REPORT SNO 5667-2008 CONFIDENTIAL

Land based testing of the 

PureBallast Treatment System 

of AlfaWall AB 

- approved test cycles

REPORT SNO 5667Ȭ2008

Confidential

Land based testing of the

PureBallast Treatment

System of AlfaWall AB

Ȭȱapproved test cycles
NIVA 5667-2008 

Norwegian Institute for Water Research 

– an institute in the Environmental Research Alliance of Norway REPORT

Main Office Regional Office, 

Sørlandet 

Regional Office, 

Østlandet 

Regional Office, 

Vestlandet 

Regional Office 

Midt-Norge 

P.O. Box 173, Kjelsås Televeien 3 Sandvikaveien 41 Nordnesboder 5 P.O. Box 1266 

N-0411 Oslo, Norway N-4879 Grimstad, Norway N-2312 Ottestad, Norway N-5008 Bergen, Norway N-7462, Norway 

Phone (47) 22 18 51 00 Phone (47) 37 29 50 55 Phone (47) 62 57 64 00 Phone (47) 55 30 22 50 Phone (47) 73 54 63 85 / 86 

Telefax (47) 22 18 52 00 Telefax (47) 37 04 45 13 Telefax (47) 62 57 66 53 Telefax (47) 55 30 22 51 Telefax (47) 73 54 63 87 

Internet: www.niva.no 

Title 

Land based testing of the PureBallast Treatment System of 

AlfaWall AB - approved test cycles 

Serial No. 

5667-2008 

Report No. Sub-No. 

O-26375 

Date 

2008 

Pages Price 

 

Author(s) 

Helge Liltved 

Christian Vogelsang 

August Tobiessen 

Tor Gunnar Jantsch 

Anne-Marie Bomo 

Topic group

Water treatment 

Geographical area

Norway 

Distribution 

Confidential 

Printed 

CopyCat 

Client(s) 

AlfaWall AB, Tumba, Sweden 

Client ref. 

Carl Tullstedt 

Malin Westberg 

Abstract 

The PureBallast Treatment System, developed by AlfaWall AB, has been land based tested according to the IMOs 

Guidelines for approval of ballast water management systems (G8), Res. MEPC.126(53) Annex 3 and Procedure 

for approval of ballast water management systems that make use of active substances (G9) Res. MEPC.126(53) 

Annex 4. The tests were conducted at NIVA’s test site located at Solbergstrand 20 km south of Oslo. Test cycles 

were conducted with moderate and high salinity water. Each cycle lasted for a period of 5 days, and a total of 10 

approved test cycles have been completed and the results of these test cycles are reported here. 

4 keywords, Norwegian 4 keywords, English 

1. Ballastvann 1. Ballast water 

2. Toksikologi 2. Toxicology 

3. Testing 3. Testing 

4. AlfaWall AB 4. AlfaWall AB 

 

Helge Liltved Jarle Nygaard 

Project Manager Strategy Director 

ISBN 978-82-577-5402-0 
NIVA 5667-2008 

Preface 

The tests were conducted in 2006 and 2007 at NIVAs test facility located 

at Solbergstrand 20 km south of Oslo. NIVA conducted the testing as a 

contract assignment for AlfaWall AB, with Det Norske Veritas (DNV) as 

verifier. 

During planning and conducting of the tests, Malin Westberg and Carl 

Tullstedt were the lead representatives of AlfaWall AB, while Hanna 

Behrens, Egil Dragsund and Håkon Øvrum have represented DNV in the 

project. From NIVA, Anne-Marie Bomo, Oddbjørn Pettersen, Per Ivar 

Johannesen, August Tobiessen, Christian Vogelsang, Tor Gunnar Jantsch 

and Helge Liltved represented were the main representatives. Several 

other AlfaWall AB and NIVA personell have been involved in the 

project. 

I will use the opportunity to thank AlfaWall AB for choosing NIVA as 

the main partner in the process of testing and verification of the 

PureBallast Treatment System, and thank all involved personell for the 

proffessionality and creativity demonstrated in completing this innovative 

project. 

Oslo, September 2008 

Helge Liltved 
NIVA 5667-2008 

Contents 

Abbreviations and acronyms 7

Summary 8

1. Background 9

2. Materials and test protocols 10

2.1 Test site 10

2.2 The evaluated ballast water treatment system 10

2.2.1 General description of the PureBallast System 10

2.3 Test waters 10

2.3.1 Overview of requirements 10

2.3.2 Fulfilment of chemical water quality test criteria 11

2.3.3 Fulfilment of biological water quality test criteria 11

Harvesting of indigenous organisms 11

Cultivation of Artemia salina and Brachiomonas submarina 12

Bacteria 12

2.4 Test procedure 12

2.4.1 Description of test cycle 12

2.4.2 Measures to avoid cross-contamination during water transfer and 

sampling 13

2.5 Sampling 13

2.5.1 Assuring the representativeness of samples 13

2.5.2 Sampling protocols 14

2.5.3 Overview of sampling equipment 14

2.5.4 Sample preservation and transportation 15

2.6 Analyses 15

2.6.1 Chemical measurements 15

Temperature 15

pH 15

Dissolved oxygen (DO) 15

Salinity 15

Dissolved organic carbon (DOC) 16

Particulate organic carbon (POC) 16

Total suspended solids (TSS) 16

2.6.2 Identification and quantification of viable organisms ≥50 μm 16

2.6.3 Identification and quantification of viable organisms >10-50 μm 16

2.6.4 Identification and quantification of viable bacteria 17

Heterotrophic bacteria 17

Thermotolerant coliform bacteria 17

Enterococcus group 17

Vibrio sp. 17

2.6.5 Toxicity measurements of treated Ballast water 18

3. Results and discussion 20

3.1 QA/QC prosedures 20

3.2 Operational performance of the PureBallast system 20
NIVA 5667-2008 

3.3 Fulfillment of chemical water quality criteria 22

3.4 Fulfillment of the biological water quality criteria 28

3.5 Biocidal effects on organisms ≥50 μm in minimum diameter 32

3.6 Biocidal effects on organisms ≥10-50 μm in minimum diameter 32

3.7 Bactericidal effects 34

3.8 Ecotoxicological responses 35

Appendix A. Total project management reports 39

Appendix B. Chemical water quality preparation 39

Appendix C. Biological water quality preparation 39

Appendix D. Collection and preservation of samples 39

Appendix E. Logging of in situ measurements 39

Appendix F. Evaluation form for organisms >50 μm 39

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

Appendix H. Evaluation form for heterotrophic bacteria, 

coliforms, E. coli, Enterococcus group, intestinal Enterococci, and 

Vibrio cholerae 39

Appendix I. Acute and chronic toxicity of the green algae 

Skeletonema costatum 39

Appendix J. Acute and chronic toxicity of marine copepods 39

Appendix K. Acute and chronic toxicity of juvenile turbot 40

Appendix L. Operational performance 40
NIVA 5667-2008 

Abbreviations and acronyms 

A. salina – Artemia salina 

AlfaWall AOT Module – AlfaWall Advanced Oxidation Technology Module 

B. submarina – Brachiomonas submarina

CFDA-AM - 5-carboxyfluorescein diacetate acetoxymethyl ester 

COD – chemical oxygen demand 

CT1 – temporary holding tank on control cycle 

CT2 – storage tank on control cycle 

DNV – Det Norske Veritas 

DO – dissolved oxygen 

DOC – dissolved organic carbon 

EC50, EC10 - the concentrations causing 50 and 10 % effect, respectively, on the test organism 

E. coli – Escherichia coli

FNU – Formazine Nephelometric Units 

GF/F – glass fiber filter grade F 

GLP – Good laboratory Practice 

IMO – International Maritime Organization 

ISO – International Organisation for Standardization 

LC50 – the concentration causing 50 % mortality of the test organism 

n – number of measurements; in calculating the standard deviation 

NIVA – Norwegian Institute for Water Research 

NS-EN ISO – Norwegian, European and International Standard 

OECD – Organisation for economic Co-operation and Development 

PBTS - PureBallast Treatment System 

POC – particulate organic carbon 

PSU – Practical Salinity Unit (= ‰) 

QAPP – quality assurance project plan 

S1-S5 – sampling points 1-5 

Std – standard deviation 

TCB – thermotolerant coliform bacteria 

TCBS – MacConkey and thiosulphate citrate bile salt agar 

TSS – total suspended solids 

TT1 – temporary holding tank on test cycle 

TT2 – storage tank on test cycle 

WST – tank with prepared test water 

Xi - individual analytical result; in calculating the standard deviation 

X – the arithmetic mean of individual analytical results; in calculating the standard deviation 
NIVA 5667-2008 

8 

Summary 

Land-based testing of the PureBallast Treatment System (PBTS) of Alfa Wall AB has been completed 

in the period of November 2006 to May 2007. The testing has been conducted according to the IMOs 

Guidelines for approval of ballast water management systems (G8), Res. MEPC.126(53) Annex 3 and 

Procedure for approval of ballast water management systems that make use of active substances (G9) 

Res. MEPC.126(53) Annex 4, hereafter referred to as G8 and G9. The tests were carried out at NIVA’s 

test site located at Solbergstrand 20 km south of Oslo, with moderate salinity and with high salinity 

water. Each cycle lasted for a period of 5 days, and a total of 10 approved test cycles have been 

completed and the results of these test cycles are reported here. All activities and collected data during 

testing of the PureBallast treatment system have been logged and reported separately (Appendix A-L).

 

The test water was prepared in a common 516 m3

 tank (WST). A combination of indigenous harvested 

organisms and cultured surrogate species (>50 μm group: Artemia salina; 10-50 μm group: 

Tetraselmis suecica and Brachiomonas submarina) and heterotrophic bacteria were added to fulfil the 

biological water quality criteria. 

The test line involved consecutive treatment of at least 200 m3

 test water by the 50 μm filter unit and 

the PureBallast System transferring the test water from WST to a temporary holding tank (TT1 with a 

volume of 230 m3

) before a second treatment by the PBTS and transfer to a storage tank (TT2 with a 

volume of 205 m3

) for at least five days storage. The control line was run by transferring at least 200 

m3

 of the same type of prepared test water from WST to a second temporary holding tank (TT1) using 

the pump of the PBTS, but in by-pass of the treatment units before transfer to a second storage tank 

(CT2 with a volume of 205 m3

) for a five days storage time. All operations were conducted with an 

average flowrate >200 m3

/h. 

In the 10 approved tests, the requirements regarding chemical composition were fulfilled in all test 

cycle, except test cycle 6 where the total suspended solid (TSS) was slightly below the required >50 

mg/l. The requirements regarding biological composition of the test water, and the biocidal effects of 

treatment and storage, were fulfilled in all 10 test cycles. 

The ecotoxicity of treated ballast water was tested at least two times with 2 different water qualities (2 

different salinities).The toxicity of undiluted (100 %) and diluted samples was determined on three 

trophic levels using marine acute and chronic tests with untreated control ballast water as control. No 

detectable toxicity was observed on the marine alga Skeletonema costatum (growth inhibition test), 

Nitocera spinipes (invertebrate reproduction test) or Scophtalmus mixmus (acute fish test and fish 

growth test). A mortality of 27% of the marine crustacean Acartia tonsa was observed at the highest 

tested concentration (100%) compared to 9% in the control. It should be commented that high deathrate in the control can be a problem with the test, as also experienced in the current test. This deathrate can give misleading results. No effects were observed in the test with the other marine crustacean 

Nitocra spinipes. It is therefore doubtful that the effect observed can be attributed to toxicity. In the 

repeated test conducted 18th Jan - 20th Jan 2007, no mortality of Acartia was observed in treated ballast 

water at 100% concentration or lower. Lack of mortality >50 % at any test concentration precludes 

estimation of LC50. 

Treated waters in test cycle 3 were also sampled and analysed for organic halogen compounds (AOX), 

extractable organic halogen compounds (EOX), trihalomethanes (THM), potential bioaccumulating 

substances (PBS) and free and total residual oxidants (TRO). All compounds, except AOX and PBS, 

were below the detection limits. However, the concentrations of AOX and PBS were not significant 

different from the concentrations found in control water. 
NIVA 5667-2008 

9 

Based on the conducted acute and chronic toxicity tests, and the chemical analysis, it is concluded that 

ballast water treated with the present ballast water technology do not provide hazard or risk to the 

marine ecosystem when released. 

1. Background 

The overall goal for AlfaWall AB is certification of the PureBallast Ballast Water Management 

System in accordance with the requirements in the IMO Convention on ballast water management and 

underlying guidelines, including the Guidelines for approval of ballast water management systems 

(G8), Res. MEPC.126(53) Annex 3 and Procedure for approval of ballast water management systems 

that make use of active substances (G9) Res. MEPC.126(53) Annex 4, hereafter referred to as G8 and 

G9. 

Pilot tests were conducted, in June 2006, for partial fulfilment of such a certification. The objective of 

the pilot testing was to verify the environmental performance characteristics of the PureBallast Pilot 

System technology relative to the biological and toxicological requirements stated in the IMO 

Guideline. By performing a pilottest, difficulties and “bottle-necks” could be detected, and measures 

could be taken to avoid problems during the full scale testing. The experience from the pilottest served 

as a fundament for large scale land-based testing of the technology. 

Land-based testing for type approval, as reported here, was conducted in the period of November 2006 

to May 2007. The tests were conducted in accordance with G8 and G9. 
NIVA 5667-2008 

10 

2. Materials and test protocols 

2.1 Test site 

The tests were conducted at NIVA’s test site located at Solbergstrand 20 km south of Oslo. Sea water 

is supplied from various depths down to 60 m in the Oslofjord, while fresh water is supplied from 

ground water bore holes or from a local creek. 

The test facility includes 4 glass-fibre reinforced polyester tanks, supplied with inlet and outlet 

arrangements and equipment for proper cleaning. The two tanks for storage are covered to prevent 

light introduction. The surfaces of the test tanks (TT1 and TT2) and the control tank (CT1) are coated 

with coatings for ships (Balloxy HB light, Jotun, Norway). Propeller devices with gentle rotation are 

mounted at the bottom and at shallow depth in WST and TT1, and at the bottom in TT2 and CT2. 

These propeller devices are used to suspend 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 PureBallast System 

The PureBallast System from AlfaWall AB is a complete ballast water treatment system composing of 

three main component ballast water treatment system; 1) a 50 μm filter for removal of larger particles 

and organisms, 2) the AlfaWall AOT unit, a patented treatment technology using the synergetic effects 

of in situ produced free radicals and direct photo-radiation to inactivate microbes and 3) control and 

auxiliary equipment including sampling points to control water flows and measure different alarm 

levels during operation as well as to take samples during operation and testing. 

2.3 Test waters 

Test water was prepared in a 516 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, 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 salina; 10-50 μm: Tetraselmis suecica and/or Brachiomonas submarina and heterotrophic 

bacteria) 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
NIVA 5667-2008 

11 

Table 2. Required biological water quality in influent test water, treated water and in control after 5 

days storage as stated in regulation G8 by IMO. 

Organism group Influent water In treated water after 

5 days storage 

In control after 

5 days storage** 

≥50 μm min. 

dimension 

Pref. 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 m3

> 10x <10 viable 

organisms per m3

Heterotrophic 

bacteria 

≥104 ml-1 

Vibrio cholerae. - <1 cfu/100 ml - 

Escherichia coli - <250 cfu/100 ml - 

Intestinal 

Enterococci

- <100 cfu/100 ml ≥10x <100 cfu/100 

ml 

** As discussed in the results and discussion section. 

2.3.2 Fulfilment of chemical water quality test criteria 

Soluble lignin (40 % solution, Borrebond FP-P, Borregaard, Norway), starch (Maizena®, Unilever 

AS, Billingstad, Norway) and kaolin clay (VWR International, Oslo Norway) 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 described in Table 1. 

The necessary additions of each compound were calculated on the basis of earlier laboratory tests and 

full scale experience. Initially it was intended to use rapid test kits to measure both DOC, POC and 

TSS right addition, however, the mandatory long heating time (2 hours) in sulphuric acid to break 

down organic compounds for the DOC and POC measurements disqualified this option due to logistic 

problems. Hence, only the TSS criteria were checked by direct measurement on site, using the HACH 

method 8006 (HACH, 2001), applicable to samples with a TSS content within the range 0-750 mg/l, 

and the pre-set calibration standards of the Odyssey DR2500 spectrophotometer (HACH). 

2.3.3 Fulfilment of biological water quality test criteria 

A combination of indigenous harvested organisms and cultured surrogate species (>50 μm: Artemia 

salina; 10-50 μm: Brachiomonas submarina and/or Tetraselmis suecica) and heterotrophic bacteria 

was added to fulfil the biological water quality criteria described in Table 2. 

Harvesting of indigenous organisms 

Fulfilment of the criteria regarding at least five 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 using a Unik 

Filter type 450 (Unik Filtersystems, Os, Norway) equipped with a 20 μm mesh size screen. The 

harvesting process had earlier been shown to be relatively gentle to the organisms; surface water (1 m 

depth) was smoothly pumped (ca. 3 m3

/h) by a jet-pump to the inlet side of the screen, and algae and 

animals were washed from the screen into a collecting tray (ca. 100 l/h, e.g. approx. 30x conc.) and 

further to a storage tank. Water from the storage tank was transported trough a pipe to WST by 

gravity. The viability and general conditions of harvested organisms were evaluated by microscopy. 
NIVA 5667-2008 

12 

Cultivation of Artemia salina and Brachiomonas submarina 

The criteria of minimum concentration of organisms ≥50 μm in minimum dimension was fulfilled by 

adding cultured Artemia salina, and the equivalent criteria for organisms ≥10-50 μm in minimum 

dimension was fulfilled by adding cultured Brachiomonas submarina and Tetraselmis suecica.

A. salina: Resting cysts of Artemia salina are available commercially. Hatching of cysts are achieved 

by adding approximately 1 g of cysts to 1 litre of 20 ‰ salinity seawater. The culture is incubated with 

a bright light source at a temperature of 22-26 °C with good aeration in the medium. Full hatching is 

usually achieved in 24-48 hours. It is possible to hatch approximately 300 000 nauplii per litre. 

Artemia nauplii are hatched with a supply of food (egg yolk) and will therefore live for up to a week 

without any external food supply. Survival length is twice that if the naupli is stored at 8-10 o

C. 

However, hatched nauplii should be used within 24 hours in order to achieve high viability. 

B. submarina and Tetraselmis suecica was grown in seawater added a commercial fertilizer (Superba) 

0,3 g/l. Cultures were grown in plexiglass sylinders with a diameter of 25 cm and height of 130 cm. 

We have been able to reach densities of 1-3x109 cells per 1 l. 

The necessary cultivation volume was calculated based on a final density of 105

 nauplii per m3 for A. 

salina and 103

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

water of 516 m3

. A critical point is the survival of the algae during the 5 days storage in the control 

water rich in grazing organisms. Initially, tests were completed to estimate and verify the survival of 

algae in the control without grazers present, a specified volume of the control water was sieved 

through a 50 μm mesh to remove the majority of grazing organisms and stored at the same 

temperature as the rest of the water and in darkness. Survival in specified volumes with and without 

grazers present was quantified to evaluate the influence of possible grazing effect during the 5 day 

storage period. 

Bacteria 

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

≥104

 ml-1. Natural heterotrophic bacteria were grown by adding growth media to a seawater sample. 

The cultivation volume was calculated based on a final density of 107

 CFU/ml in the inoculum, the 

final volume of the test water (516 m3

). 

2.4 Test procedure 

2.4.1 Description of test cycle 

The water transfer pathways between tanks and the PureBallast System during each test cycle 

(including the control) is shown in Figure 1. The test line (blue lines) involved consecutive treatment 

of at least 200 m3

 test water by the 50 μm filter unit and the AlfaWall AOT unit transferring the test 

water from tank with prepared test water (WST) to a temporary holding tank (TT1) before a second 

treatment by the AlfaWall AOT unit and transfer to a storage tank (TT2) for at least five days storage. 

The control line (red lines) was run by transferring at least 200 m3

 of the same type of prepared test 

water from WST to the temporary holding tank (TT1) using the pump of the PureBallast system, but in 

by-pass of the treatment units before transfer to a second storage tank (CT2) for a five days storage 

time. 
NIVA 5667-2008 

13 

Figure 1. Transfer of test water during one test cycle with the PureBallast system including a test line 

(blue) and a control line (red). Sampling numbers are indicated by S1-S5. 

The test cycles were conducted with high salinity water and moderate salinity water. Each cycle lasted 

for a period of 5 days. 

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

To avoid cross-contamination between consecutive test waters upon transfer between tanks, all 

pipelines and tanks were flushed for 2-3 min with sea water from 60 meters depth or ground water 

with documented quality between each test cycle, followed by rinsing with high temperature water 

(80-90°C). 

The same holding tank (TT1) is used for both treated water and control water in the same test cycle. 

To avoid contamination, treated water is always introduced to TT1 before control water. No crosscontamination will occure because the density of organisms is lower in treated water than in control 

water. 

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 

Before any samples were collected from any of the tanks at every sampling time the following 

procedure was carried out to assure that representative samples were withdrawn: 

1) The particulate content of the 516 m3 tank (WST) was homogenized using an outboard 

propeller of 3 hp and an electric mixer (Brio model 1.0, 0.75 kW). The outboard propeller was 

mounted on the edge of the tank, while the electic mixer was mounted at the bottom of the 

tank. The outboard propeller could mix alternately between the periphery and centre of the 

tank. Mixers (Brio Model 1.0, 0.75kW) are also mounted in the TT2 and CT2 to provide 

homogenity before sampling. 

WST 

516 m3 TT1 

231 m3

TT2 

205 m3

CT2 

205 m3

S1 

S2, S3 

S4, S5 

50 μm filter 

Pump 

Benrad AOT Module 
NIVA 5667-2008 

14 

2) The turbidity in different sections of each tank (upper part, middle part and bottom part, in 

both periphery and mid section) was measured by a handheld submersible probe (YSI – 600 

OMS) during homogenization. When any turbidity measurement was within a 10 % deviation 

from the average turbidity of all measurements in the tank, sampling was started. 

2.5.2 Sampling protocols 

The following procedures were used to collect samples from the different tanks and sampling times. 

All sampling were done in triplicates. 

1) Sampling of organisms ≥ 50 μm in WST (S1) and CT2 (S4 and S5). A plastic bucket was used to 

collect a 20 litre sample. The sampled water was slowly sieved through a plankton net (50 μm 

diagonal dimensions) attached to a plastic cup, collecting the organisms in the cup. 

2) Sampling of organisms ≥ 50 μm in TT2 (S2 and S3): A siphon spillway was used to collect 1 m3

test water from TT2 directly after transfer from TT1 and after 5 days storage. The water was 

sieved directly through a plankton net (50 μm diagonal dimensions) attached to a plastic cup, 

collecting and concentrating the organisms in the cup. 

3) Sampling of organisms 10-50 μm in WST (S1), TT2 (S2 and S3) and CT2 (S4 and S5): Organisms 

with a minimum diameter between 10 μm and 50 μm are sampled as 1000 ml grab samples with a 

clean glass bottle. 

4) Sampling of bacteria in WST (S1), TT2 (S2 and S3) and CT2 (S4 and S5): Bacterial samples were 

collected as 1000 ml grab samples by slowly submerging a 1000-ml sterile glass bottle. The bottle 

was closed immediately after sampling. 

2.5.3 Overview of sampling equipment 

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

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

individual parameters. 

Parameter Sampling 

equipment 

Sample container Sample volume 

WST, CT2 

Sample volume 

TT2 

Temperature Temp. meter - - - 

pH pH probe - - - 

Dissolved oxygen DO probe - - - 

Salinity Probe - - - 

DOC Directly, grab Acid washed glass bottle 100 ml 100 ml 

POC Directly, grab Acid washed glass bottle 100 ml 100 ml 

TSS Directly, grab Clean plastic bottle 1000 ml 1000 ml 

Organisms ≥50 μm Sieving* 20 l 1 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 glass bottle 1000 ml 1000 ml 

* A 20-litre grab sample (from WST and CT2) or 1 m3 collected through a siphon spillway (from TT2) 

is concentrated to a volume of 40-100 ml through a plankton net with diagonal dimensions of 50 μm. 
NIVA 5667-2008 

15 

2.5.4 Sample preservation and transportation 

Preservation methods and expected storage/holding times before measurement are shown in Table 4. 

All preservation and analysis were in accordance with the described methodology. 

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 

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 

Thermotolerant 

coliform bacteria 

Enterococcus group 

bacteria 

Vibrio sp. 

Vibrio cholerae

4o

C in darkness 6 hours < 6 h 

After sampling, samples were placed in the dark in cooling-containers and transported to the 

laboratories for analysis. 

2.6 Analyses 

2.6.1 Chemical measurements 

Temperature 

Temperature was measured in situ using a calibrated thermometer. 

pH 

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

Dissolved oxygen (DO) 

Dissolved oxygen (DO) was measured in situ using a calibrated probe and meter. DO is reported as 

mg O2/l. 

Salinity 

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

16 

Dissolved organic carbon (DOC) 

DOC was measured by an accredited method based on Norwegian Standard NS-ISO 8245 (NIVA 

method G5-1) at NIVA after filtering the sample through a GF/F filter (0.7 μm). The sample was 

acidified with phosphoric acid and aerated with oxygen to remove inorganic carbon prior to injection 

into a quartz tube filled with a platinum catalyser at 680 o

C. The organic carbon compounds were 

oxidized to CO2 which was quantified using an NDIR detector (Phoenix 8000 TOC-TC analyser with 

sample carousel STS 8000) with oxygen as carrier. Detection limit was 0.5 mg C/l. 

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 (see 4.2.1), and the measured DOC level of the same sample. 

Total suspended solids (TSS) 

A nucleopore capillary filter (0,4 μm) was dried at 40-50 o

C for 2 hours and the tara wes determined 

by weighing on a micro weight (Sartorius 4503 Micro) equipped with an ion source (Static Eliminator 

Bar Pu 210 Item no. LC 9793) removing static 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. 

Trihalomethanes (THM) and other halogenated by-products 

THMs were extracted in pentane and measured by the use of a gas chromatograph (American Public 

Health Association, 1989). Other by-products, including adsorbable organic halogen compounds 

(AOX), extractable organic halogen compounds (EOX), potential bioaccumulating substances (PBS) 

and chlorophenols, were analysed in accordance with approved methods. 

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. 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 5-

carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) according to Ganassin et al. (2000). A 

10 ml sample was incubated for 1 hour with 4 μmol of CFDA-AM. The sample was fixed with 

formalin and filtered onto black polycarbonate filters (25 mm). The filter was mounted on a glass slide 

in paraffin oil and frozen. CFDA-AM was hydrolysed only in a living cell. CFDA-AM is a marker for 

cell membrane integrity and may be measured directly in cells. In principle, the non-fluorescent 

chemicals CFDA-AM is taken up in the cytosol, where it becomes hydrolysed into fluorescent end 

products. These end products are trapped inside the cellular compartment and may be observed in an 

epifluorescence microscope using excitation filter 485 nm and emission filter of 530 nm. In the 

epifluorescence microscope viable cells are observable as brightly yellow/green coloured cells, while 

non viable cells are pale green (heterotrophic cells) or pale green with red autofluorescence of the 

chloroplast (photoautotrophs). Numbers of viable cells are counted at a magnification of 300-480 

times. 

As a complementary method to direct counting for testing of viability, the serial dilution method in 

algal growth medium was used. The serial dilution method is often referred to as the most probable 

number method. It is simply based on the fact that by diluting your sample in a sequence and 

observing in which dilutions the organisms occur (grow) in afterwards, one is able to backward 

calculate the number of cells in the original sample. The dilution series were achieved by adding 1 ml 
NIVA 5667-2008 

17 

of sample to 9 ml of media (algal growth media, 20 % Z8 seawater media). After mixing, 1 ml of this 

sample was further diluted with 9 ml. In this way a series of 10x dilution were made. The number of 

dilutions was set to cover the expected cell density range in the original sample. 3-5 parallels were 

employed in order to provide statistical significance of estimated number. 

2.6.4 Identification and quantification of viable bacteria 

Generally the samples were diluted or concentrated to achieve a quantifyable concentration of colony 

forming units on a solid growth media (agar-medium plates) or a medium-amended filterpillow. The 

dilution or concentration was based on experience and expectance of the concentration of the bacteria 

in question in the sample. Dilution was performed by stepwise 10x dilution of the sample in a dilution 

series followed by incubation on agar. 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. 

Thermotolerant coliform bacteria 

Thermotolerant coliform bacteria (TCB) were quantified according to Norwegian Standard NS-EN 

ISO 9308-1 at a temperature of 44±0.2 °C and an incubation period of 18-24 hours. 

Enterococcus group 

Total fecal Enterococci were quantified according to Norwegian Standard NS-EN ISO 7899-2 at a 

temperature of 37±0.2 °C and an incubation period of 44 hours. 

Vibrio sp. 

Total Vibrio sp. were quantified according to the method described by the American Public Health 

Association (APHA,1995). The total number of Vibrio sp., were determined by filtering of a 1-100 ml 

sample, the filter was placed on TCBS Cholera-medium agar plates (CMO333 from Oxoid), incubated 

at 35 °C, and the colonies were counted 24 hours incubation. The medium supports the growth of 

pathogenic Vibrios (Vibrio cholerae, Vibrio parahaemolyticus), some other Vibrios, and some other 

species (see table 5). Some strains of Aeromonas hydrophila grow producing yellow colonies but 

Plesimonas shigelloides does not usually grow well on TCBS. 

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

Organisms Colonies 

Vibrio cholera and El Tor type Yellow, flat, 2-3 mm diameter. 

Vibrio parahaemolyticus Blue-green, 3-5 mm diameter. 

Vibrio alginolyticus Yellow, 3-5 mm diameter. 

Vibrio metschnikovii Yellow, 3-4 mm diameter. 

Vibrio fluvialis Yellow, 2-3 mm diameter. 

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

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

Enterococcus species Yellow, 1 mm diameter. 

Proteus species Yellow-green, 1 mm diameter. 

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

18 

As indicated, only some, if any, of the colonies appearing on the agar plates in the different test cycles 

will be identified as Vibrio cholera, and the possibility of further detecting of the serotypes O1 and 

O139 is very small or abscent since non of these serotypes have been isolated from Norwegian coastal 

water. 

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

The morphology of the colonies developing on the TCBS-medium after 24 h was visually studied. 

Colonies that had distinct colors and morphology different from Vibrio cholera were not selected for 

further identification. Colonies with typical Vibrio cholera appearance were re-striked on TCBS 

medium and again inspected for growth and morphology. Traditional classical elimination or 

identification methods were used, such as appearance in culture media and physiological and 

biochemical properties. If Vibrio cholera is identified, polymerase chain reaction (PCR) will be used 

for elimination or identification of the serotypes O1 and O139. 

2.6.5 Toxicity measurements of treated Ballast water 

The following standard tests were performed on the treated ballast water. 

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

algal growth inhibition test 

• Reproductive toxicity to the marine crustacean Nitocra spinipes according to “ Forslag til 

Dansk Standard: Økotoksikologisk undersøgelse med krepsdyret Nitocra spinipes. 

Reproduksjonsmetode”. 

• Acute toxicity to the marine crustacean Acartia tonsa according to ISO 14669: Determination 

of acute lethal toxicity to marine copepods (Copepoda, Crustacea). 

• Acute toxicity towards the juvenile turbot (Scopthalmus maximus) according to OECD 

Guidelines for testing of chemicals (No. 203; Fish, acute toxicity test). 

• Chronic toxicity towards the juvenile turbot (Scopthalmus maximus) according to OECD 

Guidelines for testing of chemicals" (No. 215; Fish, juvenile growth test), adapted for marine 

species 

Growth inhibition of the marine alga Skeletonema costatum 

The inhibitory effect of Treated Ballast water on the growth of the marine diatom Skeletonema costatum, 

strain NIVA BAC1, has been investigated. The test was performed according to ISO 10253: Marine algal 

growth inhibition test with Skeletonema costatum and Phaeodactylum tricornutum.

Six replicate cultures of test algae in untreated and treated cultures were incubated on a shaking table at 20 

±2 °C, under continuous illumination. Growth was monitored by daily counting of cell numbers using a 

Coulter Multisizer. 

The growth rate in each culture was calculated from the increase in cell density from 0-48 and 0-72 

hours exposure. The analysis of difference between treatments was based on the exponential part of 

the growth curve (0-48 hours). 

Reproductive toxicity to the marine crustacean Nitocra Spinipes 

The reproductive toxicity of Treated Ballast water to the marine crustacean Nitocra spinipes has been 

investigated. The test was performed according to Draft guideline for Danish Standard: ”Økotoksikologisk 

undersøgelse med krepsdyret Nitocra spinipe. Reproduksjonsmetode”. The test was performed as a Limit 

test with 100 % Treated Ballast water and using non treated ballast water as control water. 
NIVA 5667-2008 

19 

The test was performed with 20 replicate vessels with 1 gravid female in each vessel. The vessels were 

incubated for 14 days at 20 °C. The number of alive offspring was then counted. 

Acute toxicity to the marine crustacean Acartia tonsa 

The acute toxicity of Treated Ballast water to the marine crustacean Acartia tonsa has been investigated. 

The test was performed according to ISO 14669: Determination of acute lethal toxicity to marine copepods 

(Copepoda, Crustacea). The test concentrations were in the range 32 to 100 % of Treated Ballast water. 

The test was performed with four replicate vessels with 4-8 test animals for each test concentration and 

sixteen control replicate vessels. The vessels were incubated for 48 hours at 20 ±1°C. Mortality was 

recorded after 24 and 48 hours. 

Chronic toxicity towards the juvenile turbot (Scopthalmus maximus) 

The chronic toxicity of Treated Ballast Water towards Turbot was tested in accordance with the 

"OECD Guidelines for testing of chemicals" (No. 215; Fish, juvenile growth test), adapted for marine 

fish. (ref 1). 

The testing of chronic toxicity of Treated Ballast Water is required by the IMO G-8 guidelines for 

testing of treatment technology to be used aboard ships. The fish was exposed continuously for 28 

days with water exchange 3 times per week. 

The juvenile turbot was approximately 12 weeks old at the start of the test and had a weight in the 

range of 5 to 9 g. 20 fish were used in each aquarium with 20 l of medium. The temperature in the 

water was maintained in the range of 13,4 – 18,2o

C, and a continuous light was used. 

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

temperature 

Acute toxicity towards the juvenile turbot (Scopthalmus maximus) 

The acute toxicity of Treated Ballast Water towards Turbot was tested in accordance with the draft 

procedure of Phil McWilliams (Terra Environmental Laboratory A/S) (ref 1). The procedure follows the 

general guidelines of OECD 203 "Fish, Acute toxicity test" (ref 2). 

The testing of acute toxicity of Treated Ballast Water is required by the IMO G-8 guidelines for 

testing of treatment technology to be used aboard ships. The fish was exposed continuously for 96 

hours with full water exchange every day. 

The juvenile turbot was approximately 12 weeks old at the start of the test and had a mean weight of 

7.6 g. 20 fish were used in each aquarium with 20 l of medium. The temperature in the water varied in 

the range of 13.4-14.8o

C, and a light /dark cycle of 12:12 was used. The test water was taken directly 

from the control and test tank (CT1 and TT2), after conditioning to test temperature 
NIVA 5667-2008 

20 

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/G9. All activities and collected data during testing of the 

PureBallast treatment system have been logged as summarized in Table 6. For each activity a specially 

designed log in paper and/or electronic format has been used, shown in Appendix A-L which have 

been reported separately, and was used for the respective quality assurance evaluation. 

Table 6. Log protocols for all activities of the project.

Appendix Description 

A Total project management 

B Chemical water quality preparation 

C Biological water quality preparation 

D Collection and preservation of samples 

E Logging of in situ measurements 

F Evaluation form for organisms >50 μm 

G Evaluation form for organisms ≥10-50 μm 

H Evaluation form for heterotrophic bacteria, coliforms, E. coli, Enterococcus group, 

intestinal Enterococci, and Vibrio cholerae.

I Acute and chronic toxicity of the green algae Skeletonema costatum

J Acute and chronic toxicity of marine copepods 

K Acute and chronic toxicity of juvenile turbot 

L Operational performance 

3.2 Operational performance of the PureBallast system 

The test cycles have been completed in the period of November 2006 to May 2007. The results of the 

10 approved tests are reported here. The first 5 cycles were conducted with high salinity water (>32 

PSU) and the remaining with medium salinity (3-22 PSU). Each test cycle lasted for 5 days. The dates 

of the cycles and the time for start/stopp of each step in the test cycle are given in table 7. 

A control for operation performance of the technology was performed in each test cycle by the site QA 

responsible person. Reports from these controls are included in Appendix L. 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, deballasting, 

control ballasting and control deballasting. In addition, lamp function was checked, and flow rate were 

recorded during the tests cycles. Flow rate was read on the meter mounted on the effluent pipe from 

the treatment technology. Voltage was read to 150 V for each lamp on the voltmeters mounted on the 

treatment unit. 
NIVA 5667-2008 

21 

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

Test cycle Date 

Start/stopp 

ballasting 

WST to TT1 

Start/stopp 

deballasting 

TT1 to TT2 

Start/stopp 

control 

ballasting 

WST to TT1

Start/stopp 

control 

deballasting 

TT1 to CT2 

Salinity 

PSU 

1 1-6th 

Nov 06 

14:53-16:00 

67 min 

16:40-17:40 

60 min 

17:55-19:00 

65 min 

19:15-20:15 

60 min 

32.2 

2 8-13th 

Nov 06 

12:24-13:30 

66 min 

14:01-15:00 

59 min 

15:18-16:22 

64 min 

16:35-17:35 

60 min 

32.6 

3 22-27th 

Nov 06 

12:10-13:08 

58 min 

13:22-14:21 

59 min 

14:49-15:45 

56 min 

15:46-16:35 

49 min 

32.6 

4 29th nov4th des 06 11:41-12:35 

54 min 

13:09-13:59 

50 min 

14:17-15:15 

58 min 

15:18-16:10 

52 min 

32.7 

5 6-11th 

Des 06 

10:56-11:55 

59 min 

12:15-13:01 

46 min 

13:16-14:13 

57 min 

14:17-15:06 

49 min 

32.1 

6 13-18th 

Des 06 

11:35-12:33 

58 min 

12:55-13:45 

50 min 

13:54-14:49 

55 min 

14:54-15:43 

49 min 

20.9 

7 10-15th 

Jan 07 

11:43-12:41 

58 min 

13:01-13:50 

49 min 

14:05-15:01 

56 min 

15:07-15:52 

45 min 

21.5 

8 17-22th 

jan 07 

11:00-11:58 

58 min 

12:19-13:08 

49 min 

13:24-14:20 

56 min 

14:25-15:18 

53 min 

20.8 

11 7-12th 

Mar 07 

11:10-12:07 

57 min 

12:30-13:20 

50 min 

13:40-14:35 

55 min 

14:38-15:25 

47 min 

20.5 

13 3-8th 

May 07 

11:28-12:24 

56 min 

12:42-13:32 

50 min 

13:49-14:48 

59 min 

14:51-15:38 

47 min 

21.7 

To determine the exact average flowrate during each operation, the time for start/stop and the volume 

of the tanks have been used to calculate the average flow as shown in table 8. In addition, flowrates 

were measured with a flowmeter and recorded on a regular basis during each test run. 
NIVA 5667-2008 

22 

Table 8. Calculated flowrate during each operation. 

Test cycle Dates 

Average 

flow 

ballasting 

m3

/h 

Average 

flow 

deballasting 

m3/h 

Average 

flow control 

ballasting 

m3/h 

Average 

flow control 

deballasting 

m3/h 

1 1-6th 

nov 06 

202 205 209 205 

2 8-13th 

nov 06 

205 208 211 205 

3 22-27th 

nov 06 

233 209 241 251 

4 29th nov4th des 06 250 246 233 237 

5 6-11th 

Des 06 

229 267 237 252 

6 13-18th 

Des 06 

233 246 246 251 

7 10-15th 

Jan 07 

233 251 241 273 

8 17-22th 

jan 07 

233 251 241 232 

11 7-12th 

Mar 07 

237 246 245 262 

13 3-8th 

May 07 

241 246 229 262 

3.3 Fulfillment of chemical water quality criteria 

The temperature, salinity, pH and dissolved oxygen content were measured in the test water tank 

(WST) prior to treatment and daily in the storage tanks TT2 and CT2 in each test cycle. Measurements 

were not conducted during week-ends. The results are given in Table 9. 

As shown, the temperature in the initial water (WST) varied among the tests. Small decreases in 

oxygen concentration were recorded during the test cycles. However, the oxygen content did never 

reach critical levels regarding survival of test organisms. 
NIVA 5667-2008 

23 

Table 9. Temperature, salinity and dissolved oxygen measurements in the test water tank (WST) prior 

to treatment and daily in the storage tanks TT2 and CT2 for each test cycle (measurements were not 

conducted during week-ends). 

WST TT2 CT2 

Day 0 0 1 2 3 4 5 0 1 2 3 4 5 

Parameter Unit 

Test cycle 1 

Temperature oC 9.8 9.8 8.80 8.30 7.40 7.20 6.90 - 7.90 7.50 6.70 6.50 6.30 

Salinity PSU 32.2 32.2 32.0 31.8 31.8 31.9 31.9 - 32.0 31.9 31.9 31.9 32.0 

pH 7.8 7.8 7.50 7.43 7.49 7.44 7.40 - 7.48 7.46 7.41 7.47 7.42 

Dissolved 

O2

% 85.0 85.0 83.10 78.10 78.60 73.60 72.80 - 84.30 73.20 76.50 71.10 70.40 

Test cycle 2

Temperature oC 7.60 7.60 7.00 - - 5.70 5.40 - 6.70 - - 5.30 5.00 

Salinity PSU 32.60 32.60 32.60 - - 32.40 32.30 - 32.80 - - 32.60 32.30 

pH 7.90 7.90 7.87 - - 6.80 7.88 - 8.01 - - 6.02 7.84 

Dissolved 

O2

mg/l 10.60 10.60 7.80 - - 7.80 6.28 - 6.68 - - 7.30 7.80 

Test cycle 3

Temperature oC 12.30 12.50 12.80 11.60 - - 10.60 13.00 11.50 11.10 - - 9.80 

Salinity PSU 32.60 32.60 32.60 32.60 - - 32.60 32.60 32.60 32.60 - - 32.60 

pH 8.03 8.04 8.01 7.91 - - 7.82 8.03 7.98 7.93 - - 7.87 

Dissolved 

O2

mg/l 9.90 11.00 8.90 8.70 - - 6.90 9.90 8.90 8.70 - - 6.80 

Test cycle 4

Temperature oC 12.00 11.90 11.80 11.70 - - 10.10 11.40 11.50 11.10 - - 9.80 

Salinity PSU 32.70 32.70 32.70 32.80 - - 32.70 32.70 32.70 32.70 - - 32.70 

pH 8.06 8.06 8.05 7.96 - - 7.75 8.08 8.08 7.96 - - 7.79 

Dissolved 

O2

mg/l 10.30 9.40 9.30 8.50 - - 5.10 9.60 9.10 8.50 - - 6.20 

Test cycle 5

Temperature oC 10.60 11.80 10.70 10.10 9.80 9.30 9.00 10.90 10.10 10.00 9.30 8.80 8.80 

Salinity PSU 32.10 32.10 32.10 32.20 32.20 32.00 32.10 32.10 32.10 32.20 32.10 32.10 32.10 

pH 8.09 8.08 8.03 7.96 7.86 7.89 7.78 8.10 8.00 7.97 7.91 7.93 7.77 

Dissolved 

O2

mg/l 10.00 9.80 9.80 8.90 7.60 7.10 7.00 10.00 9.10 8.50 7.80 7.40 7.10 

Test cycle 6

Temperature oC 6.6 7.4 8.0 7.5 - - 5.2 6.8 7.5 7.1 - - 4.8 

Salinity PSU 20.9 20.9 20.9 20.9 - - 20.9 20.9 20.9 20.9 - - 20.9 

pH 8.01 7.91 7.91 7.76 - - 7.47 7.96 7.88 7.78 - - 7.56 

Dissolved 

O2

mg/l 11.5 11.2 9.1 9.4 - - 7.4 11.4 9.3 9.7 - - 9.0 

Test cycle 7

Temperature oC 4.7 5.4 5.5 4.8 - - 4 4.9 4.7 4.2 - - 3.7 

Salinity PSU 21.5 21.6 21.4 21.4 - - 21.5 21.6 21.5 21.6 - - 21.5 

pH 7.88 7.81 7.87 7.82 - - 7.63 7.84 7.89 7.8 - - 7.66 

Dissolved 

O2

mg/l 11.5 11.2 9.7 10.4 - - 10 11.2 11.1 11 - - 10 

Test cycle 8

Temperature oC 4.9 5.9 4.9 4.6 - - 3.3 5.4 4.9 4.6 - - 2.6 

Salinity PSU 20.8 20.8 20.8 20.8 - - 20.7 20.8 20.8 20.8 - - 20.8 

pH 7.66 7.7 7.36 7.81 - - 7.54 7.67 7.32 7.67 - - 7.58 

Dissolved 

O2

mg/l 11.9 11.6 11.2 11.1 - - 10 11.1 11.4 11.3 - - 10.8 

Test cycle 11

Temp. oC 4.2 5.8 5.4 5.8 - - 5.4 5.4 5.4 5.8 - - 5.0 
NIVA 5667-2008 

24 

Salinity PSU 20.5 20.2 20.0 20.2 - - 20.4 20.3 20.2 20.3 - - 20.5 

pH 7.73 7.67 7.78 7.81 - - 7.75 7.75 7.82 7.82 - - 7.67 

Dissolved 

O2

mg/l 11.9 11.6 11.9 10.7 - - 10.6 11.9 11.5 10.5 - - 10.6 

Test cycle 13

Temp. oC 10.7 12.6 17.4 - - 16.8 13.2 12.6 17.4 - - 16.8 12.9 

Salinity PSU 21.7 21.7 21.8 - - 21.7 21.8 21.7 21.8 - - 21.8 21.7 

pH 8.01 7.97 7.95 - - 7.75 7.79 8.0 7.95 - - 7.75 7.78 

Dissolved 

O2

mg/l 11.3 10.8 10.51 - - 9.10 9.08 10.93 10.49 - - 9.05 8.61 

Total suspended solids (TSS), dissolved organic carbon (DOC) and particulate organic carbon (POC) 

measurements at day 0 in WST, TT2 and CT2, and at day 5 in TT2 and CT2 in the different test cycles 

are shown in Table 10 – Table 19. The required 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, while red background 

indicates failure to fulfil required level. Compounds were added to ensure fulfilment of the 

requirements; both a POC regulating compound (POCAdditive) and a DOC regulating compound 

(DOCAdditive). 

The required levels for total suspended solids (TSS), dissolved organic carbon (DOC) and particulate 

organic carbon (POC) were fulfilled in all test cycles reported, except the TSS requirement in test 6 

which was 10.4 mg/l lower than the required 50 mg/l. 

Table 10. Test 1. Chemical water quality (average and standard deviation of triplicate samples). 

TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 1 Average Stdev Average Stdev Average Stdev 

Additions to influent water - DOCAdditive = 1.2 kg. POCAdditive = 15.0 kg

Required level >1 - >1 - >1 - 

Influent water (WST) 51.3 3.9 2.6 0.2 4.8 2.8 

Treated day 0 (TT2) - - - - - - 

Treated day 5 (TT2) 6.4 - 3.5 - 0.4 - 

Control day 0 (CT2) - - - - - - 

Control day 5 (CT2) 1.6 - 3.2 - 0.0 - 

Table 11. Test 2. Chemical water quality (average and standard deviation of triplicate samples). 

TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 2 Average Stdev Average Stdev Average Stdev 

Additions to influent water - DOCAdditive = 1.2kg POCAdditive = 7.2 kg

Required level >1 - >1 - >1 - 

Influent water (WST) 6.6 2.2 2.4 0.1 1.5 1.1 

Treated day 0 (TT2) 5 - - - - - 

Treated day 5 (TT2) - 2.6 - 0.1 - 

Control day 0 (CT2) 8.8 - - - - - 

Control day 5 (CT2) 4.6 - 2.2 - 0.3 - 
NIVA 5667-2008 

25 

Table 12. Test 3. Chemical water quality (average and standard deviation of triplicate samples). 

TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 3 Average Stdev Average Stdev Average Stdev 

Additions to influent water - DOCAdditive = 1.2kg POCAdditive = 4.8 kg

Required level >1 - >1 - >1 - 

Influent water (WST) 16.5 0.9 2.0 0.1 2.9 0.1 

Treated day 0 (TT2) 15.8 - 2.6 - - - 

Treated day 5 (TT2) - - 2.0 - 0.1 - 

Control day 0 (CT2) 15.0 - 2.1 - - - 

Control day 5 (CT2) - - 2.1 - 0.3 - 

Table 13. Test 4. Chemical water quality (average and standard deviation of triplicate samples). 

TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 4 Average Stdev Average Stdev Average Stdev 

Additions to influent water - DOCAdditive = 1.2kg POCAdditive = 7.2 kg

Required level >1 - >1 - >1 - 

Influent water (WST) 18.1 1.3 3.7 0.9 3.0 1.8 

Treated day 0 (TT2) 17.1 3.4 2.9 0.4 4.7 1.7 

Treated day 5 (TT2) 2.0 0.1 2.1 0.4 

Control day 0 (CT2) 10.1 6.0 3.7 0.1 4.5 4.7 

Control day 5 (CT2) 2.0 0.1 1.3 0.4 

Table 14. Test 5. Chemical water quality (average and standard deviation of triplicate samples). 

TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 5 Average Stdev Average Stdev Average Stdev 

Additions to influent water - DOCAdditive = 1.2kg POCAdditive = 5.2 kg

Required level >1 - >1 - >1 - 

Influent water (WST) 11.9 0.2 2.0 0.3 4.9 0.1 

Treated day 0 (TT2) 11.2 0.2 2.6 0.4 4.4 0.2 

Treated day 5 (TT2) 7.6 1.1 2.4 0.3 1.0 0.2 

Control day 0 (CT2) 10.6 0.4 2.0 0.2 4.5 0.3 

Control day 5 (CT2) 11.2 4.2 2.3 0.3 1.2 0.1 

Table 15. Test 6. Chemical water quality (average and standard deviation of triplicate samples). 

TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 6 Average Stdev Average Stdev Average Stdev 

Additions to influent water - DOCAdditive = 6.0kg POCAdditive = 20 kg

Required level >50 - >5 - >5 - 

Influent water (WST) 39.6 0.7 5.1 0.1 14.7 1.1 

Treated day 0 (TT2) 37.9 0.9 5.8 0.1 12.2 0.4 

Treated day 5 (TT2) 11.8 6.8 5.9 0.2 

Control day 0 (CT2) 38.9 2.7 5.1 0.1 14.8 0.8 

Control day 5 (CT2) 14.3 1.0 4.9 0.1 
NIVA 5667-2008 

26 

Table 16. Test 7. Chemical water quality (average and standard deviation of triplicate samples). 

TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 7 Average Stdev Average Stdev Average Stdev 

Additions to influent water TSSAdditive = 10.0kg DOCAdditive = 10.0kg POCAdditive = 22 kg

Required level >50 - >5 - >5 - 

Influent water (WST) 61.6 2.1 6.2 0.1 17.1 0.1 

Treated day 0 (TT2) 59.5 0.6 7.1 0.1 15.9 0.2 

Treated day 5 (TT2) 25.7 0.3 6.5 0.1 6.5 0.4 

Control day 0 (CT2) 59.6 0.7 6.2 0.1 17.0 0.4 

Control day 5 (CT2) 22.5 0.5 6.2 0.1 5.0 0.5 

Table 17. Test 8. Chemical water quality (average and standard deviation of triplicate samples). 

TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 8 Average Stdev Average Stdev Average Stdev 

Additions to influent water TSSAdditive = 20.0kg DOCAdditive = 10.0kg POCAdditive = 14.1 kg

Required level >50 - >5 - >5 - 

Influent water (WST) 71.7 1.2 5.5 0.1 12.7 0.5 

Treated day 0 (TT2) 52.9 1.5 6.1 0.0 10.0 0.2 

Treated day 5 (TT2) 5.4 0.1 3.2 0.4 

Control day 0 (CT2) 65.5 0.2 3.8 1.6 12.4 2.0 

Control day 5 (CT2) 5.4 0.2 3.1 0.3 

Table 18. Test 11. Chemical water quality (average and standard deviation of triplicate samples). 

TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 11 Average Stdev Average Stdev Average Stdev 

Additions to influent water TSSAdditive = 20.0kg DOCAdditive = 12.0kg POCAdditive = 14.5 kg

Required level >50 - >5 - >5 - 

Influent water (WST) 67.1 1.0 7.1 0.1 10.6 2.7 

Treated day 0 (TT2) 64.3 0.3 7.2 0.1 10.8 0.7 

Treated day 5 (TT2) 29.2 1.3 6.7 0.2 3.7 0.4 

Control day 0 (CT2) 65.0 0.3 7.1 0.3 11.7 1.1 

Control day 5 (CT2) 26.0 1.5 6.4 0.2 4.5 0.7 

Table 19. Test 13. Chemical water quality (average and standard deviation of triplicate samples). 

TSS mg/l DOC, mg/l POC, mg/l TEST CYCLE 13 Average Stdev Average Stdev Average Stdev 

Additions to influent water TSSAdditive = 20.0kg DOCAdditive = 12.0kg POCAdditive = 14.5 kg

Required level >50 - >5 - >5 - 

Influent water (WST) 117.7 1.5 7.3 0.1 12.2 0.4 

Treated day 0 (TT2) 115.7 1.5 7.7 0.1 11.7 0.2 

Treated day 5 (TT2) 27.6 0.2 6.8 0.1 3.8 0.2 

Control day 0 (CT2) 117.7 2.3 7.2 0.1 12.0 0.4 

Control day 5 (CT2) 9.4 1.0 6.7 0.1 2.3 0.5 

Treated water and control water in test cycle 3 were sampled and analysed for adsorbable organic 

halogen compounds (AOX), extractable organic halogen compounds (EOX), chlorophenols and 

trihalomethanes (THM), a group of halogenated organic compounds. These compounds were analysed 

to clarify if the technology produces harmful halogenated organic by-products. 
NIVA 5667-2008 

27 

The dominating THM normally found in oxidized seawater is bromoform, which is a probable human 

carcinogen (USEPA 1996), known to bio-accumulate in aquatic animals. Bromoform is highly 

persistent in the environment. 

No THMs were found in treated water. All the congeners were below the detection limit, in both test 

waters sampeled (Table 20). 

All other compounds, except AOX, were below the detection limit (Table 20). However, AOX was 

also detected in control water. The difference between the concentration in treated water and control 

water was not significant. 

Influent water, control water and treated waters in test cycle 3 were also sampled and analysed for 

Potential Bioaccumulating Substances (PBS) in accordance with OECD 117. There were no difference 

in PBS-level between treated and control water. A value of 1.4 was measure in both waters. 

Table 20. Concentration of AOX, EOX, chlorophenols and trihalomethanes in treated water from Test 

3 in control water and treated water immediately after treatment. 

 Unit Control water Treated water 

AOX mg/l 0.45 0.57 

EOX mg/l <0.050 <0.050 

2-Monochlorophenol μg/l <0.10 <0.10 

3-Monochlorophenol μg/l <0.10 <0.10 

4-Monochlorophenol μg/l <0.10 <0.10 

2,3-Dichorophenol μg/l <0.10 <0.10 

2,4+2,5-Dichlorophenol μg/l <0.20 <0.20 

2,6-Dichlorophenol μg/l <0.10 <0.10 

3,4-Dichlorophenol μg/l <0.10 <0.10 

3,5-Dichlorophenol μg/l <0.10 <0.10 

2,3,4-Trichlorophenol μg/l <0.10 <0.10 

2,3,5-Trichlorophenol μg/l <0.10 <0.10 

2,3,6-Trichlorophenol μg/l <0.10 <0.10 

2,4,5-Trichlorophenol μg/l <0.10 <0.10 

2,4,6-Trichlorophenol μg/l <0.10 <0.10 

3,4,5-Trichlorophenol μg/l <0.10 <0.10 

2,3,4,5-Tetrachlorophenol μg/l <0.10 <0.10 

2,3,4,6-Tetrachlorophenol μg/l <0.10 <0.10 

2,3,5,6-Tetrachlorophenol μg/l <0.10 <0.10 

Pentachlorophenol μg/l <0.10 <0.10 

Trichloromethane (chloroform) μg/l <0.30 <0.30 

Tribromomethane (bromoform) μg/l <0.20 <0.20 

Dibromochloromethane μg/l <0.10 <0.10 

Bromodichloromethane μg/l <0.10 <0.10 

Treated waters in test cycle 4 were also sampled and analysed for free and total residual oxidants 

(TRO) by the DPD-colourimetric method (Hach method 8021 for free chlorine and Hach method 

8167). The results show that it is not possible to detect any production of residual oxidants measured 

as free or total chlorine in the treated water (table 21). 
NIVA 5667-2008 

28 

Table 21. Concentration of free and total residual oxidants in treated water from Test 4 in influent 

water just prior to treatment, and control water and treated water immediately after treatment. 

 Unit Influent water Control water Treated water 

Free chlorine 

Parallell 1 mg/l -0.04 0.03 0.02 

Parallell 2 mg/l 0.00 0.01 0.01 

Parallell 3 mg/l 0.05 0.02 0.02 

Parallell 4 mg/l 0.02 - - 

Parallell 5 mg/l 0.00 - - 

Average± Standard deviation 0.01±0.03 0.02±0.01 0.02±0.01 

Total chlorine 

Parallell 1 mg/l 0.00 0.00 0.01 

Parallell 2 mg/l 0.01 0.01 0.01 

Parallell 3 mg/l 0.04 0.02 0.02 

Parallell 4 mg/l 0.02 - - 

Parallell 5 mg/l 0.02 - - 

Average± Standard deviation 0.02±0.01 0.01±0.01 0.01±0.01 

3.4 Fulfillment of the biological water quality criteria 

The initial test waters’ content of organisms should comply with the requirements as given in Table 2. 

Results from quantitative measurements of the initial test waters are given in Table 22. Green 

background in table cell indicates that required level was fulfilled. 

Organisms ≥50 μm in minimum diameter 

The requirements regarding density of the ≥50 μm group was met in all test cycles. The requirements 

regarding the biological diversity within the population were fulfilled. 

Organisms ≥10-50 μm in minimum diameter 

The quantifications of individuals in the ≥10-50 μm group were based on growth in dilution series and 

microscope counts of CFDA-AM stained cells. In all tests, the requirements regarding influent density 

of the ≥10-50 μm group was met. The requirements regarding the biological diversity within the 

population were also fulfilled. 

Heterotrophic bacteria

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

present in a density of ≥104

 ml-1. This requirement was fulfilled in all tests as shown in Table 22. 

The IMO G-8 guideline 2.3.20 also specifies that “...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 22. Vibrio cholerae was not detected 

in any of the test cycles. 
NIVA 5667-2008 

29 

Table 22. Initial content of organisms within the defined test organism groups (ref. Table 2) in the test 

water. Green background indicates that required level was fulfilled. 

Test organism Method Influent Requirement 

Test 1 

Microscope counts 12.3 ± 2.7 x 104 m-3 ≥105 m-3

Phyla 5 ≥3 different Organisms 

≥50 μm Species >7 ≥5 different 

1700 ml-1 Dilution method 

95 % conf. interval 700-4800 ml-1

Microscope counts 1213 ± 41 ml-1

≥1000 ml-1

Phyla 5 ≥3 different 

Organisms 

≥10-50 μm 

Species >9 ≥5 different 

Hetero. bact. Bacterial counts 4.2 ± 1.4 x 105 ml-1 ≥104 ml-1

Coliform bacteria Bacterial counts 3 ± 2 x 101/100 ml - 

Vibrio sp. Bacterial counts >100/100 ml - 

Vibrio cholerae Bacterial counts, elimination method Not detected - 

Enterococcus group Bacterial counts 2 ± 0 x 103/100 ml - 

Test 2 

Microscope counts 16.0 ± 3.7 x 104 m-3 ≥105 m-3

Phyla 4 ≥3 different Organisms 

≥50 μm Species >5 ≥5 different 

3000 ml-1 Dilution method 

95 % conf. interval 1000-13000 ml-1

Microscope counts 1351 ± 156 ml-1

≥1000 ml-1

Phyla 4 ≥3 different 

Organisms 

≥10-50 μm 

Species >8 ≥5 different 

Hetero. bact. Bacterial counts 1.4 ± 0.53 x 105 ml-1 ≥104 ml-1

Coliform bacteria Bacterial counts 2.6 ± 1.2 x 102/100 ml - 

Vibrio sp. Bacterial counts >100/100 ml - 

Vibrio cholerae Bacterial counts, elimination method Not detected - 

Enterococcus group Bacterial counts 1.2 ± 0.41 x 103/100 ml - 

Test 3 

Microscope counts 21.5 ± 1.9 x 104 m-3 ≥105 m-3

Phyla 6 ≥3 different Organisms 

≥50 μm Species >5 ≥5 different 

3000 ml-1 Dilution method 

95 % conf. interval 1000 - 13000 ml-1

Microscope counts 1409 ± 30 ml-1

≥1000 ml-1

Phyla 3 ≥3 different 

Organisms 

≥10-50 μm 

Species >9 ≥5 different 

Hetero. bact. Bacterial counts 1.6 ± 0.21 x 106 ml-1 ≥104 ml-1

Coliform bacteria Bacterial counts 3.7 ± 1.2 x 101/100 ml - 

Vibrio sp. Bacterial counts 2.3 ± 0.50 x 104/100 ml - 

Vibrio cholerae Bacterial counts, elimination method Not detected - 

Enterococcus group Bacterial counts 4.3 ± 0.70 x 101/100 ml - 

Test 4 

Microscope counts 12.6 ± 9.7 x 104 m-3 ≥105 m-3

Phyla 4 ≥3 different Organisms 

≥50 μm Species >8 ≥5 different 

2400 ml-1 Organisms Dilution method 

95 % conf. interval 1000 - 9500 ml-1

≥1000 ml-1
NIVA 5667-2008 

30 

Microscope counts 1691 ± 212 ml-1

Phyla 3 ≥3 different 

≥10-50 μm 

Species >8 ≥5 different 

Hetero. bact. Bacterial counts 6.9 ± 1.0 x105 ml-1 ≥104 ml-1

Coliform bacteria Bacterial counts 500 ± 265 /100 ml - 

Vibrio sp. Bacterial counts 5.3 ± 4.6 x 105/100 ml - 

Vibrio cholerae Bacterial counts, elimination method Not detected - 

Enterococcus group Bacterial counts 2.5 ± 1.9 x 103/100 ml - 

Test 5 

Microscope counts 16.2 ± 2.4 x 104 m-3 ≥105 m-3

Phyla 6 ≥3 different Organisms 

≥50 μm Species >12 ≥5 different 

1700 ml-1 Dilution method 

95 % conf. interval 700 - 4800 ml-1

Microscope counts 1411 ± 89 ml-1

≥1000 ml-1

Phyla 3 ≥3 different 

Organisms 

≥10-50 μm 

Species >10 ≥5 different 

Hetero. bact. Bacterial counts 6.5 ± 0.9 x 105 ml-1 ≥104 ml-1

Coliform bacteria Bacterial counts 6070 ± 306 /100 ml - 

Vibrio sp. Bacterial counts 3.6 ± 3.4 x 105/100 ml - 

Vibrio cholerae Bacterial counts, elimination method Not detected - 

Enterococcus group Bacterial counts 337 ± 293 /100 ml - 

Test 6 

Microscope counts 17.1± 1.5 x 104 m-3 ≥105 m-3

Phyla 8 ≥3 different Organisms 

≥50 μm Species >10 ≥5 different 

5000 ml-1 Dilution method 

95 % conf. interval 2000 - 20000 ml-1

Microscope counts 2199 ± 173 ml-1

≥1000 ml-1

Phyla 7 ≥3 different 

Organisms 

≥10-50 μm 

Species >11 ≥5 different 

Hetero. bact. Bacterial counts 3.4 ± 1.5 x 105 ml-1 ≥104 ml-1

Coliform bacteria Bacterial counts 1970 ± 153 /100 ml - 

Vibrio sp. Bacterial counts 2.3 ± 1.4 x 105/100 ml - 

Vibrio cholerae Bacterial counts, elimination method Not detected - 

Enterococcus group Bacterial counts 43 ± 15 /100 ml - 

Test 7 

Microscope counts 15.4 ± 1.0 x 104 m-3 ≥105 m-3

Phyla 5 ≥3 different Organisms 

≥50 μm Species >6 ≥5 different 

2400 ml-1 Dilution method 

95 % conf. interval 1000 - 9500 ml-1

Microscope counts 2266 ± 336 ml-1

≥1000 ml-1

Phyla 4 ≥3 different 

Organisms 

≥10-50 μm 

Species >6 ≥5 different 

Hetero. bact. Bacterial counts 2.6 ± 1.4 x 105 ml-1 ≥104 ml-1

Coliform bacteria Bacterial counts 2730 ± 321 /100 ml - 

Vibrio sp. Bacterial counts 3.7 ± 2.5 x 103/100 ml - 

Vibrio cholerae Bacterial counts, elimination method Not detected - 

Enterococcus group Bacterial counts 31 ± 7 /100 ml - 

Test 8 
NIVA 5667-2008 

31 

Microscope counts 14.7 ± 1.3 x 104 m-3 ≥105 m-3

Phyla 4 ≥3 different Organisms 

≥50 μm Species >6 ≥5 different 

3000 ml-1 Dilution method 

95 % conf. interval 1000 - 1300 ml-1

Microscope counts 2558 ± 132 ml-1

≥1000 ml-1

Phyla 4 ≥3 different 

Organisms 

≥10-50 μm 

Species >10 ≥5 different 

Hetero. bact. Bacterial counts 1.6±0.9 x 104 ml-1 ≥104 ml-1

Coliform bacteria Bacterial counts 1130 ± 493 /100 ml - 

Vibrio sp. Bacterial counts 5.0±0.5 x 103/100 ml - 

Vibrio cholerae Bacterial counts, elimination method Not detected - 

Enterococcus group Bacterial counts 66 ± 37 /100 ml - 

Test 11 

Microscope counts 13.8 ± 1.8 x 104 m-3 ≥105 m-3

Phyla 5 ≥3 different Organisms 

≥50 μm Species >9 ≥5 different 

Dilution method 

95 % conf. interval 

5000 ml-1

2000 – 20000 ml-1 ≥1000 ml-1

Microscope counts 2795 ± 87 ml-1 

Phyla 3 ≥3 different 

Organisms 

≥10-50 μm 

Species 10 ≥5 different 

Hetero. bact. Bacterial counts 2.9 ± 1.71 × 104/ ml ≥104 ml-1

Coliform bacteria Bacterial counts 3.7 ± 1.5 / 100 ml - 

Vibrio sp. Bacterial counts 4.8 ± 2.4 × 104/100 ml - 

Vibrio cholerae Bacterial counts, elimination method Not detected - 

Enterococcus group Bacterial counts 1.2 ± 0.3 × 103/ 100 ml - 

Test 13 

Microscope counts 24.7 ± 1.5 x 104 m-3 ≥105 m-3

Phyla 3 ≥3 different 

Organisms 

≥50 μm 

Species >9 ≥5 different 

Dilution method 

95 % conf. interval 

3000 ml-1

1000-13000 ml-1

≥1000 ml-1

Microscope counts 1576 ± 147 ml-1 

Phyla 4 ≥3 different 

Organisms 

≥10-50 μm 

Species >10 ≥5 different 

Hetero. bact. Bacterial counts 5.1 ± 1.1 × 104/ ml ≥104 ml-1

Coliform bacteria Bacterial counts > 8 × 103/100 ml - 

Vibrio sp. Bacterial counts 3.9 ±3.9 × 103/100 ml - 

Vibrio cholerae Bacterial counts, elimination method Not detected - 

Enterococcus group Bacterial counts > 8 × 103/ 100 ml - 
NIVA 5667-2008 

32 

3.5 Biocidal effects on organisms ≥50 μm in minimum diameter 

The number of viable organisms ≥50 μm in minimum diameter, as determined on the basis of motility 

and integrity by microcope examination, in treated test water and control immediately after treatment 

and after five days of storage is given in Table 23. For the performance of the ballast water treatment 

system to pass the regulation D-2 of the IMO Guidelines, as given in Table 2, less than 10 viable 

organisms ≥50 μm in minimum diameter per m3

 should be present in the treated water after five days 

storage. 

As can be seen in Table 23 and indicated by the green background colour in the table cells, this 

requirement was fulfilled in all test reported. 

The equivalent requirement for the non-treated control test water is stated as: “If in any test cycle the 

average discharge results from the control water is a concentration less than or equal to 10 times the 

values in Regulation D-2.1, the test cycle is invalid.” It may be interpreted that the minimum level of 

viable organisms in the control test-water at the time of discharge (e.g. after 5 days storage) should be 

higher than 100 m-3. This requirement was fulfilled in all tests. 

Table 23. Viable organisms ≥50 μm in minimum diameter in treated test water and control 

immediately after treatment and after five days of storage. Green background indicates that required 

level was fulfilled. 

Treated water Control water 

Day 0 Day 5 Day 0 Day 5 

Organisms ≥50 μm in minimum diameter (individuals/m3) 

Requirement - <10 - >100 

Test cycle 1 0 ± 0 0 ± 0 5.6 ± 2.4 x 103 1.7 ± 0.5 x 103

Test cycle 2 0 ± 0 0.3 ± 1.0 7.8 ± 1.7 x 103 1.0 ± 0.3 x 103

Test cycle 3 1.3 ± 0.6 0.3 ± 1.0 4.3 ± 1.4 x 103 2.6 ± 1.7 x 102

Test cycle 4 0 ± 0 0 ± 0 4.3 ± 0.6 x 103 1.5 ± 0.7 x 102

Test cycle 5 0 ± 0 0 ± 0* 11.0 ± 3.3 x 103 5.4 ± 0.4 x 102

Test cycle 6 4.3 ± 2.5 0 ± 0 32.2 ± 7.9 x 103 6.8 ± 2.0 x 103

Test cycle 7 0.3 ± 1.0 0 ± 0 3.1 ± 1.0 x 103 6.3 ± 1.2 x 102

Test cycle 8 0 ± 0 0.3 ± 1.0 11.7 ± 0.4 x 103 3.3 ± 0.7 x 103

Test cycle 11 9.3 ± 1.2 0.0 ± 0 3.1 ± 1.0 x 103 7.6 ± 0.8 x 102

Test cycle 13 26 ± 3.5 0.7 ± 1.2 8.6 ± 3.8x 103 1.4 ± 0.5 x 103

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

The number of viable organisms ≥10-50 μm in minimum diameter, as determined by the serial dilution 

method in algal growth medium and by microscopy examination after incubation with CFDA-AM, in 

treated test water and control immediately after treatment and after five days of storage is given in 

Table 24. 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 

ml should be present in the treated water after five days storage. 

As can be seen in Table 24 and indicated by the green background colour in the table cells, this 

requirement was fulfilled in all 10 test cycles reported here, both when based on the dilution method 

and on microscope counts. 
NIVA 5667-2008 

33 

Comments should be made regarding the results for test cycle 3, 4, 5 and 6 (lower part of table 27). 

Allready in test cycle 3, ciliates were recorded in TT2 day 5 sample. The TT2 day 5 samples in the 

following test cycles (4, 5 and 6) all recorded the same ciliate. The ciliate is a benthic elongated 

sidmoid shaped cell, resembling the genus Colpidium sp. The species was not observed in any samples 

from WST. It was also recorded in TT2 day 0 test cycle 6, but not in the other TT2 day 0 samples. It 

was concluded that this ciliate was a contamination occurring in the ballast water tanks, specifically in 

the drain pipes from tanks. We improved the cleaning procedures of these drains with hot pressure 

washer between each run and have not experienced this problem since. 

Table 24. Viable organisms ≥10-50 μm in minimum diameter in treated test water and control 

immediately after treatment and after five days of storage. Green background indicates that required 

level was fulfilled. 

Treated water Control water 

Day 0 Day 5 Day 0 Day 5 

Organisms ≥10-50 μm in minimum diameter (individuals/ml) 

Requirement - <10 - >100 

Dilution method 

95 % confidence interval 

Test cycle 1 <0.2 0.4 2000 240 

<0.1-1.0 0.1-1.7 1000-24000 100-950 

Test cycle 2 <0.2 <0.2 2000 1300 

<0.1-1.1 <0.1-1.1 1000-14000 500-3900 

Test cycle 3 0.7 0.2 2000 2400 

0.2-2.1 <0.1-1.1 1000-14000 1000-9500 

Test cycle 4 1.3 0.2 2000 1600 

0.5-3.9 <0.1-1.1 1000-14000 600-5300 

Test cycle 5 0.7 0.2 2000 2400 

0.2-2.1 <0.1-1.0 1000-14000 1000-9600 

Test cycle 6 0.8 0 2000 1600 

0.3-2.5 <0.1-1.0 1000-14000 600-5300 

Test cycle 7 5 0.8 1100 500 

2-17 0.3-2.5 300-4800 200-2000 

Test cycle 8 0.2 <0.2 300 900 

<0.1-1.1 <0.1-1.1 100-1300 300-300 

Test cycle 11 11 0.8 5000 1600 

4-30 0.3-2.5 2000-24000 600-5300 

Test cycle 13 2.3 0.4 5000 2400 

0.9-8.7 0.1-1.7 2000-24000 1000-9500 

Microscope counts 

Test cycle 1 0 ± 0 0 ± 0 1748 ± 321 842 ± 65 

Test cycle 2 0.3 ± 0.6 0.7 ± 1.2 1368 ± 209 1372 ± 93 

Test cycle 3* 0.3 ± 0.6 0.3 ± 0.6 1490 ± 128 1311 ± 156

Test cycle 4* 1.7 ± 0.6 0.7 ± 1.2 1527 ± 212 1349 ± 156

Test cycle 5* 0.7 ± 0.6 0 ± 0 2302 ± 162 1865 ± 108

Test cycle 6* 92.5 ± 13 2.7 ± 0.6 2016 ± 150 1237 ± 133

Test cycle 7 5.7 ± 1.5 0.7 ± 0.6 2620 ± 213 2193 ± 43 

Test cycle 8 5.3 ± 1.5 1.3 ± 0.6 3087 ± 205 1587 ± 377

Test cycle 11 20 ± 2.6 1.7 ± 0.6 2895 ± 95 1256 ± 175

Test cycle 13 13.7 ± 5.9 1.7 ± 0.6 1558 ± 85 741 ± 92 
NIVA 5667-2008 

34 

Microscope counts with contamination of Ciliates 

Test cycle 3 0.3 ± 0.6 5.7 ± 2 

Test cycle 4 1.7 ± 0.6 12.7 ± 1.5 

Test cycle 5 0.7 ± 0.6 10.3 ± 2.1 

Test cycle 6 95 ± 13 16.7 ± 2.9 

The equivalent requirement for the non-treated control test water is stated as: “If in any test cycle the 

average discharge results from the control water is a concentration less than or equal to 10 times the 

values in Regulation D-2.1, the test cycle is invalid.” It may be interpreted that the minimum level of 

viable organisms in the control test water at the time of discharge (e.g. after 5 days storage) should be 

higher than 100 per ml. This requirement was fulfilled in all test cycles. 

3.7 Bactericidal effects 

The numbers of thermotolerant coliform bacteria, Vibrio sp., Vibrio cholerae and intestinal 

Enterococci, determined as described in section 2.6.4, in treated test water and control water 

immediately after treatment and after five days of storage for test cycles are given in Table 25. 

Regulation D-2 only requires documentation of the bactericidal effect of the ballast water treatment 

system on Escherichia coli (a thermotolerant coliform bacteria), Vibrio cholerae (toxicogenic 

serotypes O1 and O139) and Enterococci given as a maximum allowable residual concentration after 

five days storage; <250 cfu/100ml, <1 cfu/100ml and <100 cfu/100ml, respectively. 

As seen from Table 25, and indicated by the green background colour in table cells, these 

requirements are fulfilled for E. coli (since the requirement is fulfilled for thermotolerant coliform 

bacteria as a group) and Enterococci. However, there are no required additions of these bacterial 

groups. Vibrio sp. are common in salt and brackish surface water, while the toxicogenic Vibrio 

cholerae serotypes O1 and O139 have not been found in Norwegian coastal waters. Vibrio cholerae

was not found in any of the samples from treated water. 

Table 25. Culturable thermotolerant coliform bacteria, Vibrio sp. and intestinal Enterococci in treated 

test water and control water immediately after treatment and after five days of storage. Green 

background indicates that required level was fulfilled. 

Treated water Control water 

Day 0 Day 5 Day 0 Day 5 

Heterotrophic bacteria (cfu/ml) 

Requirement - - - - 

Test cycle 1 - - - - 

Test cycle 2 - - - - 

Test cycle 3 1.5 ± 0.7 x 105 3.0 ± 1.5 x 105 2.6 ± 0.5 x 106 4.8 ± 6.5 x 105

Test cycle 4 3.9 ± 6.5 x 104 6.3 ± 3.7 x 105 1.0 ± 0.7 x 106 3.5 ± 2.1 x 105

Test cycle 5 4.6 ± 4.4 x 104 5.4 ± 1.8 x 105 1.4 ± 0.3 x 106 2.9 ± 2.1 x 105

Test cycle 6 6.4 ± 7.2 x 103 7.7 ± 6.0 x 105 6.5 ± 1.3 x 105 2.5 ± 3.1 x 105

Test cycle 7 8.2 ± 3.5 x 102 2.6 ± 0.4 x 106 3.4 ± 0.9 x 105 9.6 ± 2.0 x 105

Test cycle 8 2.1 ± 0.6 x 103 2.8 ± 0.6 x 106 2.8 ± 1.8 x 104 9.4 ± 1.9 x 105

Test cycle 11 1.1 ± 1.2 x 103 1.1 ± 0.3 x 106 3.9 ± 2.1 x 104 4.1 ±1.6 x 105

Test cycle 13 2.9 ± 1.9 x 103 2.4 ± 1.7 x 106 7.7 ± 2.8 x 104 5.0 ± 2.9 x 104

Coliform bacteria and Escherichia coli* (cfu/100 ml) 

Requirement - - <250* - - 

Test cycle 1 21 ± 19 40 ± 6 0 ± 0* 17 ± 5 0 ± 0 

Test cycle 2 0 ± 0 0 ± 0 0 ± 0* 67 ± 57 133 ± 231 
NIVA 5667-2008 

35 

Test cycle 3 0 ± 0 0 ± 0 0 ± 0* 23 ± 6 0 ± 0 

Test cycle 4 0 ± 0 0 ± 0 0 ± 0* 767 ± 231 0 ± 0 

Test cycle 5 0 ± 0 0 ± 0 0 ± 0* 8670 ± 611 70 ± 27 

Test cycle 6 0 ± 0 0 ± 0 0 ± 0* 3630 ± 321 17 ± 6 

Test cycle 7 0 ± 0 7 ± 2 0 ± 0* 3500 ± 900 867 ± 416 

Test cycle 8 0 ± 0 0.3 ± 0.6 0 ± 0* 1370 ± 231 447 ± 653 

Test cycle 11 0 ± 0 0 ± 0 0 ± 0* 17 ± 4 1 ± 1 

Test cycle 13 6.7 ± 0.6 4 ± 1 0 ± 0* > 8000 > 8000 

Vibrio sp. and Vibrio cholerae** (cfu/100 ml) 

Requirement - - <1** - - 

Test cycle 1 - >100 <1** >100 >100 

Test cycle 2 - >100 <1** >100 >100 

Test cycle 3 12 ± 10 31 ± 13 <1** 3.5 ± 1.6 x 104 2.7 ± 1.2 x 104

Test cycle 4 6.7 ± 5.8 59 ± 27 <1** 2.1 ± 0.9 x 106 4.4 ± 2.8 x 106

Test cycle 5 22 ± 33 98 ± 10 <1** 1.8 ± 1.2 x 106 2.9 ± 0.8 x 106

Test cycle 6 3 ± 6 6 ± 3 <1** 9.3 ± 9.5 x 104 6.5 ± 5.1 x 104

Test cycle 7 9 ± 1 9 ± 9 <1** 3.3 ± 2.1 x 103 3.2 ± 4.5 x 103 

Test cycle 8 0.3 ± 0.6 3 ± 3 <1** 6.0 ± 1.0 x 103 8.5 ± 5.6 x 103 

Test cycle 11 0 ± 0 4 ± 0 <1** 1.4 ± 0.5 x 104 1.8 ± 1.4 x 104

Test cycle 13 0.7 ± 0.6 830 ± 840 <1** 4.1 ± 3.6 x 103 8.0 ± 2.5 x 102

Enterococcus group and Intestinal Enterococci*** (cfu/100 ml) 

Requirement - - <100*** - -

Test cycle 1 1.3 ± 2.3 2 ± 1 2 ± 1*** 1250 ± 383 194 ± 317 

Test cycle 2 0.0 ± 0.0 1 ± 0 1 ± 1*** 197 ± 29 147 ± 23 

Test cycle 3 0.0 ± 0.0 0 ± 0 0 ± 0*** 15 ± 12 0.0 ± 0.0 

Test cycle 4 0.0 ± 0.0 0.3 ± 0.6 0 ± 0*** 2.7 ± 2.2 x 103 2.4 ± 1.3 x 103

Test cycle 5 9 ± 16 0 ± 0 0 ± 0*** 2.1 ± 0.7 x 102 8.0 ± 6.1 x 102

Test cycle 6 0.3 ± 0.6 0 ± 0 0 ± 0*** 49 ± 38 9 ± 4 

Test cycle 7 0.0 ± 0.0 0 ± 0 0 ± 0*** 41 ± 3 17 ± 1 

Test cycle 8 0.0 ± 0.0 0.7 ± 1.2 0.7 ± 1.2*** 76 ± 40 11 ± 2 

Test cycle 11 0 ± 0 0 ± 0 0 ± 0*** 9.8 ± 3.4 x 102 8.3 ± 2.1 x 103

Test cycle 13 2.7 ± 0.6 0.3 ± 0.6 0.3 ± 0.6*** > 8000 1.1 ± 0.4 x 104 

* The figures refer to the number identified as Escherichia coli/100 ml within the group of coliform 

bacteria. There is a requirement for Escherichia coli being <250 cfu/100ml after five days storage (D-2). 

** The figures refer to the number of Vibrio cholerae/100 ml after five days storage. There is a requirement 

for toxicogenic Vibrio cholerae (serotypes O1 and O139) being <1 cfu/100 ml after five days storage (D-2). 

*** The figures refer to the number identified as intestinal Enterococci/100 ml within the group of 

Enterococcus. There is a requirement for intestinal Enterococci being <100 cfu/100ml after five days 

storage (D-2). 

3.8 Ecotoxicological responses 

The ecotoxicity of treated ballast water was tested at least two times with 2 different water qualities (2 

different salinities). The toxicity of undiluted (100 %) and diluted samples was determined on three 

trophic levels using marine acute and chronic tests with untreated control ballast water as control. The 

tests and test results are summarized below. The complete test reports are presented in the 

Appendixes. 
NIVA 5667-2008 

36 

Growth inhibition of the marine alga Skeletonema costatum 

Treated water from TT1 and untreated control water were used in the tests, which were conducted 3 times 

and included both water qualities (high and moderate salinity). An overview of the tests and duration is 

shown in Table 26. 

Table 26. Overview of growth inhibiton tests with marine algae Skeletonema costatum 

NIVA study number Duration Water quality 

B484/c 30th Nov - 3th Dec 2006 High salinity (32.7 PSU) 

B484/d 6th Dec - 9th Dec 2007 High salinity (32.1 PSU) 

B493 14th Feb -17th Feb 2007 Moderate salinity (21.0 PSU) 

There was no significant difference in growth rates between the untreated and treated ballast water, and the 

control sea water in any of the tests. Hence, for the treated Ballast water, the results are given in table 27. 

Table 27. Growth inhibition of the marine algae Skeletonema costatum by treated ballast water 

Effects on growth rate Concentration 

% 

48 h ErC50

1

 >100 

NOEC2 ≥100 1

 ErC50: Concentration that causes 50 % reduction of the growth rate as compared to controls 1

 NOEC: No Observable Effect Concentration, i.e. the highest concentration that does not cause a significant inhibition of the 

growth rate 

It was concluded that no growth inhibiting effect on the marine alga Skeletonema costatum was 

caused by the treated ballast water. 

The complete test reports can be found in Appendix I. 

Reproductive toxicity to the marine crustacean Nitocra spinipes 

Treated water from TT1 and untreated control water were used in the tests. The test was conducted 2 times 

with the 2 different water qualities (high and moderate salinity). An overview of the tests and duration is 

shown in Table 28. 

Table 28. Overview of reproductive toxicity tests to the marine crustacean Nitocra spinipes 

NIVA study number Duration Water quality 

B484/b 10th Nov - 27th Nov 2006 High salinity (32.7 PSU) 

B489/b 8th Mar - 20th Mar 2007 Moderate salinity (20.5 PSU) 

It was concluded that the reproduction of Nitocra spinipes is not affected by exposure of treated ballast 

water for 14 days. There was no significant difference in the average number of offspring in females 

exposed to Treated Ballast water when compared to non treated ballast water of the same quality in any of 

the tests. The main results are given in the table 29. 

 

1

 ErC50: Concentration that causes 50 % reduction of the growth rate as compared to controls 2

 NOEC: No Observable Effect Concentration, i.e. the highest concentration that does not cause a significant 

inhibition of the growth rate 
NIVA 5667-2008 

37 

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

The complete test reports can be found in Appendix J. 

Acute toxicity to the marine crustacean Acartia tonsa 

Treated water from TT1 and untreated control water were used in the tests. The test was conducted 2 times 

with the 2 different water qualities (high and moderate salinity). An overview of the tests and duration is 

shown in Table 30. 

Table 30. Overview of acute toxicity tests with the marine crustacean Acartia tonsa 

NIVA study number Duration Water quality 

B484 30th Nov - 2th Des 2006 High salinity (32.7 PSU) 

B489 18th Jan - 20th Jan 2007 Moderate salinity (20.5 PSU) 

In the test conducted 30th Nov - 2th Des 2006, a mortality of 27% was observed at the highest tested 

concentration (100%). It should be commented that high death-rate in the control can be a general problem 

with the Acartia tonsa test, as also experienced in the current test. This death-rate can give misleading 

results. No effects were observed in the test with the other marine crustacean Nitocra spinipes. It is 

therefore doubtful that the effect observed can be attributed to toxicity. 

In the test conducted 18th Jan - 20th Jan 2007, no mortality of Acartia was observed in treated ballast water 

at 100% concentration or lower. 

Lack of mortality >50 % at any test concentration precludes estimation of LC50. The results are given in 

Table 31. 

Table 31. Acute toxicity of treated ballast water on Acartia tonsa. 

NIVA study number Concentration (%) 95 % confidence limits 

B484 48h LC50 * >100 - 

B489 48h LC50 * >100 - 

* LC = Lethal Concentration LC50 represents the concentration that results in lethality of 50 % of the test animals. 

The complete test reports can be found in Appendix J. 

Chronic toxicity towards the juvenile turbot (Scopthalmus maximus) 

Treated water from TT1 and untreated control water were used in the tests. The test was conducted 2 times 

with the 2 different water qualities (high and moderate salinity). An overview of the tests and duration is 

shown in Table 32. 

Table 32. Overview of chronic toxicity tests with the juvenile turbot (Scopthalmus maximus) 

NIVA study number Duration Water quality 

B484/f 1th Nov - 5th Des 2006 High salinity (32.2 PSU) 

B489 10th Jan - 2th Feb 2007 Moderate salinity (21.5 PSU) 

NIVA Study number Treated Ballast water Control 

B484/b 20.5 ± 9.1 21.9 ± 9.4 

B489/b 11.8 ± 9.3 11.1 ± 11.7 
NIVA 5667-2008 

38 

There was no mortality observed in any of the test, and the exposed fish did not behave or look different 

from control fish. Treated Ballast Water did not exert any significant effects on the growth rate of juvenile 

turbot as determined using ANOVA statistics. 

The results of the test are given in table 33. 

Table 33. Chronic toxicity tests of treated ballast water to the juvenile turbot (Scopthalmus maximus)

NIVA study number Parameter Control Treated Ballast water 

B484/f Average growth rate d-1 0.0063 0.0059 

B489 Average growth rate d-1 0.0067 0.0071 

The complete test reports can be found in Appendix K. 

Acute toxicity towards the juvenile turbot (Scopthalmus maximus) 

Treated water from TT1 and untreated control water were used in the tests. The test was conducted 2 times 

with the 2 different water qualities (high and moderate salinity). An overview of the tests and duration is 

shown in Table 34. 

Table 34. Overview of acute toxicity tests with the juvenile turbot (Scopthalmus maximus) 

NIVA study number Duration Water quality 

B495 1th Nov - 4th Nov 2006 High salinity (32.2 PSU) 

B489 10th Jan - 15th Jan 2007 Moderate salinity (21.5 PSU) 

Treated Ballast Water did not cause mortality to juvenile turbot. As no mortality was observed, an 

exact LC50 cannot be estimated. NOEC in this test is >100 %. The NOEC is the highest concentration 

without observed toxic effects. The results of the tests are given in Table 35. 

Table 35. Acute toxicity of treated ballast water to the juvenile turbot (Scopthalmus maximus)

Parameter Concentration (%) 95% confidence limits 

LC50 (96 h): >100 - 

NOEC based on mortality: ≥100 

LC50 - the concentration causing 50 % mortality 

NOEC - no observed effect concentration 

The complete test reports can be found in Appendix K. 

Summary of toxicological tests 

Based on the conducted acute and chronic toxicity tests it is concluded that ballast water treated with 

the present technology do not provide hazard or risk to the marine ecosystem when released. 
NIVA 5667-2008 

39 

Appendix A. Total project management reports 

Appendix B. Chemical water quality preparation 

Appendix C. Biological water quality preparation 

Appendix D. Collection and preservation of samples 

Appendix E. Logging of in situ measurements 

Appendix F. Evaluation form for organisms >50 μm 

Appendix G. Evaluation form for organisms ≥10-50 

μm 

Appendix H. Evaluation form for heterotrophic 

bacteria, coliforms, E. coli, Enterococcus group, 

intestinal Enterococci, and Vibrio cholerae 

Appendix I. Acute and chronic toxicity of the green 

algae Skeletonema costatum 

Appendix J. Acute and chronic toxicity of marine 

copepods 
NIVA 5667-2008 

40 

Appendix K. Acute and chronic toxicity of juvenile 

turbot 

Appendix L. Operational performance 
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