### REPORT SNO 5659-2008 CONFIDENTIAL



# Land based testing of the OptiMarin ballast water management system of OptiMarin AS - Treatment Effect Studies Final ReportNIVA 5659-2008



## 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
Gaustadalleén 21 Televeien 3 Sandvikaveien 41 Nordnesboder 5 P.O. Box 1266 0349 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) 22 18 51 00 Phone (47) 22 18 51 00 Phone (47) 22 18 51 00 Phone (47) 22 18 51 00 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 OptiMarin ballast water management system of OptiMarin AS - treatment effect studies - final report - confidential Serial No. 5659-2008

Report No. Sub-No. O-27432 Date 27 th of August, 2008

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

Geographical area Norway Distribution Confidential

Printed CopyCat AS

Client(s) OptiMarin AS Client ref. Birgir Nilsen

Abstract The Optimarin ballast water management system of Optimarin AS, has been land based tested according to the IMOs Guidelines for approval of ballast water management systems (G8), Res. MEPC.125(53) Annex 3 and Procedure for approval of ballast water management systems that make use of active substances (G9) Res. MEPC.125(53) Annex 4. Only the biological effect studies according to G8 are reported here. The tests were conducted at NIVA’s test site located at Solbergstrand 20 km south of Oslo. Test cycles were conducted with medium and high salinity water. Each cycle lasted for a period of 5 days, and a tota l of 12 test cycles have been completed.

4 keywords, Norwegian 4 keywords, English

1. Ballastvann 1. Ballast water
2. Testing 2. Testing
3. Optimarin 3. Optimarin
4. Biologisk effektivitet 4. Biolgical efficacy

Helge Liltved Tor Gunnar Jantsch Jarle Nygard
Forskningsleder Prosjektleder Fag- og markedsdirektør ISBN 978-82-577-5394-8NIVA 5659-2008



# Preface

The tests were conducted in 2008 at NIVAs test facility located at Solbergstrand 20 km south of Oslo. NIVA conducted the testing as a contract assignment for OptiMarin AS, with Det Norske Veritas (DNV) as verifier.

During planning and conducting of the tests, Svein Berglia was the lead representative of OptiMarin AS, while Håkon Øvrum Hansen represented DNV classification in the project. From NIVA, Anne-Marie Bomo, Oddbjørn Pettersen, Per Ivar Joha nnesen, August Tobiessen, Christian Vogelsang, Tor Gunnar Jantsch and Helge Liltved were the main representatives. Several other OptiMar in AS and NIVA personell have been involved in the project.

We will use the opportunity to thank OptiMarin AS for choosing NIVA as the main partner in the process of testing and verification of the OptiMarin ballast water management system, and thank all involved personell for the professionalism and creativity demonstrated in completing this project.

Oslo, August 2008

Tor Gunnar JantschNIVA 5659-2008



# Contents

Abbreviations and acronyms 7

Summary 8

1. Background 10

2. Materials and test protocols 11
2.1 Test site 11 2.2 The evaluated ballast water treatment system 11 2.2.1 General description of the OptiMarin BWMS 11 2.3 Test waters 12 2.3.1 Overview of requirements 12 2.3.2 Fulfilment of chemical water quality test criteria 13 2.3.3 Fulfilment of biological water quality test criteria 13 Harvesting of indigenous organisms 13 Cultivation of Artemia franci scana, Tetraselmis suecica and Brachiomonas submarina 13 Bacteria 13 2.4 Test procedure 13 2.4.1 Description of test cycle 13 2.4.2 Measures to avoid cross-contamination during water transfer and sampling 14 2.5 Sampling 14 2.5.1 Assuring the representativeness of samples 14 2.5.2 Sampling protocols 14 2.5.3 Overview of sampling equipment 15 2.5.4 Sample preservation and transportation 15 2.6 Analyses 16 2.6.1 Chemical measurements 16 Temperature 16 pH 16 Dissolved oxygen (DO) 16 Salinity 16 Dissolved organic carbon (DOC) 16 Particulate organic carbon (POC) 16 Total suspended solids (TSS) 16 Total residual oxidants (TRO) 17 2.6.2 Identification and quantification of viable organisms ≥ 50 μm 17 2.6.3 Identification and quantificati on of viable organisms >10-50 μm 17 2.6.4 Identification and quantification of viable bacteria 17 Heterotrophic bacteria 17 Coliform bacteria. 17 E.coli 17 Enterococcus group 18 Vibrio sp. 18 Vibrio cholerae (Serotypes O1 and O139) 18NIVA 5659-2008

3. Results and discussion 19 3.1 QA/QC prosedures 19 3.2 Operational performance of the OptiMarin BMWS 19 3.3 Fulfillment of chemical water quality criteria 21 3.4 Fulfillment of the biological water quality criteria 27 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

4. References 36NIVA 5659-2008



## Abbreviations and acronyms

A. franciscana – Artemia franciscana B. submarina – Brachiomonas submarina CFDA-AM - 5-carboxyfluorescein diacetate acetoxymethyl ester COD – chemical oxygen demand CT2 – storage tank on control cycle DNV – Det Norske Veritas DO – dissolved oxygen DOC – dissolved organic carbon E. coli – Escherichia coli FAC – Free Available chlorine FNU – Formazine Nephelometric Units GF/F – glass fiber filter grade F GLP – Good laboratory Practice IMO – International Maritime Organization ISO – International Organisation for Standardization MRV – minimum reporting value 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 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 – storage 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 5659-2008

8 

# Summary

Land-based testing of the OptiMarin ballast water management system of OptiMarin AS has been completed in the period of January to May 2008 . The testing has been conducted according to the IMOs Guidelines for approval of ballast water manage ment systems (G8), Res. MEPC.125(53) Annex 3 and Procedure for approval of ballast water managem ent systems that make u se of active substances (G9) Res. MEPC.125(53) Annex 4 , hereafter referred to as G8 and G9. Only the biological effect studies according to G8 are reported here. The tests we re carried out at NIVA’s test site located at Solbergstrand 20 km south of Oslo, with medium sa linity and with high salinity water. A total of 12 test cycles have been completed. Each cycle lasted for a period of 5 days.

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 franciscana ; 10-50 μm group: Tetraselmis suecica and Brachiomonas submarina ) were added to fulfil the biological water quality criteria. Measurements by NIVA shows that Tetraselmis suecica has an average minimum dimension of 9.3 μm and is regarded as representative of th e 10-50 μm group of organsism. In order to ensure presence of organisms >10 μm Brachiomonas submarina with an average minimum dimension of 12.5 μm was also added. One test cycle in cludes consecutive treatment of >200 m 3 test water by the filter unit and the UV-unit transferring the test water from tank with prepared test water (WST) to a ballast tank (TT1). The treated water was stored in TT1. After 5 days the water was pumped from TT1, treated in the UV-unit, and transferred to TT2 for sampling before discharge. The control cycle included transferring >200 m 3 of the same type of prepared test water from WST to a ballast tank (CT2) for at least 5 days storage using the pum p of the OptiMarin BWMS, but in by-pass of the treatment unit (filter and UV). After storage the cont rol water was pumped to TT1 for sampling before discharge.

Fulfilment of the chemical and biological requirements of the test water

• The required levels for total suspended so lids (TSS), dissolved organic carbon (DOC) and particulate organic carbon (POC) were fulf illed in all 12 test cycles reported.

• The requirements regarding influent density of the ≥ 50 μm group was met in all 12 tests reported, except in test cycle 5, where the c oncentration was below the IMO requirement. The requirements regarding the biological diversity w ithin the population were fulfilled in all tests.

• The requirements regarding influent density of the ≥ 10-50 μm group was met in all tests. The requirements regarding the biological diversity w ithin the population were fulfilled in all tests.

• The only requirement regarding bacteria in influe nt water is that heterotrophic bacteria should be present in a density of ≥ 10 4 cfu ml -1 . This requirement was fulfilled in all tests.

Biocidal effects of treatment and storage

• The required less than 10 viable organisms ≥ 50 μm in minimum diameter per m 3 in the treated water after five days storage was fulfilled in all tests. Viability was determined by observation of movement. The equivalent requirement of the non-treated control test water, stating that the level of viable organisms after 5 days storage should be higher than 100 per m 3 , was fulfilled in all 12 tests.

• The required less than 10 viable organisms ≥ 10 - 50 μm in minimum diameter per ml in the treated water after five days storage, both when based on the dilution method and on microscope counts, was reached in all tests. The equivalent requirement for the non-treated control test water after 5 days storage states that the level of viable organisms should be higher than 100 per ml. This requirement was fulfilled in all tests by both detection methods applied.

• Regulation D-2 requires documentation of maxi mum allowable effluent concentration after five days storage of Escherichia coli

, Vibrio cholerae (toxicogenic serotypes O1 and O139)NIVA 5659-2008

9 and Intestinal enterococci ; <250 cfu/100ml, <1 cfu/100ml and <100 cfu/100ml, respectively. These requirements were fulfilled for all tests.

Toxicity testing Toxicity testing according to the IMO Procedu re for approval of ballast water management systems that make use of active substances (G9) Res. M EPC.125(53) Annex 4 has been conducted by NIVA and is reported elsewhereNIVA 5659-2008

10 

# 1. Background

The goal for OptiMarin AS is certification of the OptiMarin BWMS in accordance with the requirements in the IMO Convention on ballast water management and the underlying guideline; Guidelines for approval of ballast water management systems (G8), Res. MEPC.125(53) Annex 3

. Although OptiMarin BMWS do not make use of activ e substances in their treatment process, treated ballastwater was also analysed for toxicity a nd presence of oxidation by-products, following the specifications in Procedure for approval of ballast water management systems that make use of active substances (G9) Res. MEPC.125(53) Annex 4 ., section 1-5, pages 1-7, with the results being presented elsewhere. Guidelines are hereafter referred to as G8 and G9.

Comprehensive development and pilo t testing had been conducted prior to the land based testing. The objective of the pilot testing was to verify the environmental performance characteristics of the OptiMarin BWMS technology relative to the biologica l and toxicological requirements stated in the IMO Guideline. By performing pilottests, diffi culties and “bottle-necks” could be detected, and measures could be taken to avoid problems dur ing the full scale testing. The experience from pilottesting 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 January to May 2008. The tests were conducted in accordance with G8 and G9.NIVA 5659-2008

11 

# 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 was supplied from various depths dow n to 60 m in the Oslofjord, while fresh water was 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. The three 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 OptiMarin BWMS During ballasting, the OptiMarin BWMS applie d doses of UV irradiation as the main working principle following a pre-filtration step. During de-ballasting the filter was by-passed. The UV was operated also during de-ballasting.


### Filter-unit The ballast water is filtered through a 40 μ m filter to remove solids and larger zooplankton. The filter is self cleaning through an automatic back-flushing system. The back-flushing is carried out during operation of the filter and will not hamper the ball asting operation. The back-flushing flow rate is approximately 20 m 3 /hr for a 30 sec period. The back-flushing is initiated by differential pressure over the filter (set at 0.5 bars) or on timer. If desired, the back-flushing could also be initiated manually, and even be set on continuous mode. The filter re quires a minimum back-pressure of 1.7 bars to operate, and the installation consequently also compromises a back-pressure valve.


### UV-unit The ballast water is lead to an inlet header in which the flow is split over UV chambers. After UV treatment the ballast water will go out through an outlet header. The inlet/outlet headers and the UV chambers are made of 90/10 copper-nickel. Each UV chamber has a capacity of 167 m 3 /hr for the actual water used in the tests, and the system capacity will hence be a multiple of this. During testing, two chambers at a flow-rate of approximately 334 m 3 /hr were used. Each UV chamber is equipped with a UV lamp of 35kW. Each chamber will have outlet temperature surveillance and will initiate shut-down of the system by temperatures exceeding 50 oC. The temperature is logged in a data logger. Each chamber has a sensor to measure UV intensity. This value is monitored and will initiate alarms on low intensity. The data are logged. In the test set-up, the pressure drop over the UV chambers was also manually observed by installing pressure gauges. This is only optional and not normally a part of the system.



### Electrical: The following electrical equipment is installed:

• Control/power supply for the filter. This includes control functions of the filter, operation of the filter itself and operation of the back-flushing pump.

• Control cabinet for the UV, including data logger (1 per system)

• Power supply for UV unit (1 per UV chamber)NIVA 5659-2008

12 

### Other: The following additional equipment was installed during the test at NIVA. It was not a part of the test system, but needed for the test conditions.

• Two Kolmeks 12024370 pumps arranged in series to obtain the desired flow-rate. One of the pumps is equipped with a frequency regulator. One is installed inside the container, the other outside.

• One Arkon MAG 910E electromagnetic flowmeter to monitor the flow and connected to the data logger.

Operational sequence: The Optimarin BWMS treat water upon ballasting and deballasting.



## 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 (as specified in G8), 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 organisms and cultured surrogate species (>50 μm: Artemia franciscana ; 10-50 μm: Tetraselmis suecica and Brachiomonas submarina ) were added to fulfil the biological water quality criteria, and soluble lignin, 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 within th e 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 wate rs. 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/l

Table 2. Required biological water quality in influent t est water, treated water and in control water 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 ml >10x <10 viable organisms per ml

Heterotrophic bacteria ≥ 10 4 cfu ml -1

Vibrio cholerae - <1 cfu/100 ml - Escherichia coli - <250 cfu/100 ml - Intestinal Enterococci - <100 cfu/100 ml -

** As discussed in the results and discussion section.NIVA 5659-2008

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### 2.3.2 Fulfilment of chemical water quality test criteria Chemical compounds were added to adjust the contents of dissolved organic carbon (DOC), particulate organic carbon (POC) and total suspended so lids (TSS), respectively, to within the limits of chemical water quality criteria described in Table 1. The necessary additions of each compound were calculated on the basis of earlier laboratory tests and full scale experience.



### 2.3.3 Fulfilment of biological water quality test criteria A combination of indigenous harvested orga nisms and cultured surrogate species (>50 μm: Artemia franciscana ; 10-50 μm: Brachiomonas submarina and/or Tetraselmis suecica ) were added to fulfil the biological water quality criteria described in Table 2. Measurements by NIVA shows that Tetraselmis suecica has an average minimum diameter of 9.3 μm (n=25) when growing exponentially in our cultures, the range in diameter is 7-11 μm. Brachiomonas submarina has a mean minimum diameter of 12.5 μm when growing exponentia lly with a range of 10-20 μm. B.submarina is a rather delicate organism with no protective outer shell. Even if it has the required minimum diameter it would not constitute much of a challenge for a ballast water treatment system. T. suecica on the other hand is quite robust and has an outer shell composed of cellulose-like material. It has a good survival when exposed to shear forces in pumps and a good survival in the dark. In addition it is quite tolerant with respect to survival in brackish seawater. T. suecica is therefore a robust representative of the type of organisms to be expected in the 10-50 μm size fractions of marine organisms.



### 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 out side Solbergstrand research station The species composition and viability of harvested organisms were evaluated by microscopy.



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

The required cultivation volume was calculated based on a final density of 10 5 nauplii per m 3 for A. franciscana and 10 3 per ml for B. submarina and T. suecica assuming a final volume of the test water of 516 m 3

.



### Bacteria The concentration of heterotrophic bacteria in the surface water is normally exceeding the required level of ≥ 10 4 cfu ml -1 , while the heterotrophic bacteria criteria for the high salinity test water is expected to be fulfilled by the heterotr ophic communities accompanying the cultured Tetraselmis suecica, Brachiomonas submarina and Artemia franciscana . During the tests, it was not necessary to supply any extra bacteria added as cultivated heterotrophic bacteria.



## 2.4 Test procedure

2.4.1 Description of test cycle

The different water transfers between tanks via the OptiMarin BWMS during a test cycle including the control is shown in Figure 1.NIVA 5659-2008

14 Figure 1. Transfer of test water during one test cycle with the OptiMarin BWMS including a test line (blue) and a control line (red).



### 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).

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 1) Before any samples were collected from any of the tanks at every sampling time the a procedure was carried out to assure that representative samples were withdrawn:



### 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). 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 cup, collecti ng the organisms in the cup. The content of the sampling cup was transferred to a 100 ml glass bottle for immediate counting of number of viable organisms. WST 516 m 3 S2

TT1 231 m 3

S5 UV TT2 205 m 3

CT2 205 m 3 S1 S3

S4 Filter Pump

UV unit

1 2

1 2NIVA 5659-2008

15 2) Sampling of organisms ≥ 50 μm in TT1 (S2), CT2 (S4), TT1 (S5) and TT2 (S3): A siphon spillway was used to collect 1 m 3 test water from TT1 directly after treatment and after 5 days storage (after transfer from TT1 to TT2). A similar method was u sed to collect control samples but then 45-90l were collected. The water was siev ed directly through a plankton net (50 μm diagonal dimensions) attached to a plastic cup, collecting and concentra ting the organisms in the cup. The content of the sampling cup was transferred to a 100 ml glass bottle for immediate counting of number of viable organisms. 3) Sampling of organisms 10-50 μm in WST (S1), TT1 (S2 and S5), CT2 (S4) and TT2 (S3) : Organisms with a minimum diameter between 10 μm and 50 μm were sampled as 1000 ml grab samples with a clean glass bottle. 4) Sampling of bacteria in WST (S1), TT1 (S2 and S5), CT2 (S4) and TT2 (S3) : Bacterial samples were collected as 1000 ml grab samples by slowly submerging a 1000-ml sterile bottle. The bottle was closed immediately after sampling.



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

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

Parameter Sampling equipment Sample container Sample volume WST, CT2 Sample volume TT1, 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 bottle 1000 ml 1000 ml

* A 20-litre grab sample from WST, 45-90 l from control tanks, or 1 m 3 collected through a siphon spillway (from TT1 and TT2) is concentrated to a volume of 40-100 ml through plankton net with diagonal dimensions of 50 μm.



### 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 accordance with the described methodology. After sampling, samples were placed in the dark in cooling-containe rs and transported to the laboratories for analysis.

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 - - 0NIVA 5659-2008

16 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 Coliform bacteria, E.coli Enterococcus group bacteria, Intestintal enterococci Vibrio sp. Vibrio cholerae 4 o C in darkness 24 hours 24 h



## 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.

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 through a GF/F filter (0.7 μm).

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, 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 was 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.NIVA 5659-2008

17 

### Total residual oxidants (TRO) Total residual oxidants were measured by the colo rimetric DPD-method (American Public Health Association, 1989), which is currently the me thod recommended for measurement of TRO in
seawater.



### 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. Because UV-treated plankton takes some time to die, counts were repeated after 24 hours. The initial and 24 hour counts are given in Appendix F. Viable organisms were counted and 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 a viability stain.

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 sample.

Procedure for plating Tetraselmis sp . on agar A supplementary cultivation test was also used by plating on agar plates.



### 2.6.4 Identification and quantific ation of viable bacteria Generally the samples were diluted or concentrated to achieve a quantifiable 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 the sample. Dilution was performed by stepwise 10x dilution of the sample in a dilution series followed by incubation on agar. Concentration w as 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.

Coliform bacteria. Coliform bacteria were quantified according to Norw egian Standard 4788 at a temperature of 37 ± 1.0 °C and an incubation period of 22-24 hours.

E.coli
E.coli was quantified according to Norwegian Standard 4792 or NS-EN ISO 9308-1 at a temperature of 44 ± 0.2 °C and a total incubation period of 44 hours.NIVA 5659-2008

18 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.

Intestinal Enterococci Intestinal Enterococci were quantified according to Norweg ian Standard NS-EN ISO 7899-2 or 7899 at a temperature of 44 ± 0.2 °C and an incubation period of 2 hours.

Vibrio sp. The total number of Vibrio sp., were determined by filtering of a 1-100 ml sample, and by placing the filter on TCBS Cholera-medium agar plates (CMO333 from Oxoid). Plates were incubated at 37 °C, and the colonies counted after 24 hours incubation. The medium supports the growth of pathogenic Vibrios ( Vibrio cholerae

, Vibrio parahaemolyticus ), some other Vibrios, and some other bacterial species such as some strains of Aeromonas hydrophila.

Vibrio cholerae (Serotypes O1 and O139)
Vibrio cholerae identified above will be confirmed for each colony by PCR and serotyping with specific antibodies for the outer antigens O1 and O139.NIVA 5659-2008

19 

# 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. All activities and data collected during testing of the OptiMarin treatment system have been logged as summarized in Table 5. For each activity a specially designed log in paper and/or electronic format has been u sed (Appendix A-K). These log sheets were also used as quality assurance of some of the operations perfomed during the tests.

Table 5. 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 Operational data 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 spp

.



## 3.2 Operational performance of the OptiMarin BMWS

A total of 12 test cycles have been completed in the period of January to May 2008. Test cycles 1-5, and 11 and 12 were conducted with high salinity water (>32 PSU) and the remaining with medium salinity (< 22 PSU). Each test cycle lasted for 5 days. The dates of the cycles and the time for start/stop of each step in the test cycle are given in table 6.

A control of the operation performance of the technolog y was performed in each test cycle by the site responsible person from NIVA. Reports from these c ontrols are included in Appendix D. Check points and operational parameters for documenting suffici ent operation of the treatment technology were included and monitored in this quality control, incl uding start and stop of ba llasting and deballasting of test water, and ballasting and deballasting of control water.NIVA 5659-2008

20 Table 6. Test cycles completed with dates and the time for start/stop of each step in the cycle.

Test cycle Date Start/stop ballasting WST to TT1

Day 0 Start/stop deballasting TT1 to TT2

Day 5 Start/stop control ballasting WST to CT2 Day 0 Start/stop control deballasting CT2 to TT1 Day 5 Salinity PSU

1 16-21/01/08 12:27-13:06 39 min 10:54-11:30 36 min 11:51-12:27 36 min 14:10-14:44 34 min >32

2 23-28/01/08 11:00-11:41 41 min 11:00-11:36 36 min 11:47-12:23 36 min 11:55-12:30 35 min >32

3 30/01-04/02/08 11:06-11:46 40 min 10:32-11:08 36 min 11:52-12:28 36 min 11:33-12:08 41 min >32

4 06-11/02/08 10:58-11:39 41 min 10:32-11:10 38 min 11:47-12:23 36 min 11:32-12:06 34 min >32

5 20-25/02/08 16:06-16:41 35 min 10:32-11:08 36 min 16:47-17:23 36 min 11:34-12:09 35 min >32

6 05-10/03/08 11:51-12:28 37 min 10:48-11:25 37 min 12:34-13:10 36 min 11:46-12:21 35 min <22

7 12-17/03/08 10:59-11:38 39 min 10:45-11:20 35 min 11:45-12:20 35 min 11:43-12:17 34 min <22

8 26-31/03/08 12:30-13:09 39 min 10:28-11:04 36 min 13:18-13:54 36 min 11:38-12:13 35 min <22

9 02-07/04/08 11:44-12:24 40 min 11:17-11:53 36 min 12:32-13:09 37 min 12:30-13:05 35 min <22

10 09-14/04/08 11:06-11:49 43 min 10:28-11:04 36 min 12:05-12:39 34 min 11:58-12:33 35 min <22

11 16-21/04/08 10:55-11:35 40 min 11:51-12:26 35 min 10:40-11:16 36 min 11:50-12:25 35 min >32

12 30/04-05/05/08 11:17-11:53 36 min 12:04-12:40 36 min 11:01-11:37 36 min 12:38-13:11 33 min >32

In addition, flow rate was recorded during the tests cycles. Flow meter was placed inside the OptiMarin container and was read by the Optimar in personell, and immediately communicated to NIVAs site responsible. The averages of multiple flow measurements are given in table 7. Flowrates were also calculated from the time for start/stop a nd the volume of the tanks, which give an accurate flow estimate. The ranges of calculated flowrates for a ll test cycles are given in the lower row of table 7. As observed, the measured flow rates are in general in the lower end of the calculated ranges, indicating that the flowmeter slightly underestimated the actual flows.NIVA 5659-2008

21 Table 7. Average flowrates based on measurements and calculations (lower row).

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 16-21/01/08 332 331 330 332 2 23-28/01/08 331 331 330 331 3 30/01-04/02/08 331 332 331 331 4 06-11/02/08 331 331 331 331 5 20-25/02/08 333 331 333 331 6 05-10/03/08 335 334 334 334 7 12-17/03/08 335 335 334 334 8 26-31/03/08 335 336 336 335 9 02-07/04/08 334 335 335 334 10 09-14/04/08 334 335 335 334 11 16-21/04/08 335 335 335 335 12 30/04-05/05/08 334 335 334 334 Flow range for all cycles

based on calculations 307-377 316-343 341-371 343-364



## 3.3 Fulfillment of chemical water quality criteria

The temperature, salinity, pH, dissolved oxygen cont ent and concentration of free and total chlorine (or oxidants) were measured in the test water tank (WST) prior to treatment and daily in the storage tanks TT1 and CT2 in each test cycle. Measure ments were not conducted during week-ends. The results are given in Table 8. The requirement that different test waters should be separated by 10 PSU was fulfilled. Measurements of free and total chlo rine were made and the results are below the practical detection limit of the analysis method, sh owing that no free and total chlorine, or other oxidants included in the method used, could be detected in the water.NIVA 5659-2008

22 Table 8. Temperature, salinity, pH, dissolved oxygen and free and total chlorine measurements in the test water tank (WST) prior to treatment and daily in the storage tanks TT1 (treated water) and CT2 (control water) for each test cycle (measureme nts were not conducte d during week-ends). WST TT1 CT2 Day 0 0 1 2 3 4 5 0 1 2 3 4 5 Parameter Unit Test cycle 1 Temperature oC 7.20 7.60 7.20 6.50 - - 5.70 7.30 7.00 6.20 - - 5.10 pH 7.96 8.01 8.04 8.04 - - 8.04 8.02 8.05 7.99 - - 8.03 Dissolved O 2 % 11.10 10.80 10.40 11.40 - - 10 .30 11.40 10.90 10.60 - - 10.10

Salinity PSU 32.00 32.00 32.00 32.00 - - 32.00 32.00 32.00 32.00 - - 32.00 Total chlorine mg/l - - - - - - - - - - - - -

Free chlorine mg/l - - - - - - - - - - - - -

Test cycle 2 Temperature oC 5.40 5.60 5.30 4.70 - - 4.30 5.30 5.1 4.4 - - 3.80 pH 8.01 8.06 7.98 8.00 - - 8.16 8.04 7.99 7.99 - - 8.18 Dissolved O 2 % 10.40 11.00 11.00 10.80 - - 10 .20 11.10 10.8 10.2 - - 10.90

Salinity PSU 32.10 32.10 32.10 32.10 - - 32.10 32.10 32.2 32.0 - - 32.10 Total chlorine mg/l - - - - - - - - - - - - -

Free chlorine mg/l - - - - - - - - - - - - -

Test cycle 3 Temperature oC 5.70 5.90 5.50 5.10 - - 4.20 5.80 5.50 4.90 - - 3.80 pH 8.03 8.07 8.06 8.08 - - 8.05 8.09 8.06 8.08 - - 8.05 Dissolved O 2 % 11.50 11.20 11.30 11.60 - - 11 .90 11.10 11.00 11.60 - - 12.10

Salinity PSU 32.90 32.90 32.80 32.90 - - 32.90 32.90 32.90 32.90 - - 32.90 Total chlorine mg/l - - - - - - - - - - - - -

Free chlorine mg/l - - - - - - - - - - - - -

Test cycle 4 Temperature oC 6.70 6.90 6.40 6.10 - - 5.70 6.70 6.00 5.80 - - 5.20 pH 8.04 8.04 8.02 7.91 - - 8.00 8.04 8.04 7.89 - - 7.99 Dissolved O 2 % 11.60 10.60 11.60 11.60 - - 11 .01 10.40 10.80 11.60 - - 11.20

Salinity PSU 32.60 32.60 32.60 32.60 - - 32.60 32.60 32.06 32.60 - - 32.60 Total chlorine mg/l - - <0.1 <0.1 - - <0.1 - <0.1 <0.1 - - <0.1

Free chlorine mg/l - - <0.1 <0.1 - - <0.1 - <0.1 <0.1 - - <0.1

Test cycle 5 Temperature oC 3.50 3.80 3.70 3.80 - - 4.10 3.60 3.60 3.60 - - 3.80 pH 8.04 8.03 8.01 8.00 - - 7.87 8.03 7.90 7.82 - - 7.87 Dissolved O 2 % 12.40 12.50 12.70 12.90 - - 11 .10 12.20 12.60 12.90 - - 11.50

Salinity PSU 32.70 32.70 32.70 32.70 - - 32.70 32.70 32.70 32.70 - - 32.70 Total chlorine mg/l <0.1 <0.1 <0.1 <0.1 - - <0.1 <0.1 <0.1 <0.1 - - <0.1

Free chlorine mg/l <0.1 <0.1 <0.1 <0.1 - - <0.1 <0.1 <0.1 <0.1 - - <0.1NIVA 5659-2008

23 Test cycle 6 Temperature oC 4.10 4.40 4.7 3.9 - - 4.3 4.20 4.70 3.60 - - 4.00 pH 7.83 7.68 7.73 7.81 - - 7.80 7.72 7.79 7.79 - - 7.60 Dissolved O 2 % 11.43 11.88 12.00 12.1 - - 11 .50 11.80 12.40 11.40 - - 11.40

Salinity PSU 21.70 21.70 21.70 21.70 - - 21.70 21.70 21.70 21.70 - - 21.70 Total chlorine mg/l <0.1 <0.1 <0.1 <0.1 - - <0.1 <0.1 <0.1 <0.1 - - <0.1

Free chlorine mg/l <0.1 <0.1 <0.1 <0.1 - - <0.1 <0.1 <0.1 <0.1 - - <0.1

Test cycle 7 Temperature oC 6.30 6.50 6.40 6.20 - - 5.60 6.40 6.20 6.10 - - 5.30 pH 7.89 7.76 7.77 7.85 - - 7.70 7.91 7.77 7.80 - - 7.80 Dissolved O 2 % 12.40 11.90 11.58 12.00 - - 10 .70 11.30 11.53 11.20 - - 10.20

Salinity PSU 21.50 21.50 21.50 21.50 - - 21.50 21.50 21.50 21.50 - - 21.50 Total chlorine mg/l <0.1 <0.1 <0.1 <0.1 - - <0.1 <0.1 <0.1 <0.1 - - <0.1

Free chlorine mg/l <0.1 <0.1 <0.1 <0.1 - - <0.1 <0.1 <0.1 <0.1 - - <0.1

Test cycle 8 Temperature oC 3.20 3.50 3.00 2.60 - - 3.10 3.30 2.80 2.50 - - 2.80 pH 7.82 7.84 7.76 7.84 - - 7.72 7.91 7.77 7.85 - - 7.75 Dissolved O 2 % 13.33 12.32 12.81 12.70 - - 11 .90 12.58 12.62 12.28 - - 12.50

Salinity PSU 21.40 21.40 21.40 21.40 - - 21.40 21.40 21.40 21.40 - - 21.40 Total chlorine mg/l <0.1 <0.1 <0.1 <0.1 - - <0.1 <0.1 <0.1 <0.1 - - <0.1

Free chlorine mg/l <0.1 <0.1 <0.1 <0.1 - - <0.1 <0.1 <0.1 <0.1 - - <0.1

Test cycle 9 Temperature oC 4.90 5.20 5.30 5.30 - - 5.30 5.10 5.20 5.20 - - 5.10 pH 8.01 7.02 7.99 8.02 - - 8.13 7.93 8.03 0.04 - - 8.15 Dissolved O 2 % 13.10 12.50 12.90 13.10 - - 10 .90 12.50 12.20 12.30 - - 11.10

Salinity PSU 21.60 21.60 21.50 21.40 - - 21.40 21.60 21.50 21.40 - - 21.40 Total chlorine mg/l 0.02 <0.1 <0.1 <0.1 - - <0 .1 <0.1 <0.1 <0.1 - - <0.1

Free chlorine mg/l 0.09 <0.1 <0.1 <0.1 - - <0 .1 <0.1 <0.1 <0.1 - - <0.1

Test cycle 10 Temperature oC 4.80 5.10 5.10 5.20 - - 5.50 5.00 5.00 5.10 - - 5.40 pH 8.24 8.23 8.22 8.18 - - 8.09 8.24 8.23 8.18 - - 8.11 Dissolved O 2 % 13.30 13.30 13.10 12.50 - - 11 .50 12.60 12.90 12.30 - - 12.10

Salinity PSU 20.70 20.70 20.70 20.70 - - 20.70 20.70 20.70 20.70 - - 20.70 Total chlorine mg/l <0.1 <0.1 <0.1 <0.1 - - <0.1 <0.1 <0.1 <0.1 - - <0.1

Free chlorine mg/l <0.1 <0.1 <0.1 <0.1 - - <0.1 <0.1 <0.1 <0.1 - - <0.1

Test cycle 11 Temperature oC 7.60 7.90 7.80 8.70 - - 8.30 7.80 7.70 8.50 - - 8.20 pH 8.01 8.00 8.03 7.98 - - 7.94 7.94 8.03 7.94 - - 7.91 Dissolved % 11.60 11.60 10.90 11.40 - - 10.60 10.70 10.30 11.20 - - 10.60NIVA 5659-2008

24 O 2 Salinity PSU 33.00 33.00 33.00 33.10 - - 33.00 33.00 33.00 33.10 - - 33.00 Total chlorine mg/l <0.1 <0.1 <0.1 <0.1 - - <0.1 <0.1 <0.1 <0.1 - - <0.1

Free chlorine mg/l <0.1 <0.1 <0.1 <0.1 - - <0.1 <0.1 <0.1 <0.1 - - <0.1

Test cycle 12 Temperature oC 8.90 9.30 - 9.80 - - 10.70 9.30 - 9.70 - - 10.40 pH 7.93 7.93 - 7.94 - - 7.83 7.92 - 7.93 - - 7.83 Dissolved O 2 % 11.60 8.87 - 9.10 - - 8.10 8.67 - 8.10 - - 8.10

Salinity PSU 32.40 32.40 - 32.40 - - 32.30 32.40 - 32.40 - - 32.40 Total chlorine mg/l <0.1 <0.1 - <0.1 - - <0.1 <0.1 - <0.1 - - <0.1

Free chlorine mg/l <0.1 <0.1 - <0.1 - - <0.1 <0.1 - <0.1 - - <0.1

Total suspended solids (TSS), dissolved organic carbon (DOC) and particulate organic carbon (POC) measurements at day 0 in WST, TT1 and CT2, and at day 5 in TT2 and TT1 in the different test cycles are shown in Table 9. The required level of TSS, DOC and POC in the initial test water (as defined in Table 1) are given in the line above the measured va lues for the influent water to treatment (WST). Green background indicates that required level was fulfilled, yellow background partial fulfilment, while red background indicates failure to fulfil requi red level.To ensure fulfilment of the chemical requirements of the test water; a POC regulating compound (POC Additive ), a DOC regulating compound (DOC Additive ) and a TSS regulating compound (TSS additive ), were added.

The required levels for total suspended solids ( TSS), dissolved organic carbon (DOC) and particulate organic carbon (POC) were fulfilled in all test cycles.NIVA 5659-2008

25 Table 9. Chemical water quality (average and standa rd deviation of triplicate samples). Green background indicates that required level was fulfilled, yellow background partial fulfilment, while red background indicates failure to fulfil required level. TSS mg/l DOC, mg/l POC, mg/l TEST 1 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 2.0 kg DOC Additive = 2.5 kg POC Additive = 3.0 kg Required level, influent water >1 - >1 - >1 - Influent water (WST) 13.8 0.3 2.4 0.1 2.1 0.2 Treated day 0 (TT1) 14.5 2.2 2.5 0.1 1.8 0.5 Treated day 5 (TT2) 7.5 1.0 2.3 0.1 1.0 0.1 Control day 0 (CT2) 17.7 2.8 2.4 0.1 1.8 0.1 Control day 5 (CT2) 13.9 2.6 2.2 0.0 2.2 0.1 TSS mg/l DOC, mg/l POC, mg/l TEST 2 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 2.0 kg DOC Additive = 2.5 kg POC Additive = 3.0 kg Required level, influent water >1 - >1 - >1 - Influent water (WST) 15.1 1.6 2.6 0.1 1.9 0.1 Treated day 0 (TT1) 14.6 1.1 2.7 0.1 1.7 0.1 Treated day 5 (TT2) 7.8 0.8 2.4 0.1 0.8 0.1 Control day 0 (CT2) 14.1 0.3 2.6 0.2 2.2 0.1 Control day 5 (CT2) 14.3 3.1 2.9 1.0 1.9 0.1 TSS mg/l DOC, mg/l POC, mg/l TEST 3 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 2.0 kg DOC Additive = 2.5 kg POC Additive = 3.0 kg Required level, influent water >1 - >1 - >1 - Influent water (WST) 17.8 3.3 2.3 0.1 2.5 0.4 Treated day 0 (TT1) 11.9 1.0 2.3 0.1 1.8 0.2 Treated day 5 (TT2) 9.2 5.1 2.6 0.1 0.7 0.1 Control day 0 (CT2) 16.3 1.9 2.3 0.1 3.0 0.3 Control day 5 (CT2) 11.1 0.4 2.5 0.7 2.0 0.2 TSS mg/l DOC, mg/l POC, mg/l TEST 4 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 2.0 kg DOC Additive = 2.5 kg POC Additive = 3.0 kg Required level, influent water >1 - >1 - >1 - Influent water (WST) 13.7 1.0 2.5 0.2 2.0 0.3 Treated day 0 (TT1) 13.3 2.3 2.5 0.1 2.1 0.5 Treated day 5 (TT2) 6.2 1.2 2.3 0.0 0.7 0.1 Control day 0 (CT2) 9.5 3.3 2.5 0.1 2.0 0.2 Control day 5 (CT2) 12.2 2.3 2.3 0.2 1.9 0.4 TSS mg/l DOC, mg/l POC, mg/l TEST 5 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 2.0 kg DOC Additive = 2.5 kg POC Additive = 3.0 kg Required level, influent water >1 - >1 - >1 - Influent water (WST) 12.8 1.0 2.3 0.0 2.3 0.1 Treated day 0 (TT1) 10.3 1.0 2.5 0.1 1.9 0.2 Treated day 5 (TT2) 6.5 1.1 2.3 0.1 0.7 0.1 Control day 0 (CT2) 12.5 0.3 2.3 0.1 1.9 0.0 Control day 5 (CT2) 10.3 0.4 2.1 0.0 1.8 0.1 TSS mg/l DOC, mg/l POC, mg/l TEST 6 Average Stdev Average Stdev Average StdevNIVA 5659-2008

26 Additions to influent water TSS Additive = 17.5 kg DOC Additive = 10.0 kg POC Additive = 12.5 kg Required level, influent water >50 - >5 - >5 - Influent water (WST) 59.3 1.7 5.4 0.1 6.8 1.5 Treated day 0 (TT1) 56.7 0.8 5.5 0.1 7.6 0.5 Treated day 5 (TT2) 14.8 1.1 5.5 0.2 1.1 0.2 Control day 0 (CT2) 56.8 1.3 5.3 0.1 7.5 0.5 Control day 5 (CT2) 58.7 8.2 5.3 0.1 6.1 1.5 TSS mg/l DOC, mg/l POC, mg/l TEST 7 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 17.5 kg DOC Additive = 10.0 kg POC Additive = 12.5 kg Required level, influent water >50 - >5 - >5 - Influent water (WST) 61.9 5.0 5.3 0.3 7.2 1.8 Treated day 0 (TT1) 59.2 2.5 5.3 0.1 7.7 0.2 Treated day 5 (TT2) 12.1 0.8 4.9 0.2 1.1 0.1 Control day 0 (CT2) 56.3 1.9 5.2 0.1 7.5 0.2 Control day 5 (CT2) 62.0 1.6 5.2 0.1 5.7 1.3 TSS mg/l DOC, mg/l POC, mg/l TEST 8 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 17.5 kg DOC Additive = 10.0 kg POC Additive = 12.5 kg Required level, influent water >50 - >5 - >5 - Influent water (WST) 58.0 1.0 6.1 0.3 8.7 0.3 Treated day 0 (TT1) 58.6 1.3 6.5 0.1 7.4 0.7 Treated day 5 (TT2) 18.9 3.6 6.3 0.1 1.6 0.4 Control day 0 (CT2) 57.6 0.9 6.0 0.1 7.9 1.1 Control day 5 (CT2) 70.5 4.8 5.8 0.1 8.6 0.8 TSS mg/l DOC, mg/l POC, mg/l TEST 9 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 17.5 kg DOC Additive = 10.0 kg POC Additive = 12.5 kg Required level, influent water >50 - >5 - >5 - Influent water (WST) 68.4 2.2 6.0 0.3 7.2 1.0 Treated day 0 (TT1) 67.8 3.5 6.0 0.1 7.5 0.3 Treated day 5 (TT2) 17.9 1.6 5.6 0.1 1.2 0.1 Control day 0 (CT2) 66.1 2.3 5.9 0.1 6.5 0.7 Control day 5 (CT2) 23.4 0.8 5.7 0.0 2.4 0.1 TSS mg/l DOC, mg/l POC, mg/l TEST 10 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 17.5 kg DOC Additive = 10.0 kg POC Additive = 12.5 kg Required level, influent water >50 - >5 - >5 - Influent water (WST) 57.0 1.2 6.0 0.0 6.5 0.2 Treated day 0 (TT1) 55.9 0.5 6.2 0.3 7.0 1.2 Treated day 5 (TT2) 17.0 1.2 5.5 0.2 1.3 0.1 Control day 0 (CT2) 54.1 1.6 5.9 0.2 5.8 1.2 Control day 5 (CT2) 19.6 0.3 5.7 0.1 2.0 0.0 TSS mg/l DOC, mg/l POC, mg/l TEST 11 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 2.0 kg DOC Additive = 2.5 kg POC Additive = 3.0 kg Required level, influent water >1 - >1 - >1 - Influent water (WST) 14.6 2.5 2.2 0.0 2.5 0.1 Treated day 0 (TT1) 14.4 3.2 2.2 0.1 2.2 0.3 Treated day 5 (TT2) 6.5 2.2 2.0 0.1 0.9 0.2NIVA 5659-2008

27 Control day 0 (CT2) 15.9 1.0 2.1 0.1 2.6 0.4 Control day 5 (CT2) 6.3 1.2 1.9 0.1 0.9 0.0 TSS mg/l DOC, mg/l POC, mg/l TEST 12 Average Stdev Average Stdev Average Stdev Additions to influent water TSS Additive = 2.0 kg DOC Additive = 2.5 kg POC Additive = 3.0 kg Required level, influent water >1 - >1 - >1 - Influent water (WST) 18.2 4.1 2.3 0.2 2.4 0.4 Treated day 0 (TT1) 14.6 1.7 2.3 0.1 2.0 0.4 Treated day 5 (TT2) 5.8 0.8 2.2 0.0 0.7 0.0 Control day 0 (CT2) 14.9 4.9 2.4 0.1 3.2 0.6 Control day 5 (CT2) 5.3 0.4 2.1 0.1 0.7 0.1



## 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 in itial test waters are given in Table 11.

Organisms ≥ 50 μm in minimum diameter The requirements regarding density of the ≥ 50 μm group was met in all test cycles, except in test 5 where the concentration of organisms were determin ed to be below the requirement. The requirements regarding the biological diversity within the population were fulfilled for all tests.

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. As a supplementary estimate, samples were spread on agar plates to count colony forming units. The requirements regarding influent density of the ≥ 10-50 μm group was met in all tests. The requirements regarding the biological diversity within the population were fulfilled for all tests.

Heterotrophic bacteria The only requirement regarding bacteria in influent water is that heterotrophic bacteria should be present in a density of ≥ 10 4 cfu ml -1 . This requirement was fulfilled in all tests as shown in Table 11.

The IMO G8 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 11. Vibrio cholerae was not detected in any of the test cycles.NIVA 5659-2008

28 Table 10. Initial content of organisms within the defined t est organism groups (ref. Table 2) in the test water in test 1-12. Green background indicates th at required level was fulfilled, yellow background partial fulfilment, while red background i ndicates failure to fulfil required level. Test organism Method Influent Requirement Test 1 Microscope counts 250 333 ± 16 790 ≥ 10 5 m -3 Phyla 3 ≥ 3 different Organisms
≥ 50 μm Species 7 ≥ 5 different 5000 Dilution method 95 % conf. interval 2000-20 000 Microscope counts 5705 ± 1036 Plate counts 2350 ≥ 1000 ml -1

Phyla 3 ≥ 3 different Organisms ≥ 10-50 μ m

Species 15 ≥ 5 different Marine heterotrophic bacteria Bacterial counts 3.0 ± 0.5 x 10 4 ≥ 10 4 cfu ml -1
Coliform bacteria Bacterial counts 3.7 ± 0.6 x 10 0

- Vibrio sp. Bacterial counts 0 ± 0 - Vibrio cholerae Bacterial counts, elimination method - - Enterococcus group Bacterial counts 1.3 ± 1.2 x 10 0

- Test 2 Microscope counts 219 696 ± 21 082 ≥ 10 5 m -3 Phyla 3 ≥ 3 different Organisms
≥ 50 μm Species 9 ≥ 5 different 2400 Dilution method 95 % conf. interval 1000-9500 Microscope counts 2299 ± 286 Plate counts 1550 ≥ 1000 ml -1

Phyla 3 ≥ 3 different Organisms ≥ 10-50 μ m

Species 12 ≥ 5 different Marine heterotrophic bacteria Bacterial counts 2.1 ± 0.05 x 10 4 ≥ 10 4 cfu ml -1
Coliform bacteria Bacterial counts 2.3 ± 2.3 x 10 0

- Vibrio sp. Bacterial counts 4.1 ± 0.06 x 10 3

- Vibrio cholerae Bacterial counts, elimination method - - Enterococcus group Bacterial counts 1.0 ± 1.0 x 10 0

- Test 3 Microscope counts 146 375 ± 8420 ≥ 10 5 m -3 Phyla 3 ≥ 3 different Organisms
≥ 50 μm Species 8 ≥ 5 different 2400 Dilution method 95 % conf. interval 1000-9500 Microscope counts 1556 ± 412 Plate counts 1350 ≥ 1000 ml -1

Phyla 3 ≥ 3 different Organisms ≥ 10-50 μ m

Species 10 ≥ 5 different Marine heterotrophic bacteria Bacterial counts 3.1 ± 0.2 x 10 4 ≥ 10 4 cfu ml -1
Coliform bacteria Bacterial counts 0 ± 0 - Vibrio sp. Bacterial counts 3.7 ± 2.8 x 10 3

- Vibrio cholerae Bacterial counts, elimination method - - Enterococcus group Bacterial counts 0 ± 0 - Test 4 Organisms Microscope counts 117 767 ± 10 572 ≥ 10 5 m -3NIVA 5659-2008

29 Phyla 3 ≥ 3 different ≥ 50 μm Species 7 ≥ 5 different 2200 Dilution method 95 % conf. interval 900 ± 5900 Microscope counts 2072 ± 110 Plate counts 1500 ≥ 1000 ml -1

Phyla 4 ≥ 3 different Organisms ≥ 10-50 μ m

Species 16 ≥ 5 different Marine heterotrophic bacteria Bacterial counts 2.4 ± 0.3 x 10 4 ≥ 10 4 cfu ml -1
Coliform bacteria Bacterial counts 1.0 ± 1.7 x 10 0

- Vibrio sp. Bacterial counts 6.6 ± 1.4 x 10 3

- Vibrio cholerae Bacterial counts, elimination method - - Enterococcus group Bacterial counts 1.3 ± 1.5 x 10 0

- Test 5 Microscope counts 71 229 ± 14 144 ≥ 10 5 m -3 Phyla 3 ≥ 3 different Organisms
≥ 50 μm Species 6 ≥ 5 different 2400 Dilution method 95 % conf. interval 1000-9500 Microscope counts 1781 ± 143 Plate counts 1000 ≥ 1000 ml -1

Phyla 3 ≥ 3 different Organisms ≥ 10-50 μ m

Species 7 ≥ 5 different Marine heterotrophic bacteria Bacterial counts 1.7 ± 0.5 x 10 4 ≥ 10 4 cfu ml -1
Coliform bacteria Bacterial counts 0 ± 0 - Vibrio sp. Bacterial counts 1.2 ± 0.2 x 10 3

- Vibrio cholerae Bacterial counts, elimination method - - Enterococcus group Bacterial counts 0 ± 0 - Test 6 Microscope counts 232 708 ± 29 189 ≥ 10 5 m -3 Phyla 3 ≥ 3 different Organisms
≥ 50 μm Species 8 ≥ 5 different 1700 Dilution method 95 % conf. interval 700-4800 Microscope counts 2109 ± 277 Plate counts 1600 ≥ 1000 ml -1

Phyla 3 ≥ 3 different Organisms ≥ 10-50 μ m

Species 9 ≥ 5 different Marine heterotrophic bacteria Bacterial counts 3.0 ± 0.3 x 10 4 ≥ 10 4 cfu ml -1
Coliform bacteria Bacterial counts 2.3 ± 2.5 x 10 0

- Vibrio sp. Bacterial counts 1.2 ± 0.4 x 10 4

- Vibrio cholerae Bacterial counts, elimination method - - Enterococcus group Bacterial counts 0.3 ± 0.6 x 10 0

- Test 7 Microscope counts 253 438 ± 8083 ≥ 10 5 m -3 Phyla 3 ≥ 3 different Organisms
≥ 50 μm Species 8 ≥ 5 different 2400 Dilution method 95 % conf. interval 1000-9500 Microscope counts 1776 ± 202 Organisms ≥ 10-50 μ m

Plate counts 1175 ≥ 1000 ml -1NIVA 5659-2008

30 Phyla 3 ≥ 3 different Species 13 ≥ 5 different Marine heterotrophic bacteria Bacterial counts 2.3 ± 0.6 x 10 4 ≥ 10 4 cfu ml -1
Coliform bacteria Bacterial counts 1.7 ± 0.6 x 10 0

- Vibrio sp. Bacterial counts 2.5 ± 0.2 x 10 3

- Vibrio cholerae Bacterial counts, elimination method - - Enterococcus group Bacterial counts 1.0 ± 1.0 x 10 0

- Test 8 Microscope counts 265 083 ± 17 924 ≥ 10 5 m -3 Phyla 5 ≥ 3 different Organisms
≥ 50 μm Species 6 ≥ 5 different 3000 Dilution method 95 % conf. interval 1000-13000 Microscope counts 2800 ± 494 Plate counts 3550 ≥ 1000 ml -1

Phyla 3 ≥ 3 different Organisms ≥ 10-50 μ m

Species 7 ≥ 5 different Marine heterotrophic bacteria Bacterial counts 1.8 ± 0.2 x 10 4 ≥ 10 4 cfu ml -1
Coliform bacteria Bacterial counts 2.0 ± 1.0 x 10 0

- Vibrio sp. Bacterial counts 1.9 ± 0.1 x 10 3

- Vibrio cholerae Bacterial counts, elimination method - - Enterococcus group Bacterial counts 1.7 ± 0.6 x 10 0

- Test 9 Microscope counts 262 167 ± 42 253 ≥ 10 5 m -3 Phyla 7 ≥ 3 different Organisms
≥ 50 μm Species 8 ≥ 5 different 2400 Dilution method 95 % conf. interval 1000-9500 Microscope counts 2117 ± 264 Plate counts 1725 ≥ 1000 ml -1

Phyla 4 ≥ 3 different Organisms ≥ 10-50 μ m

Species 8 ≥ 5 different Marine heterotrophic bacteria Bacterial counts 8.6 ± 0.9 x 10 4 ≥ 10 4 cfu ml -1
Coliform bacteria Bacterial counts 3.3 ± 2.1 x 10 1

- Vibrio sp. Bacterial counts 3.1 ± 0.04 x 10 3

- Vibrio cholerae Bacterial counts, elimination method - - Enterococcus group Bacterial counts 3.3 ± 1.2 x 10 0

- Test 10 Microscope counts 260 292 ±31 001 ≥ 10 5 m -3 Phyla 6 ≥ 3 different Organisms
≥ 50 μm Species 5 ≥ 5 different 1300 Dilution method 95 % conf. interval 500-3900 Microscope counts 1927 ± 182 Plate counts 1700 ≥ 1000 ml -1

Phyla 3 ≥ 3 different Organisms ≥ 10-50 μ m

Species 9 ≥ 5 different Marine heterotrophic bacteria Bacterial counts 1.2 ± 0.006 x 10 5 ≥ 10 4 cfu ml -1
Coliform bacteria Bacterial counts 3.0 ± 5.2 x 10 0

- Vibrio sp. Bacterial counts 1.4 ± 0.1 x 10 3

- Vibrio cholerae Bacterial counts, elimination method - -NIVA 5659-2008

31 Enterococcus group Bacterial counts 1.3 ± 1.5 x 10 0

- Test 11 Microscope counts 248 008 ± 35 848 ≥ 10 5 m -3 Phyla 4 ≥ 3 different Organisms
≥ 50 μm Species 9 ≥ 5 different 5000 Dilution method 95 % conf. interval 2000-20000 Microscope counts 2013 ± 54 Plate counts 1400 ≥ 1000 ml -1

Phyla 3 ≥ 3 different Organisms ≥ 10-50 μ m

Species 20 ≥ 5 different Marine heterotrophic bacteria Bacterial counts 1.8 ± 0.2 x 10 5 ≥ 10 4 cfu ml -1
Coliform bacteria Bacterial counts 1.3 ± 0.6 x 10 0

- Vibrio sp. Bacterial counts 1.3 ± 1.1 x 10 4

- Vibrio cholerae Bacterial counts, elimination method - - Enterococcus group Bacterial counts 0 ± 0 - Test 12 Microscope counts 200 404 ± 6019 ≥ 10 5 m -3 Phyla 3 ≥ 3 different Organisms
≥ 50 μm Species 6 ≥ 5 different 5000 Dilution method 95 % conf. interval 2000-20000 Microscope counts 4010 ± 353 Plate counts 3050 ≥ 1000 ml -1

Phyla 3 ≥ 3 different Organisms ≥ 10-50 μ m

Species 8 ≥ 5 different Marine heterotrophic bacteria Bacterial counts 4.0 ± 0.0 x 10 4 ≥ 10 4 cfu ml -1
Coliform bacteria Bacterial counts 0 ± 0 - Vibrio sp. Bacterial counts 6.9 ± 0.8 x 10 4

- Vibrio cholerae Bacterial counts, elimination method - - Enterococcus group Bacterial counts 1.3 ± 1.15 x 10 0

-NIVA 5659-2008

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 12. Because UV- treated plankton takes some time to die counts were repeated after 24 hours. It is these counts that are reported in Table 12. 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 storag e. This requirement was fulfilled in all tests.

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 .” This has been interpreted to 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 organisms m -3 . This requirement was fulfilled in all tests.

Table 11. 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, yellow background partial fulf ilment, while red background indicates failure to fulfil required level. 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 7.7 ± 3.2 0 ± 0 86 407 ± 13 848 59 278 ± 11 484 Test cycle 2 19.0 ± 20.8 0 ± 0 88 250 ± 5987 58 083 ± 7062 Test cycle 3 9.0 ± 5.3 0 ± 0 67 870 ± 5271 44 963 ± 4470 Test cycle 4 7.7 ± 5.7 0 ± 0 53 313 ± 5164 48 032 ± 5853 Test cycle 5 1.3 ± 2.3 0 ± 0 9246 ± 871 7245 ± 2408 Test cycle 6 144.0 ± 55.6 1.0 ± 1.0 110 333 ± 19 332 64 819 ± 18 440 Test cycle 7 6.7 ± 9.9 1.3 ± 0.6 133 676 ± 9303 58 574 ± 16 971 Test cycle 8 74.3 ± 18.1 1.0 ± 0 165 389 ± 35 011 49 032 ± 12 602 Test cycle 9 51.0 ± 68.1 1.0 ± 1.0 132 185 ± 4720 66 093 ± 2360 Test cycle 10 64.3 ± 23.9 0 ± 0 122 241 ± 37 182 14 852 ± 1587 Test cycle 11 1.0 ± 1.7 0 ± 0 72 657 ± 13 845 21 319 ± 1553 Test cycle 12 2.3 ± 2.5 0 ± 0 69 148 ± 6625 31 142 ± 12 158
* With UV treatment, it takes some time be fore death manifest itself. Counting wa s therefore repeated after 24 hours and these are the values that are reported in the table.



## 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, by microscopy examination after incubation with CFDA-AM and plating on seawater agar plates, in treated test water and control immediately after treatment and after five days of storage is given in Table 13. For the performance of the ballast water treatment system to pass the regulation D-2 of the IMO Guidelines, as gi ven in Table 2, less than 10 viable organisms ≥ 10- 50 μm in minimum diameter per ml should be present in the treated water after five days storage. This requirement was fulfilled in all test cy cles using all three detection methods.

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 theNIVA 5659-2008

33 values in Regulation D-2.1, the test cycle is invalid.” It was interpreted to be 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 require ment was fulfilled in all test cycles.

Table 12. 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, yellow background partial fulf ilment, while red background indicates failure to fulfil required level. Treated water Control water Day 0 Day 5 Day 0 Day 5 Organisms ≥ 10-50 μm in minimum diameter (individuals ml -1 ) Requirement - <10 - >100 Dilution method 95 % confidence interval

<0.2 <0.2 2000 2400 Test cycle 1 <0.1-1.0 <0.1-1.0 1-14 x 10 3 1-9.5 x 10 3
<0.2 <0.2 5000 2400 Test cycle 2 <0.1-1.0 <0.1-1.0 2-24 x 10 3 1-9.5 x 10 3
<0.2 <0.2 2000 2400 Test cycle 3 <0.1-1.0 <0.1-1.0 1-14 x 10 3 1-9.5 x 10 3
<0.2 <0.2 2000 2400 Test cycle 4 <0.1-1.0 <0.1-1.0 1-14 x 10 3 1-9.5 x 10 3
<0.2 <0.2 2000 2400 Test cycle 5 <0.1-1.0 <0.1-1.0 1-14 x 10 3 1-9.5 x 10 3
13 <0.2 2000 2200 Test cycle 6 5-33 <0.1-1.0 1-14 x 10 3 0.9-5.9 x 10 3
50 <0.2 5000 2400 Test cycle 7 20-200 <0.1-1.0 2-20 x 10 3 1-9.5 x 10 3
3 <0.2 5000 3000 Test cycle 8 1-12 <0.1-1.0 2-24 x 10 3 1-13 x 10 3
22 <0.2 1500 2400 Test cycle 9 9-59 <0.1-1.0 0.5-5.1 x 10 3 1-9.5 x 10 3
30 <0.2 5000 2400 Test cycle 10 10-130 <0.1-1.0 2-24 x 10 3 1-9.5 x 10 3
0.2 <0.2 1500 2400 Test cycle 11 0.1-1.7 <0.1-1.0 0.5-5.1 x 10 3 1-9.5 x 10 3
1.7 <0.2 2000 2400 Test cycle 12 0.7-4.7 <0.1-1.0 1-14 x 10 3 1-9.5 x 10 3 Microscope counts Test cycle 1 17.7 ± 4.7 0.7 ± 0.6 5169 ± 931 5324 ± 158 Test cycle 2 11.3 ± 4.0 0 ± 0 2576 ± 337 2489 ± 180 Test cycle 3 13.3 ± 3.8 0 ± 0 1106 ± 131 2437 ± 207 Test cycle 4 25.3 ± 5.7 0 ± 0 1953 ± 40 2057 ± 79 Test cycle 5 29.0 ± 7.0 0 ± 0 1348 ± 182 2351 ± 376 Test cycle 6 66.3 ± 14.0 3.3 ± 0.6 2195 ± 152 2083 ± 526 Test cycle 7 53.3 ± 12.0 4.7 ± 2.1 1927 ± 143 2610 ± 252 Test cycle 8 82.7 ± 36.1 7.7 ± 0.6 3007 ± 510 2964 ± 226 Test cycle 9 92 ± 14.4 5.3 ± 2.1 2186 ± 220 1555 ± 233 Test cycle 10 66.0 ± 2.0 5.3 ± 2.9 1996 ± 186 1279 ± 104 Test cycle 11 44.7 ± 15.0 0 ± 0 1987 ± 166 1357 ± 91 Test cycle 12 42.7 ± 1.2 0 ± 0 3699 ± 559 3474 ± 323NIVA 5659-2008

34 Plate counts Test cycle 1 <10 <10 NA 3100 Test cycle 2 <10 <10 NA 1100 Test cycle 3 <10 <10 NA 2200 Test cycle 4 <10 <10 NA 2250 Test cycle 5 <10 <10 NA 2150 Test cycle 6 10 <10 NA 900 Test cycle 7 50 <10 NA 1760 Test cycle 8 106 <10 NA 1317 Test cycle 9 103 <10 NA 1750 Test cycle 10 40 <10 NA 1450 Test cycle 11 <10 <10 NA 1350 Test cycle 12 <10 <10 NA 3700



## 3.7 Bactericidal effects

The numbers of coliform bacteria, E.coli, Vibrio sp., Vibrio cholerae, Enterococcus group 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 are given in Table 14. Regulation D-2 only requires documentation of the bactericidal effect of the ballast water treatment system on E .coli

, Vibrio cholerae (toxicogenic serotypes O1 and O139) and intestinal Enterococci given as a maximum allowable concentration after five days storage of <250 cfu/100ml, <1 cfu/100ml and <100 cfu/100ml, respectively. These requirements were fu lfilled for all bacterial species.

Vibrio sp. is common in salt and brackish surface water. However, the possibility of further detecting of the serotypes O1 and O139 is very small or abscen t since none of these serotypes have been isolated from Norwegian coastal water. Vibrio cholerae was not found in any of the samples from treated water during the 12 test cycles.

Table 13. Coliform bacteria, E.coli, Vibrio sp. Vibrio cholera , Enterococcus group and intestinal Enterococci in treated test water and control water immediately after treatment and after five days of storage for test cycles 1-12. Green background indicates that required level was fulfilled, yellow background partial fulfilment, while red backgroun d indicates failure to fulfil required level. Treated water Control water Day 0 Day 5 Day 0 Day 5 Marine heterotrophic bacteria (cfu ml -1 ) Requirement - - - - Test cycle 1 0.9 ± 0.9 x 10 1 1.2 ± 0.5 x 10 1 6.2 ± 4.8 x 10 4 2.9 ± 2.9 x 10 5 Test cycle 2 2.4 ± 0.2 x 10 1 6.1 ± 0.7 x 10 1 2.4 ± 0.7 x 10 4 2.1 ± 0.4 x 10 5 Test cycle 3 1.2 ± 0.06 x 10 2 5.9 ± 0.4 x 10 1 3.6 ± 0.2 x 10 4 3.7 ± 0.5 x 10 5 Test cycle 4 2.2 ± 0.2 x 10 2 5.7 ± 1.6 x 10 1 2.4 ± 0.3 x 10 4 3.2 ± 0.9 x 10 5 Test cycle 5 4.9 ± 0.7 x 10 1 2.0 ± 0.6 x 10 1 1.2 ± 0.2 x 10 4 1.3 ± 0.2 x 10 5 Test cycle 6 1.9 ± 0.2 x 10 2 2.1 ± 0.4 x 10 2 3.3 ± 0.6 x 10 4 1.4 ± 0.8 x 10 6 Test cycle 7 8.3 ± 2.7 x 10 1 3.4 ± 0.3 x 10 1 3.3 ± 1.1 x 10 4 6.0 ± 0.9 x 10 5 Test cycle 8 3.4 ± 0.4 x 10 1 2.0 ± 0.9 x 10 3 1.8 ± 0.3 x 10 4 4.1 ± 1.0 x 10 5 Test cycle 9 2.2 ± 0.9 x 10 3 1.1 ± 0.2 x 10 2 8.9 ± 1.2 x 10 4 4.3 ± 0.5 x 10 4 Test cycle 10 2.2 ± 0.2 x 10 2 3.9 ± 1.9 x 10 0 1.3 ± 0.1 x 10 5 3.6 ± 1.5 x 10 4 Test cycle 11 3.3 ± 0.8 x 10 2 1.5 ± 0.3 x 10 2 1.6 ± 0.2 x 10 5 2.9 ± 0.8 x 10 5 Test cycle 12 6.3 ± 1.1 x 10 2 3.1 ± 1.2 x 10 1 4.6 ± 0.9 x 10 4 9.2 ± 0.6 x 10 4 Coliform bacteria (Coli.) and Escherichia coli (E. coli) * (cfu 100 ml -1 )NIVA 5659-2008

35 Coli. Coli. E. coli Coli. Coli. Requirement - - <250* - - Test cycle 1 0 ± 0 0 ± 0 0 ± 0 5.3 ± 0.6 x 10 0 0 ± 0 Test cycle 2 0 ± 0 0 ± 0 0 ± 0 4.3 ± 1.2 x 10 0 0 ± 0 Test cycle 3 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Test cycle 4 0 ± 0 0 ± 0 0 ± 0 1.7 ± 2.9 x 10 0 0 ± 0 Test cycle 5 0 ± 0 0 ± 0 0 ± 0 0.7 ± 1.2 x 10 0 0 ± 0 Test cycle 6 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Test cycle 7 0 ± 0 0 ± 0 0 ± 0 1.3 ± 0.6 x 10 0 0 ± 0 Test cycle 8 0 ± 0 0 ± 0 0 ± 0 1.7 ± 0.6 x 10 0 0.7 ± 0.6 x 10 0 Test cycle 9 0 ± 0 0 ± 0 0 ± 0 2.0 ± 1.0 x 10 1 2.3 ± 2.1 x 10 0 Test cycle 10 0 ± 0 2 ±1 0 ± 0 1.1 ± 0.8 x 10 1 0.3 ± 0.6 x 10 0 Test cycle 11 0 ± 0 0 ± 0 0 ± 0 2.0 ± 0.0 x 10 1 0.7 ± 0.6 x 10 0 Test cycle 12 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Vibrio sp. and Vibrio cholerae ** ( V. cholerae ) (cfu 100 ml -1 ) Vibrio sp. Vibrio sp V. cholerae Vibrio sp. Vibrio sp. Requirement - - <1** - - Test cycle 1 0 ± 0 6.8 ± 3.4 x 10 1 < 1 ** 0.6 ± 1.0 x 10 3 5.6 ± 0.5 x 10 2 Test cycle 2 0.9 ± 0.9 x 10 0 0.6 ± 0.5 x10 0 < 1** 4.8 ± 0.5 x 10 3 4.4 ± 0.3 x 10 3 Test cycle 3 0.3 ± 0.5 x 10 0 0.6 ± 1.0 x 10 1 < 1** 5.6 ± 5.6 x 10 3 5.6 ± 1.6 x 10 3 Test cycle 4 0 ± 0 2.4 ± 0.5 x 10 0 < 1** 8.3 ± 1.0 x 10 3 5.6 ± 1.0 x 10 3 Test cycle 5 1.8 ± 0.9 x 10 0 0 ± 0 < 1** 1.5 ± 0.3 x 10 3 1.0 ± 0.2 x 10 3 Test cycle 6 0.3 ± 0.5 x 10 0 0.9 ± 0.9 x 10 0 < 1 ** 2.0 ± 0.8 x 10 4 3.3 ± 0.1 x 10 3 Test cycle 7 0.3 ± 0.5 x 10 0 1.2 ± 0.5 x 10 0 < 1 ** 5.0 ± 0.3 x 10 3 2.3 ± 0.5 x 10 3 Test cycle 8 0.6 ± 0.5 x 10 0 0 ± 0 < 1 ** 2.3 ± 1.0 x 10 3 3.0 ± 0.2 x 10 3 Test cycle 9 0.3 ± 0.5 x 10 0 1.8 ± 2.4 x 10 0 < 1 ** 3.2 ± 0.1 x 10 3 1.6 ± 0.3 x 10 3 Test cycle 10 3.6 ± 4.8 x 10 0 0 ± 0 < 1 ** 2.1 ± 0.1 x 10 3 7.0 ± 1.6 x 10 2 Test cycle 11 4.8 ± 3.2 x 10 0 0.3 ± 0.5 x 10 0 < 1** 1. 6 ± 0.4 x 10 4 9.9 ± 0.6 x 10 3 Test cycle 12 8.3 ± 0.6 x 10 1 2.6 ± 2.0 x 10 1 < 1** 8.3 ± 0.2 x 10 4 2.9 ± 2.6 x 10 4 Enterococcus group (Ent. gr.) and Intestinal Enterococci ( Int. Ent.) *** (cfu 100 ml -1 ) Ent. gr. Ent. gr. Int. Ent. Ent. gr. Ent. gr. Requirement - - <100*** - - Test cycle 1 0 ±1 0 ± 0 0 ± 0 3.3 ± 2.1 x 10 0 0 ± 0 Test cycle 2 1.7 ± 2.4 x 10 1 0 ± 0 0 ± 0 1.7 ± 2.1 x 10 0 2.0 ± 1.0 x 10 0 Test cycle 3 0 ± 0 0 ± 0 0 ± 0 0.3 ± 0.6 x 10 0 1.0 ± 1.0 x 10 0 Test cycle 4 0 ± 0 0 ± 0 0 ± 0 0 ± 0 2.0 ± 1.0 x 10 0 Test cycle 5 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Test cycle 6 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Test cycle 7 0 ± 0 0 ± 0 0 ± 0 0.3 ± 0.6 x 10 0 0.3 ± 0.6 x 10 0 Test cycle 8 0 ± 0 0 ± 0 0 ± 0 0.3 ± 0.6 x 10 0 1.0 ± 0.0 x 10 0 Test cycle 9 0 ± 0 0 ± 0 0 ± 0 5.0 ± 1.7 x 10 0 2.0 ± 2.0 x 10 0 Test cycle 10 0 ± 0 0 ± 0 0 ± 0 1.0 ± 1.0 x 10 0 0.3 ± 0.6 x 10 0 Test cycle 11 0 ± 0 0 ± 0 0 ± 0 0.7 ± 1.2 x 10 0 0 ± 0 Test cycle 12 0 ± 0 0 ± 0 0 ± 0 0.7 ± 0.6 x 10 0 0 ± 0 * 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 identified as 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).NIVA 5659-2008

36 

# 4. References

APHA (1995) Standard Methods for the Examination of Water and Wastewater , 19 th ed. American Public Health Association, Washington, DC.

APHA (1995) 2540 D Total suspended solids dried at 103 – 105 °C. Standard Methods for the Examination of Water and Wastewater , 19 th ed., American Public Health Association, Washington, DC. pp. 2-54.

HACH (2001) Odyssey DR/2500 Spectrophotometer Procedure manual, HACH Company. US EPADPD method nr. 8167 and nr. 8021.

IMO (2004) International Convention for the Contro l and Management of Sh ips' Ballast Water and Sediments, the International Maritime Orga nisation (IMO), adopted 13 February 2004.

International Standard NS-ISO 5667-3:2001.Water quality – Sampling – Part 3: Guidance on the preservation and handling of samples. 2. ed. 2004, Norges Standardiseringsforbund, Oslo.

International Standard NS-EN ISO 19458:2006. Wate r quality- Sampling for microbiological analysis. 1. ed. 2006, Norges Standardiseringsforbund, Oslo.

International Standard NS-EN ISO 9308-1:2000. Water quality-Detection and enumeration of Escherichia coli and coliform bacteria. Part 1: membrane filtration method. 1. ed. 2000, Norges Standardiseringsforbund, Oslo.

MEPC (2004) Harmful aquatic organisms in ballast water. Report of the Ballast Water Working Group. Prepared by the Marine Environment Pr otection Committee (MEPC) for the International Maritime Organisation (IMO), MEPC 52/WP.7, 13 October 2004.

Norwegian Accreditation No. TEST 009 (1993) for NIVA laboratory. Accreditation of chemical testing, taxonomy, and sampling according to Norwegian Standard NS-EN ISO/IEC 17025.

Norwegian Standard NS 4792:1990. Water qualit y - Thermotolerant coliform bacteria and presumptive E.coli - Membrane filtration method. 1.ed. 1990, Norges Standardiseringsforbund, Oslo.

Norwegian Standard NS-EN ISO 6222:1999. Wate r analysis - Enumeration of culturable microorganisms (bacterial counts) - Colony count by inoculation in a nutrient agar culture medium. 1. ed. 1999. Norges Standardiseringsforbund, Oslo.

Norwegian Standard NS-EN ISO 7899-2:2000. Wa ter quality - Detection and enumeration of intestinal enterococci - Part 2. Membrane filtration method. 1. ed. 2000, Norges Standardiseringsforbund, Oslo.

Norwegian Standard NS 4788 Water examination - co liform bacteria – membrane filtration method. 1. ed. 1990. Norges Standardiseringsforbund, Oslo.

Norwegian Standard ISO 8245:1991. Water quality- Guid elines for the determination of total organic carbon. 1. ed. 1991, Norges Standardiseringsforbund, Oslo.NIVA 5659-2008

37 OECD (1985). OECD Test Guideline for Testing of Chemicals 202; Daphnia sp. Acute immobolisation test and reproduction test.

Resolution MEPC.125(53). Annex 3 of MEPC 53/24/Add1. Adopted on 22 July 2005. Guidelines for approval of ballast water management systems (G8).

Resolution MEPC.125(53). Annex 4 of MEPC 53/24/Add1. Adopted on 22 July 2005. Procedure for approval of ballast water management systems that make use of active substances (G).

Throndsen, J (1978). Chapter 7.6: The dilution-culture method. p. 218-224. In: Phytoplankton manual. Ed: Sourina, A. Published by UNESCO, France.

UNESCO (1981) UNESCO Technical Papers in Marine Science No. 39 and No. 40.Gaustadalléen 21 • NO-0349 Oslo, Norway Telephone: +47 22 18 51 00 • Fax: 22 18 52 00 www.niva.no • post@niva.no