REPORT SNO 5617-2008 

Shipboard testing of the 

PureBallast Treatment 

System of AlfaWall AB 

Confidential 
NIVA 5617-2008 

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 

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Internet: www.niva.no 

Title 

Shipboard testing of the PureBallast Treatment System of 

AlfaWall AB 

Serial No. 

5617-2008 

Report No. Sub-No. 

O-27170 

Date 

19th May 2008 

Pages Price 

?? 

Author(s) 

Tor Gunnar Jantsch 

August Tobiessen 

Helge Liltved 

Topic group

Water treatment 

Geographical area

Norway 

Distribution 

Confidential 

Printed 

NIVA 

Client(s) 

AlfaWall AB, Tumba, Sweden 

Client ref. 

Carl Tullstedt 

Abstract 

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

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

conducted on board M/V Aida and a total of 4 test cycles have been completed. 

 

4 keywords, Norwegian 4 keywords, English 

1. Ballastvann 1. Ballast water 

2. Ombord 2. Shipboard 

3. Testing 3. Testing 

4. AlfaWall AB 4. AlfaWall AB 

 Tor Gunnar Jantsch Helge Liltved Jarle Nygard 

Project Manager Research Manager Strategy director

ISBN 978-82-577-5352-8
NIVA 5617-2008 

Preface 

The tests were conducted in 2007 and 2008 on board M/V Aida. 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 Per 

Borin were the lead representatives of AlfaWall AB, while Hanna 

Behrens, Egil Dragsund, Håkon Øvrum and Erik Nyquist have 

represented DNV in the project. From NIVA, August Tobiesen, Tor 

Gunnar Jantsch and Helge Liltved 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 demonstrated in completing this project. 

Oslo, May 2008 

Tor Gunnar Jantsch 
NIVA 5617-2008 

Contents 

Abbreviations and acronyms 7

Summary 8

1. Background 9

2. Infrastructure and test protocols 10

2.1 Description of ship and technology set-up 10

2.1.1 Ship and shipboard installation 10

2.1.2 The PureBallast ballast water management system 12

2.1.3 Overview of water quality requirements of shipboard testing 12

2.2 Running test cycles 13

2.3 Sampling 17

2.4 Sample analysis 20

3. Sampling procedures 21

3.1 Representativeness of samples 21

3.2 Sampling of test waters 21

3.3 Sample preservation 22

3.4 Measures to avoid cross-contamination during test water transfer 

and sampling 22

4. Testing and measurement protocols 23

4.1 Biological measurements – initial quick tests 23

4.1.1 Rapid method of evaluation of density of 10-50 μm organisms 23

4.2 In situ measurements 23

4.2.1 Temperature 23

4.3 Discrete samples 23

4.3.1 Salinity 23

4.3.2 Particulate organic carbon (POC), Dissolved organic carbon 

(DOC) and Total organic carbon (TOC) 23

4.3.3 Total suspended solids (TSS) 24

4.3.4 Organisms >50 μm 24

4.3.5 Organisms ≥10-50 μm 24

4.3.6 E. Coli 25

4.3.7 Intestinal Enterococci 25

4.3.8 Vibrio spp. and Vibrio cholerae (Serotypes O1 and O139) 25

4.3.9 Sample transportation 26

5. Results and discussion 28

5.1 QA/QC prosedures 28

5.2 Operational performance of the PureBallast system 28

5.3 Chemical water quality 29

5.4 Fulfillment of the biological water quality criteria and biocidal 

effects on organisms ≥50 μm in minimum diameter 31
NIVA 5617-2008 

5.5 Fulfillment of the biological water quality criteria and biocidal 

effects on organisms ≥10-50 μm in minimum diameter 31

5.6 Bactericidal effects 32

Appendix A. Total project management reports 34

Appendix B. Collection and preservation of samples 34

Appendix C. Logging of in situ measurements 34

Appendix D. Evaluation form for organisms >50 μm 34

Appendix E. Evaluation form for organisms ≥10-50 μm 34

Appendix F. Evaluation form for coliforms, E. coli, Enterococcus 

group, intestinal Enterococci, and Vibrio cholerae 34

Appendix G. Operational performance 34
NIVA 5617-2008 

Abbreviations and acronyms 

Wallenius AOT Module – Wallenius Advanced Oxidation Technology Module 

CFDA-AM - 5-carboxyfluorescein diacetate acetoxymethyl ester 

DNV – Det Norske Veritas 

DO – dissolved oxygen 

DOC – dissolved organic carbon 

E. coli – Escherichia coli

GF/F – glass fiber filter grade F 

GLP – Good laboratory Practice 

IMO – International Maritime Organization 

ISO – International Organisation for Standardization 

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-S3 – sampling points 1-3 

Std – standard deviation 

TCB – thermotolerant coliform bacteria 

TCBS – MacConkey and thiosulphate citrate bile salt agar 

TSS – total suspended solids 

Xi

 - individual analytical result; in calculating the standard deviation 

X – the arithmetic mean of individual analytical results; in calculating the standard deviation 
NIVA 5617-2008 

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Summary 

Shipboard testing of the PureBallast Treatment System (PBTS) of AlfaWall AB has been completed in 

the period of October 2007 to April 2008, a period exceeding 6 months. The testing has been 

conducted according to the IMOs Guidelines for approval of ballast water management systems (G8), 

Res. MEPC.126(53) Annex 3, hereafter referred to as G8. The tests were carried out onboard the vessel 

M/V Aida. A total of 4 successful test cycles have been completed, out of which three were valid with 

regards to organism concentration in the uptake water. 

Fulfilment of chemical requirements of uptake ballast water 

• There are no requirements regarding chemical water quality. Uptake water was analysed with 

respect to temperature, salinity, particulate organic carbon and total suspended solids as 

required. The analyses show that a wide variety of water qualities were covered by the 

conducted tests, with variations in temperature from approximately 2 to 12 oC, in salinity from 

7 to 33 PSU, in particulate organic carbon from <0.5 to 25 mg/l, and suspended solid from 1 

to 14 mg/l. 

Fulfilment of the biological requirements of the uptake ballast water 

The required density of the ≥50 μm group (viable organism concentration exceeding 10 times 

the values of Regulation D-2.1) was met in all 4 tests cycles. 

• The requirement in the ≥10-50 μm group (viable organism concentration exceeding 10 times 

the values of Regulation D-2.1) was met in 3 test cycles when assayed by microscope counts 

and in 2 test cycles when assayed by the dilution-culture method.

Biocidal effects of treatment 

• The required “less than 10 viable organisms ≥50 μm in minimum diameter per m3

 in the 

treated water” was fulfilled in all 4 tests cycles. The equivalent requirement of the non-treated 

control water, stating that the level of viable organisms at deballasting should be higher than 

10 per m3, was fulfilled in all 4 test cycles. 

• The required “less than 10 viable organisms ≥10 - 50 μm in minimum diameter per ml in 

treated water on deballasting”, was reached in all 4 test cycles, both when assayed by 

microscope counts and when assayed by the dilution-culture method. The equivalent 

requirement for the non-treated control water upon deballasting states that the level of viable 

organisms should be higher than 10 per ml. This requirement was fulfilled in all test cycles by 

both detection methods. 

• Regulation D-2 requires the maximum concentrations of Escherichia coli, Vibrio cholerae

(toxicogenic serotypes O1 and O139) and Intestinal Enterococci to be; <250 cfu/100ml, <1 

cfu/100ml and <100 cfu/100ml, respectively, in the treated ballast water upon discharge. 

These requirements were fulfilled for all the bacteria in all 4 test cycles. 
NIVA 5617-2008 

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1. Background 

The overall goal for AlfaWall AB is to aquire certification of the PureBallast Treatment System 

(PBTS) 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. 

Land-based pilot tests were conducted by NIVA 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. The experience from the pilottest served as a fundament for 

large scale land-based and shipboard testing of the technology. 

Land-based testing for type approval was conducted by NIVA in the period of November 2006 to May 

2007. The tests were conducted in accordance with G8 and G9. 

Shipboard testing for type approval, as reported here, was conducted in the period of October 2007 to 

April 2008. The tests were conducted in accordance with G8. 
NIVA 5617-2008 

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2. Infrastructure and test protocols 

2.1 Description of ship and technology set-up 

2.1.1 Ship and shipboard installation 

The shipboard testing of the PureBallast Treatment System of Alfa Wall AB has been conducted on 

board the Wallenius Marines car carrier M/V Aida (see Figure 1). 

Figure 1 Wallenius Car Carrier M/V Aida. 

Configuration of the test tanks with pipes, valves and devices is shown in Figure 2 and Figure 3. The 

tanks have the following labelling (Table 1): 

Table 1 Labelling of ballast tanks used for testing. 

Description 

Tank for control water (TCW1) No.3 D.B (P) 

Tank for control water (TCW2) No.3 D.B (S) 

Tank for treated water (TTW1) No. 4 D.B (P)

Tank for treated water (TTW2) No. 4 D.B (S)
NIVA 5617-2008 

11 

Figure 2. Configuration of the test tanks with pipes, valves and devices. 

Figure 3. Overview of Ballast Water Tanks for M/V Aida. 
NIVA 5617-2008 

12 

2.1.2 The PureBallast ballast water management system 

System and component description 

The PureBallast Treatment System of 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 Wallenius AOT (Advanced Oxidation Technology) Module, a 

patented treatment system using the synergetic effects of in situ produced free radicals and direct 

photo-radiation to inactivate microbes and 3) control and auxiliary equipment including , cleaning in 

place (CIP), sampling points to control water flows and measure different alarm levels during 

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

Units of the PureBallast Treatment System 

The test equipment is a complete PureBallast Treatment System with a flow rate of max 1000 m3/h. 

The system has one pre-filter and four Wallenius AOT units with capacity of 250 m3

/h each and a 

CIP-unit for cleaning the AOT units. 

Figure 4. The PureBallast Treatment System. AOT units to the left and pre-filter and CIP-unit to the 

right.

2.1.3 Overview of water quality requirements of shipboard testing 

Table 2 summarizes the biological requirements regarding biological water quality in uptake ballast 

water, in treated ballast water, and in control ballast water on discharge, as stated in regulation G8 by 

IMO. 

Wallenius AOT

Filter

CIP
NIVA 5617-2008 

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Table 2 Required biological water quality in uptake ballast water, in treated ballast water, and in 

control ballast water on discharge, as stated in regulation G8 by IMO. 

Organism group Uptake water for both 

the control tank and the 

ballast water to be 

treated 

In treated ballast 

water on discharge 

In control ballast water 

on discharge 

≥50 μm min. 

dimension 

Viable organism 

concentration exceeding 

10 times the values of 

Regulation D-2.1 

(Regulation D-2.1: <10 

viable organisms per m3) 

Concentration to be 

achieved: >100 viable 

organisms per m3

In compliance with 

Regulation D-2 

(Regulation D-2: <10 

viable organisms per 

m

3

) 

Concentration to be 

achieved: <10 viable 

organisms per m3

Viable organism 

concentration exceeding 

the values of Regulation 

D-2.1 

(Regulation D-2.1: <10 

viable organisms per m3

) 

Concentration to be 

achieved: >10 viable 

organisms per m3

≥10-50 μm min. 

dimension 

Viable organism 

concentration exceeding 

10 times the values of 

Regulation D-2.1 

(Regulation D-2.1: <10 

viable organisms per ml) 

Concentration to be 

achieved: >90 viable 

organisms per ml 

In compliance with 

Regulation D-2 

(Regulation D-2: <10 

viable organisms per 

ml) 

Concentration to be 

achieved: <10 viable 

organisms per ml 

Viable organism 

concentration exceeding 

the values of Regulation 

D-2.1 

(Regulation D-2.1: <10 

viable organisms per ml) 

Concentration to be 

achieved: >10 viable 

organisms per ml 

Vibrio cholerae - <1 cfu/100 ml - 

Escherichia coli - <250 cfu/100 ml - 

Intestinal 

Enterococci 

(Serotype O1 and 

O139)

- 

 

<100 cfu/100 ml - 

There are no requirements regarding chemical water quality. Uptake water should be analysed with 

respect to temperature, salinity, particulate organic carbon and total suspended solids. 

2.2 Running test cycles 

The different water transfers between tanks via the PureBallast Treatment System during a test cycle 

including the control is shown in Figure 5. One test cycle involves consecutive treatment of test water 

during ballasting by the 50 μm filter unit and the Wallenius AOT Module of the PureBallast 

Treatment System (green lines), transferring the test water from the sea to a tank for treated water 

(TTW) (Figure 5, upper left). A second treatment is conducted by the Wallenius AOT unit during 

deballasting (Figure 5, lower right). A control cycle (red lines) is run by ballasting seawater to a tank 

for control water (TCW) using the ballast water pump of the vessel, but in by-pass of the PureBallast 

system (Figure 5, upper right). The deballasting of control water is also conducted by pumping only, 

by-passing the PureBallast system (Figure 5, lower left). 
NIVA 5617-2008 

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Ballast of treated water: Ballast of control water: 

AOT

Q1

Q3

Q2

PureBallast

1 m

1 m

1 m3

S1

Control tank(s)

TCW

Treated tank(s)

TTW

Seachest

 

AOT

Q1

Q3

Q2

PureBallast

Control tank(s)

TCW

Treated tank(s)

TTW

1 m

1 m

1 m3

S2

Seachest

 

Deballast of control water: Deballast of treated water: 

 

Figure 5 Illustration of ballasting and deballasting of treated water and control water, including 

sampling valves Q1-Q3 and samples S1-S4. 

The figures 6-10 give a schematic overview of the different phases of a ballasting and deballasting 

cycle, including cleaning of the ballast water piping system prior to running the cycle (figure 6), 

ballast of treated water (figure 7), ballast of control water (figure 8), deballast of control water (figure 

9), and deballast of treated water (figue 9). 
NIVA 5617-2008 

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Figure 6 Cleaning of BW piping system prior to test. 

Figure 7 Ballast of treated water. 
NIVA 5617-2008 

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Figure 8 Ballast of control water. 

Figure 9 Deballast of control water. 
NIVA 5617-2008 

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Figure 10 Deballast of treated water. 

2.3 Sampling 

The various sampling points at different water transfers between tanks via the PureBallast Tratment 

System, and transfer of control water, during a test cycle are shown in Figure 5 and Table 3. The 

uptake water to the control tank was sampled (S1) from the sea chest (valve Q1) simultaneously with 

ballasting of the tank for control water (TCW) (Figure 5, upper right). The uptake water to the 

treatment process was sampled (S2) from the sea chest (valve Q1) simultaneously with ballasting (and 

treatment of the water) of the tank for treated water (TTW) (Figure 5, upper left). The discharge 

control water was sampled (S3) from valve Q2 upon deballasting of the tank for control water (TCW) 

(Figure 5, lower left). The discharge treated water was sampled (S4) from valve Q3 simultaneously 

with deballasting (and deballast treatment) of the tank for treated water (TTW) (Figure 5, lower right). 

Valve Q1, Q2 and Q3 were equipped with sampling hoses for transferring the water volume to a 

station for flow measurement and sampling. Figure 11 shows the arrangement of the flowmeter for 

measuring volume of samples. Figure 12 shows the collection of sample for analysis of organisms >50 

μm. 

Table 3 Overview of sample collection. 

Sample description Abbreviation Sampling point Number of replicates 

Influent water to control tank S1 Seachest, valve Q1 3* 

Influent water to treated tank S2 Seachest, valve Q1 3* 

Discharge control water S3 Valve Q2 3** 

Discharge treated water S4 Valve Q3 9*** 

*Three replicate samples to be collected over the period of uptake (e.g. beginning, middle, end) 

**Three replicate samples to be collected over the period of disharge (e.g. beginning, middle, end) 

***Three replicate samples to be collected at each of three times during the period of discharge (e.g. 3 

x beginning, middle, end) 
NIVA 5617-2008 

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Figure 11 Arrangement of flowmeter for measuring volume of samples. 
NIVA 5617-2008 

19 

Figure 12 Collection of samples for analysis of organisms > 50 μm. 

Samples were collected to document: 

1) the source water chemical characteristics in accordance with parameters defined by IMO, G8. 

2) that the water quality in the uptake seawater is meeting the biological water quality criteria 

defined by IMO, 

3) that the biological quality of the discharge control water is meeting the biological water 

quality criteria defined by IMO, 

4) that the biological quality of the discharge treated water is meeting the biological water quality 

criteria defined by IMO, 

5) the efficiency of the PureBallast Treatment System is in accordance with requirements for 

removing/inactivating target organisms in the ballast water upon discharge 

. 
NIVA 5617-2008 

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Sampling numbers are indicated by S1-S4 in Figure 5 as described above. 

Procedures for sampling and transfer are described in detail in section 3. 

2.4 Sample analysis 

Table 4 summarizes all types of measurements taken during the study. 

Table 4 Parameters measured during the study; type of sampling and measurement location. 

Parameter Sample number Type of sample Location for 

analysis 

Operational parameters for the PureBallast treatment system 

 

Chemical water quality measurements 

Temperature S1, S2, S3, S4 In situ, 

continuous 

M/V Aida 

Salinity S1, S2, S3, S4 Discrete grab NIVA, Norway,

Emu ltd, UK, 

Intertek 

laboratory, 

Germany 

Dissolved organic carbon, Total 

organic carbon , Particulate organic 

carbon 

S1, S2, S3, S4 Discrete grab NIVA, Norway, 

Emu ltd, UK, 

Intertek 

laboratory, 

Germany 

Total suspended solids (TSS) S1, S2, S3, S4 Discrete grab NIVA, Norway, 

Emu ltd, UK, 

Intertek 

laboratory, 

Germany 

Biological treatment performance parameters 

Organisms > 50 μm S1, S2, S3, S4 Discrete grab M/V Aida 

Organisms ≥10-50 μm S1, S2, S3, S4 Discrete grab M/V Aida and 

NIVA 

E. coli S1, S2, S3, S4 Discrete grab M/V Aida 

NIVA 

Intestinal Enterococci S1, S2, S3, S4 Discrete grab M/V Aida 

NIVA 

Vibrio sp. S1, S2, S3, S4 Discrete grab M/V Aida 

NIVA 

Vibrio cholerae (Serotype O1 and 

O139) 

S1, S2, S3, S4 Discrete grab M/V Aida 

NIVA 
NIVA 5617-2008 

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3. Sampling procedures 

3.1 Representativeness of samples 

Separate sampling lines from the valves to the sample points were used to assure that representative 

samples were drawn. Pipes were flushed with a volume corresponding to 1 times the piping volume (3 

minutes) prior to sampling. For control water and treated water, samples were collected at the 

beginning, middle and end of ballasting and deballasting. All samples were collected in triplicate, 

except for discharge treated ballast water were 9 samples were collected according to IMO G8. 

3.2 Sampling of test waters 

Table 5 gives an overview of the sampling equipment used to collect samples for the individual 

parameters. 

Table 5 Equipment and containers used for sampling and sampled volume for the individual 

parameters. 

Parameter Sampling 

equipment 

Sample container Collected 

volume 

S1, S2, S3, S4 

Temperature Temp. 

meter 

- - 

Salinity Probe - - 

POC/DOC/TOC Directly Acid washed glass 

bottle 

100 ml* 

TSS Directly Clean plastic bottle 1000 ml* 

Organisms ≥ 50 μm Sieving** Clean glass bottle 1 m3

Organisms 10-50 

μm 

Directly Clean glass bottle 1000 ml 

E. Coli 

Intestinal 

Enterococci 

Vibrio sp. 

Vibrio cholerae

Directly Sterile plastic/glass 

bottle 1000 ml 

* Might be combined depending on site of analysis. 

** A 1 m3

 sample was concentrated to a volume of 40-100 ml through a plankton net with diagonal dimensions 

of 50 μm. 

The procedures for collecting samples from sampling lines from the different sampling valves were as 

follows: 

1) Sampling of organisms ≥ 50 μm (S1, S2, S3 and S4). A plankton net was placed in a plastic 

container and was used to collect a 1 m3 sample. The sampled water was slowly sieved through a 

plankton net (50 μm mesh in diagonal dimensions) attached to a plastic cup, collecting the 

organisms in the cup. The volume of the sample was determined by measuring the volume passing 

into the sieve by a Siemens DN-50 flowmeter. 

2) Sampling of organisms 10-50 μm: Organisms with a minimum diameter between 10 μm and 50 

μm were sampled in 1000 ml bottles (S1, S2, S3 and S4). 
NIVA 5617-2008 

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3) Sampling of bacteria (S1, S2, S3 and S4): Bacterial samples were sampled in 1000 ml sterile

bottles. 

3.3 Sample preservation 

Preservation methods and expected storage times before measurement are shown in Table 6. 

Table 6 Preservation methods and expected storage times before measurement (ISO/CD 5667-3, 

2001). 

Parameter Preservation Note Maximum 

holding 

time 

Expected 

storage 

time 

Temperature - On board - 0 

Salinity - On board 

NIVA/local 

lab 

- 

0-7 days 

Particulate/Dissolved/Total 

organic carbon 

(POC/DOC/TOC) 

Acidify with 1 ml 4 M 

H2SO4 per 100 ml(pH<2), 

4

oC 

NIVA/local 

lab 7 days 

0-5 days 

Total suspended solids 

(TSS) 

4

oC On board 

NIVA/local 

lab 

24 hours 

<24 hours 

Organisms ≥ 50 μm 4oC On board 6 hours < 2 h 

Organisms 10-50 μm 4oC and freezing On 

board/NIVA 

lab 

24 hours 

< 24 h 

E. Coli

Intestinal Enterococci 

Vibrio sp. 

Vibrio cholerae

4

oC 

On 

board/NIVA 

lab/local lab 24 hours < 24 h 

3.4 Measures to avoid cross-contamination during test water transfer and 

sampling 

Manual cleaning of the ballast tanks onboard M/V Aida is very difficult to manage due to the very 

limited access of the ballast tanks during ships operation. The manholes are only available when the 

ship carries no cargo. 

Two of the ballast tanks intended to be used for testing PureBallast was visually inspected in June 

2007. Only limited water remained after draining the tanks and very little sediment was present. Some 

viable species were identified on the tanks walls. It is however estimated that the contamination is 

limited. This is due to the large volume of uptake ballast water compared to the remaining volume in 

the tanks. The potential of bloom of photosynthetic organisms in residual water and tank walls is 

absent due to lack of daylight in ballast tanks. 

To avoid cross-contamination between consecutive test waters upon transfer in the ballastsystem, all 

pipes were flushed to ensure that the entire water volume in the pipes was replaced with treated water

before each sampling, see section 2.6. The volume of the ballast piping system is about 0.1 m3

 per 

meter pipe. The total volume should therefore be less than 20 m3

 (200 m pipe). It will take less than 3 
NIVA 5617-2008 

23 

minutes to flush the entire piping system. Plankton nets were dedicated to either treated or not treated 

sample water. Plankton nets were rinsed between samples. All sample bottles were thoroughly cleaned 

before use in sea water between each sampling. 

4. Testing and measurement protocols 

An overview of all analytical measurements with instruments used, references and current uncertainty 

of the method applied is shown in Table 8 (chemical) and Table 9 (biological). 

4.1 Biological measurements – initial quick tests 

Rapid tests were performed to verify the presence of sufficient organisms according to section 2.2.2.5

in the Guideline: “Valid tests are indicated by uptake water, for both the control tank and ballast water 

to be treated, with viable organism concentration exceeding 10 times the values of Regulation D-2.1. 

This would require that there are >90 organisms/ml of size >10-50 μm and >100 organisms/m3

 of >50 

μm. Initial rapid tests are performed until the required organism concentration were approximately 

achieved. 

4.1.1 Rapid method of evaluation of density of 10-50 μm organisms 

A seawater sample was removed from sea chest Q1, 100 μl was subsampled and viewed at 40x 

magnification and cells larger than 10 μm in minimum dimension was counted. Target count was 10 

cells in 100 μl sample (target value >100 cells/ml). 

4.2 In situ measurements 

4.2.1 Temperature 

Temperature was measured in situ using a calibrated thermometer. Temperature is reported in oC. 

4.3 Discrete samples 

4.3.1 Salinity 

Salinity was measured at the respective on shore laboratory. 

4.3.2 Particulate organic carbon (POC), Dissolved organic carbon (DOC) and Total 

organic carbon (TOC) 

POC is calculated as the difference between the level of total organic carbon in the sample, measured 

on the non-filtered sample (TOC), and the measured DOC level of the same sample. DOC is 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), whilst for TOC the sample is unfiltered. The 

sample is acidified with phosphoric acid and aerated with oxygen to remove inorganic carbon (may 

also remove volatile organic carbon). The sample is injected in a quartz tube filled with a platinum 

catalyser at 680 °C. The organic carbon compounds are oxidized to CO2 which is quantified using an 

NDIR detector (Phoenix 8000 TOC-TC analyser with sample carousel STS 8000) with oxygen as 

carrier. Detection limit is 0.5 mg C/l. The Emu ltd, UK, and Intertek laboratory, Germany, uses an inhouse method for these analysis. 
NIVA 5617-2008 

24 

4.3.3 Total suspended solids (TSS) 

On board analysis was made of suspended solids by the Hach Method 8006; suspended solids, 

photometric method, 0-750 mg/L) measured by a handheld Hach spectrophotometer as the filtration, 

drying and weighing methods are considered unsuitable for use on board due to the need for 

equipment. In the laboratory analysis a nucleopore capillary filter (0,4 μm) is dried at 40-50 oC for 2 

hours and the tara is 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 

is filtered through the filter, which again is dried at 40-50 oC for 2 hours before it is weighed on the 

micro weight. The TSS is represented by the weight increase. Lowest reported value: 0.1 mg/l. Ed. 

2540 D pp 2-54: Total suspended dried at 103 – 105 °C. The method used is according to Standard 

Methods (APHA, 1995), but modified using nucleopore capillary filter in stead of glass fibre filter. 

The Emu Ltd, UK, and Intertek laboratory, Germany, uses an in-house method for these 

analysis. 

Summary of methods and equipment for chemical measurements is shown in Table 8. 

4.3.4 Organisms >50 μm 

Organisms >50 μm were inspected in microscope 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”. Viable organisms were identified to Phyla and to species level when possible. 

If organisms were found viable at the time of analysis, the samples were maintained for a period of 

time,usually 24 hours either on board or in 1 liter bottles to be transported on shore, and investigated 

for how long these organisms continued to be viable. Care was taken so that temperature was kept 

close to ambient temperature during this period. 

4.3.5 Organisms ≥10-50 μm 

CFDA-staining method 

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 is 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 and non viable cells were counted at a magnification 

of 300-480 times. 

Dilution-culture method 

The dilution-culture method was used as a complementary method for testing viability of organisms 

≥10-50 μm. The method used is based on Throndsen (1978). Briefly, the dilution series is achieved by 

adding 1 ml sample to 9 ml of algal growth media (20% Z8 seawater media). After gentle but 

thorough mixing, 1 ml of this sample is further diluted with 9 ml of growth medium. In this way, a 

series of 10x dilution series are made. Number of dilution steps is set according to the expected cell

density on the original sample. For each sample one dilution series is made. The most probable 

number (MPN) is therefore based on all 3 samples from a sample set with respect to intake water 

samples and discharge sample for control. For discharge of treated water there will be 3 sets of 3 
NIVA 5617-2008 

25 

samples. The test-tubes are sealed/corked and incubated in a room or cabinet with suitable light and 

temperature conditions on board ship until transportation in cooler chest to NIVA can be arranged 

where incubation can be continued. After two to three weeks (depending on light and temperature 

conditions) the cultures are examined microscopically and the presence of each species in the tubes is

noted. When the growth pattern (presence or absence) of each species through the culture series has 

been determined, the most probable number (MPN) per unit volume can be estimated from tables. 

Tables are given in Throndsen (1978). 

4.3.6 E. Coli

E. coli were quantified under the group Thermotolerant coliform bacteria (TCB) 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. 

4.3.7 Intestinal Enterococci

Intestinal Enterococci were quantified according to Norwegian Standard NS-EN ISO 7899-2 or 7899-

1 at a temperature of 44±0.2 °C and an incubation period of 44 hours. 

4.3.8 Vibrio spp. and Vibrio cholerae (Serotypes O1 and O139) 

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 after 24 hours of incubation. The medium supports the growth 

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

species (see table 7). Some strains of Aeromonas hydrophila grow producing yellow colonies but 

Plesimonas shigelloides does not usually grow well on TCBS. 

Table 7. 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. 

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

would be identified as Vibrio cholera. 

 

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 
NIVA 5617-2008 

26 

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

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

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

biochemical properties. If Vibrio cholera would have been identified, polymerase chain reaction 

(PCR) would have been used for elimination or identification of the serotypes O1 and O139. 

Summary of methods and equipment for biological measurements is shown in Table 9. 

4.3.9 Sample transportation 

For samples to be transported and analysed on shore the following applies: 

Frozen 10-50 μm CFDA samples and the 10-50 μm dilution series were transported to NIVA as 

luggage with frozen cooling pack. Samples for bacterial analysis were analysed within the standard 

timeframe on board the ship (filtered and incubated on board). For some samples the incubated plates 

were taken from the ship prior to completion of the incubation, the incubation period on the ship was 

extended for as long as possible, and the incubation was continued as soon as possible on-shore. 

Samples for chemical analysis were shipped in the same package but in a cooler 0-5 oC. 

Table 8. Summary of all chemical measurements. 

Parameter Units Instrument NIVA Uncertainty 

method

Reference Detection 

limit Control # of 

measurements 

Average Std 

In situ measurements

Temperature oC - Instrument 

manual 

0 

Discrete samples

Salinity PSU, 

‰ 

Autosal 

model 

8400A 

A 3 UNESCO 

(1981) 0.005 

Standard 

seawater 

IAPSO: 

34,99252 

PSU 

9 34.9934 0.00105

POC mg C/l G5-3* 

TSS mg/l 

Sartorius 

4503 

Micro 

weight 

B4 

Standard 

methods 

(1995) 

0.1 

Double 

analysis 

natural 

sample, 

TSM > 2 

mg/l 

32 0.9 % 

difference 11.1% 

* Description can be provided if requested
NIVA 5617-2008 

27 

Table 9. Summary of all biological measurements. 

Parameter Units Instrument NIVA 

method 

Reference Detection 

limit 

Organisms > 50 

μm 

#/m3 Dissecting microscope 

10-40x magnification K 9 OECD Test Guideline (1985) 1/m3

Organisms 10-

50 μm #/ml 

Epifluorescence 

microscope (excitation 

filter 485 nm; emission 

filter 530 nm) at 300-

480 times magnification 

- Ganassin et al. (2000) 1/ml 

Organisms 10-

50 μm #/ml Serial dilution technique - Throndsen (1978) 0.2/ml 

E. Coli cfu/100 

ml 

m-Endo Broth MF 

274930 (Difco) after 

concentration by 

filtration 

J2 

Norwegian Standard 

NS 4788 (expand to 

E. coli) 

1 cfu/100 

ml 

Intestinal 

Enterococci

cfu/100 

ml 

Specific agar, after 

concentration by 

filtration 44 °C, , 

verification on bileesculinagar as coloured 

colonies 

- 

NS-EN ISO 7899-2 or 

7899-1 

1 cfu/100 

ml 

Vibrio spp.

cfu/100 

ml 

T.C.B.S. Choleramedium Agar CM0333 

(Oxoid) after 

concentration by 

filtration 

- 

APHA (1995), 

terminating the 

method after 

determining total 

count of Vibrio and 

prior to specific 

identification of 

Vibrio cholerae

1 cfu/100 

ml? 

Vibrio cholerae 

(serotypes O1 

and O139)

cfu/100 

ml 

T.C.B.S. Choleramedium Agar CM0333 

(Oxoid) after 

concentration by 

filtration, PCR, 

Serotyping 

- 

APHA (1995), 

terminating the 

method after 

determining total 

count of Vibrio and 

prior to specific 

identification of 

Vibrio cholerae

1 cfu/100 

ml 
NIVA 5617-2008 

28 

5. Results and discussion 

5.1 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 collected data during testing of the PureBallast 

treatment system have been logged as summarized in Table 10. For each activity a specially designed 

log in paper and/or electronic format has been used, shown in Appendix A-G, and was used for the 

respective quality assurance evaluation. 

Table 10. Log protocols for all activities of the project.

Appendix Description 

A Total project management 

B Collection and preservation of samples 

C Logging of in situ measurements 

D Evaluation form for organisms >50 μm 

E Evaluation form for organisms ≥10-50 μm 

F Evaluation form for coliforms, E. coli, Enterococcus group, intestinal Enterococci, and 

Vibrio cholerae.

G Operational performance 

5.2 Operational performance of the PureBallast system 

A total of 4 test cycles have been completed in the period of October 2007 to April 2008. The dates of

the cycles and the time for start/stopp of each step in the test cycle are given in table 11. 
NIVA 5617-2008 

29 

Table 11. Test cycles completed with dates and the time. 

Test 

cycle Date Time Operation 

1 6th October 07 

7

th October 07 

17:17-21:00 

07:38-11:10 

Ballasting of control and treated ballast water 

Deballasting of control and treated ballast water 

2 7th October 07 

8

th October 07 

16:55-21:35 

12:50-15:58 

Ballasting of control and treated ballast water 

Deballasting of control and treated ballast water 

3 19th April 08 09:57-16:33 Ballasting of control and treated ballast water and 

deballasting of control and treated ballast water 

4 20th April 08 11:04-18:45 Ballasting of control and treated ballast water and 

deballasting of control and treated ballast water 

5.3 Chemical water quality 

The temperature, salinity, pH, POC, DOC and TOC were measured and the results are given in Table 

12. Salinity, pH, POC, DOC and TOC were measured at Emu Ltd for the first two test cycles and at 

Intertek laboratories, Germany for the last two test cycles. 

As shown in table 12, a wide span in water qualities was covered by the test cycles conducted. 

Variations in temperature from approximately 2 to 12 oC, in salinity from 7 to 33 PSU, in particulate 

organic carbon from <0.5 to 25 mg/l, and suspended solid from 1 to 14 mg/l shows that a variety of 

water qualities were included in the test cycles, and thereby also challenges for the PureBallast 

Tretment System. 
NIVA 5617-2008 

30 

Table 12. Temperature, salinity, particulate organic carbon and total suspended solids in uptake water (control and t

and treated). Triplicate samples were collected, except from discharge water treated where 9 parallell samples were 

Uptake water Disc

Control Treated Control 

 Sample 1 2 3 1 2 3 1 2 3 1 2 3 Parameter Unit 

Test cycle 1 

Temperature oC 11.7 10.7 10.3 11.2 11.0 10.9 12.8 12.7 12.6 12.4 12.3 1

Salinity PSU 30.42 32.35 32.72 21.21 29.53 32.35 31.50 31.44 31.47 26.66 26.31 25

POC mg/l NA 1.57 0.29 2.1 

TSS mg/l 12.9 7.0 12.4 7.8 

Test cycle 2 

Temperature oC 9.7 10.1 10.2 9.9 9.9 9.3 14.9 14.8 14.8 14.7 14.6 1

Salinity PSU 33.14 32.89 32.93 34.90 34.58 33.50 33.10 33.08 33.09 33.97 33.95 33

POC mg/l 2.25 2.42 1.75 1.71 

TSS mg/l 4.5 13.9 5.5 5.8 

Test cycle 3 

Temperature oC 2.3 2.3 2.5 2.5 2.4 2.4 2.5 2.6 2.7 3.2 3..3 3

Salinity PSU 7.3 7.3 7.3 7.2 7.5 7.5 10.5 9.0 9.2 10.0 9.9 9

POC mg/l 10.8 12 11.3 11.3 11.6 11.6 17.3 13.6 17.6 21.1 20.1 2

TSS mg/l 1 2 1 1 1 1 2 2 2 1 1 

Test cycle 4 

Temperature oC 6.8 6.8 7.1 6.2 6.3 6.5 7.1 6.8 6.5 6.7 6.7 6

Salinity PSU 11.2 11.0 11.3 9.4 10.8 10.9 11.3 11.3 11.1 10.6 10.5 1

POC mg/l 25.8 22.9 25.4 16.8 22.5 21.1 20.3 26.1 24.2 16.1 24.9 1

TSS mg/l 2 2 3 2 2 3 1 2 1 1 1 

NA for nonconclusive results. 
NIVA 5617-2008 

31 

5.4 Fulfillment of the biological water quality criteria and 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 microscope examination, on uptake and discharge of water for treated and control 

ballast water is given in Table 13. For a test to be valid, both the control uptake water and the ballast 

water to be treated must contain organism concentration 10 times the value of regulation D-2.1 and 

control water viable organism concentration upon discharge exceeding the values of Regulation D-2.1. 

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. 

As can be seen in Table 13 and indicated by the green background colour in the table cells, these 

requirements were fulfilled in all tests. Experience from the land based testing has shown that the 

treatement sometimes require a certain timelapse before full effect is seen on the viability of 

zooplankton. For test cycle 4, survival in some treated samples was above 10 viable organisms per m3

immediately after sampling. Notably treated sample 1 and 2 with 14 and 11 viable organisms/m3, all 

samples were reanalysed after 12 hours and then all samples had viability of <10 viable /m3 It is the 

counts performed 12 hours after the first counting that are presented in Table 13. The >50 μm treated 

samples from test cycle 4 was also counted twice although no samples had a viability of >10 

organisms/m3 in the first count. The average was reduced from 2.3 viable organisms/m3 to 1.2 viable 

organisms/m3

 after 24 hours. It is the latter value which is presented in Table 13. All other counts in 

Table 13 are from the results of the first counting of samples. 

Table 13 Viable organisms ≥50 μm in minimum diameter in uptake (treated and control) and 

discharge (treated and control) ballast water. Green background indicates that required level was 

fulfilled, while red background indicates failure to fulfil required level. 

Uptake water Discharge water 

Treated Control Treated Control 

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

Requirement >100 >100 <10 >10 

Test cycle 1 3500±636 8222±2397 2.2±2.3 3167±554 

Test cycle 2 190567±13573 79622±101739 0.7±0.9 197145±12684

Test cycle 3 8476±1821 10313±2653 0±0 1911±146 

Test cycle 4 53268±20355 60011±5442 1.2±1.5 6478±1222 

5.5 Fulfillment of the biological water quality criteria and 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, on 

uptake (treated and control) and discharge (treated and control) ballast water is given in Table 14. For 

a test to be valid, both the control uptake water and the ballast water to be treated must contain 

organism concentration 10 times the value of regulation D-2.1 and control water viable organism 

concentration upon discharge exceeding the values of Regulation D-2.1. 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. 
NIVA 5617-2008 

32 

As can be seen in Table 14 and indicated by the green background colour in the table cells, this 

requirement was fulfilled in three of the tests cycles with respect to the CFDA microscope counts 

method, but not in Test cycle nr 2 where the concentration of organisms in the uptake water was below 

the requirement for a valid test. 

For the serial dilution-culture method, the number of organisms was below the required density in test

cycle 1 and 2. The serial dilution-culture method will always represent a minimum estimate of number 

of viable cells as only those species that will actively grow in the supplied medium and at the light and 

temperature conditions given will be present in the growth tubes. The exeptions are for those species 

that significantly change size during growth. A typical example of this is the species Skeletonema 

costatum. This species was present in Test cycle 3 and 4 but was not included in the estimate of >10-

50 μm cells when analysed in microscope, however in the serial dilution cultures cells >10 μm was 

frequently observed, and it is mainly these observations that give the observed relative high cell 

concentrations. As microscope counts enable counting of all organisms present within a specific size 

range, microscope counts give the best estimate of initial concentration of >10-50 μm organisms. 

However the microscope method may overestimate number of viable cells in treated ballast water 

when UV is used as treatment technology, because there is a time lag between treatment and actual 

death of organisms. This time lag can be up to 24 hours or more if temperature is very low. 

Table 14 Summary of results for viable organisms ≥10-50 μm in minimum diameter in uptake and 

discharge treated and control ballast water. 95 % confidens interval is given below in brackets for the 

dilution series estimates. For microscope counts the Standard deviation is given. Green background 

indicates that required level was fulfilled, while red background indicates failure to fulfil required

level. 

Uptake water Discharge water 

Treated Control Treated Control 

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

Requirement >90 >90 <10 >10 

Dilution-culture method 

95 % confidence interval 

Test cycle 1 4 17 <0.2 17 

(95 % conf. int.) (1-17) (4-43) (<0.1-1.0) (8-43) 

Test cycle 2 27 17 <0.2 13 

(95 % conf. int.) (13-86) (7-47) (<0.2-1.0) (5-39) 

Test cycle 3 110 110 <0.2 200 

(95 % conf. int.) (30-480) (30-480) (<0.2-1.0) (100-1400)

Test cycle 4 110 110 <0.2 20 

(95 % conf. int.) (30-480) (30-480) (<0.2-1.0) (10-140) 

CFDA Microscope counts 

Test cycle 1 91±22 92±43 0.7±0.7 86±34 

Test cycle 2 43±30 14±3 0.0±0 15±3 

Test cycle 3 330±159 302±90 1.7±1.3 163±23 

Test cycle 4 118±31 97±42 0.9±0.9 84±16 

5.6 Bactericidal effects 

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

Enterococci, determined as described in section 2.6.4, on uptake and discharge of water for treated and 

control ballast water is given in Table 15. Regulation D-2 only requires discharge standards to be met
NIVA 5617-2008 

33 

on Escherichia coli (a thermotolerant coliform bacteria), Vibrio cholerae (toxicogenic serotypes O1 

and O139) and Enterococci given as a maximum allowable residual concentration at disharge; <250 

cfu/100ml, <1 cfu/100ml and <100 cfu/100ml, respectively. 

As seen from Table 15, 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. E. coli, Intestinal Enterococci and Vibrio cholerae (serotype O1 

and O139) were not found in any of the samples. 

Table 15 Culturable thermotolerant coliform bacteria, Vibrio sp. and intestinal Enterococci in uptake 

and discharge treated and control ballast water. Green background indicates that required level was 

fulfilled, while red background indicates failure to fulfil required level. 

Uptake water Discharge water 

Treated Control Treated Control 

Thermotolerant coliform bacteria and Escherichia coli* (cfu/100 ml)

Requirement - - - <250*- - 

Test cycle 1 0 ± 0 0 ± 0 0 ± 0 0 ± 0* 0 ± 0 

Test cycle 2 0 ± 0 0 ± 0 0 ± 0 0 ± 0* 0 ± 0 

Test cycle 3 0 ± 0 0 ± 0 0 ± 0 0 ± 0* 0 ± 0 

Test cycle 4 0 ± 0 0 ± 0 0 ± 0 0 ± 0* 0 ± 0 

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

Requirement - - - <1** - 

Test cycle 1 130 ± 46 46 ± 7 0 ± 0 <1** 170 ± 27 

Test cycle 2 29 ± 13 17 ± 5 0 ± 0 <1** 130 ± 25 

Test cycle 3 5 ± 3 37 ± 55 0 ± 0 <1** 16 ± 10 

Test cycle 4 2 ± 2 1 ± 1 0 ± 0 <1** 4 ± 5 

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

Requirement - - - <100*** -

Test cycle 1 0 ± 0 0 ± 0 0 ± 0 0 ± 0*** 0 ± 0 

Test cycle 2 0 ± 0 0 ± 0 0 ± 0 0 ± 0*** 0 ± 0 

Test cycle 3 0 ± 0 0 ± 0 0 ± 0 0 ± 0*** 0 ± 0 

Test cycle 4 0 ± 0 0 ± 0 0 ± 0 0 ± 0*** 0 ± 0 

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

coliform bacteria. There is a requirement for Escherichia coli being <250 cfu/100ml in discharge treated 

water (Regulation D-2). 

** The figures refer to the number of Vibrio cholerae/100 ml. There is a requirement for toxicogenic Vibrio 

cholerae (serotypes O1 and O139) being <1 cfu/100 ml in discharge treated water (Regulation 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 in discharge treated 

water (Regulation D-2). 
NIVA 5617-2008 

34 

Appendix A. Total project management reports 

Appendix B. Collection and preservation of samples 

Appendix C. Logging of in situ measurements 

Appendix D. Evaluation form for organisms >50 μm 

Appendix E. Evaluation form for organisms ≥10-50 

μm 

Appendix F. Evaluation form for coliforms, E. coli, 

Enterococcus group, intestinal Enterococci, and 

Vibrio cholerae 

Appendix G. Operational performance