Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 1 Texel January 2010



# F INAL REPORT OF THE L AND



# BASED TESTING OF THE 

# B AL 

# P URE ®



# - B ALLAST 

# W ATER 

# T REATMENT 

# S YSTEM



# , FOR 

# T YPE 

# A PPROVAL ACCORDING TO 

# R EGULATION



# - D2 AND THE

RELEVANT 

# IMO G UIDELINE



# (A PRIL



# – J ULY 

# 200 9 )

Marcel J.W. Veldhuis Ph

- D Royal Netherlands Institute for Sea Research

P.O.Box 59 1790 AB, Den Burg, TEXEL
the Netherlands phone: +31

- 222

- 369512/300 email: Marcel.Veldhuis@nioz.nlFINALREPORTOFTHELANDBASEDTESTINGOFTHEBALPURE®~BALLASTWATERTREATMENTSYSTEM,FORTYPEAPPROVALACCORDINGTOREGULATION-D2ANDTHERELEVANTIMOGUIDELINE(APRIL—JULY2009)MarcelJ.VeldhuisPh-D4%:’Frank FuhrDipl.-Biol.Peter-PaulStehouwerMSc.Pt~RoyalNetherlandsInstituteforSeaResearchP.O.Box591790AB,DenBurg,TEXELtheNetherlandsphone:+31-222-369512/300email:Marcel.veldhuis@nioz.nl,Frank.Fuhr@nioz.nl,PeterPaul.Stehouwer@nioz.nlFinalreportoftheland-basedtestingoftheBalPure-BWT-SystemFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 3 

## Content page

1 Executive summary 4

2 Zusammenfassung 5

3 Summary table with results for the Ty pe Approval Certificate of the
BalPure ®



### - BWT system 6

4 Acknowledgements 7

5 Introduction 7

6 Description of the test facility 8

6.1 NIOZ Royal Netherlands Institute for Sea Research 8

6.2 North Sea Bal last Water Opportunity project 9

6. 3 Portrait of Severn Trent De Nora (producer of the BalPure ®



### - BWT system)

10

6. 4 The test facility 1 1

6. 5 Technical description of the BalPure ®



### - BWT system 1 3

7 Requirements to meet the D2

- Standard 1 4

7.1 Requirements to meet: G uideline G8 1 5

7.2 Experimental design 1 7

8 Results 2 4

8.1 Physical and chemical parameters 2 5

8.2 Biology 2 7

9 Environmental acceptability 3 5

10 Discussion and evaluation of results 3 8

11 References 40

Appendix 1: species list of phytoplankton and zooplankton 4 2 present during the various test runs.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 4 

## 1 Executive summary

The BALPURE® Ballast Water Treatment System (BWTS) , produced by Severn Trent De Nora (STDN, LLC US )

, was tested according to the Regulation

- D2 (D2

- Standard), the IMO Guidelines for Type Approval testing (G8) and for approval of BWT systems that make use of active substances (G9) in the spring and early summer of 200 9 in the harbour of
the Netherlands I nstitute for Sea Research. In general most of the requirements for testing were met and in several test runs
environmental conditions were harsher than strictly required

. The STDN Ballast Water Treatment System (BWTS) applies established chlorination tech nology to oxidize and
disinfect water containing aquatic ( invasive ) organisms . Prior to adding the active
substance (hypochlorite) the water was filtered through a 40 micron BallastSafeTM (BSFc)
filter

. In total 1 0 test runs were successful and the BWT syst em performed on average
much better than stated in Regulation

- D2 by achieving values for organisms well below the requirements of the D2 Standard. For many size classes of organisms the residual number of organisms was at least one order of magnitude lower than indicated numbers
in the D2 S tandard. This system should therefore be regarded as fast and an effective way of reducing the number of viable organisms in ballast water of ships, thereby minimizing the risk of new invasions originating from the ships ballast water.
The s ediment load was reduced by the self

- cleaning filter and remaining organisms were killed by the active substance chlorine

. After chlorine treatment all organisms , including a substantial fraction of the bacteria population, disintegrat ed completely and the remaining debris was found as an amorphous structure or as dissolved organic carbon.

T o minimize potential regrowth of (micro) organisms a background level of chlorine was maintained (1 mg/L) until discharge. To reduce potential toxic effects of the chlorine at discharge sodium bisulfite was added prior to discharge. No regrowth was observed for a period of 20 days in the presence of chlorine

. Environmental acceptability tests however showed that the growth of organisms, mainly plank ton, was not limited by the discharge water , after neutralization, indicating that the discharged water was still vital.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 5 

## 2 Zusammenfassung

D ie BALPURE® Ballast wasser Behandlungsanlage , von Severn Trent De Nora (STDN, LLC US) wurde im Frühjahr und Somm er 2009 gemäß Regularien

- D2 (D2

- Standard), sowie
der IMO Richtlinien zu Typzulassung stests (G8) und denen für eine Zulassung von Systemen, welche aktive Substanzen verwenden (G9) im Hafen des Königlich Niederländischen Meeresforschungsinstitut (NIOZ) getes tet. Generell wurden die allermeisten Anforderungen bezüglich der abiotischen Parameter des Testwassers und zur Organismendichte erfüllt. In den meisten Tests waren die Bedingungen schwieriger, als in den Richtlinien v orgesehen . D ie BALPURE® Ballast wasser Behandlungsanlage verwendet bekannte Chlorierungsverfahren um Wasser zu oxidieren und dadurch potentiell invasiven Organismen abzutöten. Vor Zugabe der aktiven Substanz (Hypochlorit) wird das zu behandelnde Wasser über einen Feststofffilter (BSFc,
40 Mikr ometerfilter) geleitet. Es wurden 10 voneinander unabhängige Tests nacheinander, erfolgreich durchgeführt. Das System erfüllte alle Anforderungen der Regularien

- D2 und der Richtlinie G8. Die Organismenanzahlen nach der Behandlung lagen deutlich unter den A nforderungen des

D2

- Standards, für einige Gruppen im Durchschnitt sogar um eine Größenordnung. Das System sollte demzufolge als effektive und sichere Möglichkeit zur Behandlung von Ballastwasser betrachtet werden, die dazu beitragen kann, weitere biologisc he
Invasionen zu verhindern.
Die Sedimentfracht wurde durch d ie Filtration reduziert und die verbleibenden Organismen durch die aktive Substanz (Chlorverbindung) inaktiviert. Einschließlich eines substanziellen Anteils der Bakterienpopulation wurden fast a lle Organismen vollst ändig zersetzt. Die Überreste der Organismen fanden sich lediglich in Form amorpher Strukturen und als gelöster organischer Kohlenstoff. Um Wiederaufwuchs von (Mikro

- ) Organismen zu verhindern wurde im Tank eine Konzentration der aktiv en Substanz von ca. 1 mg/ L bis zum Ablassen aufrecht erhalten. Um mögliche, negative Umwelteffekte hierdurch zu verhindern, wurde diese Restkonzentration beim Ablassen durch Zugabe

von Natriumhydrogensulfit neutralisiert.
In Inkubationse xperimenten wurde in 20 Tagen kein Planktonwachstum im behandelten Wasser bei Anwesenheit von Chlor festgestellt. Gleichzeitig durchgeführte Umweltverträglichkeitstests (Zugabe von Zeigeorganismen und Planktonkulturen, Verdünnungsrei hen) mit dem behandelte n Wasser, nach der Neutralisierung, ergaben jedoch keine negativen Auswirkungen auf das Planktonwachstum

. Diese Tests zeigten somit, dass das behandelte Wasser nach Neutralisation

, d.h. so wie es in die Umwelt entlassen wird, das Wachstum gesunder Organism en nicht beeinflusst.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 6 

### 3 Summary table with results for the Type Approval Certificate of the BALPURE® BWT System

Land

- based tests NIOZ Reference & Treated Reference Treated

salinity 23.9 PSU Intake Discharge Discharge

natural plankton Average min. max. Average min. max. Average min. max. total bacteria [counts/mL] 3.7 1 +E6 1. 78

- + E 6 5. 65 + E 6 2.1 9 +E6 0.4 1 +E6 4

. 69 +E6 4 .0 3 +E 4 0

. 61 +E 4 7

. 09 +E 4

E. coli [cfu/mL] < 0

. 1 < 0

. 1 0.14 < 0

. 1 < 0

. 1 < 0

. 1 < 0

. 1 < 0

. 1 < 0

. 1
Enterococci [ cfu/mL]



### - < 1 1 2 < 1 < 1 < 1 < 1 <1 <1 plankton <10 μm [counts/mL] 6287 3283 11554 734 130 1485 1.5 0.7 4.4
plankton 10

- 50 μm [counts/mL] 1244 1046 1737 118 104 135 0.4 n.d. 1.5 plankton >50 μm [counts/m 3 ] 8

. 8 +E5 3

. 9 +E5 14

. 8 +E5 3 .5 4 +E4 0. 98 +E4 7

. 22 +E4 0.8 n.d. 4.3



### Land

- based tests NIOZ Reference & Treated Reference Treated

salinity 33.6 PSU Intake Discharge Discharge
natural plankton Average min. max. Average min. max. Average min. max. total bacteria [counts/mL] 4

. 38 +E6 3. 57 +E6 5

. 3 2+E6 2

. 01 +E6 1

. 48 +E6 5 .0 2 +E6 1 .7 9 +E 5 1 .4 0 +E 5 2

. 73 +E 5

E. coli [cfu/mL] < 0.1 < 0.1 1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1
Enterococci [cfu/mL]



### - < 1 1 00 < 1 < 1 <1 < 1 <1 <1 plankton <10 μm [counts/mL] 2790 2082 4070 812 305 2047 1.3 n.d. 3.7
plankton 10

- 50 μm [counts/mL] 1258 1002 1799 110 96 132 0.3 n.d. 1.48
plankton >50 μm [counts/m 3 ] 5

. 8 +E5 1

. 5 +E5 1 0

. 9 +E5 1

. 2 5+E4 0.2 6 +E4 1 .8 2 +E4 0.3 n.d. 1.0

Summary table of collected data covering the major groups of organisms at two series of test

- runs for low and high salinity range, respectively.
n.d.: no n detectable in sample.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 7 

## 4 Acknowledgements

The authors thank the technical staff of the NIOZ and in particular Anna Noordeloos, Eveline Garritsen, Josje Snoek, Swier Oosterhui s, Santiago Gonzalez, Jaap Witte and Andre Smit. Their cooperation was essential for the successful completion of this certification project.
We also thank Scott Sorbet and Harold Childers for their technical assistance during the
field tests and smooth o peration of the BalPure ®

- BWT system

. We also thank Dr. Kai Trümpler

, Mrs. Karin Sigel and Miss Mareike Wendland of the Bundesambt für Seeshifffahrt und Hydrographie, acting on behalf of the German Administration, for their excellent collaboration during t his whole certification process



## 5 Introduction

Ships transport 5

- 10 billion tons of ballast water annually all over the globe (Endresen et
al. , 2004) . The ballast water is loaded with particulate sediment and an enormous variety of (living) organisms, which ranges from juvenile stages, larvae and eggs of fish and larger zooplankton (Williams et al. , 1988) ; (Carlton and Geller, 1993) to macro algae

, phytoplankton (Hamer et al. , 2000 ; Hallegraeff et al. , 1997) , bacteria and viruses ( Gollasch et al. , 1998) . In g eneral these organisms belong to the natural ecosystem in and around the port of origin but they might not be occurring naturally in the coastal waters and port of destination at the end of a ship’s journey. In hundreds of cases around the world, this has resulted in severe damage to the receiving ecosystem and to human health, because these non

- native organisms developed into a plague. This often has a high impact on the ecosystem and can cause economical damage (Hoagland et al.

, 2002) , as it results in a decrea se of stocks of commercially valuable fish and shellfish species and occasionally outbreaks of diseases such as cholera (Ruiz et al. , 2000 ; Drake
et al. , 2001) . If action is not taken the problem of invasive species will increase in an exponential manner for several reasons. Ships are getting larger, faster and the amount
of traffic across the oceans i s expected to increase rapidly during the coming decades and therefore also the change of non

- indigenous organisms to have large enough
number s f or settling and expanding. Our effort to reduce pollution of ports and coastal waters also improves the quality of the aquatic environment in these areas and therefore increases the susceptibility to invasive organisms. Originally not intentionally meant but organisms in ballast water will experience favourable conditions for settling and growing. The problem of in vasive species is considered as one of the 4 major
threats of the world’s oceans next t o land

- based marine pollution, overexploitation of living marine resources, and physical alteration/destruction of habitats

To minimize these risks for the future, the International Maritime Organization (IMO) of the United Nations has adopted the Ballast Water Convention in 2004 (Anonymous, 2005) . The Convention s tates that finally ALL ships (>50,000 in number) should install proper ballast water treatment (BWT) equipment on board between 2009 and 2016. As a temporary and intermediate solution for the time being ship s may reduce the risk of invasive species by perf orming ballast water exchange during their voyage when passing deep water (>200 m depth and 200 NM from the coast ) . Ballast water exchange faces many problems as to feasibility, safety and efficacy

. For a large part of ships’ voyages the required depth an d/or distance to shore requirements are never met; BW exchange can affect the ships construction stability and in rough seas exchange is not possible because of the risk to ship and crew. Treatment of ballast water is therefore considered
to be the best so lution of reducing the risk of invasive species.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 8 During the recent years numerous solutions for treatment of ballast water have been mentioned and tested with the ultimate goal to reduce the amount of organisms in
ballast water (Rigby and Taylor, 2001) . However, next to a high efficacy there is more needed for a BWT system to be good a system. Next to bi ologically effective the system should be practicable, environmentally acceptable and also cost effective. Despite the fact that the treatment technology for drinking

- , waste

- and process water
is well

- developed none of these techniques is directly applic able to ballast water (Rigby
et al. , 2001 ; MEPC 49/2/13, 2003 ). Besides reducing the load of organism s the sediment
load should be reduced as well . There are also considerable differences in ships operation, types of ships

, and the amount of space available for a ballast water treatment system on board and the way ships are operated. Ballast water treatme nt will develop into a new field of technology of its own with a commercial market estimated for the next 10 years in the order of 8 billion Euro (Haskoning, 2001)

. As a primarily scientific research institute NIOZ is defining its role in the certification

process of land

- based trials of BWT systems as to study 1) the numerical abundance and biodiversity of organisms prior, during and after

a tr eatment with the BalPure ®

- BWT system (efficacy of the BWT system), 2) to determine the viability status of the remaining organisms during discharge, 3) to assess possible environmental risks of discharging chlorine

- treated ballast water by measuring resi dual effluent toxicity in order to determine latent effects, other than measured in specific toxicity tests conducted for the G9 (environmental impact).

This research strategy allows for more in depth testing, while it includes ALL organisms (from virus t ill whale) and not only the size classes as specified in the Convention D2

- standard.



## 6 Description of the treatment facility

6.1 NIOZ Royal Netherlands Institute for Sea Research

NIOZ Royal Netherlands Institute for Sea Research is the National Oceano graphic Institute of the Netherlands. NIOZ is part of the Netherlands Organization for Scientific Research (NWO). The institute employs around 200 people and the annual budget is approximately €20 million. The mission of NIOZ is to gain and communicate scientific knowledge on seas and oceans for the understanding and sustainability of our planet. The institute also facilitates and supports marin e research and education in the Netherlands and in Europe.

In order to fulfil its mission, the institute performs tasks in three specific fields.
Research: The emphasis is on innovative and independent fundamental research in continental seas and open oce ans. The institute also carries out research based on societal questions when this merges well with its fundamental work. The senior scientists at NIOZ all participate in international research projects.
Education: The institute educates PhD and other stud ents of universities and schools for professional education. Together with universities NIOZ also organises courses for PhD
students and master students in the marine sciences. A number of our senior scientists
of NIOZ are also appointed as professor s at t he Dutch and foreign universities. Facilitary services: NIOZ invites marine scientists from Dutch and foreign institutes and universities to write scientific proposals involving the institute’s research vessels,Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 9 laboratories, and the large research equipme nt, which is often designed and built by the institute’s own technical department. The basic oceanographic disciplines studied at NIOZ are physics, chemistry, biology and geology. Multidisciplinary research is regarded as one of the main strengths of NIOZ.

More information on www.nioz.nl

Figure 1 : aerial view of the NIOZ harbour (lower right), NIOZ laboratories (upper left) and TESO ferry (top).



## 6.2 North Sea Ballast Water Opportunity project

The Ballast Water Oppo rtunity project ( www.NorthSeaBallast.eu ) , originally an initiative of the BSH (Federal Maritime and Hydrographic Agency, Germany) and the Royal Netherlands Institute for Sea Research (NIOZ, Netherlands), involv es all relevant stakeholders within the maritime sector in the North Sea region like governmental institutions, inter

- governmental and non

- governmental organisations, industry and scientific and technological institutes. This structure and participation of fers a broad and sound base for the project in support of a succesful implementatin of the IMO Convention in the region, as North Sea, Skagerrak, and Kattegat form one ecological zone. Moreover, the project being one of the largest and most integrative in its kind, the objectives (investments) will become available as a model for other european maritime regions as well as across the globe. To facilitate this initiative funding was received from the North Sea InterregIVB (an ERDF program). For the embedding in a more global strategy the project is liaising with the Globallast II initiative of the IMO.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 10 

## 6. 3 Portrait of Severn Trent De Nora, LLC., (producer of the
BalPure ® 

## BWT

- s ystem)

More information on: www.severntrentdenora.com

Severn Trent De Nora, LLC., (STDN), is a joint venture subsidiary of Severn Trent Services and Gruppo De Nora. STDN is the world leader in the supply of on

- site generation of biocide solutions to the power, marine, offshore industrial water disinfection markets. Over the past t hirty years, STDN organizations have supplied 65% of the worldwide
installed base of on

- site generated hypochlorite. STDN draws upon the strength and global resources of Severn Trent Services and Gruppo De Nora. The joint venture was formed in 2001 by the combination of their respective subsidiaries, Exceltec International Corp. and DeNora Seaclor, S.r.l. The history of STDN includes: 1974

- Diamond Shamrock Corporation, Electrolytic Systems Division formed. 1986

- Acquisition of OMNIPURE by Diamond Shamrock Corporation. 1987

- Formation of Eltech International Corp. (Sugar Land, TX). 1997

- Eltech International Corp. name changed to Exceltec International Corp. 1998

- Exceltec International Corp. acquired by Severn Trent

Services. 2001

- Severn Trent Services ent ers into joint venture with Gruppo De Nora.

2002

- Severn Trent De Nora is formed. Established in 1923 by Oronzio De Nora, Gruppo De Nora is recognized worldwide as a leading supplier of electrochemical technologies for the production of chlorine, caustic s oda and derivatives, as well as the largest worldwide supplier of noble metal

- coated electrodes for the chlor

- alkali industry and for the electrochemical industry in general. Energy saving and environmental protection are the Group's distinguishing technol ogies. Gruppo De Nora has built more than 500 plants for the production of chlorine and caustic soda and for the electrochlorination of
industrial waters in 60 different countries.

Ballast water treatment

- Technical Overview of B AL P URE®

STDN has develop ed proprietary ballast water treatment systems that have been specifically designed to safely and economically eliminate the worldwide transfer of aquatic invasive species. BALPURE® technology is patented, proven, well understood and uniquely competitive b ased on thirty years of on

- site hypochlorite generation equipment manufacturing and service to the off

- shore and marine markets. The company has an experienced management team and has established key partnerships in naval architecture, marine engineering, systems engineering, fabrication, science, chemicals, distribution, and service. In research underwritten by STDN at the University of Washington, the use of chlorination was proven effective in seawater on representative groups of all target organisms. Additional extensive shipboard testing is in progress conducted under a grant from the United States National Oceanographic and Atmospheric Administration (NOAA). Commercial land based testing has also been conducted on the BALPURE® system at the United St ates Naval Research Laboratory in Key West Florida as part of the Environmental Protection Agencies ETV program. STDN as a United States based company is working to achieve global certification via

two avenues. In the United States, the U.S. Coast Guard (U SCG) Shipboard Technology Evaluation Program (STEP) is the first hurdle toward certification and STDN received acceptance in early 2009. European countries and much of the world is a pplying IMO proposed standards. STDN is pleased to work with the Royal Net herlands Institute for Sea Research (NIOZ) to conduct certification testing and the German Federal MaritimeFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 11 Agency (BSH) who will act as the administrating agency during the certification and Type Approval process.



## 6. 4 The te s t facility

The land

- based tests were carried out at the Royal Netherlands Institute for Sea Research (NIOZ , www.nioz.nl ), Landsdiep 4, 1797 SZ 't Horntje, Texel, the Netherlands,
from March through July 200 9

.

The NIOZ test site is equipped with 3 coated concrete tanks of 300 m3 v olume each to simulate the ballast water tanks of the ship (Figure 2). The tanks were cleaned using

pressure

- washing after each run. Water samples were taken from bypasses of the standard piping used to fill and to empty the tanks or directly from the tank at outflow

at ca. 30 c m from the bottom.

Figure 2 : Inside views of one of three
subterranean water tanks

According to the requirements of the Guidelines G8, sampling points are fitted before the treatment system and directly after the system. S amples varying in volume from 500 mL up to 1 m 3 (clean IBCs) were taken using clean sampling containers. Sampling containers and all further handling of the samples were separated in a control and a treated set to avoid cross contamination by the active su bstance. The basic handling, such as concentrating, filtration and chemical analysis was done at the test site. Additional samples (1 to 10 L) were transported to the institute’s laboratories for further special analysis. For re

- growth experiments 10 L of sample was transported (Nalgene bottle) to a climate room for incubation experiments (ca. 12

– 15 °C; a light; dark regime of 16:8 h and 100 **μ** mol quanta. m - 2 .s - 1 )

The B AL P URE® BWTS was connected to a typical ballast water pump which was located in
the NIO Z harbour. This is a pristine harbour with a direct access to the Wadden Sea and the origin of the test water changes with the tide. Furthermore, provisions were made to allow the addition of salt water and / or freshwater in order to adjust the salinity o f the natural water of the NIOZ harbour to the required test conditions of brackish waterFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 12 and marine water with a minimum of 10 PSU difference. A detailed description of the test installation is presented in figure 3

Figure 3 : P&I diagram of the B AL P U RE® BWTS installation at NIOZ with points of samplingFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 13 

## 6. 5 Technical description of the B AL 

## P URE 

## ® BWTS The STDN Ballast Water Treatment System (BWTS) applies established chlorination technology to oxidize and disinfect aquatic invasive species (AIS). Chlo rination has a long history (80+ years) of being applied safely and economically for the control of microorganisms in industrial and municipal waters. The technology has found to be effective against a wide range of nuisance causing organisms. It is simila r to a technology already approved by the IMO and type approved by the Republic of Korea.
The B AL P URE® BWTS provides a safe and simple electrolytic process for the on

- site generation of dilute hypochlorite on demand without storage during the ballasting operation and the neutralization of residual oxidants during the de

- ballasting operation. Sodium hypochlorite is generated by means of electrolysis of seawater. In this process, seawater flows through the electrolytic cells in a ratio of 1 part for every 1 00 parts of ballast water. The system can operate eff ectively in brackish water to 8 PSU. Because the amount of seawater required to effectively treat ballast water is small even fresh water ballast can be treated by B AL P URE® BWT with supplemental salt or stored seawater in designated ballast tanks such as aft peak tanks. The current passing through the seawater causes the salt (NaCl) and water (H 2 O) to form sodium hypochlorite (NaOCl) and hydrogen (H 2 ) as a secondary by

- product. Hypochlorite solution and H ydrogen are produced and separated immediately upon exit from the electrolytic cells. The weak hypochlorite solution (1 g /L ) is injected back into the ballast stream. Hydrogen that is separated from the hypochlorite is immediately diluted to less than 1% h ydrogen by forced air blowers and discharged to a safe location. The hypochlorite is generated automatically on demand and is matched to the ballast flow rate and the oxidant
demand of the ballast water. Initial oxidant concentration is dosed at 1.5 times the TOC concentration which remains effective for several days. T o minimize potential regrowth of (micro) organisms a background level of chlorine is maintained (1 mg/L) until discharge. This will affect toxicity and by

- product concentrations at compul sory sampling at day 1 and day 5 of the tests. To reduce potential toxic effects of the chlorine at discharge sodium sulfite or bisulfite is added prior to discharge. Sulfite when reacted forms sulfate which is present in seawater at concentrations 4,000 times stronger than
the treated ballast water.
For this land b ased test, the water is filtered through a 40 micron BallastSafeTM BSFc Automatic Electric Filter, Model BSFc

- H

- 1.6 prior to treatment. The unique sintered stainless steel screen technology enables it to have an unparalleled zooplankton
removal rate. Ballas tSafe’s filter features continuous cleaning of large volumes of dirt during ballasting without interruption, and a reversible screw system for smooth, reliable and rapid cleaning of the entire screen surface. The effectiveness of this filter
has been succe ssfully demonstrated in previous trials at NIOZ.

Figure 4 : outline of B AL P URE® BWT
systemFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 14 The BALPURE® Ballast Water Treatment System at NIOZ for land based testing is a full scale commercial available system of STDN and has not been downsized. The BALPURE®

BWTS is a self

- monitoring system with an automated bypass, and has been reviewed for compliance with the class requirements of the ABS Americas Division classification society. All materials and specifications were selected in order to meet t he class requirements of this classification society for ballast water systems and electric
installations. Subsequently, a second BALPURE® BWTS has been installed on a test ship (SeaRiver/Exxon

- Mobil cargo tanker; American Progress).

The process for Ba sic Approval has recently started and a formal request to the MEPC

has be en made by the BSH.

The applied test protocols were communicated with the German Administration ( Federal Maritime and Hydrographic Agency of Germany ; BSH ; Anonymous, 2009) and a
shor t description of the various applied methods is included in the next section. During the certification process the whole practical procedure of intake and discharge has been
witnessed at several occasions by national and international agencies

.



## 7 Require ments to meet the Regulation

- D2

According to D2

- Standard of the IMO/MEPC Convention of 2004 (Anonymous, 2005) ships that meet the requirements of t he Convention by meeting the ballast water performance standard must discharge:

T h e present Standard

- D2 is defined as a standard for the water characteristics at discharge . Furthermore, with exception of some indicator microbes ( point 3) orga nisms

< 10 **μ** m are completely excluded . This is certainly an omission since this size class in particular includes numerous phytoplankton species characterized as Harmful Algal Blooms (HABs)

. Nevertheless, t he standard is clear with respect to the maximum number of organisms remaining present. On the other hand a proper definition of the dimensions of organisms is still subject of (academic) discussion. Moreover, a s an operational
definition for viable organisms the IMO is using : organism s and any life stag es thereof that are living , but a more adequate (scientific) definition is: an organism that is able to complete its life

- cycle, including reproduction (DNA replication). During the land

- based tests cell viability in the context of both definitions was stu died.

In addition to the (basic) requirements for the D2

- Standard we have adopted a variety of methods and techniques to determine abundance , sizes and the viability status of

1) Less than 10 viable organisms per cubic metre greater than or equal to 50 micrometers in minimum dimension; 2) Less than 10 viable organisms less than 50 micrometers in minimum dimension and greater than or equal to 10 micrometers in minimum dimension and 3) Less than the following concentrations of indicator microbes, as a human health
standard: 1. Toxicogenic Vibrio chlorerae (serotypes O1 and O139) with less than 1 colony forming unit (cfu) per 100 millilitres or less than 1 cfu per 1 gramme (wet weight) of zooplankton samples:

2. Escherichia coli less than 250 cfu 100 millilitres ; 3. intestinal Enterococci less than 100 cfu per 100 millilitres

.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 15 different types of organisms. This includes also plankton < 10 μm other than b acteria (i.e. phytoplankton and viruses). Moreover, we extended our research effort to examin e not only the fate of the organisms in the large

- scale ballast water basins but also took subsamples for incubation under optimal growth conditions in our testing to study the growth potential of potentially remaining organisms or survival stages such as eggs, cysts or dormant cells over a longer period than the recommended 5 days. These later experiments allowed us also to study po tential latent toxicity effects a nd therefore to determine the vitality of the treated water prior and after discharge.



## 7.1 Requirements to meet : guideline G8

Next to the D2

- Standard two guidelines were developed by the IMO as a framework for approval of ballast water treatment systems (G8) and approval of the use of active substances in ballast water treatment systems (G9). For land

- based testing MEPC

53 /Annex 3 (Anonymous, 2005) and modifications as adapted at MEPC.174.58 (Anonymous 2008) were the basic framework of the present study. The most relevant parts will be presented below. These guidelines were generically designed to meet the conditions of a broad range of potentially e ffective treatment techniques to be tested in typical port and environmental conditions found across the globe. Most test protocols therefore require extensions of the test design to cover the specific aspects of the
treatment. The land

- based testing serve s to determine the biological efficacy of the BWT systems under consideration for Type Approval under more or less controlled and replicable conditions. This is intended to ensure that the efficacy of the equipment is consistent and can be shown repeatedly . The test set

- up should therefore be representative of the characteristics of the arrangements used and the type of environment the BWT system was designed for.

One of the main criteria in t he G8 test requirements is the salinity range and related to
thi s the differences in Total Suspended Solids (TSS), Particulate Organic Carbon (POC) and Dissolved Organic Carbon (DOC). This resulted in three main categories of test conditions (Table 1).

Table 1 : Three different salinity ranges and minimum concentratio ns of TSS, POC and
DOC in the water.

Salinity

Parameter > 32 PSU 3

– 32 PSU < 3 PSU unit Total Suspended
Solids > 1 > 50 > 50 mg/L

Particulate Organic
Carbon > 1 > 5 > 5 mg/L

Dissolved Organic
Carbon > 1 > 5 > 5 mg/L

Previous experiments and ad ditional tests , as documented in the application for Basic Approval; (MEPC 58/2/2 ) showed that the BALPURE® BWT

- System was not affected by differences in salinity. The physical step, filtration, will certainly not be affected by salinity but also the react ivity of the active compound ( c hlorine ) will not be altered in the presence of high concentrations of salt. It was for this reason that the Type Approval tests were conducted at the intermediate (3

– 32 PSU) and high salinity (>32 PSU) regions. Moreover, t he only difference in composition of the test water betweenFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 16 the freshwater and intermediate salinity water is the presence o r absence of seasalt. All other minimum requirements for TSS, POC and DOC for these two water types were identical (Table 1 ).
The B AL P URE® BWTS can treat ballast water of any quality listed in Table 1 and can use ballast water with salinity of > 8 PSU to generate the necessary oxidants for ballast

water treatment. A small s upplemental salt or stored seawater source is required f or ba llast water with < 8 PSU for the slip stream to generate required oxidant amounts. A further requirement is that the difference between the two salinity regimes should be
at least 10 PSU

. The test water

, originating from the Wadden Sea

, and the actual

samp ling did vary with the tide and as a result salinity was subject to variations . To
assure the 10 PSU salinity differences it was decided to have the possibility of adding
fresh surface water and upgrade coastal water of the North Sea by enhanc ing the
salin ity (brine solution of commercially available sea salt; ca. 18%). As target number the freshwater addition was adjusted to a salinity of ca. 23 PSU for the low salinity regime and ca. 33 for the high salinity regime. In practice ca. 15 % (v/v) of freshwate r was added during the low salinity tests and about 4% of brine solution (Instant Ocean ® ), during the high salinity test runs. In order to compensate the dilution of the TSS by the freshwater some extra sediment (taking from a nearby mudflat) was added as well.
These additions were made close at the pump site , to ensure proper mixing, with a constant flow rate and done during filling of the control and the treated ballast tank.

Biology

The guideline G8 also defines criteria for the number and diversity of the organisms to be met during Type Approval testing (Table 2 )

. These criteria should be met for all three salinity regions.

Table 2 : Minimal numbers and species diversity required at intake for different size classes and groups of organisms

.

Influent test water

Parameter unit Remarks

organisms ≥ 50 micron > 10 5 / m 3 at least 5 species from at least 3 different phyla/divisions

10 ≤ organism size ≤ 50
micron > 10 3 / mL at least 5 species from at least 3 different phyla/divisions

heterotrophic bacteria > 10 4 / mL not further defined

The test water should contain minimum densities of plankton which are typical
densities encountered in the Wadden Sea during the annual spring bloom in April/May. With respect to the species diversity, the Wadden Sea is kn own for its natur al richness in organisms and during the test period (April

– July) indeed a large diversity in organisms, adults, juveniles, larvae, eggs , resting stages etc

. was encountered. An important aspect, so far not recognized in the guidelines ( G 8 )

, when dealing only with natural populations of organisms in the influent of the test water is the natural seasonality of species and blooms. The actual onset of the spring bloom is characterized by a dominance in phytoplankton, but usually lacks high zo oplankton abundance

. Only

at a later stage zooplankton starts to increase in abundance, subsequently due toFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 17 predation it will diminish the numerical abundance of (smaller size) phytoplankton
component

. Furthermore, for the high salinity range, the composi tion of the organisms in the water resembles that of a typical oceanic environment. This implies an increase of smaller
sized cells , down to the micrometer scale, and also a dramatic decline in the number of larger (>10 μ m) organisms. So far this shift in community structure has not been accounted for when using natural plankton for testing.

Human pathogens

Table 3 : Maximum allowed numbers of 3 groups of indicator microbes in the effluent test water on discharge. cfu: colony forming units

Effluent test water

Parameter unit Remarks

Toxicogenic
Vibrio chlorerae < 1 cfu/100 mL or < 1 cfu/ g wet weight of zooplankton serotypes O1 and O139

Escherichia coli <250 cfu/ 100 mL

intestinal

Enterococci <100 cfu/ 100 mL

Within the group of prokaryotic micr obes only bacteria and more specifically the heterotrophic group (Table 2) has been defined by the standard but for completeness
this should include all bacteria and presently also Archea e . While these microbes are part of the natural community in the aqua tic environment the indicator microbes (Table 3) , human pathogens are introduced as part of human activity and often associated
with sewage discharge. In the present research all microbes have been included as a bulk parameter , the number of heterotrophs a s a viable component as well as the viability of the whole microbial community ha s been determined

. Within the whole microbial community the number of heterotrophic bacteria was
determined as well as E. coli and total enterococci. The test area of the ins titute is part of a tidal estuary of the Wadden Sea, which is essentially a pristine environment. Moreover, waste water treatment is highly developed in the Netherlands. Therefore, numbers of these human pathogens during the tests were to be expected to be low for most of the sampling period

. On the other hand during the different treatment steps a significant amount of particulate organic material is transferred into dissolved organic carbon (DOC) which acts as an excellent substrate for stimulating growth of (heterotrophic) bacteria.



## 7.2 **Experimental design**

A variety of methods w ere applied to examine the biological efficacy of the BALPURE®

BWT

- system for the different categories of organisms during the two test series. For detailed description we refer to the outline of the official test protocols for the

BALPURE® BWT

- System (Anonymous, 2009) . Sampl e h andling and volumes were according to the description of the guideline for BWT testing (G8) or described in detail
when these guidelines were insufficient or other consideration s were taken into

account

. Subsamples were taken randomly or throughout the who le filling procedure of
the tanks. As indicated previously there was great emphasis on analysing the freshlyFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 18 collected samples and having multiple methods to examine numerical abundance and viability. Besides various biological samples there was also a bas ic set of physical and chemical parameters which were monitored prior, during and after discharge. A short description of each parameter and how it has been analysed is given below.

P hysical and chemical properties of test water

Temperature

The water temperature wa s measured using a calibrated thermometer.

pH

The pH

- level is measured using a calibrated pH

- meter.

Salinity

For salinity ca. 250

- 500 mL water is sampled and stored at room temperature (glass bottles) until analysis by dire ct measurement in the laboratory at NIOZ. Salinity of the water

wa s measured after each test cycle using a refractometer (Atago) calibrated against 0 and 33 PSU standard (sea)water. The accuracy of the salinity measurement is 0.5 PSU.

Dissolved Oxygen

T he s pectrophotometric method of the Winkler method (Winkler, 1888 ; Pai et al. , 1993) ; Reinthaler, 2006) was used to determine the oxygen concentration in the water . Samples were taken using gastight tubing which was specially fitted to the sampling tubing that was

u sed to sample the ballast simulating tanks. The coded glass bottles are flushed at least three times their volume (ca. 120 mL ) with water. The sample bottles were stored in a dark container filled with water of the same temperature as the samples until fu rther analysis at the laboratory. In the laboratory 1 mL

H 2 SO 4 is added prior to measuring the OD at 456 nm with a Hitachi U

- 3010 Spectrophotometer. The oxygen concentration was calculated using standards and expressed as **μ** M O 2 /L (or mg O 2 /L = **μ** M O 2 * 0.03 2) . Since both salinity and temperature change over the season the oxygen concentrations were expressed as percentage relative to the natural saturation value for the given temperature and salinity.

DOC The concentration of dissolved organic carbon (DOC) was m easured according to Reintaler
& Herndl (Reinthaler and Herndl, 2005)

. Samples for DOC (15 mL) wer e filtered through GF/C filters and sealed in pre

- combusted glass ampoules after adding 50 **μ** l of phosphoric acid (H 3 PO 4 ). Sealed ampoules are stored at 4 ° C. The DOC concentration wa s determined in the laboratory by the high temperature combustion method using a Shimadzu TOC

- Vcpn analyzer. Standards we re prepared with potassium hydrogen phthalate (Nacalao Tesque, Inc, Kioto, Japan). The mean concentration of triplicate injections of each sample (three in total) is calculated. The average analytical precision of the instrument is < 3 %.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 19 Figure 5 : the challenge water with a high particle and sediment load.

TSS / POC (total suspended solids and particulate organic Carbon)

For TSS/POC pre

- weighted g lass fibre filter s (GF/C) are used. Each filter wa s coded and
stored in a clean Petri dish. The filtered volume wa s dependent on the particle load and concentration and type of organisms present in the water. The higher the total particle load in the sampl e, the smaller the volume that c ould be filtered before the filter clogs. Practical
volumes we re between 100 and 1000 mL per sample.
After filtration the filter wa s rinsed with fresh water (MiliQ) to remove sea salt. Filters we re dried overnight at 60 °C and allowed to cool in a vacuum exicator before weighing. The total amount of suspended solids wa s calculated from the weight increase of the filter and averaged for the three replicates (mg/ L )

. Next, the filter is combusted at 500°C (overnight) and allowe d to cool in a vacuum exicator and weighted again. The POC wa s calculated from the weight decrease between this measurement and the TSS weight.

Analytical determination of chlorine and related compounds

During each test cycle, t he concentration of the ac tive compound ( Total Chlorine ) w as measured as soon as possible after filling of the treated ballast water tank , also at regular
intervals during the 5 day hold period, and during discharge.

Chemistries that have been tested and verified by the United St ates Environmental Protection Agency (USEPA) are used to analyze the treated ballast water produced by the

B AL P URE® BWT

- System

. Total Residual Oxidant (TRO) was measured as total chlorine using the Hach 2100p hand
held Pocket C olorimeter II

. The device e mploys the DPD Method accepted for reporting water analyses by the USEPA and is equivalent to USEPA method 330.5 for waste water
and Standard Method 4500

- Cl G for drinking water.

Figure 6 : Example of sampl e collection point at tank 3.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 20 Biology

The majority, but not the entire large size fraction (> 50 μm) consists of zooplankton, while the majority of the small size (10

- 50 μm) fraction consists of phytoplankton. Organisms > 50 μm are retained as recommended in the G8 guidelines MEPC 54/Inf.3 ( using a modified Hydrobios net).

. Samples for the 10

- 50 μm fraction were collected from the effluent of the Hydrobios net. These samples we re then filtered over a 10 μm sieve and fixated. A second set of samples for this size class wa s taken and not sepa rated from the organisms < 10 μm in order to include the fate of the smaller sized (phyto) plankton community as well and to avoid further damage of the plankton. The results of these samples we re compared to the ones from the double filtered samples to eva luate the loss of organisms caused by processing the samples.

Sample sizes

During the land

- based tests container s from 1 to 10 00 L were used for sampling and /or storage. Samples were taken continuously and evenly during the whole process of filling

or em ptying the ballast water tanks. These containers were thoroughly rinsed or heat

treated prior to use. Samples for the human pathogens were taken in sterile (bar

coded) bottles provided by the bacterial test laboratory.

Figure 7 : 1000 L container (IB C) with a 50 **μ** m Hydrobios sampling net

Organisms > 50 μm

The samples are pre

- concentrated over a Hydrobios 50 **μ** m net resulting in a n end
volume of approximately 100 to 200 mL . The samples we re transferred to the lab directly after sampling and Ne utral Red wa s added in a ratio that yields an end concentration of 1:50,000. Staining time is 2+ hours. Neutral Red stains living organisms (Crippen, 1974 ; Fleming and Coughlan, 1978) distinctively and quite rapidly ( less than
one hour , figure 6 ). Therefore the viability assessment remains unaffected by the possible death of organisms during the staining or during sample analysis. It is assumed that dead but physically intact organisms will also be found. Consequently a detailed inspection of eac h intact individual is needed to assess viability. This includes the staining as well as the detection of internal (heart, gills) movement. Organisms
which were not intact are assumed to be dead.
Neutral Red is a reliable staining method for all major gro ups of organisms but inconsistent staining was found for bivalves. For this latter group movement (including
internal such as h eart and gills for juvenile mussels) has to be used obligator il y toFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 21 determine viability. This is dependent on the expertise of th e person analyzing the samples. Therefore the same person analyzed all samples.

Figure 8 : (top) live copepod (left) and nauplia (right) from a Neutral Red stained sample. (bottom) dead copepod (left) and nauplia (right) from a NR stained sampl e.

The samples are analyzed manually using a binocular with a 20x magnification for counting and up to 50x for species identification and measurements when necessary. For inter comparison a subset of samples was also analyzed using a semi

- automated
tool (FlowCam, Fluid Imaging Technologies ; (Anonymous, 2001)

. Organisms need to be counted according to their size. Here organisms of 50 **μ** m in minimu m dimension are relevant. Several tests have shown that a single size bar is not efficient as viable organisms move in the counting chamber. Better results are achieved when the entire field of view is equipped with a size grid

. The minimum dimension to me asure will be adjusted to the specific organism groups.

Organisms < 50 **μ** m

S amples for visual inspection of species number and diversity we re pre

- concentrated using a sieve made of a Hydrobios 10 **μ** m mesh net using the 50 **μ** m prefiltered sample (e ffective size range is 10

– 50 **μ** m )

. The retained organisms we re flushed into 50 mL

Greiner tubes using filtered seawater and fixed with Lugols solution. Sample analysis

wa s conducted by microscopic count with an inverted microscope at 200x magnification (method by Utermöhl). Since the Utermöhl is not suitable to asses viability counting was
restricted to the structural integrity of organisms and therefore the presence of intact
cells (Paerl, 1978) . This method works for both zoo

- and phytoplankton.
This size fraction wa s also covered by flow cytometry on basis of a single cell

measurement (Veldhuis and Kraay, 2000) and PAM fluorometry , as a bulk parameter (Schreiber et al. , 1993) , using the intact and undist urbed samples. Besides numbers andFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 22 sizes these two methods can be used to assess the cell viability (Veldhuis et al. , 2001 ; Veldhuis et al. , 2006) or in case of the PAM fluorometry also the photosynthetic efficiency of the phytoplankton.

F low cytometry: For total organisms counts 3 mL of unfiltered sample water ( reference
and treated, each in triplicate) we re analyzed using a calibrated flow cytometer. This yields the total number of particles (dead and live organisms as well as detritus) as well as their size range and the presence o r absence of chlorophyll

. For the counts ex actly 1

mL was analyzed. The size of the plankton was determined by comparison to standardized beads (10 and

50 **μ** m). These beads we re also used as standards to calibrate the performance of the flow cytometer. For organism viability testing, on the level of the individual cell, SYTOX G reen wa s added

to 1 mL of sample water (control and treated, each in triplicate). After 15 minutes samples we re analyzed using the flow cytometer for the presence of dead and/or li v e organisms (cf. Veldhuis et al, 2001 , Caso t ti et al, 2005 ).



### nucleus B A

Fig ure 9 : (A) E pifluorescence microscopic picture of a li v e phytoplankton cell

. The red signal is due to the presence of chlorophyll and (B) a dead phytoplankton cell with a yellow/green fluorescence of the nucleus after staining with SY TOX Green.

PAM fluorometry: The photochemical efficiency of photosystem II of phytoplankton ( providing a n estimate of the general health of the algae) can be addressed using Pulse

Amplitude Modulated fluorometer (PAM

- fluorometry) WALTZ

- water PAM (Schreiber et
al. , 1993) . For this 3 mL of unfiltered sample water (control and treated, each in triplicate) are filled into a glass cuvette and analysed using the Pulse

- Amplitude
Modulated fluorometer. The instrument was calibrated against filtered seawater and a healthy fast

- gr owing population of phytoplankton.

B acteria

The classical method for count ing bacteria in many applications is based on plating on selective media. Unfortunately, for studies in the aquatic environment this approach is by far insufficient for various rea sons (Gasol and Del

- Giorgio, 2000)

. As a result t otal
bacteria were now determined by flow cytometry, using DNA

- specific stains to get a
more accurate bacteria number . In addition samples were taken at discharge for specific human pathogens and heterotrophic bacteria using a plate method.

A 1.5 mL water sample wa s taken and pipetted in a Cryovial (in tri plicate) and formaldehyde was added as a preservative

. Samples were frozen and stored at

- 50 ° C until further analysis.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 23 Upon analysis the sample is allowed to thaw completely. A subsample of 100 **μ** l is ta ken,
diluted with a TE

- buffer, and the nucleic acid dye PicoGreen (MP) wa s added. Within 5 to 15 minutes after the addition of the stain the sample is analyzed using a flow
cytometer (cf. (Gasol et al. , 2000 ; Veldhuis et al. , 1997) . A know n bacterial standard is
used for calibration and counting

. The number of total heterotrophic bacteria was determined using a plate method a s the number of colony forming units (cfu’s) after incubation of the water at intake and discharge according to an international standard (NEN

- EN

- ISO 6222 :1999 “Vitens laboratory bv” at Leeuwarden, RvA lab. no. L043) ).

Human pathogens

The samples for microbiological analysis of the presence and number of human pathogens were taken in special bottles of 600 mL and send to “ eurofins/C. mark b v” at

Heerenveen (accreditation certificate: RvA lab. no. L043). All analyses were carried out according to NEN

- EN

- ISO standards. These samples are sent to the laboratory immediately after sampling using a cooled transport container (4 ° C ) . The analysis is carried out according NEN

- EN

- ISO 7899

- 2 for
intestinal enterococci and NEN

- EN

- ISO 9308

- 1 for E. coli and related bacteria of the coli

group as adopted for surface and waste water analysis in the Netherlands.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 24 

## 8 Results

The present section is a compilatio n of all relevant information needed for Type Approval Certification tests according to the Guidelines (G8)

, but also includes some relevant results of experiments conducted to assess the environmental effect of the active substance and /or

its by

- products in the environment upon discharge (vitality of the treated water)

. It should however, be noted that d etailed studies regarding potential toxic ity according to standard procedures have also been carried out . The se results were submitted to IMO/ GESAMP

BWWG

. Data are presented as averages or ranges separated for the two salinity regimes
tested. In Annex 1 a detailed species list of organisms observed is presented

.

Figure 10 : overview of the B AL P URE® BWTS ( chlorination unit ) , discharge pump (left) and sampl e containers (right ) in the NIOZ harbour (top) . Overview of the external BsF filter (bottom).Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 25 The tests were carried out at two different salinity regimes (Tables 4 , 5 and 6 ) hereafter referred to as low and high salinity test series.



## 8.1 **Physical a**  **nd chemical parameters** 

Table 4 : A verage salinity and temperature of water at intake during the low salinity tests of
control and treated tanks for test runs I

– V I

.

test run salinity [PSU] range temperature [°C]

I

– V I 2 3

. 9 22

- 26 8. 8

- 1 2

. 8

To the low salinity test cycles brackish water from the NIOZ harbour was collected during low tide and fresh water, taken from a fresh water source was added to a maximum of 15% (v/v).

Table 5 : A verage salinity and temperature of water at intake during the high sal inity tests of
control and treated tanks for test runs V I I

– X I

.

test run salinity [PSU] range temperature [°C]

VI I

- X I 3 3

. 6 30

- 36 1 3

. 2

- 1 5

. 7

To the high salinity test cycles coastal water from the North Sea was collected during high tide and a brine solution made from natural sea salt was added to a maximum of 4 % (v/v).

In regards to salinity

, the difference between both the high and low salinity regimes is slightly less than 10 PSU (0.3 PSU difference) . However, given the standard variation
and th e fact that tidal effects and prevailing winds occasionally disturbed the ideal

situation

, the minor deviation is considered to be of minor importance. Moreover

, efficacy of the B AL P URE® BWT

- System is not largely affected by changes in terms of initial
dos age, final (T5) and residual values of TRO within the salinity regime tested (Table 6)

.

Table 6: Average oxidant and sodium bisulfite values (mg/L) in treated ballast water for 6 low salinity test cycles and 5 high salinity test cycles . Test runs I

- VI l ow salinity and VII

- XI high salinity.

Test

run BalPure

Treatment
d ose TRO (T0) TR (T1) TRO (T5) Post

n eutralization

TRO Residual Sodium Bisulfite @ Discharge

I

- VI 14.83 14.80 7.19 4.46 Not Detected 5.00

VII

- XI 13.17 12.36 4.97 2.98 Not Detected 3.83

T0 = immediately after BalPure treatment (day 0) T1 = one day after BalPure treatment (day 1) T5 = five days after BalPure treatment (day 5) Neutralization occurred on Day 5 during discharge of BalPure treated water.
Not Detected = <0.0 2 mg/LFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 26 Low salinity

A summary of the r esults of the basic parameters (oxygen concentration, pH, TSS, POC and DOC) is presented in T able 7 for the reference and treated water sample at intake and discharge.

Table 7 : average or range of o xygen, pH, T SS, POC and DOC concentrations of test series run at low salinity (6 test runs in total ; I

- VI ) at intake and discharge;

1 : relative to saturation value for given salinity and temperature ; the oxygen concentrations

were not correct ed for interference of the active substances or derivates of it.

Parameter Intake s.d. Discharge (day 5) s.d. unit

O 2 reference 1 103

- 119 85

- 104 %

O 2 treated 1 130

- 146 109

- 134 %
pH reference 8.30 0.09 8.25 0.10

pH

- treated 8.52 0.10 7.90 0.15

TSS

- reference 46.8 6.2 16.2 3.5 mg/L

TSS

- treated 41.9 18.3 12.6 4.4 mg/L

POC

- reference 16.3 2.0 9.1 3.3 mg/L

POC

- treated 15.3 5.3 5.6 1.8 mg/L

DOC

- reference 2.8 0.2 2.7 0.3 mg

- C/L

DOC

- treated 3.0 0.4 3.5 0.3 mg

- C/L

Table 7 c learly demonstrates that as far as the majo rity of the basic parameters are concerned the values were in acc ordance or very close with the criteria as indicated in the guidelines (G8, Table 1)

. On average TSS values were slightly less than the recommended 50 mg/L but on the other hand POC , the more reactive component of the particles in seawater, was three fold higher than the required minimum value. As far as the oxygen values of the reference tanks are concerned values at intake were closely corresponding to a saturation values (100%) for the give n salinity and temperature.

Since the water temperature increase d steadily during the test series values are presented as a range and not averaged. At discharge on day 5, the oxygen concentration in the reference tank declined by a maximum of 19 % compare d to intake
values

. The oxygen concentration in the treated tank tended to be higher, by as much

as 26 % as compared to the reference tank . This apparent super

- saturation is partly an artefact caused by the active substance chlorine interfering with the Win kler method
for O 2 measurements

. Separate tests showed that for the app lied concentrations of
chlorine added at least half of the observed increase in the oxygen concentration could
be accounted for. After the required incubation period of 5 days the oxyge n concentration dropped only slightly (ca. 10 % ).

The total suspended solids (TSS) and particulate organic carbon (POC) concentrations were sufficiently high for appropriate testing at intake but these values varied considerably among the different test r uns. At discharge the sediment concentration
declined significantly , mainly because of sedimentation in the tank during the holding period of 5 days. At discharge on day 5, both TSS and POC were reduced in the treat ed tank, but the reduction was higher tha n was measured in the reference tank. Based on the observed increase in DOC it can be assumed that a significant part of the POC in the treated tank was decomposed and converted into DOC.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 27 High salinity

Table 8 : average or range of o xygen, pH, TSS, POC a nd DOC concentrations of test series run at high salinity (5 test runs in total ; VII

- XI ) at intake and discharge;

1 : relative to saturation value for given salinity and temperature ; the oxygen

concentrations were not correct ed for interference of the acti ve substances or
derivates of it.

Parameter Intake s.d. Discharge (day 5) s.d. unit

O 2 reference 1 76

- 109 53

- 81 %

O 2 treated 1 112

- 141 98

- 120 %
pH reference 8.05 0.20 7.92 0.27

pH

- treated 8.33 0.12 7.50 0.40

TSS

- reference 33.9 37.10 9.7 0 3.10 mg/L

TSS

- treated 15.6 3.60 9.60 1.30 mg/L

POC

- reference 7.6 2.1 5.0 1.20 mg/L

POC

- treated 8.1 4.7 3.7 1.10 mg/L

DOC

- reference 3.0 0.5 2.8 0.5 mg

- C/L

DOC

- treated 3.3 0.3 3.7 0.4 mg

- C/L

The results of the basic parameters at the high sali nity range were , with a few exceptions, on the whole the same as for the low salinity range (Table 8 ). A t intake the
ambient water was , with exception of a single test run, saturated with oxygen and at discharge oxygen concentrations in the both control an d treated tanks declined only moderately . Oxygen values were still far from depleted. Partly because of the increase in seawater from the open North Sea sediment, TSS values were lower but POC remained high, as a result of the high content of organisms, as compared to the test
series conducted at low salinity. Mainly due to sedimentation the residual TSS load and
POC after the 5 day holding period was reduced to ca. 9.7 mg/L and 5.0 mg/L, respectively.



## 8.2 **Biology**

Organisms > 50 μm

For the land based t ests natural plankton was used and the required diversity of organisms (5 species of at least 3 different phyla) was easily fulfilled for all tests (Table

9 ) . On average at least 10 different species of 4 to 5 different phyla were present in each sample (f ull details on species present see Annex 1 ). Regarding the required minimal numbers of organisms per volume (Table 2) , the values for the low salinity series ( test series I

- VI ) were well above the minimum requirements

. In some cases the numbers in the >50 **μ** m size fraction were 1 4 times higher ( test run V ) than the minimum required number of 100,000 m - 3

. At the high salinity range the water
at intake contained also, and in contrast to previous years when numbers were occasionally slightly less than the minim um required

, sufficient numbers

. The combination of prefiltration (filter BSFc, 40 μm filter) and chlorine addition turned out to be an effective way of eliminating the larger fraction of organisms. For both salinity regimes the remaining number of organ isms in the size class > 50 **μ** m were at least one order of magnitude lower than the Standard

- D2 (Table 9)

. At seven out of 11

test runs no living organisms w ere observed in the 3 replicate 1 m 3 subsamples.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 28 As opposed to the treated tank

, numbers of organis ms in the reference tank were still significantly above the G8 requirement during discharge and visual inspection and viability measurement indicated that t hese organisms were both intact and viable.

Table 9 : N umber of organisms > 50 **μ** m (average of 3 samp les) at intake and on discharge at day 5 of the reference and treated tank. All number s are presented per m 3

.



### total plankton > 50 μm numbers/m 3

low salinity Intake Reference Treated

test run C

- T0 C

- T5 STDN

- T5

I 0

. 65 E+6 2

. 58 E+4 4

. 3

II 1

. 26 E+6 0

. 98 E+ 4 0.3

III 0

. 39 E+6 2

. 36 E+ 4 0

IV 1

. 06 E+6 6

. 4 8E+ 4 0

V 1.48E+6 7.22E+4 0

VI 0.43E+6 1.61E+4 0

average 0

. 88 E+6 3. 54 E+ 4 0.8

s.d. 0

. 43 E+6 2 .6 4 E+ 4 1.7



### total plankton > 50 μm numbers/m 3

high salinity Intake Reference Treated

te st run C

- T0 C

- T5 STDN

- T5

VI I 0.3 1E+ 6 1. 9 1E+4 0

VII I 0.15 E+ 6 0

. 26 E+4 0

IX 0.39 E+ 6 1 .5 0 E+ 4 0

X 1

. 09 E+ 6 1. 82 E+4 0.3

X I 0

. 97 E+ 6 0 .7 7 E+4 1 .0

average 0

. 58 E+ 6 1. 2 5E+4 0.3

s.d. 0

. 42 E+ 6 0

. 71 E+3 0.4

Organisms 10

– 50 μm

This size class wa s dominated by ph ytoplankton, although heterotrophic organisms like ciliates and flagellates were occasionally present i n high numbers at intake

. Additional studies indicated that even gentle pumping of water into the reference tank resulted in a substantial damage of the fragile fraction of the plankton community like flagellates
and ciliates. For that reason the flow cytometric analysis of water samples was focussed on the phytoplankton component using chlorophyll a auto fluorescence as a n extra selection parameter. Moreov er, the photosynthetic efficiency of the phytoplankton community was measured, as a tool to determine the efficacy of the treatment on this plankton group more specifically

. Finally, a 10 L subsample of both the reference and
treated water was collected at intake and incubated under optimal growth conditions in
order to determine potential growth of cells which have survived the treatment or hatching of resting stages/spores. Results of plankton numbers in the 10

– 50 **μ** m of
both reference and treated incub ation are included in the table for comparison with the actual measurement of the tanks (Table 10 ).Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 29 Table 10 shows that the phytoplankton community in the reference tank responded as was observed in earlier experiments. Pumping the water into the tanks an d the prolonged dark period reduced the total number of plankton to 10% of the value at intake. However, the growth potential of this phytoplankton was very high as can be derived from the increase in the numerical abundance when a subsample of the water

a t intake was incubated under optimal growth conditions (C

- Inc

- T5). For the low salinity range there was an average 10 fold increase in numerical abundance as compared with the numbers at intake and at the high salinity this factor was 3.6.

In contrast in t he treated tank the number of intact cells immediately after intake ( STDN

- T0) declined to an extreme low value. At this stage the remaining cells do meet the requirements as defined in the Standard

- D2. The incubation period of 5 days
resulted in a further reduction of the cell numbers and on average the remaining number of viable cells was < 0.1 per mL. Testing for regrowth also indicated that the treatment was very effective and as no viable cells were observed in the incubated samples ( STDN

- inc

- T5). Also after neutralising treated water with bisulfite at discharge and incubated for another 5 to 7 days no regrowth of phytoplankton was observed (STDN

- dis

- Tx). As expected and corroborating with the results for the larger organisms no effect was
observed of th e difference in salinity regime.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 30 Table 10 : Total plankton number of plankton in the 10

– 50 **μ** m size range at intake WS= Wadden Sea) , reference (C) and treated tank ( STDN ), reference and treated incubated samples (Inc.) at day 5 (T5) during discharge , inc ubated and neutralized discharge treated water (STDN

Dis

- Tx) . Runs I to VI at low salinity range and VII to XI at high salinity range. All numbers are presented per mL as total counts or of viable cells (*). Tx= varying sampling day (5 to 7 days).

10

- 50 μm cell numbers cells/ml STDN

low salinity Intake Reference Treated total viable

test run WS C

- T5 C

- inc

- T5 STDN

- T0 STDN

- T5 STDN

- T5 * STDN

- inc

- T5 STDN

Dis

- T x

I 1046 117 8145 14.8 1.5 <0.1 1.5 0.0

II 1477 104 8459 7.4 0.7 <0.1 0. 0 0.7

III 1047 135 41137 9.6 0.0 <0.1 0.0 0.0

IV 1109 119 4850 5.9 0.0 <0.1 2.2 1.5

V 1048 124 3420 4.4 0.0 0.0 0.0

VI 1737 107 13101 7.4 0.0 <0.1 2.2 0

average 1244 118 13185 8.3 0.4 <0.1 1.0 0.4

high salinity

VII 1023 108 2998 6.66 0 <0.1 3.7 0

VI II 1002 112 11734 5.18 0 <0.1 1.48 0

I X 1406 132 2098 5.92 1.48 <0.1 0.74 0

X 1799 103 3519 9.62 0 <0.1 0.74 3.7

X I 1058 96 2152 4.44 0 <0.1 2.22 0

average 1258 110 4500 6.4 0.3 <0.1 1.8 0.7

Organisms <10 μm

In order to have a more complete insight of the fate of all organisms, i.e. also fraction the planktonic fraction < 10 **μ** m in diameter, a selective population of phytoplankton in the size range of ca. 6 **μ** m was monitored in both the reference and treated water (Table 1 1 ). Although no clear criteria are, yet, defined for this size class it was clear that
the B AL P URE® BWT

- System was also effective in reducing organisms in the smaller than 10

micron range. Flow cytometr ic analysis showed the presence of a varying but high
number of cells at intake but extreme ly low number of intact cells in the treated tank immediately after intake (T0) . After 5 days of holding the numbers declined even
further and the presence of viable cells resulted in a number < 0.1 per mL at discharge

. Incubation of chlorine treated water under ideal growth conditions did not alter the general observation since no regrowth of phytoplankton was observed ( STDN

- inc

- T5). Also after neutralisation of the chlorine treated water at discharge regrowth of nanoph ytoplankton was not observed within five to seven days of incubation.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 31 Table 1 1 : Phytoplankton number of a dominant phytoplankton species present ( Phaeocystis globosa ; ca. 6 **μ** m in cell diameter ) at i ntake, reference (C) and treated tank ( STDN ), reference and treated incubated samples (Inc.) at day 5 (T5) during discharge , incubated and neutralized discharge treated water (STDN

- Dis

- Tx)

. Test r uns I to VI at low salinity range and VII to XI at high salinity range. All numbers are presented per mL as total co unts or of viable cells (*). Tx= varying sampling day (5 to 7 days).

Phaeocysti s ~ 6 μm cells/ml STDN

low salinity Intake Reference Treated viable

test run C

- T0 C

- T5 C

- inc

- T5 STDN

- T0 STDN

- T5 STDN

- T5* STDN

- inc

- T5 STDN

- Dis

- T x

I 3283 1009 8589 16.3 0.7 <0.1 0.7 1.5

II 4216 130 11500 11.8 0.7 <0.1 0.0 0.0

III 3735 664 24017 14.8 0.7 <0.1 1.5 1.5

I V 11554 217 3493 14.1 4.4 <0.1 9.6 19.2

V 10048 1485 11020 22.2 1.5 <0.1 58.5 10.4

VI 4887 901 16177 22.9 0.7 <0.1 3.7 0.74

ave rage 6287 734 12466 17.0 1.5 <0.1 12.3 5.6
high salinity

VII 2471 305 2372 9.62 0 <0.1 1.48 0

VI II 4070 2047 28450 2.96 1.48 <0.1 2.96 0.74

I X 3068 999 2744 2.22 0.74 <0.1 1.48 0

X 2258 354 3159 4.44 3.7 <0.1 2.22 1.48

X I 2082 363 1730 3.7 0.74 <0.1 5.18 0.74

average 2790 814 7691 4.6 1.3 <0.1 2.7 0.6

Photosynthetic efficiency

Another approach to gain insight in the physiological condition and therefore the growth potential of the phytoplankton community is by measuring the photosyn thetic efficiency (F

v /F

m ) of the cells. This was done for the whole community (<50 **μ** m ) , but when required is also possible for different size fractions. For a clear understanding it
must be noted that values of F
v /F

m > 0.4 are indicative of a healthy phyto plankton population; a F
v /F

m < 0.4 indicates that the phytoplankton community is experiencing severe stress and a value < 0.1 is typically observed in decaying phytoplankton populations.
Table 1 2 shows that, with one exception (test run VIII ) during intak e the whole phytoplankton community in the water was in a physiologically healthy condition, i.e. containing mostly photosynthetic active and therefore consisting of viable phytoplankton cells. Prolonged incubation of the control in the dark (5 days of dar k is a considerable stress condition for algae) resulted in a considerable reduction of the photosynthetic efficiency to values indicative of severe stress and occasionally values were measured typical of a decaying population. In contrast the photosynthet ic efficiency of a subset of phytoplankton of the control taken at intake and incubated under optimal growth conditions was typical for a healthy and viable phytoplankton community (C

- inc

- T5). This is not surprising sinceFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 32 phytoplankton also increased in nu merical abundance in these incubated water samples (Tables 10 and 1 1 ). After passing the treatment system no distinct phytoplankton cells were observed and
therefore F
v /F

m was typical of a decaying algal community. Prolonged storage in ballast
tanks showe d that there was no recovery of the phytoplankton population. A lso in the incubated sample , taken at intake as well as after the neutralization step at discharge, and kept under optimal conditions enabling growth, no recovery of the phytoplankton community was observed.

Table 1 2 : Photosynthetic efficiency (Fv/Fm) of the whole phytoplankton community (2

- > 50

**μ** m cell diameter ) at i ntake, reference (C) and treated tank ( STDN ), reference and treated incubated samples (Inc.) at day 5 (T5) during discharge , in cubated and neutralized discharge treated water (STDN

- Dis

- Tx) . Runs I to VI at low salinity range and VII to XI at high salinity

range. Tx= varying sampling day (5 to 7 days).

F v /F

m Intake Reference Treated

low salinity C

- T0 C

- T5 C

- in c

- T5 STDN

- T0 STDN

- T5 STDN

- inc

- T5 STDN

- Dis

- T x

I 0.67 0.31 0.54 0.04 0.02 0.08 0.09

II 0.67 0.21 0.31 0.04 0.05 0.12 0.03

III 0.52 0.16 0.47 0.01 0.00 0.19 0.15

I V 0.54 0.01 0.48 0.04 0.10 0.27 0.03

V 0.55 0.12 0.44 0.07 0.11 0.05 0.05

VI 0.54 0.28 0.51 0.02 0.06 0.21 0.07

average 0.58 0.18 0.46 0.04 0.06 0.15 0.07

s.d. 0.07 0.11 0.08 0.02 0.04 0.08 0.05
high salinity

VII 0.61 0.14 0.48 0.02 0.06 0.04 0.05

VI II 0.46 0.21 0.55 0.15 0.17 0.19 0.10

I X 0.63 0.25 0.64 0.00 0.08 0.06 0.12

X 0.55 0.15 0.66 0.04 0.06 0.06 0.04

X I 0.53 0.12 0.60 0.00 0.03 0.03 0.10

average 0.56 0.17 0.59 0.04 0.08 0.08 0.08

s.d. 0.07 0.06 0.07 0.06 0.05 0.07 0.04

B acteria

For the microbial community the presence/absence of two types of human pathogens ( E. coli and enterococci) was monitored prior to and after treatment and after
neutralisation of the chlorine with bisulfite

, while the response of the whole microbial community was also assessed

. Table 1 3 shows that even during intake (C

- T0) the number of both target microorganisms was well below the standard as indicated in the Standard

- D2

. I n five

test runs the numbers of human pathogens were above the detection limit for one or both target microbes (test run s IV, VI, VIII, X and XI ). The reason for this is that the NIOZ harbour is located in a pristine environment with little or no urban activity. Subsequently, also the number of human pathogens in the reference and treated tanks were below detection limit during discharge.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 33 During test run IX water from a sewage plant was spike d in to the test set up in order to examine the effect of the BWT system on the E. coli and enterococci. At intake cfu’s for both groups of pathogens, as measured in the control tanks, were 0.12 and 57 cfu’s per mL, respectively. In the treated tank (T0) both groups of pathogens were already below
the detection limit.

Table 1 3 : plate c ounts of the human pathogens E. coli and total enterococci (as cfu’s) at intake and during discharge of the reference and treated tank and neutralized discharge treated water (STDN

- Dis

- Tx) . nd; no data. Test runs XI

, XIII and XV share the same reference since this
was a combined test run. Tx= varying sampling day (5 to 7 days). nd: not determined

human pathogens Intake Reference Treated

counts /mL C

- T0 C

- T0 C

- T5 C

- T5 STDN

T5 STDN

T5 STDN

Dis

- Tx STDN

Dis

- Tx

low salinity E.coli Enterococci E.coli Enterococci E.coli Enterococci E.coli Enterococci

I nd <1 nd <1 nd <1 nd <1

II <0.1 <1 <0.1 <1 <0.1 <1 <0.1 <1

III <0.1 <1 <0.1 <1 <0.1 <1 <0.1 <1

IV <0.1 2 <0.1 <1 <0.1 <1 <0.1 <1

V <0.1 <1 <0.1 nd <0.1 nd <0.1 <1

VI 0.14 12 <0.1 <1 <0.1 <1 <0.1 <1

range <0.1

- 0.14 <1

- 12 <0.1 <1 <0.1 <1 <0.1 <1

hig h salinity

VII <0.1 <1 <0.1 <1 <0.1 <1 <0.1 <1

VIII 1 100 <0.1 <1 <0.1 <1 <0.1 <1

IX <0.1 <1 <0.1 <1 <0.1 <1 <0.1 <1

X <0.1 2 <0.1 <1 <0.1 <1 <0.1 <1

XI <0.1 4 <0.1 <1 <0.1 <1 <0.1 <1

range <0.1

- 1 <1

- 100 <0.1 <1 <0.1 <1 <0.1 <1

In contrast to the near absence of human pathogens, the typical marine microbial community was abundantly present. The total bacteria l community is well studied in the Wadden Sea, using a method based on staining the nucleic acid of the cells for many years. The currently observed numbers ranging from 1

. 78 to 5

. 65 10 6 per m L (variation
of factor 3

. 1 ) are numb ers typically observed in spring and early summer situation in
the area (Table 1 4 )

. Of this total bacterial population only a very small fraction could be identified as heterotrophic ones using the plate assay method (10 to 170 per mL ). Only

at one test ru n (VIII) a cfu count > 3000 was measured. Therefore, if only plating would
be used as criteria for heterotrophic bacteria number this would result in a severe underestimation of the actual bacteria numbers. After 5 days of incubation total
bacteria numbers on average declined significantly , by as much as 45% in the reference tank but there was considerable variation between the different test runs (C

- T5). Due to the presence of labile organic carbon (DOC) growth of in particular the heterotrophic
bacteria w as stimulated explaining the increase in cfu ’s in the reference tank at day 5.

Bacteria numbers in the incubated water of the reference tanks also declined numerically but numbers were in between the counts measured at intake and those of the discharge wa ter of the main tank (C

- inc

- T5).Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 34 In the treated tanks and in the incubation bottles of th e same water the total bacteria numbers at discharge (STDN

- T5 and STDN

- inc

- T5) declined by one (high salinity) to two orders of magnitude (low salinity) relative to the original numbers at intake. In addition, with exception of test run IX, also the number of colony forming units declined to below the detection limit (<1). The chlorine treatment act ed initially as a disinfection agent of the microbial community, ther efore preventing (re) growth, but resulted also in a massive disintegration of the bacteria cells.
However, after neutralisation and a second incubation the remaining bacteria population recovered to some extend resulting in a slight increase in total numer ical abundance. In particular the group of heterotrophic bacteria thriving on the high dissolved organic carbon load of the water did benefit most

.

Table 13 : Total bacteria number (total bact.) and colony forming heterotrophs (colony = cfu numbers per mL ) at intake (C

- T0) and during discharge of the reference (C

- T5) and treated (E

T5) tank as well as the total bacteria numbers in the incubated water samples (inc.). Test runs

XI

, XIII and XV share the same reference since this was a combined test run. * ra nge of values

. Tx= varying sampling day (5 to 7 days). Nd: no data.

bacteria Intake Reference Treated

C

- T0 C

- T0 C

- T5 C

- T5 C

- inc

T5 STDN

- T0 STDN

- T5 STDN

T5

- Inc STDN

- T5 STDN

Dis

- Tx STDN

Dis

- Tx high salinity total
bact. colony total
bact. colony total
bact. total
bact. total
bact. total
bact. colony total
bact. colony

I 1.78E+6 22 0.41E+6 220 1.95E+6 0.49E+4 0.67E+4 12.7E+4 <1 11.6E+5 290

II 1.90E+6 27 0.81E+6 320 2.60E+6 0.5.7E+4 0.61E+4 1.29E+4 <1 7.75E+5 420

III 3.67E+6 10 1.58E+6 840 3.47E+6 7.15E+4 4.50E+4 6.18E+4 <1 7.91E+5 23

IV 5.65E+6 13 2.21E+6 70 3.37E+6 10.2E+4 5.77E+4 6.06E+4 <1 5.17E+5 840

V 5.08E+6 9 3.84E+6 330 4.69E+6 7.92E+4 5.55E+4 7.15E+4 <1 9.82E+5 540

VI 4.20E+6 130 4.25E+6 310 2.50E+6 3.31E+4 7.09E+4 6.52E+4 <1 6 .12E+5 3000 average/

range 3.71E+6 * 9

- 130 2.19E+6 * 70

- 840 3.10E+6 4.94E+4 4.03E+4 6.65E+4 <1 8.06E+5 * 23

- 3000

low salinity

VII 3.93E+6 11 2.63E+6 30 4.28E+6 1.49E+5 1.40E+5 1.61E+5 <1 10.7E+5 nd

VIII 5.32E+6 >3000 2.56E+6 2800 5.02E+6 1 .09E+5 2.73E+5 2.69E+5 <1 9.63E+5 600

IX 4.19E+6 170 1.80E+6 200 1.48E+6 1.17E+5 1.59E+5 1.65E+5 330 1.21E+5 270

X 4.89E+6 57 1.60E+6 640 4.63E+6 0.88E+5 1.56E+5 1.69E+5 <1 11.0E+5 250

XI 3.57E+6 30 1.48E+6 200 1.78E+6 1.42E+5 1.69E+5 2.12E+5 <1 8.02E+5 270 average/

range 4.38E+6 * 11

- >3000 2.01E+6 * 30

- 2800 3.44E+6 1.21E+5 1.79E+5 1.95E+5 * <1

- 330 8.10E+5 * 250

- 600Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 35 

## 9 Environmental acceptability

Ballast water treatment system s applying active substances, and also systems not using
active substances acco rding to the latest version of the guidelines G8 (MEPC 174. 58)

, should demonstrate that the treated water upon discharge is not harmful to the
environment and aquatic organisms. Although not obligatory for the present system a series of studies were carried out to examine regrowth of remaining phyto

- and bacterioplankton on the long

- term (2 5 days)
and to determine the vitality of the discharged treated water

. In total two long

- term incubation experiments ( one at low salinity and one at high salinity range) w ere conducted with treated water which was in collected in a clean container (10 L) and incubated under optimal growth conditions in a climate room. To stimulate regrowth of planktonic organism extra nutrients were added ( nitrate ; 30 μM, phosphate; 2 μM,

s ilicate; 20 μM) . In particular at the peak of the spring bloom and thereafter the ambient nutrient concentration (mainly silicate and phosphate) can be extremely low, which may prevent growth of phytoplankton and to some extend also the bacteria community

. The high concentration of DOC was considered to be a sufficient pool of organic material for the (heterotrophic) bacteria.

II large

time [day] 0 2 4 6 8 10 12 14 16 18 20 22 24 26cell counts [number/mL]

0.01 0.1

1 10 100 1000 10000

control
STDN intake STDN discharge

Figure 11 : time course of phytoplankton numbers (10

– 50 μm size range) during a maximum of

2 5 day incubation period, control and treated water collected at intake and neutralised treated water at discharge ( STDN) . Test run II , values are mean of three replicates

.

Figure 11 shows that in the reference incubation growth of phytoplankton, in this case the fraction of 10

- 50 μm started immediately from day one onwards resulting in an numerical increase of nearly two orders of magnitude. Numbers increased mainly in the first few days but this increase continued until day 20. In contrast in the treated water, taken at intake, cell numbers declined to less than 10 per mL within the first two days and remained fairly constant in the subsequent days

. Also the smaller phytoplankton community responded in a similar way (data not shown).
Incubation of neutra lized treated water on the other hand showed a different response. Initially, numbers of intact but still chlorophyll containing phytoplankton cells declinedFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 36 to a nearly undetectable number (<0.01 cell/mL) . However, after a lag phase of 10 days
there was r ecovery of the phytoplankton community.
These results indicate that the treatment appears to be very effective in killing or deactivating phytoplankton but also the residual chlorine concentration prevent ed hatching of resting stages or cysts for a s long as the chemical was present

. However, after neutralizing the water quality improved in such a manner that growth of phytoplankton was again possible.

Based on the two long

- term incubation of experiments it was evident that as far as phytoplankton was con cerned the treated water was free of viable phytoplankton cells for a considerable period of time. The virtual absence of organism s for a prolonged period would suggest that the treated water would be non

- vital or ‘ dead

- water ’. Nevertheless, the decontamin ation of the treated water with bisulfite increased t he vitality of the water in such a manner that phytoplankton growth was possible

.



### II total Bacteria

time [day] 0 2 4 6 8 10 12 14 16 18 20 22 24 26bacteria [number/mL]



### 1e+3 1e+4 1e+5 1e+6 1e+7

control
STDN intake STDN discharge

Figure 1 2 : time course of total bacteria numbers during a maximum of 2 5 day incu bation period,
control and treated water collected at intake and neutralised treated water at discharge ( STDN)

. Test run II , values are mean of three replicates

.

With respect to bacteria numbers control and treated water showed a distinct different
patter n (fig. 1 2 ) . In the control bottle bacteria first increased by a factor five which was followed by a rapid decline. Gradual regrowth occurred in the following 10 days to and declining again to values corresponding to the initial numbers at intake towards t he end of the incubation period. In contrast bacteria numbers in the treated water declined by almost three order of magnitude in the first two days of the incubation period. There was some recovery from day two till day 4, followed by a second decline. Fr om day 6 onwards bacteria numbers increased gradually but were at day 20 still one order magnitude lower than the number measured at intake. In the neutralized treated water bacteria recovered apparently rapidly resulting in a rapid numerical increase unti l day 10. Thereafter bacteria growth reached a more stationary phase followed by a decline and second growth period starting at day 19.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 37 Similar to the phytoplankton component of the planktonic organisms also bacteria were extremely disseminated and deactiv ated in terms of numbers and growth potential after chlorine treatment. The neutralization step largely reduced the chlorine stressor and remaining cells recovered almost instantly. With ample dissolved organic carbon and inorganic nutrients present this r esulted in a rapid increase in bacteria numbers.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 38 

## 10 Discussion and evaluation of results

The present ed data show that the experimental design, holistic approach and additional long

- term incubation experiments provide a sound data base of information on the performance of the B AL P URE® BWT

- System under semi

- in situ conditions

. The initial idea of a treatment system based on the use of the active substance chlorine as the secondary treatment, after a course filtration step, and keeping the in tank

concentr ation high enough to prevent regrowth turned out to very effective. With only some minor deviations the present 11 test runs were conducted according to
the IMO’s G8 guidelines. T he main conclusion is , as far as the biology is concerned, that
the residual number of organisms at discharge are well in compliance with the
Standard

- D2. Often the numbers on discharge were at least one order of magnitude
lower than the Standard

- D2. The results of the various ecotoxicity studies and chemical analysis of treated a nd neutralized water at discharge , conducted concurrently with the
present test runs, are presented in the dossier for IMO G9 Final Approval. The treated water remained stable for most of the parameter s over the whole holding period of 5 days. Concentra tio ns of dissolved oxygen (DO) and dissolved organic carbon (DOC) hardly varied. Only the total suspended solids and the particulate organic carbon declined slightly mainly as a result of sedimentation. In this respect it should be noted that the application of the self

- cleaning filter effectively removed larger particles and the load of sediment in the treated tanks was minimal compared to the control tank. The stable DO and DOC were indicative of a reduced biological activity. Indeed t he
treated water was no t only void of organisms for the recommended period of 5 days
but the long

- term incubations also indicated that there was no regrowth of phytoplankton and even minimal growth of bacteria

. This implies that the active substance is also actively preventing g ermination of resting stages, cysts or other type of dormant cells/organisms even under optimal growth conditions as applied in the

climate rooms. Nevertheless, in the presence of chlorine recovery of the remaining organisms was not possible until the tre ated water was revitalized during the neutralization step. It should be noted that the toxicological and chemical aspects of the treated water are discussed
in detail in the environmental assessment studies as part of the IMO G9 application for Final Appro val. T he present growth experiments, as part of a community large recovery study including several environmental acceptability aspects, clearly demonstrate that chlorine detoxified water just prior to discharge contains no detrimental effect of the
treated water to the organisms in the basin receiving this water in high quantities.

During the testing period we encountered some issues which need attention for future testing and legislation

. 1. T he number of organisms in the < 10 **μ** m is a biologically relevant fraction to be taken into account in BWT system test studies.

2. The long

- term incubations (ca. 20 days) and the community large environmental acceptability studies provide good insight in the response of the whole community to the growth potential/limitati on of the treated water when
released in to environment. These experiments should be conducted next to the standard set of toxicology and residual chemistry tests of the treated ballast

water.
3. T esting for the presence of human pathogens strongly depends on the natural
abundance of the se microbes in the natural environment. Since these pathogens

can not be supplemented for health and safety reasons

, accurate testing isFinal report of the land

- based testing of the BalPure ®

- BWT

- S ystem 39 therefore not possible. Moreover, viability tests of the total bacteria community
showed th at not all bacteria were effectively killed. Therefore, at least in theory

, the human pathogens would remain a potential risk. This is a factor of concern
for the land

- based tests as well as for the ship

- board trials when the ship remains in fairly clean p orts for intake and discharge. In the case of B AL P URE® BWT

- System its active compound ( chlorine ) has been evaluated for total and faecal coliforms ( Escherichia coli O157:H7, Koski et al, 1966; Rice et al, 1999) as well as with V.

c holerea (Anonymous, 1999; Nair ) and proven to be a n effective method of
disinfection.

In conclusion, the present configuration of the B AL P URE® BWT

- System offers a reliable and environmentally safe cleaning of the ballast water resulting in organism numbers
well below the Standard of the IMO Regulation

- D2.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 40 

## 11 References
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- 420.Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 42 

## Appendix 1

Phytoplankton Marsdiep April

July 200 9 +: present but rare, ++: present; +++: dominant;

data by Jolanda van Iperen ++++: very abun dant; +++++: massively present

group species name relative
dominance autotrophic flagellate Phaeocystis globosa colony cell +++++ Phaeocystis globosa colonies < 50 μm ++++ Phaeocystis globosa colonies > 50 μm +++++ autotrophic flagellate P haeocystis globosa flagellate cell +++++ diatom pennate Pseudonitzschia delicatissima group ++++ autotrophic flagellate Prymnesiales indet. <10 μm ++++ diatom centricate Thalassiosira spp. 10 μm ++++ diatom centricate Chaetoceros socialis ++++

a utotrophic flagellate Hemiselmis group +++ diatom centricate Skeletonema "costatum" group +++ autotrophic flagellate Plagioselmis group +++ autotrophic flagellate Pyramimonas spp. <10 μm ++ autotrophic flagellate Teleaulax acuta group ++ diatom centricate Minutocellus group ++ diatom centricate Thalassiosira spp. 10

- 30 μm ++ diatom centricate Guinardia delicatula ++ freshwater green alga Pediastrum spp. ++ autotrophic flagellate Prasinophyceae indet. <10 μm ++ hetero/auto dinoflagellate Gymnodiniaceae indet. 10

- 30 μm ++ diatom centricate Leptocylindrus danicus ++ diatom centricate Brockmanniella brockmannii ++ diatom centricate Cerataulina pelagica + autotrophic flagellate autotrophic flagellate indet. <10 μm + diatom centricat e Chaetoceros spp. <10 μm solitairy cells + autotrophic flagellate Chlorophyceae indet. + freshwater green alga Crucigenia spp. + freshwater green alga Chlorophyta fresh indet. + diatom centricate Skeletonema "costatum" lenses +

heterotrop hic flagellate parasite cyst of Ochromonas group ++++ heterotrophic flagellate heterotrophic flagellate indet. <10 μm ++++ heterotrophic flagellate Paulinella spp. +++ heterotrophic flagellate heterotrophic flagellate indet. 10

- 30 μm ++ heterotrop hic flagellate Choanoflagellatea indet. ++ hetero/auto flagellate Cryptophyceae "light" group ++ heterotrophic dinoflagellate Oxyrrhis marina ++ heterotrophic dinoflagellate Katodinium glaucum ++ heterotrophic flagellate Ciliophrys group ++ heter otrophic flagellate Chrysophyceae indet. + heterotrophic flagellate Telonema spp. +Final report of the land

- based testing of the BalPure ®

- BWT

- S ystem 43 Species list zooplankton and some larger (atypical) plankton



## Appendix 1 continued

Phylum Class Subclass, Order, etc. species

no. Identified

genera most likely

presen t

Sarcomastigophora Dinoflagellida 2 Noctiluca, Protoperidinium

Bacillariophyceae 3+ Bidulphia,
Coscinodiscus

Cnidaria Hydrozoa 2+ Obelia Scyphozoa 2 Aurelia, Cyanea

Ctenophora 2+ Pleurobra chia,
Beroe, Mnemiopsis

Nemathelminthes Rotatoria 1+ Asplanchna

Nematoda 1+

Annelida Polychaeta 2+

Arthropoda Crustacea Order
Calanoida 4+ Temora, Acartia, Centropages,
Calanus and/or

Pseudocalanus Oithona

Order Harpacticoida 2+ Tigriopus
Subclass Cirripedia 1+ Semibalanus

Suborder
Cladocera 2 Podon, Evadne

Subclass
Malacostraca 2+ Carcinus (zoea larvae)

Mollusca Gastropoda 1+ Littorina
Lamellibranchia 2+ Cerastoderma Mya

Echinodermata Ophiuroidea and/or 2+ Ophiothrix,
Echinocardium

Echinoidea

Urochordata Larvacea 1 Oikopleura Minimum number of species encountered (10 phyla): 32