# REPORT SNO 5617-2008



# S hipboard testing of the

PureBallast Treatment System of AlfaWall AB

ConfidentialNIVA 5617-2008



## Norwegian Institute for Water Research

– an institute in the Environmental Research Allian ce 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, Norw ay 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 T elefax (47) 62 57 66 53 Telefax (47) 55 30 22 51 Te lefax (47) 73 54 63 87
Internet: www.niva.no

Title Shipboard testing of the PureBallast Treatment Syst em of

AlfaWall AB Serial No.
5617-2008

Report No. Sub-No.
O-27170 Date
19 th 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 Alfa Wall 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 c ycles 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 978-82-577-5352-8NIVA 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 Nyquis t have represented DNV in the project. From NIVA, August T obiesen, Tor Gunnar Jantsch and Helge Liltved were the main repr esentatives. Several other AlfaWall AB and NIVA personell have been invo lved 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 veri fication of the PureBallast Treatment System, and thank all involve d personell for the proffessionality demonstrated in completing this pr oject.

Oslo, May 2008

Tor Gunnar JantschNIVA 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 syst em 12 2.1.3 Overview of water quality requirements of shi pboard 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 te st water transfer and sampling 22

4. Testing and measurement protocols 23
4.1 Biological measurements – initial quick tests 2 3 4.1.1 Rapid method of evaluation of density of 10-5 0 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 o rganic carbon (DOC) and Total organic carbon (TOC) 23 4.3.3 Total suspended solids (TSS) 24 4.3.4 Organisms >50 μ m 24 4.3.5 Organisms ≥ 10-50 μ m 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 syst em 28 5.3 Chemical water quality 29 5.4 Fulfillment of the biological water quality cri teria and biocidal effects on organisms ≥ 50 μm in minimum diameter 31NIVA 5617-2008

5.5 Fulfillment of the biological water quality cri teria 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 34NIVA 5617-2008



## Abbreviations and acronyms

Wallenius AOT Module – Wallenius Advanced Oxidation Technology Module CFDA-AM - 5-carboxyfluorescein diacetate acetoxymet hyl 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 Standardizatio n n – number of measurements; in calculating the stan dard deviation NIVA – Norwegian Institute for Water Research NS-EN ISO – Norwegian, European and International S tandard OECD – Organisation for economic Co-operation and D evelopment 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

X i - individual analytical result; in calculating the standard deviation X – the arithmetic mean of individual analytical re sults; in calculating the standard deviationNIVA 5617-2008

8 

# Summary

Shipboard testing of the PureBallast Treatment Syst em (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 carri ed 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 wat er.

Fulfilment of chemical requirements of uptake balla st water

• There are no requirements regarding chemical water quality. Uptake water was analysed with respect to temperature, salinity, particulate organ ic 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 fro m 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 up take ballast water The required density of the ≥ 50 μm group (viable organism concentration exceedin g 10 times the values of Regulation D-2.1) was met in all 4 te sts cycles.

• The requirement in the ≥ 10-50 μm group (viable organism concentration excee ding 10 times the values of Regulation D-2.1) was met in 3 test c ycles when assayed by microscope counts and in 2 test cycles when assayed by the dilution-c ulture method.

Biocidal effects of treatment

• The required “less than 10 viable organisms ≥ 50 μm in minimum diameter per m 3 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 org anisms at deballasting should be higher than 10 per m 3 , 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 req uirement 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 bacte ria in all 4 test cycles.NIVA 5617-2008

9 

# 1. Background

The overall goal for AlfaWall AB is to aquire certi fication of the PureBallast Treatment System (PBTS) in accordance with the requirements in the I MO 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.1 26(53) Annex 4 , hereafter referred to as G8 and

G9.

Land-based pilot tests were conducted by NIVA in Ju ne 2006 for partial fulfilment of such a certification. The objective of the pilot testing w as to verify the environmental performance characteristics of the PureBallast Pilot System tec hnology relative to the biological and toxicologica l requirements stated in the IMO Guideline. The exper ience 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 G 8 and G9.

Shipboard testing for type approval, as reported he re, was conducted in the period of October 2007 to April 2008. The tests were conducted in accordance with G8.NIVA 5617-2008

10 

# 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 (s ee 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 pipe s, valves and devices.

Figure 3. Overview of Ballast Water Tanks for M/V A ida.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 tre atment system; 1) a 50 μm filter for removal of larger particles and organisms, 2) the Wallenius AO T (Advanced Oxidation Technology) Module, a patented treatment system using the synergetic effe cts of in situ produced free radicals and direct photo-radiation to inactivate microbes and 3) contr ol 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 operati on and testing.



### Units of the PureBallast Treatment System

The test equipment is a complete PureBallast Treatm ent System with a flow rate of max 1000 m 3 /h. The system has one pre-filter and four Wallenius AOT units wit h capacity of 250 m 3 /h each and a CIP-unit for cleaning the AOT units.

Figure 4. The PureBallast Treatment System. AOT uni ts 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 rega rding biological water quality in uptake ballast water, in treated ballast water, and in control bal last water on discharge, as stated in regulation G8 by

IMO. Wallenius AOT Filter

CIPNIVA 5617-2008

13 Table 2 Required biological water quality in uptake ballas t water, in treated ballast water, and in control ballast water on discharge, as stated in re gulation 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 m 3 )

Concentration to be
achieved: >100 viable organisms per m 3 In compliance with Regulation D-2

(Regulation D-2: <10 viable organisms per

m 3 )

Concentration to be
achieved: <10 viable organisms per m 3 Viable organism

concentration exceeding the values of Regulation

D-2.1

(Regulation D-2.1: <10 viable organisms per m 3 )

Concentration to be
achieved: >10 viable organisms per m 3

≥ 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 organ ic 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 tes t cycle involves consecutive treatment of test wate r during ballasting by the 50 μm filter unit and the Wallenius AOT Module of the PureBallast Treatment System (green lines), transferring the te st 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 cyc le (red lines) is run by ballasting seawater to a t ank for control water (TCW) using the ballast water pum p 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

14 Ballast of treated water: Ballast of control wa ter:

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 deballastin g 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 d ifferent phases of a ballasting and deballasting cycle, including cleaning of the ballast water pip ing system prior to running the cycle (figure 6), ballast of treated water (figure 7), ballast of con trol water (figure 8), deballast of control water ( figure 9), and deballast of treated water (figue 9).NIVA 5617-2008

15 Figure 6 Cleaning of BW piping system prior to test

.

Figure 7 Ballast of treated water.NIVA 5617-2008

16 Figure 8 Ballast of control water.

Figure 9 Deballast of control water.NIVA 5617-2008

17 Figure 10 Deballast of treated water.



## 2.3 Sampling

The various sampling points at different water tran sfers between tanks via the PureBallast Tratment System, and transfer of control water, during a tes t cycle are shown in Figure 5 and Table 3. The uptake water to the control tank was sampled (S1) f rom the sea chest (valve Q1) simultaneously with ballasting of the tank for control water (TCW) (Fig ure 5, upper right). The uptake water to the treatment process was sampled (S2) from the sea che st (valve Q1) simultaneously with ballasting (and treatment of the water) of the tank for treated wat er (TTW) (Figure 5, upper left). The discharge control water was sampled (S3) from valve Q2 upon d eballasting 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 hos es for transferring the water volume to a station for flow measurement and sampling. Figure 1 1 shows the arrangement of the flowmeter for measuring volume of samples. Figure 12 shows the co llection 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 Q 1 3* Influent water to treated tank S2 Seachest, valve Q 1 3* Discharge control water S3 Valve Q2 3** Discharge treated water S4 Valve Q3 9*** *Three replicate samples to be collected over the p eriod 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

18 Figure 11 Arrangement of flowmeter for measuring vo lume of samples.NIVA 5617-2008

19 Figure 12 Collection of samples for analysis of org anisms > 50 μm.

Samples were collected to document: 1) the source water chemical characteristics in accord ance with parameters defined by IMO, G8. 2) that the water quality in the uptake seawater is me eting the biological water quality criteria defined by IMO, 3) that the biological quality of the discharge contro l water is meeting the biological water quality criteria defined by IMO, 4) that the biological quality of the discharge treate d 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 balla st water upon discharge

.NIVA 5617-2008

20 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 samp ling and measurement location.

Parameter Sample number Type of sample Location for analysis Operational parameters for the PureBallast treatmen t 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 Discret e 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

NIVANIVA 5617-2008

21 

# 3. Sampling procedures

3.1 Representativeness of samples

Separate sampling lines from the valves to the samp le points were used to assure that representative samples were drawn. Pipes were flushed with a volum e corresponding to 1 times the piping volume (3 minutes) prior to sampling. For control water and t reated water, samples were collected at the beginning, middle and end of ballasting and deballa sting. All samples were collected in triplicate, except for discharge treated ballast water were 9 s amples 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 samp led 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 m 3
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 m 3 sample was concentrated to a volume of 40-100 ml t hrough 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 m 3 sample. The sampled water was slowly sieved throug h a plankton net (50 μm mesh in diagonal dimensions) at tached 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

22 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 bef ore measurement are shown in Table 6.

Table 6 Preservation methods and expected storage times bef ore 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

H 2 SO 4 per 100 ml(pH<2),

4 o C NIVA/local

lab 7 days 0-5 days

Total suspended solids (TSS) 4 o C On board
NIVA/local

lab 24 hours <24 hours

Organisms ≥ 50 μm 4 o C On board 6 hours < 2 h Organisms 10-50 μm 4 o C and freezing On
board/NIVA

lab 24 hours < 24 h

E. Coli

Intestinal Enterococci

Vibrio sp. Vibrio cholerae 4 o C On

board/NIVA
lab/local lab 24 hours < 24 h



## 3.4 Measures to avoid cross-contamination during test w ater transfer and sampling

Manual cleaning of the ballast tanks onboard M/V Ai da is very difficult to manage due to the very limited access of the ballast tanks during ships op eration. The manholes are only available when the ship carries no cargo.

Two of the ballast tanks intended to be used for te sting PureBallast was visually inspected in June 2007. Only limited water remained after draining th e 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 te st 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 s ystem is about 0.1 m 3 per meter pipe. The total volume should therefore be le ss than 20 m 3 (200 m pipe). It will take less than 3NIVA 5617-2008

23 minutes to flush the entire piping system. Plankton nets were dedicated to either treated or not treat ed sample water. Plankton nets were rinsed between sam ples. 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 ins truments 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 o f sufficient organisms according to section 2.2.2.5 in the Guideline: “Valid tests are indicated by upt ake water, for both the control tank and ballast wa ter to be treated, with viable organism concentration e xceeding 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/m 3 of >50 μm. Initial rapid tests are performed until the req uired organism concentration were approximately
achieved.



### 4.1.1 Rapid method of evaluation of density of 10-50 μm o rganisms

A seawater sample was removed from sea chest Q1, 10 0 μl was subsampled and viewed at 40x magnification and cells larger than 10 μm in minimu m 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 rep orted in o C.



## 4.3 Discrete samples

4.3.1 Salinity

Salinity was measured at the respective on shore la boratory.



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

POC is calculated as the difference between the lev el 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. Th e sample is acidified with phosphoric acid and aerate d 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 a re oxidized to CO 2 which is quantified using an NDIR detector (Phoenix 8000 TOC-TC analyser with sa mple 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 t he Hach Method 8006; suspended solids, photometric method, 0-750 mg/L) measured by a handh eld Hach spectrophotometer as the filtration, drying and weighing methods are considered unsuitab le 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 o C for 2 hours and the tara is determined by weighing on a m icro 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 drie d at 40-50 o C 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 measu rements is shown in Table 8.

4.3.4 Organisms >50 μ m
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 Chemical s 202, “ Daphnia sp. acute immobilisation test and reproduction test”. Viable organisms were ident ified to Phyla and to species level when possible. If organisms were found viable at the time of analy sis, 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 investigat ed 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 μ m
CFDA-staining method The viability of the micro-plankton (>10-50 μm) was determined by observing cells incubated with 5- carboxyfluorescein diacetate acetoxymethyl ester (C FDA-AM) according to Ganassin et al. (2000). A 10 ml sample was incubated for 1 hour with 4 μmol o f CFDA-AM. The sample was fixed with formalin and filtered onto black polycarbonate filt ers (25 mm). The filter was mounted on a glass slid e in paraffin oil and frozen. CFDA-AM is hydrolysed o nly in a living cell. CFDA-AM is a marker for cell membrane integrity and may be measured directl y 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 observa ble as brightly yellow/green coloured cells, while non viable cells are pale green (heterotrophic cell s) or pale green with red autofluorescence of the chloroplast (photoautotrophs). Numbers of viable an d non viable cells were counted at a magnification

of 300-480 times.

Dilution-culture method The dilution-culture method was used as a complemen tary method for testing viability of organisms

≥ 10-50 μ m. The method used is based on Throndsen (1978). B riefly, the dilution series is achieved by adding 1 ml sample to 9 ml of algal growth media (2 0% Z8 seawater media). After gentle but thorough mixing, 1 ml of this sample is further dil uted with 9 ml of growth medium. In this way, a
series of 10x dilution series are made. Number of d ilution 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 fr om a sample set with respect to intake water samples and discharge sample for control. For disch arge of treated water there will be 3 sets of 3NIVA 5617-2008

25 samples. The test-tubes are sealed/corked and incub ated in a room or cabinet with suitable light and temperature conditions on board ship until transpor tation in cooler chest to NIVA can be arranged

where incubation can be continued. After two to thr ee weeks (depending on light and temperature conditions) the cultures are examined microscopical ly 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 coli form bacteria (TCB) according to Norwegian Standard NS-EN ISO 9308-1 at a temperatur e 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 p eriod 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 b y 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-mediu m agar plates (CMO333 from Oxoid), incubated at 35 °C, and the colonies were counted after 24 ho urs 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 af ter 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 ap pearing on the agar plates in the different test cy cles

would be identified as Vibrio cholera

.

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

The morphology of the colonies developing on the TC BS-medium after 24 h was visually studied. Colonies that had distinct colors and morphology di fferent from Vibrio cholera were not selected forNIVA 5617-2008

26 further identification. Colonies with typical Vibrio cholera appearance would be re-striked on TCBS medium and again inspected for growth and morpholog y. Traditional classical elimination or identification methods would be used, such as appea rance in culture media and physiological and biochemical properties. If Vibrio cholera would have been identified, polymerase chain react ion (PCR) would have been used for elimination or ident ification of the serotypes O1 and O139.

Summary of methods and equipment for biological mea surements 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 dilut ion series were transported to NIVA as luggage with frozen cooling pack. Samples for bacte rial 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 incubatio n was continued as soon as possible on-shore. Samples for chemical analysis were shipped in the s ame package but in a cooler 0-5 o C.

Table 8. Summary of all chemical measurements. Uncertainty Parameter Units Instrument NIVA

method Reference Detection
limit Control # of

measurements Average Std

In situ measurements Temperature o C - 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 requestedNIVA 5617-2008

27 Table 9. Summary of all biological measurements.

Parameter Units Instrument NIVA

method Reference Detection
limit Organisms > 50

μm #/m 3 Dissecting microscope 10-40x magnification K 9 OECD Test Guideline (1985) 1/m 3

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

mlNIVA 5617-2008

28 

# 5. Results and discussion

5.1 QA/QC prosedures

Quality assurance and quality control have been per formed 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 ste p 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 6 th

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 7 th

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 19 th

April 08 09:57-16:33 Ballasting of control and tre ated ballast water and deballasting of control and treated ballast water

4 20 th

April 08 11:04-18:45 Ballasting of control and tre ated ballast water and deballasting of control and treated ballast water



## 5.3 Chemical water quality

The temperature, salinity, pH, POC, DOC and TOC wer e 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 tes t cycles.

As shown in table 12, a wide span in water qualitie s was covered by the test cycles conducted. Variations in temperature from approximately 2 to 1 2 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, a nd thereby also challenges for the PureBallast Tretment System.NIVA 5617-200830Table 12.Temperature, salinity, particulate organic carbon and total suspended solids in uptake water (controland treated) and discharge water (controland treated). Triplicate samples were collected, except from discharge water treated where 9 parallellsamples were collected.Uptake waterDischarge waterControlTreatedControlTreatedSample123123123123456789Parameter UnitTest cycle 1TemperatureoC11.710.710.311.211.010.912.812.712.612.412.312.211.911.811.711.311.211.2Salinity PSU30.4232.3532.7221.2129.5332.3531.5031.4431.4726.6626.3125.9725.3125.2425.2625.6825.6625.57POC mg/lNA1.570.292.1NA0.29TSS mg/l12.97.012.47.82.49.2Test cycle 2TemperatureoC9.710.110.29.99.99.314.914.814.814.714.614.614.013.813.713.612.912.7Salinity PSU33.1432.8932.9334.9034.5833.5033.1033.0833.0933.9733.9533.9533.9033.9233.9533.9333.9533.94POC mg/l2.252.421.751.71-0.010.05TSS mg/l4.513.95.55.87.44.4Test cycle 3TemperatureoC2.32.32.52.52.42.42.52.62.73.23..33.43.43.53.63.63.43.4Salinity PSU7.37.37.37.27.57.510.59.09.210.09.99.69.69.59.49.39.69.5POC mg/l10.81211.311.311.611.617.313.617.621.120.126.912.719.721.917.716.414.9TSS mg/l121111222112112122Test cycle 4TemperatureoC6.86.87.16.26.36.57.16.86.56.76.76.35.85.65.65.14.7-Salinity PSU11.211.011.39.410.810.911.311.311.110.610.510.510.410.410.410.510.510.5POC mg/l25.822.925.416.822.521.120.326.124.216.124.9171715.718.818.521.919TSS mg/l223223121112112111NA for nonconclusive results.NIVA 5617-2008

31 

## 5.4 Fulfillment of the biological water quality criteri a and biocidal effects on organisms ≥ 50 μm in minimum diameter

The number of viable organisms ≥ 50 μm in minimum diameter, as determined on the bas is 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 b e valid, both the control uptake water and the ball ast water to be treated must contain organism concentra tion 10 times the value of regulation D-2.1 and control water viable organism concentration upon di scharge 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 viabl e organisms ≥ 50 μm in minimum diameter per m 3
should be present.

As can be seen in Table 13 and indicated by the gre en background colour in the table cells, these requirements were fulfilled in all tests. Experienc e from the land based testing has shown that the treatement sometimes require a certain timelapse be fore full effect is seen on the viability of zooplankton. For test cycle 4, survival in some tr eated samples was above 10 viable organisms per m 3
immediately after sampling. Notably treated sample 1 and 2 with 14 and 11 viable organisms/m 3 , all samples were reanalysed after 12 hours and then all samples had viability of <10 viable /m 3 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 al though no samples had a viability of >10 organisms/m 3 in the first count. The average was reduced from 2 .3 viable organisms/m 3 to 1.2 viable organisms/m 3 after 24 hours. It is the latter value which is pr esented 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 co ntrol) and discharge (treated and control) ballast water. Gree n background indicates that required level was fulfilled, while red background indicates failure t o fulfil required level.

Uptake water Discharge water

Treated Control Treated Control Organisms ≥ 50 μm in minimum diameter (individuals/m 3 ) 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 criter ia 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 exa mination after incubation with CFDA-AM, on uptake (treated and control) and discharge (treated and control) ballast water is given in Table 14. F or a test to be valid, both the control uptake water a nd the ballast water to be treated must contain organism concentration 10 times the value of regula tion D-2.1 and control water viable organism concentration upon discharge exceeding the values o f Regulation D-2.1. For the performance of the ballast water treatment system to pass the regulati on 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 gre en background colour in the table cells, this requirement was fulfilled in three of the tests cyc les with respect to the CFDA microscope counts method, but not in Test cycle nr 2 where the concen tration 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 w ill always represent a minimum estimate of number of viable cells as only those species that will act ively 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 typ ical example of this is the species Skeletonema

costatum . This species was present in Test cycle 3 and 4 bu t 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 observa tions that give the observed relative high cell concentrations. As microscope counts enable countin g 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 numb er of viable cells in treated ballast water when UV is used as treatment technology, because th ere is a time lag between treatment and actual death of organisms. This time lag can be up to 24 h ours 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 % c onfidens interval is given below in brackets for th e dilution series estimates. For microscope counts th e 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

4 17 <0.2 17 Test cycle 1 (95 % conf. int.) (1-17) (4-43) (<0.1-1.0) (8-43)

27 17 <0.2 13 Test cycle 2 (95 % conf. int.) (13-86) (7-47) (<0.2-1.0) (5-39)

110 110 <0.2 200 Test cycle 3 (95 % conf. int.) (30-480) (30-480) (<0.2-1.0) (100-1400)

110 110 <0.2 20 Test cycle 4 (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 upta ke and discharge of water for treated and control ballast water is given in Table 15. Regulat ion D-2 only requires discharge standards to be metNIVA 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 concentratio n at disharge; <250 cfu/100ml, <1 cfu/100ml and <100 cfu/100ml, respect ively.

As seen from Table 15, and indicated by the green b ackground colour in table cells, these requirements are fulfilled for E. coli (since the requirement is fulfilled for thermotole rant 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. Gr een background indicates that required level was fulfilled, while red background indicates failure t o 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 dis charge treated water (Regulation D-2). *** The figures refer to the number identified as i ntestinal 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