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

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

kindly asked to bring their copies to meetings and not to request additional copies.

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 ORGANISMS IN BALLAST WATER 

Basic Approval of Active Substances used 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 – Ballast 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 International 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 approved by the Organization. 

2 The Procedure for approval of Ballast Water 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/Add.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 Working Group. In the meantime, a 

document on the proposal for approval containing all the non-confidential data related to the 

respecitve Active Substance needs 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 the data requirements of the Procedure (G9). 
MEPC 55/2/5 - 2 - 

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5 Sweden therefore submits the non-confidential part of the applicant’s dossier in the annex 

of this document to the Organization for evaluation according to the Procedure (G9). The 

complete dossier will be made available to the 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 proposal for approval and decide as appropriate. 

*** 
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ANNEX 

PROPOSAL FOR APPROVAL 

ALFA LAVAL BALLAST WATER MANAGEMENT SYSTEM - PureBallast

This report is structured in accordance with the 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 Ballast 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 organisms enter into the ballast water tanks and reduces 

sediment built-up in the tanks, which is a potential area for survival of organisms. The water is 

treated again at discharge to ensure that any potential 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 system, 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 Technology) Unit - A patented system product 

using naturally occurring photo-processes in the surface layer of sea-water. The 

Benrad AOT process acts on two levels: in-situ formation of reactive free radicals, 

and direct photo-effects. These two levels synergistically inactivate 

micro-organisms. A special UV light wavelength spectrum is used in combination 

with a catalyst which is based on TiO2. The TiO2 acts as a true catalyst and is not 

consumed in the process. No substances are added to the process. 

• Control and Auxiliary Equipment including sampling points - The control system 

and transformer control the system and supply all items with power. The auxiliary 

equipment controls the flow and measure different 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. 
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• Sampling points - Allows for accurate sampling before and after treatment system. 

• CIP (Cleaning in Place) - Support equipment for automatic 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 toxicity 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 supplied upon request to the IMO Technical Group 

(GESAMP-BWWG). 

All tests are performed with the following properties of the system: 

Specified flow rate 25 m3

/h 

A complete treatment cycle is two stage treatments which correspond to Ballast as well as 

Deballast. A complete treatment cycle is used for 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-extraction, see appendix 1. 

The following property is relevant for the full scale test system: 

Specified flow rate 250 m3

/h 

Description of the pilot scale test setup 

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

Flow rate 25 m3/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.
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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 process description, which has the action against 

Harmful Aquatic Organisms and Pathogens. 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 expressed 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 whether such a system required Active Substance 

approval.” However, the word “residual” seemed undefined to a number of delegations, which 

suggested that further consideration of this particular aspect should be given in due course. 

Despite that within a EU definition in which the use of TiO2 in combination with UV-light could 

be defined as a biocide, at the 21st Competent Authority meeting for the implementation of 

Directive 98/8/EC concerning the placing on the market of biocidal products (BPD) in 

April 2006, an agreement was reached among Member States, that the use of TiO2 as part of the 

Advanced Oxidation Technology shall not fall within 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 the treatment process, all “Relevant Chemicals” as 

defined by IMO is the potential transformation or reaction products in the treated water. 

The above interpretation of definitions indicates that the terms “Active Substance”, “Ballast 

Water Discharge”, “Preparation” and “Relevant Chemicals” all refers to the treated ballast water 

from the Benrad AOT process. This means that it is the treated water that is considered in this 

application and thus tested regarding toxicity and content of chemicals. 

3 PRINCIPLES 

No comment. 
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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, TiO2 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). TiO2 is also a widely used 

component in paints and cosmetic products. The action of reactive free radicals is of temporal 

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

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

NDRL/NIST Solution Kinetics Database 2005). 

The only environmental concern of the PureBallast treatment system might be attributed to the 

reaction products. To elucidate whether any environmental effects are to be expected, a series of 

chemical analysis and ecotoxicological tests of possible formation of toxic reaction products and 

their potential effects is relevant. Of primary interest would be the slight possibility of formation 

of halogenated organics or toxic oxidation products (for example bromate). 

A problem related to Ozone treatment of sea water 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 process formation of bromate, BrO3

-

, is unlikely to occur, since this would 

require multiple one-electron oxidation steps. Moreover, the utilization of photocatalysis is 

one of the attractive methods for the removal of BrO3

-

. The reduction of BrO3

-

 on a 

TiO2 photocatalyst under UV irradiation is a well-known reaction, and bromate has even been 

used as an additive to improve the oxidation efficiency of TiO2 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 produced 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 chemical properties, specifically for: 

• Chloro-organic compounds; 

• Bromo-organic compounds; 

• A general toxicity analysis (aggregate acute and chronic toxicity); 

• Bromate formation; 

• THM formation (Tri halo methane); and 

• Octanol/Water partition coefficient. 
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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 information on the properties or actions of the 

Preparation including any of its components as follows: 

.1 Data on effects on aquatic plants, invertebrates, fish, and other biota, including 

sensitive and representative organisms: 

• acute aquatic toxicity; 

No acute toxicity was observed. See report in appendix 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 juvenile development and reproduction in 

an invertebrate test organism were not found to be affected (see section 8.4 

in the Toxicity Bioassays Report). Moreover, no chemicals identified as 

potential endocrine affecting substances 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/biomagnification/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. 
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.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 and effect under aerobic and anaerobic 

conditions: 

• Modes of degradation (biotic; abiotic); 

The primary active species are of sufficient 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 will 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 changes 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 (Br2.-, CO3.-, Cl2.- radicals), hydrogen 

abstraction (hydroxyl, CO3.- radicals) or addition reactions 

(hydroxyl radicals). 
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All these radicals are naturally formed in the surfaces of (sea) 

water so any reaction products can be expected to be of a character 

similar to those of “natural” decomposition in water. It should be 

noted that the reactive free radicals will react indiscriminately 

forming an array of different reaction 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 affect physical properties of the 

water, see appendix 3. No potential 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 and Preparations and 

the treated ballast water, if applicable: 

This technology is a physical-chemical process and does not involve the addition 

of any substances. The in-situ generated free radicals have life times (half life) of 

micro or milli seconds and are consumed 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 the pKa or buffer properties of the 

treated water. 
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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 consumption of dissolved oxygen 

whereas it may increase slightly due to the formation of acidic 

groups and residual H2O2. To be investigated further. 

• autoignition temperature; and 

Not applicable. 

• other known relevant physical or chemical hazards. 

None at this date. 

.5 Analytical methods at environmentally relevant concentrations. 

4.2.2 A proposal for approval should include the above data set either for the Preparation or for 

each component separately, and a list of the name and relative quantities (in volumetric 

percentages) of the components 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 Preparations should be carried out in accordance with 

internationally recognized guidelines. 

4.2.4 The testing process should contain a rigorous quality control/quality assurance program 

consisting of: 

All analyses are done according to standard methods and toxicity testing is performed under 

supervision by the Department of Systems Ecology, Stockholm University. The testing 

laboratory is accredited by SWEDAC for chemical analysis of environmental samples and 

phytoplankton monitoring. Although the laboratory is not accredited specifically for 

toxicological testing, the Department 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 these plans, along with other guidance 
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documents and other general quality control information are available for 

download from the International Organization 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 management structure and policies of the 

Test Organization (including 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 chemicals can be submitted by the applicant to 

satisfy the required data needed for the evaluation 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 required dosage and retention time. 

4.2.7 A proposal for approval should include (Material) 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 test reports, the risk characterization and a consideration of the 

uncertainty associated with the assessment. 

The analysis performed is preliminary and will be complemented with further tests both with 

regards to toxicity as well as chemical properties of treated water. The preliminary test results 

found no measurable levels of potentially toxic substances and no acute or chronic effects of the 

full strength effluent on aquatic organisms. Therefore, no apparent environmental risks could be 

identified. 

All analyses are done according to standard methods and toxicity testing is performed under 

supervision by the Department of Systems Ecology, Stockholm University. The testing 

laboratory is accredited by SWEDAC for chemical analysis of environmental samples and 

phytoplankton monitoring. Although the laboratory is not accredited specifically for 

toxicological testing, the Department has 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. 

Further tests will be performed on full scale system using natural seawater according to the 

QAPP in appendix 1. 
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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 should be conducted (see Table 1 in section 6). 

.1 Persistence tests: 

Persistence should preferably be assessed 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 their expected lifetime”.

.2 Bioaccumulation tests: 

The assessment of the (potential for) bioaccumulation should use measured 

bioconcentration factors in marine (or freshwater) organisms. Where these tests 

are not applicable, or if logPow <3, Bio Concentration Factor (BCF) values may 

be estimated using (Quantitative) Structure-Activity Relationship ((Q)SAR) 

models. 

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. The octanol/water 

partitioning values do not suggest bioaccumulation/biomagnification/bioconcentration 

to occur. 

.3 Toxicity tests: 

Acute and/or chronic ecotoxicity data, ideally covering the sensitive life stages, 

should in principle be used for the assessment of the toxicity criterion. 

Acute and chronic tests covering survival 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 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. 

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 Discharge is that it integrates and addresses the 

potential for interactions of the Active Substances and Preparations with the possible 

by-products. 
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.1 For the basic approval process, the discharge testing should be performed in a 

laboratory using techniques and equipment to simulate Ballast Water Discharge 

following treatment by the Preparation. 

.2 For final approval, the discharge testing should be performed as part of the 

land-based type approval process using the treated ballast water discharge. 

5.2.2 The applicant should provide both acute and chronic toxicity test data using standardized 

test procedures to determine the toxicity of the Preparation and Relevant Chemicals as used in 

conjunction with the Ballast Water Management System. This testing approach should be 

performed on the treated Ballast Water Discharge, as the Ballast Water Management system 

could either mitigate or enhance the adverse effects 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 the 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 sensitive life-stage. The preference is to include both 

a sub-lethal endpoint (growth) and a survival endpoint. Either freshwater 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 organisms die (LCx), No Observed Adverse Effect 

Concentrations (NOAECs), chronic No Observed Effect Concentration (NOEC) and/or Effect 

Concentration at which x % of test organisms show effect (ECx), as appropriate based on the 

experimental design. 

5.2.6 A dilution series including a 100% ballast water 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 the dilution capacity would 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 multiple discharges into one location (even though 

this is not necessarily the case). 

The actual NOEC were not measurable; the statistically 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 holding time necessary to achieve the no adverse 

effect concentration upon discharge. Knowing the half-life (days), decay rate, dosage rate, 

volume of system and toxicity tests with time series, then a computational model can be used to 

determine the amount of time needed to hold the 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 preliminary risk characterization. 
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5.3.2 Both the Active Substance or Preparation as well as the treated Ballast Water Discharge 

should be subject to toxicity testing in order to protect the receiving environment from toxic 

effects due to discharges. 

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

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

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

environment. 

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

5.3.4 The rate of abiotic and biotic degradation of the Active Substances and Preparations 

under aerobic and anaerobic conditions should be assessed, resulting in the identification of 

relevant metabolites in the relevant 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 degradation of the Active Substances and Preparations 

under aerobic and anaerobic conditions should be assessed, resulting in the characterization of 

the persistence of the Active Substances, Preparations and Relevant Chemicals in terms of 

degradation rates under specified conditions (e.g. pH, redox, temperature). 

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

5.3.6 The partition coefficients (solids-water partition 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 chemicals and therefore this is not applicable. 

5.3.7 For Active Substances and Preparations, the 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 potential for bioaccumulation of 

the treated water was assessed by measurement of possible (if any) changes 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 toxicity testing there will be no residuals 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 Substances, Preparations and Relevant Chemicals is 

initially based on a dataset of acute and/or chronic ecotoxicity data for aquatic organisms, being 

primary producers (algae or sea grasses), consumers (crustaceans), predators (fish), and should 

include secondary poisoning to mammalian and avian top-predators, as well as data for sediment 

species. 
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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 potential (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 redundant if the potential of the substance of 

concern to partition into the sediment is low (e.g., Koc <500 L/kg). 

The partition into the sediment cannot exceed the 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, mutagenic and endocrine disruptive properties. If the 

screening results give rise to concerns, this should give rise to a further effect assessment. 

As no carcinogenic, mutagenic and endocrine disruptive substances were found in the analyzed 

water after the treatment, the assessment is not relevant. 

5.3.15 An analytical method suitable for monitoring Active Substances and Preparations in 

ballast water discharges should be available. 

As showed in the chemical and toxicity testing there 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 safety 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 technical 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 ensure 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 treatment unit, as described in section 4.1, headline 

“Radicals formed and their 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 with regards to ship and crew 

safety, see appendix 2. 
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6.3.2 For the protection of personnel involved in the handling and storage of the Active 

Substances and Preparations, the proposal should include relevant ((M)SDS). The Organization 

should evaluate (M)SDS, mammalian toxicity data and chemical properties hazards (see 

paragraphs 4.2.1.2 and 4.2.1.4) and ensure that potential hazardous properties of the Active 

Substances, Preparations or Relevant Chemicals 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 trade (e.g., ice, tropical, humidity, etc.). 

Not applicable. 

6.4 Environmental protection 

6.4.1 In order to approve the application, the Organization should determine that the Active 

Substances, Preparations or Relevant Chemicals are not Persistent, Bioaccumulative and Toxic 

(PBT). Preparations that exceed all these criteria (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 substances 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 aquatic 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 

LogPoctanol/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 assessment of PBT and the discharge with scientific knowledge of 

the Active Substances, Preparations and Relevant Chemicals concerned. The risk evaluation 

should qualitatively take into account 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. 
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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 Substances and Preparations should include 

information on their intended use and application. The quantity 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 number 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 maximum 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)SDS should describe 

appropriate storage and handling together with the effects of degradation 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 Labelling of Chemicals (GHS) and the relevant IMO 

regulations (e.g. the IMDG Code) and guidelines (e.g. the GESAMP Hazard Evaluation 

Procedure). Where these regimes are not applicable, relevant national 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 comply with the approval conditions such as 

maximum allowable concentration and maximum 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 involved, and the evaluating regulatory scientists, if 

any. 

8.1.2 Procedure to be followed: 

.1 The manufacturer should evaluate the Active Substances or Preparations and the 

potential discharge in accordance with the approval criteria specified in this 

procedure. 
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.2 Upon completion, the manufacturer should prepare an Application on the Active 

Substances and Preparations and submit it to the Member of the Organization 

concerned. 

.3 The Administration having received a satisfactory application should as soon as 

possible propose an approval to the Organization. 

.4 Members of the Organization may propose an approval. 

.5 The Organization should announce and set the time frame for the evaluation of 

Active Substances and Preparations. 

.6 Parties, Members of the Organization, the United Nations and its Specialized 

Agencies, intergovernmental organizations having agreements with the 

Organization and non-governmental organizations in consultative status with the 

Organization may submit information that is relevant to the evaluation. 

.7 The Organization should establish a Technical Group in accordance with its rules 

of procedure ensuring that proprietary data should be treated as confidential. 

.8 The Technical Group should review the comprehensive proposal along with any 

additional data submitted and report to the Organization whether the proposal has 

demonstrated a potential for unreasonable risk for environment, human health, 

property or resources in accordance with the 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 and its Specialized 

Agencies, intergovernmental organizations having agreements with the 

Organization and non-governmental organizations in consultative status with the 

Organization, prior to its consideration by the competent Committee. 

.10 The Committee of the Organization should decide whether to approve any 

proposal, introduce any modifications thereto, if appropriate, taking into account 

the Technical Group’s report. 

.11 The Member of the Organization that submitted the application to the 

Organization should inform in writing the applicant about the decision made with 

regard to the respective Active Substance or Preparation and their manner of 

application. 

.12 Active Substances or Preparations receiving basic approval by the Organization 

may be used for prototype or type approval testing based on the guidelines 

developed by the Organization. An Active Substance or Preparation may be used 

for Prototype or Type Approval testing for the approval of different BWMS 

without going through basic 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 received basic approval) 
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must be approved by the Organization. For this purpose, the Member of the Organization 

submitting an application should conduct the Type Approval tests in accordance with Guidelines 

for Approval of Ballast Water Management Systems. 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 received Basic Approval by the Organization may be used for evaluation 

of Ballast Water Management systems using Active 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 systems 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 specifications, 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 Substances and Preparations in the Ballast Water 

Management systems approved by the Organization, to the Member of the Organization. The 

Member of the Organization should inform the Organization accordingly. 

8.4.2 Manufacturers intending to significantly change any part of a Ballast Water Management 

System that has been approved by the Organization 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 approval in the following circumstances: 

.1 If the Active Substances and Preparations 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 materially 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 approval 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 Organization or observer to have been caused 

by the approved Ballast Water Management system that make use of Active 

Substances or Preparations. 
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9 CONCLUSION 

The PureBallast process uses UV-energy together with catalysts in order to produce free radicals 

which will react with and destroy the cell membranes of the microorganisms causing their death. 

The process simulates and enhances the natural process which occurs when the sunlight hits the 

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

can enter the ballast tanks. 

The chemical testing made before and after the treatment has not indicated any residual 

substances. Preliminary results from 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 further 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. 
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REFERENCES (FOR PUREBALLAST PROCESS DESCRIPTION) 

Ahmed S., Jones C.E., Kemp T.J. and Unwin P.R. (1999) “The role of mass transfer in solution 

photocatalysis at a supported titanium dioxide surface” Phys. Chem. Chem. Phys., (1):5229-5233 

Al-Ekabi H., Butters B., Delaney D., Ireland J., Lewis N., Powwell T. and Story J. (1993) 

“Photocatalytic purification and treatment of water and air” Ed. Ollis D.F. and Al-Ekabi H., 

Elsevier, Amsterdam, 321-335. 

Baxendale J.H. and Busi F. (1982) “The study of fast and transient 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 Hydrated Electrons, Hydrogen Atoms 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 photocatalysis” 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) “Stimulation of Artemia salina nauplius emergence 

under the action of gamma irradiation” Radiobiologiia:18(1):135-8. 

Kamat P.V. (1993) “Photochemistry on Nonreactive 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 degradation 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), National Institute of Standards and Technology, 

webresource http://kinetics.nist.gov/solution/index.php 

Nogueira F. et al. (1998) “Radio resistance studies in Methylobacterium” Radiat. Phys. Chem. 

52(1-6):15-19. 

Noguchi H., Nakajima A., Watanabe T. and Hashimoto K. (2003) “Design of a photocatalyst for 

bromate decomposition: Surface modification of TiO2 by pseudo-boehmite” Environ. Sci. 

Technol. 37:153-157. 
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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) “Hydrogen peroxide toward enhanced oxide growth on 

titaniumin PBS solution: Blue coloration and clinical 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 solution” The European Physical Journal A - Hadrons and Nuclei 

(Historical Archive), 138:411-431. 

Rijstenbil J.W. (2003) “Effects of UVB radiation and salt stress on growth, pigments and 

antioxidative defence of the marine diatom Cylindrotheca closterium” Mar. Ecol. Prog. Ser. 

254:37-48. 

Rijstenbil J.W. (2005) “UV- and salinity-induced oxidative effects in the marine diatom 

Cylindrotheca closterium during simulated emersion” Marine 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 processes at titanium dioxide surfaces: enhancing 

effects of surface modification with oxide-(sub) monolayers on photocatalytic activity” 

Encyclopedia of surface and colloid science, Marcel Dekker Inc., 4043-4058. 

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

Correlation of the Electronic Transition Energies of Iodine, Bromine, Chlorine, Hydroxyl, and 

Hydrogen Radicals with Their Electron Affinities” Journal of the American Chemical 

Society 97(7):1716-1721. 

Xiao-Ying Yu (2004) “Critical Evaluation of Rate Constants of Hydrogen 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 the rpoS gene on the survival of Salmonella 

typhimurium in sea water exposed to sunlight” Canadian Journal of Microbiology 50(5):341-350. 
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APPENDIX 1 

EXTRACTION FROM QAPP FOR 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 includes biological performance testing as well as 

toxicity testing for “Final Approval” 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 quality assurance and quality control activities 

during the evaluation of the Alfa Laval PureBallast 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) (MEPC 53/24/Add.1, annex 3). The requirements 

involve all parts and processes of the test as listed in: 

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

• Part 2 – Test and performance specifications 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 the determination of biological constituents 

in ballast water. 

• Section 4.1 – 4.8 (page 24-26). 

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

Substance (G9) 

• Section 1-5 (pages 1-7). 

The QAPP is a mean to reveal any problems before start-up and during execution of the project 

at as early stage as possible to minimize any potential procedural, technical 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, both preparations, execution of tests and reporting 

of results; 
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1.2.1 Preparations 

 Construction and preparation of the Test Site according to the IMO 

Guidelines (MEPC 53/24/Add.1) and advices and instructions from the US 

Coast Guard/ETV (Battelle Duxbury, 2004). 

 Installation and preparation of the Alfa Laval ballast water management 

system according to its operating manual. 

 Preparation of test waters according 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 management 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 toxicological analyses of treated test 

waters. 

 Performing chemical analyses. 

 Performing biological analyses. 

 Performing toxicological analyses. 

 Performing risk characterisation 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 improvements of products and services. 

• Quality manual for accredited laboratory analyses and tests. In August 

1993 NIVA’s Biological Laboratory was the first laboratory in Norway 

accredited by Norwegian Accreditation for ecotoxicological tests. At the 

same time NIVA’s Chemical Laboratory, which provides the widest scope 
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of analyses of environmental and 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 according to internationally 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 registered as a GLP laboratory in Norway 

(reg. no GLP 007) and is regularly inspected by the Norwegian Metrology 

and Accreditation Services. 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 effluents 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 responsibility 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 m3 test water by the 50 μm filter unit and the Benrad AOT Module of the 

PureBallast system transferring the test water from tank with prepared test water (WST1) to a 

temporary holding tank (TT1) before a second treatment by the Benrad AOT unit and transfer to 

a storage tank (TT2) for at least five days storage. A control cycle (red lines) is run by 

transferring 200 m3

 of the same type of prepared test water from WST2 to a second temporary 

holding tank (CT1) using the pump of the PureBallast system, but in by-pass of the treatment 

units before transfer to a second storage tank for at least five days storage (CT2). One cycle 

including one control will be run for each of the test waters (high and medium salinity). 
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WST1 

200 m3

TT1 

200 m3

TT2 

200 m3

CT2 

200 m3

CT1 

200 m3

S1 

S2, S3 

S5, S6 

50 μm filter 

Pump 

Benrad AOT Module 

WST2 

200 m3

S4 

Figure 1 Transfer of test water during 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 test waters (WST1 and WST2) is meeting the 

chemical and biological water quality criteria defined by IMO; 

.2 the efficiency of the Alfa Laval PureBallast 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 environmental toxicity of the water directly after 

treatment (at the time of deballasting). 

Sampling numbers are indicated by S1-S6 in Figure 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 – early, 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 sampling and transfer are described in detail in 

section 3. 
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Table 1. Parameters to be measured during the 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 (TSS) S1, S2, S3, S4, S5, 

S6 

Discrete grab Solbergstrand, 

NIVA Oslo 

Biological treatment performance parameters 

Organisms > 50 μm S1, S2, S3, S4, S5, 

S6 

Discrete grab Solbergstrand 

Organisms ≥10-50 μm 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 Oslo 
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4.4 Toxicity testing of treated ballast water 

The treated ballast water will be tested for chronic or sub-chronic toxicity using three test 

organisms representing algae, crustacean (copepods) and fish. The tests with copepods and fish 

involve long term exposure with frequent renewal. For these tests a batch of the treated ballast 

water will be stored for the time necessary to complete the tests. Changes of the composition of 

this sample that may occur during storage is considered as a simulation of changes that will occur 

after discharge of treated ballast water and, therefore, do not invalidate the tests for this particular 

purpose. The tests will be conducted for basic and final approval. 

4.4.1 Growth inhibition of marine algae 

The algae growth inhibition test will be performed according to International Standard 

ISO 10253: Water Quality – Marine algal growth inhibition 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 fluorescent tubes providing approximately 

75 μM 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 particle counter (Coulter Multisizer) after 24, 

48 and 72 hours. The growth rate of each culture is calculated and expressed as percentage of the 

growth rate of control cultures in pure seawater growth medium. If more than 50% growth 

inhibition is observed in the ballast water, the EC50 and the EC10 (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 concentration of ballast water. 

4.4.2 Reproduction of marine copepods 

A reproduction test covering the early life stages of the marine 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 exposed in ballast water and ballast water 

diluted in pure seawater for 13 days. 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 water 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 EC50 and the EC10 (i.e. the concentrations causing 50 and 10% effect, 

respectively) will be calculated using non linear regression analysis 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 three times per week. Fish are fed daily. The fish 

are weighed at the start and end of the test. Effects on growth rates at each treatment will be 
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compared to the control (exposed in pure seawater). If more than 50% growth inhibition is 

observed in the ballast water, the EC50 and the EC10 (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 concentration of ballast water. 

Reference: OECD 203 Modified for turbot juveniles, NIVA method K19. 
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APPENDIX 2 

 LLOYDS APPROVAL OF FULL SCALE PROTOTYPE SYSTEM ONBOARD M/V 

DON QUIJOTE 
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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 μg Cl/L 77 μg Cl/L 

Extractable organic halogens <2 μg/L <2 μg/L 

Chlorophenols < 0,01 μg/L < 0,01 μg/L 

Trihalomethanes 

Chloroform (CHCl3) 2 μg/L 1 μg/L 

Bromoform (CHBr3) <1 μg/L <1 μg/L 

Dichlorobromomethane (CHCl2Br) <1 μg/L <1 μg/L 

Dibromochloromethane (CHClBr2) <1 μg/L <1 μg/L 

Potential bioaccumulative substance <0 (Log Pow) <0 (Log Pow) 
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APPENDIX 4 

Alfa Laval 21-04-2006 

TOXICITY BIOASSAYS 

for Alfa Laval Ballast Water 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, 2006
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SUMMARY 

Laboratory 24, 48, and 72-h tests were conducted at Department of Systems Ecology, Stockholm 

University, testing aggregate toxic effects of artificial 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: survival (24-h test; algae, rotifers, 

Artemia), reproduction (48-h test, rotifers), and growth (72-h test; algae). A dilution series 

of 6.25%, 12.5%, 25%, 50% and 100% effluent was tested in each bioassay. Toxicity testing was 

conducted on April 18-21, 2006, to determine: (1) LOEC (the Lowest Observed Effect 

Concentration) for survival and reproduction; (2) the IC25 (the Inhibition Concentration of the 

effluent at which there is a 25% reduction in the growth), and (3) the NOEC (the No Observed 

Effect Concentration, or the highest concentration tested that did not exhibit any toxicity) for 

survival, reproduction, and growth. 

Test results indicated no acute or chronic toxicity to survival, growth or reproduction in any test 

organisms at any effluent concentrations as tested. The statistically determined LOEC and IC25

and actual NOEC were not measurable; the statistically determined NOEC was 100%. 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. 
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TEST REVIEW 

1 Laboratory accreditation 

Laboratory: Systems Ecology, Stockholm University, 

 Sv. Arrhenius v. 21A, SE-10691, 

 Stockholm, Sweden 

Accreditor: Swedish Board for Accreditation and Conformity Assessment 

(SWEDAC) 

Accreditation ID: 1449 

Category: testing laboratory (analyses of environmental 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 guides 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 collection of the large volume samples (10 L, 15 oC), 

whole water samples were taken as sub-samples for 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 toxicological tests included 3 representative 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-μm filter to remove algae used in the 

test system, and prepared for testing within 2 hours after collection. 

4 Tests organisms and assays 

Four species were used for the toxicity evaluation 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 for marine algal growth inhibition tests; 

ISO/CD 10253). The kit is supplied ready-to-use 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 1962), 20‰ artificial seawater (Instant OceanTM, 
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Aquarium Systems), supplied with constant aeration, illumination ~35 μmol 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 (MicroBioTests 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, eliminating the need for 

maintaining cultures and providing highly standardized 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, Crustacea). Artoxkit MTM 

(MicroBioTests Inc., Nazareth, Belgium), acute toxicity screening 24-h test for estuarine/marine 

environments (adheres to ASTM Standard Guide E1440-91). Hatching and test setup were 

conducted according to the manufacturer’s standard procedures. 

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

Test 

organism 

Protocol, 

source 

Organism 

type, size 

Assay Endpoint Summary 

section 

Phaeodactylum 

tricornutum

Algaltoxkit 

MTM 

Unicellular 

alga, 15-20 

μm 

Growth 

inhibition 

test 

Intrinsic growth 

rate (r, d-1) 

8.1 

Dunaliella 

tertiolecta

MFE80805: 

Appendix 1 

(NIWA, 

1998) 

Unicellular 

alga, 10-12 

μm 

Growth 

inhibition 

test 

Intrinsic growth 

rate (r, d-1) 

8.2 

Brachionus 

plicatilis

Rotoxkit 

MTM 

Rotifer, 

planktonic 

invertebrate, 

110-130 μm 

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 assayed 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 the checkpoint and were counted as eggs. Growth 

rate was calculated as: 
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r (d-1) = (ln Nt – ln No)/t, where Nt and No – 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 sample yielding 3 control 

replicates and 3 replicates for each effluent dilution tested. The test methods for acute toxicity 

evaluation followed standard toxicity methods (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 manuals (USEPA 1993, 1994, 2000). Dilution water 

used in all bioassays was control water obtained from the test system. 

6.3 Controls. As dilution water was different from the water used to culture algae and hatch 

zooplankters, an additional control was set with sterilized f/2 media that was routinely used as 

growth media in algal cultures (Guillard and Ryther 1962). Thus, the dual control system 

included a dilution water control and a culture water control that were run concurrently in all 

bioassays and underwent the same test conditions. 

7 Statistics 

Statistical evaluation was performed according to existing 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 differences between the 

controls were observed, the dilution 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 controls (Prism 4.0, GraphPad Software Inc.). 

Proportions (i.e. survival data) were arcsine square root transformed 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 toxicity to survival, growth or reproduction in any test organisms 

at any effluent concentrations as tested. The statistically determined LOEC and IC25 were not 

measurable; the statistically determined NOEC was 100%. Since the full strength effluent 

(100%) did not have any deleterious effects on any of the test species and chemical analyses 

found no measurable levels of potentially dangerous compounds (see Chemical Analysis Report, 

the effluent would not be expected to be toxic to aquatic organisms and have negative effects on 

ecological communities at environmentally relevant concentrations. 
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Test summaries 

8.1 Test conducted: Algaltoxkit MTM, 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 ·106

 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% 

IC25 >100% (could not be determined)

NOEC 100% 

Conclusion: Test results indicated no chronic toxicity to P. tricornutum

growth at any of the tested concentrations. The IC25 and 

actual NOEC could not be determined. 

Quality assurance: P. tricornutum; K2Cr2O7 – Reference toxicant, tested 18-21 

April, 2006. Dilution series: 1.8, 3.2, 5.6, 10, 18 mg L-1. 

Growth: 72-h IC25 3.8 mg L-1 (PASS); 72-h IC25 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. 
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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: 106

 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% 

IC25 >100% (could not be determined)

NOEC 100% 

Conclusion: Test results indicated no chronic toxicity to D. tertiolecta

growth at any of the tested concentrations. The IC25 and 

actual NOEC could not be determined. 

Quality assurance: 1). D. tertiolecta; K2Cr2O7 – Reference toxicant, tested 

18-21 April, 2006. Dilution series: 1.8, 3.2, 5.6, 10, 18 mg 

L-1. Growth: 72-h IC25 2.3 mg L-1 (PASS); 72-h IC25 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. 
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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 observed. Although survival at 12.5% 

was below MSD value, the ANOVA followed by Dunnett’s 

test indicated that the difference was not significant. The 

LOEC and actual NOEC could not be determined. 

Quality assurance: B. plicatilis; K2Cr2O7 – Reference toxicant, tested 18-19 

April, 2006. Dilution series: 1000, 560, 320, 180, 100 

mg L-1. Survival: 24-h LC50 350 (PASS); 24-h LC50 Range 

of Acceptability: 200 – 750 mg L-1 mg L-1. 

Test acceptability: Mortality did not exceed 10% in controls (Table 4). 
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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, age 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 production, rotifers were provided 

with food (Tetraselmis suecica, 105 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. 
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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; K2Cr2O7 – Reference toxicant, tested 19-20 

April, 2006. Dilution series: 1000, 560, 320, 180, 100 mg 

L-1. Survival: 24-h LC50 460 mg L-1 (PASS); 24-h LC50

Range of Acceptability: 200 – 480 mg L-1. 

Test acceptability: Mortality did not exceed 10% in controls (Table 6). 
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_____________________