I:\MEPC\55\2-5.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

55th session Agenda item 2 MEPC 55/2/5 21 April 2006 Original: ENGLISH

HARMFUL AQUATIC ORGANI SMS IN BALLAST WATER

Basic Approval of Active Substances us ed by PureBallast management system

Submitted by Sweden

SUMMARY

Executive summary: This document contains non-confidential information for Basic Approval of Active Substances used by PureBallast management system as required by the Procedure for approval of Ballast 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 – Ba llast Water Working Group in accordance with the provisions of the above mentioned procedure

Action to be taken: Paragraph 6

Related documents: BWM/CONF/36, MEPC 53/24/Add.1, BWM.2/Circ.4

Introduction

1 Regulation D-3.2 of the Inte rnational Convention for the Control and Management of Ships’ Ballast Water and Sediments stipulates that Ballast Water Management sytems that make use of Active Substances to comply with the Convention shall be appr oved by the Organization.

2 The Procedure for approval of Ballast Wate r Management systems that make use of Active Substances (G9) adopted by resolution MEPC.126(53) defines principle aspects to be documented by data or testing (MEPC 53/24/A dd.1, annex 4, paragraph 4.2.1) and some basic principles for risk evaluation (MEPC 53/24/ Add.1, annex 4, paragraph 5.3). According to section 6 of the Procedure (G9) the Organization should evaluate the information provided in the application.

3 According to BWM.2/Circ.4 proposals for approval of Ballast Water Management systems

that make use of Active Substances are to be submitted to Marine Environment Division of the Organization to be reviewed by the GESAMP-Ballast Water Work ing Group. In the meantime, a document on the proposal for approval containing all the non-confidential data related to the respecitve Active Substance need s to be submitted to MEPC 55.

4 The competent authority in Sweden has verified the application dossiers prepared by ALFA LAVAL and believes them to satisfy th e data requirements of the Procedure (G9).MEPC 55/2/5 - 2 -

I:\MEPC\55\2-5.doc 5 Sweden therefore submits the non-confidential part of the applicant’s do ssier in the annex of this document to the Organi zation for evaluation according to the Procedure (G9). The complete dossier will be made available to th e experts of the GESAMP-Ballast Water Working Group with the understanding of confidential treatment.

Action requested of the Committee

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

***MEPC 55/2/5

I:\MEPC\55\2-5.doc ANNEX

PROPOSAL FOR APPROVAL

ALFA LAVAL BALLAST WATER MANAGEMENT SYSTEM - PureBallast

This report is structured in accordance with th e headlines in the “Procedure for approval of Ballast Water Management systems that make use of Active Substances (G9)”.

1 INTRODUCTION

Working principles of the Alfa Laval Balla st Water Management System (PureBallast)

The ballast water is treated at intake and once again at discharge. The treatment on intake ensures that a minimal amount of viable organism s enter into the ballast water tanks and reduces sediment built-up in the tanks, whic h is a potential area for survival of organisms. The water is treated again at discharge to ensure that any pot ential re-growth of organisms in the ballast water tanks is prohibited.

Description of the PureBallast system and its components

The PureBallast product is a complete syst em, composing of three main functions:

• Filter (50 μm) - An automatic back flushing filter for removal of larger particles and organisms as well as to reduce the level of sediments in the ballast water

tanks.

• Benrad AOT (Advanced Oxidation Technol ogy) Unit - A patented system product using naturally occurring phot o-processes in the surface layer of sea-water. The Benrad AOT process acts on two levels: in-s itu formation of reactive free radicals, and direct photo-effects. These tw o levels synergistically inactivate micro-organisms. A special UV light wave length spectrum is used in combination with a catalyst wh ich is based on TiO 2 . The TiO 2 acts as a true catalyst and is not consumed in the process. No subs tances are added to the process.

• Control and Auxiliary Equipment includi ng sampling points - The control system and transformer control the system and s upply all items with power. The auxiliary equipment controls the flow and measure di fferent alarm levels. The system also composes a CIP unit which is an automatic service device that cleans the quartz sleeves covering the UV-lamps after each Ballast or Deballast sequence. This operation is done to avoid scaling from seawater contaminants which would otherwise reduce the efficiency of the Benrad AOT Module.

This application will focus on the Benrad AOT treatment unit . The filter and other components are not considered to be relevant for Active Substance approval.

Control and auxiliary equipment including sampling points

• Control System - An overall control system that controls the process, log all data and give alarms.

• Transformer - Supplies all units with power independent of available power properties on ship.MEPC 55/2/5

ANNEX Page 2

I:\MEPC\55\2-5.doc • Sampling points - Allows for accurate samp ling before and after treatment system.

• CIP (Cleaning in Place) - Support equipment for automa tic cleaning of the quartz sleeves protecting the UV-lamps.

Description of the Benrad AOT unit in the pilot scale test system

The pilot scale test system is used for the toxici ty testing as well as analyses regarding chemical water properties. The pilot scale is designed to be similar to the full scale system with regards to:

• Lamp type and power relative to flow rate and geometry;

• Number of lamps;

• Geometry of channels (Properties of flow is equal to full scale); and

• Geometry of catalyst surfaces.

Detailed information about this can be s upplied upon request to th e IMO Technical Group (GESAMP-BWWG).

All tests are performed with the fo llowing properties of the system:

Specified flow rate 25 m 3 /h

A complete treatment cycle is two stage treatments which correspond to Ballast as well as Deballast. A complete treatment cycle is used fo r all toxicity and chemical water property tests which is presented in this application.

Description of the full scale test system

The full scale test system is designed for the full scale land based testing and will also be used for the “Final Approval”-test program as specified in the attached QAPP-ex traction, see appendix 1.

The following property is relevant for the full scale test system: Specified flow rate 250 m 3 /h

Description of the pilot scale test setup

The toxicity testing in this application is performed with the following parameters:

Flow rate 25 m 3 /h Treatments Ballast + Deballast (Two treatments)

Treated volume 1 600 litre Salinity 22 PSU (Instant Ocean © synthetic sea salt) Temperature 18 ° C Volume of samples 3 x 10 l before treatment *

3 x 10 l after treatment *
Organisms Alga Tetraselmis suecica at a density of about 1000 org/ ml

* ) From these samples sub-samples were taken for toxicity testing as well as for chemical testing.MEPC 55/2/5

ANNEX

Page 3

I:\MEPC\55\2-5.doc 2 DEFINITIONS

Interpretation for the PureBallast system (focus on Benrad AOT unit)

An “Active Substance” as defined by IMO is a substance or organism, including a virus or a fungus that has a general or specific action on or against Harmful Aquatic Organisms and Pathogens. In this process, it is the complex chemistry in the treated ballast water from the Benrad AOT process, as described in the pr ocess description, which has the action against Harmful Aquatic Organisms and Pa thogens. The process utilizes free radicals to treat the organisms.

A previous submission from Sweden raised the question if the PureBallast process is to be

defined as an Active Substance or not. In this respect, an opinion was e xpressed by the Ballast Water Review Group established during MEPC 53 as follows: “provided it could be

demonstrated to the Administration that there was no residual in the ballast water discharge, then

it would be for the Administration to decide wh ether such a system required Active Substance approval.” However, the word “residual” seemed undefined to a number of delegations, which suggested that further considera tion of this particular aspect should be given in due course.

Despite that within a EU defi nition in which the use of TiO 2 in combination with UV-light could be defined as a biocide, at the 21st Competen t Authority meeting for the implementation of Directive 98/8/EC concerning the placing on th e market of biocidal products (BPD) in April 2006, an agreement was reached among Member States, that the use of TiO 2 as part of the Advanced Oxidation Technology shall not fall w ithin the scope of the BPD (see Manual of Decisions for Implementation of Directive 98 /8/EC concerning the placing on the market of biocidal products, point 2.2.1.3).

“Ballast Water Discharge” is the treated ballast water that has passed the system twice, first during Ballast and then after Deballast.

The term “Preparation” refers to the extremely short lived free radicals generated in treated ballast water from the Benrad AOT process.

Due to the short life span of the chemistry in th e treatment process, all “Relevant Chemicals” as defined by IMO is the potential transformation or reaction products in the treated water.

The above interpretation of defi nitions indicates that the term s “Active Substance”, “Ballast Water Discharge”, “Preparation” and “Relevant Chem icals” all refers to the treated ballast water from the Benrad AOT process. This means that it is the treated wate r that is considered in this application and thus tested regarding toxicity and content of chemicals.

3 PRINCIPLES

No comment.MEPC 55/2/5

ANNEX Page 4

I:\MEPC\55\2-5.doc 4 GENERAL REQUIREMENTS

4.1 Identification

Discharge characteristics

The basic process operates without addition of chemicals using the synergistic effects of in situ produced free radicals and UV-photons to inactivate micro-organisms. Per se , TiO 2 is non-toxic as shown by its use in re-constructive surgery to obtain bone-compatible links for Ti-implants such as teeth and hip-joints (Pan et al. 1996, Diebold 2002). TiO 2 is also a widely used component in paints and cosmetic products. The ac tion of reactive free radicals is of temporal

nature , i.e. these species are so short-lived (micro-m illi second time-scale) that they are not

observable outside the AOT-treatment unit (Baxendale and Busi 1982, Buxton et al. 1988,

NDRL/NIST Solution Ki netics Database 2005).

The only environmental concern of the PureBallast treatment system might be attributed to the reaction products. To elucidate whether any environm ental effects are to be expected, a series of chemical analysis and ecotoxicological tests of possible formation of t oxic reaction products and their potential effects is relevant. Of primary inte rest would be the slight possibility of formation of halogenated organics or toxic oxi dation products (for example bromate).

A problem related to Ozone treatment of sea wa ter is a significant formation of bromate, a suspected carcinogen. This reaction may proceed by way of an oxygen atom transfer mechanism. In the photo-catalytic proce ss formation of bromate, BrO 3 - , is unlikely to occur, since this would require multiple one-electron oxidation steps. Mo reover, the utilization of photocatalysis is

one of the attractive methods for the removal of BrO 3 - . The reduction of BrO 3 - on a TiO 2 photocatalyst under UV irradiation is a well -known reaction, and bromate has even been used as an additive to improve the oxidation efficiency of TiO 2 photocatalysts (Oosawa and Gretzel 1988, Al-Ekabi et al. 1993, Mills et al . 1996, Lindner et al. 1997, Noguchi et al. 2003)

It should be pointed out that the radicals produc ed in the AOT-treatment unit are not different from those naturally formed by sun light in the surface layer of seawater. In this case, photo-cleavage of hydrogen peroxide and/or photo- oxidation of nitrate/nitrite ions may be the

source of radical formation.

Therefore, treated ballast water is tested for toxicity and chemi cal properties, specifically for:

• Chloro-organic compounds;

• Bromo-organic compounds;

• A general toxicity anal ysis (aggregate acute and chronic toxicity);

• Bromate formation;

• THM formation (Tri halo methane); and

• Octanol/Water partition coefficient.MEPC 55/2/5

ANNEX

Page 5

I:\MEPC\55\2-5.doc 4.2 Data-set for Active Substances and Preparations

The potential toxic effects of the treatment were tested in a set of bioassays; see Appendix 4 for the tests review and test reports.

4.2.1 A proposal for approval should include inform ation on the properties or actions of the Preparation including any of its components as follows:

.1 Data on effects on aquatic plants, inverteb rates, fish, and other biota, including sensitive and representative organisms:

• acute aquatic toxicity;

No acute toxicity was observed. See report in appendi x 4, section 8.3

and 8.5.

• chronic aquatic toxicity;

No chronic toxicity was observed. See report in appendix 4, section 8.1-8.3.

• endocrine disruption;

Not tested specifically, however juve nile development and reproduction in an invertebrate test orga nism were not found to be affected (see section 8.4 in the Toxicity Bioassays Report). Moreover, no chemicals identified as potential endocrine affecting substan ces were found in the treated water (See Chemical Analyses Report). This suggests endocrine disruption as being unlikely to occur.

• sediment toxicity;

As showed in the chemical and toxicity testing there will be no residuals resulting from the treatment and therefore no particle flux to the sediment. Sediment toxicity is therefore not expected.

• bioavailability/biomagni fication/bioconcentration;

Octanol/water partitioning does not suggest bioaccumulation/biomagnification/ bioconcentration to occur, see appendix 3.

• food web/population effects.

Since the full strength effluent (100%) did not have any deleterious effects on any of the test species, the effluent would not be expected to be toxic to aquatic organisms and have negative effects on ecological communities at environmentally relevant concentrations.MEPC 55/2/5

ANNEX Page 6

I:\MEPC\55\2-5.doc .2 Data on mammalian toxicity:

Not relevant according to the results above, and because the Active

Substances are short-lived.

• acute toxicity;

• effects on skin and eye;

• chronic and long-term toxicity;

• developmental and reproductive toxicity;

• carcinogenicity; and

• mutagenicity.

.3 Data on environmental fate a nd effect under aerobic and anaerobic

conditions:

• Modes of degradati on (biotic; abiotic);

The primary active species are of suff icient reactivity to either react

or terminate with each other well within the milli-second to second time scales and will thus be consumed before they can leave the reactor. The primary residues wi ll therefore be reaction products stemming from the oxidation (major) or reduction (minor) caused by the free radicals initially formed. As these are formed analogously to natural decomposition processes they can be expected to be naturally degradable by natural biotic or abiotic paths.

• Bioaccumulation, partition coefficient, octanol/water coefficient;

Potential changes in hydrophobicity have been analyzed on the

treated water and no ch anges have been found.

• Persistence and identification of the main metabolites in the relevant media (ballast water, marine and fresh waters);

As no oxidation products were identified and no measurable quantities of those were found, the persistence assessment is not

relevant.

• Reaction with organic matter;

The process generates free radicals of sufficient reactivity able to react with most organic compounds . The major modes of reaction will be one-electron transfer (Br 2 .-, CO 3 .-, Cl 2 .- radicals), hydrogen abstraction (hydroxyl, CO 3 .- radicals) or addition reactions (hydroxyl radicals).MEPC 55/2/5

ANNEX

Page 7

I:\MEPC\55\2-5.doc All these radicals are naturally fo rmed in the surfaces of (sea) water so any reaction products can be expected to be of a character similar to those of “natural” decomposition in water. It should be

noted that the reactive free radica ls will react indiscriminately forming an array of different r eaction products by various reaction paths, making it difficult to predict specific products. Generally these kinds of processes are discussed for environmental applications to reduce the amount of hazardous material in water

streams.

• potential physical effects on wildlife & benthic habitats;

Not tested. Treatment does no a ffect physical properties of the water, see appendix 3. No poten tial physical effects could be expected.

• potential residues in seafood; and

Since bioaccumulation effects are not expected, no residues resulting from the treatment are likely to occur.

• any known interactive effects.

No interactive effects are expected.

.4 Physical and chemical properties for the Active Substances a nd Preparations and the treated ballast water, if applicable:

This technology is a physical-chemical pr ocess and does not i nvolve the addition of any substances. The in-situ generated free radicals have life times (half life) of

micro or milli seconds and are c onsumed inside the reactor.

• melting point; Not applicable.

• boiling point; Not applicable.

• flammability; Not applicable.

• density (relative density); Not applicable.

• vapour pressure, vapour density; Not applicable.

• water solubility/dissociation constant (pKa);

The process may increase the amount of oxidized groups in the dissolved organic matter, leading to a decrease in COD, i.e. increase in oxidized groups like: acidic, alcoholic, aldehyde and ketostructures. This may alter th e pKa or buffer properties of the

treated water.MEPC 55/2/5

ANNEX Page 8

I:\MEPC\55\2-5.doc The treated water are assessed by measurement of possible changes in the octanol/water partition values of treated water. No changes has been found, see test results for chemical properties in appendix 3.

• oxidation/reduction potential;

The potential for bioaccumulation of the treated water are assessed by measurement of possible changes in the octanol/water partition values of treated water. No changes has been found, see test results for chemical properties in appendix 3.

• corrosivity to the materials or equipment of normal ship construction;

This may decrease due to c onsumption of dissolved oxygen whereas it may increase slightly due to the formation of acidic groups and residual H 2 O 2 . To be investigated further.

• autoignition temperature; and Not applicable.

• other known relevant physic al or chemical hazards.

None at this date.

.5 Analytical methods at environm entally relevant concentrations.

4.2.2 A proposal for approval should include the above data set eith er for the Preparation or for each component separately, and a list of the name and relative quantities (in volumetric percentages) of the component s should be also attached. As described in section 8.1, all proprietary data should be treated as confidential.

Not applicable.

4.2.3 The tests for Active Substances and Preparati ons should be carried ou t in accordance with internationally recognized guidelines.

4.2.4 The testing process should contain a rigor ous quality control/quality assurance program consisting of:

All analyses are done according to standard methods and toxic ity testing is performed under supervision by the Department of Systems Ecology, Stockholm University. The testing laboratory is accredited by SWEDAC for chemi cal analysis of environmental samples and phytoplankton monitoring. Although the laborat ory is not accredited specifically for toxicological testing, the Depart ment has an Ecotoxicology Division, personnel with a solid experience in toxicological research, and all tests were performed according to good laboratory practice. The data on quality assurance are provided in each test report.

.1 Both a Quality Management Plan (QMP) and a Quality Assurance Project Plan (QAPP). Guidance on preparation of th ese plans, along with other guidanceMEPC 55/2/5

ANNEX

Page 9

I:\MEPC\55\2-5.doc documents and other general quality control information are available for download from the International Orga nization for Standardization (ISO) (www.iso.org ).

An extraction of a QAPP for further testing is attached in appendix 1.

.2 The QMP addresses the quality control ma nagement structure and policies of the Test Organization (inclu ding subcontractors and outside laboratories).

.3 The QAPP is a project specific technical document reflecting the specifics of the system to be tested, the test facility, and other conditions affecting the actual design and implementation of the required experiments.

4.2.5 Dossiers already used for registration of ch emicals can be submitted by the applicant to satisfy the required data needed for the eval uation of Active Substances and Preparations according to this procedure.

4.2.6 The proposal should describe the manner of application of the Preparation for Ballast Water Management, including requir ed dosage and retention time.

4.2.7 A proposal for approval should include (Mat erial) Safety Data Sheets ((M)SDS).

4.3 Assessment report

4.3.1 A proposal for approval should include an assessment report. The assessment report should address the quality of the te st reports, the risk characteri zation and a consid eration of the uncertainty associated with the assessment.

The analysis performed is prelim inary and will be complemented with further tests both with regards to toxicity as well as ch emical properties of tr eated water. The preliminary test results found no measurable levels of poten tially toxic substances and no acu te or chronic effects of the full strength effluent on aquatic organisms. Therefore, no appare nt environmental risks could be

identified.

All analyses are done according to standard methods and toxic ity testing is performed under supervision by the Department of Systems Ecology, Stockholm University. The testing laboratory is accredited by SWEDAC for chemi cal analysis of environmental samples and phytoplankton monitoring. Although the laborat ory is not accredited specifically for toxicological testing, the De partment has Ecotoxicology Di vision, personnel with a solid experience in toxicological research, and all tests were performed according to good laboratory practice. The data on quality assurance are provided in each test report.

Further tests will be performed on full scale system using natural seawater according to the QAPP in appendix 1.MEPC 55/2/5

ANNEX Page 10

I:\MEPC\55\2-5.doc 5 RISK CHARACTERIZATION

5.1 Screening for persistency, bioaccumulation and toxicity

5.1.1 An assessment on the intrinsic properties of the Active Substance and/or Preparation such as persistency, bioaccumulation and toxicity shou ld be conducted (see Table 1 in section 6).

.1 Persistence tests:

Persistence should preferably be assess ed in simulation test systems that determine the half-life under relevant conditions. Biodegradation screening tests may be used to show that the substances are readily biodegradable. The

determination of the half-life should include assessment of relevant chemicals.

See description in section 4.1 “ Radicals formed and th eir expected lifetime”.

.2 Bioaccumulation tests:

The assessment of the (potential fo r) bioaccumulation should use measured bioconcentration factors in marine (or freshwater) organisms. Where these tests are not applicable, or if logPow <3, Bi o Concentration Factor (BCF) values may be estimated using (Quantitative) Structure-Activity Relationship ((Q)SAR)

models.

The potential for bioaccumulation of the trea ted water are assessed by measurement of possible changes in the octanol/w ater partition values of trea ted water. No changes has been found, see test results for chemical pr operties in appendix 3. The octanol/water partitioning values do not suggest bioaccumulation/biomagnification/bioconcentration

to occur.

.3 Toxicity tests:

Acute and/or chronic ecotoxicity data, id eally covering the sensitive life stages, should in principle be used for the assessment of the to xicity criterion.

Acute and chronic tests covering surviv al of juveniles and reproduction of zooplankton (2 species) and growth in algae (2 species) indicated no acute or chronic toxicity to any of these endpoints in any test organisms at any effluent

concentrations as tested. Since the full st rength effluent (100%) did not have any deleterious effects on any of the test species , the effluent would not be expected to be toxic to aquatic organisms and have negative effects on ecological communities at environmentally relevant concentrations.

5.2 Toxicity testing of the treated Ballast Water

5.2.1 Toxicity testing is necessary for the Active Substance, or Preparations (see sections 4.2.1 and 5.3) and the treated Ballast Water Discharge as covered in this section. The advantage of conducting toxicity testing on the Ballast Water Disc harge is that it integrates and addresses the potential for interactions of the Active Subs tances and Preparations with the possible by-products.MEPC 55/2/5

ANNEX Page 11

I:\MEPC\55\2-5.doc .1 For the basic approval process, the disc harge testing should be performed in a laboratory using techniques and equipmen t to simulate Ballast Water Discharge following treatment by the Preparation.

.2 For final approval, the discharge testi ng should be performe d as part of the land-based type approval process using th e treated ballast water discharge.

5.2.2 The applicant should provide both acute and chr onic toxicity test data using standardized test procedures to determine the toxicity of the Preparation and Relevant Chemicals as used in conjunction with the Ballast Wa ter Management System. This testing approach should be performed on the treated Ballast Water Discharg e, as the Ballast Water Management system could either mitigate or enhance the adverse effe cts of the Preparation or Relevant Chemicals.

5.2.3 The discharge toxicity tests should be conducted on samples drawn from the land-based test set-up, which would be representative of th e discharge from the Ballast Water Management
system.

5.2.4 These toxicity tests should include chronic test methods with multiple test species (a fish, an invertebrate and a plant) that address the sensi tive life-stage. The prefer ence is to include both a sub-lethal endpoint (growth) and a survival endpoint. Either fres hwater or marine test methods

should be tested.

5.2.5 The test results to be provided include : acute 24-hour, 48-hour, 72-hour, and 96-hour

Lethal Concentration at which x % of the test or ganisms die (LCx), No Observed Adverse Effect Concentrations (NOAECs), chronic No Observed Effect Concentration (NOEC) and/or Effect Concentration at which x % of test organisms s how effect (ECx), as appropriate based on the experimental design.

5.2.6 A dilution series including a 100% ballast wa ter discharge would be tested to determine the no adverse effect level using the statistical endpoints (NOEC or ECx). An initial analysis could use a conservative approach where th e dilution capacity woul d not be taken into consideration (no modeling or plumes analysis would be used). The rationale for taking a conservative approach is that there could be mu ltiple discharges into one location (even though this is not necessarily the case).

The actual NOEC were not measurable; the st atistically determined NOEC was 100%.

5.2.7 The acute and chronic toxicity test data in conjunction with the information in

Section 4.2.1 should be used to determine the ho lding time necessary to achieve the no adverse effect concentration upon discha rge. Knowing the ha lf-life (days), decay rate, dosage rate, volume of system and toxicity tests with time seri es, then a computational model can be used to

determine the amount of time needed to hold th e treated ballast water before discharge.

5.3 Risk characterization and analysis

5.3.1 For the basic approval process, fate and effect testing should be performed in the laboratory with Active Substances and Preparations. This section lists information that could be useful for a preliminar y risk characterization.MEPC 55/2/5

ANNEX Page 12

I:\MEPC\55\2-5.doc 5.3.2 Both the Active Substance or Preparation as well as the treated Ballast Water Discharge should be subject to toxi city testing in order to protect the receiving environment from toxic effects due to discharges.

These are all the same substances as di scussed in Interpretation of Definition.

5.3.3 The reaction with organic matter of Active S ubstances and Preparations that produce free

radicals should be addressed qualitatively so as to identif y products of concern to the

environment.

See description in section 4.1 “ Radicals formed and th eir expected lifetime”.

5.3.4 The rate of abiotic and biotic degradati on of the Active Substances and Preparations

under aerobic and anaerobic conditions should be assess ed, resulting in th e identification of

relevant metabolites in the re levant media (ballast water, marine and fresh waters).

This process does not involve the addition of chemicals.

5.3.5 The rate of abiotic and biotic degradati on of the Active Substances and Preparations

under aerobic and anaerobic conditions should be a ssessed, resulting in th e characterization of the persistence of the Active Substances, Prepar ations and Relevant Chemicals in terms of degradation rates under specified co nditions (e.g. pH, redox, temperature).

See description in section 4.1 “ Radicals formed and th eir expected lifetime”.

5.3.6 The partition coefficients (solids-water part ition coefficient (Kd) and/or organic carbon normalized distribution coefficient (Koc)) of the Active Substances, Preparations and Relevant

Chemicals should be determined.

This process does not involve the addition of chem icals and therefore this is not applicable.

5.3.7 For Active Substances and Preparations, th e potential for bioaccumulation should be

assessed in marine or freshwater organisms (fish or bivalves) if the logarithm octanol/water partition coefficient (logPow) is >3.

This process does not involve the addition of chemicals. The pot ential for bioaccumulation of the treated water was assessed by measurement of possible (if any) change s in the octanol/water partition values of treated water. This analysis showed that no changes occurred in the log Pow

before and after treatment.

5.3.8 Based on the information on fate and behavior of Active Substances and Preparations, the discharge concentrations at selected time intervals should be predicted.

As shown in the chemical and toxi city testing there will be no resi duals or effects directly after the Benrad AOT unit and therefore discharge concentrations at different time intervals is not

relevant.

5.3.9 The effect assessment of the Active Substanc es, Preparations and Re levant Chemicals is initially based on a dataset of acu te and/or chronic ecot oxicity data for aquatic organisms, being primary producers (algae or sea grasses), cons umers (crustaceans), predators (fish), and should include secondary poisoning to mammalian and avian top-predators, as well as data for sediment species.MEPC 55/2/5

ANNEX Page 13

I:\MEPC\55\2-5.doc The initial assessment was based on acute and chronic toxicity to primary producers (algae) and herbivore consumers (rotifers, crustaceans).

5.3.10 An assessment of secondary poisoning is redundant if the substance of concern demonstrates a lack of bioaccumulation potenti al (e.g., BCF <500 L/kg wet weight for the whole organism at 6% fat).

Secondary poisoning testing is not relevant as octanol/water partitioning does not suggest

bioaccumulation to occur.

5.3.11 An assessment of sediment species is redund ant if the potential of the substance of concern to partition into the sedi ment is low (e.g., Koc <500 L/kg).

The partition into the sediment cannot exceed th e indicated value; the assessment is therefore

redundant.

5.3.12 The effect assessment of the Active Substances, Preparations and Relevant Chemicals should include a screening on carcinogenic, mutage nic and endocrine disruptive properties. If the screening results give rise to concerns, this s hould give rise to a further effect assessment.

As no carcinogenic, mutagenic and endocrine disr uptive substances were found in the analyzed water after the treatment, the assessment is not relevant.

5.3.15 An analytical method suitable for monitori ng Active Substances a nd Preparations in ballast water discharges should be available.

As showed in the chemical and toxicity testing th ere will be no residuals or effects directly after the Benrad AOT unit and therefore monitoring of “Active Substances” or “Preparations” in the discharge is not relevant.

6.3 Ship and personnel safety

6.3.1 In order to protect the ship and personnel sa fety the technical group should evaluate the physical and chemical hazards (see paragraph 4.2.1.4) to ensure that potential hazardous properties of the Active Substances, Preparations or Relevant Chemicals formed in the treated

ballast water should not create any unreasonable risk to the ship and personnel. Proposed procedures for the use and tec hnical equipment introduced needs to be taken into account.

The only potential risk present in the PureBallast system identified, which is related to the Benrad AOT process, is the UV- irradiation from the UV-lamps and overheating in the Benrad AOT treatment unit. If the flow is cut off the lamps will be automatically shut-off so there is no risk of overheating. Several independent actions en sure that the shut-off is made. The lamps are completely enclosed in the Benrad AOT chambers and can not be operated outside the chamber. There is therefore no risk that any personnel are exposed to the UV-irradiation. The free radicals

will not be able to leave the Benrad AOT treatm ent unit, as described in section 4.1, headline

“ Radicals formed and th eir expected lifetime”.

A full scale prototype system has been placed onboard a car carrier. The system has been evaluated by Lloyd’s classification society and has been approved w ith regards to ship and crew safety, see appendix 2.MEPC 55/2/5

ANNEX Page 14

I:\MEPC\55\2-5.doc 6.3.2 For the protection of personnel involved in the handling and storage of the Active Substances and Preparations, the proposal should incl ude relevant ((M)SDS). The Organization should evaluate (M)SDS, mammalian toxicity data and chemical pr operties hazards (see paragraphs 4.2.1.2 and 4.2.1.4) and ensure that pot ential hazardous prope rties of the Active Substances, Preparations or Relevant Chemical s should not create any unreasonable risk to the ship or personnel. This evaluation should take into account the different circumstances that a ship or personnel may face in its trad e (e.g., ice, tropical, humidity, etc.).

Not applicable.

6.4 Environmental protection

6.4.1 In order to approve the a pplication, the Organization s hould determine that the Active Substances, Preparations or Re levant Chemicals are not Persis tent, Bioaccumulative and Toxic (PBT). Preparations that exceed all these crit eria (Persistence, Bioaccumulation and Toxicity) in

the table below are considered PBT.

The chemical analyses and toxicity assays results give no rise to concern on potential risks to the aquatic environment. As no potentially dangerous s ubstances were identified in the water and the full strength effluent did not have any deleterious effects on any of the test species, the effluent would not be expected to be harmful to aq uatic organisms and have negative effects on ecological communities.

Table 1. Criteria for identification of PBT substances

Criterion PBT criteria

Persistence Half-life: > 60 days in marine water, or > 40 days in freshwater*, or > 180 days in marine sediment, or > 120 days in freshwater sediment*

Bioaccumulation BCF > 2,000 or LogP octanol/water ≥ 3

Toxicity Chronic NOEC < 0.01 mg/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.

6.4.2 The Organization should determine the overall acceptability of the risk the Preparation may pose in its use for Ballast Water Management . It should do so by comparing the information provided and the undertaken assess ment of PBT and the discharge with scientific knowledge of the Active Substances, Preparations and Relevant Chemicals concerned. The risk evaluation should qualitatively take into acco unt cumulative effects that may occur due to the nature of shipping and port operations.

6.4.3 The risk evaluation should consider the uncertainties involved in the application for approval, and as appropriate, provide advice on how these uncertainties can be dealt with.MEPC 55/2/5

ANNEX Page 15

I:\MEPC\55\2-5.doc 7 REGULATION OF THE USE OF ACTIVE SUBSTANCES AND PREPARATIONS

7.1 Handling of Active Substances and Preparations

7.1.1 The proposal for approval of Active Subs tances and Preparations should include

information on their intended us e and application. The quanti ty of Active Substances and Preparations to be added to the ballast water and the maximum allowable concentration of the

Active Substances therein should be described in the instructions provided by the manufacturer.

The system is designed with a specified numb er of UV-lamps and with specified catalyst geometry. The same power will be used regardless of the water quality. The toxicity testing and chemical properties of the water are tested on this fixed power and at specified flow rate. The system can not generate more free radicals than what is achieved with the energy used.

The system should ensure that the maximu m dosage and maximum allowable discharge concentration are not exceeded at any time.

See 7.1.1.

7.2 Hazard documentation and labeling

7.2.1 The proposal should include ((M)SDS) as required. The (M)S DS should describe appropriate storage and handling to gether with the effects of degr adation and chemical reactivity during storage and should be included in the instructions provided by the manufacturer.

Not applicable.

7.2.2 Documentation of hazards or the (M )SDS should conform to the UN Globally Harmonized System of Classification and Label ling of Chemicals (GHS) and the relevant IMO regulations (e.g. the IMDG Code) and guidelin es (e.g. the GESAMP Hazard Evaluation Procedure). Where these regimes are not applicable, relevant na tional or regional regimes should

be followed.

7.3 Procedures and use

7.3.1 Detailed procedures and information for safe application of Active Substances and Preparations on board should be supplied and co mply with the approval conditions such as

maximum allowable concentration and maxi mum discharge concentration, if any.

8 APPROVAL

8.1 Basic approval

8.1.1 All proprietary data should be treated as confidential by the Organization and its Technical Group, the Competent Authorities involve d, and the evaluating regulatory scientists, if

any.

8.1.2 Procedure to be followed:

.1 The manufacturer should evaluate the Activ e Substances or Preparations and the potential discharge in accordance with th e approval criteria specified in this procedure.MEPC 55/2/5

ANNEX Page 16

I:\MEPC\55\2-5.doc .2 Upon completion, the manufacturer should prepare an Applicat ion on the Active Substances and Preparations and submit it to the Member of the Organization

concerned.

.3 The Administration having received a sa tisfactory application should as soon as possible propose an appr oval to the Organization.

.4 Members of the Organization may propose an approval.

.5 The Organization should announce and se t the time frame for the evaluation of Active Substances and Preparations.

.6 Parties, Members of the Organizati on, the United Nations and its Specialized Agencies, intergovernmental organiza tions having agreements with the Organization and non-governmental organizatio ns in consultative status with the Organization may submit in formation that is rele vant to the evaluation.

.7 The Organization should establish a Tec hnical Group in accordance with its rules of procedure ensuring that proprietary da ta should be treated as confidential.

.8 The Technical Group should review the comprehensive proposal along with any additional data submitted and report to th e Organization whether the proposal has demonstrated a potential for unreasonable risk for environment, human health, property or resources in accordance with th e criteria specified in this procedure.

.9 The Technical Group’s report should be in written form and circulated to the Parties, Members of the Organization, the United Nations a nd its Specialized Agencies, intergovernmental organiza tions having agreements with the Organization and non-governmental organizatio ns in consultative status with the Organization, prior to its consider ation by the competent Committee.

.10 The Committee of the Organization s hould decide whether to approve any proposal, introduce any modifications theret o, if appropriate, taking into account the Technical Group’s report.

.11 The Member of the Organization th at submitted the application to the Organization should inform in writing the applicant about the decision made with regard to the respective Active Substan ce or Preparation and their manner of application.

.12 Active Substances or Preparations r eceiving basic approval by the Organization may be used for prototype or type a pproval testing based on the guidelines developed by the Organization. An Active Substance or Preparation may be used for Prototype or Type Approval testi ng for the approval of different BWMS without going through ba sic approval again.

8.2 Final approval

8.2.1 In accordance with Regulation D-3.2, a Ballast Water Management system using an Active Substance or Preparation to comply with the Convention (which r eceived basic approval)MEPC 55/2/5

ANNEX Page 17

I:\MEPC\55\2-5.doc must be approved by the Organi zation. For this purpose, th e Member of the Organization submitting an application should conduct the Type Approval tests in accordance with Guidelines for Approval of Ballast Water Management System s. The results should be conveyed to the Organization for confirmation that the residual toxicity of the discharge conforms to the

evaluation undertaken for Basic Approval. This would result in Final Approval of the Ballast Water Management system in accordance with Regulation D-3.2. Active Substances or Preparations that have receive d Basic Approval by the Organizati on may be used for evaluation of Ballast Water Management systems using Ac tive Substances or Preparations for Final Approval.

8.3 Notification of approval

8.3.1 The Organization will record the Basic and Final Approval of Active Substances and Preparations and Ballast Water Management syst ems that make use of Active Substances and circulate the list once a year including the following information:

• Name of Ballast Water Management system that make use of Active Substances and Preparations; • Date of approval; • Name of manufacturer; and • Any other specifica tions, if necessary.

8.4 Modification

8.4.1 Manufacturers should report any modifications in names, including trade and technical name, composition or use of the Active Substan ces and Preparations in the Ballast Water Management systems approved by the Organization, to the Member of the Organization. The Member of the Organization should in form the Organization accordingly.

8.4.2 Manufacturers intending to sign ificantly change any part of a Ballast Water Management System that has been approved by the Organizati on or the Active Substances and Preparations used in it should submit a new application.

8.5 Withdrawal of approval

8.5.1 The Organization may withdraw any appr oval in the following circumstances:

.1 If the Active Substances and Preparati ons or Ballast Water Management system

that make use of Active Substances no longer conforms to requirements due to

amendments of the Convention.

.2 If any data or test records differ mate rially from data relied upon at the time of approval and is deemed not to satisfy the approval condition.

.3 If a request for withdrawal of a pproval is made by the Member of the Organization on behalf of the manufacturer.

.4 If unreasonable harm to environment, human health, property or resources is demonstrated by any Member of the Organi zation or observer to have been caused by the approved Ballast Water Management system that make use of Active Substances or Preparations.MEPC 55/2/5

ANNEX Page 18

I:\MEPC\55\2-5.doc 9 CONCLUSION

The PureBallast process uses UV-en ergy together with catalysts in order to produce free radicals which will react with and destroy the cell membra nes of the microorganisms causing their death. The process simulates and enhances the natural pr ocess which occurs when the sunlight hits the

surface of the seawater. There are no chemical s added and there are no chemicals formed which

can enter the ballast tanks.

The chemical testing made before and after the treatment has not i ndicated any residual substances. Preliminary results fr om acute and chronic toxicity tests show that there is no toxic

effect detected in the treated water.

The continued test program will include further toxicity testing as defined in the attached QAPP-extraction found as an appendix. This test program will include furt her toxicity testing on both pilot and full scale system for the “Final Approval”.

The system placed onboard will not require any handling of chemicals onboard; neither will it compromise the safety for the ship or the crew. As showed in the chemical tests the properties/reactivity of the water is not changed compared to the inlet seawater and will not

affect the corrosion rate in the BW tanks.MEPC 55/2/5

ANNEX Page 19

I:\MEPC\55\2-5.doc REFERENCES (FOR PUREBALLAST PROCESS DESCRIPTION)

Ahmed S., Jones C.E., Kemp T.J. and Unwin P.R. (1999) “The role of ma ss transfer in solution photocatalysis at a supported titanium dioxide surface” Phys. Chem. Chem. Phys., (1):5229-5233

Al-Ekabi H., Butters B., Dela ney D., Ireland J., Lewis N., Powwell T. and Story J. (1993) “Photocatalytic purification and treatment of water and air” E d. Ollis D.F. and Al-Ekabi H., Elsevier, Amsterdam, 321-335.

Baxendale J.H. and Busi F. (1982) “The study of fast and transi ent species by pulse radiolysis” NATO Advanced Study Institute Series , Reidel Publ. Company, Vol. D

Buxton G.V., Greenstock C.L., Helman W.P. and Ross A.B. (1988) “Critical Review of Rate

Constants for Reactions of H ydrated Electrons, Hydrogen At oms and Hydroxyl Radicals in Aqueous Solutions” J. of Phys. and Chem. Ref. Data, 2(17):513-886.

Diebold U. (2003) “The surface science of titanium dioxide” Surface Science Reports 48:53-229.

Engel D.W. et al. (1974) “The effect of ionizing radiation and salinity on the grass shrimp Palaemonetes pugio” Radiation Research 59.

Fujishima A., Rao T.N. and Tryk D.A. (2000) “Titanium dioxide photoc atalysis” Journal of Photochemistry and Photobiology C: Photochemistry Reviews 1, 1–21.

Horne R. A. (1969) in Marine Chemistry , Wiley-Interscience, N.Y., 153-155.

Ivanovskii IuA. and Mitrofanov IuA (1978) “Stimu lation of Artemia salina nauplius emergence under the action of gamma irradia tion” Radiobiologiia:18(1):135-8.

Kamat P.V. (1993) “Photochemistry on Nonre active and Reactive (Semiconductor) Surfaces”

Chem. Rev. 93:267-300.

Legrini O., Oliveros E. and Braun A.M. (1993) “Photochemical Processes for Water Treatment”

Chem. Rev. 93:671-698.

Lindner M., Theurich J. and Bahnemann D.W. (1997) “Photocatalytic de gradation of organic compounds: accelerating the process efficiency ” Water Science and Technology, 35(4):79-86.

Mills A., Belghazi A. and Rodman D. (1996) “Bromate removal from drinking water by semiconductor photocatalysis” Wat. Res. 30(9):1973-1978.

NDRL/NIST Solution Kinetics Database (2005), Na tional Institute of Standards and Technology, webresource http://kinetic s.nist.gov/solution/index.php

Nogueira F. et al. (1998) “Radio resistance studies in Methylobacterium” Ra diat. Phys. Chem. 52(1-6):15-19.

Noguchi H., Nakajima A., Watanabe T. and Hash imoto K. (2003) “Design of a photocatalyst for bromate decomposition: Surface modification of TiO2 by pseudo-boehmite” Environ. Sci.

Technol. 37:153-157.MEPC 55/2/5

ANNEX Page 20

I:\MEPC\55\2-5.doc Oosawa Y. and Gretzel M. (1988) “Effect of surface hydroxyl density on photocatalytic oxygen generation in aqueous TiO2 suspensions” Journal of the Chemical Society, Faraday Transactions 1, 84(1), 197 – 205.

Pan J., Thierry D. and Leygraf C. (1996) “Hydroge n peroxide toward enhanced oxide growth on

titaniumin PBS solution: Blue coloration and c linical relevance” J. Biomedical Materials Research, Vol 30. 393-402.

Platzman R. and Frank J. (1954) “The role of the hydration configuration in electronic processes involving ions in aqueous solu tion” The European Physical J ournal A - Hadrons and Nuclei (Historical Archive), 138:411-431.

Rijstenbil J.W. (2003) “Effects of UVB radia tion and salt stress on growth, pigments and antioxidative defence of the marine diatom Cy lindrotheca closterium” Mar. Ecol. Prog. Ser.

254:37-48.

Rijstenbil J.W. (2005) “UV- and salinity-in duced oxidative effects in the marine diatom Cylindrotheca closterium during simula ted emersion” Mari ne Biology 147:1063-1073.

Rothenberger G., Moser J., Grätzel. M., Serpone . N. and Sharma. D.K. (1985) “Charge Carrier Trapping and Recombination Dynamics in Small Semiconductor Particles” J. Am. Chem. Soc.

107:8054.

Tada H. (2002) “Photocatalytic oxidation proc esses at titanium dioxi de surfaces: enhancing

effects of surface modificati on with oxide-(sub) monolayer s on photocatalytic activity” Encyclopedia of surface and colloid sc ience, Marcel Dekker Inc., 4043-4058.

Treinin A. and Hayon E. (1975) “Charge Transf er Spectra of Halogen Atoms in Water.

Correlation of the Electronic Tran sition Energies of Iodine, Bromine, Chlorine, Hydroxyl, and Hydrogen Radicals with Their Electron Affin ities” Journal of the American Chemical Society 97(7):1716-1721.

Xiao-Ying Yu (2004) “Critical Eval uation of Rate Constants of H ydrogen Peroxide Photolysis in Acidic Aqueous Solutions Containing Chloride Ions” J. Phys. Chem. Ref. Data, Vol. 33, No 3.

Zaafrane S., Maatouk K., Gauthier J.M. and Bakhrouf A. (2004) “Influence of preliminary cultivation conditions and the presence of th e rpoS gene on the su rvival of Salmonella typhimurium in sea water exposed to sunlight” Canadian Journal of Microbiology 50(5):341-350.MEPC 55/2/5

ANNEX Page 21

I:\MEPC\55\2-5.doc APPENDIX 1

EXTRACTION FROM QAPP FO R FULL SCALE TEST PROGRAM

The appended text below is only an extraction of the complete QAPP for the complete full scale land based test program. The test program incl udes biological performance testing as well as toxicity testing for “Final Approva l” according to the Procedure (G9).

Extraction of QAPP...

1 Project description, objectives and organization

1.1 Address of QAPP

This QAPP describes the implementation of qual ity assurance and quality control activities during the evaluation of the Alfa Laval PureBalla st water management system according to the requirements for biological and toxicity testing stated in the IMO Guidelines for Approval of Ballast Water Management Systems (G8) (M EPC 53/24/Add.1, annex 3). The requirements involve all parts and processes of the test as listed in:

Annex - Guidelines for approval of ba llast water management systems (G8)

• Part 2 – Test and performance specifica tions for approval of ballast water
management systems.

• 2.1 Quality assurance and quality control procedures (page 15).

• 2.3 Land-based Testing (pages 17-22).

• 2.4 Reporting of Test Results (page 22).

• Part 4 – Sample analysis methods for th e determination of biological constituents

in ballast water.

• Section 4.1 – 4.8 (page 24-26).

Procedure for approval of ballast water ma nagement systems that make use of Active Substance (G9)

• Section 1-5 (pages 1-7).

The QAPP is a mean to reveal any problems befo re start-up and during ex ecution of the project at as early stage as possible to minimize any potential procedural, tec hnical and scientific inadequacies and time- and economic losses. This QAPP will be used for biological testing in accordance with the IMO guidelines as well as for the final and basic approval testing of Active

Substances.

1.2 Processes to be evaluated

The QAPP will cover all parts of the project, bot h preparations, execution of tests and reporting of results;MEPC 55/2/5

ANNEX Page 22

I:\MEPC\55\2-5.doc 1.2.1 Preparations

 Construction and preparation of the Test Site according to the IMO Guidelines (MEPC 53/24/Add.1) and advi ces and instructions from the US Coast Guard/ETV (Battelle Duxbury, 2004).

 Installation and preparation of the Al fa Laval ballast water management system according to its operating manual.

 Preparation of test waters accordi ng to chemical quality criteria.

 Preparation of test waters according to biological quality criteria including harvesting and culturing of test organisms.

1.2.2 Execution of tests

 Running the Alfa Laval ballast water ma nagement system according to its operating manual.

 Monitoring the operational performance of the ballast water management system itself.

 Sampling for chemical and biological analyses of initial test waters.

 Sampling for chemical, biological and toxi cological analyses of treated test

waters.

 Performing chemical analyses.

 Performing biological analyses.

 Performing toxicological analyses.

 Performing risk characteri sation of treated waters.

 Preparing for and performing discharge of control water and treated test water to receiving waters.

1.4.2 Quality management plan (QMP) of NIVA

The following documents describe NIVA’s quality system:

• Quality manual for NIVA. The quality manual for NIVA is the primary document describing the quality system that is implemented for quality assurance and quality improvement s of products and services.

• Quality manual for accredited laboratory analyses and tests. In August 1993 NIVA’s Biological La boratory was the first laboratory in Norway accredited by Norwegian Accreditation fo r ecotoxicological tests. At the same time NIVA’s Chemical Laboratory, which provides th e widest scopeMEPC 55/2/5

ANNEX Page 23

I:\MEPC\55\2-5.doc of analyses of environmental an d water samples in Norway, was accredited according to Norwegian Standard NS-EN ISO/IEC 17025 (Norwegian Accreditation, 1993). NIVA performs annually 150.000 analyses of water quality including nutrients, metals, unspecified organic material, organic micropollutants and test of marine soft-bottom fauna. The accreditation gives the customer a warranty that NIVA’s laboratories use analytical procedures accordi ng to internationa lly acknowledged quality systems (NS-EN ISO/IEC 17025).

• Quality manual for ecological testing based on OECD Principles of Good Laboratory Practice . NIVA now performs ecotoxicity testing according to international standards and is regist ered as a GLP laboratory in Norway (reg. no GLP 007) and is regularly in spected by the Norwegian Metrology

and Accreditation Servi ces. Our ecotoxicological laboratory performs standardized tests (e.g. in accordance with OECD guidelines or ISO

standards ) as well as special services concerning toxicity, biodegradability

and bioaccumulation of chemical substances, products and complex mixtures, e.g. industrial effluent s and environmental samples.

• Quality manual for internal control (health, environment and safety)

• Quality manual for management of projects. A project manual has been developed and has been in use from 2005. The project manual contains checklists for activities and responsibi lity covering all parts of project accomplishment. One person is independently responsible for quality control of each project.

2.5 Running test cycles

The different water transfers between tanks via the PureBallast system of Alfa Laval during a test cycle including the control is shown in Figure 3. One test cycle (blue lines) involves consecutive

treatment of 200 m 3 test water by the 50 μ m filter unit and the Be nrad AOT Module of the PureBallast system transferring the test water fr om tank with prepared test water (WST1) to a temporary holding tank (TT1) before a second treat ment by the Benrad AOT unit and transfer to a storage tank (TT2) for at least five days st orage. A control cycle (red lines) is run by transferring 200 m 3 of the same type of prepared test water from WST2 to a second temporary holding tank (CT1) using the pump of the PureBa llast system, but in by-pass of the treatment units before transfer to a second storage tank fo r at least five days storage (CT2). One cycle including one control will be run for each of th e test waters (high and medium salinity).MEPC 55/2/5

ANNEX Page 24

I:\MEPC\55\2-5.doc WST1

200 m 3 TT1

200 m 3 TT2

200 m 3

CT2

200 m 3 CT1

200 m 3 S1 S2, S3

S5, S6 50 μ m filter Pump

Benrad AOT Module

WST2

200 m 3 S4

Figure 1 Transfer of test water duri ng one test cycle with the PureBallast system including a test line (blue) and a control line (red). Sampling numbers are indicated by S1-S6.

2.6 Sampling and measurements

Table 2 summarizes all types of measurements to be taken during the study. Samples are

withdrawn to document:

.1 that the water quality in the initial te st waters (WST1 and WST2) is meeting the chemical and biological water qua lity criteria defined by IMO;

.2 the efficiency of the Alfa Laval PureBalla st system in removing/inactivating target organisms (TT2) immediately and after 5 days storage;

.3 the water quality in the control tank (CT2) as a reference to the water quality in TT2; and

.4 the chronic and sub-chronic environmenta l toxicity of the water directly after treatment (at the time of deballasting).

Sampling numbers are indicated by S1-S6 in Fi gure 1. All grab samples (see Table 4) will be collected in triplicates. S1 will be collected in WST1 directly before treatment, S2 will be collected in TT2 immediately after treatment – ea rly, middle and late in the test cycle – and S3 after at least 5 days storage in TT2. The same procedure will be used when sampling from the control line (S4, S5 and S6). Procedures for sa mpling and transfer are described in detail in

section 3.MEPC 55/2/5

ANNEX Page 25

I:\MEPC\55\2-5.doc Table 1. Parameters to be measured during th e study; time and type of sampling and

measurement location.

Parameter Sample number Type of sample Location Operational parameters for the PureBallast treatment system

Chemical water quality measurements Temperature S1, S2, S3, S4, S5,

S6 In situ , continuous Solbergstrand

pH S1, S2, S3, S4, S5,

S6 In situ , continuous Solbergstrand

Dissolved oxygen S1, S2, S3, S4, S5,

S6 In situ , continuous Solbergstrand

Salinity S1, S2, S3, S4, S5,

S6 In situ , continuous Solbergstrand

Dissolved organic carbon (DOC) S1, S2, S3, S4, S5,

S6 Discrete grab Solbergstrand,

NIVA Oslo Particulate organic carbon (POC) S1, S2, S3, S4, S5,

S6 Discrete grab Solbergstrand,

NIVA Oslo Total suspended solids (T SS) S1, S2, S3, S4, S5,

S6 Discrete grab Solbergstrand,

NIVA Oslo Biological treatment performance parameters Organisms > 50 μ m S1, S2, S3, S4, S5,

S6 Discrete grab Solbergstrand

Organisms ≥ 10-50 μ m S1, S2, S3, S4, S5,

S6 Discrete grab NIVA Oslo

Heterotrophic bacteria S1, S2, S3, S4, S5,

S6 Discrete grab NIVA Oslo

Coliform bacteria S1, S2, S3, S4, S5,

S6 Discrete grab NIVA Oslo

Enterococcus group bacteria S1, S2, S3, S4, S5,

S6 Discrete grab NIVA Oslo

Vibrio sp. S1, S2, S3, S4, S5,

S6 Discrete grab NIVA Oslo

Vibrio cholerae S1, S2, S3, S4, S5,

S6 Discrete grab NIVA Oslo

Environmental toxicity evaluation parameters Toxicity; growth inhibition of green algae S2, S5 Discrete grab NIVA Oslo

Toxicity; reproduction of marine copepods S2, S5 Discrete grab NIVA Oslo

Toxicity; Juvenile fish growth

test S2, S5 Discrete grab NIVA OsloMEPC 55/2/5

ANNEX Page 26

I:\MEPC\55\2-5.doc 4.4 Toxicity testing of treated ballast water

The treated ballast water will be tested for ch ronic or sub-chronic toxi city using three test organisms representing algae, crustacean (copepods) and fish. The tests with copepods and fish involve long term exposure with fr equent renewal. For these tests a batch of the treated ballast water will be stored for the time necessary to co mplete the tests. Changes of the composition of this sample that may occur during storage is consid ered as a simulation of changes that will occur after discharge of treated ballast water and, therefore, do not invalidat e the tests for this particular purpose. The tests will be conducte d for basic and final approval.

4.4.1 Growth inhibition of marine algae

The algae growth inhibition test will be pe rformed according to International Standard ISO 10253: Water Quality – Marine al gal growth inhibi tion test with Skeletonema costatum and Phaeodactylum tricornutum . The diatom S. costatum (NIVA-strain BAC 1) will be used as test organism. The ballast water sample will be filtered to remove indigenous algae and spiked with a growth medium concentrate. A dilution series of the sample in pure seawater containing the same nutrient concentrate will be prepared. All batches will be inoculated with S. costatum from an exponentially growing laboratory culture and incubated in glass flasks on a shaking table

at 20 ° C under continuous illumination from fl uorescent tubes providing approximately

75 μ M m -2 s -1 of photosynthetic active radiation (PAR). The test will be performed with three replicates at each concentration of ballast water and six control replicates. The cell density will be determined by counting with an electronic pa rticle counter (Coulter Multisizer) after 24, 48 and 72 hours. The growth rate of each culture is calculated and expresse d as percentage of the growth rate of contro l cultures in pure seawater growth medium. If more than 50% growth

inhibition is observed in the ballast water, the EC 50 and the EC 10 (i.e. the concentrations causing 50 and 10% growth inhibiti on respectively) will be calculated using non linear regression analysis of the growth rate against log concentration of ballast water.

4.4.2 Reproduction of marine copepods

A reproduction test covering the early life stages of the ma rine and brackish harpacticoid Copepod Nitocra spinipes will be carried out according to Bengtsson and Bergström (1987) and Tarkpea et al. (1999). Newly fertilised females will be e xposed in ballast water and ballast water diluted in pure seawater for 13 da ys. A semi-static procedure will be used in which test water is changed every second day. The animals are fed with live algae ( Rhodomonas baltica ). Ten animals are exposed individually at each ballast wa ter concentration and in the seawater control. Survival of the parent females and the number of offspring produced by each female is recorded and compared to the control. If more than 50 % reduction in production of offspring is observed in the ballast water, the EC 50 and the EC 10 (i.e. the concentrations causing 50 and 10% effect, respectively) will be calculated using non linear regression anal ysis of number of offspring against log concentration of ballast water.

4.4.3 Juvenile fish growth test

A prolonged exposure test with juvenile turbot ( Scopthalmus maximus ) will be carried out in accordance with the OECD Guideline for Testing of Chemicals 215: Fish Juvenile Growth Test. Juvenile fish in exponential growth phase are placed in test chambers and are exposed to ballast

water and ballast water diluted with pure seawater under semi -static conditions. The test duration is 28 days with renewal of the water thr ee times per week. Fish are fed daily. The fish are weighed at the start and end of the test. Ef fects on growth rates at each treatment will beMEPC 55/2/5

ANNEX Page 27

I:\MEPC\55\2-5.doc compared to the control (exposed in pure seawater ). If more than 50% growth inhibition is observed in the ballast water, the EC 50 and the EC 10 (i.e. the concentrations causing 50 and 10% growth inhibition respectively) will be calculated using non linear regression analysis of the growth rate against log con centration of ballast water.

Reference: OECD 203 Modified for tu rbot juveniles, NIVA method K19.MEPC 55/2/5

ANNEX Page 28

I:\MEPC\55\2-5.doc APPENDIX 2

LLOYDS APPROVAL OF FULL SCA LE PROTOTYPE SYSTEM ONBOARD M/V DON QUIJOTEMEPC 55/2/5

ANNEX Page 29

I:\MEPC\55\2-5.doc APPENDIX 3

TEST REPORTS FROM ANALYSIS OF CHEMICAL SUBSTANCES IN SEAWATER

Below follows a summary of the chemical analysis of untreated and treated water. The sampling was supervised by Stockholm University and the analysis was performed by approved

laboratories.

Test codes

Test Before treatment After treatment Bromate 83Br1, 83Br2 86Br1, 86Br2 Absorbed organic halogens 83AOX 86AOX Extractable organic halogens 83EOX 86EOX Chlorophenols 83KL 86KL

Trihalomethanes 83THM 86THM

Potential bioaccumulative substance V007237-06 V007238-06

Test results

Test Before treatment After treatment Bromate <0,01 mg/L <0,01 mg/L <0,01 mg/L <0,01 mg/L Absorbed organic halogens 76 μ g Cl/L 77 μ g Cl/L Extractable organic halogens <2 μ g/L <2 μ g/L Chlorophenols < 0,01 μ g/L < 0,01 μ g/L

Trihalomethanes Chloroform (CHCl 3 ) 2 μ g/L 1 μ g/L Bromoform (CHBr 3 ) <1 μ g/L <1 μ g/L Dichlorobromomethane (CHCl 2 Br) <1 μ g/L <1 μ g/L Dibromochloromethane (CHClBr 2 ) <1 μ g/L <1 μ g/L Potential bioaccumulative substance <0 (Log Pow) <0 (Log Pow)MEPC 55/2/5

ANNEX Page 30

I:\MEPC\55\2-5.doc APPENDIX 4

Alfa Laval 21-04-2006

TOXICITY BIOASSAYS

for Alfa Laval Ballast Wat er Management System ( PureBallast )

Report

Prepared for:

Alfa Laval Tumba AB and Benrad AB Stockholm, Sweden

Prepared by: Elena Gorokhova, PhD

Systems Ecology, Stockholm University, Sweden April, 2006MEPC 55/2/5

ANNEX Page 31

I:\MEPC\55\2-5.doc SUMMARY

Laboratory 24, 48, and 72-h tests were conducted at Departme nt of Systems Ecology, Stockholm University, testing aggregate toxic effects of artif icial seawater after processing in the PureBallst treatment system. Four species were used for the toxicity evaluation: two algae ( Phaeodactylum

tricornutum and Dunaliella tertiolecta ), a rotifer ( Brachionus plicatilis ), and an arthropod (nauplii of Artemia franciscana ). The endpoints assayed were: surviv al (24-h test; al gae, rotifers,

Artemia ), reproduction (48-h test, rotifers), and grow th (72-h test; algae). A dilution series of 6.25%, 12.5%, 25%, 50% and 100% e ffluent was tested in each bi oassay. Toxicity testing was conducted on April 18-21, 2006, to determine: (1) LOEC (the Lowest Observed Effect Concentration) for survival and reproduction; (2) the IC 25 (the Inhibition Concentration of the

effluent at which there is a 25% reduction in th e growth), and (3) the NOEC (the No Observed Effect Concentration, or the highest concentrati on tested that did not e xhibit any toxicity) for survival, reproduction, and growth.

Test results indicated no acute or chronic toxicity to survival, gr owth or reproduction in any test organisms at any effluent concentrations as te sted. The statistically determined LOEC and IC 25 and actual NOEC were not measurable; the sta tistically determined NOEC was 100%. Since the full strength effluent (100%) did not have any de leterious effects on any of the test species, the effluent would not be expected to be toxic to aquatic organism s and have negative effects on ecological communities at environmentally relevant concentrations.MEPC 55/2/5

ANNEX Page 32

I:\MEPC\55\2-5.doc TEST REVIEW

1 Laboratory accreditation

Laboratory: Systems Ecol ogy, Stockholm University, Sv. Arrhenius v. 21A, SE-10691, Stockholm, Sweden Accreditor: Swedish Board for Accreditation and Conformity Assessment (SWEDAC)

Accreditation ID: 1449 Category: testing laborator y (analyses of environmen tal samples, phytoplankton analysis) Effective: 3 May, 1996

2 Test purpose

The basic test protocol was to characterize effluent by evaluating acute and chronic toxic effects of artificial seawater after processing in the PureBallast treatment system. The test layout and interpretation were based on standard guide s and recommendations (USEPA 1994, 2000, 2002).

3 Test water – collection, transport and handling

Control and test water samples were obtained at Alfa Laval laboratory test facilities on 18 April, 2006, 10.30 to 11.00 am, and transported to the laboratory at Systems Ecology, which was contracted to perform ecotoxicity tests. The samples were obtained by Alfa Laval staff in the presence and under supervision of an assistant from the testing laboratory as follows. Water pumped from the tank without the treatment was used as control samples, while water passing twice through the PureBallast treatment system (i.e., full ballasting-deballasting cycle) was tested as the effluent. Immediately after collect ion of the large volume samples (10 L, 15 oC), whole water samples were taken as sub-samples fo r the chemical and toxicological analyses and sent to respective laboratories. The results from chemicals analyses are summarized in Appendix I of this report. Samples for toxicologi cal tests included 3 repres entative controls and 3 samples of the treated water (1 L each; stored in glass screw-cap bottles). They were transported

in an insulated container to the laboratory, filtered with 2- μ m filter to remove algae used in the test system, and prepared for test ing within 2 hours after collection.

4 Tests organisms and assays

Four species were used for the toxicity ev aluation of the same effluent (Table 1):

4.1 Phaeodactylum tricornutum , Algaltoxkit MTM (MicroBioTests Inc., Nazareth, Belgium), standardized 72-h algal growth inhibition test for toxicity screening in estuarine/marine environments (adheres to ISO Guideline fo r marine algal growth inhibition tests; ISO/CD 10253). The kit is supplied ready-to-u se with algal beads, providing usiform test organisms; the algae were reared prior the test and inoculated according to the manufacturer’s

standard procedures.

4.2 Dunaliella tertiolecta (Chlorophyceae), strain CCMP366, obtained from The CCMP

National Center for Culture of Marine Phytoplankton (USA; more information at http//ccmp.bigelow.org ). In-house cultures maintained since December 2005 in standard f/2 enriched medium (Guillard and Ryther 196 2), 20‰ artificial seawater (Instant Ocean TM

,MEPC 55/2/5

ANNEX Page 33

I:\MEPC\55\2-5.doc Aquarium Systems), supplied with constant aeration, illumination ~35 μ mol quanta m -2 s -1 , at 21 °C and 12/12 light/dark cycle. Used in standard growth inhibition test for Dunaliella (MFE80805: Appendix 1; Hall and Golding 1998).

4.3 Brachionus plicatilis (Rotatoria). Rotoxkit MTM (MicroBi oTests Inc., Nazareth, Belgium), toxicity screening 24-48 h test for estuarine/marine environments (adheres to ASTM Standard Guide E1440-91). Standard acute toxicity test for Brachionus (ASTM 1998). This is a commonly used test organism, a multicellular invertebrate highly relevant for estuarine and marine environment (Ruck 1998). The kit is supplied ready- to-use with cysts, e liminating the need for maintaining cultures and providing highly standard ized test animals. Hatching methods, rearing, and test setup followed recommendations of Snell and Moffat (1992) and the manufacturer’s standard procedures.

4.4 Artemia franciscana (formerly Artemia salina ) (Branchiopoda, Crus tacea). Artoxkit MTM (MicroBioTests Inc., Nazareth, Belgium), acute toxi city screening 24-h test for estuarine/marine environments (adheres to ASTM Standard Guid e E1440-91). Hatching and test setup were conducted according to the manuf acturer’s standard procedures.

Table 1. Summary of bioassays, test species, te st protocols, and endpoints measured.

Test organism Protocol,

source Organism type, size Assay Endpoint Summary

section

Phaeodactylum

tricornutum Algaltoxkit

MTM Unicellular alga, 15-20

μ m Growth

inhibition

test Intrinsic growth rate (r, d -1 ) 8.1

Dunaliella

tertiolecta MFE80805: Appendix 1 (NIWA, 1998) Unicellular alga, 10-12

μ m Growth

inhibition

test Intrinsic growth rate (r, d -1 ) 8.2

Brachionus plicatilis Rotoxkit

MTM Rotifer, planktonic invertebrate,

110-130 μ m 24-h acute

static test

48-h shortterm static

test Survival (%)

Reproduction (eggs female -1 ) 8.3

8.4

Artemia sp. Artoxkit MTM Planktonic arthropod, 360-410 μm 24-h acute

static test Survival (%) 8.5

5 Endpoints assayed

5.1 Lethal endpoint (i.e., survival) was assayed in acute 24-h tests for Brachionus and

Artemia (Table 1). Animals were considered dead if they did not exhibit any internal or external

movements in 5 s of observation.

5.2 Sub-lethal endpoints were assayed for Brachionus (reproduction; 48-h test) and algae (growth; 72-h test). Reproduction activity was a ssayed as number of eggs per female in each replicate. If number of rotifers observed were higher than number of individuals at the start of the incubation, they were assumed to hatch before th e checkpoint and were c ounted as eggs. Growth

rate was calculated as:MEPC 55/2/5

ANNEX Page 34

I:\MEPC\55\2-5.doc r

(d -1 ) = ( ln N t – ln N
o )/ t , where N t and N

o – algal density at checkpoint t and at the start of the test, respectively; t – incubation time (days).

6 Test methods and procedures

Five protocols were used for the same effluent with one test per sa mple yielding 3 control replicates and 3 replicates for each effluent dilu tion tested. The test methods for acute toxicity evaluation followed standard toxicity meth ods (USEPA 1994, 2002); particular protocols specified below for each bioassay.

6.1 Tests setup. The experiments were initiated around 13.00 on the same day. For each sample and each bioassay 3 repeats were performed.

6.2 Tested concentrations. A dilution series of 6.25%, 12.5%, 25%, 50% and 100% effluent was used as recommended by WET method ma nuals (USEPA 1993, 1994, 2000). Dilution water used in all bioassays was control wa ter obtained from the test system.

6.3 Controls. As dilution water was different from th e water used to culture algae and hatch zooplankters, an additional control was set with st erilized f/2 media that was routinely used as growth media in algal culture s (Guillard and Ryther 1962). Thus, the dual control system

included a dilution water control and a culture wate r control that were r un concurrently in all bioassays and underwent the same test conditions.

7 Statistics

Statistical evaluation was performed according to ex isting methods for lethal and sub-lethal tests (Environment Canada 2005). A nominal error rate of 0.05 was applied when testing hypothesis to determine the test results. Prior to the analysis of the test treatment data the two controls were compared using a t-test (USEPA 1994). Since at no occasion any diffe rences between the controls were observed, the d ilution water control was used in data reporting and statistical comparisons. In all bioassays, one-way ANOVA followed by Dunnett’s post test was used to

evaluate differences between treatments and c ontrols (Prism 4.0, GraphPad Software Inc.). Proportions (i.e. survival data) were arcsine square root tran sformed prior to statistical comparisons. Minimal significant difference (MSD) was calculated as specified in USEPA (2000) and reported for each bioassay.

8 Results

There were no acute or chronic t oxicity to survival, gr owth or reproduction in any test organisms at any effluent concentrations as teste d. The statistically determined LOEC and IC 25 were not measurable; the statistically determined NO EC was 100%. Since the full strength effluent (100%) did not have any de leterious effects on a ny of the test species and chemical analyses

found no measurable levels of potentially dangerous compounds (see Chemical Analysis Report, the effluent would not be expect ed to be toxic to aquatic orga nisms and have negative effects on ecological communities at environmentally relevant concentrations.MEPC 55/2/5

ANNEX Page 35

I:\MEPC\55\2-5.doc Test summaries

8.1 Test conducted: Algaltoxkit M TM

, Phaeodactylum tricornutum 72-h chronic toxicity test;

Test dates: 18-21 April, 2006;

Source/Age of organisms: MicroBioTests Inc./4-days culture;

Test concentrations: see section 5.2;

Dilution/control water: see section 5.3;

Deviation from test protocol: 4-days culture was used (as opposed to 3-d) because of the lower rearing temperature (18 oC as opposed to 20 oC);

Test conditions: 1) Static, non-renewal; 2) Starting density: 2 ·10 6 cell mL -1 ; 3) Multiwell testplates, 0.3 mL testvolume per replicate; 4) temperature: 17±1 oC, 12:12 light/dark cycle; 6) pH: 6.5 – 7.8;

Test endpoints: growth rate;

Results: Table 2. Intrinsic growth rate for Phaeodactylum

tricornutum exposed for 72 h to PureBallast-treated water

Concentration r , d -1 (mean±SD)

Dilution control 0.83±0.11

6.25 0.82±0.04

12.5 0.81±0.06

25 0.82±0.08

50 0.82±0.03

100 0.82±0.01

MSD 19%

IC 25 >100% (could not be determined)

NOEC 100%

Conclusion: Test results indicated no chronic toxicity to P. tricornutum growth at any of the test ed concentrations. The IC 25 and

actual NOEC could not be determined.

Quality assurance: P. tricornutum ; K 2 Cr 2 O 7 – Reference toxicant, tested 18-21 April, 2006. Dilution seri es: 1.8, 3.2, 5.6, 10, 18 mg L -1

. Growth: 72-h IC 25 3.8 mg L -1 (PASS); 72-h IC 25 Range of Acceptability: 2.8 – 11 mg L -1

.

Test acceptability: Increase in cell number (after 72 h) in control replicates by

a factor of >16.MEPC 55/2/5

ANNEX Page 36

I:\MEPC\55\2-5.doc 8.2 Test conducted: Dunaliella tertiolecta (MFE80805, NIWA, 1998)

72-h chronic toxicity test;

Test dates: 18-21 April, 2006;

Source/Age of organisms: strain CCMP366, The CCMP National Center for Culture of Marine Phytoplankton /exponentially growing culture;

Test concentrations: see section 5.2;

Dilution/control water: see section 5.3;

Deviation from test protocol: as a measure of cell density, OD at 670 nm (measured with spectrofluorometer Fluorostar Optima, BMG) was used as opposed to particle counter indicated in the protocol (i.e.,

the same method as recommended for P. tricornutum in Algaltoxkit MTM);

Test conditions: 1) Static, non-renewal; 2) Starting density: 10 6 cell mL -1 ; 3) multiwell testplates, 0.3 mL testvolume per replicate; 4) temperature: 17±1 oC, 12:12 light/dark cycle; 6) pH: 6.5 – 7.8;

Test endpoints: growth rate;

Results: Table 3. Intrinsic growth rate for D. tertiolecta exposed

for 72 h to PureBallast-treated water

Concentration r , d -1 (mean±SD)

Dilution control 0.89±0.09

6.25 0.86±0.07

12.5 0.89±0.12

25 0.84±0.10

50 0.88±0.06

100 0.87±0.06

MSD 17%

IC 25 >100% (could not be determined)

NOEC 100%

Conclusion: Test results indicated no chronic toxicity to D. tertiolecta growth at any of the test ed concentrations. The IC 25 and

actual NOEC could not be determined.

Quality assurance: 1). D. tertiolecta ; K 2 Cr 2 O 7 – Reference toxicant, tested 18-21 April, 2006. Dilution se ries: 1.8, 3.2, 5.6, 10, 18 mg

L -1 . Growth: 72-h IC 25 2.3 mg L -1 (PASS); 72-h IC 25 Range of Acceptability: 2.8 – 11 mg L -1

.
Test acceptability: Increase in cell number (after 72 h) in control replicates by

a factor of >16.MEPC 55/2/5

ANNEX Page 37

I:\MEPC\55\2-5.doc 8.3 Test conducted: Rotoxkit MTM

, Brachionus plicatilis 24-h acute toxicity test;

Test dates: 18-19 April, 2006;

Source/Age of organisms: MicroBioTests Inc./<2 h old;

Test concentrations: see section 5.2;

Dilution/control water: see section 5.3;

Deviation from test protocol: none;

Test conditions: 1) Static, non-renewal; 2) 5 organisms per group; 3) multiwell testplates, 5 mL testvolume per group; 4) no feeding during incubation; 5) temperature: 23±1 oC, darkness 6) pH: 6.2 – 7.2; 7) aeration: none;

Test endpoints: mortality (=immobility);

Results: Table 4. Survival (%) of juvenile B. plicatilis exposed for

24 h to PureBallast-treated water

Concentration Survival% (mean±SD)

Dilution control 100±0.0

6.25 100±0.0

12.5 92±11.8

25 100±0.00

50 100±0.00

100 100±0.00

MSD 6% LOEC >100% (could not be determined)

NOEC 100%

Conclusion: No acute toxicity to B. plicatilis at any of the tested

concentrations was observe d. Although survival at 12.5% was below MSD value, the ANOVA followed by Dunnett’s

test indicated that the diffe rence was not significant. The

LOEC and actual NOEC could not be determined.

Quality assurance: B. plicatilis ; K 2 Cr 2 O 7 – Reference toxican t, tested 18-19 April, 2006. Dilution se ries: 1000, 560, 320, 180, 100 mg L -1 . Survival: 24-h LC 50 350 (PASS); 24-h LC 50 Range of Acceptability: 200 – 750 mg L -1 mg L -1

.

Test acceptability: Mortality did not exceed 10% in controls (Table 4).MEPC 55/2/5

ANNEX Page 38

I:\MEPC\55\2-5.doc 8.4 Test conducted: Rotoxkit MTM

, Brachionus plicatilis 48-h short-term toxicity test;

Test dates: 18-20 April, 2006;

Source/Age of organisms: MicroBioTests Inc ./<3 h old;

Test concentrations: see section 5.2;

Dilution/control water: see section 5.2;

Deviation from test protocol: Egg production in B. plicatilis exposed to the effluent for 48 h was determined in the same way as specified for

freshwater B. calyciflorus (Rotoxkit FTM). At the laboratory conditions during the tests, ag e at first egg production was 36.7±3.5 h and egg development time was 15.3±3.5 h. This implies that at after 48 h normally developing rotifers had their first clutch of eggs.

Test conditions: 1) Static, non-renewal; 2) 5 organisms per group; 3) multiwell testplates, 5 mL testvolume per group; 4) to ensure egg producti on, rotifers were provided with food ( Tetraselmis suecica , 10 5 cells mL -1 ) at the start; 5) temperature: 25±1 oC, darkness, no aeration; 6) pH: 6.2 – 7.2;

Test endpoints: egg production rate (=fecundity, egg female -1 );

Results: Table 5. Fecundity (egg female -1 ) of B. plicatilis exposed

for 48 h to PureBallast-treated water Concentration Fecundity (mean±SD)

Dilution control 1.07±0.09

6.25 1.00±0.16

12.5 1.00±0.16

25 1.00±0.16

50 0.93±0.18

100 1.07±0.09

MSD 32% LOEC >100% (could not be determined)

NOEC 100%

Conclusion: Test results indicated no toxicity to B. plicatilis reproduction at any of the tested concentrations. The LOEC

and actual NOEC could not be determined.

Quality assurance: see section 8.3;

Test acceptability: Mortality did not exceed 10% in controls.MEPC 55/2/5

ANNEX Page 39

I:\MEPC\55\2-5.doc 8.5 Test conducted: Artoxkit MTM

, Artemia sp

. 24-h acute toxicity test;

Test dates: 18-19 April, 2006;

Source/Age of organisms: MicroBioTests Inc./<4-h old;

Test concentrations: see section 5.2;

Dilution/control water: see section 5.3;

Deviation from test protocol: none;

Test conditions: 1) Static, non-renewal; 2) 5 organisms per group; 3) multiwell testplates, 5 mL testvolume per group; 4) no food during incubation; 5) temperature: 23±1 oC, darkness 6) pH: 6.2 – 7.2; 7) aeration: none;

Test endpoints: mortality (=immobility);

Results: Table 6. Survival (%) of juvenile Artemia exposed for 24 h

to PureBallast-treated water

Concentration Survival% (mean±SD)

Dilution control 91.7±11.8

6.25 100±0.0

12.5 91.7±11.8

25 100±0.0

50 100±0.0

100 91.7±11.8

MSD 30% LOEC >100% (could not be determined)

NOEC 100%

Conclusion: No acute toxicity to Artemia at any of the tested

concentrations was observed. The LOEC and actual NOEC

could not be determined.

Quality assurance: 1). Artemia ; K 2 Cr 2 O 7 – Reference toxican t, tested 19-20 April, 2006. Dilution se ries: 1000, 560, 320, 180, 100 mg

L -1 . Survival: 24-h LC 50 460 mg L -1 (PASS); 24-h LC 50 Range of Acceptability: 200 – 480 mg L -1

.

Test acceptability: Mortality did not exceed 10% in controls (Table 6).MEPC 55/2/5

ANNEX Page 40

I:\MEPC\55\2-5.doc References

ASTM (1993). Guide for conducting acute toxicity tests on aqueous effl uents with fishes, macroinvertebrates, and amphibians. Annual Book on ASTM Standards, pp. 866-882. Designation E1192-88, Phyladelphia, American Society for Testing and Materials, West Conshohocken, PA.

ASTM (1998). Standard guide for acut e toxicity test with the rotifer Brachionus . In: Allen RF (ed.) Annual Book on ASTM Standards, Section II – Water and Environmental Technology, pp. 837-843. American Society for Testing and Materials, West Conshohocken, PA.

Environment Canada (2005). Guidance document on statistical methods for environmental toxicity tests. Report EPS 1/RM/46. Met hod Development and Application Section, Environmental Technology Centre, E nvironment Canada, Ottawa, Ontario.

Guillard RRI and Ryther JH (1962). Studi es on marine planktonic diatoms. 1. Cyclotella nana Hustedt, and Detonula confervacia (Cleve). Can. J. Microbiol. 8: 229-239.

Hall JA and Golding LA (1998). Standard methods for whole e ffluent toxicity testing: development and application. Report no. MF E80205. NIWA report for the Ministry for the Environment, Wellington, New Zealand.

Snell TW and Moffat BD (1992). A 2-d life cycle test wi th the rotifer Brachionus calyciflorus

. Environ. Toxicol. Chem. 11: 1249-1257.

Ruck JG (1998) Toxkit technology and New Zealand Water Quality Guidelines. Environ. Toxicol. Water Qual. 13: 337-345.

USEPA (1994) Short-term Methods for Estimati ng the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, 2 nd ed., EPA/600/4-91/003. United States Environmental Protection Agency, E nvironmental Monitoring Systems Laboratory, Cincinnati, OH.

USEPA (2000) Method Guidance and Recomme ndations for Whole Effluent Toxicity (WET) Testing (40 CFR, Part 136). EPA/821/B00/004.United States Environmental Protection Agency, Office of Water ( 4303), NW Washington DC.

USEPA (2002) Methods for Measuring the Acute T oxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, 5 th ed., EPA-821-R-02-012. United States Environmental Protection Agency, Office of Water (4303T), NW Washington DC.

_____________________