### REPORT SNO 5667-2008 CONFIDENTIAL



# Land based testing of the PureBallast Treatment System of AlfaWall AB - approved test cycles 

# REPORT SNO 5667

Ȭ 2008 Confidential



# L and based testing of the PureBallast Treatment System of AlfaWall AB

Ȭȱ approved test cyclesNIVA 5667-2008



## Norwegian Institute for Water Research

– an institute in the Environmen tal Research Alliance of Norway 

# REPORT

Main Office Regional Office, Sørlandet Regional Office, Østlandet Regional Office, Vestlandet Regional Office Midt-Norge P.O. Box 173, Kjelsås Televeien 3 Sandv ikaveien 41 Nordnesboder 5 P.O. Box 1266 N-0411 Oslo, Norway N-4879 Grimstad, Norway N-2312 Ottestad, Norway N-5008 Bergen, Norway N-7462, Norway Phone (47) 22 18 51 00 Phone (47) 37 29 50 55 Phone (47) 62 57 64 00 Phone (47) 55 30 22 50 Phone (47) 73 54 63 85 / 86 Telefax (47) 22 18 52 00 Telefax (47) 37 04 45 13 Telefax (47) 62 57 66 53 Telefax (47) 55 30 22 51 Telefax (47) 73 54 63 87 Internet: www.niva.no

Title Land based testing of the PureBallast Treatment System of AlfaWall AB - approved test cycles Serial No. 5667-2008

Report No. Sub-No. O-26375 Date 2008

Pages Price

Author(s) Helge Liltved Christian Vogelsang August Tobiessen Tor Gunnar Jantsch Anne-Marie Bomo Topic group Water treatment

Geographical area Norway Distribution Confidential

Printed CopyCat

Client(s) AlfaWall AB, Tumba, Sweden Client ref. Carl Tullstedt Malin Westberg

Abstract

The PureBallast Treatment System, developed by AlfaWall AB, has been land based tested according to the IMOs Guidelines for approval of ballast water management systems (G8), Res. MEPC.126(53) Annex 3 and Procedure for approval of ballast water management systems that make use of active substances (G9) Res. MEPC.126(53) Annex 4. The tests were conducted at NIVA’s test site located at Solbergstrand 20 km south of Oslo. Test cycles were conducted with moderate and high salinity water. Each cycle lasted for a period of 5 days, and a total of 10 approved test cycles have been completed and the re sults of these test cycles are reported here.

4 keywords, Norwegian 4 keywords, English

1. Ballastvann 1. Ballast water

2. Toksikologi 2. Toxicology

3. Testing 3. Testing

4. AlfaWall AB 4. AlfaWall AB

Helge Liltved Jarle Nygaard
Project Manager Strategy Director ISBN 978-82-577-5402-0NIVA 5667-2008



# Preface

The tests were conducted in 2006 and 2007 at NIVAs test facility located at Solbergstrand 20 km south of Oslo. NIVA conducted the testing as a contract assignment for AlfaWall AB, with Det Norske Veritas (DNV) as verifier.

During planning and conducting of the tests, Malin Westberg and Carl Tullstedt were the lead representatives of AlfaWall AB, while Hanna Behrens, Egil Dragsund and Håkon Øv rum have represented DNV in the project. From NIVA, Anne-Marie Bomo, Oddbjørn Pettersen, Per Ivar Johannesen, August Tobiessen, Christia n Vogelsang, Tor Gunnar Jantsch and Helge Liltved represented were the main representatives. Several other AlfaWall AB and NIVA persone ll have been involved in the project.

I will use the opportunity to thank AlfaWall AB for choosing NIVA as the main partner in the process of testing and verification of the PureBallast Treatment System, and th ank all involved personell for the proffessionality and creativity demonstr ated in completing this innovative project.

Oslo, September 2008

Helge LiltvedNIVA 5667-2008



# Contents

Abbreviations and acronyms 7

Summary 8

1. Background 9

2. Materials and test protocols 10
2.1 Test site 10 2.2 The evaluated ballast water treatment system 10 2.2.1 General description of the PureBallast System 10 2.3 Test waters 10 2.3.1 Overview of requirements 10 2.3.2 Fulfilment of chemical water quality test criteria 11 2.3.3 Fulfilment of biological water quality test criteria 11 Harvesting of indigenous organisms 11 Cultivation of Artemia salina and Brachiomonas submarina 12 Bacteria 12 2.4 Test procedure 12 2.4.1 Description of test cycle 12 2.4.2 Measures to avoid cross-contamination during water transfer and sampling 13 2.5 Sampling 13 2.5.1 Assuring the representativeness of samples 13 2.5.2 Sampling protocols 14 2.5.3 Overview of sampling equipment 14 2.5.4 Sample preservation and transportation 15 2.6 Analyses 15 2.6.1 Chemical measurements 15 Temperature 15 pH 15 Dissolved oxygen (DO) 15 Salinity 15 Dissolved organic carbon (DOC) 16 Particulate organic carbon (POC) 16 Total suspended solids (TSS) 16 2.6.2 Identification and quantification of viable organisms ≥ 50 μm 16 2.6.3 Identification and quantificati on of viable organisms >10-50 μm 16 2.6.4 Identification and quantification of viable bacteria 17 Heterotrophic bacteria 17 Thermotolerant coliform bacteria 17 Enterococcus group 17 Vibrio sp. 17 2.6.5 Toxicity measurements of treated Ballast water 18

3. Results and discussion 20 3.1 QA/QC prosedures 20 3.2 Operational performance of the PureBallast system 20NIVA 5667-2008

3.3 Fulfillment of chemical water quality criteria 22 3.4 Fulfillment of the biological water quality criteria 28 3.5 Biocidal effects on organisms ≥ 50 μm in minimum diameter 32 3.6 Biocidal effects on organisms ≥ 10-50 μm in minimum diameter 32 3.7 Bactericidal effects 34 3.8 Ecotoxicological responses 35

Appendix A. Total project management reports 39

Appendix B. Chemical water quality preparation 39

Appendix C. Biological water quality preparation 39

Appendix D. Collection and preservation of samples 39

Appendix E. Logging of in situ measurements 39

Appendix F. Evaluation form for organisms >50 μm 39

Appendix G. Evaluation form for organisms ≥ 10-50 μm 39

Appendix H. Evaluation form for heterotrophic bacteria, coliforms, E. coli , Enterococcus group, intestinal Enterococci , and Vibrio cholerae 39

Appendix I. Acute and chronic toxicity of the green algae Skeletonema costatum 39

Appendix J. Acute and chronic toxicity of marine copepods 39

Appendix K. Acute and chronic toxicity of juvenile turbot 40

Appendix L. Operational performance 40NIVA 5667-2008



## Abbreviations and acronyms

A. salina – Artemia salina AlfaWall AOT Module – AlfaWall Advanced Oxidation Technology Module B. submarina – Brachiomonas submarina CFDA-AM - 5-carboxyfluorescein diacetate acetoxymethyl ester COD – chemical oxygen demand CT1 – temporary holding tank on control cycle CT2 – storage tank on control cycle DNV – Det Norske Veritas DO – dissolved oxygen DOC – dissolved organic carbon EC 50 , EC 10 - the concentrations causing 50 and 10 % effect, respectively, on the test organism E. coli – Escherichia coli FNU – Formazine Nephelometric Units GF/F – glass fiber filter grade F GLP – Good laboratory Practice IMO – International Maritime Organization ISO – International Organisation for Standardization LC 50 – the concentration causing 50 % mortality of the test organism n – number of measurements; in calculating the standard deviation NIVA – Norwegian Institute for Water Research NS-EN ISO – Norwegian, European and International Standard OECD – Organisation for economic Co-operation and Development PBTS - PureBallast Treatment System POC – particulate organic carbon PSU – Practical Salinity Unit (= ‰) QAPP – quality assurance project plan S1-S5 – sampling points 1-5 Std – standard deviation TCB – thermotolerant coliform bacteria TCBS – MacConkey and thiosulphate citrate bile salt agar TSS – total suspended solids TT1 – temporary holding tank on test cycle TT2 – storage tank on test cycle WST – tank with prepared test water X i - individual analytical result; in calculating the standard deviation X – the arithmetic mean of individual analytical results; in calculating the standard deviationNIVA 5667-2008

8 

# Summary

Land-based testing of the PureBallast Treatment Syst em (PBTS) of Alfa Wall AB has been completed in the period of November 2006 to May 2007. The testing has been conducted according to the IMOs Guidelines for approval of ballast water management systems (G8), Res. MEPC.126(53) Annex 3 and Procedure for approval of ballast water management systems that make use of active substances (G9) Res. MEPC.126(53) Annex 4 , hereafter referred to as G8 and G9. The tests were carried out at NIVA’s test site located at Solbergstrand 20 km south of Oslo, with moderate salinity and with high salinity water. Each cycle lasted for a period of 5 days, and a total of 10 approved test cycles have been completed and the results of these test cycles are reported here. All activities and collected data during testing of the PureBallast treatment system have been logged and reported separately (Appendix A-L).

The test water was prepared in a common 516 m 3 tank (WST). A combination of indigenous harvested organisms and cultured surroga te species (>50 μm group: Artemia salina ; 10-50 μm group: Tetraselmis suecica and Brachiomonas submarina ) and heterotrophic bacteria were added to fulfil the biological water quality criteria.

The test line involved consecutive treatment of at least 200 m 3 test water by the 50 μm filter unit and the PureBallast System transferring the test water from WST to a temporary holding tank (TT1 with a volume of 230 m 3 ) before a second treatment by the PBTS and transfer to a storage tank (TT2 with a volume of 205 m 3 ) for at least five days storage. The control line was run by transferring at least 200

m 3 of the same type of prepared test water fro m WST to a second temporary holding tank (TT1) using the pump of the PBTS, but in by-pass of the treatment units before transfer to a second storage tank (CT2 with a volume of 205 m 3 ) for a five days storage time. A ll operations were conducted with an average flowrate >200 m 3 /h.

In the 10 approved tests, the requirements regardi ng chemical composition were fulfilled in all test cycle, except test cycle 6 where the total suspende d solid (TSS) was slightly below the required >50 mg/l. The requirements regarding biological compositi on of the test water, and the biocidal effects of treatment and storage, were fulfilled in all 10 test cycles.

The ecotoxicity of treated ballast water was tested at least two times with 2 different water qualities (2 different salinities).The toxicity of undiluted (100 %) and diluted samples was determined on three trophic levels using marine acute and chronic tests w ith untreated control ballast water as control. No detectable toxicity was observed on the marine alga Skeletonema costatum (growth inhibition test), Nitocera spinipes (invertebrate reproduction test) or Scophtalmus mixmus (acute fish test and fish growth test). A mortality of 27% of the marine crustacean Acartia tonsa was observed at the highest tested concentration (100%) compared to 9% in th e control. It should be commented that high deathrate in the control can be a problem with the test, as also experienced in the current test. This deathrate can give misleading results. No effects were obser ved in the test with the other marine crustacean Nitocra spinipes . It is therefore doubtful that the effect observed can be attributed to toxicity. In the repeated test conducted 18 th Jan - 20 th Jan 2007, no mortality of Acartia was observed in treated ballast water at 100% concentration or lower. Lack of mo rtality >50 % at any test concentration precludes estimation of LC 50 . Treated waters in test cycle 3 were also sample d and analysed for organic halogen compounds (AOX), extractable organic halogen compounds (EOX), trihalomethanes (THM), potential bioaccumulating substances (PBS) and free and total residual oxi dants (TRO). All compounds, except AOX and PBS, were below the detection limits. However, the c oncentrations of AOX and PBS were not significant different from the concentrations found in control water.NIVA 5667-2008

9 Based on the conducted acute and chronic toxicity tests, and the chemical analysis, it is concluded that ballast water treated with the present ballast wate r technology do not provide hazard or risk to the marine ecosystem when released.



# 1. Background

The overall goal for AlfaWall AB is certificati on of the PureBallast Ballast Water Management System in accordance with the requirements in th e IMO Convention on ballast water management and underlying guidelines, including the Guidelines for approval of ballast water management systems (G8), Res. MEPC.126(53) Annex 3 and Procedure for approval of ballast water management systems that make use of active substances (G9) Res. MEPC.126(53) Annex 4 , hereafter referred to as G8 and G9.

Pilot tests were conducted, in June 2006, for partia l fulfilment of such a certification. The objective of the pilot testing was to verify the environmental performance characteristics of the PureBallast Pilot System technology relative to the biological a nd toxicological requirements stated in the IMO Guideline. By performing a pilottest, difficulties and “bottle-necks” could be detected, and measures could be taken to avoid problems during the full scale testing. The experience from the pilottest served as a fundament for large scale land-based testing of the technology.

Land-based testing for type approval, as reported he re, was conducted in the period of November 2006 to May 2007. The tests were conducted in accordance with G8 and G9.NIVA 5667-2008

10 

# 2. Materials and test protocols

2.1 Test site

The tests were conducted at NIVA’s test site located at Solbergstrand 20 km south of Oslo. Sea water is supplied from various depths down to 60 m in the Oslofjord, while fresh water is supplied from ground water bore holes or from a local creek.

The test facility includes 4 glass-fibre reinforced polyester tanks, supplied with inlet and outlet arrangements and equipment for proper cleaning. Th e two tanks for storage are covered to prevent light introduction. The surfaces of the test tanks ( TT1 and TT2) and the control tank (CT1) are coated with coatings for ships (Balloxy HB light, Jotun, Norway). Propeller devices with gentle rotation are mounted at the bottom and at shallow depth in WST and TT1, and at the bottom in TT2 and CT2. These propeller devices are used to suspend particl es, including algae and zooplankton, evenly and homogenise the content. A test is conducted to verify homogenisation prior to each test and prior to sampling.



## 2.2 The evaluated ballast wa ter treatment system

2.2.1 General description of the PureBallast System The PureBallast System from AlfaWall AB is a complete ballast water treatment system composing of three main component ballast water treatment system; 1) a 50 μm filter for removal of larger particles and organisms, 2) the AlfaWall AOT unit, a patented treatment technology using the synergetic effects of in situ produced free radicals and direct photo-radia tion to inactivate microbes and 3) control and auxiliary equipment including sampling points to c ontrol water flows and measure different alarm levels during operation as well as to take samples during operation and testing.



## 2.3 Test waters

Test water was prepared in a 516 m 3 tank (WST) from high salinity sea water from 60 meters depth or from brackish surface water depending on the require d salinity (>32 PSU or 3-32 PSU, respectively, with a minimum difference of 10 PSU). The 516 m 3 of test water was used for both testing and control. A combination of harvested indigenous or ganisms and cultured surrogate species (>50 μm: Artemia salina ; 10-50 μm: Tetraselmis suecica and/or Brachiomonas submarina and heterotrophic bacteria) were added to fulfil the biological water qua lity criteria, and soluble lig nin, starch and kaolin was added to adjust the contents of dissolved organic carbon (DOC), particulate organic carbon (POC) and total suspended solids (TSS), respectively, to w ithin the limits of chemical water quality criteria.



### 2.3.1 Overview of requirements Table 1 summarizes the chemical requirements of th e test waters used, while Table 2 summarizes the biological requirements.

Table 1. Required chemical water quality of test waters . Salinities should be separated by at least 10 PSU Salinity DOC POC TSS

Test water 1 >32 PSU >1 mg/l >1 mg/l >1 mg/l Test water 2 3-32 PSU >5 mg/l >5 mg/l >50 mg/lNIVA 5667-2008

11 Table 2. Required biological water quality in influent test water, treated water and in control after 5 days storage as stated in regulation G8 by IMO.

Organism group Influent water In treated water after 5 days storage In control after 5 days storage** ≥ 50 μm min. dimension Pref. 10 6 m -3

, ≥ 10 5 m -3 Min. 5 species from 3 diff. phyla/divisions <10 viable organisms
per m 3 > 10x <10 viable organisms per m 3

≥ 10-50 μm min. dimension 10 4 ml -1

, ≥ 10 3 ml -1 Min. 5 species from 3 diff. phyla/divisions <10 viable organisms
per m 3 > 10x <10 viable organisms per m 3

Heterotrophic bacteria ≥ 10 4 ml -1

Vibrio cholerae . - <1 cfu/100 ml - Escherichia coli - <250 cfu/100 ml - Intestinal Enterococci - <100 cfu/100 ml ≥ 10x <100 cfu/100 ml ** As discussed in the results and discussion section.



### 2.3.2 Fulfilment of chemical water quality test criteria Soluble lignin (40 % solution, Borrebond FP-P, Bo rregaard, Norway), starch (Maizena®, Unilever AS, Billingstad, Norway) and kaolin clay (VWR Inte rnational, Oslo Norway) was added to adjust the contents of dissolved organic carbon (DOC), par ticulate organic carbon (POC) and total suspended solids (TSS), respectively, to within the limits of ch emical water quality criteria described in Table 1. The necessary additions of each compound were calcula ted on the basis of earlier laboratory tests and full scale experience. Initially it was intended to u se rapid test kits to measure both DOC, POC and TSS right addition, however, the mandatory long heating time (2 hours) in sulphuric acid to break down organic compounds for the DOC and POC measurem ents disqualified this option due to logistic problems. Hence, only the TSS criteria were checke d by direct measurement on site, using the HACH method 8006 (HACH, 2001), applicable to samples with a TSS content within the range 0-750 mg/l, and the pre-set calibration standards of the Odyssey DR2500 spectrophotometer (HACH).



### 2.3.3 Fulfilment of biological water quality test criteria A combination of indigenous harvested orga nisms and cultured surrogate species (>50 μm: Artemia salina ; 10-50 μm: Brachiomonas submarina and/or Tetraselmis suecica ) and heterotrophic bacteria was added to fulfil the biological water quality criteria described in Table 2.



### Harvesting of indigenous organisms Fulfilment of the criteria regarding at least five speci es from three different phyla/divisions of both test groups of organisms ≥ 10 μm in minimum dimensions was assu red by harvesting indigenous algae and planktonic animal species from surface water outsi de Solbergstrand research station using a Unik Filter type 450 (Unik Filtersystems, Os, Norway ) equipped with a 20 μm mesh size screen. The harvesting process had earlier been s hown to be relatively gentle to the organisms; surface water (1 m depth) was smoothly pumped (ca. 3 m 3 /h) by a jet-pump to the inlet side of the screen, and algae and animals were washed from the screen into a collecting tray (ca. 100 l/h, e.g. approx. 30x conc.) and further to a storage tank. Water from the storag e tank was transported trough a pipe to WST by gravity. The viability and general conditions of harv ested organisms were evaluated by microscopy.NIVA 5667-2008

12 

### Cultivation of Artemia salina and Brachiomonas submarina The criteria of minimum concentration of organisms ≥ 50 μm in minimum dimension was fulfilled by adding cultured Artemia salina , and the equivalent criteria for organisms ≥ 10-50 μm in minimum dimension was fulfilled by adding cultured Brachiomonas submarina and Tetraselmis suecica.

A. salina : Resting cysts of Artemia salina are available commercially. Hatching of cysts are achieved by adding approximately 1 g of cysts to 1 litre of 20 ‰ salinity seawater. The culture is incubated with a bright light source at a temperature of 22-26 ° C with good aeration in the medium. Full hatching is usually achieved in 24-48 hours. It is possible to hatch approximately 300 000 nauplii per litre. Artemia nauplii are hatched with a supply of food (egg yolk) and will therefore live for up to a week without any external food supply. Survival length is twice that if the naupli is stored at 8-10 o C. However, hatched nauplii should be used within 24 hours in order to achieve high viability. B. submarina and Tetraselmis suecica was grown in seawater added a commercial fertilizer (Superba) 0,3 g/l. Cultures were grown in plexiglass sylinders with a diameter of 25 cm and height of 130 cm. We have been able to reach densities of 1-3x10 9 cells per 1 l.

The necessary cultivation volume was calcul ated based on a final density of 10 5 nauplii per m 3 for A. salina and 10 3 per ml for B. submarina and Tetraselmis suecica assuming a final volume of the test water of 516 m 3 . A critical point is the survival of the al gae during the 5 days storage in the control water rich in grazing organisms. Initially, tests were completed to estimate and verify the survival of algae in the control without grazers present, a sp ecified volume of the control water was sieved through a 50 μm mesh to remove the majority of grazing organisms and stored at the same temperature as the rest of the water and in darkn ess. Survival in specified volumes with and without grazers present was quantified to evaluate the infl uence of possible grazing effect during the 5 day storage period.



### Bacteria The concentration of heterotrophic bacteria in the surface water is normally exceeding the required ≥ 10 4 ml -1 . Natural heterotrophic bacteria were grown by adding growth media to a seawater sample. The cultivation volume was calculated based on a final density of 10 7 CFU/ml in the inoculum, the final volume of the test water (516 m 3 ).



## 2.4 Test procedure

2.4.1 Description of test cycle The water transfer pathways between tanks and the PureBallast System during each test cycle (including the control) is shown in Figure 1. Th e test line (blue lines) involved consecutive treatment of at least 200 m 3 test water by the 50 μm filter unit and the AlfaWall AOT unit transferring the test water from tank with prepared test water (WST) to a temporary holding tank (TT1) before a second treatment by the AlfaWall AOT unit and transfer to a storage tank (TT2) for at least five days storage. The control line (red lines) was run by transferring at least 200 m 3 of the same type of prepared test water from WST to the temporary holding tank (TT1) using the pump of the PureBallast system, but in by-pass of the treatment units before transfer to a second storage tank (CT2) for a five days storage time.NIVA 5667-2008

13 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-S5.

The test cycles were conducted with high salinity wa ter and moderate salinity water. Each cycle lasted for a period of 5 days.



### 2.4.2 Measures to avoid cross-contamination during water transfer and sampling

To avoid cross-contamination between consecutive test waters upon transfer between tanks, all pipelines and tanks were flushed for 2-3 min with sea water from 60 meters depth or ground water with documented quality between each test cycle, followed by rinsing with high temperature water (80-90 ° C).

The same holding tank (TT1) is used for both treated water and control water in the same test cycle. To avoid contamination, treated water is always in troduced to TT1 before control water. No crosscontamination will occure because the density of orga nisms is lower in treated water than in control
water.

To avoid cross-contamination during sampling, th e buckets, siphon overflow and plankton net were rinsed in sea water from 60 meters between each sampling.



## 2.5 Sampling

2.5.1 Assuring the representativeness of samples Before any samples were collected from any of the tanks at every sampling time the following procedure was carried out to assure that representative samples were withdrawn: 1) The particulate content of the 516 m3 tank (WST) was homogenized using an outboard propeller of 3 hp and an electric mixer (Brio m odel 1.0, 0.75 kW). The outboard propeller was mounted on the edge of the tank, while the electic mixer was mounted at the bottom of the tank. The outboard propeller could mix alternat ely between the periphery and centre of the tank. Mixers (Brio Model 1.0, 0.75kW) are also mounted in the TT2 and CT2 to provide homogenity before sampling. WST 516 m 3 TT1 231 m 3 TT2 205 m 3

CT2 205 m 3 S1 S2, S3

S4, S5 50 μm filter Pump Benrad AOT ModuleNIVA 5667-2008

14 2) The turbidity in different sections of each tank (upper part, middle part and bottom part, in both periphery and mid section) was measured by a handheld submersible probe (YSI – 600 OMS) during homogenization. When any turbidit y measurement was within a 10 % deviation from the average turbidity of all measureme nts in the tank, sampling was started.



### 2.5.2 Sampling protocols The following procedures were used to collect samples from the different tanks and sampling times. All sampling were done in triplicates.

1) Sampling of organisms ≥ 50 μm in WST (S1) and CT2 (S4 and S5). A plastic bucket was used to collect a 20 litre sample. The sampled water was slowly sieved through a plankton net (50 μm diagonal dimensions) attached to a plastic c up, collecting the organisms in the cup. 2) Sampling of organisms ≥ 50 μm in TT2 (S2 and S3): A siphon spillway was used to collect 1 m 3 test water from TT2 directly after transfer from TT1 and after 5 days storage. The water was sieved directly through a plankton net (50 μm di agonal dimensions) attached to a plastic cup, collecting and concentrating the organisms in the cup. 3) Sampling of organisms 10-50 μm in WST (S1), TT2 (S2 and S3) and CT2 (S4 and S5) : Organisms with a minimum diameter between 10 μm and 50 μm are sampled as 1000 ml grab samples with a clean glass bottle. 4) Sampling of bacteria in WST (S1), TT2 (S2 and S3) and CT2 (S4 and S5) : Bacterial samples were collected as 1000 ml grab samples by slowly submerging a 1000-ml sterile glass bottle. The bottle was closed immediately after sampling.



### 2.5.3 Overview of sampling equipment An overview of sampling equipment and containers used and sampled volumes are given in Table 3.

Table 3. Equipment and containers used for sampling and necessary and sampled volume for the individual parameters.

Parameter Sampling equipment Sample container Sample volume WST, CT2 Sample volume TT2 Temperature Temp. meter - - - pH pH probe - - - Dissolved oxygen DO probe - - - Salinity Probe - - - DOC Directly, grab Acid washed glass bottle 100 ml 100 ml POC Directly, grab Acid washed glass bottle 100 ml 100 ml TSS Directly, grab Clean plastic bottle 1000 ml 1000 ml Organisms ≥ 50 μm Sieving* 20 l 1 m 3 Organisms ≥ 10-50 μm Directly, grab Clean glass bottle 1000 ml 1000 ml Heterotrophic bacteria Coliform bacteria Enterococcus group bacteria Vibrio sp. Vibrio cholerae Directly, grab Sterile glass bottle 1000 ml 1000 ml

* A 20-litre grab sample (from WST and CT2) or 1 m 3 collected through a siphon spillway (from TT2) is concentrated to a volume of 40-100 ml through a plankton net with diagonal dimensions of 50 μm.NIVA 5667-2008

15 

### 2.5.4 Sample preservation and transportation Preservation methods and expected storage/holding ti mes before measurement are shown in Table 4. All preservation and analysis were in a ccordance with the described methodology.

Table 4. Preservation methods and expected storage/holdi ng times before measurement (ISO/CD 5667-3, 2001).

Parameter Preservation Max holding

time Expected storage

time Temperature - - 0 pH - - 0 Dissolved oxygen - - 0 Salinity - - 0 Dissolved organic carbon (DOC) Acidify to pH<2 with 1 ml 4 M H 2 SO 4 per 100 ml, stored at 4 o C 7 days 0-5 days

Particulate organic carbon (POC) Acidify to pH<2 with with 1 ml 4 M H 2 SO 4 per 100 ml,stored at 4 o C in darkness 7 days 0-5 days

Total suspended solids (TSS) Stored at 4 o C in darkness 24 hours <24 hours

Organisms ≥ 50 μm Stored at 4 o C in darkness 6 hours < 2 h Organisms ≥ 10-50 μm Stored at 4 o C in darkness 6 hours < 6 h Heterotrophic bacteria Thermotolerant coliform bacteria Enterococcus group bacteria Vibrio sp. Vibrio cholerae 4 o C in darkness 6 hours < 6 h

After sampling, samples were placed in the dark in cooling-containers and transported to the laboratories for analysis.



## 2.6 Analyses

2.6.1 Chemical measurements Temperature Temperature was measured in situ using a calibrated thermometer.

pH pH was measured in situ using a calibrated probe and pH-meter.

Dissolved oxygen (DO) Dissolved oxygen (DO) was measured in situ using a calibrated probe a nd meter. DO is reported as mg O 2 /l.

Salinity Salinity was measured in situ using a calibrated salioterm.NIVA 5667-2008

16 Dissolved organic carbon (DOC) DOC was measured by an accredited method based on Norwegian Standard NS-ISO 8245 (NIVA method G5-1) at NIVA after filtering the sample th rough a GF/F filter (0.7 μm). The sample was acidified with phosphoric acid and aerated with ox ygen to remove inorganic carbon prior to injection into a quartz tube filled with a platinum catalyser at 680 o C. The organic carbon compounds were oxidized to CO 2 which was quantified using an NDIR detect or (Phoenix 8000 TOC-TC analyser with sample carousel STS 8000) with oxygen as carri er. Detection limit was 0.5 mg C/l.

Particulate organic carbon (POC)

POC is calculated as the difference between the leve l of total organic carbon in the sample, measured on the non-filtered sample (see 4.2.1), and the measured DOC level of the same sample.

Total suspended solids (TSS) A nucleopore capillary filter (0,4 μm) was dried at 40-50 o C for 2 hours and the tara wes determined by weighing on a micro weight (Sartorius 4503 Micro) equipped with an ion source (Static Eliminator Bar Pu 210 Item no. LC 9793) removing static elec tricity. The sample was filtered by suction through the filter, which again was dried at 40-50 o C for 2 hours before being weighed on the micro weight. The TSS is represented by the weight increas e. Lowest reported value: 0.1 mg/l.

Trihalomethanes (THM) and other halogenated by-products THMs were extracted in pentane and measured by the use of a gas chromatograph (American Public Health Association, 1989). Other by-products, including adsorbable organic halogen compounds (AOX), extractable organic halogen compounds (E OX), potential bioaccumulating substances (PBS) and chlorophenols, were analysed in accordance with approved methods.



### 2.6.2 Identification and quantific ation of viable organisms ≥ 50 μm Counting of organisms ≥ 50 μm was performed by microscopy at 10-40x magnification within 6 hours of sampling. Viable organisms were counted a nd identified based on motility and integrity according to OECD (1985): OECD Test Guideline for Testing of Chemicals 202, “ Daphnia sp. acute immobilisation test and reproduction test”.



### 2.6.3 Identification and quantification of viable organisms >10-50 μm The viability of the micro-plankton (>10-50 μm) wa s determined by observing cells incubated with 5- carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) according to Ganassin et al. (2000). A 10 ml sample was incubated for 1 hour with 4 μmol of CFDA-AM. The sample was fixed with formalin and filtered onto black pol ycarbonate filters (25 mm). The filter was mounted on a glass slide in paraffin oil and frozen. CFDA-AM was hydrolysed only in a living cell. CFDA-AM is a marker for cell membrane integrity and may be measured dir ectly in cells. In principle, the non-fluorescent chemicals CFDA-AM is taken up in the cytosol, where it becomes hydrolysed into fluorescent end products. These end products are trapped inside the cellular compartment and may be observed in an epifluorescence microscope using excitation filter 485 nm and emission filter of 530 nm. In the epifluorescence microscope viable cells are observabl e as brightly yellow/green coloured cells, while non viable cells are pale green (heterotrophic cells) or pale green with red autofluorescence of the chloroplast (photoautotrophs). Numbers of viable cells are counted at a magnification of 300-480 times.

As a complementary method to direct counting for testing of viability, the serial dilution method in algal growth medium was used. The serial dilution method is often referred to as the most probable number method. It is simply based on the fact that by diluting your sample in a sequence and observing in which dilutions the organisms occur (g row) in afterwards, one is able to backward calculate the number of cells in the original samp le. The dilution series were achieved by adding 1 mlNIVA 5667-2008

17 of sample to 9 ml of media (algal growth media, 20 % Z8 seawater media). After mixing, 1 ml of this sample was further diluted with 9 ml. In this wa y a series of 10x dilution were made. The number of dilutions was set to cover the expected cell density range in the original sample. 3-5 parallels were employed in order to provide statistical significance of estimated number.



### 2.6.4 Identification and quantific ation of viable bacteria Generally the samples were diluted or concentrated to achieve a quantifyable concentration of colony forming units on a solid growth media (agar-medi um plates) or a medium-amended filterpillow. The dilution or concentration was based on experience and expectance of the concentration of the bacteria in question in the sample. Dilution was performed by stepwise 10x dilution of the sample in a dilution series followed by incubation on agar. Concentra tion was performed by filtering a predetermined sample volume through a sterile filter followed by incubation of the filter on growth media.

Heterotrophic bacteria Heterotrophic bacteria were quantified according to a modified version of Norwegian Standard NS-EN 6222/NS 4791 by growth on a marine agar at 22° C for two to three days.

Thermotolerant coliform bacteria Thermotolerant coliform bacteria (TCB) were qua ntified according to Norwegian Standard NS-EN ISO 9308-1 at a temperature of 44±0.2 °C and an incubation period of 18-24 hours.

Enterococcus group

Total fecal Enterococci were quantified according to Norwegian Standard NS-EN ISO 7899-2 at a temperature of 37±0.2 °C and an incubation period of 44 hours.

Vibrio sp.

Total Vibrio sp

. were quantified according to the method descr ibed by the American Public Health Association (APHA,1995). The total number of Vibrio sp., were determined by filtering of a 1-100 ml sample, the filter was placed on TCBS Cholera-me dium agar plates (CMO333 from Oxoid), incubated at 35 °C, and the colonies were counted 24 hours incubation. The medium supports the growth of pathogenic Vibrios ( Vibrio cholerae

, Vibrio parahaemolyticus) , some other Vibrios , and some other species (see table 5). Some strains of Aeromonas hydrophila grow producing yellow colonies but Plesimonas shigelloides does not usually grow well on TCBS.

Table 5. Colonial appearance of organisms on TCBS Medium after 24 hours incubation at 35°C. Organisms Colonies

Vibrio cholera and El Tor type Yellow, flat, 2-3 mm diameter. Vibrio parahaemolyticus Blue-green, 3-5 mm diameter. Vibrio alginolyticus Yellow, 3-5 mm diameter.

Vibrio metschnikovii Yellow, 3-4 mm diameter.

Vibrio fluvialis Yellow, 2-3 mm diameter.

Vibrio vulnificus Blue-green, 2-3 mm diameter.

Vibrio mimicus Blue-green, 2-3 mm diameter.

Enterococcus species Yellow, 1 mm diameter.

Proteus species Yellow-green, 1 mm diameter.

Pseudomonas species Blue-green, 1 mm diameter.NIVA 5667-2008

18 As indicated, only some, if any, of the colonies appear ing on the agar plates in the different test cycles will be identified as Vibrio cholera , and the possibility of further detecting of the serotypes O1 and O139 is very small or abscent since non of these ser otypes have been isolated from Norwegian coastal
water.

The strategy for elimination or identification of serotypes O1 and O139 was as follows:

The morphology of the colonies developing on the TCBS-medium after 24 h was visually studied. Colonies that had distinct colors and morphology different from Vibrio cholera were not selected for further identification. Colonies with typical Vibrio cholera appearance were re-striked on TCBS medium and again inspected for growth and mo rphology. Traditional classical elimination or identification methods were used, such as app earance in culture media and physiological and biochemical properties. If Vibrio cholera is identified, polymerase chain reaction (PCR) will be used for elimination or identification of the serotypes O1 and O139.



### 2.6.5 Toxicity measurements of treated Ballast water The following standard tests were pe rformed on the treated ballast water.

• Growth inhibition of the marine alga Skeletonema costatum according to ISO 10253: Marine algal growth inhibition test

• Reproductive toxicity to the marine crustacean Nitocra spinipes according to “ Forslag til Dansk Standard: Økotoksikologisk undersøgelse med krepsdyret Nitocra spinipes

. Reproduksjonsmetode”.

• Acute toxicity to the marine crustacean Acartia tonsa according to ISO 14669: Determination of acute lethal toxicity to marine copepods ( Copepoda, Crustacea ).

• Acute toxicity towards the juvenile turbot ( Scopthalmus maximus ) according to OECD Guidelines for testing of chemicals (No. 203; Fish, acute toxicity test).

• Chronic toxicity towards the juvenile turbot ( Scopthalmus maximus ) according to OECD Guidelines for testing of chemicals" (No. 215; Fi sh, juvenile growth test), adapted for marine species

Growth inhibition of the marine alga Skeletonema costatum The inhibitory effect of Treated Ballast water on the growth of the marine diatom Skeletonema costatum

, strain NIVA BAC1, has been inves tigated. The test was performed according to ISO 10253: Marine algal growth inhibition test with Skeletonema costatum and Phaeodactylum tricornutum.

Six replicate cultures of test algae in untreated and treated cultures were incubated on a shaking table at 20
± 2 ° C, under continuous illumination. Growth was mon itored by daily counting of cell numbers using a Coulter Multisizer.

The growth rate in each culture was calculated from the increase in cell density from 0-48 and 0-72 hours exposure. The analysis of difference betwee n treatments was based on the exponential part of the growth curve (0-48 hours).

Reproductive toxicity to the marine crustacean Nitocra Spinipes The reproductive toxicity of Treated Ball ast water to the marine crustacean Nitocra spinipes has been investigated. The test was performed according to Draft guideline for Danish Standard: ”Økotoksikologisk undersøgelse med krepsdyret Nitocra spinipe . Reproduksjonsmetode”. The test was performed as a Limit test with 100 % Treated Ballast water and using non treated ballast water as control water.NIVA 5667-2008

19 The test was performed with 20 replicate vessels with 1 gravid female in each vessel. The vessels were incubated for 14 days at 20 ° C. The number of alive offspring was then counted.

Acute toxicity to the marine crustacean Acartia tonsa The acute toxicity of Treated Ballast water to the marine crustacean Acartia tonsa has been investigated. The test was performed according to ISO 14669: Determin ation of acute lethal toxicity to marine copepods ( Copepoda, Crustacea ). The test concentrations were in the ra nge 32 to 100 % of Treated Ballast water.

The test was performed with four replicate vessels wi th 4-8 test animals for each test concentration and sixteen control replicate vessels. The v essels were incubated for 48 hours at 20 ± 1 ° C. Mortality was recorded after 24 and 48 hours.

Chronic toxicity towards the juvenile turbot ( Scopthalmus maximus ) The chronic toxicity of Treated Ballast Water to wards Turbot was tested in accordance with the "OECD Guidelines for testing of chemicals" (No. 215; Fish, juvenile growth test), adapted for marine fish. (ref 1).

The testing of chronic toxicity of Treated Ballast Water is required by the IMO G-8 guidelines for testing of treatment technology to be used aboard ships. The fish was exposed continuously for 28 days with water exchange 3 times per week.

The juvenile turbot was approximately 12 weeks old at the start of the test and had a weight in the range of 5 to 9 g. 20 fish were used in each a quarium with 20 l of medium. The temperature in the water was maintained in the range of 13,4 – 18,2 o C, and a continuous light was used.

The test water was taken directly from control and test tank (CT1 and TT2), after condition to test temperature

Acute toxicity towards the juvenile turbot ( Scopthalmus maximus ) The acute toxicity of Treated Ballast Water toward s Turbot was tested in accordance with the draft procedure of Phil McWilliams (Terra Environmental Laboratory A/S) (ref 1). The procedure follows the general guidelines of OECD 203 "Fish, Acute toxicity test" (ref 2).

The testing of acute toxicity of Treated Ballast Water is required by the IMO G-8 guidelines for testing of treatment technology to be used aboard ships. The fish was exposed continuously for 96 hours with full water exchange every day.

The juvenile turbot was approximately 12 weeks old at the start of the test and had a mean weight of 7.6 g. 20 fish were used in each aquarium with 20 l of medium. The temperature in the water varied in the range of 13.4-14.8 o C, and a light /dark cycle of 12:12 was u sed. The test water was taken directly from the control and test tank (CT1 and TT2 ), after conditioning to test temperatureNIVA 5667-2008

20 

# 3. Results and discussion

3.1 QA/QC prosedures

Quality assurance and quality control have been perfo rmed during the testing according to Chapter 5 in the QAPP and according to G8/G9. All activities and collected data during testing of the PureBallast treatment system have been logged as su mmarized in Table 6. For each activity a specially designed log in paper and/or electronic format h as been used, shown in Appendix A-L which have been reported separately, and was used for the respective quality assurance evaluation.

Table 6. Log protocols for all activities of the project. Appendix Description A Total project management B Chemical water quality preparation C Biological water quality preparation D Collection and preservation of samples E Logging of in situ measurements F Evaluation form for organisms >50 μm G Evaluation form for organisms ≥ 10-50 μm H Evaluation form for heterotrophic bacteria, coliforms, E. coli , Enterococcus group, intestinal Enterococci , and Vibrio cholerae. I Acute and chronic toxicity of the green algae Skeletonema costatum J Acute and chronic toxicity of marine copepods K Acute and chronic toxicity of juvenile turbot L Operational performance



## 3.2 Operational performance of the PureBallast system

The test cycles have been completed in the period of November 2006 to May 2007. The results of the 10 approved tests are reported here. The first 5 cy cles were conducted with high salinity water (>32 PSU) and the remaining with medium salinity (3-22 PS U). Each test cycle lasted for 5 days. The dates of the cycles and the time for start/stopp of each step in the test cycle are given in table 7.

A control for operation performance of the technology was performed in each test cycle by the site QA responsible person. Reports from these controls are included in Appendi x L. Check points and operational parameters for documenting sufficien t operation of the treatment technology were included and monitored in this quality control, incl uding start and stop of ballasting, deballasting, control ballasting and control deball asting. In addition, lamp function was checked, and flow rate were recorded during the tests cycles. Flow rate was read on the meter mounted on the effluent pipe from the treatment technology. Voltage was read to 150 V for each lamp on the voltmeters mounted on the treatment unit.NIVA 5667-2008

21 Table 7. T est cycles completed with dates and the time for start/stopp of each step in the cycle.

Test cycle Date Start/stopp ballasting

WST to TT1 Start/stopp deballasting

TT1 to TT2 Start/stopp control ballasting WST to TT1 Start/stopp control deballasting TT1 to CT2 Salinity PSU

1 1-6th Nov 06 14:53-16:00 67 min 16:40-17:40 60 min 17:55-19:00 65 min 19:15-20:15 60 min 32.2

2 8-13th Nov 06 12:24-13:30 66 min 14:01-15:00 59 min 15:18-16:22 64 min 16:35-17:35 60 min 32.6

3 22-27th Nov 06 12:10-13:08 58 min 13:22-14:21 59 min 14:49-15:45 56 min 15:46-16:35 49 min 32.6

4 29th nov4th des 06 11:41-12:35 54 min 13:09-13:59 50 min 14:17-15:15 58 min 15:18-16:10 52 min 32.7

5 6-11th Des 06 10:56-11:55 59 min 12:15-13:01 46 min 13:16-14:13 57 min 14:17-15:06 49 min 32.1

6 13-18th Des 06 11:35-12:33 58 min 12:55-13:45 50 min 13:54-14:49 55 min 14:54-15:43 49 min 20.9

7 10-15th Jan 07 11:43-12:41 58 min 13:01-13:50 49 min 14:05-15:01 56 min 15:07-15:52 45 min 21.5

8 17-22th jan 07 11:00-11:58 58 min 12:19-13:08 49 min 13:24-14:20 56 min 14:25-15:18 53 min 20.8

11 7-12th Mar 07 11:10-12:07 57 min 12:30-13:20 50 min 13:40-14:35 55 min 14:38-15:25 47 min 20.5

13 3-8th May 07 11:28-12:24 56 min 12:42-13:32 50 min 13:49-14:48 59 min 14:51-15:38 47 min 21.7

To determine the exact average flowrate during each operation, the time for start/stop and the volume of the tanks have been used to calculate the averag e flow as shown in table 8. In addition, flowrates were measured with a flowmeter and recorded on a regular basis during each test run.NIVA 5667-2008

22 Table 8. Calculated flowrate during each operation.

Test cycle Dates Average flow ballasting

m 3 /h Average flow deballasting

m 3 /h Average flow control

ballasting

m 3 /h Average flow control deballasting

m 3 /h 1 1-6th nov 06 202 205 209 205

2 8-13th nov 06 205 208 211 205

3 22-27th nov 06 233 209 241 251

4 29th nov4th des 06 250 246 233 237

5 6-11th Des 06 229 267 237 252

6 13-18th Des 06 233 246 246 251

7 10-15th Jan 07 233 251 241 273

8 17-22th jan 07 233 251 241 232

11 7-12th Mar 07 237 246 245 262

13 3-8th May 07 241 246 229 262



## 3.3 Fulfillment of chemical water quality criteria

The temperature, salinity, pH and dissolved oxygen content were measured in the test water tank (WST) prior to treatment and daily in the storage tanks TT2 and CT2 in each test cycle. Measurements were not conducted during week-ends. Th e results are given in Table 9.

As shown, the temperature in the initial water (WST) varied among the tests. Small decreases in oxygen concentration were recorded during the t est cycles. However, the oxygen content did never reach critical levels regarding survival of test organisms.NIVA 5667-2008

23 Table 9. Temperature, salinity and dissolved oxygen measur ements in the test water tank (WST) prior to treatment and daily in the storage tanks TT2 a nd CT2 for each test cycle (measurements were not conducted during week-ends). WST TT2 CT2 Day 0 0 1 2 3 4 5 0 1 2 3 4 5 Parameter Unit Test cycle 1 Temperature oC 9.8 9.8 8.80 8.30 7.40 7.20 6.90 - 7.90 7.50 6.70 6.50 6.30 Salinity PSU 32.2 32.2 32.0 31.8 31.8 31.9 31.9 - 32.0 31.9 31.9 31.9 32.0 pH 7.8 7.8 7.50 7.43 7.49 7.44 7.40 - 7.48 7.46 7.41 7.47 7.42 Dissolved O 2 % 85.0 85.0 83.10 78.10 78.60 73.60 72.80 - 84.30 73.20 76.50 71.10 70.40

Test cycle 2 Temperature oC 7.60 7.60 7.00 - - 5.70 5.40 - 6.70 - - 5.30 5.00 Salinity PSU 32.60 32.60 32.60 - - 32 .40 32.30 - 32.80 - - 32.60 32.30 pH 7.90 7.90 7.87 - - 6.80 7.88 - 8.01 - - 6.02 7.84 Dissolved O 2 mg/l 10.60 10.60 7.80 - - 7.80 6.28 - 6.68 - - 7.30 7.80

Test cycle 3 Temperature oC 12.30 12.50 12.80 11. 60 - - 10.60 13.00 11.50 11.10 - - 9.80 Salinity PSU 32.60 32.60 32.60 32.60 - - 32.60 32.60 32.60 32.60 - - 32.60 pH 8.03 8.04 8.01 7.91 - - 7.82 8.03 7.98 7.93 - - 7.87 Dissolved O 2 mg/l 9.90 11.00 8.90 8.70 - - 6.90 9.90 8.90 8.70 - - 6.80

Test cycle 4 Temperature oC 12.00 11.90 11.80 11. 70 - - 10.10 11.40 11.50 11.10 - - 9.80 Salinity PSU 32.70 32.70 32.70 32.80 - - 32.70 32.70 32.70 32.70 - - 32.70 pH 8.06 8.06 8.05 7.96 - - 7.75 8.08 8.08 7.96 - - 7.79 Dissolved O 2 mg/l 10.30 9.40 9.30 8.50 - - 5.10 9.60 9.10 8.50 - - 6.20

Test cycle 5 Temperature oC 10.60 11.80 10.70 10.10 9.80 9.30 9.00 10.90 10.10 10.00 9.30 8.80 8.80 Salinity PSU 32.10 32.10 32.10 32.20 32.20 32. 00 32.10 32.10 32.10 32 .20 32.10 32.10 32.10 pH 8.09 8.08 8.03 7.96 7.86 7.89 7.78 8.10 8.00 7.97 7.91 7.93 7.77 Dissolved O 2 mg/l 10.00 9.80 9.80 8.90 7.60 7.10 7.00 10.00 9.10 8.50 7.80 7.40 7.10

Test cycle 6 Temperature oC 6.6 7.4 8.0 7.5 - - 5.2 6.8 7.5 7.1 - - 4.8 Salinity PSU 20.9 20.9 20.9 20.9 - - 20.9 20.9 20.9 20.9 - - 20.9 pH 8.01 7.91 7.91 7.76 - - 7.47 7.96 7.88 7.78 - - 7.56 Dissolved O 2 mg/l 11.5 11.2 9.1 9.4 - - 7.4 11.4 9.3 9.7 - - 9.0

Test cycle 7 Temperature oC 4.7 5.4 5.5 4.8 - - 4 4.9 4.7 4.2 - - 3.7 Salinity PSU 21.5 21.6 21.4 21.4 - - 21.5 21.6 21.5 21.6 - - 21.5 pH 7.88 7.81 7.87 7.82 - - 7.63 7.84 7.89 7.8 - - 7.66 Dissolved O 2 mg/l 11.5 11.2 9.7 10.4 - - 10 11.2 11.1 11 - - 10

Test cycle 8 Temperature oC 4.9 5.9 4.9 4.6 - - 3.3 5.4 4.9 4.6 - - 2.6 Salinity PSU 20.8 20.8 20.8 20.8 - - 20.7 20.8 20.8 20.8 - - 20.8 pH 7.66 7.7 7.36 7.81 - - 7.54 7.67 7.32 7.67 - - 7.58 Dissolved O 2 mg/l 11.9 11.6 11.2 11.1 - - 10 11.1 11.4 11.3 - - 10.8

Test cycle 11 Temp. oC 4.2 5.8 5.4 5.8 - - 5.4 5.4 5.4 5.8 - - 5.0NIVA 5667-2008

24 Salinity PSU 20.5 20.2 20.0 20.2 - - 20.4 20.3 20.2 20.3 - - 20.5 pH 7.73 7.67 7.78 7.81 - - 7.75 7.75 7.82 7.82 - - 7.67 Dissolved O 2 mg/l 11.9 11.6 11.9 10.7 - - 10.6 11.9 11.5 10.5 - - 10.6

Test cycle 13 Temp. oC 10.7 12.6 17 .4 - - 16.8 13.2 12.6 17.4 - - 16.8 12.9 Salinity PSU 21.7 21.7 21.8 - - 21.7 21.8 21.7 21.8 - - 21.8 21.7 pH 8.01 7.97 7.95 - - 7.75 7.79 8.0 7.95 - - 7.75 7.78 Dissolved O 2 mg/l 11.3 10.8 10.51 - - 9.10 9.08 10.93 10.49 - - 9.05 8.61

Total suspended solids (TSS), dissolved organic carbon (DOC) and particulate organic carbon (POC) measurements at day 0 in WST, TT2 and CT2, and at day 5 in TT2 and CT2 in the different test cycles are shown in Table 10 – Table 19. The required leve l of TSS, DOC and POC in the initial test water (as defined in Table 1) are given in the line a bove the measured values for the influent water to treatment (WST). Green background indicates that re quired level was fulfilled, while red background indicates failure to fulfil required level. Compounds were added to ensure fulfilment of the requirements; both a POC regulating compound (POC Additive ) and a DOC regulating compound (DOC Additive ).

The required levels for total suspended solids ( TSS), dissolved organic carbon (DOC) and particulate organic carbon (POC) were fulfilled in all test cycles reported, except the TSS requirement in test 6 which was 10.4 mg/l lower than the required 50 mg/l.

Table 10. Test 1. Chemical water quality (average and st andard deviation of triplicate samples). TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 1 Average Stdev Average Stdev Average Stdev Additions to influent water - DOC Additive = 1.2 kg. POC Additive = 15.0 kg Required level >1 - >1 - >1 - Influent water (WST) 51.3 3.9 2.6 0.2 4.8 2.8 Treated day 0 (TT2) - - - - - - Treated day 5 (TT2) 6.4 - 3.5 - 0.4 - Control day 0 (CT2) - - - - - - Control day 5 (CT2) 1.6 - 3.2 - 0.0 -

Table 11. Test 2. Chemical water quality (average and st andard deviation of triplicate samples). TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 2 Average Stdev Average Stdev Average Stdev Additions to influent water - DOC Additive = 1.2kg POC Additive = 7.2 kg Required level >1 - >1 - >1 - Influent water (WST) 6.6 2.2 2.4 0.1 1.5 1.1 Treated day 0 (TT2) 5 - - - - - Treated day 5 (TT2) - 2.6 - 0.1 - Control day 0 (CT2) 8.8 - - - - - Control day 5 (CT2) 4.6 - 2.2 - 0.3 -NIVA 5667-2008

25 Table 12. Test 3. Chemical water quality (average and st andard deviation of triplicate samples). TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 3 Average Stdev Average Stdev Average Stdev Additions to influent water - DOC Additive = 1.2kg POC Additive = 4.8 kg Required level >1 - >1 - >1 - Influent water (WST) 16.5 0.9 2.0 0.1 2.9 0.1 Treated day 0 (TT2) 15.8 - 2.6 - - - Treated day 5 (TT2) - - 2.0 - 0.1 - Control day 0 (CT2) 15.0 - 2.1 - - - Control day 5 (CT2) - - 2.1 - 0.3 -

Table 13. Test 4. Chemical water quality (average and st andard deviation of triplicate samples). TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 4 Average Stdev Average Stdev Average Stdev Additions to influent water - DOC Additive = 1.2kg POC Additive = 7.2 kg Required level >1 - >1 - >1 - Influent water (WST) 18.1 1.3 3.7 0.9 3.0 1.8 Treated day 0 (TT2) 17.1 3.4 2.9 0.4 4.7 1.7 Treated day 5 (TT2) 2.0 0.1 2.1 0.4 Control day 0 (CT2) 10.1 6.0 3.7 0.1 4.5 4.7 Control day 5 (CT2) 2.0 0.1 1.3 0.4

Table 14. Test 5. Chemical water quality (average and st andard deviation of triplicate samples). TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 5 Average Stdev Average Stdev Average Stdev Additions to influent water - DOC Additive = 1.2kg POC Additive = 5.2 kg Required level >1 - >1 - >1 - Influent water (WST) 11.9 0.2 2.0 0.3 4.9 0.1 Treated day 0 (TT2) 11.2 0.2 2.6 0.4 4.4 0.2 Treated day 5 (TT2) 7.6 1.1 2.4 0.3 1.0 0.2 Control day 0 (CT2) 10.6 0.4 2.0 0.2 4.5 0.3 Control day 5 (CT2) 11.2 4.2 2.3 0.3 1.2 0.1

Table 15. Test 6. Chemical water quality (average and st andard deviation of triplicate samples). TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 6 Average Stdev Average Stdev Average Stdev Additions to influent water - DOC Additive = 6.0kg POC Additive = 20 kg Required level >50 - >5 - >5 - Influent water (WST) 39.6 0.7 5.1 0.1 14.7 1.1 Treated day 0 (TT2) 37.9 0.9 5.8 0.1 12.2 0.4 Treated day 5 (TT2) 11.8 6.8 5.9 0.2 Control day 0 (CT2) 38.9 2.7 5.1 0.1 14.8 0.8 Control day 5 (CT2) 14.3 1.0 4.9 0.1NIVA 5667-2008

26 Table 16. Test 7. Chemical water quality (average and st andard deviation of triplicate samples). TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 7 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 10.0kg DOC Additive = 10.0kg POC Additive = 22 kg Required level >50 - >5 - >5 - Influent water (WST) 61.6 2.1 6.2 0.1 17.1 0.1 Treated day 0 (TT2) 59.5 0.6 7.1 0.1 15.9 0.2 Treated day 5 (TT2) 25.7 0.3 6.5 0.1 6.5 0.4 Control day 0 (CT2) 59.6 0.7 6.2 0.1 17.0 0.4 Control day 5 (CT2) 22.5 0.5 6.2 0.1 5.0 0.5

Table 17. Test 8. Chemical water quality (average and st andard deviation of triplicate samples). TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 8 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 20.0kg DOC Additive = 10.0kg POC Additive = 14.1 kg Required level >50 - >5 - >5 - Influent water (WST) 71.7 1.2 5.5 0.1 12.7 0.5 Treated day 0 (TT2) 52.9 1.5 6.1 0.0 10.0 0.2 Treated day 5 (TT2) 5.4 0.1 3.2 0.4 Control day 0 (CT2) 65.5 0.2 3.8 1.6 12.4 2.0 Control day 5 (CT2) 5.4 0.2 3.1 0.3

Table 18. Test 11. Chemical water quality (average and st andard deviation of triplicate samples). TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 11 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 20.0kg DOC Additive = 12.0kg POC Additive = 14.5 kg Required level >50 - >5 - >5 - Influent water (WST) 67.1 1.0 7.1 0.1 10.6 2.7 Treated day 0 (TT2) 64.3 0.3 7.2 0.1 10.8 0.7 Treated day 5 (TT2) 29.2 1.3 6.7 0.2 3.7 0.4 Control day 0 (CT2) 65.0 0.3 7.1 0.3 11.7 1.1 Control day 5 (CT2) 26.0 1.5 6.4 0.2 4.5 0.7

Table 19. Test 13. Chemical water quality (average and st andard deviation of triplicate samples). TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 13 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 20.0kg DOC Additive = 12.0kg POC Additive = 14.5 kg Required level >50 - >5 - >5 - Influent water (WST) 117.7 1.5 7.3 0.1 12.2 0.4 Treated day 0 (TT2) 115.7 1.5 7.7 0.1 11.7 0.2 Treated day 5 (TT2) 27.6 0.2 6.8 0.1 3.8 0.2 Control day 0 (CT2) 117.7 2.3 7.2 0.1 12.0 0.4 Control day 5 (CT2) 9.4 1.0 6.7 0.1 2.3 0.5

Treated water and control water in test cycle 3 we re sampled and analysed for adsorbable organic halogen compounds (AOX), extractable organi c halogen compounds (EOX), chlorophenols and trihalomethanes (THM), a group of halogenated organic compounds. These compounds were analysed to clarify if the technology produces harm ful halogenated organic by-products.NIVA 5667-2008

27 The dominating THM normally found in oxidized seawat er is bromoform, which is a probable human carcinogen (USEPA 1996), known to bio-accumulate in aquatic animals. Bromoform is highly persistent in the environment.

No THMs were found in treated water. All the congene rs were below the detection limit, in both test waters sampeled (Table 20).

All other compounds, except AOX, were below the detection limit (Table 20). However, AOX was also detected in control water. The difference betw een the concentration in treated water and control water was not significant.

Influent water, control water and treated waters in test cycle 3 were also sampled and analysed for Potential Bioaccumulating Substances (PBS) in acco rdance with OECD 117. There were no difference in PBS-level between treated and control water. A value of 1.4 was measure in both waters.

Table 20. Concentration of AOX, EOX, chlorophenols and tr ihalomethanes in treated water from Test 3 in control water and treated water immediately after treatment. Unit Control water Treated water AOX mg/l 0.45 0.57 EOX mg/l <0.050 <0.050 2-Monochlorophenol μg/l <0.10 <0.10 3-Monochlorophenol μg/l <0.10 <0.10 4-Monochlorophenol μg/l <0.10 <0.10 2,3-Dichorophenol μg/l <0.10 <0.10 2,4+2,5-Dichlorophenol μg/l <0.20 <0.20 2,6-Dichlorophenol μg/l <0.10 <0.10 3,4-Dichlorophenol μg/l <0.10 <0.10 3,5-Dichlorophenol μg/l <0.10 <0.10 2,3,4-Trichlorophenol μg/l <0.10 <0.10 2,3,5-Trichlorophenol μg/l <0.10 <0.10 2,3,6-Trichlorophenol μg/l <0.10 <0.10 2,4,5-Trichlorophenol μg/l <0.10 <0.10 2,4,6-Trichlorophenol μg/l <0.10 <0.10 3,4,5-Trichlorophenol μg/l <0.10 <0.10 2,3,4,5-Tetrachlorophenol μg/l <0.10 <0.10 2,3,4,6-Tetrachlorophenol μg/l <0.10 <0.10 2,3,5,6-Tetrachlorophenol μg/l <0.10 <0.10 Pentachlorophenol μg/l <0.10 <0.10 Trichloromethane (chloroform) μg/l <0.30 <0.30 Tribromomethane (bromoform) μg/l <0.20 <0.20 Dibromochloromethane μg/l <0.10 <0.10 Bromodichloromethane μg/l <0.10 <0.10

Treated waters in test cycle 4 were also sampled and analysed for free and total residual oxidants (TRO) by the DPD-colourimetric method (Hach method 8021 for free chlorine and Hach method 8167). The results show that it is not possible to de tect any production of residual oxidants measured as free or total chlorine in the treated water (table 21).NIVA 5667-2008

28 Table 21. Concentration of free and total residual oxidants in treated water from Test 4 in influent water just prior to treatment, and control wate r and treated water immediately after treatment. Unit Influent water Control water Treated water Free chlorine Parallell 1 mg/l -0.04 0.03 0.02 Parallell 2 mg/l 0.00 0.01 0.01 Parallell 3 mg/l 0.05 0.02 0.02 Parallell 4 mg/l 0.02 - - Parallell 5 mg/l 0.00 - - Average± Standard deviation 0.01±0.03 0.02±0.01 0.02±0.01 Total chlorine Parallell 1 mg/l 0.00 0.00 0.01 Parallell 2 mg/l 0.01 0.01 0.01 Parallell 3 mg/l 0.04 0.02 0.02 Parallell 4 mg/l 0.02 - - Parallell 5 mg/l 0.02 - - Average± Standard deviation 0.02±0.01 0.01±0.01 0.01±0.01



## 3.4 Fulfillment of the biological water quality criteria

The initial test waters’ content of organisms should comply with the requirements as given in Table 2. Results from quantitative measurements of the initia l test waters are given in Table 22. Green background in table cell indicates that required level was fulfilled.

Organisms ≥ 50 μm in minimum diameter The requirements regarding density of the ≥ 50 μm group was met in all test cycles. The requirements regarding the biological diversity within the population were fulfilled.

Organisms ≥ 10-50 μm in minimum diameter The quantifications of individuals in the ≥ 10-50 μm group were based on growth in dilution series and microscope counts of CFDA-AM stained cells. In all tests, the requirements regarding influent density of the ≥ 10-50 μm group was met. The requirements regarding the biological diversity within the population were also fulfilled.

Heterotrophic bacteria The only requirement regarding bacteria in influent water is that heterotrophic bacteria should be present in a density of ≥ 10 4 ml -1 . This requirement was fulfilled in all tests as shown in Table 22.

The IMO G-8 guideline 2.3.20 also specifies that “...th e following bacteria do not need to be added to the influent water, but should be measured at the influent and at the time of discharge: 1. Coliform; 2. Enterococcus group; 3. Vibrio cholerae and 4. Heterotrophic bacteria.” All these bacteria groups were measured in the influent water and the results are given in Table 22. Vibrio cholerae was not detected in any of the test cycles.NIVA 5667-2008

29 Table 22. Initial content of organisms within the defined test organism groups (ref. Table 2) in the test water. Green background indicates th at required level was fulfilled. Test organism Method Influent Requirement Test 1 Microscope counts 12.3 ± 2.7 x 10 4 m -3 ≥ 10 5 m -3 Phyla 5 ≥ 3 different Organisms
≥ 50 μm Species >7 ≥ 5 different 1700 ml -1 Dilution method 95 % conf. interval 700-4800 ml -1 Microscope counts 1213 ± 41 ml -1 ≥ 1000 ml -1

Phyla 5 ≥ 3 different Organisms ≥ 10-50 μ m Species >9 ≥ 5 different Hetero. bact. Bacterial counts 4.2 ± 1.4 x 10 5 ml -1 ≥ 10 4 ml -1
Coliform bacteria Bacterial counts 3 ± 2 x 10 1 /100 ml - Vibrio sp. Bacterial counts >100/100 ml - Vibrio cholerae Bacterial counts, elimination method Not detected - Enterococcus group Bacterial counts 2 ± 0 x 10 3 /100 ml - Test 2 Microscope counts 16.0 ± 3.7 x 10 4 m -3 ≥ 10 5 m -3 Phyla 4 ≥ 3 different Organisms
≥ 50 μm Species >5 ≥ 5 different 3000 ml -1 Dilution method 95 % conf. interval 1000-13000 ml -1 Microscope counts 1351 ± 156 ml -1 ≥ 1000 ml -1

Phyla 4 ≥ 3 different Organisms ≥ 10-50 μ m Species >8 ≥ 5 different Hetero. bact. Bacterial counts 1.4 ± 0.53 x 10 5 ml -1 ≥ 10 4 ml -1
Coliform bacteria Bacterial counts 2.6 ± 1.2 x 10 2 /100 ml - Vibrio sp. Bacterial counts >100/100 ml - Vibrio cholerae Bacterial counts, elimination method Not detected - Enterococcus group Bacterial counts 1.2 ± 0.41 x 10 3 /100 ml - Test 3 Microscope counts 21.5 ± 1.9 x 10 4 m -3 ≥ 10 5 m -3 Phyla 6 ≥ 3 different Organisms
≥ 50 μm Species >5 ≥ 5 different 3000 ml -1 Dilution method 95 % conf. interval 1000 - 13000 ml -1 Microscope counts 1409 ± 30 ml -1 ≥ 1000 ml -1

Phyla 3 ≥ 3 different Organisms ≥ 10-50 μ m Species >9 ≥ 5 different Hetero. bact. Bacterial counts 1.6 ± 0.21 x 10 6 ml -1 ≥ 10 4 ml -1
Coliform bacteria Bacterial counts 3.7 ± 1.2 x 10 1 /100 ml - Vibrio sp. Bacterial counts 2.3 ± 0.50 x 10 4 /100 ml - Vibrio cholerae Bacterial counts, elimination method Not detected - Enterococcus group Bacterial counts 4.3 ± 0.70 x 10 1 /100 ml - Test 4 Microscope counts 12.6 ± 9.7 x 10 4 m -3 ≥ 10 5 m -3 Phyla 4 ≥ 3 different Organisms
≥ 50 μm Species >8 ≥ 5 different 2400 ml -1 Organisms Dilution method 95 % conf. interval 1000 - 9500 ml -1 ≥ 1000 ml -1NIVA 5667-2008

30 Microscope counts 1691 ± 212 ml -1 Phyla 3 ≥ 3 different ≥ 10-50 μ m

Species >8 ≥ 5 different Hetero. bact. Bacterial counts 6.9 ± 1.0 x10 5 ml -1 ≥ 10 4 ml -1
Coliform bacteria Bacterial counts 500 ± 265 /100 ml - Vibrio sp. Bacterial counts 5.3 ± 4.6 x 10 5 /100 ml - Vibrio cholerae Bacterial counts, elimination method Not detected - Enterococcus group Bacterial counts 2.5 ± 1.9 x 10 3 /100 ml - Test 5 Microscope counts 16.2 ± 2.4 x 10 4 m -3 ≥ 10 5 m -3 Phyla 6 ≥ 3 different Organisms
≥ 50 μm Species >12 ≥ 5 different 1700 ml -1 Dilution method 95 % conf. interval 700 - 4800 ml -1 Microscope counts 1411 ± 89 ml -1 ≥ 1000 ml -1

Phyla 3 ≥ 3 different Organisms ≥ 10-50 μ m Species >10 ≥ 5 different Hetero. bact. Bacterial counts 6.5 ± 0.9 x 10 5 ml -1 ≥ 10 4 ml -1
Coliform bacteria Bacterial counts 6070 ± 306 /100 ml - Vibrio sp. Bacterial counts 3.6 ± 3.4 x 10 5 /100 ml - Vibrio cholerae Bacterial counts, elimination method Not detected - Enterococcus group Bacterial counts 337 ± 293 /100 ml - Test 6 Microscope counts 17.1± 1.5 x 10 4 m -3 ≥ 10 5 m -3 Phyla 8 ≥ 3 different Organisms
≥ 50 μm Species >10 ≥ 5 different 5000 ml -1 Dilution method 95 % conf. interval 2000 - 20000 ml -1 Microscope counts 2199 ± 173 ml -1 ≥ 1000 ml -1

Phyla 7 ≥ 3 different Organisms ≥ 10-50 μ m Species >11 ≥ 5 different Hetero. bact. Bacterial counts 3.4 ± 1.5 x 10 5 ml -1 ≥ 10 4 ml -1
Coliform bacteria Bacterial counts 1970 ± 153 /100 ml - Vibrio sp. Bacterial counts 2.3 ± 1.4 x 10 5 /100 ml - Vibrio cholerae Bacterial counts, elimination method Not detected - Enterococcus group Bacterial counts 43 ± 15 /100 ml - Test 7 Microscope counts 15.4 ± 1.0 x 10 4 m -3 ≥ 10 5 m -3 Phyla 5 ≥ 3 different Organisms
≥ 50 μm Species >6 ≥ 5 different 2400 ml -1 Dilution method 95 % conf. interval 1000 - 9500 ml -1 Microscope counts 2266 ± 336 ml -1 ≥ 1000 ml -1

Phyla 4 ≥ 3 different Organisms ≥ 10-50 μ m Species >6 ≥ 5 different Hetero. bact. Bacterial counts 2.6 ± 1.4 x 10 5 ml -1 ≥ 10 4 ml -1
Coliform bacteria Bacterial counts 2730 ± 321 /100 ml - Vibrio sp. Bacterial counts 3.7 ± 2.5 x 10 3 /100 ml - Vibrio cholerae Bacterial counts, elimination method Not detected - Enterococcus group Bacterial counts 31 ± 7 /100 ml - Test 8NIVA 5667-2008

31 Microscope counts 14.7 ± 1.3 x 10 4 m -3 ≥ 10 5 m -3 Phyla 4 ≥ 3 different Organisms
≥ 50 μm Species >6 ≥ 5 different 3000 ml -1 Dilution method 95 % conf. interval 1000 - 1300 ml -1 Microscope counts 2558 ± 132 ml -1 ≥ 1000 ml -1

Phyla 4 ≥ 3 different Organisms ≥ 10-50 μ m Species >10 ≥ 5 different Hetero. bact. Bacterial counts 1.6±0.9 x 10 4 ml -1 ≥ 10 4 ml -1
Coliform bacteria Bacterial counts 1130 ± 493 /100 ml - Vibrio sp. Bacterial counts 5.0±0.5 x 10 3 /100 ml - Vibrio cholerae Bacterial counts, elimination method Not detected - Enterococcus group Bacterial counts 66 ± 37 /100 ml - Test 11 Microscope counts 13.8 ± 1.8 x 10 4 m -3 ≥ 10 5 m -3 Phyla 5 ≥ 3 different Organisms
≥ 50 μm Species >9 ≥ 5 different Dilution method 95 % conf. interval 5000 ml -1 2000 – 20000 ml -1 ≥ 1000 ml -1

Microscope counts 2795 ± 87 ml -1 Phyla 3 ≥ 3 different Organisms ≥ 10-50 μ m Species 10 ≥ 5 different Hetero. bact. Bacterial counts 2.9 ± 1.71 × 10 4 / ml ≥ 10 4 ml -1
Coliform bacteria Bacterial counts 3.7 ± 1.5 / 100 ml - Vibrio sp. Bacterial counts 4.8 ± 2.4 × 10 4 /100 ml - Vibrio cholerae Bacterial counts, elimination method Not detected - Enterococcus group Bacterial counts 1.2 ± 0.3 × 10 3 / 100 ml - Test 13 Microscope counts 24.7 ± 1.5 x 10 4 m -3 ≥ 10 5 m -3 Phyla 3 ≥ 3 different Organisms
≥ 50 μm Species >9 ≥ 5 different Dilution method 95 % conf. interval 3000 ml -1 1000-13000 ml -1 ≥ 1000 ml -1

Microscope counts 1576 ± 147 ml -1 Phyla 4 ≥ 3 different Organisms ≥ 10-50 μ m

Species >10 ≥ 5 different Hetero. bact. Bacterial counts 5.1 ± 1.1 × 10 4 / ml ≥ 10 4 ml -1
Coliform bacteria Bacterial counts > 8 × 10 3 /100 ml - Vibrio sp. Bacterial counts 3.9 ±3.9 × 10 3 /100 ml - Vibrio cholerae Bacterial counts, elimination method Not detected - Enterococcus group Bacterial counts > 8 × 10 3 / 100 ml -NIVA 5667-2008

32 

## 3.5 Biocidal effects on organisms ≥ 50 μm in minimum diameter

The number of viable organisms ≥ 50 μm in minimum diameter, as determined on the basis of motility and integrity by microcope examination, in treated test water and control immediately after treatment and after five days of storage is given in Table 23. For the performance of the ballast water treatment system to pass the regulation D-2 of the IMO Guidelines, as given in Table 2, less than 10 viable organisms ≥ 50 μm in minimum diameter per m 3 should be present in the treated water after five days storage.

As can be seen in Table 23 and indicated by th e green background colour in the table cells, this requirement was fulfilled in all test reported.

The equivalent requirement for the non-treated control test water is stated as: “If in any test cycle the average discharge results from the control water is a concentration less than or equal to 10 times the values in Regulation D-2.1, the test cycle is invalid.” It may be interpreted that the minimum level of viable organisms in the control test-water at the time of discharge (e.g. after 5 days storage) should be higher than 100 m -3 . This requirement was fulfilled in all tests.

Table 23. Viable organisms ≥ 50 μm in minimum diameter in treated test water and control immediately after treatment and after five days of storage. Green backgroun d indicates that required level was fulfilled. Treated water Control water Day 0 Day 5 Day 0 Day 5 Organisms ≥ 50 μm in minimum diameter (individuals/m 3 ) Requirement - <10 - >100 Test cycle 1 0 ± 0 0 ± 0 5.6 ± 2.4 x 10 3 1.7 ± 0.5 x 10 3 Test cycle 2 0 ± 0 0.3 ± 1.0 7.8 ± 1.7 x 10 3 1.0 ± 0.3 x 10 3 Test cycle 3 1.3 ± 0.6 0.3 ± 1.0 4.3 ± 1.4 x 10 3 2.6 ± 1.7 x 10 2 Test cycle 4 0 ± 0 0 ± 0 4.3 ± 0.6 x 10 3 1.5 ± 0.7 x 10 2 Test cycle 5 0 ± 0 0 ± 0* 11.0 ± 3.3 x 10 3 5.4 ± 0.4 x 10 2 Test cycle 6 4.3 ± 2.5 0 ± 0 32.2 ± 7.9 x 10 3 6.8 ± 2.0 x 10 3 Test cycle 7 0.3 ± 1.0 0 ± 0 3.1 ± 1.0 x 10 3 6.3 ± 1.2 x 10 2 Test cycle 8 0 ± 0 0.3 ± 1.0 11.7 ± 0.4 x 10 3 3.3 ± 0.7 x 10 3 Test cycle 11 9.3 ± 1.2 0.0 ± 0 3.1 ± 1.0 x 10 3 7.6 ± 0.8 x 10 2 Test cycle 13 26 ± 3.5 0.7 ± 1.2 8.6 ± 3.8x 10 3 1.4 ± 0.5 x 10 3



## 3.6 Biocidal effects on organisms ≥ 10-50 μm in minimum diameter

The number of viable organisms ≥ 10-50 μm in minimum diameter, as determined by the serial dilution method in algal growth medium and by microscopy examination after incubation with CFDA-AM, in treated test water and control immediately after treat ment and after five days of storage is given in Table 24. For the performance of the ballast water tr eatment system to pass the regulation D-2 of the IMO Guidelines, as given in Table 2, less than 10 viable organisms ≥ 50 μm in minimum diameter per ml should be present in the treated water after five days storage.

As can be seen in Table 24 and indicated by th e green background colour in the table cells, this requirement was fulfilled in all 10 test cycles re ported here, both when based on the dilution method and on microscope counts.NIVA 5667-2008

33 Comments should be made regarding the results for test cycle 3, 4, 5 and 6 (lower part of table 27). Allready in test cycle 3, ciliates were recorded in TT2 day 5 sample. The TT2 day 5 samples in the following test cycles (4, 5 and 6) all recorded th e same ciliate. The ciliate is a benthic elongated sidmoid shaped cell, resembling the genus Colpidium sp . The species was not observed in any samples from WST. It was also recorded in TT2 day 0 test cy cle 6, but not in the other TT2 day 0 samples. It was concluded that this ciliate was a contamination occurring in the ballast water tanks, specifically in the drain pipes from tanks. We improved the cleaning procedures of these drains with hot pressure washer between each run and have not experienced this problem since.

Table 24. Viable organisms ≥ 10-50 μm in minimum diameter in treated test water and control immediately after treatment and after five days of storage. Green backgroun d indicates that required level was fulfilled. Treated water Control water Day 0 Day 5 Day 0 Day 5 Organisms ≥ 10-50 μm in minimum diameter (individuals/ml) Requirement - <10 - >100 Dilution method 95 % confidence interval <0.2 0.4 2000 240 Test cycle 1 <0.1-1.0 0.1-1.7 1000-24000 100-950 <0.2 <0.2 2000 1300 Test cycle 2 <0.1-1.1 <0.1-1.1 1000-14000 500-3900 0.7 0.2 2000 2400 Test cycle 3 0.2-2.1 <0.1-1.1 1000-14000 1000-9500 1.3 0.2 2000 1600 Test cycle 4 0.5-3.9 <0.1-1.1 1000-14000 600-5300 0.7 0.2 2000 2400 Test cycle 5 0.2-2.1 <0.1-1.0 1000-14000 1000-9600 0.8 0 2000 1600 Test cycle 6 0.3-2.5 <0.1-1.0 1000-14000 600-5300 5 0.8 1100 500 Test cycle 7 2-17 0.3-2.5 300-4800 200-2000 0.2 <0.2 300 900 Test cycle 8 <0.1-1.1 <0.1-1.1 100-1300 300-300 11 0.8 5000 1600 Test cycle 11 4-30 0.3-2.5 2000-24000 600-5300 2.3 0.4 5000 2400 Test cycle 13 0.9-8.7 0.1-1.7 2000-24000 1000-9500 Microscope counts Test cycle 1 0 ± 0 0 ± 0 1748 ± 321 842 ± 65 Test cycle 2 0.3 ± 0.6 0.7 ± 1.2 1368 ± 209 1372 ± 93 Test cycle 3* 0.3 ± 0.6 0.3 ± 0.6 1490 ± 128 1311 ± 156 Test cycle 4* 1.7 ± 0.6 0.7 ± 1.2 1527 ± 212 1349 ± 156 Test cycle 5* 0.7 ± 0.6 0 ± 0 2302 ± 162 1865 ± 108 Test cycle 6* 92.5 ± 13 2.7 ± 0.6 2016 ± 150 1237 ± 133 Test cycle 7 5.7 ± 1.5 0.7 ± 0.6 2620 ± 213 2193 ± 43 Test cycle 8 5.3 ± 1.5 1.3 ± 0.6 3087 ± 205 1587 ± 377 Test cycle 11 20 ± 2.6 1.7 ± 0.6 2895 ± 95 1256 ± 175 Test cycle 13 13.7 ± 5.9 1.7 ± 0.6 1558 ± 85 741 ± 92NIVA 5667-2008

34 Microscope counts with contamination of Ciliates Test cycle 3 0.3 ± 0.6 5.7 ± 2 Test cycle 4 1.7 ± 0.6 12.7 ± 1.5 Test cycle 5 0.7 ± 0.6 10.3 ± 2.1 Test cycle 6 95 ± 13 16.7 ± 2.9

The equivalent requirement for the non-treated control test water is stated as: “If in any test cycle the average discharge results from the control water is a concentration less than or equal to 10 times the values in Regulation D-2.1, the test cycle is invalid.” It may be interpreted that the minimum level of viable organisms in the control test water at the time of discharge (e.g. after 5 days storage) should be higher than 100 per ml. This requirement was fulfilled in all test cycles.



## 3.7 Bactericidal effects

The numbers of thermotolerant coliform bacteria, Vibrio sp., Vibrio cholerae and intestinal Enterococci , determined as described in section 2.6.4, in treated test water and control water immediately after treatment and after five days of storage for test cycles are given in Table 25. Regulation D-2 only requires documentation of the bact ericidal effect of the ballast water treatment system on Escherichia coli (a thermotolerant coliform bacteria), Vibrio cholerae (toxicogenic serotypes O1 and O139) and Enterococci given as a maximum allowable residual concentration after five days storage; <250 cfu/100ml, <1 cfu/100ml and <100 cfu/100ml, respectively.

As seen from Table 25, and indicated by the green background colour in table cells, these requirements are fulfilled for E. coli (since the requirement is fulfilled for thermotolerant coliform bacteria as a group) and Enterococci . However, there are no required additions of these bacterial
groups. Vibrio sp. are common in salt and brackish surface water, while the toxicogenic Vibrio cholerae serotypes O1 and O139 have not been found in Norwegian coastal waters. Vibrio cholerae was not found in any of the samples from treated water.

Table 25. Culturable thermotolerant coliform bacteria, Vibrio sp. and intestinal Enterococci in treated test water and control water immediately after tr eatment and after five days of storage. Green background indicates that required level was fulfilled. Treated water Control water Day 0 Day 5 Day 0 Day 5 Heterotrophic bacteria (cfu/ml) Requirement - - - - Test cycle 1 - - - - Test cycle 2 - - - - Test cycle 3 1.5 ± 0.7 x 10 5 3.0 ± 1.5 x 10 5 2.6 ± 0.5 x 10 6 4.8 ± 6.5 x 10 5 Test cycle 4 3.9 ± 6.5 x 10 4 6.3 ± 3.7 x 10 5 1.0 ± 0.7 x 10 6 3.5 ± 2.1 x 10 5 Test cycle 5 4.6 ± 4.4 x 10 4 5.4 ± 1.8 x 10 5 1.4 ± 0.3 x 10 6 2.9 ± 2.1 x 10 5 Test cycle 6 6.4 ± 7.2 x 10 3 7.7 ± 6.0 x 10 5 6.5 ± 1.3 x 10 5 2.5 ± 3.1 x 10 5 Test cycle 7 8.2 ± 3.5 x 10 2 2.6 ± 0.4 x 10 6 3.4 ± 0.9 x 10 5 9.6 ± 2.0 x 10 5 Test cycle 8 2.1 ± 0.6 x 10 3 2.8 ± 0.6 x 10 6 2.8 ± 1.8 x 10 4 9.4 ± 1.9 x 10 5 Test cycle 11 1.1 ± 1.2 x 10 3 1.1 ± 0.3 x 10 6 3.9 ± 2.1 x 10 4 4.1 ±1.6 x 10 5 Test cycle 13 2.9 ± 1.9 x 10 3 2.4 ± 1.7 x 10 6 7.7 ± 2.8 x 10 4 5.0 ± 2.9 x 10 4
Coliform bacteria and Escherichia coli * (cfu/100 ml) Requirement - - <250* - - Test cycle 1 21 ± 19 40 ± 6 0 ± 0* 17 ± 5 0 ± 0 Test cycle 2 0 ± 0 0 ± 0 0 ± 0* 67 ± 57 133 ± 231NIVA 5667-2008

35 Test cycle 3 0 ± 0 0 ± 0 0 ± 0* 23 ± 6 0 ± 0 Test cycle 4 0 ± 0 0 ± 0 0 ± 0* 767 ± 231 0 ± 0 Test cycle 5 0 ± 0 0 ± 0 0 ± 0* 8670 ± 611 70 ± 27 Test cycle 6 0 ± 0 0 ± 0 0 ± 0* 3630 ± 321 17 ± 6 Test cycle 7 0 ± 0 7 ± 2 0 ± 0* 3500 ± 900 867 ± 416 Test cycle 8 0 ± 0 0.3 ± 0.6 0 ± 0* 1370 ± 231 447 ± 653 Test cycle 11 0 ± 0 0 ± 0 0 ± 0* 17 ± 4 1 ± 1 Test cycle 13 6.7 ± 0.6 4 ± 1 0 ± 0* > 8000 > 8000 Vibrio sp. and Vibrio cholerae ** (cfu/100 ml) Requirement - - <1** - - Test cycle 1 - >100 <1** >100 >100 Test cycle 2 - >100 <1** >100 >100 Test cycle 3 12 ± 10 31 ± 13 <1** 3.5 ± 1.6 x 10 4 2.7 ± 1.2 x 10 4 Test cycle 4 6.7 ± 5.8 59 ± 27 <1** 2.1 ± 0.9 x 10 6 4.4 ± 2.8 x 10 6 Test cycle 5 22 ± 33 98 ± 10 <1** 1.8 ± 1.2 x 10 6 2.9 ± 0.8 x 10 6 Test cycle 6 3 ± 6 6 ± 3 <1** 9.3 ± 9.5 x 10 4 6.5 ± 5.1 x 10 4 Test cycle 7 9 ± 1 9 ± 9 <1** 3.3 ± 2.1 x 10 3 3.2 ± 4.5 x 10 3 Test cycle 8 0.3 ± 0.6 3 ± 3 <1** 6.0 ± 1.0 x 10 3 8.5 ± 5.6 x 10 3 Test cycle 11 0 ± 0 4 ± 0 <1** 1.4 ± 0.5 x 10 4 1.8 ± 1.4 x 10 4 Test cycle 13 0.7 ± 0.6 830 ± 840 <1** 4.1 ± 3.6 x 10 3 8.0 ± 2.5 x 10 2 Enterococcus group and Intestinal Enterococci *** (cfu/100 ml) Requirement - - <100*** - - Test cycle 1 1.3 ± 2.3 2 ± 1 2 ± 1*** 1250 ± 383 194 ± 317 Test cycle 2 0.0 ± 0.0 1 ± 0 1 ± 1*** 197 ± 29 147 ± 23 Test cycle 3 0.0 ± 0.0 0 ± 0 0 ± 0*** 15 ± 12 0.0 ± 0.0 Test cycle 4 0.0 ± 0.0 0.3 ± 0.6 0 ± 0*** 2.7 ± 2.2 x 10 3 2.4 ± 1.3 x 10 3 Test cycle 5 9 ± 16 0 ± 0 0 ± 0*** 2.1 ± 0.7 x 10 2 8.0 ± 6.1 x 10 2 Test cycle 6 0.3 ± 0.6 0 ± 0 0 ± 0*** 49 ± 38 9 ± 4 Test cycle 7 0.0 ± 0.0 0 ± 0 0 ± 0*** 41 ± 3 17 ± 1 Test cycle 8 0.0 ± 0.0 0.7 ± 1.2 0.7 ± 1.2*** 76 ± 40 11 ± 2 Test cycle 11 0 ± 0 0 ± 0 0 ± 0*** 9.8 ± 3.4 x 10 2 8.3 ± 2.1 x 10 3 Test cycle 13 2.7 ± 0.6 0.3 ± 0.6 0.3 ± 0.6*** > 8000 1.1 ± 0.4 x 10 4 * The figures refer to the number identified as Escherichia coli /100 ml within the group of coliform bacteria. There is a requirement for Escherichia coli being <250 cfu/100ml after five days storage (D-2). ** The figures refer to the number of Vibrio cholerae /100 ml after five days storage. There is a requirement for toxicogenic Vibrio cholerae (serotypes O1 and O139) being <1 cfu/100 ml after five days storage (D-2). *** The figures refer to the number identified as intestinal Enterococci /100 ml within the group of Enterococcus. There is a requirement for intestinal Enterococci being <100 cfu/100ml after five days storage (D-2).



## 3.8 Ecotoxicological responses

The ecotoxicity of treated ballast water was tested at least two times with 2 different water qualities (2 different salinities). The toxicity of undiluted (100 %) and diluted samples was determined on three trophic levels using marine acute and chronic tests w ith untreated control ballast water as control. The tests and test results are summarized below. Th e complete test reports are presented in the Appendixes.NIVA 5667-2008

36 Growth inhibition of the marine alga Skeletonema costatum

Treated water from TT1 and untreated control water were used in the tests, which were conducted 3 times and included both water qualities (hi gh and moderate salinity). An overv iew of the tests and duration is shown in Table 26.

Table 26. Overview of growth inhibiton tests with marine algae Skeletonema costatum NIVA study number Duration Water quality B484/c 30 th Nov - 3th Dec 2006 High salinity (32.7 PSU) B484/d 6 th Dec - 9 th Dec 2007 High salinity (32.1 PSU) B493 14 th Feb -17 th Feb 2007 Moderate salinity (21.0 PSU)

There was no significant difference in growth rates betw een the untreated and treated ballast water, and the control sea water in any of the tests. Hence, for the tr eated Ballast water, the results are given in table 27.

Table 27. Growth inhibition of the marine algae Skeletonema costatum by treated ballast water Effects on growth rate Concentration %

48 h E r C 50 1 >100

NOEC 2 ≥ 100

1 E r C 50 : Concentration that causes 50 % reduction of th e growth rate as compared to controls

1 NOEC: No Observable Effect Concentration, i.e. the highest co ncentration that does not cause a significant inhibition of the growth rate

It was concluded that no growth inhibiting effect on the marine alga Skeletonema costatum was caused by the treated ballast water.

The complete test reports can be found in Appendix I.

Reproductive toxicity to the marine crustacean Nitocra spinipes

Treated water from TT1 and untreated control water were used in the tests. The test was conducted 2 times with the 2 different water qualities (high and moderate salinity). An overview of the tests and duration is shown in Table 28.

Table 28. Overview of reproductive toxicity tests to the marine crustacean Nitocra spinipes NIVA study number Duration Water quality B484/b 10 th Nov - 27 th Nov 2006 High salinity (32.7 PSU) B489/b 8 th Mar - 20 th Mar 2007 Moderate salinity (20.5 PSU)

It was concluded that the reproduction of Nitocra spinipes is not affected by exposure of treated ballast water for 14 days. There was no significant difference in the average number of offspring in females exposed to Treated Ballast water when compared to non treated ballast wa ter of the same quality in any of the tests. The main results are given in the table 29.

1 E r C 50 : Concentration that causes 50 % reduction of the growth rate as compared to controls

2 NOEC: No Observable Effect Concen tration, i.e. the highest concentration that does not cause a significant inhibition of the growth rateNIVA 5667-2008

37 Table 29. Average number of offspring of the marine crustacean Nitocra spinipes ± standard deviation

The complete test reports can be found in Appendix J.

Acute toxicity to the marine crustacean Acartia tonsa

Treated water from TT1 and untreated control water were used in the tests. The test was conducted 2 times with the 2 different water qualities (high and moderate salinity). An overview of the tests and duration is shown in Table 30.

Table 30. Overview of acute toxicity tests with the marine crustacean Acartia tonsa NIVA study number Duration Water quality B484 30 th Nov - 2 th Des 2006 High salinity (32.7 PSU) B489 18 th Jan - 20 th Jan 2007 Moderate salinity (20.5 PSU)

In the test conducted 30 th Nov - 2 th Des 2006, a mortality of 27% was observed at the highest tested concentration (100%). It should be commented that high death-rate in the control can be a general problem with the Acartia tonsa test, as also experienced in the current test. This death-rate can give misleading results. No effects were observed in the test with the other marine crustacean Nitocra spinipes . It is therefore doubtful that the effect obser ved can be attributed to toxicity.

In the test conducted 18 th Jan - 20 th Jan 2007, no mortality of Acartia was observed in treated ballast water at 100% concentration or lower.

Lack of mortality >50 % at any test concentration precludes estimation of LC 50 . The results are given in Table 31.

Table 31. Acute toxicity of treated ballast water on Acartia tonsa. NIVA study number Concentration (%) 95 % confidence limits

B484 48h LC 50 * >100 -

B489 48h LC 50 * >100 -

* LC = Lethal Concentration LC 50 represents the concentration that results in lethality of 50 % of the test animals.

The complete test reports can be found in Appendix J.

Chronic toxicity towards the juvenile turbot ( Scopthalmus maximus )

Treated water from TT1 and untreated control water were used in the tests. The test was conducted 2 times with the 2 different water qualities (high and moderate salinity). An overview of the tests and duration is shown in Table 32.

Table 32. Overview of chronic toxicity t ests with the juvenile turbot ( Scopthalmus maximus ) NIVA study number Duration Water quality B484/f 1 th Nov - 5 th Des 2006 High salinity (32.2 PSU) B489 10 th Jan - 2 th Feb 2007 Moderate salinity (21.5 PSU) NIVA Study number Treated Ballast water Control B484/b 20.5 ± 9.1 21.9 ± 9.4 B489/b 11.8 ± 9.3 11.1 ± 11.7NIVA 5667-2008

38 There was no mortality observed in any of the test, a nd the exposed fish did not behave or look different from control fish. Treated Ballast Water did not exert an y significant effects on the growth rate of juvenile turbot as determined using ANOVA statistics.

The results of the test are given in table 33.

Table 33. Chronic toxicity tests of treated ballast water to the juvenile turbot ( Scopthalmus maximus ) NIVA study number Paramete r Control Treated Ballast water

B484/f Average growth rate d -1 0.0063 0.0059

B489 Average growth rate d -1 0.0067 0.0071

The complete test reports can be found in Appendix K.

Acute toxicity towards the juvenile turbot ( Scopthalmus maximus )

Treated water from TT1 and untreated control water were used in the tests. The test was conducted 2 times with the 2 different water qualities (high and moderate salinity). An overview of the tests and duration is shown in Table 34.

Table 34. Overview of acute toxicity tests with the juvenile turbot ( Scopthalmus maximus ) NIVA study number Duration Water quality B495 1 th Nov - 4 th Nov 2006 High salinity (32.2 PSU) B489 10 th Jan - 15 th Jan 2007 Moderate salinity (21.5 PSU)

Treated Ballast Water did not cause mortality to ju venile turbot. As no mortality was observed, an exact LC 50 cannot be estimated. NOEC in this test is >100 %. The NOEC is the highest concentration without observed toxic effects. The results of the tests are given in Table 35.

Table 35. Acute toxicity of treated ballast water to the juvenile turbot ( Scopthalmus maximus ) Parameter Concentration (%) 95% confidence limits

LC 50 (96 h): >100 - NOEC based on mortality: ≥ 100

LC 50 - the concentration causing 50 % mortality NOEC - no observed effect concentration

The complete test reports can be found in Appendix K.

Summary of toxicological tests

Based on the conducted acute and chronic toxicity test s it is concluded that ballast water treated with the present technology do not provide hazard or risk to the marine ecosystem when released.NIVA 5667-2008

39 

# Appendix A. Total project management reports

Appendix B. Chemical water quality preparation

Appendix C. Biological water quality preparation

Appendix D. Collection and preservation of samples

Appendix E. Logging of in situ measurements

Appendix F. Evaluation form for organisms >50 μm

Appendix G. Evaluation form for organisms ≥ 10-50
μm

Appendix H. Evaluation form for heterotrophic bacteria, coliforms, E. coli , Enterococcus group, intestinal Enterococci , and Vibrio cholerae

Appendix I. Acute and chronic toxicity of the green algae Skeletonema costatum

Appendix J. Acute and chronic toxicity of marine copepodsNIVA 5667-2008

40 

# Appendix K. Acute and chronic toxicity of juvenile turbot

Appendix L. Operational performanceGaustadalléen 21 • NO-0349 Oslo, Norway Telephone: +47 22 18 51 00 • Fax: 22 18 52 00 www.niva.no • post@niva.no