For reasons of economy, this document is pr inted in a limited number. Delegates are kindly asked to bring their copies to mee tings and not to request additional copies.

I:\MEPC\58\2-3.doc

INTERNATIONAL MARITIME ORGANIZATION

IMO 

# E

MARINE ENVIRONMENT PROTECTION COMMITTEE 58th session Agenda item 2 MEPC 58/2/3 21 March 2008 Original: ENGLISH

HARMFUL AQUATIC ORGANI SMS IN BALLAST WATER

Application for Final Approval of th e NK-O3 BlueBallast System (Ozone)

Submitted by the Republic of Korea

SUMMARY

Executive summary: This document contains the non-confidential information related to the application for Final A pproval of the NK-O3 BlueBallast System (Ozone) under the Procedure for approval of ballast water management systems that make use of Active Substances (G9) adopted by resolution MEPC.126(53)

Strategic Direction: 7.1 High-level Action: 7.1.2 Planned output: 7.1.2.4

Action to be taken: Paragraph 6

Related documents BWM/CONF/36; MEPC 53/24/Add.1; MEPC 55/2/3, MEPC 55/2/27; MEPC 56/2/2 and BWM.2/Circ.12

Introduction

1 Regulation D-3.2 of the International Conve ntion for the Control and Management of Ships’ Ballast Water and Sediments stipulates th at ballast water management systems that make use of Active Substances to comply with the Convention shall be appr oved by the Organization.

2 The Procedure for approval of ballast water management systems that make use of Active Substances (G9) contains the in formation that should be included into a proposal for approval (MEPC 53/24/Add.1, annex 4, paragraph 4.2.1) and prov isions for risk charac terization and analysis (MEPC 53/24/Add.1, annex 4, para graph 5.3). According to sect ion 6 of Procedure (G9), the Organization should evaluate the info rmation provided in the application.MEPC 58/2/3 - 2 -

I:\MEPC\58\2-3.doc 3 According to BWM.2/Circ.12, a document on the proposal for approva l containing all the non-confidential data related to the respective ballast water mana gement systems that make use of Active Substances needs to be submitted to MEPC 58.

4 The competent authority in the Republic of Ko rea has verified the application dossier and believes that it satisfies the data requirements of Procedure (G9) adopted by resolution MEPC.126(53).

5 The Republic of Korea therefore submits the non-confidential part of the manufacturer’s application dossier in the annex to this documen t to the Organization for evaluation according to Procedure (G9). The complete dossier will be made available to the experts of the GESAMP-BWWG with th e understanding of conf idential treatment.

Action requested of the Committee

6 The Committee is invited to consider the proposal for approval and to decide as appropriate.

***MEPC 58/2/3

I:\MEPC\58\2-3.doc ANNEX

NON-CONFIDENTIAL INFORMATION ON THE NK-O3 BLUEBALLAST SYSTEM (THE REPUBLIC OF KOREA)

This Summary of Non-Confidential Information is structured in accordance with the headings in the Procedure for approval of ballast water ma nagement systems that make use of Active Substances (G9).

It includes the follo wing appendices:

.1 Approval from Korea Register of Shipping for full-scal e shipboard test system installed on board MV Hyundai Hong Kong ; .2 Extracts (title structures) of QMP/QAPPs for both the G9 and G8 testing; and

.3 Whole-Effluent Toxicity (WET) testi ng report by Dong-A University Hospital.

A full dossier including the full Application for Final Approval, structured according to the headings contained in the draf t Methodology for information gather ing and the conduct of work of the GESAMP-BWWG, including all test re sults, confidential information and supporting technical manuals and references, has been submitted to the GESAMP-BWWG in accordance with Procedure (G9).

In summary, the Type Approval testing conducted acco rding to Guidelines (G8) and the Basic and Final Approval testing condu cted according to Procedure (G9), shows that the NK-O3 BlueBallast system using Ozone meets the D-2 standard of the IMO Ballast Water Management Convention and has no significant toxic effects or residuals.

1 INTRODUCTION

1.1 Previous applications and purpose of this application

The Republic of Korea has made two previous applications to the GESAMP-BWWG in relation to the NK-O3 system, as follows:

– application as Ozone (concept) to GESAMP-BWWG-2 held in May 2006; and

– application as NK-O3 BWTS (Ozone) to GESAMP-BWWG-3 held in February 2007.

The results of these applications were as follows:

– GESAMP-BWWG-2: The review as contained in GESAMP-BWWG 2/9 noted that the application took the form of a generic concept only. Due to a lack of information on a variety of topics the GESAMP-BWWG did not recommend the application for Basic Approval; and

– GESAMP-BWWG-3: The review reported in GESAMP-BWWG 3/9 (as contained in document MEPC 56/2/2, anne x 6) recommended that Basic Approval be granted to the NK-O3 system and further recommended that additional information on a number of topics shoul d be provided in the Final Approval application.MEPC 58/2/3 ANNEX Page 2

I:\MEPC\58\2-3.doc The NK-O3 system was subsequently granted Basic Approval by MEPC 56 held in July 2007.

The purpose of this applicati on to GESAMP-BWWG-6 (to be held in May 2008) is to seek Final Approval for the NK-O3 system by MEPC 58 (to be held in October 08).



### Note:

For branding and marketing purposes, the brand-name NK-O3 BWTS used in the submission to GESAMP-BWWG-3 has been updated/changed to NK-O3 BlueBallast system.

This brand-name applies hereinafter and is used alternately with the simpler name NK-O3 System.

1.2 Working principles of th e NK-O3 BlueBallast System

In the NK-O3 BlueBallast System, a shipboard Ozone generator take s ambient air and strips away the nitrogen, concentrating the oxygen content – which is then passed through a high frequency electrical field to produce Ozone (O 3 ). The Ozone is then injected into the incoming ballast water to oxidize and neutralize en trained aquatic species.

Although Ozone has an extremely short half-lif e (5.8 seconds in seaw ater, 30 minutes in fresh water and in gaseous form 0.9 hour), it is one of the most powerful oxidizing agents produced – effectively neutralizing endo-toxins, vi ruses, bacteria, fungi and organic material extremely rapidly. For this reason, Ozone has been widely used in the medical sterilization and water treatment industr ies for many years.

When Ozone is injected into influent ballast water by the NK-O3 System, a percentage of the entrained aquatic species are killed by direct contact with the Ozone . The remainder are killed or neutralized when the Ozone reacts with other chemicals that occur naturally in seawater, to form TROs (Total Residual Oxidants: hypobromous acid and hypobromide ion) , highly effective disinfectants in their own right. Both Ozone and hypobromous acid di sintegrate extremely rapidly – ensuring that there is no damage to the receiving waters into which the treated ballast water is discharged.

In cases where ships take on fresh water as balla st (e.g., ports located in lakes or rivers), brominated compounds are not formed and the Ozone alone acts as the Active Substance.

Further details on these Active Substances are provided in section 4.1.

1.3 Main system components

The NK-O3 System is composed of four integrated modules:

– Oxygen Generator – Ozone Generator – Ozone Injector – Monitoring and control system

The Oxygen Generator is based on standard, off-the-shelf technology, thereby keeping costs very low compared to other systems. The Oxygen Ge nerator takes air from a compressor and strips away the nitrogen, concentrating the oxygen and providing the raw material to produce Ozone.MEPC 58/2/3 ANNEX

Page 3

I:\MEPC\58\2-3.doc The Ozone Generator is also based on standard , off-the-shelf technolog y, again keeping costs very low. The Ozone Generator passes the in coming oxygen through a high frequency electrical field to produce Ozone.

The Ozone Injector is based on a purpose-built, pa tented ‘side-stream injector’. This diverts incoming ballast water from the main ballast pipes in to a ‘side-stream’, wher e it is injected with Ozone before re-entering the main ballast str eam. The side-stream injector ensures a high kill-rate through optim al saturation of the incoming ballast.

The side-stream injector also ensures a restricted dosage area – compared to trying to inject Ozone into the large volumes of the ballast tank s. This eliminates potential for corrosion, by concentrating the Ozone in the injector area only , which is made from high-grade, non-corroding stainless steal.

The monitoring and control system again uses standard, off-the-shelf technology, and includes a variety of sensors, alarms, meters, valves and sw itches connected to central control software, and integrated with the ship’s overall ballast manage ment system. This allows all aspects of the system to be monitored and controlled, and for all data on system operation to be kept electronically and printed as required by inspecto rs. The system includes safety alarms and automatic cut-off switches.

There are also several items of ancill ary equipment which support these modules:

– Power Supply Unit (PSU) to provide overall power to the system;

– Compressor to feed air to the oxygen gene rator (in some cases this can be based on the ship’s existing air compressors);

– Refrigerated Dryer (RD) to de-humidif y air being fed to the oxygen generator;

– Chiller for the Closed Loop Cooling Sy stem (CLCS), which keeps the Ozone Generator cool; and

– Ozone Destruct System (ODS) to convert any unused Ozone back to oxygen before release to the atmosphere.MEPC 58/2/3 ANNEX Page 4

I:\MEPC\58\2-3.doc The overall layout of the NK-O3 System is show n in Figure 1. Full technical details of the NK-O3 BlueBallast System are contained in the Full Dossier submitted to GESAMP-BWWG.

Figure 1: Schematic of overall Layout of the NK-O3 BlueBallast System OZONE ANALYSER OZONE MONITOR

OXYGEN GENERATOR OZONE INJECTOR OZONE GENERATORMEPC 58/2/3 ANNEX

Page 5

I:\MEPC\58\2-3.doc 1.4 Description of tests undertaken

NK Co. Ltd of Busan, Republic of Korea (NK), has maintained a dedicated in-house research and development (R&D) team for over 20 years. Un der a comprehensive and rigorous R&D program involving scientists and engineers from NK, as well as a variety of national and international partners and external experts, including several major universitie s, the Korean Marine Equipment Research Institute (KOMERI) and other reputable, independent institutions; NK has conducted R&D on several types of ballast water treatment systems since 2002, including:

– Ozone (O 3 ) disinfection;

– Ultraviolet (UV) disinfection;

– Electrolysis (chlorinatio n) disinfection; and

– the above technologies in co mbination with pre-filtration.

This R&D led NK to decide to initially focu s on seeking IMO’s approval for an Ozone-based system (NK-O3) from October 2005, while continuing to develop the other t echnologies as well. The reasons for focusing on Ozone initially are:

– the R&D results show that the NK-O3 System is capable of meeting (and exceeding) the Ballast Water Perfor mance Standard under regulation D-2 of the International Convention for the Control and Manage ment of Ships’ Ballast Water and Sediments, and also the more stringent standards being proposed under the United States legislation;

– with appropriate monitoring and control systems; it is an inherently safe and environmental-friendly system, which does not require ch emicals or other potentially harmful substances to be carried on the ship;

– it can be based on existing, readil y available, off-the-shelf technology (oxygen generator, Ozone generator, monito ring and control systems, etc.), fitted together in modular, plug- and-play format; thereby keeping costs very low compared to many other systems;

– it can be easily scaled, using the modul ar components; to suit different vessel types, sizes and ballast capacities;

– it has a compact design requi ring minimum on-board space;

– it can be easily integrated into ships’ existing ballast systems;

– it has low capital cost an d low operating costs; and

– it is easy to maintain and is simple to operate.

NK is also confident that th rough applying its extensive engi neering, manufacturing and R&D expertise and vast experience with ship-board sy stems, it will also be able to achieve similar results with the other technologies.MEPC 58/2/3 ANNEX Page 6

I:\MEPC\58\2-3.doc NK has proceeded with testing of the Ozone system with a view to obtaining Type Approval from the Korean Administration; in accordan ce with the Guidelines for approva l of ballast water management systems (G8); and also Basic and Final Approval fr om IMO; in accordance with the Procedure for approval of ballast water management systems that make use of Active Substances (G9).

Comprehensive testing has included:

– lab-based bench tests;

– land-based tests on a pur pose-built test barge that can be moved to different locations for testing under different conditions;

– shipboard tests using a full-scale NK-O3 System; installed on the 82,000 DWT/6,800 TEU container ship M.V. Hyundai Hong Kong ; tested during real-life operational voyages; and

– toxicity testing of the treated ballast water in accordance with Procedure (G9).

Full descriptions of thes e tests and their results are provided in the respective re ports on each, as contained in the full dossier.

2 DEFINITIONS

For the purpose of this report, terms and definiti ons are the same as specified in Procedure (G9), and are therefore not repeated here, except for th e following which are specific to this report:

“ Active Substance ” refers to the extremely short-lived Ozone and Total Residual Oxidants (TROs) generated in treated ballast water from the NK-O3 treatment process.

“ Ballast Water Discharge ” is the treated ballast water that has passed through the NK-O3 System during ballasting and has then b een stored in the ballast tanks prior to discharge.

“ Preparation ” has the same meaning as “Active Substance” for the NK-O3 System.

“ Relevant Chemicals ” has the same meaning as “Ac tive Substance” for the NK-O3 System.

The above interpretations of the G9 definitions m ean that in the case of the NK-O3 System, these terms all refer to the treated ballast water that is discharged following the sequence of:

.1 ballasting;

.2 treatment;

.3 voyage; and

.4 deballasting.MEPC 58/2/3 ANNEX

Page 7

I:\MEPC\58\2-3.doc As has been applied for other ox idant-based ballast water management systems (BWMS) that has been granted Final Approval by the MEPC, it is therefore the treated ballast water that is considered in this applicati on and thus tested for toxicity and content of chemicals.

3 PRINCIPLES

Please refer to section 1.1.

4 GENERAL REQUIREMENTS

4.1 Identification of Active Substances, etc.

As outlined above and in previous applica tions to GESAMP-BWWG, the NK-O3 System does not involve the formulation of any Preparation for carriage on board ship for use in the BWMS. Rather, the Active Substance Ozone is generated on-board as required.

As outlined in previous applications, in cases where ships take on seawater as ballast, when Ozone (O 3 ) is injected into the ballast water, it reac ts with bromide that occurs naturally in seawater to produce bromide ions. In this proce ss the Ozone is destroyed extremely rapidly, and the TRO becomes the main (and highly effective) Active Substance, killi ng or neutralizing any entrained aquatic organisms.

In addition, in seawater the formed bromide ions rapidly forms hypobromous acid and hypobromite ion, and it can also react with any organic matter that may be present in the seawater/ballast water to form bromoform.

Other possible bromide related by-products in clude bromate ions, dibromoacetic acid and bromochloroacetic acid.

As stated above, in cases where ships take on fresh-water ballas t, brominated compounds are not formed, and the Ozone alone is the Active Substance.

More detailed exploration of Ozone/bromide chemis try in seawater is cont ained in the previous submission to GESAMP-BWWG, and in the re ferences provided therein (Crecelius 1979, Oemcke and van Leeuwen, 1998, Salhi and von Gunten, 1999; von Gunten and Hoigne, 1992; von Gunten et al. , 1996; von Gunten and Oliveras, 1997; Pinkernell et al. , 2000; Pinkernell and von Gunten, 2001; von Gunten et al. , 2001, Hofman and Andrews, 2001, etc.).

Strictly speaking then, for seawater ballast, Oz one and TRO are the two Active Substances of relevance to the NK-O3 System. The by-pr oducts bromoform, dibr omoacetic acid and bromochloroacetic acid are collectively referred to as Disinfection By-Products (DBPs), and may be considered as Relevant Chemicals.

However, as stated in section 2 “Definitions”, for the purposes of this application and the whole effluent toxicity (WET) testing that has been conducted to support it, “Preparation”, “Active Substance” and “Relevant Chemicals”, as well as “Ballast Water Discharge”, all have the same meaning. This “whole effluent” approach has be en adopted for other oxidant-based BWMS that has been granted Final Approval by the MEPC.

Nevertheless, relevant data as required by Procedur e (G9) are provided be low for Ozone, TRO, bromate ion and bromoform.MEPC 58/2/3 ANNEX Page 8

I:\MEPC\58\2-3.doc 4.1.1 Active Substances (Ozone and TROs)

TRO (Total Residual Oxidants) Chemical name Ozone (Triatomic Oxygen) Hypobromous acid Hypobromite ion CAS number 10028-15-6 13517-11-8 - UN number & name 1956 - - Molar mass 48.00 96.91 95.90 Empirical formula O 3 HOBr OBr Structural formula Classification (UN GHS) Purity 10~15 wt% By-product By-product

4.1.2 Relevant Chemicals (Disinfection By-Products)

Bromate ion and Bromoform

Chemical name Bromate ion Bromoform (Tribromomethane) CAS number 15541-45-4 75-25-2

Molar mass 127.90 252.73 Empirical formula BrO 3 - CHBr 3 Structural formula

Classification (UN GHS)

Bromochloroacetic acid a nd Dibromoacetic acid

Chemical name Bromochloroethanoic acid (Bromochloroacetic acid) Dibromoethanoic acid (Dibromoacetic acid) CAS number 5589-96-8 631-64-1

Molar mass 173.39 217.84 Empirical formula CHBrClCOOH CHBr 2 COOH

Structural formula

Classification (UN GHS)MEPC 58/2/3 ANNEX

Page 9

I:\MEPC\58\2-3.doc 4.1.3 Discharge characteristics

The discharge characteristics of ballast water treated by the NK-O3 System are indicated by the test results from the chemical analysis conducted during the land-based (bar ge) testing, as follows:

Ballasting

Active substance & Relevant chemicals Unit Control Treated MDL 1)

Ozone (Calculated) mg/l - 2.06~ 2.35 (avg 2.20) - TRO mg/l ND 2) 3.62~ 4.84 (avg 4.24) 0.02

Deballasting

Active substance & Relevant chemicals Unit Control Treated MDL 1)

Ozone mg/l ND ND 0.01 TRO mg/l ND ND 0.02 Bromate ion μg /l ND ND 1.00 Trichloromethane μg /l ND ND 5.00 Bromodichloromethane μg /l ND ND 0.70 Dibromochloromethane μg /l ND ND 0.70 Tribromomethane μg /l ND ND~28.4 (avg 5.58) 1.00 Monochloroacetic acid μg /l ND ND 2.13 Dichloroacetic acid μg /l ND~8.09 (avg 1.34) ND~0.85 (avg 0.14) 0.08 Trichloroacetic acid μg /l ND~0.36 (avg 0.04) ND~0.70 (avg 0.08) 0.03 Bromochloroacetic acid μg /l 0.30~9.32 (avg 4.17) 0.78~11.9 (avg 5.97) 0.03 Dibromoacetic acid μg /l ND~2.64 (avg 0.65) 0.43~8.05 (avg 3.97) 0.04

1) MDL: Method Detection Limit. 2) ND: Not detected (< MDL).

No Active Substances, Relevant Chemicals or Di sinfection By-Products we re detected in the treated discharge water.

4.2 Data-set for Active Substances and Preparations

4.2.1 Properties and Actions

As outlined above, Whole Effluent Toxici ty (WET) testing was undertaken by Dong-A University Hospital Clinical Research Centre us ing ballast water that had been treated by the NK-O3 System during land-based (barge) tests, acco rding to toxicity test methods outlined in Procedure (G9).MEPC 58/2/3 ANNEX Page 10

I:\MEPC\58\2-3.doc The full WET test results are contained in the report in appendix 3, and may be summarized in the following.

4.2.1.1 Data on effects on aquati c plants, vertebrates and fish

Acute aquatic toxicity

The full range of tests required by Procedure (G 9) was conducted and no acute aquatic toxicity was observed. Refer to appendix 3, section 3.3.

Chronic aquatic toxicity

The full range of tests required by Procedure (G9) was conducted and no ch ronic aquatic toxicity was observed. Refer to appendix 3, section 3.3.

Endocrine Disruption

Endocrine effect screening was conducted with fish according to OECD Non-spawning Fish Screening Assay (Env/JM/MOINO (2004)18). No endocrine disrup tion was detected. Refer to appendix 3, section 3.3.

Endocrine effect screening was al so conducted with Cell lining ac cording to Standard Operating Procedure outlined by Aquatic Toxicology Labor atory Michigan State University 2003 – both treated and non-treated (control) ballast water showed TCDD-EQ si gnificantly higher than the negative control solution but there was no signi ficant difference between the both treated and non-treated (control) ballast wate r; so it is concluded that el evated TCDD-EQ relative to the negative control solution did not originate from the NK-O3 treatment. Refer to appendix 3, section 3.3.

Sediment toxicity

Sediment toxicity was assessed using amphi pods in accordance with USEPA.1994, 600/R-94/025 and no toxicity was observed. Re fer to appendix 3, section 3.3.

Bioavailability biomagnification/bioconcentration

As chemical analysis of ballast water trea ted by the NK-O3 System did not detect any Active Substances, TROs or DBPs, and given the ve ry short half-lives of these substances, plus given that the WET testing did not detect any significant toxici ty, bioavailability, biomagnification and bioconcentration are not cons idered to be of relevance to this system. Refer to appendix 3, section 3.3.

Food web/population effects

As per bioavailability, biomagni fication and bioconcentration.MEPC 58/2/3 ANNEX Page 11

I:\MEPC\58\2-3.doc 4.2.1.2 Data on mammalian toxicity

Acute toxicity

Testing was conducted using an ICR Mouse acco rding to OECD Guide line TG 423 updated and adopted on 17 December 2001.

Effects on skin and eye

Standard skin sensitization te sts were not required for the NK- O3 System as outlined in the Methodology (MEPC 57/2/10, annex 4, section 3.3.2).

Repeated dose toxicity

Repeated dose toxicity tests were not requi red for the NK-O3 System as outlined in the Methodology (MEPC 57/2/10, annex 4, section 3.3.3).

Chronic toxicity

Chronic toxicity tests were not required for th e NK-O3 System as outlined in the Methodology (MEPC 57/2/10, annex 4, section 3.3.4).

Developmental and reproductive toxicity

Developmental and reproductive toxicity tests were not required for the NK-O3 System as outlined in the Methodology (MEPC 57/2/10, annex 4, section 3.3.5).

Carcinogenicity

Carcinogenicity tests were not required for the NK-O3 System as outlined in the Methodology (MEPC 57/2/10, annex 4, section 3.3.6).

Mutagenicity/genotoxicity

Mutagenicity/genotoxicity of the Whole Effluent from the NK-O3 System was assessed using:

.1 bacterial reverse mutation test;

.2 in vitro mammalian chro mosome aberration test;

.3 mammalian bone marrow micronucleus test;

.4 in vitro mammalian cell gene mutation test; and

.5 in vivo mammalian bone marrow chromosome aberration test.

No induction of mutagenicity/gen otoxicity was found in any of the tests. Refer to appendix 3, section 3.4.MEPC 58/2/3 ANNEX Page 12

I:\MEPC\58\2-3.doc Toxicokinetics

Toxicokinetics tests were not required for th e NK-O3 System as outlined in the Methodology (MEPC 57/2/10, annex 4, section 3.3.10).

4.2.1.3 Data on environmental fate and ef fects under aerobic and anaerobic conditions

Potential physical effects on wildlife and benthic habitats

As chemical analysis of ballast water treated by the NK-O3 System did not detect any Active Substances, TROs or DBPs, and given the very s hort half-lives of these substances, plus given that the WET testing did not detect any significant toxici ty, potential physical effects on wildlife and benthic habitats are not considered to be of relevance to this system.

Potential residues in seafood

As per potential physical effects on wildlife and benthic habitats.

Any known interactive effects

As per potential physical effects on wildlife and benthic habitats.

Modes of degradation (biotoc/abiotic)

As per potential physical effects on wildlife and benthic habitats.

Bioaccumulation, partition coefficient, octanol/water coefficient

As per potential physical effects on wildlife and benthic habitats.

Persistence and identificatio n of main metabolites

As per potential physical effects on wildlife and benthic habitats.

Reaction with organic matter

HOBr/OBr - will react with natural organic matter to form bromoform (CHBr 3 ) by the haloform reaction. Besides bromoform, a number of disi nfection products and ot her halogen containing compounds (THMs and HAAs) are produced when Oz one and bromide are present in natural waters. All these relevant chemi cals are naturally formed in the surfaces of (sea) water so any reaction products can be expected to be of a character similar to those of “natural” decomposition in water.MEPC 58/2/3 ANNEX Page 13

I:\MEPC\58\2-3.doc 4.2.1.4 Physical and chemical properties for the Active Substances etc.

As outlined in section 2 “Definiti ons” above, this application considers the Whole Effluent from the NK-O3 System. As the Active Substances ha ve extremely short half-lives, as chemical testing did not detect any Active Substances or DBPs in the effl uent, and as the WET testing did not detect any significant toxici ty, the physical and chemical prope rties are as pe r the range for fresh, brackish and seawater carried as ballast by ships.

However, for completeness, the table below list s the physical and chemic al properties for the two Active Substances and the three DBPs, where relevant.

Active Substance DBPs Property Ozone Hypobromous acid Bromoform Dibromoacetic acid Bromochloroacetic acid Melting point( °C ) -192.5 Not available 8.0~8.5 49 27.5~31.5 Boiling point( °C ) -111.9 Not available 149~150 195 210~215Flammability Nonflammable Not available Nonflammable Not available Not available

Density(kg/m 3 ) 2.14 Not available 2,887 No data 1,980 Vapour pressure(Pa) /vapour density 5.46x10 6

/ 1.66 Not available 747 / 8.72 3 /Not available 11 /Not available Water solubility(mg/l) /dissociation constant: 3 /Not applicable Not available / 9 3,100 /Not available 2.20 x 10 4

/ 1.48 4.40 x 10 4

/Not available

Oxidation-reduction potential: 2.07 E 0 (V) Not available Not availabl e Not available Not available

Corrosivity to materials/equipment Corrosive Not available Will attack some forms of plastic, rubber, and

coatings Not available Not available

Autoignition temperature( °C ) Not applicable Not available No da ta Not available Not available

Other known physical or chemical hazards: Colourless to blue gas, characteristic odour (freshly

mown hay) Not available Colourless to yellow liquid, sweet, suffocating,chloroformlike odor Hygroscopic crystal Hygroscopic crystalMEPC 58/2/3 ANNEX Page 14

I:\MEPC\58\2-3.doc 4.2.1.5 Analytical methods at environm entally relevant concentrations

The analytical methods at environmentally rele vant concentrations us ed for the NK-O3 WET testing are as follows:

Method Applicability Sensitivity Reference/Comments /Justificaiton Ozone Indigo Colourimetric Method Water 0.01 mg/L Standard Method 4500-O 3 TRO DPD Colourimetric Method Water 0.02 mg/L Standard Method 4500-Cl G Bromate ion Ion chromatographic Method Water 1.0 ug/L EPA Method 317.0

Bromoform Liquid-Liquid Extraction Gas Chromatographic Method Water 1.0 ug/L Standard Method 6232B

Bromochloroacetic acid 0.03 ug/L

Dibromoacetic acid Gas Chromatographic Method Water

0.04 ug/L EPA Method 552.3

Full details of each method are provided in the full Application for Final Approval submitted to the GESAMP-BWWG in accordance with Procedure (G9).

4.2.2 Data–sets for above

Please refer to the full application for Fina l Approval submitted to the GESAMP-BWWG in accordance with Procedure (G9).

4.2.3 Tests carried out in accordance with internationally recognized guidelines

All tests were carried out according to the Standa rd Operating Procedures (SOPs) of the Dong-A University Hospital Clinical Research Centre, which is certified to OECD Good Laboratory Practice (GLP) standards by:

– Korea Food and Drug Administra tion. Certification No.16, 2005 (http://www.kfda.go.kr);

– National Institute of Environmenta l Research. Certification No. 2007-12 (http://eng.nier.go.kr); and

– Rural Development Administra tor. Certification No. 2007-2 (http://www.rda.go.kr).

Full details of all test methods are contained in the full application fo r Final Approval submitted to the GESAMP-BWWG in accordance with Procedure (G9).MEPC 58/2/3 ANNEX Page 15

I:\MEPC\58\2-3.doc 4.2.4 Quality control/quality assurance

Combined Quality Management Plans (QMPs) and Quality Assurance Project Plans (QAPPs) were developed and applied for al l aspects of both the G8 and G9 testing of the NK-O3 System. Extracts (heading structures ) of both QMP/QAPPs are cont ained in appendix 2 and the full QMP/QAPPs are contained in the full application for Final Approval submitted to the GESAMP-BWWG in accordance with Procedure (G9).

4.2.5 Dossiers already submitted fo r registration of chemicals

4.2.6 Manner of application of Preparation

Please refer to sections 1.2 and 1.3 above. Fu ll technical details are contained in the full application for Final Approva l submitted to the GESAMP-BWWG.

4.2.7 Material Safety Data Sheets

Please refer to full application for Final Approval, appendix 3.

4.3 Assessment Report

The Korean Register of Shipping (KR) has performed an assessm ent of quality of te st reports (G9), risk characterization any uncertainty associat ed with the test results as addressed by Assessment Report annexed hereto.

The test results found no significan t levels of potentially toxic s ubstances and no significant acute or chronic effects of the full strength effluent on aquatic organisms. Therefore, no apparent environmental risks could be identified. See appendix.

5 RISK CHARACTERIZATION

5.1 Screening for persistency, bioaccumulation and toxicity

Please refer to above – no risk of persistency, bioaccumulation or toxicity.

5.2 Toxicity testing of th e treated ballast water

Please refer to above – no toxicity detected.

5.3 Risk characterization and analysis

Please refer to above – risk identified.MEPC 58/2/3 ANNEX Page 16

I:\MEPC\58\2-3.doc 6 EVALUATION CRITERIA

6.1 Ship and personnel safety

6.1.1 General

The NK-O3 System is inherently safe and has been certified for shipboard installation by the Korean Register of Shipping, in compliance with international design and construction standards for ship safety.

6.1.2 Corrosion

Ozone is a powerful oxidant and in general can cause increased corrosion of steel and other metals. As part of NK’s testing for Type Approval according to the IMO Guidelines (G8), corrosion tests were conducted us ing the treated effluent from the NK-O3 System, and no corrosive effects were observed. This is believed to relate to the fact that the injected Ozone disintegrates within seconds and therefore doe s not have an opportunity to cause corrosion throughout the ballast system.

In fact earlier tests conducted in the United States and reported in NK’s earlier submission, indicated reduced corrosion rates for Ozone treated ballast versus untreated controls. This is believed to relate to the fact that the treatment k ills certain species of bacteria in the water that enhance corrosion rates.

Use of the side-stream injector in the NK-O3 Sy stem ensures a restricted dosage area – compared to trying to inject Ozone into the large volumes of the ballast tanks. This eliminates potential for corrosion, by concentrating the Ozone in the inject or area only, which is made from high-grade, non-corroding stainless steal.

Full details including materials us ed for construction of the Ozone system are contained in the full Application to GESAMP-BWWG.

6.1.3 Fire and explosion

The only potential fire hazard associated with the NK-O3 System is the temporary storage of oxygen produced during opera tion of the system, which is used as the raw material to produce Ozone. The oxygen is stored in a high-pressure cylinder manufactured to international safety standards and placed within a fire-proof cabinet. Oxygen is purged from the storage system and released to atmosphere when th e system is not in operation.

In some fittings, the customer may elect to include suppleme ntary oxygen cylinders, in which case there would be permanent oxygen storage. These cylinders would also be manufactured to international safety standards and placed within a fire-proof cabinet and/or located outside of the ship’s engine room.

The whole system is equipped with fire alarm and shut-down system as standard, and is linked to the ship’s overall fire safety and suppression syst em. As stated above, the system is certified for shipboard installation by the Korean Register of Shipping in compliance with international design and construction standards for ship safety, which includes fire and explosion considerations.MEPC 58/2/3 ANNEX Page 17

I:\MEPC\58\2-3.doc 6.1.4 Contact with or inhalation of process products

In the NK-O3 System, potential exposure of ships’ crew to process produc ts may be described in the following.

6.1.4.1 Free Ozone in vicinity of the NK-O3 System

Although gaseous Ozone has a very short half-life of 0.9 hour, it is highly toxic to humans and safety precautions must be taken in the vicinity of the NK-O3 System, especially if it is installed in enclosed engine room spaces.

The NK-O3 System therefore comes with the following safety systems and procedures as standard:

– use of materials and construction that is designed to prevent any possible leakage of Ozone from the system;

– a gas separation unit together with a wast e Ozone destruction unit placed after the treatment unit, to eliminate any excess Oz one from the treatment unit and convert it back to oxygen prior to release to the atmosphere;

– an Ozone densitometer to detect any po ssible (but highly imp robable) accidental leakage from the system;

– an automatic alarm and shut-down syst em linked to the Ozone densitometer. This is designed to trigger if Ozone le vels in the vicinity of the system exceed 0.2 ppm (the recommended safe level is 0.1 ppm); and

– recommended inspection and maintenance procedures and clea rly outlined safety procedures as contained in supportin g operating manuals for personnel.

The following events will shut down the system imme diately, close all valves and stop all pumps:

– high ambient oxygen concentration (25%);

– high ambient Ozone concentration (0.2 ppm);

– no VOD available;

– emergency stop push button pressed; and

– fire alarm.

During the land-based and ship-board testing, of the NK-O3 System, conducted under real-life operating conditions, regular monitoring for po ssible Ozone leakage was conducted and at no time was any Ozone detected and at no time was the alarm or shut-down system triggered.MEPC 58/2/3 ANNEX Page 18

I:\MEPC\58\2-3.doc 6.1.4.2 Free Ozone in the ballast system/ballast tanks

The review of the previous NK-O3 submission to GESAMP-BWWG-3 raised concerns about the possibility of free Ozone gas being present in the ship’s ballast tanks when they are emptied and personnel may enter them for inspection and maintenance purposes, and also about the possibility of gaseous Ozone being emitted from the ballast tank air vents during ballasting (when Ozone treatment occurs).

The extremely short half- life of Ozone, together with safety measures outlined above, especially the waste Ozone destruction unit pl aced after the treatment unit, make this scenario implausible.

It is also highly improbable that ballast tanks would ever be opened and entered within seconds, minutes, hours or even days of the NK-O3 Syst em having been operated, which occurs during ballasting. Ballast tanks need to be emptied pr ior to entry, giving more than sufficient time for the breakdown of Ozone (which in any case is no longer present in the ballast water within seconds following injection at the point of ballasting).

Given its half-life a nd the fact that Ozone is generated and infused at th e point of ballasting, plus the size and complexity of a ship’s ballast system , it is highly improbable, if not impossible – that gaseous Ozone would persist after injection to the point of venti ng through the ballast tank vents.

However to address this percei ved concern, safety procedures have been supplemented to recommend an exclusion zone of 1 metres, to be painted on the ship’s deck around each ballast tank air vent, with clear notice for personnel to keep out of these zones during ballasting. This will be included in crew induction and safety training in ships fitted with the NK-O3 System, as reflected in the updated Op eration and Maintenance Manual and Safety Manual for Ozone system (Supporting Documents, Volume II).

6.1.4.3 Ozone in the discharged ballast water

Due to the extremely short half-life of Ozone, it is implausible that the discharged ballast water would contain any Ozone.

6.1.4.4 TRO/hypobromous acid/related compo unds in the ballast system/ballast tanks

6.1.4.5 TRO/hypobromous acid/related comp ounds in the discharged ballast water

The short half-life of these produc ts (days) makes it implausible th at the discharged ballast water would contain these products.

Testing of the chemical content of ballast wa ter treated by the NK-O3 System during land-based and shipboard tests in accordance with IMO Guid elines (G8), did not detect any of these substances.

Risk mitigation measures could include periodic sa mpling of system effluent as part of port State control compliance procedures, a lthough the test results indicate that this is not necessary.

Overall, in the NK-O3 System, ships crew are not exposed to any process products.MEPC 58/2/3 ANNEX Page 19

I:\MEPC\58\2-3.doc 6.1.5 Noise

The NK-O3 System is inherently quiet with the only main source of noise being the air-compressor used to supply air to the oxygen generator. In some installations the system my draw air from the ship’s existing air compressors. The whole system is installed in the ship’s engine room and noise levels from the system are in-discerna ble against background levels. As standard safety procedure, crew working in the engine room ar e required to wear ear-protectors. Noise is therefore not a concern in relation to the NK-O3 System.

6.2 Environmental protection

Whole Effluent Toxicity (WET) testing of ballast water treated by the NK-O3 System during land-based (barge) tests in accord ance with the IMO Procedure (G9) did not detect any toxicity on test organisms.

Testing of the chemical content of ballast wa ter treated by the NK-O3 System during land-based (barge) tests in accordance with IM O Guidelines (G8) did not detect any substances of concern.

The NK-O3 System therefore does not pose any risks to the aquatic environment – rather it helps to protect it by preventing the spre ad of invasive aquatic species.

However to confirm this, risk mitigation measures could include periodic sampling of system effluent as part of port State control complian ce procedures, although Procedure (G9) test results indicate that this is not necessary.

7 REGULATION OF THE USE OF ACTIVE SUBSTANCES

7.1 Handling of Active Substances

One of the main safety features of the NK-O3 Syst em is the fact that there is no requirement at all for storage or handling of any substances. Ozone is generated as re quired within a closed system and breaks down within seconds.

7.2 Hazard documentation and labelling

7.3 Procedures and use

Please refer to sections 1.2, 1.3 and 6.1 above.

8 APPROVAL

8.1 Basic Approval

As outlined above, the NK-O3 System received Ba sic Approval at MEPC 56 held in July 2007.

8.2 Final Approval

In order to gain Final Approval for the NK-O3 System, NK Co. Ltd. has undertaken significant further testing in accordance with Procedure (G9) and to address questions raised by GESAMP-BWWG in relation to NK’s previous submissions, and all results are contained in the full dossier submitted to GESAMP-BWWG, with this docum ent constituting a summary of non-confidential information.

* * *MEPC 58/2/3 ANNEX Page 20

I:\MEPC\58\2-3.doc APPENDIX 1

APPROVAL FROM THE KOREAN REGIS TER OF SHIPPING FOR FULL-SCALE SHIP-BOARD TEST SYSTEM INSTALLED ONBOARD MV HYUNDAI HONG KONGMEPC 58/2/3 ANNEX Page 21

I:\MEPC\58\2-3.doc * * *MEPC 58/2/3 ANNEX Page 22

I:\MEPC\58\2-3.doc APPENDIX 2

EXTRACTS (TITLE STRUCTURES ) OF QMP/QAPPS FOR BOTH THE G9 AND G8 TESTING

1. Extracts of QMP/QAPP for G9

Quality Statement

Approval Sheet

Distribution List

Table of Contents

1. Introduction and background 2. Overall Quality Management Plan (QMP) 3. Problem Definition 4. Project Management 4.1 Project Organization 4.2 Project Team Responsibilities 4.3 Project Task Description 4.4 Data Quality Objectives and Criteria for Data Measurement 4.5 Staff training and certification 4.6 Documents and records

5. Data Generation & Acquisition 5.1 Sampling design and methods 5.2 Analytical Methods 5.2.1 Aquatic Toxicity Tests 5.2.2 Mammalian Toxicity Test 5.3 Analytical Records 5.4 Quality Control 5.5 Test Facilities, In struments & Materials 5.5.1 Test Facilities 5.5.2 Waste management 5.5.3 Instruments 5.5.4 Test System 5.5.5 Materials and Reagents 5.5.6 Test and Reference Substance 5.6 Data Management 5.6.1 Laboratory Data and Information Management 5.6.2 Electronic Data Management

6. Assessment and Oversight 6.1 Assessment and Response Actions 6.1.1 Laboratory Measurements 6.1.2 System Inspections and Technical Reviews 6.1.3 QA Inspections 6.1.4 QA statement 6.1.5 Corrective Action 6.2 Reports to ManagementMEPC 58/2/3 ANNEX Page 23

I:\MEPC\58\2-3.doc 7. Data Validation and Usability 7.1 Data Review, Verification and Validation 7.1.1 Data Verification Requirements 7.1.2 Data Review Requirements 7.1.3 Data Validation Requirement 7.2 Verification and Validation Methods 7.2.1 Data Verification 7.2.2 Data Validation 7.3 Reporting of Study Results 7.3.1 Audits of Final Reports 7.3.2 Final Report 7.4 Reconciliation with User Requirements

REFERENCES

1. Toxicity test guidelines 2. GLP guidelines 3. Reference for QAPP reports

APPENDICES

Appendix 1 Copy of NK’s corporate quality assurance certification Appendix 2 List of standard operation procedures Appendix 3 List of study plans

2. Extracts of QMP/QAPP for G8

Introduction and background

Quality Management Plan (QMP)

1. NK QMP 2. KOMERI

Quality Assurance Project Plan (QAPP)

1. Project Management

1.1 Quality Statement 1.2 Approval Sheet 1.3 Distribution List 1.4 Project/Task Organization 1.5 Problem Definition 1.6 Project Task Description 1.7 Training and Certification 1.7.1 Collection and handli ng of field samples 1.7.2 Laboratory testing and analysisMEPC 58/2/3 ANNEX Page 24

I:\MEPC\58\2-3.doc 1.8 Project Team and responsibilities 1.9 Documents and records 1.9.1 QAPP 1.9.2 Field Sample Record s 1.9.3 Chain of Custody Records 1.9.4 Laboratory Raw Data Records 1.9.5 Draft Reports 1.9.6 Final Reports 1.9.7 Calibration and Quality Control Checks

2. Sampling design & methods

2.1 Types and numbers of samples 2.2 Sampling locations and frequencies 2.2.1 Land Based test 2.2.2 Shipboard test 2.3 Sampling equipment 2.4 Preparation and decontamina tion of sampling equipment 2.5 Sample containers and volumes 2.6 Sample preservation 2.6.1 Chemical water quality 2.6.2 Organisms ( ≥ 50 μm in minimum dimension) 2.6.3 Organisms (10 μm ~ 50 μm in minimum dimension) 2.6.4 Heterotrophic bacteria 2.7 Sample handling and custody 2.8 Corrective action in the event of sample failure

3. Analytical/Test Methods

4. Assessment and Oversight

4.1 Assessments and corrective actions 4.2 Reports to management

5. Data Validation and Usability

5.1 Data Review, Validation, and Verification 5.2 Validation and Verification Methods

APPENDICES

* * *MEPC 58/2/3 ANNEX Page 25

I:\MEPC\58\2-3.doc APPENDIX 3

WHOLE-EFFLUENT TOXICITY (WET) TESTING REPORT BY DONG-A UNIVERSITY HOSPITAL

These tests were conducted on the whole-effluent fr om the NK-O3 System (WET testing by DAUH-CRC).

1. Data on effects on aquatic pl ants, invertebrates and fish etc

1.1 Acute aquatic toxicity

Species LC 50 (%/duration) Reference/Comments/ Justification for missing data Fish Cyprinidon variegates Sheepshead minnow > 100% / 96 hours US EPA. (2002)

Zooplankton Brachionus plicatilis Rotifer > 100% / 24 hours ASTM E1440-91

Algae Skeletonema costatum Diatom > 100% / 96 hours ASTM E1218-97a

1.2 Chronic aquatic toxicity

Species LC50 (%/duration)

or NOEC (%/duration) Reference/Comments/ Justification for missing data

Fish Cyprinidon variegates Sheepshead minnow > 100% / 21 days US EPA. (2002)

Zooplankton Brachionus plicatilis Rotifer > 100% / 4 days Janssen C.R. et al. (1994)

1.3 Endocrine disruption

Species Information (GSI/duration) Reference/Comments/ Justification for missing data Fish Cyprinidon variegates Sheepshead minnow No Toxicity / 21 days ENV/JM/MONO (2004) 18

1.4 Sediment toxicity

Species Information (LC 50 / duration) Reference/Comments/ Justification for

missing data Crustacea Monocorophium acherusicum Amphipod > 100% / 10days US EPA. (1994)MEPC 58/2/3 ANNEX Page 26

I:\MEPC\58\2-3.doc 2. Data on mammalian toxicity

As outlined above, these tests were conducted on the whole-effluent from the NK-O3 System (WET testing by DAUH-CRC).

2.1 Acute toxicity

The acute toxicity test was performed to get th e information on the safety of test substance (untreated sea water) agai nst test animal (mouse) pr ior to conducting the study.

This information was necessary to confirm that th at the test material itself was relevant for the WET tests for mammals and applicable as undilute d state for the most appropriate starting dose.

Table 1. Mammalian acute toxicity test of the untreated seawater in ICR mouse

Assay Value (Concentration) Species Reference/Comment

Mammalian acute toxicity Oral LD 50 : Maximum concentration (100%) ICR mouse Male/Female Mukinda et al. (2007) Dadarkar et al. (2007) Eva Schlede et al . (2006) OECD Guideline TG. (2001) Erik Walum. (1998)

2.2 Mutagenicity/genotoxicity

The objective of the study was to evaluate the treated ballast water (seawater, brackish water) as whole effluent of NKO3 System for genotoxicity and they were co mpared to genotoxicity of the untreated seawater (or brackish wa ter) used ballast water material.

(1) Bacterial Reverse Mutation Test

Table 2. Mutagenicity of the bacterial rever se mutation assay with the treated ballast water (seawater, brackish water)

Assay Species Method details Value (Concentration) Result Reference /Comment

Salmonella typhimurium TA100 With and without S9 metabolic activation Maximum concentration (100%) No, there is no evidence for mutagenicity in bacterial cells Salmonella typhimurium TA1535 With and without S9 metabolic activation Maximum concentration (100%) No, there is no evidence for mutagenicity in bacterial cells Salmonella typhimurium TA98 With and without S9 metabolic activation Maximum concentration (100%) No, there is no evidence for mutagenicity in bacterial cells Bacterial

reverse mutation

assay

Salmonella typhimurium TA1537 With and without S9 metabolic activation Maximum concentration (100%) No, there is no evidence for mutagenicity in bacterial cells Aubrecht et al. (2007) Stankowski et al.

(2003) Mortelmans et al.

(2000) OECD guideline

TG 471. (1997) Ames et al. (1982)MEPC 58/2/3 ANNEX Page 27

I:\MEPC\58\2-3.doc

Assay Species Method details Value (Concentration) Result Reference /Comment

Bacterial

reverse mutation

assay Escherichia coli WP2 uvrA With and without S9 metabolic activation Maximum concentration (100%) No, there is no evidence for mutagenicity in bacterial cells Stankowski et al. (2003) OECD guideline

TG 471. (1997) Mortelmans et al.

(2000)

Table 3. Mutagenicity of the bacterial rev erse mutation assay with the untreated seawater (or brackish water)

Assay Species Method details Value (Concentration) Result Reference /Comment Salmonella typhimurium TA100 With and without S9 metabolic activation Maximum concentration (100%) No, there is no evidence for mutagenicity in bacterial cells Salmonella typhimurium TA1535 With and without S9 metabolic activation Maximum concentration (100%) No, there is no evidence for mutagenicity in bacterial cells Salmonella typhimurium TA98 With and without S9 metabolic activation Maximum concentration (100%) No, there is no evidence for mutagenicity in bacterial cells Salmonella typhimurium TA1537 With and without S9 metabolic activation Maximum concentration (100%) No, there is no evidence for mutagenicity in bacterial cells Aubrecht et al. (2007) Stankowski et al.

(2003) Mortelmans et al.

(2000) OECD guideline

TG 471. (1997) Ames et al. (1982) Bacterial

reverse mutation

assay

Escherichia coli WP2 uvrA With and without S9 metabolic activation Maximum concentration (100%) No, there is no evidence for mutagenicity in bacterial cells Stankowski et al. (2003) OECD guideline

TG 471. (1997) Mortelmans et al. (2000)

(2) In vitro Mammalian Chromosome Aberration Test

Therefore, the treated ballast water (seawater, brackish water) did not induce genotoxicity in mammalian (CHL) cells by the chromosome aberration assay.

Table 4. Mutagenicity of chromosome ab erration assay with the treated ballast water (seawater, brackish water)

Assay Species Value (Concentration) Result Reference /Comment Chromosome aberration of CHL cells CHL cell line (Chinese hamster lung) Maximum concentration (100%) No evidence for mutagenicity Asanami et al . (2001) Nestmann et al. (2007) Ogawa et al. ( 2006) US FDA guideline (2003) Galloway et al. (2000) Okumura et al. (1999) OECD guideline TG 473. (1997)MEPC 58/2/3 ANNEX Page 28

I:\MEPC\58\2-3.doc Table 5. Mutagenicity of chromo some aberration assay wi th the untreated seawater or brackish water

Assay Species Value (Concentration) Result Reference /Comment IC 50 by MTT Maximum concentration (100%) No cytotoxicity Galloway et al. (2000) Okumura et al. (1999)

Chromosome aberration of CHL cells CHL cell line (Chinese hamster lung) Maximum concentration (100%) No evidence for mutagenisity Asanami et al. (2001) Nestmann et al. (2007) Ogawa et al. (2006) US FDA guideline. (2003) Galloway et al. (2000) Okumura et al. (1999) OECD guideline TG 473. (1997)

(3) Mammalian Bone marrow Micronucleus Test

Table 6. Mutagenicity of a micronu cleus test with the treated ballast water (seawater, brackish water)

Assay Species Value (Concentration) Result Reference/Comment

MI 50 by mitotic index ICR mouse (Male) Maximum concentration (100%) No cytotoxicity

Frequency of micronuclei in bone marrow cells ICR mouse (Male) Maximum concentration (100%) No evidence for mutagenicity Poc et al. (2008) Oshiro et al. (2001) Vanhauwaert et al. (2001) OECD guideline TG 474. (1997) Gollapudi et al. (1995)

Table 7. Mutagenicity of a micronucleus test with the untreated seawater or brackish water

Assay Species Value (Concentration) Result Reference/Comment

MI 50 by mitotic index ICR mouse (Male) Maximum concentration (100%) No cytotoxicity

Frequency of micronuclei in bone marrow cells ICR mouse (Male) Maximum concentration (100%) No evidence for mutagenicity Poc et al . (2008) Oshiro et al. (2001) Vanhauwaert et al. (2001) OECD guideline TG 474. (1997) Gollapudi et al. (1995)MEPC 58/2/3 ANNEX Page 29

I:\MEPC\58\2-3.doc (4) In vitro Mammalian Cell Gene Mutation Test

Table 8. Mutagenicity of Mouse Lymphoma TK Gene Assay with the treated ballast water (seawater, brackish water)

Species Assay Value (Concentration) Result Reference/Comment

RTG 20 by survival test Maximum concentration (100%) No cytotoxicity Moore et al. (2007) Sawyer et al. (2006) Scharmke et al. (2006) Moore et al. (2000) FDA Guidelines. (2000) OECD guideline TG 475. (1997) Mitchell et al. (1997) L5178Y

mouse lymphoma cells

Mouse Lymphoma TK Gene Assay in L5178Y mouse lymphoma cells Maximum concentration (100%) No evidence for mutagenisity Moore et al . (2007) Sawyer et al. (2006) Scharmke et al. (2006) Moore et al. (2000) FDA Guidelines. (2000) OECD guideline TG 475. (1997) Mitchell et al. (1997)

Table 9. Mutagenicity of Mouse Lymphoma TK Gene Assay with the untreated seawater or brackish water

Species Assay Value (Concentration) Result Reference/Comment

RTG 10 by survival test Maximum concentration (100%) No cytotoxicity Moore et al. (2007) Sawyer et al. (2006) Scharmke et al. (2006) Moore et al. (2000) FDA Guidelines. (2000) OECD guideline TG 475. (1997) Mitchell et al. (1997) L5178Y

mouse lymphoma cells

Mouse Lymphoma TK Gene Assay in L5178Y mouse lymphoma cells Maximum concentration (100%) No evidence for mutagenicity Moore et al. (2007) Sawyer et al. (2006) Scharmke et al. (2006) Moore et al. (2000) FDA Guidelines. (2000) OECD guideline TG 475. (1997) Mitchell et al. (1997)MEPC 58/2/3 ANNEX Page 30

I:\MEPC\58\2-3.doc (5) In vivo Mammalian bone marrow Chromosome Aberration Test

Table 10. Mutagenicity of mammalian bone marrow chromosome aberration assay with the treated ballast water (seawater, brackish water)

Assay Species Value (Concentration) Result Reference/Comment

MI 50 by mitotic index ICR mouse (Male) Maximum concentration (100%) No cytotoxicity

Frequency of chromosome aberration in bone marrow cells ICR mouse (Male) Maximum concentration (100%) No evidence for mutagenicity Evangelista et al. (2006) Cheung et al . (2006) Meng et al. (2002) Shukla et al. (2002) Nakamura et al. (1997) OECD guideline TG 475. (1997)

Table 11. Mutagenicity of mammalian bone marr ow chromosome aberration assay assay with the untreated seawater or brackish water

Assay Species Value (Concentration) Result Reference/Comment

MI 50 by mitotic index ICR mouse (Male) Maximum concentration (100%) No cytotoxicity

Frequency of chromosome aberration in bone marrow cells ICR mouse (Male) Maximum concentration (100%) No evidence for mutagenicity Evangelista et al. (2006) Cheung et al. (2006) Meng et al. (2002) Shukla et al. (2002) Nakamura et al. (1997) OECD guideline TG 475. (1997)

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