Final report of the land-based testing of the BalPure®

-BWT-System 1

Texel January 2010

FINAL REPORT OF THE LANDBASED TESTING OF THE

BALPURE®-BALLAST WATER 

TREATMENT SYSTEM, FOR TYPE 

APPROVAL ACCORDING TO

REGULATION-D2 AND THE 

RELEVANT IMO GUIDELINE 

(APRIL – JULY 2009)

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.nl 
APPROVAL ACCORDING TO

REGULATION-D2 AND THE

RELEVANT IMO GUIDELINE

(APRIL — JULY 2009)

Marcel J. Veldhuis Ph-D

4%:’

Frank Fuhr Dipl.-Biol.Peter-Paul Stehouwer MSc.

Pt~

Royal Netherlands Institute for Sea ResearchP.O.Box 59

1790 AB, Den Burg, TEXEL

the Netherlands

phone: +31-222-369512/300

email: Marcel.veldhuis@nioz.nl, Frank.Fuhr@nioz.nl, PeterPaul.Stehouwer@nioz.nl

Final report of the land-based testing of the BalPure -BWT-System
Final report of the land-based testing of the BalPure®

-BWT-System 3

Content page

1 Executive summary 4

2 Zusammenfassung 5

3 Summary table with results for the Type 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 Ballast Water Opportunity project 9

6.3 Portrait of Severn Trent De Nora (producer of the BalPure®

-BWT system)

10

6.4 The test facility 11

6.5 Technical description of the BalPure®

-BWT system 13

7 Requirements to meet the D2-Standard 14

7.1 Requirements to meet: Guideline G8 15

7.2 Experimental design 17

8 Results 24

8.1 Physical and chemical parameters 25

8.2 Biology 27

9 Environmental acceptability 35

10 Discussion and evaluation of results 38

11 References 40

Appendix 1: species list of phytoplankton and zooplankton 42

present during the various test runs.
Final report of the land-based testing of the BalPure®

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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 2009 in the harbour of 

the Netherlands Institute 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 technology 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 10 test runs were successful and the BWT system 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 Standard. 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 sediment 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, disintegrated completely and the 

remaining debris was found as an amorphous structure or as dissolved organic carbon. 

To 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 

plankton, 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®

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2 Zusammenfassung 

Die BALPURE® Ballastwasser Behandlungsanlage, von Severn Trent De Nora (STDN, LLC 

US) wurde im Frühjahr und Sommer 2009 gemäß Regularien-D2 (D2-Standard), sowie 

der IMO Richtlinien zu Typzulassungstests (G8) und denen für eine Zulassung von 

Systemen, welche aktive Substanzen verwenden (G9) im Hafen des Königlich 

Niederländischen Meeresforschungsinstitut (NIOZ) getestet. 

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 vorgesehen. Die BALPURE® Ballastwasser

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 Mikrometerfilter) 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 Anforderungen 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 biologische 

Invasionen zu verhindern.

Die Sedimentfracht wurde durch die Filtration reduziert und die verbleibenden 

Organismen durch die aktive Substanz (Chlorverbindung) inaktiviert. Einschließlich eines 

substanziellen Anteils der Bakterienpopulation wurden fast alle Organismen vollständig 

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 aktiven 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 Inkubationsexperimenten wurde in 20 Tagen kein Planktonwachstum im behandelten 

Wasser bei Anwesenheit von Chlor festgestellt. Gleichzeitig durchgeführte 

Umweltverträglichkeitstests (Zugabe von Zeigeorganismen und Planktonkulturen, 

Verdünnungsreihen) mit dem behandelten 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 Organismen nicht beeinflusst.
Final report of the land-based testing of the BalPure®

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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] 3.71+E6 1.78-+E6 5.65+E6 2.19+E6 0.41+E6 4.69+E6 4.03+E4 0.61+E4 7.09+E4

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 12 < 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/m3] 8.8+E5 3.9+E5 14.8+E5 3.54+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 5.32+E6 2.01+E6 1.48+E6 5.02+E6 1.79+E5 1.40+E5 2.73+E5

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

Enterococci [cfu/mL] - < 1 100 < 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/m3] 5.8+E5 1.5+E5 10.9+E5 1.25+E4 0.26+E4 1.82+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.: non detectable in sample.
Final report of the land-based testing of the BalPure®

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4 Acknowledgements

The authors thank the technical staff of the NIOZ and in particular Anna Noordeloos, 

Eveline Garritsen, Josje Snoek, Swier Oosterhuis, 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 operation 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 this 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 macroalgae, 

phytoplankton (Hamer et al., 2000; Hallegraeff et al., 1997), bacteria and viruses 

(Gollasch et al., 1998). In general 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 decrease 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 is expected to increase rapidly during the coming decades

and therefore also the change of non-indigenous organisms to have large enough 

numbers for 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 invasive species is considered as one of the 4 major 

threats of the world’s oceans next to 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 states 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 ships may reduce the risk of 

invasive species by performing 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 and/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 solution of reducing the risk of invasive species.
Final report of the land-based testing of the BalPure®

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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 biologically 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 applicable to ballast water (Rigby

et al., 2001; MEPC 49/2/13, 2003). Besides reducing the load of organisms 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 treatment 

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 treatment 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 residual 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 till 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 Oceanographic 

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 marine 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 oceans. 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 students 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 professors at the 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®

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laboratories, and the large research equipment, 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 Opportunity 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), involves 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 offers 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®

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6.3 Portrait of Severn Trent De Nora, LLC., (producer of the 

BalPure® BWT-system)

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 thirty 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 enters 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 soda 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 technologies. 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 BALPURE®

STDN has developed 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 based 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 States 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 (USCG) 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 applying IMO 

proposed standards. STDN is pleased to work with the Royal Netherlands Institute for 

Sea Research (NIOZ) to conduct certification testing and the German Federal Maritime 
Final report of the land-based testing of the BalPure®

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Agency (BSH) who will act as the administrating agency during the certification and Type 

Approval process. 

6.4 The test 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 2009. 

The NIOZ test site is equipped with 3 coated concrete tanks of 300 m3 volume 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. Samples varying in volume from 

500 mL up to 1 m3 (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 substance. 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 μmol quanta. m-2

.s-1)

The BALPURE® BWTS was connected to a typical ballast water pump which was located in 

the NIOZ 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 of 

the natural water of the NIOZ harbour to the required test conditions of brackish water 
Final report of the land-based testing of the BalPure®

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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 BALPURE® BWTS installation at NIOZ with points of sampling 
Final report of the land-based testing of the BalPure®

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6.5 Technical description of the BALPURE® BWTS

The STDN Ballast Water Treatment System (BWTS) applies established chlorination 

technology to oxidize and disinfect aquatic invasive species (AIS). Chlorination 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 similar to a 

technology already approved by the IMO and type approved by the Republic of Korea.

The BALPURE® 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 100 parts of 

ballast water. The system can operate effectively 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 BALPURE® 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 (H2O) to form sodium hypochlorite (NaOCl) 

and hydrogen (H2) as a secondary by-product. Hypochlorite solution and Hydrogen 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% hydrogen 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. To 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 compulsory

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 based 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. BallastSafe’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 successfully demonstrated in previous trials at NIOZ. 

Figure 4: outline of BALPURE® BWT 

system
Final report of the land-based testing of the BalPure®

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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 the 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 Basic Approval has recently started and a formal request to the MEPC 

has been 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 

short 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 Requirements to meet the Regulation-D2

According to D2-Standard of the IMO/MEPC Convention of 2004 (Anonymous, 2005)

ships that meet the requirements of the Convention by meeting the ballast water 

performance standard must discharge:

The present Standard-D2 is defined as a standard for the water characteristics at 

discharge. Furthermore, with exception of some indicator microbes (point 3) organisms 

< 10 μm 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, the 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, as an operational 

definition for viable organisms the IMO is using: organisms and any life stages 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 studied. 

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®

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different types of organisms. This includes also plankton < 10 μm other than bacteria 

(i.e. phytoplankton and viruses). Moreover, we extended our research effort to examine 

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 potential latent toxicity effects and 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 effective 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 serves 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 the G8 test requirements is the salinity range and related to 

this 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 concentrations 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 additional 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 reactivity of the active compound (chlorine) 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, the only difference in composition of the test water between 
Final report of the land-based testing of the BalPure®

-BWT-System 16

the freshwater and intermediate salinity water is the presence or absence of seasalt. All 

other minimum requirements for TSS, POC and DOC for these two water types were

identical (Table 1).

The BALPURE® 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 supplemental salt or stored seawater source is required for 

ballast 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

sampling 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 enhancing the

salinity (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 freshwater 

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 > 105/ m3 at least 5 species from at least 

3 different phyla/divisions

10 ≤ organism size ≤ 50 

micron

> 103

/ mL at least 5 species from at least 

3 different phyla/divisions

heterotrophic bacteria > 104/ 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 known for its natural 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 (G8), 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 zooplankton abundance. Only 

at a later stage zooplankton starts to increase in abundance, subsequently due to 
Final report of the land-based testing of the BalPure®

-BWT-System 17

predation it will diminish the numerical abundance of (smaller size) phytoplankton

component.

Furthermore, for the high salinity range, the composition 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 μm) 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 microbes 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 Archeae. While these microbes are 

part of the natural community in the aquatic 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 as a viable component as well as the 

viability of the whole microbial community has 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 institute 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 were 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). Sample handling 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 considerations were taken into 

account. Subsamples were taken randomly or throughout the whole filling procedure of 

the tanks. As indicated previously there was great emphasis on analysing the freshly 
Final report of the land-based testing of the BalPure®

-BWT-System 18

collected samples and having multiple methods to examine numerical abundance and 

viability. Besides various biological samples there was also a basic 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.

Physical and chemical properties of test water

Temperature

The water temperature was 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 direct measurement in the laboratory at NIOZ. Salinity of the water 

was 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

The spectrophotometric 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 

used 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 further analysis at the laboratory. In the laboratory 1 mL 

H2SO4 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 μM O2/L (or mg O2/L = μM O2 * 0.032). 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 measured according to Reintaler 

& Herndl (Reinthaler and Herndl, 2005). Samples for DOC (15 mL) were filtered through 

GF/C filters and sealed in pre-combusted glass ampoules after adding 50 μl of phosphoric 

acid (H3PO4). Sealed ampoules are stored at 4 °C. The DOC concentration was determined in 

the laboratory by the high temperature combustion method using a Shimadzu TOC-Vcpn

analyzer. Standards were 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-System 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 glass fibre filters (GF/C) are used. Each filter was coded and 

stored in a clean Petri dish. The filtered volume was dependent on the particle load and 

concentration and type of organisms present in the water. The higher the total particle load 

in the sample, the smaller the volume that could be filtered before the filter clogs. Practical 

volumes were between 100 and 1000 mL per sample. 

After filtration the filter was rinsed with fresh water (MiliQ) to remove sea salt. Filters were 

dried overnight at 60 °C and allowed to cool in a vacuum exicator before weighing. The total 

amount of suspended solids was 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 allowed to cool in a vacuum exicator 

and weighted again. The POC was calculated from the weight decrease between this 

measurement and the TSS weight.

Analytical determination of chlorine and related compounds

During each test cycle, the concentration of the active compound (Total Chlorine) was

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 States Environmental 

Protection Agency (USEPA) are used to analyze the treated ballast water produced by the 

BALPURE® BWT-System. 

Total Residual Oxidant (TRO) was measured as total chlorine using the Hach 2100p hand 

held Pocket Colorimeter II. The device employs 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 sample collection 

point at tank 3.
Final report of the land-based testing of the BalPure®

-BWT-System 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 were then filtered over a 10 μm sieve and fixated.

A second set of samples for this size class was taken and not separated 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 were compared to the ones from the double filtered samples to evaluate the 

loss of organisms caused by processing the samples.

Sample sizes

During the land-based tests containers from 1 to 1000 L were used for sampling and/or

storage. Samples were taken continuously and evenly during the whole process of filling 

or emptying the ballast water tanks. These containers were thoroughly rinsed or heattreated prior to use. Samples for the human pathogens were taken in sterile (barcoded) bottles provided by the bacterial test laboratory.

Figure 7: 1000 L container (IBC) with a 50 μm Hydrobios 

sampling net 

Organisms > 50 μm

The samples are pre-concentrated over a Hydrobios 50 μm net resulting in an end 

volume of approximately 100 to 200 mL. The samples were transferred to the lab 

directly after sampling and Neutral Red was 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 each 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 groups of organisms but 

inconsistent staining was found for bivalves. For this latter group movement (including 

internal such as heart and gills for juvenile mussels) has to be used obligatorily to 
Final report of the land-based testing of the BalPure®

-BWT-System 21

determine viability. This is dependent on the expertise of the 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 sample. 

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 μm in minimum 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 measure will be 

adjusted to the specific organism groups. 

Organisms < 50 μm

Samples for visual inspection of species number and diversity were pre-concentrated 

using a sieve made of a Hydrobios 10 μm mesh net using the 50 μm prefiltered sample 

(effective size range is 10 – 50 μm). The retained organisms were flushed into 50 mL

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

was 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 was 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 undisturbed samples. Besides numbers and 
Final report of the land-based testing of the BalPure®

-BWT-System 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. 

Flow cytometry: For total organisms counts 3 mL of unfiltered sample water (reference

and treated, each in triplicate) were 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 or absence of chlorophyll. For the counts exactly 1 

mL was analyzed.

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

50 μm). These beads were also used as standards to calibrate the performance of the 

flow cytometer.

For organism viability testing, on the level of the individual cell, SYTOX Green was added 

to 1 mL of sample water (control and treated, each in triplicate). After 15 minutes 

samples were analyzed using the flow cytometer for the presence of dead and/or live 

organisms (cf. Veldhuis et al, 2001, Casotti et al, 2005).

nucleus

A B

Figure 9: (A) Epifluorescence microscopic picture of a live 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 SYTOX Green. 

PAM fluorometry: The photochemical efficiency of photosystem II of phytoplankton 

(providing an estimate of the general health of the algae) can be addressed using PulseAmplitude 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-growing population of phytoplankton.

Bacteria

The classical method for counting 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 reasons (Gasol and Del-Giorgio, 2000). As a result total 

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 was taken and pipetted in a Cryovial (in triplicate) 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-System 23

Upon analysis the sample is allowed to thaw completely. A subsample of 100 μl is taken, 

diluted with a TE-buffer, and the nucleic acid dye PicoGreen (MP) was 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 known bacterial standard is 

used for calibration and counting.

The number of total heterotrophic bacteria was determined using a plate method as 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 bv” 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-System 24

8 Results

The present section is a compilation 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 detailed studies regarding potential toxicity according to standard 

procedures have also been carried out. These results were submitted to IMO/GESAMPBWWG. 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 BALPURE® BWTS (chlorination unit), discharge pump (left) and sample 

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-System 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 and chemical parameters

Table 4: Average salinity and temperature of water at intake during the low salinity tests of 

control and treated tanks for test runs I – VI.

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

I –VI 23.9 22-26 8.8 - 12.8

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: Average salinity and temperature of water at intake during the high salinity tests of 

control and treated tanks for test runs VII – XI.

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

VII - XI 33.6 30-36 13.2 - 15.7

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 the 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 BALPURE® BWT-System is not largely affected by changes in terms of initial 

dosage, 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 low salinity and VII-XI high salinity.

Test 

run

BalPure 

Treatment 

dose

TRO 

(T0)

TR 

(T1)

TRO

(T5)

Postneutralization

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 = <0.02 mg/L 
Final report of the land-based testing of the BalPure®

-BWT-System 26

Low salinity

A summary of the results of the basic parameters (oxygen concentration, pH, TSS, POC 

and DOC) is presented in Table 7 for the reference and treated water sample at intake 

and discharge.

Table 7: average or range of oxygen, pH, TSS, 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 corrected for interference of the active substances or derivates of it.

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

O2 reference1 103-119 85-104 %

O2 treated1 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 clearly demonstrates that as far as the majority of the basic parameters are

concerned the values were in accordance 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 given salinity and temperature. 

Since the water temperature increased 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 % compared 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 Winkler method 

for O2 measurements. Separate tests showed that for the applied 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 oxygen 

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 runs. 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 treated

tank, but the reduction was higher than 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-System 27

High salinity 

Table 8: average or range of oxygen, pH, TSS, POC and 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 corrected for interference of the active substances or 

derivates of it.

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

O2 reference1 76-109 53-81 %

O2 treated1 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 9.70 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 salinity range were, with a few 

exceptions, on the whole the same as for the low salinity range (Table 8). At intake the 

ambient water was, with exception of a single test run, saturated with oxygen and at 

discharge oxygen concentrations in the both control and 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 tests 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 (full 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 μm size fraction were 14 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 minimum 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 organisms in the size class > 50 μm were at 

least one order of magnitude lower than the Standard-D2 (Table 9). At seven out of 11 

test runs no living organisms were observed in the 3 replicate 1 m3 subsamples.
Final report of the land-based testing of the BalPure®

-BWT-System 28

As opposed to the treated tank, numbers of organisms in the reference tank were still 

significantly above the G8 requirement during discharge and visual inspection and 

viability measurement indicated that these organisms were both intact and viable.

Table 9: Number of organisms > 50 μm (average of 3 samples) at intake and on discharge 

at day 5 of the reference and treated tank. All numbers are presented per m3

. 

total plankton > 50 μm numbers/m3

low salinity Intake Reference Treated

test run C-T0 C-T5 STDN-T5

I 0.65E+6 2.58E+4 4.3

II 1.26E+6 0.98E+4 0.3

III 0.39E+6 2.36E+4 0

IV 1.06E+6 6.48E+4 0

V 1.48E+6 7.22E+4 0

VI 0.43E+6 1.61E+4 0

average 0.88E+6 3.54E+4 0.8

s.d. 0.43E+6 2.64E+4 1.7

total plankton > 50 μm numbers/m3

high salinity Intake Reference Treated

test run C-T0 C-T5 STDN-T5

VII 0.31E+6 1.91E+4 0

VIII 0.15E+6 0.26E+4 0

IX 0.39E+6 1.50E+4 0

X 1.09E+6 1.82E+4 0.3

XI 0.97E+6 0.77E+4 1.0

average 0.58E+6 1.25E+4 0.3

s.d. 0.42E+6 0.71E+3 0.4

Organisms 10 – 50 μm

This size class was dominated by phytoplankton, although heterotrophic organisms like 

ciliates and flagellates were occasionally present in 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 autofluorescence as an extra

selection parameter. Moreover, 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 μm of 

both reference and treated incubation 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-System 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 and 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 

at 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 the 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 the difference in salinity regime.
Final report of the land-based testing of the BalPure®

-BWT-System 30

Table 10: Total plankton number of plankton in the 10 – 50 μm 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, incubated and neutralized discharge treated water (STDNDis-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

STDNDis-Tx

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

VIII 1002 112 11734 5.18 0 <0.1 1.48 0

IX 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

XI 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 μm in diameter, a selective population of phytoplankton in 

the size range of ca. 6 μm was monitored in both the reference and treated water 

(Table 11). Although no clear criteria are, yet, defined for this size class it was clear that 

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

micron range. Flow cytometric analysis showed the presence of a varying but high 

number of cells at intake but extremely 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 

nanophytoplankton was not observed within five to seven days of incubation.
Final report of the land-based testing of the BalPure®

-BWT-System 31

Table 11: Phytoplankton number of a dominant phytoplankton species present (Phaeocystis

globosa; ca. 6 μm in cell diameter) at intake, 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 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).

Phaeocystis ~ 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-Tx

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

IV 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

average 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

VIII 4070 2047 28450 2.96 1.48 <0.1 2.96 0.74

IX 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

XI 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 photosynthetic 

efficiency (Fv/Fm) of the cells. This was done for the whole community (<50 μm), but 

when required is also possible for different size fractions. For a clear understanding it 

must be noted that values of Fv/Fm > 0.4 are indicative of a healthy phytoplankton 

population; a Fv/Fm < 0.4 indicates that the phytoplankton community is experiencing 

severe stress and a value < 0.1 is typically observed in decaying phytoplankton 

populations. 

Table 12 shows that, with one exception (test run VIII) during intake 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 dark 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 photosynthetic 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 since 
Final report of the land-based testing of the BalPure®

-BWT-System 32

phytoplankton also increased in numerical abundance in these incubated water samples 

(Tables 10 and 11). 

After passing the treatment system no distinct phytoplankton cells were observed and 

therefore Fv/Fm was typical of a decaying algal community. Prolonged storage in ballast 

tanks showed that there was no recovery of the phytoplankton population. Also 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 12: Photosynthetic efficiency (Fv/Fm) of the whole phytoplankton community (2 - > 50 

μm cell diameter) at intake, 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). Runs I to VI at low salinity range and VII to XI at high salinity 

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

Fv/Fm Intake Reference Treated

low

salinity C-T0 C-T5 C-inc-T5

STDN -

T0

STDN

-T5

STDN -

inc-T5

STDN -

Dis-Tx

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

IV 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

VIII 0.46 0.21 0.55 0.15 0.17 0.19 0.10

IX 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

XI 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

Bacteria

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 13 shows that even during intake (C-T0) the number of both target 

microorganisms was well below the standard as indicated in the Standard-D2. In five 

test runs the numbers of human pathogens were above the detection limit for one or 

both target microbes (test runs 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-System 33

During test run IX water from a sewage plant was spiked into 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 13: plate counts 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

STDNT5STDNT5STDNDis-TxSTDNDis-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

high 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 bacterial 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 5.65 106 per mL (variation 

of factor 3.1) are numbers typically observed in spring and early summer situation in 

the area (Table 14). 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 run (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 was 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 water of the main tank (C-inc-T5). 
Final report of the land-based testing of the BalPure®

-BWT-System 34

In the treated tanks and in the incubation bottles of the 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 acted initially as a disinfection agent of 

the microbial community, therefore 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 numerical 

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 (ET5) 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. * range 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-incT5STDN

-T0

STDN

-T5

STDNT5-IncSTDN

-T5

STDNDis-TxSTDNDis-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-

600
Final report of the land-based testing of the BalPure®

-BWT-System 35

9 Environmental acceptability

Ballast water treatment systems applying active substances, and also systems not using 

active substances according to the latest version of the guidelines G8 (MEPC174.58),

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 (25 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) were 

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, 

silicate; 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 26

cell 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 

25 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 neutralized treated water on the other hand showed a different response. 

Initially, numbers of intact but still chlorophyll containing phytoplankton cells declined 
Final report of the land-based testing of the BalPure®

-BWT-System 36

to a nearly undetectable number (<0.01 cell/mL). However, after a lag phase of 10 days 

there was recovery 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 prevented

hatching of resting stages or cysts for as 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 concerned the treated water was free of viable phytoplankton cells 

for a considerable period of time. The virtual absence of organisms for a prolonged 

period would suggest that the treated water would be non-vital or ‘dead-water’. 

Nevertheless, the decontamination of the treated water with bisulfite increased the 

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 26

bacteria [number/mL]

1e+3

1e+4

1e+5

1e+6

1e+7

control

STDN intake

STDN discharge

Figure 12: time course of total bacteria numbers during a maximum of 25 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.

With respect to bacteria numbers control and treated water showed a distinct different 

pattern (fig. 12). 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 the 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. From 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 until 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-System 37

Similar to the phytoplankton component of the planktonic organisms also bacteria were 

extremely disseminated and deactivated 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 resulted in a rapid increase in bacteria numbers.
Final report of the land-based testing of the BalPure®

-BWT-System 38

10 Discussion and evaluation of results

The presented 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 BALPURE® 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 

concentration 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. The 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 and 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 parameters over the whole holding 

period of 5 days. Concentrations 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 the 

treated water was not 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 germination 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 treated 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 Approval. The 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. The number of organisms in the < 10 μm 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/limitation of the treated water when 

released into environment. These experiments should be conducted next to the 

standard set of toxicology and residual chemistry tests of the treated ballast 

water.

3. Testing for the presence of human pathogens strongly depends on the natural 

abundance of these microbes in the natural environment. Since these pathogens 

cannot be supplemented for health and safety reasons, accurate testing is 
Final report of the land-based testing of the BalPure®

-BWT-System 39

therefore not possible. Moreover, viability tests of the total bacteria community 

showed that 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 ports for intake and discharge. In the case of BALPURE® 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. 

cholerea (Anonymous, 1999; Nair) and proven to be an effective method of 

disinfection.

In conclusion, the present configuration of the BALPURE® 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-System 40

11 References

Anonymous (1999) September 1999, EPA 832-F-99-062. 

Anonymous (2001) July 11-13, 2001 Cape May FlowCAM Report. Fluid Imaging 

Technologies, Inc P.O.Box350, East Boothbay, Maine, 054544, pp 12.

Anonymous (2005) Guidelines for approval of ballast water management systems (G8). 

Annex3 Resolution MEPC.125(53), Annex, Parts 1,2,3 and 4.

Anonymous (2009) Test protocols and Quality Assurance Project Plan (QAPP) for the 

efficacy testing of the BalPure® Ballast Water Treatment System (STDN, LLC) as 

part of the type approval process under Resolution MEPC 125.53 and MEPC 

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Final report of the land-based testing of the BalPure®

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Appendix 1

Phytoplankton Marsdiep AprilJuly 2009 +: present but rare, ++: present; +++: dominant;

data by Jolanda van Iperen ++++: very abundant; +++++: 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 Phaeocystis globosa flagellate cell +++++

diatom pennate Pseudonitzschia delicatissima group ++++

autotrophic flagellate Prymnesiales indet. <10 μm ++++

diatom centricate Thalassiosira spp. 10 μm ++++

diatom centricate Chaetoceros socialis ++++

autotrophic 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 centricate 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 +

heterotrophic 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 ++

heterotrophic flagellate Choanoflagellatea indet. ++

hetero/auto flagellate Cryptophyceae "light" group ++

heterotrophic dinoflagellate Oxyrrhis marina ++

heterotrophic dinoflagellate Katodinium glaucum ++

heterotrophic flagellate Ciliophrys group ++

heterotrophic flagellate Chrysophyceae indet. +

heterotrophic flagellate Telonema spp. +
Final report of the land-based testing of the BalPure®

-BWT-System 43

Species list zooplankton and some larger (atypical) plankton

Appendix 1 continued

Phylum Class Subclass, 

Order, etc.

species 

no.

Identified 

genera

most likely 

present

Sarcomastigophora Dinoflagellida 2

Noctiluca, 

Protoperidinium

Bacillariophyceae 3+

Bidulphia, 

Coscinodiscus

Cnidaria Hydrozoa 2+ Obelia

Scyphozoa 2 Aurelia, Cyanea

Ctenophora 2+ Pleurobrachia, 

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