REPORT SNO 5659-2008 CONFIDENTIAL

Land based testing of the OptiMarin 

ballast water management system of 

OptiMarin AS - Treatment Effect Studies

Final Report
NIVA 5659-2008 

Norwegian Institute for Water Research 

– an institute in the Environmental Research Alliance of Norway REPORT

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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 

27th 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 total 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 978-82-577-5394-8 
NIVA 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 Johannesen, August Tobiessen, Christian 

Vogelsang, Tor Gunnar Jantsch and Helge Liltved were the main 

representatives. Several other OptiMarin 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 Jantsch 
NIVA 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 franciscana, 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 quantification 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) 18
NIVA 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 36
NIVA 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 

Xi - individual analytical result; in calculating the standard deviation 

X – the arithmetic mean of individual analytical results; in calculating the standard deviation 
NIVA 5659-2008 

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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 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, hereafter referred to as G8 and G9. Only the biological effect 

studies according to G8 are reported here. The tests were carried out at NIVA’s test site located at 

Solbergstrand 20 km south of Oslo, with medium salinity 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 m3

 tank (WST). A combination of indigenous harvested 

organisms and cultured surrogate 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 the 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 includes consecutive treatment of >200 m3

 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 m3

 of the same type of prepared test water from WST to a ballast tank 

(CT2) for at least 5 days storage using the pump of the OptiMarin BWMS, but in by-pass of the 

treatment unit (filter and UV). After storage the control 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 solids (TSS), dissolved organic carbon (DOC) and 

particulate organic carbon (POC) were fulfilled 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 concentration was below the IMO requirement. The 

requirements regarding the biological diversity within 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 within the population were fulfilled in all tests. 

• The only requirement regarding bacteria in influent water is that heterotrophic bacteria should 

be present in a density of ≥104

 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 m3

 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 m3

, 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 maximum 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 Procedure for approval of ballast water management systems 

that make use of active substances (G9) Res. MEPC.125(53) Annex 4 has been conducted by NIVA 

and is reported elsewhere 
NIVA 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 active substances in their treatment process, treated 

ballastwater was also analysed for toxicity and 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 pilot 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 biological and toxicological requirements stated in the 

IMO Guideline. By performing pilottests, difficulties and “bottle-necks” could be detected, and 

measures could be taken to avoid problems during 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 here, 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 down 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 particles, 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 water treatment system 

2.2.1 General description of the OptiMarin BWMS 

During ballasting, the OptiMarin BWMS applied 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 μm 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 ballasting operation. The back-flushing flow rate is 

approximately 20 m3

/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 requires 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 m3

/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 m3

/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 

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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 m3 tank (WST) from high salinity sea water from 60 meters depth or 

from brackish surface water depending on the required salinity, >32 PSU or 3-32 PSU (as specified in 

G8), respectively, with a minimum difference of 10 PSU. The 516 m3

 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 the limits of chemical water quality criteria. 

2.3.1 Overview of requirements 

Table 1 summarizes the chemical requirements of the 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/l

Table 2. Required biological water quality in influent test 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. 106 m-3, ≥105 m-3

Min. 5 species from 3 diff. 

phyla/divisions 

<10 viable organisms 

per m3

>10x <10 viable 

organisms per m3

≥10-50 μm min. 

dimension 

104 ml-1, ≥103 ml-1

Min. 5 species from 3 diff. 

phyla/divisions 

<10 viable organisms 

per ml 

>10x <10 viable 

organisms per ml 

Heterotrophic 

bacteria 

≥104 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 solids (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 organisms 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 exponentially 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 species from three different phyla/divisions of both test 

groups of organisms ≥10 μm in minimum dimensions was assured by harvesting indigenous algae and 

planktonic animal species from surface water outside Solbergstrand research station The species 

composition and viability of harvested organisms were evaluated by microscopy. 

Cultivation of Artemia franciscana, Tetraselmis 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 105

 nauplii per m3 for A. 

franciscana and 103

 per ml for B. submarina and T. suecica assuming a final volume of the test water 

of 516 m3

. 

Bacteria 

The concentration of heterotrophic bacteria in the surface water is normally exceeding the required 

level of ≥104

 cfu ml-1, while the heterotrophic bacteria criteria for the high salinity test water is 

expected to be fulfilled by the heterotrophic 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 

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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, the 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, collecting 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 m3

 S2

TT1 

231 m3 

S5

UV

TT2 

205 m3

CT2 

205 m3

S1 

 S3

S4 

Filter 

Pump 

UV unit 

1

2

1 

2
NIVA 5659-2008 

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2) Sampling of organisms ≥ 50 μm in TT1 (S2), CT2 (S4), TT1 (S5) and TT2 (S3): A siphon spillway 

was used to collect 1 m3

 test water from TT1 directly after treatment and after 5 days storage (after 

transfer from TT1 to TT2). A similar method was used to collect control samples but then 45-90l 

were collected. The water was sieved directly through a plankton net (50 μm diagonal dimensions) 

attached to a plastic cup, collecting and concentrating 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 m3

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 m3 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 times 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-containers and transported to the laboratories for analysis. 

Table 4. Preservation methods and expected storage/holding times before measurement (ISO/CD 

5667-3, 2001). 

Parameter Preservation Max 

holding 

time 

Expected 

storage 

time 

Temperature - - 0 
NIVA 5659-2008 

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pH - - 0 

Dissolved oxygen - - 0 

Salinity - - 0 

Dissolved organic carbon 

(DOC) 

Acidify to pH<2 with 1 ml 4 M H2SO4 per 

100 ml, stored at 4o

C 7 days 0-5 days 

Particulate organic carbon 

(POC) 

Acidify to pH<2 with with 1 ml 4 M 

H2SO4 per 100 ml,stored at 4o

C in 

darkness 

7 days 

0-5 days 

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

Organisms ≥50 μm Stored at 4oC in darkness 6 hours < 2 h 

Organisms ≥10-50 μm Stored at 4o

C in darkness 6 hours < 6 h 

Heterotrophic bacteria 

Coliform bacteria, E.coli 

Enterococcus group bacteria, 

Intestintal enterococci

Vibrio sp. 

Vibrio cholerae

4oC 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 and meter. DO is reported as 

mg O2/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 level 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 electricity. 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 increase. Lowest reported value: 0.1 mg/l. 
NIVA 5659-2008 

17 

Total residual oxidants (TRO) 

Total residual oxidants were measured by the colorimetric DPD-method (American Public Health 

Association, 1989), which is currently the method recommended for measurement of TRO in 

seawater. 

2.6.2 Identification and quantification 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) was 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 (grow) 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 quantification 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-medium 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 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. 

Coliform bacteria. 

Coliform bacteria were quantified according to Norwegian 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 Norwegian 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 performed 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 used (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 technology was performed in each test cycle by the site 

responsible person from NIVA. Reports from these controls are included in Appendix D. Check points 

and operational parameters for documenting sufficient operation of the treatment technology were 

included and monitored in this quality control, including start and stop of ballasting 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 Optimarin 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 and the volume of the tanks, which give an accurate 

flow estimate. The ranges of calculated flowrates for all 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 

m3

/h 

Average 

flow 

deballasting 

m3/h 

Average 

flow 

control 

ballasting 

m3

/h 

Average 

flow 

control 

deballasting 

m3

/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 content 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. Measurements 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 chlorine were made and the results are below the 

practical detection limit of the analysis method, showing 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 (measurements were not conducted 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 

O2

% 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 

O2

% 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 

O2

% 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 

O2

% 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 

O2

% 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 - - <0.1 
NIVA 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 

O2

% 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 

O2

% 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 

O2

% 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 

O2

% 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 

O2

% 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 - - 10.60 
NIVA 5659-2008 

24 

O2

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 

O2

% 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 values 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 required level.To ensure fulfilment of the chemical 

requirements of the test water; a POC regulating compound (POCAdditive), a DOC regulating compound 

(DOCAdditive) and a TSS regulating compound (TSSadditive), 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 standard 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 TSSAdditive = 2.0 kg DOCAdditive = 2.5 kg POCAdditive = 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 TSSAdditive = 2.0 kg DOCAdditive = 2.5 kg POCAdditive = 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 TSSAdditive = 2.0 kg DOCAdditive = 2.5 kg POCAdditive = 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 TSSAdditive = 2.0 kg DOCAdditive = 2.5 kg POCAdditive = 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 TSSAdditive = 2.0 kg DOCAdditive = 2.5 kg POCAdditive = 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 Stdev 
NIVA 5659-2008 

26 

Additions to influent water TSSAdditive = 17.5 kg DOCAdditive = 10.0 kg POCAdditive = 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 TSSAdditive = 17.5 kg DOCAdditive = 10.0 kg POCAdditive = 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 TSSAdditive = 17.5 kg DOCAdditive = 10.0 kg POCAdditive = 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 TSSAdditive = 17.5 kg DOCAdditive = 10.0 kg POCAdditive = 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 TSSAdditive = 17.5 kg DOCAdditive = 10.0 kg POCAdditive = 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 TSSAdditive = 2.0 kg DOCAdditive = 2.5 kg POCAdditive = 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 0.2 
NIVA 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 TSSAdditive = 2.0 kg DOCAdditive = 2.5 kg POCAdditive = 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 initial 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 determined 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 ≥104

 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 “...the 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 test organism groups (ref. Table 2) in the test 

water in test 1-12. Green background indicates that required level was fulfilled, yellow background 

partial fulfilment, while red background indicates failure to fulfil required level. 

Test organism Method Influent Requirement 

Test 1 

Microscope counts 250 333 ± 16 790 ≥105 m-3

Phyla 3 ≥3 different Organisms 

≥50 μm Species 7 ≥5 different 

Dilution method 5000 

95 % conf. interval 2000-20 000 

Microscope counts 5705 ± 1036 

Plate counts 2350 

≥1000 ml-1

Phyla 3 ≥3 different 

Organisms 

≥10-50 μm 

Species 15 ≥5 different 

Marine heterotrophic bacteria Bacterial counts 3.0 ± 0.5 x 104 ≥104 cfu ml-1

Coliform bacteria Bacterial counts 3.7 ± 0.6 x 100 - 

Vibrio sp. Bacterial counts 0 ± 0 - 

Vibrio cholerae Bacterial counts, elimination method - - 

Enterococcus group Bacterial counts 1.3 ± 1.2 x 100 - 

Test 2 

Microscope counts 219 696 ± 21 082 ≥105 m-3

Phyla 3 ≥3 different Organisms 

≥50 μm Species 9 ≥5 different 

Dilution method 2400 

95 % conf. interval 1000-9500 

Microscope counts 2299 ± 286 

Plate counts 1550 

≥1000 ml-1

Phyla 3 ≥3 different 

Organisms 

≥10-50 μm 

Species 12 ≥5 different 

Marine heterotrophic bacteria Bacterial counts 2.1 ± 0.05 x 104 ≥104 cfu ml-1

Coliform bacteria Bacterial counts 2.3 ± 2.3 x 100 - 

Vibrio sp. Bacterial counts 4.1 ± 0.06 x 103 - 

Vibrio cholerae Bacterial counts, elimination method - - 

Enterococcus group Bacterial counts 1.0 ± 1.0 x 100 - 

Test 3 

Microscope counts 146 375 ± 8420 ≥105 m-3

Phyla 3 ≥3 different Organisms 

≥50 μm Species 8 ≥5 different 

Dilution method 2400 

95 % conf. interval 1000-9500 

Microscope counts 1556 ± 412 

Plate counts 1350 

≥1000 ml-1

Phyla 3 ≥3 different 

Organisms 

≥10-50 μm 

Species 10 ≥5 different 

Marine heterotrophic bacteria Bacterial counts 3.1 ± 0.2 x 104 ≥104 cfu ml-1

Coliform bacteria Bacterial counts 0 ± 0 - 

Vibrio sp. Bacterial counts 3.7 ± 2.8 x 103 - 

Vibrio cholerae Bacterial counts, elimination method - - 

Enterococcus group Bacterial counts 0 ± 0 - 

Test 4 

Organisms Microscope counts 117 767 ± 10 572 ≥105 m-3
NIVA 5659-2008 

29 

≥50 μm Phyla 3 ≥3 different 

Species 7 ≥5 different 

Dilution method 2200 

95 % conf. interval 900 ± 5900 

Microscope counts 2072 ± 110 

Plate counts 1500 

≥1000 ml-1

Phyla 4 ≥3 different 

Organisms 

≥10-50 μm 

Species 16 ≥5 different 

Marine heterotrophic bacteria Bacterial counts 2.4 ± 0.3 x 104 ≥104 cfu ml-1

Coliform bacteria Bacterial counts 1.0 ± 1.7 x 100 - 

Vibrio sp. Bacterial counts 6.6 ± 1.4 x 103 - 

Vibrio cholerae Bacterial counts, elimination method - - 

Enterococcus group Bacterial counts 1.3 ± 1.5 x 100 - 

Test 5 

Microscope counts 71 229 ± 14 144 ≥105 m-3

Phyla 3 ≥3 different Organisms 

≥50 μm Species 6 ≥5 different 

Dilution method 2400 

95 % conf. interval 1000-9500 

Microscope counts 1781 ± 143 

Plate counts 1000 

≥1000 ml-1

Phyla 3 ≥3 different 

Organisms 

≥10-50 μm 

Species 7 ≥5 different 

Marine heterotrophic bacteria Bacterial counts 1.7 ± 0.5 x 104 ≥104 cfu ml-1

Coliform bacteria Bacterial counts 0 ± 0 - 

Vibrio sp. Bacterial counts 1.2 ± 0.2 x 103 - 

Vibrio cholerae Bacterial counts, elimination method - - 

Enterococcus group Bacterial counts 0 ± 0 - 

Test 6 

Microscope counts 232 708 ± 29 189 ≥105 m-3

Phyla 3 ≥3 different Organisms 

≥50 μm Species 8 ≥5 different 

Dilution method 1700 

95 % conf. interval 700-4800 

Microscope counts 2109 ± 277 

Plate counts 1600 

≥1000 ml-1

Phyla 3 ≥3 different 

Organisms 

≥10-50 μm 

Species 9 ≥5 different 

Marine heterotrophic bacteria Bacterial counts 3.0 ± 0.3 x 104 ≥104 cfu ml-1

Coliform bacteria Bacterial counts 2.3 ± 2.5 x 100 - 

Vibrio sp. Bacterial counts 1.2 ± 0.4 x 104 - 

Vibrio cholerae Bacterial counts, elimination method - - 

Enterococcus group Bacterial counts 0.3 ± 0.6 x 100 - 

Test 7 

Microscope counts 253 438 ± 8083 ≥105 m-3

Phyla 3 ≥3 different Organisms 

≥50 μm Species 8 ≥5 different 

Dilution method 2400 

95 % conf. interval 1000-9500 

Microscope counts 1776 ± 202 

Organisms 

≥10-50 μm 

Plate counts 1175 

≥1000 ml-1
NIVA 5659-2008 

30 

Phyla 3 ≥3 different 

Species 13 ≥5 different 

Marine heterotrophic bacteria Bacterial counts 2.3 ± 0.6 x 104 ≥104 cfu ml-1

Coliform bacteria Bacterial counts 1.7 ± 0.6 x 100 - 

Vibrio sp. Bacterial counts 2.5 ± 0.2 x 103 - 

Vibrio cholerae Bacterial counts, elimination method - - 

Enterococcus group Bacterial counts 1.0 ± 1.0 x 100 - 

Test 8 

Microscope counts 265 083 ± 17 924 ≥105 m-3

Phyla 5 ≥3 different Organisms 

≥50 μm Species 6 ≥5 different 

Dilution method 3000 

95 % conf. interval 1000-13000 

Microscope counts 2800 ± 494 

Plate counts 3550 

≥1000 ml-1

Phyla 3 ≥3 different 

Organisms 

≥10-50 μm 

Species 7 ≥5 different 

Marine heterotrophic bacteria Bacterial counts 1.8 ± 0.2 x 104 ≥104 cfu ml-1

Coliform bacteria Bacterial counts 2.0 ± 1.0 x 100 - 

Vibrio sp. Bacterial counts 1.9 ± 0.1 x 103 - 

Vibrio cholerae Bacterial counts, elimination method - - 

Enterococcus group Bacterial counts 1.7 ± 0.6 x 100 - 

Test 9 

Microscope counts 262 167 ± 42 253 ≥105 m-3

Phyla 7 ≥3 different Organisms 

≥50 μm Species 8 ≥5 different 

Dilution method 2400 

95 % conf. interval 1000-9500 

Microscope counts 2117 ± 264 

Plate counts 1725 

≥1000 ml-1

Phyla 4 ≥3 different 

Organisms 

≥10-50 μm 

Species 8 ≥5 different 

Marine heterotrophic bacteria Bacterial counts 8.6 ± 0.9 x 104 ≥104 cfu ml-1

Coliform bacteria Bacterial counts 3.3 ± 2.1 x 101 - 

Vibrio sp. Bacterial counts 3.1 ± 0.04 x 103 - 

Vibrio cholerae Bacterial counts, elimination method - - 

Enterococcus group Bacterial counts 3.3 ± 1.2 x 100 - 

Test 10 

Microscope counts 260 292 ±31 001 ≥105 m-3

Phyla 6 ≥3 different Organisms 

≥50 μm Species 5 ≥5 different 

Dilution method 1300 

95 % conf. interval 500-3900 

Microscope counts 1927 ± 182 

Plate counts 1700 

≥1000 ml-1

Phyla 3 ≥3 different 

Organisms 

≥10-50 μm 

Species 9 ≥5 different 

Marine heterotrophic bacteria Bacterial counts 1.2 ± 0.006 x 105 ≥104 cfu ml-1

Coliform bacteria Bacterial counts 3.0 ± 5.2 x 100 - 

Vibrio sp. Bacterial counts 1.4 ± 0.1 x 103 - 

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

31 

Enterococcus group Bacterial counts 1.3 ± 1.5 x 100 - 

Test 11 

Microscope counts 248 008 ± 35 848 ≥105 m-3

Phyla 4 ≥3 different Organisms 

≥50 μm Species 9 ≥5 different 

Dilution method 5000 

95 % conf. interval 2000-20000 

Microscope counts 2013 ± 54 

Plate counts 1400 

≥1000 ml-1

Phyla 3 ≥3 different 

Organisms 

≥10-50 μm 

Species 20 ≥5 different 

Marine heterotrophic bacteria Bacterial counts 1.8 ± 0.2 x 105 ≥104 cfu ml-1

Coliform bacteria Bacterial counts 1.3 ± 0.6 x 100 - 

Vibrio sp. Bacterial counts 1.3 ± 1.1 x 104 - 

Vibrio cholerae Bacterial counts, elimination method - - 

Enterococcus group Bacterial counts 0 ± 0 - 

Test 12 

Microscope counts 200 404 ± 6019 ≥105 m-3

Phyla 3 ≥3 different Organisms 

≥50 μm Species 6 ≥5 different 

Dilution method 5000 

95 % conf. interval 2000-20000 

Microscope counts 4010 ± 353 

Plate counts 3050 

≥1000 ml-1

Phyla 3 ≥3 different 

Organisms 

≥10-50 μm 

Species 8 ≥5 different 

Marine heterotrophic bacteria Bacterial counts 4.0 ± 0.0 x 104 ≥104 cfu ml-1

Coliform bacteria Bacterial counts 0 ± 0 - 

Vibrio sp. Bacterial counts 6.9 ± 0.8 x 104 - 

Vibrio cholerae Bacterial counts, elimination method - - 

Enterococcus group Bacterial counts 1.3 ± 1.15 x 100 - 
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 m3

 should be 

present in the treated water after five days storage. 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 background indicates that required 

level was fulfilled, yellow background partial fulfilment, 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 before death manifest itself. Counting was 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 given 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 cycles 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 the 
NIVA 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 requirement 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 background indicates that required 

level was fulfilled, yellow background partial fulfilment, 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 

Test cycle 1 <0.2 <0.2 2000 2400 

<0.1-1.0 <0.1-1.0 1-14 x 103 1-9.5 x 103

Test cycle 2 <0.2 <0.2 5000 2400 

<0.1-1.0 <0.1-1.0 2-24 x 103 1-9.5 x 103

Test cycle 3 <0.2 <0.2 2000 2400 

<0.1-1.0 <0.1-1.0 1-14 x 103 1-9.5 x 103

Test cycle 4 <0.2 <0.2 2000 2400 

<0.1-1.0 <0.1-1.0 1-14 x 103 1-9.5 x 103

Test cycle 5 <0.2 <0.2 2000 2400 

<0.1-1.0 <0.1-1.0 1-14 x 103 1-9.5 x 103

Test cycle 6 13 <0.2 2000 2200 

5-33 <0.1-1.0 1-14 x 103 0.9-5.9 x 103

Test cycle 7 50 <0.2 5000 2400 

20-200 <0.1-1.0 2-20 x 103 1-9.5 x 103

Test cycle 8 3 <0.2 5000 3000 

1-12 <0.1-1.0 2-24 x 103 1-13 x 103

Test cycle 9 22 <0.2 1500 2400 

9-59 <0.1-1.0 0.5-5.1 x 103 1-9.5 x 103

Test cycle 10 30 <0.2 5000 2400 

10-130 <0.1-1.0 2-24 x 103 1-9.5 x 103

Test cycle 11 0.2 <0.2 1500 2400 

0.1-1.7 <0.1-1.0 0.5-5.1 x 103 1-9.5 x 103

Test cycle 12 1.7 <0.2 2000 2400 

0.7-4.7 <0.1-1.0 1-14 x 103 1-9.5 x 103

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 ± 323 
NIVA 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 fulfilled 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 abscent 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 background 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 101 1.2 ± 0.5 x 101 6.2 ± 4.8 x 104 2.9 ± 2.9 x 105

Test cycle 2 2.4 ± 0.2 x 101 6.1 ± 0.7 x 101 2.4 ± 0.7 x 104 2.1 ± 0.4 x 105

Test cycle 3 1.2 ± 0.06 x 102 5.9 ± 0.4 x 101 3.6 ± 0.2 x 104 3.7 ± 0.5 x 105

Test cycle 4 2.2 ± 0.2 x 102 5.7 ± 1.6 x 101 2.4 ± 0.3 x 104 3.2 ± 0.9 x 105

Test cycle 5 4.9 ± 0.7 x 101 2.0 ± 0.6 x 101 1.2 ± 0.2 x 104 1.3 ± 0.2 x 105

Test cycle 6 1.9 ± 0.2 x 102 2.1 ± 0.4 x 102 3.3 ± 0.6 x 104 1.4 ± 0.8 x 106

Test cycle 7 8.3 ± 2.7 x 101 3.4 ± 0.3 x 101 3.3 ± 1.1 x 104 6.0 ± 0.9 x 105

Test cycle 8 3.4 ± 0.4 x 101 2.0 ± 0.9 x 103 1.8 ± 0.3 x 104 4.1 ± 1.0 x 105

Test cycle 9 2.2 ± 0.9 x 103 1.1 ± 0.2 x 102 8.9 ± 1.2 x 104 4.3 ± 0.5 x 104

Test cycle 10 2.2 ± 0.2 x 102 3.9 ± 1.9 x 100 1.3 ± 0.1 x 105 3.6 ± 1.5 x 104

Test cycle 11 3.3 ± 0.8 x 102 1.5 ± 0.3 x 102 1.6 ± 0.2 x 105 2.9 ± 0.8 x 105

Test cycle 12 6.3 ± 1.1 x 102 3.1 ± 1.2 x 101 4.6 ± 0.9 x 104 9.2 ± 0.6 x 104

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 100 0 ± 0 

Test cycle 2 0 ± 0 0 ± 0 0 ± 0 4.3 ± 1.2 x 100 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 100 0 ± 0 

Test cycle 5 0 ± 0 0 ± 0 0 ± 0 0.7 ± 1.2 x 100 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 100 0 ± 0 

Test cycle 8 0 ± 0 0 ± 0 0 ± 0 1.7 ± 0.6 x 100 0.7 ± 0.6 x 100

Test cycle 9 0 ± 0 0 ± 0 0 ± 0 2.0 ± 1.0 x 101 2.3 ± 2.1 x 100

Test cycle 10 0 ± 0 2 ±1 0 ± 0 1.1 ± 0.8 x 101 0.3 ± 0.6 x 100

Test cycle 11 0 ± 0 0 ± 0 0 ± 0 2.0 ± 0.0 x 101 0.7 ± 0.6 x 100

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 101 < 1 ** 0.6 ± 1.0 x 103 5.6 ± 0.5 x 102

Test cycle 2 0.9 ± 0.9 x 100 0.6 ± 0.5 x100 < 1** 4.8 ± 0.5 x 103 4.4 ± 0.3 x 103

Test cycle 3 0.3 ± 0.5 x 100 0.6 ± 1.0 x 101 < 1** 5.6 ± 5.6 x 103 5.6 ± 1.6 x 103

Test cycle 4 0 ± 0 2.4 ± 0.5 x 100 < 1** 8.3 ± 1.0 x 103 5.6 ± 1.0 x 103

Test cycle 5 1.8 ± 0.9 x 100 0 ± 0 < 1** 1.5 ± 0.3 x 103 1.0 ± 0.2 x 103

Test cycle 6 0.3 ± 0.5 x 100 0.9 ± 0.9 x 100 < 1 ** 2.0 ± 0.8 x 104 3.3 ± 0.1 x 103 

Test cycle 7 0.3 ± 0.5 x 100 1.2 ± 0.5 x 100 < 1 ** 5.0 ± 0.3 x 103 2.3 ± 0.5 x 103 

Test cycle 8 0.6 ± 0.5 x 100 0 ± 0 < 1 ** 2.3 ± 1.0 x 103 3.0 ± 0.2 x 103 

Test cycle 9 0.3 ± 0.5 x 100 1.8 ± 2.4 x 100 < 1 ** 3.2 ± 0.1 x 103 1.6 ± 0.3 x 103 

Test cycle 10 3.6 ± 4.8 x 100 0 ± 0 < 1 ** 2.1 ± 0.1 x 103 7.0 ± 1.6 x 102 

Test cycle 11 4.8 ± 3.2 x 100 0.3 ± 0.5 x 100 < 1** 1. 6 ± 0.4 x 104 9.9 ± 0.6 x 103 

Test cycle 12 8.3 ± 0.6 x 101 2.6 ± 2.0 x 101 < 1** 8.3 ± 0.2 x 104 2.9 ± 2.6 x 104

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 100 0 ± 0 

Test cycle 2 1.7 ± 2.4 x 101 0 ± 0 0 ± 0 1.7 ± 2.1 x 100 2.0 ± 1.0 x 100

Test cycle 3 0 ± 0 0 ± 0 0 ± 0 0.3 ± 0.6 x 100 1.0 ± 1.0 x 100

Test cycle 4 0 ± 0 0 ± 0 0 ± 0 0 ± 0 2.0 ± 1.0 x 100

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 100 0.3 ± 0.6 x 100

Test cycle 8 0 ± 0 0 ± 0 0 ± 0 0.3 ± 0.6 x 100 1.0 ± 0.0 x 100

Test cycle 9 0 ± 0 0 ± 0 0 ± 0 5.0 ± 1.7 x 100 2.0 ± 2.0 x 100

Test cycle 10 0 ± 0 0 ± 0 0 ± 0 1.0 ± 1.0 x 100 0.3 ± 0.6 x 100

Test cycle 11 0 ± 0 0 ± 0 0 ± 0 0.7 ± 1.2 x 100 0 ± 0 

Test cycle 12 0 ± 0 0 ± 0 0 ± 0 0.7 ± 0.6 x 100 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, 19th 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, 19th 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 Control and Management of Ships' Ballast Water and 

Sediments, the International Maritime Organisation (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. 

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