment resulted in the same efficacy pattern in all the experiments described here. A similar observation was made by Guo et al. (2012), who found an equal rate of barnacle settlement inhibition after a continuous and an intermittent US treatment with short off times. Since intermittent US assays were performed here in static conditions, avoiding any movement of the water, a potential realistic application at full scale of the system should take into account some additional aspects. For instance, results could be different in dynamic conditions as off time may decrease the treatment efficacy. Nevertheless, there is evidence that an intermittent treatment may be advantageous for an on-board application, as it would result in a much reduced power consumption and a potential extension of ultrasonic devices lifespan (Guo et al., 2012). The Experiments I and II, performed by using tanks of different materials and considering two different positions of the US transducer (in contact with the tank and not), highlighted a change in the response

Fig. 6. Effect of US source distance on A. amphitrite, B. plicatilis and A. salina mortality at 20–22 kHz, in plastic tank, after a continuous exposure (60 s) to US treatment. Results are reported as M ± SE, n = 3. Black dot (·) indicate the NOED values and crosses (+) the most sensitive organisms within each distance level (p < 0,01).

isms and Pathogens in BW not viable. In this work, different laboratory experiments were performed in order to verify the biological effectiveness of US treatment (20–22 kHz) against three representative zooplankton species. The same frequency range used here was demonstrated to be effective in the application of power US with different purposes including BWTSs (Brizzolara et al., 2006; Holm et al., 2008; Viitasalo et al., 2005), inhibition of fouling settlement (Guo et al., 2011a, 2011b; Kitamura et al., 1995; Seth et al., 2010), industrial water treatment and wastewater decontamination (Arrojo et al., 2008; Gogate, 2002; Mason et al., 2003; Sangave et al., 2007; Scherba et al., 1991), among others. In this work, the prototype used employs US technology as a single treatment to sterilize directly the BW. This aspect contrasts with the already Type Approved BWTS available on market where US technology acts a secondary treatment addressed to the self-cleaning of other treatment technologies coupled in the same BWTS. For instance, the already Type Approved BWTS OceanGuard® BWMS (Headway Technology Co., Ltd) and AquaTri6

Journal of Sea Research xxx (xxxx) xxx–xxx

N. Estévez-Calvar et al.

for all target species. In fact, this exposure method demonstrated to be the second most effective for eliminating zooplankton species. Mortality of Artemia reached the 90% after a continuous one-minute treatment. Floating net containers led to a higher exposure of organisms to US waves, and plastic was able to transmit more effectively these waves. These results contrasted with observations made by Gavand et al. (2007) who found only mortality values of 50% for Artemia larvae (inside 2-L polypropylene bottles) after a five-minute treatment with an US frequency of 1.4 kHz. Lürling and Tolman (2014) needed 135 min to achieve the 100% of mortality of 15 individuals of D. magna exposed to a 20 kHz- US treatment in Perspex tanks. These findings highlighted the importance of tanks material when using US as water treatment system. Given the efficacy observed in plastic tanks experiments with organisms inside net containers and transducer not it contact with the bottom of the tank (Experiment III.A), the same approach was applied in the experiment IV (Fig. 6). Following the highest effectiveness registered in previous experiments performed in the 60 s-US mode, the same exposure mode was used in this assay. In the attempt to elucidate the effect of horizontal distance to the transducer, we included it as an additional parameter. It was possible to observe how the effectiveness of the US treatment against the different invertebrate's species decreased with the horizontal distance. After one-minute of exposure to US, the survival of all species decreased significantly in comparison to the control, between 5 cm to 70 cm of distance. At a distance of 90 cm, Artemia reached the No Observed Effect Distance (NOED) where any significant difference is observed in comparison to the control. For barnacles and rotifers, the NOED was reached at a 40 and 80 cm-distance, respectively. Barnacles and brine shrimp larvae followed a linear relationship between distance and effect. This trend was not observed in rotifers, whose behavior was completely different. From 5 cm to 50 cm, the response was nearly invariable and rotifers did not show any decrease in the mortality. The percentage of mortality at 50 cm was higher than 50% and significantly different to barnacle and brine shrimp. Only after 50 cm distance, rotifers mortality rate decreased. It was interesting to note how at 5 cm distance from the US source, mortality values for A. amphitrite, B. plicatilis and A. salina were the same observed in experiment III.A (49%, 53% and 84%, respectively) where organisms were located inside net containers and exposed in plastic tanks with the transducer not in contact with the bottom (Fig. 5). This confirmed the consistency of the experimental set up. Except for metal-to-metal contact assay (Experiment I) and distance assay (Experiment IV) at distances between 20 and 50 cm, A. salina was the most sensitive organism in all experiments performed in this work. The brine shrimp was the largest organism among the tested ones, and this may be the reason of its higher vulnerability in response to US waves. Holm et al. (2008) suggested that an US treatment working in the range of 19–20 kHz could be more effective when treating larger planktonic organisms. Lürling and Tolman (2014) did not find any linear relationship between organism size and effect on their survival. These authors carried out US experiments with the crustacean D. magna and found that individuals of a size between 1 and 2 mm seemed more affected by US than both smaller and larger individuals. At the same time, A. amphitrite showed a halfway response between A. salina and B. plicatilis probably due to its halfway size, compared to brine shrimps and rotifers. Additional experiments would be necessary to confirm more precisely the influence of the size in the US effectiveness on planktonic organisms. In the light of the results obtained, we may hypothesize that the key factor that conditioned the sensitivity of B. plicatilis in immersion assays was the power density, which was related to the tank material: organisms inside stainless steel tubes experimented less mortality than those inside floating net containers. Indeed, the response of Brachionus, unlike that of crustaceans, did not vary significantly with exposure time. Experiments performed in this work employed organisms of size ≥ 50 μm. For this size class, regulation D-2 of the IMO

of target invertebrates exposed to US waves. Metal-to-metal contact assays (Experiment I) (Fig. 3) provided novel information on the sensitivity of zooplankton exposed to US. To our knowledge, no previous experimental data are available regarding the effectiveness of US with the emission source in direct contact with the surface of water container. Based on mortality values, this assay was the most effective in eliminating zooplankton target organisms with a maximum percentage of mortality equal to 95% and 93.33% in B. plicatilis and A. salina, respectively. It is important to underline that in comparison with immersion assays (Fig. 2, Exp. II), where a volume of 5 L of water was used, in metal-to-metal contact assays (Experiment I) only 20 mL were employed. Therefore, the higher power density could result in a higher killing efficiency. Lürling and Tolman (2014) obtained similar results when treating specimens of Daphnia magna with an US system and different volumes of water. Besides, metal-to-metal contact assay (Experiment I) (Fig. 3) pointed out the highest sensitivity of B. plicatilis in comparison with other species. After 30 s of continuous treatment, rotifers registered a mortality rate equal to 80%, whereas for crustacean a longer exposure time (60 s) was required to achieve ≥ 50% of mortality. B. plicatilis (≤250 μm of size) not only differs from A. amphitrite (270 μm) and A. salina (> 500 μm) in its dimension but also in the different biochemical composition and strength of the exoskeleton (Holm et al., 2008; Seth et al., 2010). In contrast to crustaceans, rotifers possess two different hard structures, named as lorica and mastax, which confer them a different protection. The lorica is a densely packed layer of keratin-like proteins, which forms the outer surface of the body (Fukusho, 1989). Likewise, the mastax is a calcified apparatus in the mouth region that contains about a 60% of chitin (Agrawal et al., 2010; Klusemann et al., 1990). In this sense, we may hypothesize that these hard body structures, together with the fact that rotifers show corona retraction into the trunk following mechanical or chemical contact (Clément, 1987), are processes that could facilitate or decrease their protection to US waves. Kriesel et al. (2003), who expected to observe a higher sensitivity to UV irradiation and chemical treatments in the illoricated species with respect to the loricated B. calyciflorus after exposure, similarly suggested this. Experiment I may simulate a real on-board condition such as a ship pipeline where US transducers are installed in direct contact with the external surface of the pipe. Even though, additional tests would be required in order to verify the effectiveness of the US technology in a flowing environment at real scale scenarios, it is expected that a series of transducers would be needed to guarantee the effectiveness of the treatment since an exposure of 30–60 s is short to treat large volumes of BW as those carried by commercial vessels, this jeopardizing the energy costs and making it unlikely to use such a system in the main vessel BW pipework. In immersion assays with metal tanks (Experiment II) (Fig. 4), US effectiveness considerably decreased with respect to the metal-to-metal contact assays (Fig. 3). Between trials with the transducer in contact (Fig. 2, Exp. II·B) and not in contact (Fig. 2, Exp.II.A) with the bottom of the tanks, the latter was slightly more advantageous, mainly for Artemia (Fig. 4). The transducer in contact with the bottom may have lost efficacy due to the transmission of US waves along the surface of the tank. With the transducer not in contact, the effectiveness was higher, most probably due to the propagation of waves directly through the water. Barnacles and rotifers inside net containers (Fig. 2, Exp. III.A, C) were more sensitive to US than inside steel tubes (Fig. 2, Exp. III. B, D) maybe due to a higher exposure to waves. The survival rate of Artemia did not change significantly between both tests (Fig. 5). During immersion assays in plastic tanks (Experiment III), the trend of sensitivity in organisms inside steel tubes (Experiment III·B) (Fig. 5) was nearly identical to that observed in metallic tanks (Experiment III.D). Nevertheless, when the organisms were placed inside filter containers in the plastic vessels (Experiment III.A), the US treatment was more effective than in the metallic tank (Experiment III·C) (Fig. 5), 7

Journal of Sea Research xxx (xxxx) xxx–xxx

N. Estévez-Calvar et al.

and FG have analyzed and interpreted the data. FM, MF and FG contributed to materials, facilities and analysis tools. NE, CG and FG wrote the paper. NE, CG, GP, FM, MF and FG revised the article critically for important intellectual content. All authors have approved the final article version.

Convention establishes that ships shall discharge < 10 viable organisms/m3 (IMO, 2004). According to these standards, our experiments indicate that the design of an US-based BWTS should carefully take into consideration some key parameters, such as exposure time, tank material, water volume and transducer position. Any substantial variation in one of these parameters may alter the final US system effectiveness. In general, treatment time improved the sterilization of water. In large-scale scenarios, the different sensitivity of target species towards exposure time could be faced employing an appropriate number of transducers working in alternate manner in order to guarantee a longer exposure of organisms to US waves and therefore, an increased killing efficiency. To cope with variations of effectiveness caused by other parameters such as tank material, water volume or transducer position, it would be useful that BWTSs should be set up according to the threshold of the most resistant species in order to guarantee the elimination of that and other species less resistant. In this sense, further research should include new model organisms to be tested for sensitivity following different water treatments. Additionally, at real scale scenarios, the combination of US treatment with another different BW technology may improve significantly the efficacy in removing zooplankton. For instance, filtration technologies may lead to the separation of organisms according to their size. Kriesel et al. (2003) observed how a filtration step with a 50 μm disc filter removed successfully the 96% of planktonic organisms with a size > 80 μm. Furthermore, the potential application of US technology as a single BWTS should not be rejected. Based on our results, an in-tank treatment may be plausible in vessels with relatively small ballast tanks and small volumes of BW. For instance, mega yachts have commonly 24–50 length (depending on manufacturer) with volumes of BW ranging from 5 to 10 m3. Since the distance between structural elements of the BW tank is typically < 1 m (P. Derba, Personal communication), this is an interesting point to take advantage from the highest effectiveness of the prototype in eliminating planktonic organisms as shown in Experiment IV. Logistically, the application in-tank of a series of US transducers may be plausible and would not compromise the mega yacht performance in terms of power consumption.

Acknowledgements The authors are very grateful to the company Molecolar Energy Systems (M.E.S s.r.l, Arenzano, Italy) for providing the ultrasound prototype and technical guidance during the completely experimental procedure. Authors thank Engineering Pietro Derba by his support on technical details about mega yachts ballast water tanks. References Agrawal, P., Strijkers, G.J., Nicolay, K., 2010. Chitosan-based systems for molecular imaging. Adv. Drug Deliv. Rev. 62, 42–58. http://dx.doi.org/10.1016/j.addr.2009. 09.007. Arrojo, S., Benito, Y., Martínez-Tariffa, A., 2008. A parametrical study of disinfection with hydrodynamic cavitation. Ultrason. Sonochem. 15, 903–908. http://dx.doi.org/10. 1016/j.ultsonch.2007.11.001. Brizzolara, B.A., Holm, E.R., Stamper, D.M., 2006. Disinfection of water by ultrasound: application to ballast water treatment. Naval surface warfare center Carderock division West Bethesda, MD 20817-5700. NSWCCD-61-TR–2006/16 October. In: Survivability, Structures, and Materials Department Technical Report, . Buck, E.H., 2012. Ballast water management to combat invasive species. In: Congressional Research Service, pp. 7–5700. (RL32344. http://www.crs.gov). Carlton, J.T., 1985. Transoceanic and interoceanic dispersal of coastal marine organisms: the biology of ballast water. In: Barnes, M. (Ed.), Oceanography and Marine BiologyAn Annual Review. Aberdeen University Press, Aberdeen, Scotland, UK, pp. 313–371. Carlton, J.T., 1996. Pattern, process and prediction in marine invasion ecology. Biol. Conserv. 78, 97–106. http://dx.doi.org/10.1016/0006-3207(96)00020-1. Carlton, J.T., 1999. Marine bioinvasions of New England. In: Maritimes (University of Rhode Island Sea Grant), Winter 99, . Carlton, J.T., Geller, J.B., 1993. Ecological roulette: the global transport of nonindigenous marine organisms. Science 261 (5117), 78–82. http://dx.doi.org/10.1126/science. 261.5117.78. Chu, K.H., Tam, P.F., Fung, C.H., Chen, Q.C., 1997. A biological survey of ballast water in container ships entering Hong Kong. Hydrobiologia 352, 201–206. http://dx.doi.org/ 10.1023/A:1003067105577. Clément, P., 1987. Movements in rotifers: correlations of ultrastructure and behavior. In: Rotifer Symposium IV. Developments in Hydrobiology 42. pp. 339–359. http://dx. doi.org/10.1007/BF00025764. Cope, R.C., Prowse, T.A.A., Ross, J.V., Wittmann, T.A., Cassey, P., 2015. Temporal modelling of ballast water discharge and ship-mediated invasion risk to Australia. R. Soc. Open Sci. 2, 150039. http://dx.doi.org/10.1098/rsos.150039. David, M., Gollasch, S., 2015. Ballast water management systems for vessels. In: Global Maritime Transport and Ballast Water Management. Issues and Solutions, pp. 306 (109–132). Dragsund, E., Johannessen, B.O., Andersen, A.B., Nøklebye, J.O., 2003. Corrosion effects of ballast water treatment methods. In: Proceedings, 2nd International Ballast Water Treatment R & D Symposium. Globallast Monograph Series 15. pp. 291–299. Elliot, M., 2003. Biological pollutants and biological pollution - an increasing cause for concern. Mar. Poll. Bull. 46, 275–280. http://dx.doi.org/10.1016/S0025-326X(02) 00423-X. EMSA, European Maritime Safety Agency, 2015. http://www.emsa.europa.eu/ implementation-tasks/environment/ballast-water.html. Faimali, M., Garaventa, F., Piazza, V., Greco, G., Corrà, C., Magillo, F., Pittore, M., Giacco, E., Gallus, L., Falugi, C., Tagliafierro, G., 2006. Swimming speed alteration of larvae of Balanus amphitrite (Darwin) as a behavioural end-point toxicological bioassays. Mar. Biol. 149 (1), 87–96. http://dx.doi.org/10.1007/s00227-005-0209-9. Fofonoff, P., Ruiz, G., Steves, B., Carlton, J., 2003. In ships or on ships? Mechanisms of transfer and invasion for nonnative species to the coasts of North America. Chapter 7. In: Ruiz, G., Carlton, J. (Eds.), Invasive Species Vectors and Management Strategies. Island Press, Wash.D.C. Fukusho, K., 1989. Biology and mass production of the rotifer Brachionus plicatilis. Int. J. Aquac. Fish. Technol. 1, 232–240. Garaventa, F., Gambardella, C., Di Fino, A., Pittore, M., Faimali, M., 2010. Swimming speed alteration of Artemia sp. and Brachionus plicatilis as a sub-lethal behavioural end-point for ecotoxicological surveys. Ecotoxicology 19, 512–519. http://dx.doi. org/10.1007/s10646-010-0461-8. Gavand, M.R., McClintock, J.B., Amsler, C.D., 2007. Effects of sonication and advanced chemical oxidants on the unicellular green alga Dunaliella tertiolecta and cysts, larvae and adults of the brine shrimp Artemia salina: A prospective treatment to eradicate invasive organisms from ballast water. Mar. Pollut. Bull. 54, 1777–1788. http://dx. doi.org/10.1016/j.marpolbul.2007.07.012. Globallast IMO, 2015. http://globallast.imo.org/index.asp?page=problem. htm&menu=true. Globallast IMO, 2017. http://globallast.imo.org/ballast-water-as-a-vector/. Gogate, P.R., 2002. Cavitation: An auxiliary technique in wastewater treatment schemes.

5. Conclusions This work revealed the complexity in the responses of zooplankton to a BWTS. Representative zooplanktonic species (> 50 μm of dimension) A. amphitrite, B. plicatilis and A. salina showed a different rate of sensitivity to an ultrasonic treatment system. Mortality rate for each species varied according to the different experimental set up which included changes in exposure time, tank material, water volume and position of US transducer. The results supported the potential use of an antifouling prototype system based on US-technology as a single in-tank BWTS to be applied in vessels of small length overall carrying reduced ballast water volumes, or in combination with other BW treatment technologies. Furthermore, this work outputs are of great interest and may be taken into account in the design of new BWTSs based on US. Funding sources This work was financially supported by Regione Liguria in the framework of POR-CRO FESR 2007–2013 [No specified grant number], by the “RITMARE” Flagship Project 2012–2016 supported by MIUR and coordinated by CNR, Italy [No specified grant number] and by “BALMAS Research Project” (IPA ADRIATIC CBC PROGRAMME) [Grant number 1° STR/005]. Author contributions All authors have participated in the research and article preparation. NE, CG, GP, GG, FM, MF and FG have conceived and designed the experiments. NE and CG have performed the experiments. NE, CG, GG 8

Journal of Sea Research xxx (xxxx) xxx–xxx

N. Estévez-Calvar et al.

National Research Council, 1996. Stemming the tide. In: Controlling Introductions of Nonindigenous Species by Ships' Ballast Water. National Academy Press, Washington, DC. Raikow, D.F., Reid, D.F., Blatchley III, E.R., Jacobs, G., Landrum, P.F., 2007. Effects of proposed physical ballast tank treatments on aquatic invertebrate resting eggs. Setac. Environ. Toxicol. Chem. 26 (4), 717–725. http://dx.doi.org/10.1897/06-403R.1. Raišutis, R., Voleišis, A., Kažys, R., 2008. Application of the through transmission ultrasonic technique for estimation of the phase velocity dispersion in plastic materials. Ultragarsas (Ultrasound) 63, 1392–2114. Ruiz, G.M., Rawlings, T.K., Dobbs, F.C., Drake, L.A., Mullady, T., Huq, A., Colwell, R.R., 2000. Global spread of microorganisms by ships - ballast water discharged from vessels harbours a cocktail of potential pathogens. Nature 408, 49–50. http://dx.doi. org/10.1038/35040695. Sangave, P.C., Gogate, P.R., Pandit, A.B., 2007. Ultrasound and ozone assisted biological degradation of thermally pretreated and anaerobically pretreated distillery wastewater. Chemosphere 68 (1), 42–50. http://dx.doi.org/10.1016/j.chemosphere. 2006.12.052. Sassi, J., Viitasalo, S., Rytkönen, J., Leppäkoski, E., 2005. Experiments with ultraviolet light, ultrasound and ozone technologies for onboard ballast water treatment. Espoo 2005. VTT Tiedotteita Res. Notes 2313, 1–86. Scherba, G., Weigel, R.M., O'Brien, W.D.J., 1991. Quantitative assessment of the efficacy of ultrasonic energy. Appl. Environ. Microbiol. 57 (7), 2079. Seth, N., Chakravarty, P., Khandeparker, L., Anil, A.C., Pandit, A.B., 2010. Quantification of the energy required for the destruction of Balanus amphitrite Larva by ultrasonic treatment. J. Mar. Biol. Assoc. U. K. 90 (7), 1475–1482. http://dx.doi.org/10.1017/ S0025315409991548. Taylor, A., Rigby, G., Gollasch, S., Voight, M., Hallegraeff, G.M., McCollin, T., Jelmert, A., 2002. Preventive treatment and control techniques for ballast water. In: Leppakoski, E., Gollasch, S., Olenin, S. (Eds.), Invasive Aquatic Species of Europe. Distribution, Impacts and Management. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 484–507. Underwood, A.J., Chapman, M.G., 1997. Experiments on effects of sampling biota under intertidal and shallow subtidal. J. Exp. Mar. Biol. Ecol. 207, 103–126. Vanderploeg, H.A., Nalepa, T.F., Jude, D.J., Mills, E.L., Holeck, K.T., Liebig, J.R., Grigorovich, I.A., Ovajerr, H., 2002. Dispersal and emerging ecological impacts of Ponto-Caspian species in the Laurentian Greta Lakes. Can. J. Fish. Aquat. Sci. 59, 1209–1228. Veldhuis, M.J.W., 2011. Final Report of the Land-based Testing of the Aquatricomb Ballast Water Treatment System (Aquaworx GmbH), for Type Approval According to the Regulation D-2 and the Relevant IMO Guideline (April–July 2010). Royal Netherlands Institute for Sea Research, Texel, The Netherlands. (http://www.bsh.de/ de/Meeresdaten/Umweltschutz/Ballastwasser/Dokumente/07AquaTriComb-Final_ Report_landbased_tests.pdf). Viitasalo, S., Sassi, J., Rytkönen, J., Leppäkosk, E., 2005. Ozone, ultraviolet light, ultrasound and hydrogen peroxide as ballast water treatments – Experiments with mesozooplankton in low-saline brackish water. J. Mar. Environ. Eng. 8, 35–55. Voigt, M., 2004. The Artemia test system for ballast water treatment options. In: Matheickal, J.T., Raaymakers, S. (Eds.), Proceedings of 2nd International Ballast Water Treatment R & D Symposium, IMO London, 21–23 July 2003. GloBallast Monograph Series 15 International Maritime Organization, London, pp. 321–325. Werschkun, B., Höfer, T., Greiner, M., 2012. Emerging risks from ballast water treatment. In: Federal Institute for Risk Assessment. North Sea Ballast Water. BfR Wissenschaft, pp. 1–140. Williams, R.J., Griffiths, F.B., Van der Wal, E.J., Kelly, J., 1988. Cargo vessel ballast water as a vector for the transport of non-indigenous marine species. Estuar. Coast. Shelf Sci. 26, 409–420. http://dx.doi.org/10.1016/0272-7714(88)90021-2.

Adv. Environ. Res. 6, 335–358. http://dx.doi.org/10.1016/S1093-0191(01)00067-3. Gregg, M., Rigby, G., Hallegraeff, G.M., 2009. Review of two decades of progress in the development of management options for reducing or eradicating phytoplankton, zooplankton and bacteria in ship's ballast water. Aquat. Invasions 4 (3), 521–565. http://dx.doi.org/10.3391/ai.2009.4.3.14. Guo, S.F., Lee, H.P., Chaw, K.C., Miklas, J., Teo, S.L.M., Dickinson, G.H., Birch, W.R., Khoo, B.C., 2011a. Effect of ultrasound on cyprids and juvenile barnacles. Biofouling 27 (2), 185–192. http://dx.doi.org/10.1080/08927014.2010.551535. Guo, S.F., Lee, H.P., Khoo, B.C., 2011b. Inhibitory effect of ultrasound on barnacle (Amphibalanus amphitrite) cyprid settlement. J. Exp. Mar. Biol. Ecol. 409, 253–258. http://dx.doi.org/10.1016/j.jembe.2011.09.006. Guo, S.F., Lee, H.P., Teo, S.L.M., Khoo, B.C., 2012. Inhibition of barnacle cyprid settlement using low frequency and intensity ultrasound. Biofouling 28 (2), 131–141. http://dx.doi.org/10.1080/08927014.2012.658511. Headwaytech, 2017. http://www.headwaytech.com. Holm, E.R., Stamper, D.M., Brizzolara, R.A., Barnes, L., Deamer, N., Burkholder, J.M., 2008. Sonication of bacteria, phytoplankton and zooplankton: Application to treatment of ballast water. Mar. Pollut. Bull. 56, 1201–1208. http://dx.doi.org/10. 1016/j.marpolbul.2008.02.007. IMO, 2004. International Convention for the Control and Management of Ships' Ballast Water and Sediments. International Maritime Organization, London. IMO, 2017. Current lists of ballast water management systems, which received type approval certification, basic and final approval. http://www.imo.org/en/OurWork/ Environment/BallastWaterManagement/Documents/Table%20of %20BA%20FA%20TA%20updated%20November%202016.pdf. Kitamura, H., Takahashi, K., Kanamaru, H., 1995. Inhibitory effect of ultrasound waves on the laval settlement of the barnacle, Balanus amphitrite in the laboratory. Mar. Fouling 12, 9–13. http://dx.doi.org/10.4282/sosj1979.12.9. Klusemann, J., Kleinow, W., Peters, W., 1990. The hard parts (trophi) of the rotifer mastax do contain chitin: Evidence from studies on Brachionus plicatilis. Histochemistry 94 (3), 277–283. Kriesel, I., Kolodny, Y., Cairns, W.L., Galil, B.S., Sasson, Y., Joshi, A.V., Cangelosi, A., Blatchley, E.R., TenEyck, M.C., Blacer, M.D., Brodie, P., 2003. The ternary effect for ballast water treatment. In: Proceedings, 2nd International Ballast Water Treatment R & D Symposium. Globallast Monograph Series 15. pp. 5–18. Leong, T., Ashokkumar, M., Kentish, S., 2011. The fundamentals of power ultrasound- a review. Acoust. Aust. 39 (2), 54–63. Lloyds, 2010. Ballast water treatment technology. Current status. February 2010. http:// www.psmfc.org/ballast/wordpress/wp-content/uploads/2010/06/Ballast-WaterTreatment-Technology_Feb-2010.pdf. Lloyds, 2012. Ballast water treatment technologies and current system availability. Part of Lloyd's Register's Understanding Ballast Water Management series. http://globallast. imo.org/wp-content/uploads/2015/01/BW-Treatment-Technology-Sept.-2012.pdf. Lubzens, E., Minkoff, G., Maron, S., 1985. Salinity dependence of sexual and asexual reproduction in the rotifer Brachionus plicatilis. Mar. Biol. 85, 123–126. http://dx.doi. org/10.1007/BF00397430. Lürling, M., Tolman, Y., 2014. Effects of commercially available ultrasound on the zooplankton grazer daphnia and consequent water greening in laboratory experiments. Water 6, 3247–3263. http://dx.doi.org/10.3390/w6113247. Mahvi, A.H., 2005. Application of ultrasonic Technology for Water and Wastewater Treatment. Iran. J. Public Health 38 (2), 1–17. Mason, T.J., Joyce, E., Phull, S.S., Lorimer, J.P., 2003. Potential uses of ultrasound in the biological decontamination of water. Ultrason. Sonochem. 10, 319–323. http://dx. doi.org/10.1016/S1350-4177(03)00102-0. MEPC 67/INF.28, 8 August, 2014. Marine Environment Protection Committee 67th session. http://www.bsh.de/de/Meeresdaten/Umweltschutz/Ballastwasser/MEPC/ MEPC_67-INF28.pdf.

9