I:\MEPC\56\2.doc 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. INTERNATIONAL MARITIME ORGANIZATION

IMO 

# E

MARINE ENVIRONMENT PROTECTION

COMMITTEE

56th session Agenda item 2 MEPC 56/2

14 December 2006 Original: ENGLISH

HARMFUL AQUATIC ORGANI SMS IN BALLAST WATER

Basic Approval of Active Substances used by the Hybrid Ballast Water Treatment System using Seawater Electrolytic Process

Submitted by Japan

SUMMARY

Executive summary: This document contains non-confidential information for Basic Approval of Active Substances us ed by the Hybrid Ballast Water Treatment System using Seawater Electrolytic Process as required by the Procedure for approval of Ball ast Water Management systems that make use of Active Substances (G9). Relevant documents together with the application dossier have been submitted to Marine

Environment Division of the Organization for review by the GESAMP - Ballast Water Working Group.

Action to be taken: Paragraph 3

Related documents: BWM/CONF/36, MEPC 53/24/Add.1, MEPC 55/2/15 and BWM.2/Circ.6

Introduction

1 This document contains non-confidential information for Basic Approval of Active

Substances in accordance with the Procedure fo r approval of Ballast Water Management systems

that make use of Active Substances (G9) adopted by reso lution MEPC.126(53).

2 Relevant documents and application dossier prepared by Mitsubishi Heavy Industries, Ltd. have been verified by the Minist ry of the Environment a nd the Ministry of Land, Infrastructure and Transport, the competent aut horities in Japan and the complete application

dossier will be made available to the GESA MP-Ballast Water Working Group in accordance with the provisions contained in BWM.2/Circ.6.

Action requested of the Committee

3 The Committee is invited to consider the pr oposal for approval and decide as appropriate.

***MEPC 56/2

I:\MEPC\56\2.doc ANNEX

1 INTRODUCTION

The applicant proposes a seawater electrolytic process for the deactivation of planktons and bacteria in the ballast water treatment system. This application is based on the Procedure for approval of Ballast Water Management systems that make use of Active Substances (G9).

The seawater electrolytic process is to be installed at the stag e subsequent to the mechanical treatment (filter treatment, etc.). Once the mech anical treatment removes or deactivates large organisms (the minimum dimension: 50μm or mo re), the seawater electrolytic process will deactivate the plankton and steriliz e bacteria remaining in the ballast water. Upon completion of treatment the ballast water will comply with the discharge standard of regulation D-2 of

International Convention for th e Control and Management of Ships’ Ballast Water and Sediments. The process can also be adjusted to retain enough concentration of active chlorine in the ballast water to inhibit the re-growth of organisms within ballast tanks after treatment.

This seawater electrolytic process produces active chlorine from natural se awater on-board. The process has technical achievements of the “Marine-Growth-Prevention Systems” (MGPS ® ) for ships and power plants. Mi tsubishi Heavy Industries, Ltd. has been producing MGPS ®
for 40 years and has garnered substantial e xperience in the development, design, and manufacture of ships and ship equipment. This application believes that MGPS ® is a safe and economical system for ships and crews applicab le without the storage of chemicals on board. And by designing lineup of the system capacity to respond to the different flow rates of ballast pumps, the process can be applied to ballast pumps of any flow rate for common ships.

Water chlorination is a safe and reliable disinf ection procedure used worldwide for the drinking

water sanitation.

Once active chlorine deactivates the planktons a nd bacteria in ballast water, it is easily decomposed into harmless chlorine ion, a natu ral component of seawat er, through spontaneous decomposition by reactions with organic matter in natural seawater and temperature. Active chlorine has no persisten ce or bioaccumulation.

The applicant evaluated the toxicity of seawater treated with elec trolytically generated chlorine

under the conditions that simulate ballast water treatment and then evaluated the risks using

literature data. The results of these evaluations indicate that the influence on the environment of the ballast water discharges treated by this system is acceptable.

2 DATA-SET

The data set on Active Substances and relevant chemicals used fo r this study is summarized in Appendix 1.

2.1 Identification of the Substa nce or Preparation (G9: 4.1)

2.1.1 Preparations

No commercially available preparations are used. The seawater electrolytic process produces an Active Substance on shipboard using portions of the seawater taken as ballast water.MEPC 56/2

ANNEX Page 2

I:\MEPC\56\2.doc 2.1.2 Active Substances

This application evaluates free active chlorine produced by seawater electrolysis as the Active

Substance.

Chlorine (Cl 2 ) gas is generated at seawater electrolysis. This Chlorine (Cl 2 ) gas rapidly dissolves

into seawater to form free active chlorine (a mixture of hypochlorous acid (HClO) and hypochlorite ion (ClO - )) (see section 3.3 for details). The se awater electrolytic system harnesses

the disinfectant actions of free active chlori ne to deactivate regulated organisms.

Pure free active chlorine (a mixture of hypoc hlorous acid (HClO) and hypochlorite ion (ClO - )) is not available as a preparation on the market. For this reason, there are almo st no relevant data on the free active chlorine produced by the seawater electrolytic process necessary for the evaluation in the Procedure for approval of Ballast Water Management systems that make use of Active Substances (G9). To compensate, the appli cant mainly uses data relating to sodium hypochlorite (NaClO) as reference data for the ev aluation of the free activ e chlorine in this application. NaClO rapidly hydrolyzes in water and resides there as free active chlorine. Thus,

the HClO and ClO - could be evaluated using data on NaClO.

And as mentioned in the upcoming section 2.1.3, free active chlorine rapidly reacts with nitrogen compounds and organic compounds in natural seawater to form a mixture of free active chlorine with chloramines generally composed of tota l residual chlorine (T RC) or total residual oxidant (TRO).

This makes it difficult to assess the toxicity of pure free active chlorine in toxicity tests using test animals such as aquatic organisms. In many repor ts the toxicity has been assessed as TRO or TRC. In toxicity assessment using data on TRO or TRC, however, the influence of Monochloramine is small. The applicant also a ssesses the Active Substance using data on TRC or TRO in this application.

Table 1. Active Substances

Item Information for Active Substances IUPAC name Hypochlorous acid Hypochlorite ion

CAS number 7790-92-3 7681-52-9

UN number - -

Molar mass 52.46 51.46 Empirical formula HClO ClO Structural formula O

H Cl O

- Cl

Purity of technical

material and

identification of impurities Purity is not defined so that HClO is produced with on site;

Concentration of TRC:

Effluent of the seawater electrolytic apparatus: 10 mg/l (Maximum) Effluent of electrolytic cell: 1,500 mg/l (Maximum) Stabilizers or necessary

additives Not includedMEPC 56/2

ANNEX

Page 3

I:\MEPC\56\2.doc 2.1.3 Relevant Chemicals (G9: 2.1.4)

The disinfection by-products (DBPs) are evaluate d as relevant chemical s in this seawater electrolytic system.

Most of the Active Substance decomposes to ha rmless chlorine ion, a natural component of seawater, via disinfection/deactivat ion reactions with plankton and bacteria. Small amount of the substance, however, form DB Ps by reacting with the nitr ogen compounds and organic compounds contained in natural seawater.

2.1.3.1 Reaction with nitrogen compounds and Active Substance

Inorganic nitrogen (ammonia, nitr ous acid, nitric acid) and orga nic nitrogen reside in natural seawater as nitrogen compounds. Among these, ammonia reacts with the Active Substance formed by seawater electrolytic treatment to form chloramines. The following equations show examples of reaction formul ae with HClO and ammonia:

NH 3 + HClO → NH 2 Cl + H 2 O (1)

NH 2 Cl + HClO → NHCl 2 + H 2 O (2)

NHCl 2 + HClO → NCl 3 + H 2 O (3)

Dichloramine (NHCl 2 ) formed in Equation (2) is decomposed to nitrogen gas (N 2 ) via the reactions shown by equation (4):

2NHCl 2 + H 2 O → N 2 + HClO + 3H + + 3Cl - (4)

The reaction of Equation (4) occurs in the acidic region (<pH 4.4) an d thus will never occur at the pH of seawater (around 8). The proportion of chloramines present depends on pH, as shown in Fig. 1. At pH 7 (10o C), for example, NH 2 Cl is 91% and NHCl 2 is 9% (Pain A, (1950)).

From the foregoing, we learn that monochloramine (NH 2 Cl) is one of the main chloramines present in seawater at the pH of around 8.

Monochloramine, a kind of combined chlorine, is known exert the same type of disinfectant activity as free active chlorine, but it does so t oo weakly to qualify for use as a main Active Substance in the seawater electrolytic system. The applicant therefore defines monochloramine not as an Active Substance, but as a relative ch emical, in its evaluations for this application.

But as mentioned above, chlorine-treated natural s eawater is thought to cont ain a mixture of free

active chlorine and chloramines. When the a pplicant uses data from reports on toxicity

assessments carried out as TRC or TRO and toxic ity assessment tests on tr eated seawater carried out for this application, the assessments results must therefore be considered to include the toxicity effect of chloramines.MEPC 56/2

ANNEX Page 4

I:\MEPC\56\2.doc Fig. 1. Relation between pH valu e and presence of chloramines

2.1.3.2 Reaction with organic compounds and Active Substance

DBPs are formed when active chlorine species reacts with organic compounds in natural water. Various compounds have been iden tified in studies prompted by co ncerns over the health effects of DBPs generated from the chlorination of dr inking water (U.S. EPA (1999)). However, most DBPs are present in only very small amounts in chlorinated drinking wa ter, and non-identified compounds may also be present. Thus, sufficient da ta have yet to be obtained on the toxicity and

amounts of all of the DBPs formed.

Trihalomethanes and halogenated acetic acids (chlorinated and/or br ominated compounds) are reported to be the main DBPs generated in chlori ne disinfection. But, natural seawater contains about 65mg/l of bromine ion. Thus, substitution reactions between chlori ne ion of free active chlorine and bromine ion may occur at seawater chlorination. As the results of the reactions,

chlorinated and/or brominated trihalomethanes are generated. They can be formed by the following reactions:

(1) Reaction to form trihalomethanes

RCOCH 3 + 3HOX → CHX 3 + RCOOH + 2H 2 O (X=Br or Cl) (5)

(2) Reactions to form halogenated acetic acids

RCHO + HOX → RCOOH + H + + X - (6)

RCOOH + HOX → RXCOOH + H 2 O (7)

RXCOOH + HOX → RX 2 COOH + H 2 O (8)

RX 2 COOH + HOX → RX 3 COOH + H 2 O (9)MEPC 56/2

ANNEX

Page 5

I:\MEPC\56\2.doc Table 2. Disinfection by-products for natural water

Trihalomethanes Haloacetic acids

Trichloromethane (CHCl 3 )

Bromodichloromethane (CHBrCl 2 )

Dibromochloromethane (CHBr 2 Cl)

Tribromomethane (CHBr 3 ) 2-monochloroacetic acid (CH 2 ClCOOH) 2,2-dichloroacetic acid (CHCl 2 COOH) 2,2,2-trichloroacetic acid (CCl 3 COOH)

2-monobromoacetic acid (CH 2 BrCOOH) 2,2-dibromoacetic acid (CHBr 2 COOH) 2,2,2-tribromoacetic acid (CBr 3 COOH) 2-bromo-2-chloroacetic acid (CHBrClCOOH) 2-bromo-2,2-dichloroacetic

acid (CBrCl 2 COOH) 2,2-dibromo-2-chloroacetic

acid (CBr 2 ClCOOH)

The applicant carried out preliminary tests to confirm the amount of trihalomethanes and halogenated acetic acids formed in seawater chlorination by the seawater electrolytic process.

Two types of seawater were used for the tests: on e with a relatively high concentration of organic compounds (from Yokohama Port/Tokyo Bay) and one with a low concentration of organic compounds (Oigawa Port/Shizuoka Bay). The tests were performed using a small, laboratory-sized electrolytic apparatus (capacity of the electrolytic ce ll: 500 ml) fitted with an electrode of a type similar to that used for the actual seawater electroly tic apparatus. The test seawater was placed in the electrolytic cell and el ectricity was turned on to generate a 50±3 mg FAC/l electrolytic solution. After measurement of the FAC by iodometry, the electrolytic

solution and untreated test seawater were mixed to obtain treated seawater with a FAC concentration adjusted to 5.0 mg FAC/l.

The DBPs and pH were analyzed at the Japan Food Research Laboratori es, a registered water quality examination organization under Sectio n 20, Paragraph 3 of the Water Works Law of Japan (The Ministry of H ealth, Labor and Welfare).MEPC 56/2

ANNEX Page 6

I:\MEPC\56\2.doc Table 3. Analytical results of disinfection by-products in electrolytically generated

chlorine-treated natural seawater

Item Analysis value (mg/l) Detection

limit (mg/l) Sample Condition Sampling Point Sampling Date

Initial chlorine concentration

POC

DOC TOC (=DOC+POC)

PH Tokyo Bay

2006.7 (5.0) a,b

2.49 b

0.82 b

3.31 b
8.3 (21°C) Oigawa Port

2006.9 (5.0) a,c

0.1 c

1.0 c

1.1 c 8.1 (19°C) 0.05 2(μg)

0.05

-

-

Trihalomethanes trichloromethane (CHCl 3 ) bromodichloromethane (CHBrCl 2 ) dibromochloromethane (CHBr 2 Cl) tribromomethane(CHBr 3 ) N.D.

N.D.

0.006

0.16 N.D.

N.D.

0.003

0.15 0.001

0.001

0.001

0.001

Haloacetic acids 2-monochloroacetic acid (CH 2 ClCOOH) 2,2-dichloroacetic acid (CHCl 2 COOH) 2,2,2-trichloroacetic acid (CCl 3 COOH) 2-monobromoacetic acid (CH 2 BrCOOH) 2,2-dibromoacetic acid (CHBr 2 COOH) 2,2,2-tribromoacetic acid (CBr 3 COOH) 2,2-bromochloroacetic acid (CHBrClCOOH) 2,2,2-bromodichloroacetic acid (CBrCl 2 COOH) 2,2,2-dibromochloroacetic acid (CBr 2 ClCOOH) N.D.

N.D.

N.D.

N.D.

0.02

0.03

N.D.

N.D.

N.D. N.D.

N.D.

N.D.

N.D.

N.D.

0.01

N.D.

N.D.

N.D. 0.01

0.004

0.02

0.01

0.01

0.01

0.01

0.01

0.01
a: Test seawater was electrolyzed to prepare an electrolytic solution containing about 50 mg of FAC/l. FAC was

quantitatively determined by iodometry followed by mixing of the electrolytic solution with test seawater. b: Results of analysis carried out by the applicant. c: Results of analysis carried out by the organization in toxicity tests on treated seawater (see paragraph 5.2).

N.D.:Not Detection.

Two kinds of trihalomethanes (tribromometha ne, dibromochloromethane) and two kinds of brominated acetic acids (2,2-di bromoacetic acid, 2,2,2-tribromoacetic acid) were detected. Among these, tribromomethane showed the ma ximum concentration of 0.16 mg/l. The concentrations of the other compounds were belo w the detection limits. The differences in the concentrations of the organic compounds in seaw ater conferred only small effects on the amounts of the by-products formed and composition ratios. (Japan Food Research Laboratories, Analysis Certificate, No. 106064894-003 (2006)), Japan Fo od Research Laboratories, Analysis Certificate, No. 106091274-003 (2006)).

Based on these results, four of the DBPs, na mely, tribromomethane, dibromochloromethane, 2,2-dibromoacetic acid, and 2,2,2-tribromoacetic aci d, are evaluated as re levant compounds in this application.MEPC 56/2

ANNEX

Page 7

I:\MEPC\56\2.doc 2.1.3.3 Relevant Chemicals evaluated in Basic Approval

From the foregoing, monochloramine (described in section 2.1.3.1) and four compounds of DBPs (described in section 2.1.3.2) (tribromometha ne, dibromochloromethane, 2,2-dibromoacetic acid, 2,2,2-tribromoacetic acid) are assesse d as the relevant chemicals.

The influences of other by-products (those that may be contained in the treated seawater) were

assessed as toxic effects from the treated seawater in the toxicity evaluation tests and the risk assessment of discharges from the treated ballast water.

Table 4. Relevant Chemicals in ba llast water management system

IUPAC

name monochloramine tribromomethane dibromochloromethane 2,2-dibromoacetic acid 2,2,2-tribromoacetic acid

CAS

number 10599-90-3 75-25-2 124-48-1 631-64-1 75-96-7

Molar

mass 51.48 252.73 208.8 217.8 296.7

empirical

formula NH 2 Cl CHBr 3 CHBr 2 Cl CHBr 2 COOH CBr 3 COOH

structural

formula N

H

Cl H C H

Br Br

Br C H

Cl Br

Br C H

Br Br

COOH C Br

Br Br

COOH

2.1.4 Other chemicals

Hydrogen gas is generated by the cathode reaction t hrough the electrolytic reaction of seawater. There have been no reports, however, on the toxicity of hydrogen gas against organisms (human, fish, mammalian, etc.) or other in fluences on the environment. This precludes the evaluation of hydrogen gas as a subject of toxicity assessment in this application. The safety of hydrogen gas as an inflammable gas is briefly discussed in the section on safety to the vessel body.

2.2 Effects on aquatic organism (G9:4.2.1.1)

The applicant collected information (literature) on the effective concentration of each compound against aquatic organisms and assessed the reliabi lity of the data acco rding to the method of

Klimisch et al. (1997). Klimisch et al. (1997) classifies reliability of data into four ranks shown in Table 5. Unless otherwise stated, this applica tion only describes data cl assified as rank 1 or rank 2. While almost no papers report the toxic ity values of free active chlorine (hypochlorous acid and hypochlorite ion) as they are, many have reported the re sults of toxicity tests using sodium hypochlorite to form free active chlorine. Thus, the applicant collected the data on free active chlorine mainly from the literature on sodium hypochlorite.

The concentration of free active chlorine (hypoc hlorous acid (HClO) and hypochlorite ion (ClO - )) is written as FAC.MEPC 56/2

ANNEX Page 8

I:\MEPC\56\2.doc Given that free active chlorine are liable to form oxidizers in natural water, most of the Active

Substance concentrations are arra nged in terms of total residual chlorine (TRC), total residual oxidant (TRO), or chlorine -produced oxidant (CPO).

The value of TRC includes that of free activ e chlorine and combined active chlorine (chloramines etc.). Both The value of TRO a nd CPO include that of TRC and other oxidants.

Concentrations of oxidants ex cept TRC are very low at chlo rination; both TRO and CPO are

about the same of TRC.

Table 5. Categories/codes of reliability (Klimisch et al. 1997)

Code Category

1 Reliable without restriction

2 Reliable with restrictions

3 Not reliable 4 Not assignable

2.2.1 Acute aquatic toxicity

2.2.1.1 Free active chlorine

In an acute aquatic toxicity test using Oncorhynchus kisutch (fish/saltwater), 96h LC 50 was 0.032mg TRO/l (Thatcher (1978)). According to the report using Ceriodaphnia dubia (crustaceans/freshwater), 24h LC 50 was 0.005 mg FAC/l according to running water system giving no feed (Taylor (1993)). In an acute aquatic toxicity test using Thalassiosira pseudonana (algae/saltwater), 24h IC 50 (growth rate) was 0.075 mg TRC/l (Gentile et al. (1976)). Further, in an acute aquatic toxicity test using Crassostrea virginica (mollusks/ estuarinewater) in which chlorine (Cl 2 ) was added to free active chlorine. As a result, 96h EC 50 was 0.023 mg TRC/l (Roberts and Gleeson (1978)).

2.2.1.2 Relevant chemicals

(1) Monochloramine

In an acute aquatic toxicity test using Oncorhynchus kisutch (fish/saltwater), 96h LC 50 was 0.079-0.083 mg/l (Larson, G.L. et al. (1978)). Further, in an acute aquatic toxicity test using

Gammarus pseudolimnaeus (crustaceans/freshwater), 96h LC 50 was 0.22-0.235 mg/l (Arthur,

J.W et al. (1971)). There has been no report on acute aquatic toxi city value in terms of EC 50 of monochloramine against algae.

(2) Tribromomethane

In an acute aquatic toxicity test using Cyprinodon variegatus (fish/saltwater), 96h LC 50 was 7.1 mg/l (Ward et al. (1981)) according to running water. Further, statistically computed 96h NOEC was 2.9 mg/l (Heitmuller et al. (1981)). In an acute aq uatic toxicity test using Daphnia magna (crustaceans/freshwater), 24-48h LC 50 was 46-56 mg/l and 48h NOEC was less than 7.8 mg/l (LeBlanc, G.A.(1980)). Furthermore, in an acute aquatic toxicity test using

Skeletonema costatum (algae/saltwater), 96h EC 50 was 12.3 mg/l (ECOTOX (2005)).MEPC 56/2

ANNEX

Page 9

I:\MEPC\56\2.doc (3) Dibromochloromethane

Only freshwater organism tests of dibromochloromethane are reported.

In an acute aquatic toxicity test using Oryzias Latipes (fish/freshwater), 96h LC 50 was 79mg/l (NITE(2006a)). In an acute aquatic toxicity test (growth) using Selenastrum capricornutum (algae/freshwater), 72h EC 50 was 6.1mg/l and 72h NOEC was 2.9 mg/l (NITE(2006a)). In an acute aquatic toxicity te st (immobilization) using Daphnia magna (crustacean/freshwater),

48h EC 50 was 27mg/l (NITE(2006a)). Further, in an acute aquatic toxicity test using Tetrahymena pyriformis (protozoa/freshwater), 24h LC 50 was 65 mg/l (Yoshioka, Y. et al. (1985)).

(4) 2,2-dibromoacetic acid

Only freshwater organism tests of 2,2-dibromoacetic acid are reported.

An aquatic toxicity test on 2,2-dibromoacetic aci d as a decomposed matter of dibromoacetamide was carried out, and in an acute aquatic toxicity test using Pimephales promelas (fish/freshwater), 96h LC 50 was 6.9-mg/l (Mayes, M.A. et al. (1985)).

(5) 2,2,2-tribromoacetic acid

No report on acute aquatic toxicity of 2,2,2-trib romoacetic acid against aquatic organisms is

available.

2.2.2 Chronic aquatic toxicity

2.2.2.1 Free active chlorine

In a chronic aquatic toxicity test using Menidia peninsulae (fish/saltwater), 28d NOEC was 0.04 mg CPO/l (CPO: chlorine produced oxi dant) (Goodman (1983)). In a test using

Pandalus danae (crustaceans/saltwater), 1month NOEC was 0.05 mg FAC/l (Gibson (1976)). In a chronic aquatic toxicity test with Microcosm (algae/freshwater), 7d NOEC (biomass) was 0.0027 mg TRC/l (Cairns (1990)). Further, in a chronic aquatic toxicity test using Crassostrea virginica (mollusk/brackish water), 15d LOEC (14% effect) was 0.014 mg TRO/l (Liden (1980)). The NOEC of free active ch lorine can be estimated to be LOEC/2, and 15d NOEC is to be 0.007 mg TRO/l.

2.2.2.2 Relevant chemicals

(1) Monochloramine

There is no report on the chr onic toxicity of monochloramin e and the NOEC has not been reported.

(2) Tribromomethane

There are only a few reports on the chronic aquatic toxicity of tribromomethane against aquatic organisms. Ward et. al. (1981) carried out a test against Cyprinodon variegates (fish/saltwater)

and obtained a result of 28d NOEC (juvenile mortality) = 4.8-8.5 mg/l.MEPC 56/2

ANNEX Page 10

I:\MEPC\56\2.doc (3) Dibromochloromethane

Only acute aquatic toxicity tests with freshw ater organism of Dibromochloromethane are reported.

In a reproduction t oxicity test using Daphnia magna (crustacean/freshwater), 21d EC 50 was 1.5mg/l and 21d NOEC was 0.063mg/l. In a prolonged toxic ity test using Oryzias latipes (fish/freshwater), 21d NOEC was 3.2 mg/l (NITE (2006)).

(4) 2,2-dibromoacetic acid

No report on the chronic aquatic toxicity of 2,2-dibromoacetic acid is available.

(5) 2,2,2-tribromoacetic acid

No report on the chronic aquatic toxicity of 2,2,2-tribromoacetic acid is available.

2.2.3 Endocrine disruption effects

There are no report showing that free active chlori ne and the relevant chemicals have endocrine disruption effects on aquatic organisms.

2.2.4 Bioaccumulation

2.2.4.1 Free active chlorine

There is no report on the observed value of BCF and Log Pow of free active chlorine.

According to the calculated value using the calculation program (EPI Suite TM , U.S.EPA) based on QSAR, the Log Pow value of HClO is -0.87(esti mated) (that of NaClO is -3.42(estimated)), the value smaller than Log Pow ≥ 3, which is the rough indication fo r bioaccumulation potential. In addition, free active chlorine bioaccumulati on potential easily decomposes by contact with organic compounds, it is speculated that free active chlorine is no t high bioaccumulation potential compound.

2.2.4.2 Relevant chemicals

(1) Monochloramine

There is no report on the observed value of BCF and Log Pow of monochloramine.

According to the calculated value using the calculation program (EPI Suite TM , U.S.EPA) based on QSAR, the Log Pow value of monochl oramine is -1.19, the value smaller than Log Pow=3 which is the rough indication for bioaccu mulation potential, it is speculated that this chemical is not high bioaccumulation potential compound.

(2) Tribromomethane

According to ATSDR (2005), the Log Pow va lue of tribromomethane was 2.4(observed), because the value smaller than Log Pow=3, which is the rough indication for bioaccumulation potential, it is considered that this chemical is not high bi oaccumulation potential compound.MEPC 56/2

ANNEX Page 11

I:\MEPC\56\2.doc Further, the calculated value of BCF us ing the calculation program (EPI Suite TM , U.S.EPA) based on QSAR is 14. This value is smaller th an BCF>2000, which is the rough indication for bioaccumulation potential. This result also supports that this chemical is not high bioaccumulation potential compound.

(3) Dibromochloromethane

There is no report on the observed value of BCF. According to ATSDR (2005), the Log Pow of tribromomethane is 2.16(observed), because the value smaller than Log Pow=3, which is the rough indication for bioaccumulation potential, it is considered that this chemical is not high bioaccumulation potential compound.

Further, the calculated value of BCF us ing the calculation program (EPI Suite TM , U.S.EPA) based on QSAR is 9.2. This value is smaller than BCF>2000, which is the rough indication for bioaccumulation potential. This result also supports that this chemical is not high bioaccumulation potential compound.

(4) 2,2-dibromoacetic acid

No report on BCF of 2,2-dibromoacetic acid is availableAccording to Schultz et al. (1999), Log Pow value is 1.22(observed), because the value smaller than Log Pow=3 which is the rough indication for bioaccumulation potential, it is considered that this chemical is not high bioaccumulation potential compound.

Further, the calculated value of BCF us ing the calculation program (EPI Suite TM , U.S.EPA) based on QSAR is 3.2. This value is smaller than BCF>2000, which is the rough indication for bioaccumulation potential. This result also supports that this chemical is not high bioaccumulation potential compound.

(5) 2,2,2-tribromoacetic acid

No report on BCF of 2,2,2-tribromoacetic acid is available. According to Schultz et al. (1999), Log Pow value is 2.07(observed), because the value smaller than Log Pow=3, which is the rough indication for bioaccumulation potential, it is considered that this chemical is not high bioaccumulation potential compound.

Further, the calculated value of BCF us ing the calculation program (EPI Suite TM , U.S.EPA) based on QSAR is 3.2. This value is smaller than BCF>2000, which is the rough indication for bioaccumulation potential. This result also supports that this chemical is not high bioaccumulation potential compound.

2.2.5 Sediment toxicity and F ood web/population effects

2.2.5.1 Free active chlorine

As the Log Pow value of hypochlorous acid is -0.87 (estimated) (that of sodium hypochlorite is - 3.42(estimated)), which is smaller than the rough indication value for bioaccumulation potential of Log Pow=3, free active chlorine is not considered to have high bioaccumulation potential. Therefore the possibility of accumulati on to the higher-level predator in the food

web is low.MEPC 56/2

ANNEX Page 12

I:\MEPC\56\2.doc The Koc value of hypochlorous acid is 14.3l/kg (estimated by EPI Suite TM ) (that of sodium hypochlorite is 14.3(estimated by EPI Suite TM )). Because Koc is smaller than 500l/kg, it is considered that soil adsorption potential of free active chlorine is low.

Also, environmental distribution ratio of hypochlorous acid and sodium hypochlorite are calculated according Level III Fugacity model. Th e value of environmenta l distribution ratio of hypochlorous acid and sodium hypochlorite are both 0.0713% (estimated by EPI Suite TM ). Therefore, hypochlorous acid and sodium hypochlorite are consid ered to have low sediment persistence. Consequently, the risk of exposur e to benthic organism is considered low.

2.2.5.2 Relevant chemicals

(1) Monochloramine

As the Log Pow value of monochloramine is –1.19 (estimated), which is smaller than the rough indication value for bioaccumulation potential of Log Pow=3, monochloramine is not considered to have high bioaccumulation potential. Therefor e the possibility of accumulation to the higher level predator in the food web is low.

The Koc value of monochloramine is 14.3l/kg (estimated by EPI Suite TM ). Because Koc is smaller than 500l/kg, it is considered that soil ad sorption potential of monochloramine is low.

Also, environmental distribution ratio of monochloramine is calculated according Level III Fugacity model. Th e value of environmental dist ribution ratio monochloramine is e both 0.0679% (estimated by EPI Suite TM ) . Therefore, monochloramine is considered to have low sediment persistence. Consequently, the risk of exposure to benthic organism is

considered low.

(2) Tribromomethane

As the Log Pow value of tribromomethane is 2.4(observed), which is smaller than the rough indication value for bioaccumulation potential of Log Pow=3, tribromomethane is not considered to have high bioaccumulation potential. Therefor e the possibility of accumulation to the higher level predator in the food web is low.

The Koc value of tribromomethan e is 218.8l/kg (observed, DiToro et al. (1991)). Because Koc is smaller than 500l/kg, it is considered that so il adsorption potential of tribromomethane is relatively low.

Also, environmental distribution ratio of trib romomethane is calculated according Level III Fugacity model. The value of environmenta l distribution ratio tribromomethane is e both 0.0165% (estimated by EPI Suite TM ). Therefore, tribromomethane is considered to have low sediment persistence. Consequently, the risk of exposure to benthic organism is

considered low.

(3) Dibromochloromethane

As the Log Pow value of dibromochloromethane is 2.16 (observed), which is smaller than the rough indication value for bioaccumulation potent ial of Log Pow=3, dibromochloromethane is not considered to have high bioaccumulation poten tial. Therefore the possibility of accumulation to the higher-level predator in the food web is low.MEPC 56/2

ANNEX Page 13

I:\MEPC\56\2.doc The Koc value of dibromochloromethane is 84l/kg (ATSDR(2005)). Because Koc is smaller than 500l/kg, it is considered that soil adsorption potential of dibromochloromethane is relatively low.

Also, environmental distribution ratio of dibromoc hloromethane is calculated according Level III Fugacity model. The value of environmental distribution ratio dibromochloromethane is e both 0.0149% (estimated by EPI Suite TM ). Therefore, dibromochlorom ethane is considered to have low sediment persistence. Consequently, the risk of exposure to benthic organism is

considered low.

(4) 2,2-dibromoacetic acid

As the Log Pow value of 2,2-dibromoacetic acid is 1.22 (estimated), which is smaller than the rough indication value for bioaccumulation potent ial of Log Pow=3, 2,2-dibromoacetic acid is not considered to have high bioaccumulation poten tial. Therefore the possibility of accumulation to the higher-level predator in the food web is low.

The Koc value of 2,2-dibromoacetic acid is 1.90l/kg (estimated by EPI Suite TM ). Because Koc is smaller than 500l/kg, it is considered that soil adsorption potential of 2,2-dibromoacetic acid is relatively low.

Also, environmental distribution ra tio of 2,2-dibromoacetic acid is calculated according Level III Fugacity model. The value of environmental distribution ratio 2,2-dibromoacetic acid is e both 0.0703% (estimated by EPI Suite TM ). Therefore, 2,2-dibromoacetic acid is considered to have low sediment persistence. Consequently, the risk of exposure to benthic organism is

considered low.

(5) 2,2,2-tribromoacetic acid

As the Log Pow value of 2,2,2-tribromoacetic acid is 2.07 (estimated), which is smaller than the rough indication value for bioaccumulation potentia l of Log Pow=3, 2,2,2-tribromoacetic acid is not considered to have high bioaccumulation poten tial. Therefore the possibility of accumulation to the higher-level predator in the food web is low.

The Koc value of 2,2,2-tribromoacetic acid is 2.74l/kg (estimated by EPI Suite TM ). Because Koc is smaller than 500l/kg, it is considered that soil adsorption potential of 2,2,2-tribromoacetic acid is relatively low.

Also, environmental distribution ratio of 2,2, 2-tribromoacetic acid is calculated according Level III Fugacity model. The value of environm ental distribution ratio 2,2,2-tribromoacetic acid is e both 0.0715% (estimated by EPI Suite TM ). Therefore, 2,2,2-tribromo acetic acid is considered to have low sediment persistence. Consequentl y, the risk of exposure to benthic organism is

considered low.MEPC 56/2

ANNEX Page 14

I:\MEPC\56\2.doc 2.2.6 Determination of PNEC

2.2.6.1 Free active chlorine

The sensibility value of Ceriodaphnia dubia (freshwater organism) was the highest (24h LC 50 =0.005 mg FAC/l) on valid short-term toxicity data of free active chlorine. However, the value is not adapted, because this ballast water treatment system designed for seawater.

96h EC 50 =0.023mg TRC/l of mollusks is adapted, because the value is high next except that of Ceriodaphnia dubia

.

The sensibility value of micr ocosm (freshwater organism) was the 7d NOEC=0.0027 mg TRC/l on valid long-term toxicity data of free active chlorine. Howeve r, the value is not adapted, because this ballast water treatment system designed for seawater. 15d NOEC=0.007 mg TRO/l of mollusks is adapted, because the value is clearly higher than that of fish (28d NOEC=0.04mgCPO/L).

Because there is no valid long-term toxicity da ta of marine algae, Assessment Factor is

considered 100 on the determinatio n of PNEC of free ac tive chlorine in seaw ater. Therefore, PNEC is calculated using valid long-term toxici ty data of mollusks. The value of PNEC is 0.007 mg TRO/l/100 = 0.07 ц g TRO/l.

When the concentration of free ac tive chlorine in the discharged ballast water is in the range of 0.1-3.0mg TRC/l (paragraph 3.1), it is considered that free activ e chlorine in the discharged

ballast water has acute aquatic toxicity.

2.2.6.2 Relevant chemicals

(1) Monochloramine

Only an acute toxicity data of fish (96h LC 50 = 0.079-0.083mg/l) was reported as valid toxicity

data of monochloramine. Monochloramine is less sensibility than free active chlorine (96h EC 50 =0.023mg TRC/l). Therefore, even if the t oxicity of Active Substance is evaluated using TRC or TRO, there is no significant influen ce of toxicity of monochloramine. Then, it is

considered that the result of evaluati on is almost about free active chlorine.

(2) Tribromomethane

The value of 96h LC 50 =7.1mg/l of fish is the most hi ghest value of sensibility of tribromomethane. Then, it is adapted as valid s hort-term toxicity data of tribromomethane in

seawater.

The valid data using fish, crustaceans and al gae are reported, and 96 h NOEC=2.9mg/l that is acute toxicity data of fish is adopted for determination of PNEC. Assessment factor should be 100 due to applying the valid acute toxicity da ta to the NOEC, but 10 was applied to the

assessment factor. Because the acu te toxicity data was lower th an the chronic toxicity data. PNEC of tribromomethane was NOEC/10 = 0.29mg/l.

When the concentration of tribromomethane in the discharged ballast water is 0.16mg/l (see paragraph 2.1.3.2, table3), it is considered that tribromomethane in the discharged ballast water has no acute aquatic toxicity.MEPC 56/2

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I:\MEPC\56\2.doc (3) Dibromochloromethane

PNEC of dibromochloromethane c ould not be determined, because th ere is no report on toxicity data of marine organism.

(4) 2,2-dibromoacetic acid

PNEC of 2,2-dibromoacetic acid could not be de termined, because there is no report on toxicity data of marine organism.

(5) 2,2,2-tribromoacetic acid

PNEC of 2,2,2-tribromoacetic acid could not be determined, because there is no report on toxicity data of marine organism.

2.3 Mammalian toxicity (G9:4.2.1.2)

2.3.1 Acute toxicity

2.3.1.1 Free active chlorine

WHO has reported the oral LD 50 for calcium hypochlorite in rats as 850 mg/kg (WHO (2003)).

2.3.1.2 Relevant chemicals

(1) Monochloramine

Abdel-Rahman, M.S. et al. carried out a gavage te st in Sprague-Dawley rats and reports that at a dosage of 0.18-0.8 mg/kg, elevation of blood glut athione level was observed 15 to 30 minutes after the administration, but it re turned to normal level after more than 120 minutes. No effect was found in a blood osmotic fragility test (Abdel-Rahman, M.S. et al. (1984)).

(2) Tribromomethane

Tribromomethane is known to have the lowest or al acute toxicity among trihalomethanes. The

oral LD 50 for tribromomethane in ICR Swiss mice ( both sexes) was reported as 1400-1550 mg/kg (Bowman, F.J. et al. (1978)) and that in Sp rague-Dawley rats (both sexes) as 1147-1338 mg/kg (Chu, I. et al. (1980)).

(3) Dibromochloromethane

The oral LD 50 in ICR Swiss mice is reported as 800-1200 mg/kg (Bowman, F.J. et al. (1978)) and that in Sprague-Dawley rats as 848-1186 mg/kg (Chu, I. et al. (1980)).

(4) 2,2-dibromoacetic acid

The oral LD 50 in Sprague-Dawley rats is reported as 1737 mg/kg (Linder et al. (1994a)).MEPC 56/2

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I:\MEPC\56\2.doc (5) 2,2,2-tribromoacetic acid

No report is available concerning acut e toxicity of 2,2,2-tribromoacetic acid.

2.3.2 Effects on skin and eye

2.3.2.1 Free active chlorine

Loden carried out skin irrita tion tests on sodium hypochlorite (2, 20, 35, 50% solutions) in

rabbits in accordance with OECD Guideline 404, and reports of irritation index 1.2 with

the 2% solution and irritation index 5.2-5.3 with the 20, 35 and 50% solutions (IUCLID (2000)).

Pashley conducted eye irritation tests on sodium hypochlorite (0.005%, 0.052%, 0.525%. 5.25% solutions) in New Zealand white rabbits (8 ra bbits of assorted sexes). The 5.25% solution showed an irritating effect, which were not h ealed in 7 days. Effect was observed with

the 0.525% solution until after 2 and 6 hours. No effect was observed with the 0.052% and 0.005% solutions (Pashley (1985)).

No effect was found in the skin sensitization test with mixture of 5.65% NaClO solution and water (50/50 mixture) agains t guinea pigs (IUCLID(2000)).

2.3.2.2 Relevant chemicals

(1) Monochloramine

Monochloramine showed an irritating effect on the respiratory system (HSDS (2006)).

(2) Tribromomethane

Tribromomethane is known to have moderate irrita ting effects on respiratory organ, skin and eye. It is reported that an eye drop test with non-dilution so lution and a repeated sk in contact test in

rabbits showed moderate irr itation, which, however, were heal ed after one to two days (Torkelson, T.R. et al. (1994)).

According to ACGIH, the time weighted averag e (TWA) concentration for skin is 0.5 mg/l.

No report is available concerning human inhalation toxicity of tribromomethane. Dogs exposed

to ≥ 7000 ppm became deeply anesthetized after 8 minutes and died after a 1-hour exposure (RAIS, (2006)).

(3) Dibromochloromethane

Dibromochloromethane is known to have irritating effects on respiratory or gan, skin and eye. No report is available concerning human inhala tion toxicity of dibromochloromethane.

(4) 2,2-dibromoacetic acid

As 2,2-dibromoacetic acid is a strong acid, unne utralized solution may causes serious burn to akin and eye.MEPC 56/2

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I:\MEPC\56\2.doc (5) 2,2,2-tribromoacetic acid

As 2,2,2-tribromoacetic acid is a strong acid, unne utralized solution may causes serious burn to akin and eye.

2.3.3 Repeated-dose toxicity

2.3.3.1 Free active chlorine

As a result of 90-day administration of free act ive chlorine (0-250 mg/l) through drinking water to Sprague-Dawley rats, no consiste nt change in organ weight or tissues was observed. The “no observed adverse effect level (NOAEL)” was determined as 250 mg/l (16.7-24.9 mg/kg/d) (Daniel, F.B. et al. (1990a)).

In a 90-day administration test (0-200 mg/l) through drinking water to B6CF3F 1 mice, decrease of the weight of pancreas and liver was obs erved in 100 and 200 mg/l-administered groups of male mice. With female mice, increase of h eart weight was observed in 200 mg/l-administered group. The NOAEL was determined as 50 mg/l (10-12 mg/kg/d) (Daniel, F.B. et al. (1991)).

2.3.3.2 Relevant chemicals

(1) Monochloramine

As a result of 90-day administration (0-200 mg/l) of monochloramine through drinking water to Sprague-Dawley rats, decrease of water intake at all dose levels and decrease of feed intake in male rats at 200mg/l dose were observed. However, as remarkable decrease in body weight gain was observed only in 200 mg/l-administered group and for other reasons, the NOAEL was determined as 100mg/l (5.8-7.7 mg/kg/d) (Daniel, F.B. et al. (1990a)).

In a 90-day administration test (0-200 mg/l) through drinking water to B6CF3F 1 mice, decrease of feed intake was observed in both sexes (parti cularly female) at 200mg/l dose. Likewise, water

intake decreased in both male and female mice at all dose levels, with the effect on female mice being bigger. Decrease in body weight gain wa s observed in both sexes at 200mg/l dose. The NOAEL was determined as 50 mg/l (8.6-9.2 mg/kg/d) (Daniel, F.B. et al. (1991)).

(2) Tribromomethane

As a result of 13-week gavage tests on corn o il including tribromomethane using F344/N rats and

B6CF3F 1 mice, hepatocyte vacuolation was observed only in male rats administered with 50 mg/kg/d or more and in male mice admi nistered with 200 and 400 mg/kg/d. This phenomenon was not observed in female rats and female mice. In this study, the NOAEL for rat was computed as 25 mg/kg/d and that for mice as 100 mg/kg/d (NTP (1989)).

(3) Dibromochloromethane

As a result of a 90-day gavage test on corn o il including Dibromochloromet hane (0-200 mg/kg/d) using Sprague-Dawley rats, de crease in body weight gain was observed at 200mg/kg/d dose. The NOAEL for gavage of dbromochloromethane was determined as 50 mg/kg/d (Daniel,

F.B. et al. (1990b)).MEPC 56/2

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I:\MEPC\56\2.doc (4) 2,2-dibromoacetic acid

In a 28-day administrati on test in mice (B6CF3F 1 mice) through drinking water, the NOAEL is computed as 38 mg/kg/d (WHO (2004)).

In a 3-month administrati on test in mice (B6CF3F 1 mice) through drinking water (0-2,000 mg/l),

all the mice survived until the end of the test period. However, decrease in body weight gain and that in water intake were observed in 1,000 and 2,000 mg/l-administered groups. In groups administered with 500 mg/l or more, substantia l increase of liver weight was found in both sexes (NTP (2005)).

Likewise, in a 3-month administra tion test in rats (F 344/N rats) through drinki ng water, decrease in body weight gain, decrease in wa ter intake and substa ntial increase of liv er weight were found (NTP (2005)).

(5) 2,2,2-tribromoacetic acid

No report is available on a repeated dose test on 2,2,2-tribromoacetic acid.

2.3.4 Chronic toxicity

2.3.4.1 Free active chlorine

A test was carried out in which drinking water treated with sodium hypochlorite was administered to F344 rats for 2 years. To ma le rats, 0, 4.2, 7.3 or 13.6 mg/kg/d was administered and to female rats, 0, 4.2, 7.8 or 14.4 mg/kg/d wa s administered. Water intake decreased depending upon the dosage. No effect was observe d on body weight and surviv al rate. Also, no significant effect was observed on carcinogenicity (NTP (1992)).

In a test using B6C3F 1 mice, 0, 7.3, 14.0 or 23.8 mg/kg/d wa s administered to male mice and 0, 7.6, 14.2 or24.2 mg/kg/d was administered to female mice for 2 years. Water intake decreased depending upon the dosage. No effect was observed on body weight and survival rate. Also, no effect was observed on ca rcinogenicity (NTP (1992)).

From the above, the NOAEL was determined as 14.4 mg/kg/d.

2.3.4.2 Relevant chemicals

(1) Monochloramine

A test was carried out in which drinking water treated with chloramine was administered to F344 rats for 2 years. To male rats was administ ered 0, 2.1, 4.8 or 8.7 mg/kg/d and to female rats, 0, 2.8, 5.3 or 9.5 mg/kg/d was administered. Alt hough decrease in water and feed intake and a slight decrease in body weight we re observed, no clinical change was found at the end of the test (NTP (1992)).

In a test using B6C3F 1 mice, 0, 5.0, 8.9 or 15.9 mg/kg/d was administered to male mice, and 0, 4.9, 9.0 or 17.2 mg/kg/d to female mice for 2 y ears. Although decrease in water and feed intake, and body weight decrease were observed, ther e was no change in the survival rate. As a result of histopathological examination, no clinical effect was found (NTP (1992)).MEPC 56/2

ANNEX Page 19

I:\MEPC\56\2.doc (2) Tribromomethane

Drinking water to which tribromomethane was a dded was administered to male F344 rats and

B6C3F 1 mice for one year and clinical indicators of kidney toxicity was examined. The dose level of tribromomethane was 6.2, 29 or 57 mg/kg/ d for rats and 8.3, 39 or 73 mg/kg/d for mice.

Increase in clinical indicators relating to tubular and glomerular injuries was shown in mice of all the treatment level. Urine N-acetyl- β -glucosamidase increased at 39 and 78mg/kg/d dose. As in mice, urinary protein increased in rats of all the test groups. However, no evidence showing loss

of tubular function has been ob tained. Urine aspartate-tran saminase increased at 29 and 57 mg/kg/d dose. NOEC is not reported. (Moore, T.C. et al. (1994)).

(3) Dibromochloromethane

Gavage tests on corn oil including dibromochlor omethane were carried out in F344/N rats and

B6C3F 1 mice. Liver lesions were observed in all the test groups of rats (0, 40, 80 mg/kg/d) and mice (0, 50, 100 mg/kg/d). Hepa tic focal necrosis and liver calcification were observed at 100mg/kg/d, and in female mice, fatty changes we re observed in all dose levels. NOEC is not reported (NTP (1985)).

(4) 2,2-dibromoacetic acid

In a 2-year administration te st (0-1,000 mg/l) in B6CF3F 1 mice through drinking water, increase in body weight gain was observed at the dosag es of 50 mg/l and 500 mg/l in male mice. However, no difference was found between cont rol groups in female mice (NTP (2005)).

In the case of a 2-year administra tion test in F344/N rats through dr inking water, survival rate of test groups at the end of the te st period showed no difference be tween control groups. Decrease in body weight gain was observed at the dosag e of 500 or 1,000 mg/l, whereas water intake decreased at the dosage of 1,000 mg/l (NTP (2005)).

(5) 2,2,2-tribromoacetic acid

No report relating to chronic toxicity of 2,2,2-tribromoacetic acid is available.

2.3.5 Developmental and reproductive toxicity

2.3.5.1 Free active chlorine

No information relating to the re sults of tests carried out in accordance w ith OECD 414 or 416 is

available.

A reproductive toxicity test wa s carried out in which drinking water subjected to chlorine treatment with sodium hypochlorite (100 mg FAC /l) was administered by drinking to BDII rats over 7 generations. No effect on fertiliz ing ability was observed (Druckrey (1968)).

Reproductive toxicity tests were carried out in which drinking water subjected to chlorine treatment with sodium hypochlorite (10 mg TRC /l) was administered by drinking to C3H/BL

mice and C57BL/6J mice for 6 months. No effe ct on reproduction was observed (Les (1968)).MEPC 56/2

ANNEX Page 20

I:\MEPC\56\2.doc 2.3.5.2 Relevant chemicals

(1) Monochloramine

A reproductive toxicity (fertility ) test was carried out in wh ich chloramine-treated water (0-10 mg/kg/d) was administered by gavage to Long-Evans rats for 66 to76 days (including breeding period). No change was observed in fe rtility, viability, litter size and day of eye opening. Furthermore, no effect on sperm count , direct progressive sp erm movement, percent sperm mobility and sperm morphologic characte ristics was observed. The NOAEL was estimated as 10 mg/kg/d in this study (Carlton, B. et al. (1986)).

(2) Tribromomethane

According to the report of Ruddick, J.A. et al. , fetotoxicity was observed in Sprague-Dawley rats administered with 50-200 mg/kg of tribromometh ane by gavage, but no fetal malformation was observed (Ruddick J.A. et al. (1983)).

In a 105-day gavage test in Swiss CD-1 mice, no effect was observed on fertility, litters per pair, live pups per litter, sex of live pups and pup body weights (WHO (2000)).

(3) Dibromochloromethane

A water drinking test on dibromochloromethane (0, 0.1, 1.0, 4.0 mg/l) added water was carried out in ICR Swiss mice over 2 generations. In high dose level (4.0mg/L) of the F 1 generation, decrease in fertility and gestational index were observed, whereas in the F 2 generation, reduction in fertility was only observed in highly dose level (4.0mg/L). In moderately dose level (1.0mg/L) and highly dose level (4.0mg/L) of the F 1 and F 2 generations, decrease of litter size and that of viability index were shown. Neither dominant lethal nor teratogenic effects were observed in both generations (WHO (2000)).

(4) 2,2-dibromoacetic acid

A 9-day or 20-day gavage test on 2,2-dibrom oacetic acid (0, 62. 5, 125, 250 mg/kg/d) in Holtzman rats was carried out, and E2 was increas ed remarkably in 250 mg/kg/d-administered groups. The NOAEL was estimated as 125 mg/kg/d in this study (Cummings et al. (1998)).

(5) 2,2,2-tribromoacetic acid

No report relating to developmental and repro ductive toxicity of 2,2,2-tribromoacetic acid is

available.

2.3.6 Carcinogenicity

2.3.6.1 Free active chlorine

A test in which drinking water subjected to ch lorine treatment with sodium hypochlorite was administered by water drinking to F344 rats for 104 weeks was carried out. To male rats was administered 0, 4.2, 7.3 or 13.6 mg/kg/d and to female rats, 0, 4.2, 7.8 or 14.4 mg/kg/d was administered. No carcinogenicity was shown in ma le rats. In female rats, mononuclear leukemia slightly increased in 7.8 and 14.4 mg/kg/d groups. Ho wever, it was considered that this could not be regarded as the evidence of carcinogenicity (NTP (1992)).MEPC 56/2

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I:\MEPC\56\2.doc In a test using B6C3F 1 mice, as a result of administering 0, 7.3, 14.0 or 23.8 mg/kg/d to male mice, and 0, 7.6, 14.2 or 24.2 mg/kg/d to female mice for 104 weeks, no carcinogenicity was observed (NTP (1992)).

According to the carcinogenicity assessment of International Agency for Research on Cancer (IARC), sodium hypochlorite is classified as Group 3 (not classifiable as to carcinogenicity to humans) (IRIS).

2.3.6.2 Relevant chemicals

(1) Monochloramine

As no data showing carcinogenicity of monochloramine to humans is available, monochloramine is classified with Group 3 (not classifiable as to carcinogenici ty to humans) according to the carcinogenicity assessment of IA RC and Group D (not cl assifiable as to human carcinogenicity) by U.S. Environmental Protection Agency (U.S. EPA).

(2) Tribromomethane

IARC assessed carcinogenicity of tribromomethane and concluded that only insufficient evidence is available as to the carcinoge nicity to humans and that the ev idence is limited regarding the carcinogenicity to experimental animals. As a result, tribromomethane was classified as Group 3 (not classifiable as to carcinoge nicity to humans) (IARC (1991, 1999)).

U.S. EPA classifies tribromomethane as Group B2 (probable human carcinogen) (IRIS).

(3) Dibromochloromethane

IARC assessed carcinogenicity of dibromochlorom ethane and concluded that only insufficient evidence is available as to the carcinogenicity to humans and that the evidence is limited regarding the carcinogenicity to ex perimental animals. As a re sult, dibromochloromethane was classified as Group 3 (not cla ssifiable as to carcinogenici ty to humans) (IARC (1991, 1999)).

U.S. EPA classifies this compound as Gr oup C (probable human carcinogen) (IRIS).

(4) 2,2-dibromoacetic acid

In a 2-year administration test (0- 1,000 mg/l) through drinking water in B6CF3F 1 mice, incidence of liver neoplasms increased in both sexes at all dose levels and that of multiple hepatocellular adenoma, hepatocellular adenoma or cancer remarkably increased in male of all the administration groups and female of 500 mg/l and 1,000 mg/l-administered groups (NTP (2005)).

In the case of a 2-year administration test thro ugh drinking water in F344/N rats, incidence of malignant mesothelioma consider ably increased in male mice of 1,000 mg/l-administered group.

Incidence of mononuclear cell leuk emia was elevated in female mice and particularly, high

incidence was shown in female mice of 1,000 mg/l-administered group (NTP (2005)).

2,2-Dibromoacetic acid is not included in the s ubstances subject of carc inogenicity assessment by IARC.MEPC 56/2

ANNEX Page 22

I:\MEPC\56\2.doc (5) 2,2,2-tribromoacetic acid

No report on carcinogenicity of 2,2,2-tribromoacetic acid is available. This compound is not included in the substances subject of carcinogenicity assessment by IARC.

2.3.7 Mutagenicity/genotoxicity

2.3.7.1 Free active chlorine

Sodium hypochlorite show ed mutagenicity in Salmonella tymphimurium TA100 ( in vitro ) in the presence of a metabolic activation system (S9mix) (Ishidate (1984)).

A cytogenetic assay carried out in human embryo stem cell ( in vitro ) showed mutagenicity (Sasaki (1980)).

A micronucleus assay was carri ed out in male ddY mice ( in vivo ), but mutagenicity was not observed (Hayashi (1988)).

A chromosomal aberration test was carried out in CD-1 Swiss mice ( in vivo ), but mutagenicity was not observed (Meier et al. (1985)).

2.3.7.2 Relevant chemicals

(1) Monochloramine

In a reverse mutation test using Salmonella tymphimurium , slight increase of reverse mutation is reported (Thomas,E.L. et al. (1987)). However, in a test using mice (CD-1 mice), mutagenicity was not shown (Meier, J.R. et al. (1985)).

(2) Tribromomethane

As regards the mutagenicity of tribromomethane, there are reports with different results and conclusion is yet to be made. In reverse mutation tests using Salmonella tymphimurium , some reports conclude that tribromomethane has mutagenicity (Simmon ,V.F. et al. (1978) and Simmon V.F. (1977)), and others show that the compound has no e ffect on mutagenicity (NTP (1989a), Rapson W.H.8 (1980), Varma M.M. (1988)).

Likewise, concerning mutagenicity tests in mammal s, there are two kinds of report, one showed that the compound has mutagenicity (Morimoto, K. (1983), Fujie, K. (1993)) and the other report

showed that it has no effect thereon (Galloways, S.M. et al. (1985)).

(3) Dibromochloromethane

As regards the mutagenicity of dibromochlorometha ne in bacteria, there ar e reports with different results as in the case of tribromomethane. In reverse mutation tests using Salmonella tymphimurium , some reports showed that the com pound has mutagenicity (Simmon, V.F. et al. (1978), Simmon V.F. (1977), Varma M.M. (1988)) and others showed that it has no effect thereon (NTP (1985), Zeiger, E. et al. (1987)).MEPC 56/2

ANNEX Page 23

I:\MEPC\56\2.doc On the other hand, many reports show the results that dibromochloromethane has carcinogenicity in mammals such as human lymphocytes (Morimoto, K. et al. (1983)), rat erythroblastic

leukemia K 3 D cells (Fujie, K (1993)), etc.

(4) 2,2-dibromoacetic acid

As a result of a reverse mutation test using Salmonella tymphimurium TA100, mutagenicity was observed in the range of 10-3,000 mg/l (Giller, et al. (1997)). Although NTP reported mutagenicity of 2,2-dibromoacetic acid as a result of a reverse mutation test using Salmonella tymphimurium TA100, no mutagenicity was observed in the test using Salmonella tymphimurium TA98 (NTP (2005)).

(5) 2,2,2-tribromoacetic acid

As a result of a reverse mutation test using Salmonella tymphimurium , mutagenicity was observed in the range of 2,000-10,000 mg/l (Giller, et al. (1997)).

2.4 Data on environmental fate and effect under aerobic and anaerobic conditions (G9: 4.2.1.3)

2.4.1 Modes of degradation (biotic; abiotic)

2.4.1.1 Free active chlorine

Gaseous chlorine (generated on the anodal surface of an electrolysis vessel ) rapidly hydrolyzes in water with a pH more than 5 and forms hypochlorous acid as shown in E quation (10). Hydrolytic constant of Equation (10) are 1.5 x 10 -4 (mol/l) 2 (0°C) and 4.0 x 10 -4 (mol/l) 2 (25°C). More than 99% in terms of a formula weight concentration of chlorine of 10 -3 mol/l in pure water is hydrolyzed (Morris (1978)).

Cl 2 + H 2 O ↔ HClO + H + + Cl - (10)

On the other hand, hypochlorous acid is an oxidiz er, a disinfectant and pr imarily an inorganic substance. Therefore, it is said that it is not proper to discuss its biodegradability (IUCLID (2000)).

Taylor measured decay rate of hypochlorous acid and hypochlorite ion in the acute aquatic toxicity test. The decay rate wa re reported as follows; the half life at pH 7.0 (main formula is HClO) is about 0.8hour and the half life at pH 8.0 (main formula is ClO - ) is about 1.4 hour (Taylor et al. (1993)).

2.4.1.2 Relevant chemicals

(1) Monochloramine

Decomposition rate of monochloramine in water is slow and it is relatively a stable compound (Rice, R.G. et al. (1986)). The half-life of monochlorami ne by hydrolysis is re ported as 10 hours (Margerum et al. , (1978)).

No report on biodegradation of monochloramine is available.MEPC 56/2

ANNEX Page 24

I:\MEPC\56\2.doc (2) Tribromomethane

The rate constant of hydrolysis for tribromomethane is estimated as 3.2x10 -11 /sec (Mabey, W.

et al. (1978)), and this is a stab le compound in water (estimated half life is 686 years).

In the long-term risk assessmen t of the treated ballast wate r discharges (section 5.3), the behaviors of tribromomethane concentration in natural seawater were considered for degradation rate that is k = 0.00978 (day -1 ) = 0.693/t 1/2 (decay) set in the databa se of CHEMMAP (Science and technology Co., Ltd. and Asia-Pacific Appl ied Science Associates (2006), Appendix B).

Tribromomethane is considered a volatile com pound. According to trial ca lculation, the half-life of tribromomethane is 1h (riffle)-581 h (pool) (Kaczmar, S.W. et al. (1984).

The rate constant for the vapor-phase reaction of tribromomethane with photochemically-produced hydroxyl radi cals has been estimated as 4.94x10 -14 cm 3 /molecule-sec at 25 °C using a structure estimation method. This corresponds to an atmospheric half life of about 325 days at an atmospheric concentration of 5x10 5 hydroxyl radicals per cu cm. (HSDB,(2006))

Although aerobic biodegradation of tribromometh ane is limitative, it is reported that tribromomethane is easily degraded anaerobicall y, for example, by methanogenic bacteria or denitrifying bacteria. In denitr ifying condition, 60 ug/l of tribro momethane was degraded to 59, 37, 35, and 2 ug/l after 2, 3, 4, and 6 weeks, respectively (HSDB (2006)). Through field investigation, the half-life of trihalomethane by anaerobic degradation is estimated as 21-42 days.

(3) Dibromochloromethane

The rate constant of hydrolysis for di bromochloromethane is estimated as 8.0x10 -11 /sec (Mabey,

W. et al. (1978)), and dibromochloromethane is a st able compound in water (estimated half life is 274 year).

However, dibromochloromethane is consider ed a volatile compound. According to trial calculation, the half-life of dibromochloromethane is 0.7h (ri ffle)-398 h (pool) (Kaczmar, S.W.

et al. (1984).

The rate constant for the vapor-phase reaction of chlorodibromomethane with photochemically-produced hydroxyl radi cals has been estimated as 5.8x10 -14 cm 3 /molecule-sec at 25 °C using a structure estimation method. This co rresponds to an atmospheric half life of about 280 days at an atmospheri c concentration of 5x10 5 hydroxyl radicals per cm 3 . (HSDB,(2006))

Same as tribromomethane, this compound hardly undergoes aerobic biodegradation but is easily biodegraded anaerobically. In an anaerobically experiment w ith methanogenic mixed culture, initial concentration of 19, 25 and 205 ug/l of dibr omochloromethane were degraded to less than detection limit after 2 weeks incubation (Bouwer, E.J. et al. (1981)).

(4) 2,2-dibromoacetic acid

Haloacid dehalogenase (EC3.8.1.3) has been f ound in many kind of bacteria such as Alcaligenes

sp., Arthrobacter sp., Moraxella sp., Pseudomonas sp. or Xanthobacter sp., which are widely distributed in environment (soil, inland water, seawater). The enzymes degrade halogen (Cl orMEPC 56/2

ANNEX Page 25

I:\MEPC\56\2.doc Br or F)-substituted short-chain fatty acid (IU BMB (2006)). After 10 days aerobic cultivation

of Xanthobacter authotrophicus GJ10, 2,2-dibromoacetic acid was degraded and released

8.8mM bromine ion from initial concentration of 5mM of 2,2-dibromoa cetic acid. (Janssen,

D.B. et al. (1985)).

(5) 2,2,2-tribromoacetic acid

Like 2,2,2-dibromoacetic acid, 2,2,2-tribromoacetic ac id is degraded by haloacid dehalogenase (EC3.8.1.3). An enzyme activities assay of ha loacid dehalogenase, whish purified from

Rhizobium sp., was carried out in Tris buffer (pH7.5). It is reported that the catalytic efficiency (=Kcat/Km) of 2,2,2-tribromoacetic acid was 0.64 x 10 4 /M/sec, this value is lower than that of 2,2-dibromoacetic acid (1.64 x 10 4 /M/sec)(Huyop, F.Z. (2004)).

2.4.2 Bioaccumulation, partition coefficient

2.4.2.1 Free active chlorine

As the calculated Log Pow value of hypochlorous acid is -0.87 (that of sodium hypochlorite is -3.42), which is smaller than the rough indi cation value for bioaccumulation potential of Log Pow=3, free active chlorine is not considered to have high bioaccumulation potential. Therefore the possibility of accumulation to the higher- level predator in the food web is low.

Also, the Koc value of hypochlorous acid is 14.3l/kg (estimated by EPI Suite TM ) (that of sodium hypochlorite is 14.3(estimated by EPI Suite TM )). Because Koc is smaller than 500l/kg, it is considered that soil adsorption potential of free active chlorine is low. Therefore, free active

chlorine is considered to have low sediment toxicity.

2.4.2.2 Relevant chemicals

(1) Monochloramine

As the calculated Log Pow valu e of monochloramine is –1.19, whic h is smaller than the rough indication value for bioaccumulation potential of Log Pow=3, monochloramine is not considered to have high bioaccumulation potential. Ther efore the possibility of accumulation to the higher-level predator in the food web is low.

Also, the Koc value of monochloramin e is 14.3l/kg (estimated by EPI Suite TM ). Because Koc is smaller than 500l/kg, it is considered that soil adsorption potential of monochloramine is low. Therefore, free active chlorine is consid ered to have low sediment toxicity.

(2) Tribromomethane

As the Log Pow value of tribromomethane is 2.4(observed), which is smaller than the rough indication value for bioaccumulation potential of Log Pow=3, tribromomethane is not considered to have high bioaccumulation potential. Ther efore the possibility of accumulation to the higher-level predator in the food web is low.

Also, the Koc value of tribromometh ane is 218.8l/kg ( observed, DiToro et al. (1991)). Because Koc is smaller than 500l/kg, it is considered that soil adsorption potential of tribromomethane is relatively low. Therefore, free active chlorine is considered to have low sediment toxicity.MEPC 56/2

ANNEX Page 26

I:\MEPC\56\2.doc (3) Dibromochloromethane

As the Log Pow value of dibromochloromethane is 2.16 (observed), which is smaller than the rough indication value for bioaccumulation potent ial of Log Pow=3, dibromochloromethane is not considered to have high bioaccumulation poten tial. Therefore the possibility of accumulation to the higher-level predator in the food web is low.

Also, the Koc value of dibromochloromethane is 84l/kg (ATSDR (2005)). Because Koc is smaller than 500l/kg, it is considered that soil ad sorption potential of dibromochloromethane is relatively low. Therefore, free active chlorine is considered to have low sediment toxicity.

(4) 2,2-dibromoacetic acid

As the calculated Log Pow value of 2,2-dibromo acetic acid is 1.22, which is smaller than the rough indication value for bioaccumulation potent ial of Log Pow=3, 2,2-dibromoacetic acid is not considered to have high bioaccumulation poten tial. Therefore the possibility of accumulation to the higher-level predator in the food web is low.

Also, the Koc value of 2,2-dibromoacetic acid is 1.90l/kg (estimated by EPI Suite TM ). Because Koc is smaller than 500l/kg, it is considered that soil adsorption potential of 2,2-dibromoacetic acid is relatively low. Therefore, free active chlori ne is considered to have low sediment toxicity.

(5) 2,2,2-tribromoacetic acid

As the calculated Log Pow value of 2,2,2-tribro moacetic acid is 2.07, which is smaller than the rough indication value for bioaccumulation potentia l of Log Pow=3, 2,2,2-tribromoacetic acid is not considered to have high bioaccumulation poten tial. Therefore the possibility of accumulation to the higher-level predator in the food web is low.

Also, the Koc value of 2,2,2-tribromoacetic acid is 2.74l/kg (estimated by EPI Suite TM ). Because Koc is smaller than 500l/kg, it is considered th at soil adsorption potentia l of 2,2,2-tribromoacetic acid is relatively low. Therefore, free active chlori ne is considered to have low sediment toxicity.

2.4.3 Main metabolites

2.4.3.1 Free active chlorine

The metabolites of free active chlorine are H + , Cl and O 2 , and no persistent substance is formed.

2.4.3.2 Relevant chemicals

(1) Monochloramine

The metabolites of monochloramine are H + , Cl - , NH 3 and N 2 , and no persistent substance is

formed.

(2) Tribromomethane

The metabolites of tribromomethane are H + , Br - and CO 2 , and no persistent substance is formed.MEPC 56/2

ANNEX Page 27

I:\MEPC\56\2.doc (3) Dibromochloromethane

The metabolites of monochloramine are H + , Br - , Cl - and CO 2 , and no persistent substance is

formed.

(4) 2,2-dibromoacetic acid

The metabolites of 2,2-dibromoacetic acid are H + , Br - , CH 3 COOH and CO 2 , and no persistent

substance is formed.

(5) 2,2,2-tribromoacetic acid

The metabolites of 2,2,2-triib romoacetic acid are H + , Br - , CH 3 COOH and CO 2 , and no persistent

substance is formed.

2.4.4 Reaction with organic matter

2.4.4.1 Free active chlorine

It is known that when free active chlorine reacts with organic matters in water, DBPs such as trihalomethane and halogenated acetic acids are formed. In this app lication, 4 substances (tribromomethane, dibromochloromethane, 2,2 -dibromoacetic acid an d 2,2,2-tribromoacetic acid) detected by preliminary experiments are de signated as by-products from the ballast water management system. As to by-products resulted from disinfection of drinking water by chlorine, presence of various subs tances has been reported due to concern about th e influence on the health of humans. However, most of the DBPs in dr inking water are present in a very small amount. Moreover, presence of non-identified compounds may be possible. Thus, there have been obtained no sufficient data on the toxicity a nd the amount of formation of all the DBPs (see paragraph 2.1.3.2).

2.4.4.2 Relevant chemicals

(1) Monochloramine

Trihalomethanes are formed by the reaction of m onochloramine with an organic matter, but the amount of formation is very small compared w ith that formed by the reaction of hypochlorous acid. Rick Baugh, J.F. et al. carried out a test using the water of the Ohio River. As a result, the amount of trihalomethanes formed by monochl oramine treatment is reduced by 90.7 to 99.9% compared with the case where the water is treated with the same amount of chlorine (Rickabaugh, J.F. et al. (1978)).

(2) Tribromomethane

No reports on the reaction of tribromomethane with organic matters are available.

(3) Dibromochloromethane

No reports on the reaction of dibromochlorom ethane with organic matters are available.MEPC 56/2

ANNEX Page 28

I:\MEPC\56\2.doc (4) 2,2-dibromoacetic acid

No reports on the reaction of 2,2-dibromoacetic acid with organic matters are available.

(5) 2,2,2-tribromoacetic acid

No reports on the reaction of 2,2,2-tribromoacetic acid with organic matters are available.

2.4.5 Potential physical effects on wildlife and benthic habitats

2.4.5.1 Free active chlorine

Free active chlorine is highly soluble in water (IUCLID (2000)) and does not have a property of floating on the surface or sedimenting to the bo ttom. So, the compound is not the subject of evaluation of potential physical effects on wild life and benthic habitant s under GESAMP hazard evaluation (GESAMP (2002)).

2.4.5.2 Relevant chemicals

(1) Monochloramine

Monochloramine is highly soluble in water and does not have a property of floating on the surface or sedimenting to the bottom. So, th e compound is not the subject of evaluation of potential physical effects on wildlife and bent hic habitants under GESAMP hazard evaluation (GESAMP (2002)).

(2) Tribromomethane

Tribromomethane has a higher specific gravity th an seawater (2.899 (20°C)) and a low solubility (3.1g/l (20°C)). Therefore, according to evaluation based on GESAMP hazard evaluation (GESAMP (2002)), the compound is classified as Rating S (substances having a potential effect on benthic habitats).

(3) Dibromochloromethane

Dibromochloromethane has a higher specific gravity than seawater (2.451 (20°C)) and a low solubility (2.7g/l (20°C)). Therefore, according to eval uation based on GESAMP hazard evaluation (GESAMP (2002)), the compound is cl assified as Rating S (substances having a potential effect on benthic habitats).

(4) 2,2-dibromoacetic acid

2,2-dibromoacetic acid is highly soluble in water (2100g/l) and does not have a property of floating on the surface or sedimenting to the bo ttom. So, the compound is not the subject of evaluation of potential physical effects on wild life and benthic habitant s under GESAMP hazard evaluation (GESAMP (2002)).MEPC 56/2

ANNEX Page 29

I:\MEPC\56\2.doc (5) 2,2,2-tribromoacetic acid

2,2,2-tribromoacetic acid is highly soluble in water (2000g/l) and does not have a property of floating on the surface or sedimenting to the bo ttom. So, the compound is not the subject of evaluation of potential physical effects on wild life and benthic habitant s under GESAMP hazard evaluation (GESAMP (2002)).

2.4.6 Potential residuals in seafood

2.4.6.1 Free active chlorine

No report concerning persistence of free active chlorine in seafood is available. As free active

chlorine is considered to have low bioaccumu lation potential, its persistence in seafood is speculated to be low.

2.4.6.2 Relevant chemicals

(1) Monochloramine

No report concerning persistence of monochl oramine in seafood is available. As

monochloramine is considered to have low bio accumulation potential, its persistence in seafood is speculated to be low.

(2) Tribromomethane

As tribromomethane is considered to have lo w bioaccumulation potential, its persistence in seafood is speculated to be low. A ccording to the report of Gotoh, M. et al. (1992), tribromomethane was not detected in oysters, clam and the sediment of the Ariho River and the Yoshinaga River.

(3) Dibromochloromethane

As dibromochloromethane is considered to have low bioaccumulation potential, its persistence in seafood is al speculated to be low. According to the report of Gotoh, M. et al. (1992), dibromochloromethane was not detected in oyste rs, clam and the sediment of the Ariho River and the Yoshinaga River.

(4) 2,2-dibromoacetic acid

No report concerning persistence of 2,2-dibr omoacetic acid in seafood is available. As 2,2-dibromoacetic acid is considered to have low bioaccumulation potential, its persistence in seafood is speculated to be low.

(5) 2,2,2-tribromoacetic acid

No report concerning persistence of 2,2,2-triibromoacetic acid in seafood is available. As 2,2,2-tribromoacetic acid is considered to have low bioaccumulation pot ential, its persistence in seafood is speculated to be low.MEPC 56/2

ANNEX Page 30

I:\MEPC\56\2.doc 2.4.7 Any known interactive effects

2.4.7.1 Free active chlorine

Free active chlorine may genera te corrosive gas including gaseous chlorine under high temperature or low pH condition (<pH4).

2.5 Physical and chemical properties (G9: 4.2.1.4)

As the physical and chemical properties for free active chlorine, property values of HClO,

Cl 2 and NaClO are shown in Table 6. Property values concerning HClO have not been reported

almost at all because HClO itself is an unstable substance.

Table 6. Physical and chemical pr operties for free active chlorine Properties Value Reference Melting point (°C) -101 (Cl 2 ) Weast, R. C. (1988-1989) Boiling point (°C) -34.6 (Cl 2 ) Weast, R. C. (1988-1989) Flammability (flash point for liquids; °C) Nonflammable (Cl 2 ) HSDB

3.214 (Cl 2 , 0°C and 101.3kPa) Weast, R. C. (1988-1989) Density (20°C;kg/m 3 )

1.21x10 3 (NaClO: 14%-aqueous solution) HSDB

7.77 x10 5 (Cl 2 , 25 °C) HSDB Vapor pressure (20 °C; Pa)

17.4 - 20 x10 2 (NaClO) IUCLID (2000) Vapor density (air = 1) 2.5 (Cl 2 ,-34.6°C) HSDB 14.6 (Cl 2 , 0 °C) 5.7 (Cl 2 , 30°C) Weast, R. C. (1988-1989) Weast, R. C. (1988-1989) Water solubility (temp.; effect of pH; g/l) 293 (NaClO, 0 °C) HSDB 7.5 (HClO, 25°C) Morris (1966) Dissociation constant (pKa) 3.4 (Cl 2 , 25°C) Morris (1978) Autoignition temperature (°C) NA

Surface tension NA Viscosity NA

2.6 Analytical methods at environmenta lly relevant concentrations (G9: 4.2.1.5)

2.6.1 Free active chlorine

Analytical methods of free active chlorine is described in Ameri can Public Health Association (APHA)’s Standard Methods for the Ex amination of Water and Wastewater 19 th , 4500-Cl Chlorine (Residual). Typi cal analytical methods are as follows (APHA (1995)):

(1) DPD/Spectrophotometric colorimetr y (APHA Standard method 4500-Cl G)

According to this method, pinkish red color deve loped by the reaction of free active chlorine or total residual chlorine with N, N-diethyl-p-phe nylene diamine (DPD) is measured, and free active chlorine or total residual chlorine is quantit atively determined by comparison with standard colorimetric specimen.MEPC 56/2

ANNEX Page 31

I:\MEPC\56\2.doc The determination limit is 0.01mg FAC/l (or mg TRC/l) under ideal measurement conditions, but is higher under ordinary operation conditions.

i) Measurement of free act ive chlorine concentration

To a test tube to which 0.5 ml of phosphate bu ffer and 0.5 ml of a DPD solution (1 g/l DPD oxalate, 1.5 g/l DPD sulfate pentahydrate or 1.1 g/l anhydrous DPD sulfate) are pipetted is added 10 ml of sample with mixing. Thereafter, red color developed is measured by a spetctrophotometer, and the concentration of free ac tive chlorine is obtaine d from the calibration curve preliminarily prepared using standard specimen.

ii) Measurement of total residual chlorine concentration

To the solution undergoing th e color development in i) above is added 0.1 g of potassium iodide to be dissolved. After the mixture is kept st anding still for 2 minutes, red color developed is measured using a spectrophotometer, and the concen tration of total residua l chlorine is obtained from the calibration curve preliminarily prepared using standard materials.

This method is generally used for the measuremen t of residual chlorine co ncentration in drinking water because its measurement range is 0.05 to 2.0 mg TRC/l and the appara tus for analysis is

small.

(2) Iodometry (APHA Standard method 4500-Cl B)

According to this method, total residual chlorine is quantitativel y determined by titrating iodine liberated by the reaction of total residual ch lorine with potassium iodide using sodium

thiosulfate.

Water is added to an appropriate amount of a sa mple to make the volum e of 500 ml, followed by

the addition of 5 ml of acetic acid to adjust the pH in the range 3-4. To the resulting solution is added 1 g of potassium iodide with mixing. The liberated iodine is titrated with 10 mmol/l

sodium thiosulfate solution and the concentration of total residual chlorine in water is calculated from the volume (ml) of sodium th iosulfate required for the titration.

total residual chlorine (m g TRC/l)=(d-a) x f x test water (ml)/1000 x 0.3545 d:titer of sodium thiosulfate (ml)

a:titer of sodium thiosulfate for blank test (water) (ml)

f:factor of sodium thiosulfate

Although the detection limit is 0.04 mg TRC/l accord ing to this method, because the precision of values below 1 mg/l is low, this method is used for the measurement of the concentration of high

concentration total residual chlorine.

(3) Amperometry/ Titration (A PHA Standard method 4500-Cl D)

When iodine is liberated by the reaction of free ac tive chlorine or total residual chlorine with potassium iodide, direct current electricity flows through the solution. By tit rating the water with a reducing agent, iodine is reduced and the cu rrent value goes down. The point at which the current value no more goes down is the end point of titration. The concentration of free active

chlorine or total residual chlorine is calculate d from the amount of the reducing agent required

for the titration.MEPC 56/2

ANNEX Page 32

I:\MEPC\56\2.doc i) Measurement of free act ive chlorine concentration

To an appropriate amount of sample is added 1 ml of phosphate buffer (pH 7), and titration is carried out with 0.00564mol/l phenylarsenoxide solu tion using an amperometric titrator. The concentration of free active chlorine sample is calculated from the volume (ml) of phenylarsenoxide solution (0.00564mol/l) (d) according to the following formula:

Free active chlorine (mg FAC/l) = d x 0.2 x 1000/sample volume (ml)

ii) Measurement of total residual chlorine concentration

After adding 1 ml of potassium iodide solutio n (5w/v%) and 1 ml of phosphate buffer (pH4) to an appropriate amount of sample th e concentration of total residual chlorine in the test water is calculated through simila r operations as in i) above us ing an amperometric titrator.

2.6.2 Relevant chemicals

(1) Monochloramine

As in the case of free active ch lorine, monochloramine can be analyzed as combined chlorine according to the DPD method (APHA Method 4500-Cl G).

In the DPD method, total chlorine (free active chlo rine plus combined chlorine) is measured by the addition of potassium iodide in a simila r manner as in the measurement of free active chlorine, and the amount of combined chlorine (amount of chloramines) is calculated by subtracting the amount of free active chlorine.

(2) Tribromomethane

Analytical method of tribromomethane is described in METHOD 551 of U.S. EPA (1990).

A sample (35 ml) is extracted with 2 ml of me thyl-tert-butyl ether (MTBE), and 2 μl of the extraction solution is injected to GC provided with a silica glass cap illary column and an electron capture detector (ECD). Standard samples can be analyzed in 40 to 50 minutes using a column

used for this method. Detected substances can be identified using a separate column or GC-MS.

(3) Dibromochloromethane

Dibromochloromethane can be analyzed usi ng GC/ECD or GC/MS according to METHOD 551 of U.S. EPA (1990) as same as tribromomethane.

(4) 2,2-dibromoacetic acid

Analytical method of halogenated acetic acids including 2,2-dibrom oacetic acid is described in METHOD 552.3 of U.S. EPA (2003).

A sample (40 ml) is adjusted to lower pH value than 0.5 and extracted with 4 ml of methyl-tert-butyl ether (MTBE) or tert-amyl methyl ether containi ng internal standard. To the extracted halogenated acetic acid is added me thanol, followed by heating for 2 hours for methyl-esterification. The mixture is then dehydrat ed and neutralized to be used for analysis. Halogenated acetic acids are identified and quantit atively determined using GC provided with a capillary column and ECD.MEPC 56/2

ANNEX Page 33

I:\MEPC\56\2.doc (5) 2,2,2-tribromoacetic acid

2,2,2-tribromoacetic acid can be analyzed acco rding to METHOD 552.3 of U.S. EPA (2003) using GC/ECD as same as in the case of 2,2-tribromoacetic acid.

3 USE OF THE ACTIVE SUBSTANCE

The following describes in this section, the operation method fo r the seawater electrolytic process. “mg FAC(t)/l” means the theoretical concentration of free active chlorine without consideration of initial decay after mixing electrolyzed seaw ater and untreated seawater. “mg TRC/l” means the measured concen tration of total residual chlorine.

3.1 Seawater Electrolytic Process on Hybr id Ballast Water Management System

The ballast water management system applied is a hybrid management system composed of a mechanical treatment process and seawater electro lytic process (Fig. 2 shows the treatment flow in the case of seawater intake). The mechanical treatment proce ss treats mainly large organisms (minimum dimension: 50 μm or more) using mechanical treatment apparatus such as strainers to remove of impurities, filters to remove or d eactivate large organisms, etc. The seawater electrolytic process is installed in the stage immediately followi ng mechanical treatment in order to treat small organisms (the minimum dimension: less than 50 μm), bacteria, etc. that elude the

mechanical treatment. The free active chlorine generated in the seawater electrolytic process

derives its bactericidal effects from the chlorine ion, a main component of seawater generated via seawater electrolysis.

The seawater electrolysis equipment consists ma inly of an electrolytic cell with built-in electrodes, a DC power supply uni t, and current contro l equipment. Together these components are installed in intake/discharge lines of the ba llast water. The system can be applied during intake or discharge of ballast wa ter or during navigation (part of ba llast water is circulated in the seawater electrolytic process). As a rule, however , the treatment is conducted during ballast water

intake.

The allowable concentration range of FAC and TRC is summarized in Table 7.

Seawater Electrolytic

Cell

Ballast

Water
Tanks P P

Discharged

Ballast

Water Ballast Pump Strainer DC Power Supply Unit

and Control Equipment

FI

02

FI

01

V-1 V-6

V-2

V-7 V-4

V-5 CI

01

V-8 V-3

V-9 Mechanical

Treatment

Process

Hybrid Ballast Water Management System Seawater Electrolysis

Process V-10 V-11

Seawater Electrolytic

Cell

Ballast

Water
Tanks P P

Discharged

Ballast

Water Ballast Pump Strainer DC Power Supply Unit

and Control Equipment

FI

02

FI

01

V-1 V-6

V-2

V-7 V-4

V-5 CI

01

V-8 V-3

V-9 Mechanical

Treatment

Process

Hybrid Ballast Water Management System Seawater Electrolysis

Process V-10 V-11

Fig.2. Flow diagram of Hybrid Ballast Water Management System (at Intaking)MEPC 56/2

ANNEX Page 34

I:\MEPC\56\2.doc Table 7. Allowable FAC and TRC concentration range of Hybrid Ballast Water Management System

Allowable concentration range

Item

Concentration* Deversion ratio of treated

seawater 16 mg FAC(t)/l (minimum) 30% of intake seawater Efflent of Electrolytic Cell

1,500 mg FAC(t)/l (maximum) 0.33% of intake seawater

Efflent of Seawater Electrolysis Process 5.0 mg FAC(t)/l -

Discharged ballast water 0.1-3.0 mg TRC/l -

* mg FAC(t)/l : The theoretical c oncentration of free active chlorine w ithout consideration of initial decay.
mg TRC/l : The measured concentration of total residual chlorine

.

3.2 Active Substance generation mechanism by seawater electrolysis

The Active Substance is generate d by electrochemical reactions in the seawater electrolytic process. The details are described below.

3.2.1 Anode reaction

If the water containing chlorine ion such as seaw ater, blackish water and etc. is electrolyzed, the following reactions progress normally on the anode surface:

2Cl - → Cl 2 + 2e - (E 0 = 1.359V at 25°C) (11)

2H 2 O → O 2 + 4H + + 4e - (E 0 = 1.229V at 25°C) (12)

This seawater electrolysis system, however, uses catalyst-coated electrodes that suppress oxygen generation. Thus, the reaction of Equation (12) hardly occurs under high Cl ion concentration conditions such as those of seawater, and the chlorine can be produced selectively.

3.2.2 Cathode reaction

The following reaction occurs on the cathode surface:

2H + + 2 e - → H 2 (E 0 = 0V at 25°C) (13)

2H 2 O + 2e - → H 2 + 2OH - (E 0 = -0.828V at 25°C) (14)

If we assume that chlorine of 1 mg FAC(t)/l is produced by the electrolysis, then 0.028 mg-H 2 /l (0.315 ml-H 2 /l) of hydrogen (H 2 ) is generated on the cathode.

3.3 Existence form of Active Substance in seawater

Cl 2 gas is generated by the anode reaction. Cl 2 gas is thought as highly soluble compound (0.64

ml/ml-H 2 O). Cl 2 gas rapidly dissolves into the seawater and becomes hypochlorous acid (HClO) (Equation (15)). 1 mole of hypochlorous acid (HClO) is generated from 1 mole of chlorine gas.MEPC 56/2

ANNEX Page 35

I:\MEPC\56\2.doc Additionally, hypochlorous aci d (HClO) is hydrolyzed and hypochlorite ion (ClO - ) is generated (Equation (16). Chlorine gas (Cl 2 ) /hypochlorous acid (HCl O)/hypochlorite ion (ClO - ) are in equilibrium and the existence forms depend on th e pH. The mixture of hypochlorous acid (HClO) /hypochlorite ion (ClO - ) is called “free active chlorine”.

Cl 2 + H 2 O ↔ HClO + H + + Cl - (15)

HClO ↔ H + + ClO - (16)

As natural seawater is normally of pH 8, the dissolved chlorine takes the form of hypochlorous acid (HClO) or hypochlorite ion (ClO - ).

Fig.3. Simulated variation in composition of a ch lorine solution with de gree of acidity or alkalinity for 0.1 M Cl 2 in water at standard temperat ure and pressure (AISE (1997)).

As shown in Equation (11), 2 moles of electr on are required for 1 mo le of chlorine gas generation. As shown in Equation (15) and (16), 1 mole of free act ive chlorine (the mixture of hypochlorous acid (HClO) /hypochlorite ion (ClO - )) is generated from 1 mole of chlorine gas (Cl 2 ). The quantity of electric ch arge of 1 mole of electron is 96500 coulomb. When 1 A of current is applied during 1 second, the quantity of electric charge is defined as 1 coulomb. Then,

theoretical free active chlori ne (FAC) generation is e xpressed in Equation (17).

Theoretical FAC generation[mol] = Applied current[A] x applied tim e[sec] / 96500[A·sec/mol] / 2 (17)

Theoretical FAC generation at 1 A current ap plied during 1 hour (3600second) is calculated using Equation(18).MEPC 56/2

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I:\MEPC\56\2.doc Theoretical FAC generation[mol] = 1 [A] x 3600 [sec] / 96500 [A·sec/mol] / 2

= 1.87 x 10 -2 [mol] (18)

Theoretical FAC generation per unit current an d time is expressed in Equation (19) using the result of Equation (18).

Theoretical FAC generation[mol/A/h] = 1.87 x 10 -2 [mol/A/h] (19)

Current efficiency( η ) means generation efficiency of FA C per applied curr ent and it is defined as Equation (20).

Current efficiency( η )[%]

= actual amount of FAC generation[mol]/theoretical am ount of FAC generation[mol] x 100 (20)

3.4 Safety of the seawater electrolysis system

The seawater electrolytic process does not require the loading or storage of the Active Substance

on board. This eliminates all hazar ds entailed in the handling of chemicals by the ship and the crew (poisonings, burns, fires, etc.).

In this system, the amount of electrolytic chlori ne generation is controlled by the applied current. Then, no excess chlorine is generated at norma l operating condition. If the emergency condition (power failure, electrolytic leakage etc) occurs, curre nt applying to electrolytic cell is turned off. When the flow rate of injection seawater to el ectrolytic cell comes down below the preset value, the electrolytic cell is turned off automatically to prevent excess chlorine generation. The flow rate of injection seawater to electrolyt ic cell is monitored using flow meters.

4 MATERIAL SAFETY DATA SHEET (G9:4.2.7)

Material Safety Data Sheet (MSDS) in conf ormity with JIS Z 7250 (2005) is described (APPENDIX 2). JIS Z 7250 is a Japane se Industrial Standard estab lished based on part 1 of the ISO 11014-1 Safety data sheet for chemical produc ts. The Content and orde r of the section were published as the first edition in 1994 with modifications of the technical contents for harmonization with “GHS, Part 1, Chapter 1.5 Hazard communication: Safety data sheet” (implemented by resolution of the United Nati ons Economic and Social Council in July 2003).MEPC 56/2

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I:\MEPC\56\2.doc 5 RISK CHARACTERIZATION

5.1 Screening for persistence, bioaccu mulation and toxicity (G9: 5.1)

5.1.1 PBT criteria

The criteria of PBT characteristics defined by th e Procedure (G9) are as follows (Table 8):

Table 8. PBT criteria

Criterion PBT criteria

Persistence Half-life: > 60 days in marine water > 40 days in freshwater* > 180 days in marine sediment > 120 days in freshwater sediment* Bioaccumulation Experimentally determined BCF > 2,000, or if no experimentally BCF has been determined, Log Pow ≥ 3 Toxicity Chronic toxicity NOEC <0.01mg/l

*For the purpose of marine environmental risk assessment half-life data in freshwater and freshwater sediment can be overruled by data obtained under marine conditions.

5.1.2 PBT character of Active Substance and relevant chemicals

5.1.2.1 Free active chlorine

i) Persistence

As free active chlorine is unstable substance and is easily decomposed by light, heat or reaction with organic compound, it is considered that it does not po ssess persistence.

Hypochlorous acid is an oxidizer and a disinfectan t and, is primarily an inorganic substance. Therefore, it is said that it is not proper to discuss its bi odegradability (IUCLID (2000)).

Also, the Koc value of hypochlorous acid is 14.3l/kg (estimated by EPI Suite TM ) (that of sodium hypochlorite is 14.3(estimated by EPI Suite TM )). Because Koc is smaller than 500l/kg, it is considered that soil adsorption potential of free active chlorine is low. Therefore, free active

chlorine is considered to have low sediment toxicity.

ii) Bioaccumulation

Log Pow of hypochlorous acid is -0.87 (estim ated) (that of sodium hypochlorite -3.42 (estimated)) and this value is smaller than PBT criteria (Log Pow ≥ 3). In addition, it is easily decomposable inorganic substance. It is speculated that this chemical is not high bioaccumulation potential compound.

iii) Toxicity

As the chronic aquatic t oxicity value of free active chlorine is presumed to be 7d NOEC=0.0027 mg TRC/l from the results of chronic aquatic toxi city test for algae microcosm, it is evaluated based on PBT criteria (NOEC<0.01mg/l) that free active chlorine has some toxicities.MEPC 56/2

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I:\MEPC\56\2.doc 5.1.2.2 Relevant chemicals

(1) Monochloramine

i) Persistence

As the half-life of monochloramin e by hydrolysis is 10 h (Margerum et al. (1978)), it is considered that there is no persistence in the environment.

Koc calculated from the structure of monochl oramine with calculation program (EPI Suite TM ) of U.S. EPA based on QSAR is 14.3l/kg, and it is be lieved that the adsorption amount to soil/sludge

is small.

There is no report on biodegra dation of monochloramine.

ii) Bioaccumulation

The data on Log Pow and BCF of monochloramine have not been reported. As the calculation results by EPI Suite TM , Log Pow of monochloramine is -1. 19 and is smaller than PBT criteria (Log Pow ≥ 3). Therefore, the bioaccumulation is considered low.

Furthermore, the calculation result of BCF is BCF=3.2, and is sufficiently low value in comparison with the PBT criteria (BCF > 2,000).

iii) Toxicity

Since the evaluation as single substance is difficult, the toxicity of monochloramine has not been

clarified.

(2) Tribromomethane

i) Persistence

Though the half-life of tribromometh ane in the water calculated fr om hydrolysis rate constant (3.2x10 -11 /sec) is 686 years and tribromomethane is relatively stable compound in the water (Mabey, W. et al. (1978)). The half-life by vo latilization is calculated as 1h (riffle)-581h (pool) (Kaczmar, S.W. et al. (1984).

As described in paragraph 2.4.1.2, aerobic biodegrad ation of tribromometh ane is limitative, however, it is reported that tribromomethane is easily degraded anaerobically, for example, by methanogenic bacteria or denitrifying bacteria.

Koc of tribromomethane is 218.8l/kg (observe d), and the adsorption to soil/sediment is considered to be small (DiToro et al. (1991)).

ii) Bioaccumulation

Log Pow of tribromomethane is 2.4 (observed) and is smaller than PBT criteria (Log Pow ≥ 3). Therefore, the bioaccumulation is considered to be low.MEPC 56/2

ANNEX Page 39

I:\MEPC\56\2.doc Though there is no report on BCF, the calculation result by BCF calculation program of U.S. EPA (EPI Suite TM ) based on QSAR is BCF=14, and it is a sufficiently low value in comparison with the PBT criteria (BCF > 2,000).

iii) Toxicity

From the report that NOEC (28 days) to Cyprinodon variegates was 4.8 mg/l (Ward et. al. (1981)), it is evaluated based on PBT criteria (NOEC<0.01mg/l) that tribromomethane has no toxicity.

(3) Dibromochloromethane

i) Persistence

Though the half-life of dibromochloromethane in the water calculated fro m the hydrolysis rate constant (8.0x10 -11 /sec) is 686 years and dibromochlorometha ne is relatively stable compound in the water (Mabey, W. et al. (1978)). The half-life by volatili zation is tentatively calculated as 0.7h(riffle)-398h (pool) (Kaczmar, S.W. et al. (1984).

As described in paragraph 2.4.1.2, aerobic biodegradat ion of dibromochloromethane is limitative, however, it is reported that dibromochlorom ethane is easily degraded anaerobically.

Koc of dibromochloromethane is 84l/kg (observe d), and the adsorption amount to soil/sludge is considered to be small (HSDB (2006b)).

ii) Bioaccumulation

Log Pow of dibromochloromethane is 2.16 (observe d) and is smaller than PBT criteria (Log

Pow ≥ 3). Therefore, the bioaccumulation is considered to be low.

Though there is not a report on BCF, the calcula tion result by BCF calculation program of U.S. EPA (EPI Suite TM ) based on the QSAR is BCF=9, and it is a sufficiently low value in comparison with the PBT criteria (BCF > 2,000).

iii) Toxicity

From the report (NITE (2006)) th at NOEC (21 days) to daphnia magna was 0.063mg/l and that to Oryzias latipes was 3.2 mg/l (NITE (2006)), it is evaluated based on the PBT criteria (NOEC<0.01mg/l) that dibromoc hloromethane has no toxicity.

(4) 2,2-dibromoacetic acid

i) Persistence

As 2,2-dibromoacetic acid is easily subjected to biodegradation in th e environment, it is considered that the persistence is not shown.

As described in paragraph 2.4.1.2, biodegradations by haloacid dehalogenase of aerobic bacteria are reported.MEPC 56/2

ANNEX Page 40

I:\MEPC\56\2.doc Also, the Koc value of 2,2-dibromoacetic acid is 1.90l/kg (estimated by EPI Suite TM ). Because Koc is smaller than 500l/kg, it is considered that soil adsorption potential of 2,2-dibromoacetic acid is relatively low. Therefore, free active chlori ne is considered to have low sediment toxicity.

ii) Bioaccumulation

Log Pow of 2,2-dibromoacetic acid is 1.22 (estimated value) and is smaller than PBT criteria (Log Pow ≥ 3). Thus, the bioaccumulation is considered to be low.

Though there is no report on the BCF, the calculati on result by BCF calcula tion program of U.S. EPA (EPI Suite TM ) based on QSAR is BCF=3.2, and it is a sufficiently low value in comparison with the PBT criteria (BCF > 2,000).

iii) Toxicity

There is no report on the toxicity of 2,2-dibromo acetic acid, and the toxicity evaluation is still

uncertain.

(5) 2,2,2-tribromoacetic acid

i) Persistence

As 2,2-dibromoacetic acid is readily subjected to biodegradation in the environment, it is supposed that the pers istence is not shown.

As described in paragraph 2.4.1.2, biodegradations by haloacid dehalogenase of aerobic bacteria are reported.

Also, the Koc value of 2,2,2-tribromoacetic acid is 2.74l/kg (estimated by EPI Suite TM ). Because Koc is smaller than 500l/kg, it is considered th at soil adsorption potentia l of 2,2,2-tribromoacetic acid is relatively low. Therefore, free active chlori ne is considered to have low sediment toxicity.

ii) Bioaccumulation

Log Pow of 2,2,2-tribromoacetic acid is 1.71 (estimated value) and is smaller than PBT criteria (Log Pow ≥ 3). Thus, the bioaccumulation is considered to be low.

Though there is no report on the BCF, the calculati on result by BCF calcula tion program of U.S. EPA (EPI Suite TM ) based on QSAR is BCF=3.2, and it is a sufficiently low value in comparison with the PBT criteria (BCF > 2,000).

iii) Toxicity

There is no report on the toxicity of 2,2,2-tribromo acetic acid, and the toxici ty evaluation is still

uncertain.

5.1.3 Summary of PBT evaluation

The persistence and bioaccumulation of hypochlorous acid, an Active Substance, satisfy the PBT criteria. The toxicity, on the other hand, fails to satisfy the PBT criteria (NOEC<0.01 mg/l). The hypochlorous acid must therefore be assumed to be toxic.MEPC 56/2

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I:\MEPC\56\2.doc But as shown by the toxicity evaluation test of th e treated ballast water (S ection 5.2) and the risk evaluation of the discharge from that water (S ection 5.3), the free active chlorine acting within this seawater electrolysis system is not toxic under actual operating conditions. The toxicity is nullified by the rapid dilution and decomposition of th e free active chlorine after the discharge of the free active chlorine together with the treated ballast water.

Out of the relevant chemicals, the persistenc e and bioaccumulation of monochloramine satisfy PBT criteria by margins comparable to those of free active chlorine. The difficulty in evaluating monochloramine as a single substance precludes conclusive evaluations of its toxicity.

Tribromomethane and dibromochloromethane satisfy all the PBT criteria and are considered to have very small effects on the environment.

2,2-dibromoacetic acid and 2,2,2-tribromoacetic acid sa tisfy the PBT criteria for persistence and

bioaccumulation. A lack of data makes it diffi cult to clearly eval uate their toxicity.

5.2 Toxicity evaluation of th e treated ballast water (G9:5.2 )

Seawater electrolysis may very well generate substances other than free active chlorine, monochloramine, tribromomethane, dibrom ochloromethane, 2,2-dibromoacetic acid and 2,2,2-tribromoacetic acid (lis ted in Section 2) in water. It would be very difficult, however, to identify all of the relevant chemicals, in light of their minute quantities and widely variable types. Section 5.2 of the Procedure (G9) (MEPC 53/24/ Add.1) requires a toxicity test of discharge ballast water after treatment. Owing to this, the po ssibility of interaction of the Active Substance can be evaluated integrally together with by-products.

The chronic toxicity for the test organisms was th erefore evaluated in a s eawater chlorinated by a simulated ballast water management system, using a small device fitted with the same types of electrodes used for the actual seawater electrolytic process. IDEA Consultants, Inc. carried out the toxicity test in accordance with GLP Crite ria of the OECD (OECD Principles of Good Laboratory Practice, ENV/MC/CHE M (98)17, 1997) (IDEA Consulta nts, Inc., Early life stage toxicity test for the electrolyte-chlor inated seawater using Java medaka, Oryzias javanicus

, BWF-0601 (2006), IDEA Consultants, Inc., Chronic to xicity test for the el ectrolyte-chlorinated seawater using marine Gammarid, Hyale barbicorins , BWG-0601 (2006), ID EA Consultants, Inc., Growth inhibition test fo r the electrolyte-chlorinated seawater using marine diatom,

Skeletonema costatum , BWA-0601 (2006)).

5.2.1 Test method and conditions

(1) Preparation of test seawater

Natural seawater filtered with 10 μ m pore diameter filter was electrolyzed, and electrolyzed seawater at 50±3 mg FAC/l was produced. The unit (mg FAC/l) means concentration of free active chlorine. FAC of the electrolyzed seawater and the test seawater, treated seawater held for 2 days, were analyzed by iodometry and DPD/ Spectrophotometric colori metry, respectively. Then, the electrolyzed seawater was added to unt reated filtered seawater to prepare treated seawater, active chlorine concentration of 5.0 mg FAC/l. This treated seawater was put into a glass container and was held in darkness at 25°C for two days, then, it was used for the exposure of test organism. The test seawater, treated seaw ater held for 2 days, was gradually diluted with dilution seawater (natural seawat er filtered by activated carbon f ilter) to 1/1 (no dilution), 1/3.2, 1/10, 1/32, 1/100, and 1/320, and of each was ap plied to the exposure in this study.MEPC 56/2

ANNEX Page 42

I:\MEPC\56\2.doc (2) Early life stage toxicity test for the treated seawater using Java medaka, Oryzias javanicus

This study was conducted in accordance with the OECD guideline for testing of chemicals No.210, “Fish early-life stag e toxicity test”, 1992.

Java medaka, Oryzias javanicus , was used for this study as test species. The exposure of test organism, Oryzias javanicus , was conducted under condition shown below. During the exposure duration, hatchability and the number of days to hatching in the exposed fe rtilized eggs, and the post-hatch mortality, were measur ed. Also, total length and body we ight of all survived animals were measured at the completion of exposure. For each parameter, the data set of calculated values was checked for homogeneity of varian ces across control and treatment groups by

Bartlett’s test. The differences between cont rol group and treatment groups were determined using one-way analysis of va riance (ANOVA) followed by Dunne tt's multiple comparison tests when homogeneity was observed, or by the n onparametric Kruskal-Wallis test followed by Welch’s t-test with Bonferroni-Holm’s adju stment when no homogeneity was observed.

The residual chlorine concentration, as an Active Substance concentration, was measured twice a week in the newly prepared test solution, an d was measured once a week in test solution immediately prior to the renewal (aft er a day from previous replacement).

Type of exposure : Semi-static, w hole amount renewal every 24 hours Exposure duration : From embryo stage at 1 day post-fertiliza tion to 28 days post-hatch (40 days)

Number of treatment : 6 treatment groups (1/1, 1/3.2, 1/10, 1/32, 1/100 and 1/320 group), and a control group Number of replicates : 3 repl icates per each treatment and 3 replicates for control group

Volume of test solution : 100ml/vessel in embryo phase and 500ml/v essel in post-hatch phase Test organism : Fertilized egg 1 da y post-fertilization of Java medaka, Oryzias j avanicus Number of animals : 60 animals/group (20 animals per replicate vessel) Temperature : 26±1 oC Illumination : Cool-white type fluores cent bulbs, 12 hours light/12 hours dark Feeding (pos-hatch phase) : Brine shrimp nauplii once daily

(3) Chronic toxicity test for the tr eated seawater using marine Gammarid, Hyale barbicorins

This study was conducted in accordance w ith the test method reported by Anasco et al. using marine gammarid, Hyale barbicorins , as a test organism. In this study, cumulative mortality, molting frequency, head length and body weight of the su rvived animals at the completion of the exposure were applied for endpoints, instea d of cumulative mortality and body length.

The exposure of test organism, Hyale barbicorins , was conducted under condition shown below. During the exposure duration, mortality and the fr equency of molting were measured. Also, the head length and body weight (dry weight) of all survived an imals were measured at the completion of the 21 days of exposure.MEPC 56/2

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I:\MEPC\56\2.doc The differences between the treatment groups and the control group were determined by chi-squared test, with Bonferroni-Holm adju stment, for cumulative mortality and molting frequency: the data set of surviving number and cumulative number of dead animals at the completion of exposure, and the cumulative molting frequency for surviving animals was applied, respectively. The homogeneity of variance across the groups for the data set for head length and body weight of surviv ing animals were observed by Ba rtlett’s test, and thus the differences between the treatment groups and th e control group were determined by one-way analysis of variance (ANOVA) , followed by Dunnett’s multiple comparison test.

The residual chlorine concentration, as an Active Substance concentration, was measured twice a week in the newly prepared test solution, an d was measured once a week in test solution immediately prior to the renewal (aft er a day from previous replacement).

Type of exposure : Semi-static, w hole amount renewal every 24 hours Exposure duration : 21 days Number of treatment : 6 treatment groups (1/1, 1/3.2, 1/10, 1/32, 1/100 and 1/320 group), and a control group Number of replicates : 20 replicates per tr eatment group and 20 replicates for control

group Volume test solution : 10ml/vessel Test animal : Marine Gammarid, Hyale barbicorins, within 24 hours post-hatch Number of animals : 20 animals/group (1 animal per vessel) Temperature : 24±1 oC Illumination : Cool-white type fluores cent bulbs, 12 hours light/12 hours dark Feeding : Artificial flak food, TetraMarine ® , < 0.5mm crushed pieces, once daily

(4) Growth inhibition test for the tr eated seawater using marine diatom, Skeletonema

costatum

This study was conducted in accordance with th e International standard ISO 10253: “Water quality -Marine algae grow th inhibition test with Skeletonema costatum and Phaeodactylum
tricornutum”, 1995.

Marine diatom, Skeletonema costatum, was used for this study as te st species. The exposure of test organism, Skeletonema costatum , was conducted under condition shown below. The cell density in each test vessel was measured at 24, 48 and 72 hours after the start of exposure, and area under the growth curve and the growth rate over the exposure durati on was calculated. For each parameter, the data set of calculated va lues was checked for homogeneity of variances across control and treatment groups by Bartlett’s test. The differences between control group and treatment groups were determined using one-w ay analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests when homoge neity was observed, or by the nonparametric Kruskal-Wallis test followed by Welch’s t-test with Bonferroni-Holm’s adjustment when no homogeneity was observed.

The residual chlorine concentrati on, as an Active Substance con centration, in test solution was measured in each treatment group at the beginning and the completion of the exposure.

Type of exposure : Static, shaking at 100 rpm Exposure duration : 72 hoursMEPC 56/2

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I:\MEPC\56\2.doc

Number of treatment : 6 treatment gr oups (1/1, 1/3.2, 1/10, 1/32, 1/100 and 1/320 group), and a control group Number of replicates : 3 replicates per each treatment and 6 replicates for control

Volume of test solution : 30 ml/vessel Initial cell density : 1 x 10 4 cells/ml Temperature : 20±2 oC Illumination : Cool-white type fluor escent bulbs, continuous at 7,000 Lux ±

20% pH adjustment of test

solution : None

5.2.2 Results and conclusions

(1) Early life stage toxicity test for the treated seawater using Java medaka, Oryzias javanicus

The cumulative hatchability of exposed eggs and the post-hatch cumulative mortality in control group were 95% and 3.5%, respectively, satisfying the validity criteria in this study (more than 70% in the hatchability, and less than 30 % in the mortality). In addition, the test conditions (water temperature, salinity, pH value, and diss olved oxygen concentration of test solution) had been maintained at an adequate level throughout the exposure dur ation, and thus there was no factor, which may have influenced the re liability of the results of this study.

The free active chlorine (FAC) concentrations in the newly prepared test solutions were 3.7 - 3.9 mg FAC/l in 1/1 group, and 0.39 - 0.89 mg FAC/l in 1/3.2 group, whereas the

FAC concentrations were lower than determ ination limit of 0.05 mg FAC/l (ND) in other treatment groups and control group at all measurem ent days. The FAC in th e test solution just before the replacement were 1.9 - 2.4 mg FAC/l in 1/1 group, ND - 0.07 mg FAC/l in 1/3.2 group, and ND at all measurement days in other treatment groups and control group.

In regard to the cumulative hatchability of fertilized eggs, 1/1 gr oup was 0%, showing a significant decrease compared to the control gr oup at p<0.05. As for the cumulative post-hatch mortality, 1/3.2 group resulted in 100%, and a significant increase compared to the control group at p<0.05 was observed. No significant effect was observed on the cumulative hatchability, the days to hatching, and the cumulative post-hatch mortality, in othe r treatment groups. A statistical significant difference was observed for the total le ngth of survived animals at completion of the exposure of 1/100 group compared to control group at p<0.05. However, it is considered that the total length in 1/100 group was a contingent alteration with no relationship w ith the toxicity of test seawater, because no clear tendency was observed in average of total length between 1/10 and 1/320 group. Also, no significan t difference on the body weight of the survived animals was statistically observed in all treatment groups, compared to the control group.

From the results of this study, the le thal or sub-lethal effect of test seawater to th e early life stage Java medaka, Oryzias javanicus , which was treated at 5.0 mg FAC /l and held in the darkness for two days, was concluded as follows.

No observed effect dilution ratio: 1/10

Lowest observed effect dilution ratio: 1/3.2MEPC 56/2

ANNEX Page 45

I:\MEPC\56\2.doc (2) Chronic toxicity test for the tr eated seawater using marine Gammarid, Hyale barbicorins

The cumulative mortality in control group was 5% at the completion of e xposure, satisfying the validity criterion of this study (le ss than 30% at the comp letion of exposure). In addition, the test conditions (water temperature, salinity, pH valu e, and dissolved oxygen concentration of the test solution) had been maintained at an adequate level throughout the exposure duration, and thus there was no factor, which may have influenced the reliability of the results of this study.

The FAC concentrations in the newly prepared test solutions were 3.6 mg FAC/ml in 1/1 group, which was measured only once, and 0.39-0.50 mg FAC/l in 1/3.2 group, whereas, the FAC were

lower than determination limit of 0.05 mg FAC/ml (ND: lower than 0.05 mg FAC/ml) in other treatment and control groups at al l measurement days. In addition, the FAC of the test solution just before the replacement were 2.0 mg FAC/l in 1/1 group, which was measured only once, and ND at all measurement days in other treatment and control groups.

The cumulative mortality at the completion of the exposure was 100% in 1/1 and 1/3.2 group, and a significant increase, compared to contro l group, was observed for these groups at p<0.05. The cumulative mortality for groups 1/10, 1/32, 1/100, and 1/320 was 5% or below, and no statistical significance was obser ved between the control group. In regards to the cumulative frequency of molting the completion of exposure, no statistical significan ce was statistically observed in each treatment group compared to c ontrol group. Also, no statistical significant

difference was observed between each treatmen t group and control group for both the head length and the body weight of the survived an imals at the completion of the exposure.

From the results of this study, the lethal or sub-lethal effect of test seawat er, which was treated at 5.0 mg FAC/l and held in the darkne ss for two days, to the marine Gammarid, Hyale

barbicorins , was concluded as follows.

No observed effect dilution ratio: 1/10

Lowest observed effect dilution ratio: 1/3.2

(3) Growth inhibition test for the tr eated seawater using marine diatom, Skeletonema

costatum

The average cell density in control gr oup at the end of the exposure was 57.6×10 4 cells/mL, satisfying the validity criteria in this study (m ore than 16 times the in itial cell density in 72 hours). In addition, the environmental condition had been maintained at an adequate level throughout the exposure duration, and thus there wa s no factor, which may have influenced the reliability of the results of this study.

The FAC in test solution was 2.4 mg FAC/l in 1/1 group, whereas, the FAC was lower than determination limit of 0.05 mg FAC/l in all of other treatment groups and control, at beginning of the exposure. The TRC at the completion of expos ure was lower than determination limit in all treatment and control groups.

In the area under the gr owth curve over exposure duration (0 to 72 hours), a clear declining trend which depend on the dilution ratio of test seawater was observed, and a significant difference, or a significant reduction of biomass, was statis tically observed at 1/10, 1/3.2, and 1/1 group, compared with control group at p<0.05. A simila r tendency was observed in the growth rate from 0 to 72 hours, and likewise, a significant difference from control group, or a significantMEPC 56/2

ANNEX Page 46

I:\MEPC\56\2.doc decline in growth rate, was observed at 1/1, 1/3.2, and 1/10 group at p<0.05. Neither the significant reduces of growth rate, nor the area under the grow th curve, was observed in treatment groups with dilution rate of 1/32 or below.

From the results of this study, th e influence of test seawater to the growth of marine diatom

Skeletonema costatum , which was treated at 5.0mg/ FAC/l and held in the darkness for two days,

was concluded as follows:

No observed effect dilution ratio: 1/32

Lowest observed effect dilution ratio: 1/10

5.2.3 Conclusion of toxicity test of the treated ballast water

The toxicity was evaluated on the test organism s in the seawater chlorinated with the method simulating the ballast water management system, with a small device usi ng the electrodes of the same type as those of the seawater electrolyti c process. IDEA Consultants, Inc. carried out the toxicity test in accordance with the test plan and standard operation procedure (SOP), and GLP Criteria of OECD (OECD Principl es of Good Labor atory Practice, ENV/MC/CHEM (98) 17, 1997).

The results of the toxicity test of the treated ballast water are summarized below (Table 9). Dilution ratio (treated ballast water/natural seawater) was used for No Observed Effect Concentration (NOEC) and Lowest Observed Eff ect Concentration (LOEC) of treated ballast water. From these results, the results of the growth inhibition test with skeletonema costatum were adopted as the highest no observable effect concentration a nd the lowest observable effect concentration of the treated ballast water, whic h were determined to be NOEC = 3.1% (v/v) and LOEC = 10%(v/v), respectively.

Table 9. Results of toxicity evalua tion of the treated ballast water

Species NOEC* LOEC* Oryzias javanicus (fish, 28d) 10%(v/v) 31%(v/v)

Hyale barbicorins (crustaceans, 21d) 10%(v/v) 31%(v/v)

Skeletonema costatum (algae, 72h) 3.1%(v/v) 10%(v/v)

* Dilution rate with treated balla st water and dilution seawater.

5.3 Risk assessment of treate d ballast water discharges

It is necessary to understand the effect of the treated ballast water discharges on marine organism. In this section, the short- and long-term (for 1 year) risks of the treated ballast water discharges have been assessed.

The short-term risk of the treated ballast water discharges has been assessed with the treated

ballast water to estimate the effect of free activ e chlorine and relevant chemicals in parallel. Dilution ratio (treated ballast water/natural seaw ater) has been used for No Observed Effect Concentration (NOEC) of the treated ballast wa ter, and NOEC = 3.1% (v/v). The results of the chronic toxicity tests (fish, crustacean, algae) of the treated ballast water have been applied for the NOEC (section 5.2). Acute toxicity value is usua lly applied in short-term risk assessment, but the chronic toxicity value has be en adopted because th e chronic toxicity value is mostly lower than the acute toxicity value re sulting in the safe side evaluation. Degrad ation of the treatedMEPC 56/2

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I:\MEPC\56\2.doc ballast water has not been considered due to the same reason as above. Therefore, assessment factor has not been considered. The time period when dilution ratio of the treated ballast water had reached 3.1%(v/v) has been estimated in the short-term risk assessment.

The long-term risk of the treated ballast water discharges has been assessed with tribromomethane. The long-term risk should be assessed with the treat ed ballast water under normal circumstances. However, the half-life value of the treated ballast water can not be defined

because the treated ballast water contains varied components (e.g., free active chlorine, tribromomethane, etc.). And the treated ballast wa ter must continue to be accumulated in natural seawater. Accordingly, the long-term risk can not be assessed with reasonable accuracy without considering the half-life value. Therefore, the risk assessment for tribromomethane is conducted because it has the highest concentration among rele vant chemicals in the treated ballast water and its accumulation in natural seawater has to be considered due to low degradation rate. The long-term risk of free active chlori ne, which has very low half-life value, is considered to be very low, so it hasn’t been assessed. The concentr ation of tribromomethane was 0.16 mg/l as the analytical result (section 2.1.3.2). 96h NOEC = 2.9 mg/l ( Cyprinodon variegates /fish) was applied to the NOEC of tribromo methane (section 2.2.6). The NOEC is valid acute toxicity data. Although chronic toxicity data shou ld be applied to long-term risk assessment, the acute toxicity data was applied to the NOEC so that it was lo wer than the chronic toxicity data (28d NOEC (juvenile mortality) = 4.8-8.5 mg/l ( Cyprinodon variegates /fish)) (section 2.2.2.2) . Evaluation by the acute toxicity data is on the safe side. A ssessment factor should be 100 due to applying the valid acute toxicity data to the NOEC, but 10 wa s applied to the assessment factor. Because the acute toxicity data was lower than the chro nic toxicity data. PNEC of tribromomethane was NOEC/10 = 0.29mg/l.

Then, the behaviors of the discharge from the treated ballast water and residual chemical components must be properly understood in order to assess the potential risks they pose after discharge to the marine environment. For th is investigation the applicant developed a high-resolution hydrodynamic model of Tokyo Bay using HYDROMAP (Zigic et al. (2003)), a computerized system from Applied Science Associates. The hydrodynamic simulations were linked to a comprehensive chemical di spersion model known as CHEMMAP (French McCay, 2001; French McCay and Isaji, 2004; French McCay et al. , 2006), another application from Applied Science Associates, in order to qu antify the fate and disp ersion of the discharge and tribromomethane within Tokyo Bay.

Exposure assessments were carried out by expe rt marine and freshwater modelers from Asia-Pacific Applied Science Associates (APA SA). Effect assessments were conducted by Science and Technology Co., Ltd. (SAT), a compa ny with expertise and research experience in assessing the effects of antif ouling paints (Science and Tec hnology Co., Ltd. and Asia-Pacific Applied Science Associates. “Risk evaluation of discharge treated ball ast water in Yokohama Port (2006)).

5.3.1 Environmental information

The effect of bathymetry, tides, salinities and water temperatures, all play a role in the fate of any

ballast water release. Historical measurements of these parameters in the Yokohama Port were used as input data into the simulation models to determine the range of possible concentrations, which might result under the influence of all these factors.

In particular, water currents within the region ar e tidally driven as shown in Fig. 3 and were included into the study to account for their dynamic nature, since these tidal currents are time-varying. These tidal currents were created using a detailed high-resolution model of Tokyo Bay. Fig. 3 demonstrates that the tidal range in Yokohama Port is < 2 m during spring tides.MEPC 56/2

ANNEX Page 48

I:\MEPC\56\2.doc The tides demonstrate a diurnal ineq uality (that is, tidal ranges dur ing the day can be significantly less than the tidal range at night). During neap tides, the tides change only once a day with a range < 1 m.

-1.0 -0.5 -0.0 0.5

-0.10 -0.05 0.00 0.05 0.10

0.000 0.025 0.050 0.075

1 Aug Aug 2006 8 Tue 15 Tue 22 Tue 1 Sep 8 Fri 15 Fri 22 Fri 1 Oct Oceanographic Conditions at Discharge LocationEle.(m)Stick(kt)Speed(kt)

Date-Time

Fig.3. Example currents within Yokohama Po rt from a hydrodynamic model of Tokyo Bay demonstrating the range of tidal co nditions possible for Yokohama Port.

Publicly available water quality data for Yokohama Port also show that water temperatures are known to range between 10 - 28 oC and that salinities are known to range from 18 ppt to 31 ppt.

5.3.2 Treated Ballast Water Release Information

Shipping statistics for Yokohama Port were examined to quantif y the amount of ballast water brought into the port on an annual ba sis. From these statistics, tw o case studies or scenarios were developed for the modelling study being:

(1) In the short-term risk assessment, the dist ribution and short-term fa te of treated ballast water discharged from one ship in Yokohama Port (least-case and worst-case rates and volumes) were simulated. From the shipping statistics, the least-case discharge volume from one ship is 3,000 m 3 over 6 hours (discharge rate being 500 m 3 /hour) while the worst-case discharge volume from one ship is 72,000 m 3 over 12 hours (discharge rate being 6,000 m 3 /hour).

(2) In the long-term risk assessment, the pot ential for the accumulation of tribromomethane

concentration in the treated ballast water discharged at a rate of 212,329 m 3 /day for 365 days representing the total possible volume from a ll ships in Yokohama Port over a one-year period was simulated. Statistical data of ship arriva ls at Yokohama Port (200 4) and estimated annual amounts of ballast water discharg ed in Yokohama Port, used for the long-term evaluation, are as

follows:MEPC 56/2

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I:\MEPC\56\2.doc Statistical data of ship a rrivals at Yokohama Port (2004) Number of incoming ships: 11,214/annum, 31/day Total gross tonnage: 193,647,519 GT/annum, 530,541 GT/day Percentage of total gross tonna ge: Full container ship: 56.3%

Car carrier: 14.6%

Tanker: 12.2% General cargo ship: 9.5%

Others: 7.4%

Table 10. Condition of long-term risk evaluation at Yokohama Port

(1) Since Japan is a country importing fossil fuels, such as crude oil, etc., it is assumed that all the tankers are inward ships (which do not discharge ballast water), the total of gross tonnage was deducted from th e statistical data (2004).

(2) Ballast water amount was estimated from class of ship and gross tonnage.

Further, in order to determin e the ‘worst-case’ risk to ma rine organisms from in-water concentrations of discharged the treated ballast water in Y okohama Port, three inner harbour

locations were selected in order to find a ‘worst-case’ location in terms of dilution efficiency.

These three test locations are shown in Fig. 4. Consequently, the CHEMMAP modelling simulated scenario 1 at these three locations until discharge dilutions were reduced to below the NOEC (that is 3.1% (v/v) of the tr eated ballast water in Port Wate rs). The NOEC is dilution rate

with treated ballast water and dilution seawater. For scenario 2, only one location was selected as a ‘worst-case’ assumption a nd the CHEMMAP model simulated the discharge of the treated ballast water from all ships over a one year period at this location and calculated

tribromomethane concentrations from an initial concentration of 0.16 mg/L before discharge to below PNEC (that is 0.29 mg/L assuming an a ssessment factor of 10) for the long-term

simulation. Annual Daily Annual Daily Annual Daily

Full Container Ship 5,500 15 110,000,000 301,370 55,000,000 150,684

Car Carrier 1,500 4 37,500,000 102,740 7,500,000 20,548

General Cargo Ship 3,000 8 30,000,000 82,192 15,000,000 41,096

Total 10,000 27 177,500,000 486,302 77,500,000 212,328 Ship Type Number of

Shi p s Gross Tonnage[t] Amount of Ballast

Water [ t ]MEPC 56/2

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I:\MEPC\56\2.doc Fig.4. Location map showing the simulated inne r-harbour discharge locations used in the CHEMMAP modelling.

5.3.2.1 Short-Term modelling

For the short-term case study, the results of the modelling, given different combinations of the tides, release volume (least-case and worst-case) a nd release location (1, 2 and 3), were analysed

to determine the time taken for treated ballast wa ter to completely dilute to less than the NOEC (that is 3.1% (v/v) of the treated ballast water in Port Waters) for each different scenario tested (detail in section 5.3). These results are shown in Table 11.

From Table 11 it can be concluded that volume is the most significant variable in determining the time frame for discharge dilution ratio to naturally disperse below NOEC while the effect of location and tidal strengt h demonstrated only a slight infl uence. Consequently, the longest period of time taken for discha rged calculations to fall below NOEC was estimated by the model to be 24 hours after discharge ceases, that is, Test Number 7, being a worst-case discharge volume at Location 2 during neap tides. Fig. 5 shows the expect ed concentration distribution after 36 hours given a 12 hours discharge from a single ship.

It is important to note that the calculated time frames (Time period) in Table 11 do not include the period of discharge, being 12 hours for the wo rst-case volume and 6 hours for the least-case

volume.

The NOEC (that is the dilution ratio (3.1% (v/v)) with treated ballast water and dilution seawater) is 72h NOEC (algae) of the chronic toxicity tests (sect ion 5.2) of the treated ballast water. It is

showed that the value of PEC/NOEC reached le ss than 1 at 24 hours after discharge (at 36 hours after discharge start time).MEPC 56/2

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I:\MEPC\56\2.doc Table 11. Model calculated time period fo r a discharge of treated ballast water from a single ship to completely dilute to levels below NOEC in Yokohama Port after discharge stops.

Test
Number Location Tides Volume

(m 3 ) Discharge period (hours) Time period (hours)

1 1 Neap 3,000 6 1.25

2 1 Spring 3,000 6 1.00

3 2 Neap 3,000 6 1.50

4 2 Spring 3,000 6 1.25

5 3 Neap 3,000 6 1.50

6 1 Neap 72,000 12 23.00

7 2 Neap 72,000 12 24.00

8 3 Neap 72,000 12 23.00

9 3 Spring 72,000 12 21.50

Fig.5. The expected concentrati on distribution after 36 hours given a 12 hours discharge from a single ship (Test Number 7).

5.3.2.2 Long-Term modelling

Given that the short-term results above show no exceedance of NOEC from a single discharge of

the treated ballast water after 24 hours, the only other consideration was to investigate the effect of all ships discharging treated ballast water on a daily basis to see if th ere is the potential for multi-dose accumulation of tribromomethane. Multiple dosing occurs when subsequent discharge occurs at the same location as previ ous discharge and before the previous discharge have completely diluted.MEPC 56/2

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I:\MEPC\56\2.doc Fig. 6 shows an example of the effect of this multiple dosing when assuming that all ships discharge at the one location, which is a worst-case assumption. The long-term risk of the treated ballast water discharges has been assessed w ith tribromomethane (initial concentration of 0.16 mg/L and PNEC = 0.29mg/l, detail in sec tion 5.3). The behaviors of tribromomethane

concentration in natural seawater were considered for Henry’s law (volatil e) and degradation rate that is k = 0.00978 (day -1 ) = 0.693/t 1/2 (decay) set in the database of CHEMMAP (Science and technology Co., Ltd. and Asia-P acific Applied Science Asso ciates (2006),APPENDIX B). In Fig.6, it can be seen that from all ships disc harging over 12 months, the distribution of

tribromomethane concentrations do not exceed PN EC at any time during that period. The time series graph in Fig. 6 shows the peak concentrations of tribromo methane will initially increase from background levels, but this in crease in concentration is lim ited due to its volatility and decay. The fluctuations in Hazard Quotient over time in Fig. 6 also demonstrate the influence of current speed on dilution; during spring tides mi xing increases and diluti on is maximised while during neap tides, mixing decreases and concen trations reach there peak at this time.

Consequently, in conclusion, th e CHEMMAP model results, shown in Fig. 6, demonstrate that this ‘worst-case’ scenario did not produce any accumulation that exceeded PNEC even with an assessment factor of 10 and all possible tidal regimes.

Fig.6. The expected concentrati on distribution for a long-term simulation of a discharge of treated ballast water showing that accumula tion is limited and that tribromomethane

concentrations do not exceed PNEC even if all ships discharge at one location over a 12 month period. Insert time-series graph shows example concentrations 100m fr om discharge location.MEPC 56/2

ANNEX Page 53

I:\MEPC\56\2.doc 6 RISK ASSESMENT

6.1 Risk to safety of ship

6.1.1 General

The system is subject to the risks of excessive production of Active Substance, leaks from the apparatus resulting from system runaways cau sed by accidents or failures of the system.

In this system, the amount of electrolytic chlorine generation is controlled by the applied current. Then, no excess chlorine is generated at norma l operating condition. If the emergency condition (power failures, electrolytic leakage etc) occurs , current applying to electrolytic cell turns off. When the flow rate of injection seawater to el ectrolytic cell comes down below preset value, the electrolytic cell is turned off au tomatically to prevent excess ch lorine generation. The flow rate of injection seawater to electrolytic cell is monitored using flow meters.

6.1.2 Corrosion

Sodium hypochlorite has chemical properties similar to those of the Active Substance and is

corrosive to metals. It can therefore be assumed to increase the risk of corrosion of the piping or

ballast tank.

The intake/discharge line and ballast tank for the ballast water are normally protected by plating, paint, polymer linings, etc. to safeguard agains t corrosion. Direct contact between metal materials

and the Active Substance is considered low. Th ere are few risk of corrosion, however, only a low concentration (5.0 mg FAC/l) of the Active Substance is measured during treatment of this
system.

The polyethylene-lined steel pipe, FRP pipe, rubber lining steel pipe, etc. used for the electrolysis cell and piping around also reduce the risk of corrosion by conferring resistance against hypochlorous acid with high concentra tions of the Active Substance ( ≤ 1,500 mg FAC/l). These piping materials have a long hist ory of use in land-used MGPS.

6.1.3 Generation of hydrogen gas

As described in Section 3.2.2, the following reac tions occur on the cathode surface during the generation of the Active Substance by the seawater electrolysis. This leads to a risk of explosion by accumulation of the resulting hydrogen gas.

2H + + 2 e - → H 2 (21)

2H 2 O + 2e - → H 2 + 2OH - (22)

If the theoretical amount of hydrogen gas is assumed to be generated in this seawater electrolysis system, hydrogen gas of 0.14 mg-H 2 /l (1.58 ml-H 2 /l) will be generated agai nst chlorine of 5.0 mg FAC/l, the maximum amount required for typical s eawater treatment. This is very small in comparison with the solubility of hydrogen in water (1.62 mg-H 2 /l-H 2 O; freshwater, 21°C, 1 atm) (HSDB, 2006), hence most of th e generated hydrogen gas is thoug ht to be dissolved in the

seawater.MEPC 56/2

ANNEX Page 54

I:\MEPC\56\2.doc The hydrogen gas in the ballast water treatment equi pment is also unlikely to accumulate, as the seawater electrolysis system uses a flowing type electrolytic cell wi thout a hold-up portion for liquid/gas.

Furthermore, only low concentration of the hydrogen gas shifts to the gas phase. The hydrogen gas is discharged from the vent ilation hole of the ballast tank.

6.2 Risk to human health

Details on the handling of the electrolyzed seawater containing the Active Substance are described in the MSDS attached as Appendix 2.

Human contact with the electrolyzed seawater and with the chloride gas generated from hypochlorous acid, an Active Substance, are assumed to be very rare. The possibility of contact is very low under normal operation conditions and operation works. There have been no reported cases of disasters of this nature with affects on human health in the service history of

MGPS ® for ships.

This seawater electrolysis system produces the Active Substance on board. Its adoption eliminates the need to load and store chemicals, as well as the risks related thereto. The Active Substance is produced inside sealed equipment, eliminating the danger of direct contact with personnel directly under normal operati ng conditions and operation works.

The acute aquatic toxicity value of the electrolyzed seawater (Rats; Oral LD 50 =850 mg/kg, see Appendix 2) is low even in the worst case (leak of electrolyzed seawater from the electrolytic cell, ≤ 1500 mg FAC/L). The seawater has no chronic aq uatic toxicity, mutage nicity/genotoxicity, carcinogenicity, developmental/reproduction toxi city, or endocrine disturbance effect.

In the unlikely event that the treatment water leak s, the safety of the crew can be ensured by sufficiently ventilating the room and washing the site of the leak with ample water while wearing protective equipment (goggles, rubber gloves, masks for halogens, etc.).

The concentration of Active Substa nce of treatment water in the ballast tank is lower (0.1-3.0 mg TRC/l) than the drinking water guideline of WHO (5mg TRC/l). Therefore, it is speculated that the hazard of the treatment water may near level to that of chlorinated drinking water. However,

chronic toxicities of some of relevant chemicals are not evaluated. Then, at the time of a periodic inspection, it is preferable that the worker puts on personal pr otective equipment (goggles, rubber gloves, masks for halogens, etc.) to preven t direct contact with treatment water.

The chlorine gas poses a higher ri sk to health than the electrol yzed seawater, but the conditions leading to chlorine gas generation (rise of temp erature, decrease of pH, etc.) differ from normal operation conditions and the ri sk of generation are low.

Thus, the methods commonly used to prevent oxyge n deficiency (ventilati on fan, compressed air, etc.) during the routine inspection of the ballast water tank are t hought to secure the safety of

the crew.MEPC 56/2

ANNEX Page 55

I:\MEPC\56\2.doc 6.3 Risk to the aquatic environment

As this system produces the Active Substance on boa rd using seawater, there is no need to stow harmful chemicals on the ship. The amount of Ac tive Substance produced can be controlled by the electric power applied to the electrolysis cell. And the electro lysis equipment is automatically shut down by the automatic safety devices, ha lting the production of Active Substance, in the rare event of an accident involving the system or ship. These safeguards ensure that Active Substance will not be released in high concentr ations into the aquatic environment or cause

adverse effects.

The discharge of the ballast water treated with this system poses no hazard to the aquatic environment. As already described in the sec tion on risk assessment (section 5.3), the discharge is rapidly diluted, rapidly diffuses, and it will not accumulate. This satisfies the provisions stipulated in the related regulations.

7 ASSESMENT REPORT (G9:4.3)

The persistence, bioaccumulation, an d toxicity were assessed based on reliable bibliographic data on the physicochemical properties and environmenta l toxicity of the Activ e Substance generated with seawater electrolysis process and the re levant chemicals genera ted from ballast water treatment by the Active Substance.

Neither the Active Substance no r relevant the chemical has persistence or bioaccumulation.

On the other hand, the data indicate that hypochlorous acid/ hypochlorite ion, both Active Substances, has toxicity. Furthermore, reliable da ta have yet to be reported on 2,2-dibromoacetic acid and 2,2,2-tribromoacetic acid, and neither of these brominated acetic acids could be definitively evaluated for toxicity . Notwithstanding, their toxicity in the electrolyzed seawater is

much lower than the concentrations measured in documented cases of toxicity. The other relevant

chemicals were found not to be toxic.

Given the Active Substance has toxicity and that the toxicity of several of the other chemicals cannot be determined, the applican t executed a toxicity evaluation te st and risk assessment of the treated ballast water discharges for the treated ba llast water under the cond itions of actual use.

The NOEC of the treated ballast wa ter has been determined to be 3.1% (v/v), based on the results of the chronic toxicity tests of the treated ball ast water, water that had been chlorinated by a method simulating the ballast water treatment syst em according to the OECD test to evaluate toxicity for fishes, crustacean, and algae in the aq uatic environment. Diluti on ratio (treated ballast water/natural seawater) has been used fo r the NOEC of the treated ballast water.

The transport, diffusion and fate of the discharge of ballast water treated by this treatment system were predicted using NOEC (reported by the toxi city tests), the high -resolution flow model (HYDROMAP), and the chemical com pounds diffusion model (CHEMSAP).

In the short-term risk assessment of the treate d ballast water discharges , the estimation of the time required for the toxicity to fall below th e NOEC (3.1% (v/v) as di lution ratio) presupposed

different conditions of the di scharge point, tide, and discharge amount. The time required for

elimination of the risk was 1.00 - 1.25 hours for a ship with a ballast tank capacity of 3,000 m 3
and 21.50 -24.00 hours for a ship with a ballast tank cap acity of 72,000 m 3

.MEPC 56/2

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I:\MEPC\56\2.doc In the long-term risk assessment, the applicant carried out a long-term simulation assuming the worst case, all ships anchored in Yokohama Port discharge ball ast water at one point, using tribromomethane, the substance considered to have the highest persistence in the treated ballast water, as an indicator. The simulation estimated a tribromomethane concentration below PNEC (0.29 mg/l; assessment factor =10) all year.

The evaluations of the ballast water management system, including the seawater electrolytic process, indicate that the system can satisfy the provisions stipulated in the related regulations, and further, that the system in no way poses risk s to ships, human, the a quatic environment, or

marine resources.

The data used in this application concerning the seawater electrolytic process and Active Substance are based mainly on the results obtained by the laboratory test and from small pilot-scale test equipment. This raises unans wered questions about the scale-up effect, the fluctuation of seawater properties, etc. in rela tion to the current data and its certainty (G9:6.4.3). Plans are in place to continuously survey these effects in land-based te sts and shipboard tests with the goal of developing systems to be approved in the future.

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9 ABBREVIATION

ACGIH American Conferen ce of Governmental I ndustrial Hygienists

AF Assessment Factor

AISE Association Internationale de la Savonnerie, de la Détergence et des Produits

d’Entretien ATSDR Agency for Toxic Substances and Disease Registry

CPO Chlorine Produced Oxidant DBP Disinfection By-Product

BCF Bioconcentration Factor DOC Dissolved Organic Carbon

EC 50 Median Effective Concentration

FAC Free active chlorine GHS The Globally Harmonized System of Classification a nd Labelling of Chemicals GLP Good Laboratory Practices

HSDB Hazard Substances Data Bank IARC International Agency for Research on CancerMEPC 56/2

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I:\MEPC\56\2.doc IC 50 Median Inhibition Concentration IMO International Maritime Organization

IUCLID International Uniform Ch emicaL Information Database Koc adsorption coefficient

LC 50 Median Lethal Concentration

LD 50 Median Lethal Dose

LOEC Lowest Observed Effect Concentration

NA Not Availabel

ND Not Determined

NITE National Institute of Technology and Evaluation, Japan NTP National Toxicology Program

NOAEL No Observed Adverse Effect Level

NOEC No Observed Effect Concentration

NOEL No Observed Effect Level OECD Organization for Economic Cooperation and Development PBT Persistence, Bioaccumulation, Toxicity

PEC Predicted Environmental Concentration

PNEC Predicted No Effect Concentration POC Particulate Organic Carbon

Pow octanol/water coefficient PSU Practical Salinity Units QSAR Quantitative Structure-Activity Relationship

THMs Trihalomethanes TOC Total Organic carbon

TRC Total Residual Chlorine

TRO Total Residual oxidant U.S.EPA United States Environmental Protection Agency WHO World Health Organization

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