For reasons of economy, this document is printed in a limited number. Delegates are 

kindly asked to bring their copies to meetings 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 ORGANISMS IN BALLAST WATER

Application for Final Approval of the 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 Approval 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 Convention for the Control and Management of 

Ships’ Ballast Water and Sediments stipulates that ballast water management systems that make 

use of Active Substances to comply with the Convention shall be approved by the Organization. 

2 The Procedure for approval of ballast water management systems that make use of Active 

Substances (G9) contains the information that should be included into a proposal for approval 

(MEPC 53/24/Add.1, annex 4, paragraph 4.2.1) and provisions for risk characterization and analysis 

(MEPC 53/24/Add.1, annex 4, paragraph 5.3). According to section 6 of Procedure (G9), the 

Organization should evaluate the information 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 approval containing all the 

non-confidential data related to the respective ballast water management systems that make use 

of Active Substances needs to be submitted to MEPC 58. 

4 The competent authority in the Republic of Korea 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 document to the Organization for evaluation according to 

Procedure (G9). The complete dossier will be made available to the experts of the 

GESAMP-BWWG with the understanding of confidential 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 management systems that make use of Active 

Substances (G9). 

It includes the following appendices: 

.1 Approval from Korea Register of Shipping for full-scale 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) testing report by Dong-A University Hospital. 

A full dossier including the full Application for Final Approval, structured according to the 

headings contained in the draft Methodology for information gathering and the conduct of work 

of the GESAMP-BWWG, including all test results, 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 according to Guidelines (G8) and the Basic 

and Final Approval testing conducted 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, annex 6) recommended that Basic Approval 

be granted to the NK-O3 system and further recommended that additional 

information on a number of topics should be provided in the Final Approval 

application. 
MEPC 58/2/3 

ANNEX 

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The NK-O3 system was subsequently granted Basic Approval by MEPC 56 held in July 2007. 

The purpose of this application 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 the NK-O3 BlueBallast System 

In the NK-O3 BlueBallast System, a shipboard Ozone generator takes ambient air and strips away 

the nitrogen, concentrating the oxygen content – which is then passed through a high frequency 

electrical field to produce Ozone (O3). The Ozone is then injected into the incoming ballast water 

to oxidize and neutralize entrained aquatic species. 

Although Ozone has an extremely short half-life (5.8 seconds in seawater, 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, viruses, bacteria, fungi and organic material 

extremely rapidly. For this reason, Ozone has been widely used in the medical sterilization and 

water treatment industries 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 disintegrate 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 ballast (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 Generator 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 

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The Ozone Generator is also based on standard, off-the-shelf technology, again keeping costs 

very low. The Ozone Generator passes the incoming oxygen through a high frequency electrical 

field to produce Ozone. 

The Ozone Injector is based on a purpose-built, patented ‘side-stream injector’. This diverts 

incoming ballast water from the main ballast pipes into a ‘side-stream’, where it is injected with 

Ozone before re-entering the main ballast stream. The side-stream injector ensures a high 

kill-rate through optimal 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 tanks. 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 switches connected to central control software, and 

integrated with the ship’s overall ballast management 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 inspectors. The system includes safety alarms and 

automatic cut-off switches. 

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

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

– Compressor to feed air to the oxygen generator (in some cases this can be based 

on the ship’s existing air compressors); 

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

– Chiller for the Closed Loop Cooling System (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 

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The overall layout of the NK-O3 System is shown 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 MONITOR OZONE ANALYSER

OXYGEN GENERATOR OZONE INJECTOR OZONE GENERATOR
MEPC 58/2/3 

ANNEX 

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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. Under 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 universities, 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 (O3) disinfection; 

– Ultraviolet (UV) disinfection; 

– Electrolysis (chlorination) disinfection; and 

– the above technologies in combination with pre-filtration. 

This R&D led NK to decide to initially focus on seeking IMO’s approval for an Ozone-based 

system (NK-O3) from October 2005, while continuing to develop the other technologies 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 Performance Standard under regulation D-2 of 

the International Convention for the Control and Management 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 chemicals or other 

potentially harmful substances to be carried on the ship; 

– it can be based on existing, readily available, off-the-shelf technology 

(oxygen generator, Ozone generator, monitoring 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 modular components; to suit different vessel 

types, sizes and ballast capacities; 

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

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

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

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

NK is also confident that through applying its extensive engineering, manufacturing and R&D 

expertise and vast experience with ship-board systems, it will also be able to achieve similar 

results with the other technologies. 
MEPC 58/2/3 

ANNEX 

Page 6 

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NK has proceeded with testing of the Ozone system with a view to obtaining Type Approval from the 

Korean Administration; in accordance with the Guidelines for approval of ballast water management 

systems (G8); and also Basic and Final Approval from 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 purpose-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 these tests and their results are provided in the respective reports on each, as 

contained in the full dossier. 

2 DEFINITIONS 

For the purpose of this report, terms and definitions are the same as specified in Procedure (G9),

and are therefore not repeated here, except for the 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 been 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 “Active Substance” for the NK-O3 

System. 

The above interpretations of the G9 definitions mean 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 

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As has been applied for other oxidant-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 application 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 applications 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 (O3) is injected into the ballast water, it reacts with bromide that occurs naturally in 

seawater to produce bromide ions. In this process the Ozone is destroyed extremely rapidly, and 

the TRO becomes the main (and highly effective) Active Substance, killing 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 include bromate ions, dibromoacetic acid and 

bromochloroacetic acid. 

As stated above, in cases where ships take on fresh-water ballast, brominated compounds are not 

formed, and the Ozone alone is the Active Substance. 

More detailed exploration of Ozone/bromide chemistry in seawater is contained in the previous 

submission to GESAMP-BWWG, and in the references 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, Ozone and TRO are the two Active Substances of 

relevance to the NK-O3 System. The by-products bromoform, dibromoacetic 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 been adopted for other oxidant-based BWMS that 

has been granted Final Approval by the MEPC. 

Nevertheless, relevant data as required by Procedure (G9) are provided below for Ozone, TRO, 

bromate ion and bromoform. 
MEPC 58/2/3 

ANNEX 

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4.1.1 Active Substances (Ozone and TROs) 

Chemical name Ozone TRO (Total Residual Oxidants) 

(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 O3 HOBr OBrStructural 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 BrO3

-

 CHBr3

Structural formula 

Classification (UN GHS) 

Bromochloroacetic acid and 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 CHBr2COOH 

Structural formula 

Classification (UN GHS) 
MEPC 58/2/3 

ANNEX 

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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 (barge) testing, as 

follows: 

Ballasting 

Active substance & 

Relevant chemicals 

Unit Control Treated MDL1)

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 MDL1)

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 Disinfection By-Products were 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 Toxicity (WET) testing was undertaken by Dong-A 

University Hospital Clinical Research Centre using ballast water that had been treated by the 

NK-O3 System during land-based (barge) tests, according to toxicity test methods outlined in 

Procedure (G9). 
MEPC 58/2/3 

ANNEX 

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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 aquatic plants, vertebrates and fish 

Acute aquatic toxicity 

The full range of tests required by Procedure (G9) 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 chronic 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 disruption was detected. Refer to 

appendix 3, section 3.3. 

Endocrine effect screening was also conducted with Cell lining according to Standard Operating 

Procedure outlined by Aquatic Toxicology Laboratory Michigan State University 2003 – both 

treated and non-treated (control) ballast water showed TCDD-EQ significantly higher than the 

negative control solution but there was no significant difference between the both treated and 

non-treated (control) ballast water; so it is concluded that elevated 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 amphipods in accordance with USEPA.1994, 600/R-94/025 

and no toxicity was observed. Refer to appendix 3, section 3.3. 

Bioavailability biomagnification/bioconcentration 

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 short half-lives of these substances, plus 

given that the WET testing did not detect any significant toxicity, bioavailability, 

biomagnification and bioconcentration are not considered to be of relevance to this system. 

Refer to appendix 3, section 3.3. 

Food web/population effects 

As per bioavailability, biomagnification and bioconcentration. 
MEPC 58/2/3 

ANNEX 

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4.2.1.2 Data on mammalian toxicity 

Acute toxicity 

Testing was conducted using an ICR Mouse according to OECD Guideline TG 423 updated and 

adopted on 17 December 2001. 

Effects on skin and eye 

Standard skin sensitization tests 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 required 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 the 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 chromosome 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/genotoxicity was found in any of the tests. Refer to appendix 3, 

section 3.4. 
MEPC 58/2/3 

ANNEX 

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Toxicokinetics 

Toxicokinetics tests were not required for the 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 effects 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 short half-lives of these substances, plus given 

that the WET testing did not detect any significant toxicity, 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 identification 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 (CHBr3) by the haloform 

reaction. Besides bromoform, a number of disinfection products and other halogen containing 

compounds (THMs and HAAs) are produced when Ozone and bromide are present in natural 

waters. All these relevant chemicals 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 

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4.2.1.4 Physical and chemical properties for the Active Substances etc. 

As outlined in section 2 “Definitions” above, this application considers the Whole Effluent from 

the NK-O3 System. As the Active Substances have extremely short half-lives, as chemical 

testing did not detect any Active Substances or DBPs in the effluent, and as the WET testing did 

not detect any significant toxicity, the physical and chemical properties are as per the range for 

fresh, brackish and seawater carried as ballast by ships. 

However, for completeness, the table below lists the physical and chemical properties for the 

two Active Substances and the three DBPs, where relevant. 

Property Active Substance DBPs 

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 149~150 195 210~215 

Flammability Nonflammable Not available Nonflammable Not available Not available 

Density(kg/m3) 2.14 Not available 2,887 No data 1,980 

Vapour pressure(Pa) 

/vapour density 

5.46x106

/ 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 

2.20x104 

/ 1.48 

4.40x104 

/Not available 

Oxidation-reduction 

potential: 

2.07 E0(V) Not available Not available 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 data 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 

crystal 
MEPC 58/2/3 

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4.2.1.5 Analytical methods at environmentally relevant concentrations 

The analytical methods at environmentally relevant concentrations used 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-O3

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 Final 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 Standard 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 Administration. Certification No.16, 2005 

(http://www.kfda.go.kr); 

– National Institute of Environmental Research. Certification No. 2007-12 

(http://eng.nier.go.kr); and 

– Rural Development Administrator. Certification No. 2007-2 

(http://www.rda.go.kr). 

Full details of all test methods are contained in the full application for Final Approval submitted 

to the GESAMP-BWWG in accordance with Procedure (G9). 
MEPC 58/2/3 

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4.2.4 Quality control/quality assurance 

Combined Quality Management Plans (QMPs) and Quality Assurance Project Plans (QAPPs) 

were developed and applied for all aspects of both the G8 and G9 testing of the NK-O3 System. 

Extracts (heading structures) of both QMP/QAPPs are contained 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 for registration of chemicals 

4.2.6 Manner of application of Preparation

Please refer to sections 1.2 and 1.3 above. Full technical details are contained in the 

full application for Final Approval 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 assessment of quality of test reports (G9), 

risk characterization any uncertainty associated with the test results as addressed by 

Assessment Report annexed hereto. 

The test results found no significant levels of potentially toxic substances 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 the 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 

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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 using 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 does 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 kills certain species of bacteria in the water that 

enhance corrosion rates. 

Use of the side-stream injector in the NK-O3 System 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 injector area only, which is made from high-grade, 

non-corroding stainless steal. 

Full details including materials used 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 operation 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 the system is not in operation. 

In some fittings, the customer may elect to include supplementary 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 system. 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 

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6.1.4 Contact with or inhalation of process products 

In the NK-O3 System, potential exposure of ships’ crew to process products 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 waste Ozone destruction unit placed after the 

treatment unit, to eliminate any excess Ozone from the treatment unit and convert 

it back to oxygen prior to release to the atmosphere; 

– an Ozone densitometer to detect any possible (but highly improbable) accidental 

leakage from the system; 

– an automatic alarm and shut-down system linked to the Ozone densitometer. 

This is designed to trigger if Ozone levels 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 clearly outlined safety 

procedures as contained in supporting operating manuals for personnel. 

The following events will shut down the system immediately, 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 possible 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 

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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 placed 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 System having been operated, which occurs during 

ballasting. Ballast tanks need to be emptied prior 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 and the fact that Ozone is generated and infused at the 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 venting through the ballast tank vents. 

However to address this perceived 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 Operation 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 compounds in the ballast system/ballast tanks 

6.1.4.5 TRO/hypobromous acid/related compounds in the discharged ballast water

The short half-life of these products (days) makes it implausible that the discharged ballast water 

would contain these products. 

Testing of the chemical content of ballast water treated by the NK-O3 System during land-based 

and shipboard tests in accordance with IMO Guidelines (G8), did not detect any of these 

substances. 

Risk mitigation measures could include periodic sampling of system effluent as part of port State 

control compliance procedures, although 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 

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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-discernable against background levels. 

As standard safety procedure, crew working in the engine room are 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 accordance with the IMO Procedure (G9) did not detect any toxicity 

on test organisms. 

Testing of the chemical content of ballast water treated by the NK-O3 System during land-based 

(barge) tests in accordance with IMO 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 spread of invasive aquatic species. 

However to confirm this, risk mitigation measures could include periodic sampling of system 

effluent as part of port State control compliance 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 System is the fact that there is no requirement at 

all for storage or handling of any substances. Ozone is generated as required 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 Basic 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 document constituting a summary of non-confidential information. 

* * * 
MEPC 58/2/3 

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APPENDIX 1 

APPROVAL FROM THE KOREAN REGISTER OF SHIPPING FOR FULL-SCALE 

SHIP-BOARD TEST SYSTEM INSTALLED ONBOARD MV HYUNDAI HONG KONG 
MEPC 58/2/3 

ANNEX 

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* * * 
MEPC 58/2/3 

ANNEX 

Page 22 

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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, Instruments & 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 Management 
MEPC 58/2/3 

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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 handling of field samples

1.7.2 Laboratory testing and analysis 
MEPC 58/2/3 

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1.8 Project Team and responsibilities

1.9 Documents and records 

1.9.1 QAPP 

1.9.2 Field Sample Records 

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 decontamination 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 

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APPENDIX 3 

WHOLE-EFFLUENT TOXICITY (WET) TESTING REPORT 

BY DONG-A UNIVERSITY HOSPITAL 

These tests were conducted on the whole-effluent from the NK-O3 System 

(WET testing by DAUH-CRC). 

1. Data on effects on aquatic plants, invertebrates and fish etc 

1.1 Acute aquatic toxicity 

 Species LC50

(%/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 

(LC50 / duration) 

Reference/Comments/ 

Justification for 

missing data 

Crustacea Monocorophium acherusicum 

Amphipod 

> 100% / 10days US EPA. (1994) 
MEPC 58/2/3 

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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 the information on the safety of test substance 

(untreated sea water) against test animal (mouse) prior to conducting the study. 

This information was necessary to confirm that that the test material itself was relevant for the 

WET tests for mammals and applicable as undiluted 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 LD50 : 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 compared to genotoxicity of the 

untreated seawater (or brackish water) used ballast water material. 

(1) Bacterial Reverse Mutation Test 

Table 2. Mutagenicity of the bacterial reverse 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 

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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 reverse 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 aberration 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 

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Table 5. Mutagenicity of chromosome aberration assay with the untreated seawater or brackish water 

Assay Species Value 

(Concentration) 

Result Reference 

/Comment 

IC50 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 micronucleus test with the treated ballast water (seawater, brackish water) 

Assay Species Value 

(Concentration) 

Result Reference/Comment 

MI50 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 

MI50 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 

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(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 

RTG20 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 

RTG10 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 

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(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 

MI50 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 marrow chromosome aberration assay assay with the 

untreated seawater or brackish water 

Assay Species Value 

(Concentration) 

Result Reference/Comment 

MI50 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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