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

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Texel February 2009

FINAL REPORT OF THE LANDBASED TESTING OF THE

ECOCHLOR®-SYSTEM, FOR TYPE 

APPROVAL ACCORDING TO

REGULATION-D2 AND THE 

RELEVANT IMO GUIDELINE 

(APRIL – JULY 2008)

Marcel J.W. Veldhuis Ph-D

Frank Fuhr Dipl.-Biol.

Peter Paul Stehouwer MSc.

Royal Netherlands Institute for Sea Research

P.O.Box 59

1790 AB, Den Burg, TEXEL

the Netherlands

phone: +31-222-369512/300

email: veldhuis@nioz.nl, ffuhr@nioz.nl, pstehouwer@nioz.nl
Final report of the land-based testing of the ECOCHLOR®

-System 2

FINAL REPORT OF THE LANDBASED TESTING OF THE

ECOCHLOR®-SYSTEM, FOR TYPE 

APPROVAL ACCORDING TO

REGULATION-D2 AND THE 

RELEVANT IMO GUIDELINE 

(APRIL – JULY 2008)

Royal Netherlands Institute for Sea Research

P.O.Box 59

1790 AB, Den Burg, TEXEL

the Netherlands

phone: +31-222-369512/300

email: veldhuis@nioz.nl, ffuhr@nioz.nl, pstehouwer@nioz.nl
Final report of the land-based testing of the ECOCHLOR®

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Content page

1 Executive summary 4

2 Zusammenfassung 5

3 Summary table with results for the Type Approval Certificate of the 

ECOCHLOR® 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 Portrait of Ecochlor (producer of the Ecochlor®

-system) 10

6.3 The test facility 10

6.4 Technical description of the Ecochlor®

-system 12

7 Requirements to meet the D2 Standard 14

7.1 Requirements to meet: guideline G8 14

7.2 Experimental design 17

8 Results 23

8.1 Physical and chemical parameters 24

8.2 Biology 26

9 Environmental acceptability 34

10 Discussion and evaluation of results 37

11 References 39

Appendix 1: species list of phytoplankton and zooplankton 41

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

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1 Executive summary

The ECOCHLOR®-System, a ballast water treatment system, 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 2008 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 original ECOCHLOR®

-

System was designed without a mechanical filtration step but the system performed much 

better with the addition of a coarse filter (BSFc, 40 micron filter). In total 11 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 Standard-D2. This system 

should therefore be regarded as an effective way of cleaning ballast water in 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 organisms were killed by the 

active substance chlorine-dioxide. Except for bacteria nearly all organisms disintegrated 

completely and the remaining debris was found as an amorphous structure or as dissolved 

organic carbon. 

No regrowth was observed for a period exceeding at least 15 days. Environmental 

acceptability tests however showed that the growth of organisms, mainly plankton, was not 

limited by the discharge water indicating that the discharged water was still vital.

Upon discharge no residual effects of the chemical treatment to the receiving environment 

were observed. The active substance decomposed within the treated ballast tank. 
Final report of the land-based testing of the ECOCHLOR®

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2 Zusammenfassung 

Das ECOCHLOR®-System zur Behandlung von Ballastwasser wurde im Frühjahr und Sommer 

2008 gemäß Regularien-D2 (D2-Standard), sowie der IMO Richtlinien zu Tests für eine 

Typzulassung (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 

sogar schwieriger, als in den Richtlinien verlangt. Das ECOCHLOR®

-System war ursprünglich 

ohne mechanische Filtration entworfen, aber das System arbeitete deutlich besser nach dem 

Zufügen eines Feststofffilters (BSFc, 40 Mikrometerfilter). Es wurden 11 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 den Vorbehandlungsschritt reduziert (selbstreinigender 

Filter) und die Organismen durch die aktive Substanz Chlordioxid inaktiviert. Mit der 

Ausnahme von Bakterien wurden fast alle Organismen vollständig zersetzt. Die Überreste 

der Organismen fanden sich lediglich in Form amorpher Strukturen und als gelöster 

organischer Kohlenstoff.

In Inkubationsexperimenten über Zeiträume von mehr als 15 Tagen unter vorteilhaften 

Umweltbedingungen konnte kein Planktonwachstum im behandelten Wasser festgestellt 

werden. Gleichzeitig durchgeführte Umweltverträglichkeitstests (Zugabe von 

Zeigeorganismen und Planktonkulturen, Verdünnungsreihen) mit dem behandelten Wasser 

ergaben jedoch keine negativen Auswirkungen auf das Planktonwachstum und zeigten 

somit, dass das behandelte Wasser selbst das Wachstum gesunder Organismen nicht 

beeinflusst.

Bei der Abgabe des behandelten Wassers wurden keine negativen Effekte auf den 

empfangenden Wasserkörper festgestellt. Die aktive Substanz wurde in den Tanks gemäß 

der Erwartungen aus den Vorversuchen abgebaut.
Final report of the land-based testing of the ECOCHLOR®

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3 Summary table with results for the Type Approval Certificate of the ECOCHLOR® BWT-System

Land-based tests NIOZ Reference & Treated Reference

 

Treated

salinity 31.9 PSU Intake Discharge Discharge

natural plankton Average min. max. Average min. max. Average min. max.

total bacteria [counts/mL] 3.79+E6 1.25-+E6 5.24+E6 2.15+E6 0.94+E6 5.37+E6 4.90+E6 0.48+E6 8.24+E6

E. coli [cfu/mL] < 0,1 < 0,1 < 0,1 < 0,1 < 0,1 < 0,1 < 0,1 < 0,1 < 0,1

Enterococci [cfu/mL] 8.3 < 1 17 < 1 < 0,1 1 < 0,1 < 0,1 < 0,1

plankton <10 μm [counts/mL] 2264 1004 4556 290 173 632 1.0 n.d 3

plankton 10-50 μm [counts/mL] 1628 730 3207 140 121 151 0.7 n.d. 3.7

plankton >50 μm [counts/m3] 1.78+E5 0.52+E5 4.11+E5 0.15+E4 0.25+E4 2.76+E4 0.9 n.d. 3.7

Land-based tests NIOZ Reference & Treated Reference Treated

salinity 23,1 PSU Intake Discharge Discharge

natural plankton Average min. max. Average min. max. Average min. max.

total bacteria [counts/mL] 6.47+E6 3.66+E6 9.02+E6 1.89+E6 0.86+E6 2.90+E6 4.74+E6 2.54+E6 7.86+E6

E. coli [cfu/mL] 0.5 < 0,1 1.4 < 0,1 < 0,1 < 0,1 < 0,1 < 0,1 < 0,1

Enterococci [cfu/mL] 8 < 1 27 < 0,1 < 0,1 1 < 1 < 1 < 1

plankton <10 μm [counts/mL] 8945 3278 15096 712 548 1195 1.6 n.d. 6.7

plankton 10-50 μm [counts/mL] 1326 719 1746 157 130 175 0.1 n.d. 0.7

plankton >50 μm [counts/m3] 1.76+E5 0.36+E5 1.32+E5 0.65+E4 0.27+E4 0.84+E4 n.d. n.d. n.d.

Summary table of collected data covering the major groups of organisms at two series of 6 and 5 test-runs for low and high salinity range, respectively.

n.d.: non detectable in sample.
Final report of the land-based testing of the ECOCHLOR®

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4 Acknowledgements

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

Garritsen, Swier Oosterhuis, Siem Groot, Robert Lakeman, Santiago Gonzalez and Pieter van 

Kralingen. Their cooperation was essential for the successful completion of this certification 

project.

Special thanks also to Yolanda van Iperen for providing the species list of plankton in the 

Wadden Sea.

We also thank Mr. Rolf von Ostrowski, Dr. Kai Trümpler and Mrs. Karin Sigler 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 (Gollash 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 ship 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 ECOCHLOR®

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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 

as to study 

1) the numerical abundance and biodiversity of organisms prior, during and after a 

treatment with the ECOCHLOR Ballast Water Treatment 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-dioxide-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 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 is also appointed as professor at 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, 

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.
Final report of the land-based testing of the ECOCHLOR®

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More information on www.nioz.nl

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

TESO ferry (top).
Final report of the land-based testing of the ECOCHLOR®

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6.2 Portrait of ECOCHLOR (producer of the ECOCHLOR-system)

More information on: 

Ecochlor, Inc., 

www.ecochlor.com

Ballast water treatment - The ECOCHLOR-System

The ECOCHLOR®-System operates in-line during the uptake of the ballast water. It is based 

on a two-step treatment process which includes physical separation (self-cleaning filter) as 

well as secondary treatment with chlorine-dioxide as the active substance. 

The Ecochlor® BWTS is viable for all ballast water capacity ranges, and is distinguished by the 

system’s capability to treat at high ballast water flow rates. The Ecochlor® BWTS can be 

installed as a retrofit on existing vessels or during a new construction, with the latter being 

the most straightforward and cost effective. Additionally, Ecochlor’s treatment system can 

be easily incorporated into a land-based system to treat ballast water land-side.

www.ecochlor.com, founded in 2001, has developed proprietary ballast 

water treatment systems that have been specifically designed to safely and economically 

eliminate the worldwide transfer of aquatic invasive species. Ecochlor’s technology is 

patented, proven, well understood and uniquely competitive. 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. Ecochlor is based in the United States with capabilities to install, service, and 

support its systems throughout the world.

In research underwritten by Ecochlor at the University of Rhode Island, Graduate School of 

Oceanography (URI), the use of chlorine dioxide was proven effective in seawater on 

representative groups of all target organisms. Additional extensive shipboard testing has 

been conducted by URI under a grant from the United States National Oceanographic and 

Atmospheric Administration (NOAA).

Ecochlor has installed two commercial systems to date. In July 2004, the company installed 

its first system on Atlantic Container Lines Atlantic Compass, one of the world’s largest 

combined Container/RORO ships. This ship is a Swedish flagged vessel that carries cargo 

between Europe and North America. In July 2005, the company installed its second system 

on the bulker M/V Moku Pahu, operated by Matson Navigation that carries sugar from 

Hawaii to the West Coast, USA and grain to Asia. 

International approval is mandatory, and Ecochlor has undertaken a two-pronged global 

approach to certification. In the United States, the two Ecochlor systems installed onboard 

have been accepted into the U.S. Coast Guard (USCG) Shipboard Technology Evaluation 

Program (STEP). European countries are ahead of the U.S. in the approvals process for 

ballast water treatment systems. Ecochlor was granted IMO Basic Approval in July 2008, 

and an application for IMO Final Approval has been submitted. Ecochlor is pleased to work 

with the Royal Netherlands Institute for Sea Research (NIOZ) to conduct certification testing 

and the German Federal Maritime Agency (BSH) who is acting as the administrating agency 

during the certification and Type Approval process. 

6.3 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 2008. 
Final report of the land-based testing of the ECOCHLOR®

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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. 1 m 

from the bottom.

Figure 2: Inside view of one of 

three subterranean water tanks

The Ecochlor-BWT-System 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 and 

marine water with a minimum of 10 PSU difference. A detailed description of the test 

installation is presented in figure 3.

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 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)
Final report of the land-based testing of the ECOCHLOR®

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Figure 3: P&I diagram of the Ecochlor installation at NIOZ with points of sampling 

6.4 Technical description of the ECOCHLOR-System

The Ecochlor® Ballast Water Treatment System (BWTS) applies established chlorine dioxide 

technology to oxidize and disinfect aquatic invasive species (AIS). Chlorine dioxide has a 

long history (50+ years) of being applied safely and economically for the control of 

microorganisms in industrial and municipal waters. What is unique about chlorine dioxide is 

its ability to be effective against a wide range of troublesome organisms. A second unique 

feature of chlorine dioxide as compared to other oxidants (such as chlorine) is the 

minimization of unwanted by-products. For example, chlorine reacts by both an oxidative 

and a substitution pathway. Because of this, unwanted chlorinated by-products such as 

trihalomethanes (THMs) typically accompany chlorine applications. In contrast, chlorine 

dioxide reacts only by an oxidative pathway thereby eliminating the potential to form 

unwanted and regulated chlorinated organic by-products even in the most heavily 

contaminated waters. 

The Ecochlor® BWTS generates chlorine dioxide using the Eka Chemicals Purate® technology, 

a patent-protected chlorine dioxide generation method. Eka’s Purate® technology is a 

chlorate-based chlorine dioxide generation process. This method differs from other 

conventional chlorine dioxide generation methods that involve use of aqueous or gaseous 

chlorine, making the Purate® method environmentally superior. Additionally, Eka’s Purate®

technology produces chlorine dioxide efficiently, thereby controlling the required input of 

precursor chemicals and eliminating production of unwanted by-products. 

The Ecochlor® BWTS generates and injects a dilute solution of chlorine dioxide during the 

uptake of ballast water. Chlorine dioxide is not stored as a concentrated chemical at any 

Location of 

Ecochlor System
Final report of the land-based testing of the ECOCHLOR®

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time. It is generated automatically on demand based on the amount of ballast water being 

pumped. After injection into the incoming ballast water, chlorine dioxide remains effective 

in the ballast water tanks for a short period (2 – 20 hours) in order to neutralize ballast tank 

biofilm that can cause bacterial re-growth. Over time, the chlorine dioxide continues to 

react and/or decay in the ballast tanks so that at the time of discharge the ballast water 

contains an undetectable concentration of chlorine dioxide.

Prior to treatment, the water is filtered through a 40 micron BallastSafeTM BSFc Automatic 

Electric Filter, Model BSFc-H-1.6. 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 sediment 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.

During all test cycles, with the exception of high salinity test run 6, chlorine dioxide 

treatment was applied to incoming ballast water at 5 mg/L. During test cycle 6, chlorine 

dioxide was applied at a dose of 4 mg/L. This was the first test run after the addition of the 

filtration phase and Ecochlor wanted to determine the effectiveness of 4 mg/L treatment. 

Although the IMO D-2 standard was met using 4 mg/L, this treatment was not enough to 

demonstrate compliance with more stringent standards that are being proposed in the 

United States. Therefore, the remaining test cycles were performed using a 5 mg/L dose of 

chlorine dioxide. 

The Ecochlor® Ballast Water Treatment System at NIOZ for land based testing is Ecochlor's 

smallest commercial system and has not been downsized. The Ecochlor® BWTS is a selfmonitoring system and has been reviewed for compliance with the class requirements of 

the Lloyd’s Register North America and ABS Americas Division classification societies. All 

materials and specifications were selected in order to meet the class requirements of these 

classification societies for ballast water systems and electric installations. 

Subsequently, the prototype Ecochlor® BWTS that is installed on two test ships (M/V Atlantic 

Compass and M/V Moku Pahu) have received full plan approval. The Atlantic Compass

system was approved by Lloyd’s Register North America (Atlantic Compass LR No. 8214176, 

letter dated 26 April 2004, Ref. NAO/0402535/001/GP) and the Moku Pahu system was 

approved by ABS Americas Division (M/V Moku Pahu ABSID 8208482, letters dated 8 July 

2005 REF 503135 and 15 August 2005 REF 510359). 

The applied test protocols were communicated with the German Administration (Federal 

Maritime and Hydrographic Agency of Germany; BSH) 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 by the classification society 

(Lloyds Register) national and international agencies. 

NIOZ is currently acting as an official certification organization for land-based testing for the 

German and UK administration according to approved protocols and final reports will be 

made public at time of awarding the certificate.
Final report of the land-based testing of the ECOCHLOR®

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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).

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 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 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.

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) was compiled of which 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.

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 milliliters or less than 1 cfu per 1 

gramme (wet weight) of zooplankton samples:

2. Escherichia coli less than 250 cfu 100 milliliters;

3. intestinal Enterococci less than 100 cfu per 100 milliliters.
Final report of the land-based testing of the ECOCHLOR®

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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 ECOCHLOR®-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-dioxide) 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 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).

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. 
Final report of the land-based testing of the ECOCHLOR®

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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, 

eggs, 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 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), i.e. the 
Final report of the land-based testing of the ECOCHLOR®

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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 ECOCHLOR®-

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

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Spectrophotometer. The oxygen concentration was calculated using standards and 

expressed as μM O2/L (or mg O2/L; = μM O2 * 0.032)

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-5000 

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 %.

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 was 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-dioxide

During each test cycle, the concentration of the active compound (chlorine dioxide, ClO2) 

was measured as soon as possible after filling of the treated ballast water tank and also at 

regular intervals until the detection limit of the applied method was reached.

Chemistries that have been tested and verified by the United States Environmental 

Protection Agency (USEPA) are used to analyze the chlorine dioxide treated ballast water 

produced by the Ecochlor® BWTS. Chlorine dioxide residuals are measured using the 

lissamine green B (LGB) chemistry (USEPA Method 327).

Figure 4: Example of sample collection 

point at tank 3.

Chlorine dioxide measurements are performed using the Palin-test 1000 Chlordiox-Duo 

Photometer with prepackaged reagent kits to measure chlorine dioxide in the 0.1 to 20 

mg/L concentration range. The LGB detection chemistry is highly selective for chlorine 

dioxide in the presence of chlorite ion. The prepared reagents establish the proper pH to 
Final report of the land-based testing of the ECOCHLOR®

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minimize any potential reaction with chlorite ion. The reagent kit also includes a chemical 

mask to react with chlorine if it is present in the sample. Under the measurement 

conditions, studies indicate that the seawater matrix does not create interference issues 

that might lead to inaccurate measurements. The recommended operating range is 0 – 40 

°C, and the test reagents are calibrated for use at 15 – 25 °C. 

The sensitivity of the Palin-test LGB analytical method provides field verification of the 

expected decay of chlorine dioxide in treated ballast water at levels below environmentally 

relevant concentrations. 

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.

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 heat-treated 

prior to use. Samples for the human pathogens were taken in sterile (bar-coded) bottles 

provided by the bacterial test laboratory.

Figure 5: 1000 L container 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 
Final report of the land-based testing of the ECOCHLOR®

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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 determine viability. This is 

dependent on the expertise of the person analyzing the samples. Therefore the same 

person analyzed all samples.

Figure 6: (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. 
Final report of the land-based testing of the ECOCHLOR®

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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 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).

nucleus

A B

Figure 7: (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.

Next to cell specific analysis Plant-pigments and chlorophyll a were determined to assess 

the fate of the whole phytoplankton community (Jeffrey et al., 1997; Kraay et al., 1992). For 
Final report of the land-based testing of the ECOCHLOR®

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this purpose water samples of 0.2 to 1.5 L (GF/C filters) were taken. The samples were 

frozen until further analysis. 

The system used is a Dionex HPLC system equipped with a C18 separation column. The 

different algal pigments can be separated according to their polarity. The following solvents 

are used as elutes in the HPLC gradient: A 0.5M ammoniumacetate in methanol and water 

(85:15), B acetonitril and water (90:10) and C ethylacetate 100%.

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 oC until further 

analysis. 

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).

Human pathogens

The samples for microbiological analysis are taken in special bottles of 600 mL and send to a 

special laboratory for further analysis. This laboratory was “Vitens laboratory bv” at 

Leeuwarden (accreditation certificate: NEN/ISO/IEC 17025; lab. no. L043). All analysis’ are 

carried out according to NEN-EN-ISO standards.

These samples are sent to the laboratory immediately after sampling using a cooled 

transport container (4 oC). 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 ECOCHLOR®

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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. 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/GESAMP-BWWG. Data are 

presented as averages or ranges separated for the two salinity regimes tested. In Annex 1 a 

detailed species list of organisms observed is presented.

Figure 8: overview of the ECOCHLOR®-System (filter and container) in the NIOZ harbour (top) 

and detail of the internal disinfection unit (bottom). 
Final report of the land-based testing of the ECOCHLOR®

-System 24

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 first low salinity tests 

of control and treated tanks for test runs I – III.

test run salinity [PSU] s.d. temperature [°C]

I –III 22.1 0.92 8.5 - 10.4

To the low salinity test cycles brackish water from the NIOZ harbour was collected during 

low tide and fresh water, taken from Lake Ijssel 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 VI – X.

test run salinity [PSU] s.d. temperature [°C]

VI - X 31.9 1.3 14.3 - 17.5

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).

During the first three test runs it became clear that with respect of the organism > 50 μm 

the ECOCHLOR®-System was not in compliance with the Standard-D2. The test runs at low 

salinity were repeated after the high salinity test series (Table 6). All data therefore exclude 

the results of the first test-runs (table 4) unless indicated otherwise.

Table 6: Average salinity and temperature of water at intake during the second low salinity 

tests of control and treated tanks for test runs XI – XVI.

test run salinity [PSU] s.d. temperature [°C]

XI - XVI 23.1 0.65 16.7 - 18.8

In regards to salinity, the difference between both the high and low salinity regimes is 

slightly less than 10 PSU. 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 EcochlorBWT-system is not affected by changes in the salinity.
Final report of the land-based testing of the ECOCHLOR®

-System 25

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: Oxygen, pH, TSS, POC and DOC concentrations of test series run at low salinity (6 test 

runs in total; XI-XVI) at intake and discharge; 1

: range of values; the oxygen concentrations are corrected for the interference of the active

substances or derivates of it.

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

O2 reference1 5.7-8.5 3.5-5.9 mg/L

O2 treated1 10.6-13.3 9.9-12.0 mg/L

pH reference 7.95 0.09 n.d

pH-treated 6.91 - 6.92 -

TSS-reference 75.3 23.6 14.0 9.8 mg/L

TSS-treated 19.3 9.50 13.0 2.1 mg/L

POC -reference 11.6 1.50 7.60 3.0 mg/L

POC-treated 9.0 3.90 6.60 1.4 mg/L

DOC -reference 3.2 0.47 2.8 0.37 mg-C/L

DOC-treated 3.4 0.36 3.5 0.43 mg-C/L

Table 7 clearly demonstrates that as far as the basic parameters are concerned the values 

were in accordance with the criteria as indicated in the guidelines (G8, Table 1). 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 with 

exception of 1 test run (69% saturation level). 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 ca. 33% 

compared to intake values. In contrast, oxygen concentration in the treated tank tended to 

be higher, by as much as 60% as compared to the reference tank. This apparent supersaturation is partly an artefact caused by the active substance chlorine-dioxide interfering 

with the Winkler method for O2 measurements. Separate tests showed that for the applied 

concentrations of chlorine-dioxide 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 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. Compared to the reference 

tank, the applied filter of the Ecochlor system effectively reduced the amount of TSS at 

intake and partly also the POC. Most of this material remained in suspension. At discharge 

on day 5, both TSS and POC were reduced in the treated tank, but the reduction was far less 

than was measured in the reference tank. 
Final report of the land-based testing of the ECOCHLOR®

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High salinity 

Table 8: Oxygen, pH, TSS, POC and DOC concentrations of test series run at high salinity 

(5 test runs in total; VI-X) at intake and discharge; 1

: range of values; the oxygen concentrations are corrected for the interference of the 

active substances or derivates of it.

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

O2 reference1 4.7-9.2 2.6-6.1 mg/L

O2 treated1 8.8-12.0 8.0-10.9 mg/L

pH reference 8.07 0.05 n.d.

pH-treated 6.81 - 6.89

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 10.2 5.30 4.80 1.30 mg/L

POC-treated 7.8 2.10 4.80 0.50 mg/L

DOC -reference 4.0 1.02 3.3 0.51 mg-C/L

DOC-treated 4.3 0.76 4.3 0.93 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 slightly. 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. 10 mg/L and 4.8 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 8). 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 (both test series runs 1 - 3 and 11 - 16) were, with exception of run 

11/12, well above the minimum requirements. In some cases the numbers in the >50 μm 

size fraction were 48 times higher (test run II) than the minimum required number of 

100,000 m-3

. At the high salinity range the water at intake contained in three cases 

insufficient numbers (test runs 8/9 and 10) although on average the numbers did meet the 

minimum requirement. As explained earlier this is mainly due to the fact that natural 

seawater was used without any addition of organisms and that on average numbers of 

larger sized organisms is known to be much lower in high saline water than brackish water.

During the first three test runs measurements indicated that the numbers of the organisms 

at discharge exceeded the maximum allowed number of 10 organisms m-3

. Close 

observation showed that in particular the hard shelled plankton (cypris larvae of barnacles) 

was only partly affected by the chemical treatment and a significant number were still alive 

at discharge. Barnacles are found world-wide in costal habitats. They form a major part of 
Final report of the land-based testing of the ECOCHLOR®

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the biomass on many hard-substrates in coastal areas. Most barnacles undergo the stage of 

cypris larvae during their life cycle. In this life stage the larvae posses a hard shell which they 

can close entirely. They do not take up food during this stage and for some species have 

been shown to survive up to 20 days as cypris. A number of barnacle species has already 

been identified as invading new habitats using ships as vectors. This group therefore is 

important in the scope of ballast water treatment and any system must be able to 

successfully deal with them.

This resulted in a modification of the treatment system, which added a self-cleaning filter 

(BSFc, 40 μm filter) phase prior to treatment with chlorine dioxide. 

As a result the whole set of test runs at the lower salinity range were repeated (test runs 11 

– 16). The installation of the filter 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. 

Moreover, at the low salinity range no living organisms were observed in the 6 replicate 

tests.

As mentioned above in Section 6.4, a 4 mg/L treatment of chlorine dioxide was applied 

during test run 6. This was done to determine if the addition of the filtration phase would 

allow the chlorine dioxide dosage to be reduced. The results for test run 6 clearly meet the 

IMO D-2 standard. However, the remaining treatment cycles were performed with 5 mg/L 

chlorine dioxide treatment to demonstrate that the more stringent discharge standards 

being proposed in the United States can be achieved with the Ecochlor System. 

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 8: Number of organisms > 50 μm at intake and on discharge at day 5 of the 

reference and treated tank. For test run XI, XIII and XV the same control was used, since 

this was a combined test run. All numbers are presented per m3

. nd: no data 

total plankton > 50 μm numbers/m3

low salinity Intake Reference Treated

test run C-T0 C-T5 E-T5

I 1.24E+06 1.08E+04 3.0

II 4.83E+06 6.17E+02 81.0

III 1.01E+06 2.18E+03 25.3

IV 3.12E+06 2.18E+03 nd

V nd

average 2.55E+06 3.95E+03 36.4

s.d. 1.79E+06 4.65E+03 40.2
Final report of the land-based testing of the ECOCHLOR®

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Table 8: continued

total plankton > 50 μm numbers/m3

high salinity Intake Reference Treated

test run C-T0 C-T5 E-T5

VI 4.11E+05 1.31E+04 3.7

VII 2.75E+05 1.67E+04 0.0

VIII 8.57E+04 2.45E+03 0.3

IX 5.15E+04 1.73E+04 0.3

X 6.85E+04 2.76E+04 0.0

average 1.78E+05 1.54E+04 0.9

s.d. 1.58E+05 9.05E+03 1.6

total plankton > 50 μm numbers/m3

low salinity Intake Reference Treated

test run C-T0 C-T5 E-T5

XI 3.57E+04 8.38E+03 0.0

XII 0.0

XIII 3.61E+05 8.57E+03 0.0

XIV 0.0

XV 1.32E+05 2.70E+03 0.0

XVI 0.0

average 1.76E+05 6.55E+03 0.0

s.d. 1.67E+05 3.34E+03 0.0

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. For that reason 

the flow cytometric analysis included chlorophyll a fluorescence 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 9).

Table 9 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). 

In contrast in the treated tank the number of intact cells immediately after intake (E-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 
Final report of the land-based testing of the ECOCHLOR®

-System 29

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 (E-inc-T5).

As expected and corroborating with the results for the larger organisms no effect was 

observed of the difference in salinity regime.

Table 9: Total plankton number of plankton in the 10 – 50 μm size range at intake, reference

(C) and treated tank (E), reference and treated incubated samples (Inc.) at day 5 (T5) during 

discharge. Runs VI to X at high salinity range and XI to XVI at low salinity range. Test runs XI,

XIII and XV share the same reference since this was a combined test run. All numbers are 

presented per mL as total counts or of viable cells (*).

10- 50 μm cell numbers cells/ml Ecochlor

high salinity Intake Reference Treated total viable

test run C-T0 C-T5 C-inc-T5 E-T0 E-T5 E-T5* E-inc-T5

VI 1470 121 9391 4.4 3.7 <0.1 1.5

VII 1345 149 6007 0.0 0.0 <0.1 0.0

VIII 3207 149 7774 0.0 0.0 <0.1 0.0

IX 730 130 5754 8.1 0.0 <0.1 0.0

X 1385 151 5941 0.7 0.0 <0.1 0.7

average 1628 140 6973 2.7 0.7 <0.1 0.4

Low salinity

XI 1746 175 3766 5.9 0.0 <0.1 0.0

XII 0.0 0.0 <0.1 0.0

XIII 1512 130 7776 0.7 0.7 <0.1 0.0

XIV 2.2 0.0 <0.1 0.0

XV 719 165 38544 2.2 0.0 <0.1 0.0

XVI 0.0 0.0 <0.1 0.0

average 1326 157 16695 1.9 0.1 <0.1 0.0

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 group of phytoplankton in the size range of ca. 6 

μm was monitored in the control and treated water (Table 10). Although no clear criteria 

are, yet, defined for this size class it was clear that the Ecochlor®

-System was also effective 

in reducing organisms in this size range. Flow cytometric analysis showed the presence of a 

extreme low number of intact cells in the treated tanks immediately after intake. After 5 

days of holding the numbers declined even further and in terms of viable cells the number 

was < 0.1 per mL at discharge. Incubation of chlorine dioxide treated water under ideal 

growth conditions did not alter the general observation since no regrowth of phytoplankton 

was observed (E-inc-T5).
Final report of the land-based testing of the ECOCHLOR®

-System 30

Table 10: Phytoplankton number of a dominant phytoplankton species present (Phaeocystis

globosa; ca. 6 μm in size) at intake, reference and treated tank, reference and treated 

incubated samples (Inc.) at day 5 during discharge. Runs VI to X were test runs at high salinity 

range and XI to XVI at low salinity range. Test runs XI, XIII and XV share the same reference

since this was a combined test run. All numbers are presented per mL as phytoplankton counts 

or of viable cells (*).

Phaeocystis ~ 6 μm cells/ml Ecochlor

high salinity Intake Reference Treated total viable

test run C-T0 C-T5 C-inc-T5 E-T0 E-T5 E-T5* E-inc-T5

VI 4556 632 9458 0 3.0 <0.1 0.7

VII 1795 204 4364 0 0 <0.1 0

VIII 2106 204 6650 2.2 2.2 <0.1 2.2

IX 1004 236 21473 0.7 0 <0.1 0

X 1857 173 14440 4.4 0 <0.1 1.5

average 2264 290 11277 1.5 1.0 <0.1 0.9

low salinity

XI 8460 393 8574 76.2 0.0 <0.1 0.0

XII 4.4 0.0 <0.1 0.7

XIII 15096 1195 27619 5.2 1.5 <0.1 0.0

XIV 9.6 0.0 <0.1 1.5

XV 3278 548 36220 3.0 6.7 <0.1 0.7

XVI 46.6 4.4 <0.1 0.7

average 8945 712 24138 24.2 1.6 <0.1 0.6

Photosynthetic efficiency

Another approach to gain insight in the physiological condition and therefore the growth 

response of the phytoplankton community is by measuring the photosynthetic efficiency 

(Fv/Fm) of the cells. This was done for the whole community, but is also possible for each of 

the 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; an 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 11 shows that, with one exception (test run 7) during intake the whole phytoplankton 

community in the water was in a physiologically healthy condition, i.e. containing mostly 

photosynthetic active and therefore 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 phytoplankton also 

increased in numerical abundance in these subsamples (Tables 9 and 10). 

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, but also in the incubated sample under optimal growth conditions, showed that there 

was no recovery of the phytoplankton population.
Final report of the land-based testing of the ECOCHLOR®

-System 31

Table 11: Photosynthetic efficiency (Fv/Fm) of the whole phytoplankton community (2 - > 50 

μm size range) at intake, in the reference and treated tanks at discharge and in the incubated 

samples taken from the reference and treated tank after days of incubation. Runs VI to X were 

test runs at high salinity range and XI to XVI at low salinity range. Test runs XI, XIII and XV share 

the same reference since this was a combined test run.

high salinity Intake Reference Treated

Fv/Fm C-T0 C-T5 C-inc-T5 E-T0 E-T5 E-inc-T5

VI 0.59 0.13 0.63 0.00 0.00 0.00

VII 0.44 0.11 0.65 0.00 0.00 0.00

VIII 0.67 0.16 0.60 0.00 0.00 0.00

IX 0.62 0.28 0.72 0.00 0.00 0.00

X 0.66 0.13 0.64 0.00 0.00 0.00

average 0.60 0.16 0.65 0.00 0.00 0.00

s.d. 0.09 0.07 0.04 0.00 0.00 0.00

low salinity Reference Treated

XI 0.56 0.10 0.73 0.13 0.00 0.00

XII 0.00 0.00 0.00

XIII 0.65 0.42 0.72 0.00 0.00 0.00

XIV 0.00 0.00 0.00

XV 0.51 0.24 0.63 0.02 0.00 0.00

XVI 0.00 0.00 0.00

average 0.57 0.25 0.69 0.03 0.00 0.00

s.d. 0.07 0.16 0.05 0.05 0.00 0.00

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, while the response of the 

whole microbial community was also assessed.

Table 12 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 7, 8, 9, 11, and 15). 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 ECOCHLOR®

-System 32

Table 12: Counts of the human pathogens E. coli and total enterococci (as cfu’s) at intake and 

during discharge of the reference and treated tank. nd; no data. Test runs XI, XIII and XV share 

the same reference since this was a combined test run.

human pathogens Intake Reference Treated

counts/mL C-T0 C-T0 C-T5 C-T5 E-T5 E-T5

high 

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

VI <0.1 <1 <0.1 <1 <0.1 <1

VII nd 1 <1 <1

VIII <0.1 17 <0.1 <1 <0.1 <1

IX <0.1 7 <0.1 <1 <0.1 <1

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

average <0.1 8.3 <0.1 <1 <0.1 <1

low salinity

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

XII <0.1 <1

XIII <0.1 <1 <0.1 <1 <0.1 <1

XIV <0.1 <1

XV 1.4 27 <0.1 1 <0.1 <1

XVI <0.1 <1

average 0.5 <1 - 27 <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 1.25 to 9.02 106 per mL (variation of factor 

7.2) are numbers typically observed in spring and early summer (Table 13). Of this total 

bacterial population only a very small fraction could be identified as heterotrophic ones

using the plate assay method (<10 to 50 per mL). 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 in the control tank but there was considerable variation between the different test 

runs (C-T5). 

Bacteria numbers in the incubated water of the reference tanks did not follow the pattern 

observed for the treated tank samples. In the high salinity test series there was on average a 

slight increase in bacteria numbers after 5 days whereas during the low salinity test series 

average number was comparable to the values observed for the tank.

In the treated tanks and in the incubation bottles of the same water the total bacteria 

numbers at discharge varied from the original numbers during intake in an inconsistent 

manner. During the high salinity test run there was in nearly all experiments a considerable 

increase in bacterial number (on average 2 fold increase relative to intake). In contrast to 

the low salinity test there was considerable variation between the different test runs but on 

average numbers in the treated water were lower than in the control. The apparent 

increase in bacterial numbers at the high salinity test runs is most likely due the presence of 

chloroplasts and mitochondria which are liberated during the disintegration of the larger 
Final report of the land-based testing of the ECOCHLOR®

-System 33

organisms. The size and the DNA content of these cell constituents are matching that of free 

living marine bacteria.

The treatment with chlorine dioxide was acting as a disinfection agent since bacteria 

numbers in both the tank and in the incubation bottle showed only moderate changes 

during the 5 days of incubation. 

This disinfection also resulted in a decline in the number of colony-forming heterotrophs, 

measured as cfu’s. However, in two tests the chemical treatment could not prevent 

bacterial activity resulting in high increase in heterotrophic bacteria thriving on the high 

dissolved organic carbon load of the water. 

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

bacteria Intake Reference Treated

C-T0 C-T0 C-T5 C-inc-T5 C-T5 E-T0 E-T5 E-inc-T5 E-T5

high 

salinity

total 

bact. colony

total 

bact.

total 

bact. colony

total 

bact.

total 

bact.

total 

bact. colony

VI 5.42E+6 20 9.49E+5 6.11E+6 10 7.94E+6 4.80E+5 3.28E+6 1700

VII 1.25E+6 50 5.37E+6 8.17E+6 20 9.54E+6 6.15E+6 9.74E+6 <10

VIII 4.64E+6 20 1.68E+6 5.15E+6 30 3.03E+6 2.86E+6 5.87E+6 40

IX 3.21E+6 30 3.29E+5 3.00E+6 <10 1.13E+7 8.24E+6 1.15E+7 <10

X 4.45E+6 10 2.40E+6 6.26E+6 60 7.11E+6 6.77E+6 7.65E+6 <10

average 3.79E+6 26 2.15E+6 5.74E+6 26 7.78E+6 4.90E+6 7.61E+6

<10-

1700*

low salinity

XI 3.66E+6 45 1.89E+6 6.24E+5 700 4.50E+6 3.69E+6 6.29E+6 >1000

XII 3.37E+6 3.14E+6 3.74E+6 <10

XIII 6.72E+6 <10 2.90E+6 3.56E+6 20 7.95E+6 7.86E+6 6.83E+6 <10

XIV <10 5.30E+6 5.07E+6 5.39E+6 <10

XV 9.02E+6 20 8.67E+5 2.52E+6 8.58E+6 6.13E+6 8.82E+5 <10

XVI 3.10E+6 2.54E+6 3.72E+6 <10

average 6.47E+6 25 1.89E+6 2.23E+6 243 5.47E+6 4.74E+6 4.47E+6

<10-

>1000*
Final report of the land-based testing of the ECOCHLOR®

-System 34

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 long-term (20 days) regrowth and vitality experiments. In total 3 long-term 

incubation experiments (2 at low salinity and 1 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 also bacteria. 

time [days]

0 2 4 6 8 10 12 14 16 18 20 22

phytoplankton 10-50 

μm [cells/mL]

0.01

0.1

1

10

100

1000

10000

control

Ecochlor

Figure 9: time course of phytoplankton numbers (10 – 50 μm size range) during a 20 day 

incubation period, control and treated water (Ecochlor). Test run X, values are mean of three

replicates.

Figure 9 shows that regrowth of phytoplankton, in this case only the fraction of 10-50 μm is 

shown was found after 15 days of incubation. In the two other studies no phytoplankton 

regrowth was observed, of any size class, during the whole incubation period of 20 days. 

These results indicate that the treatment was very effective in killing or deactivating 

phytoplankton but also in preventing hatching of resting stages or cysts for a considerable 

period.

Based on the first two similar types 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’. Specific toxicity studies 

and the presence of undesirable chemical end- or by-products, associated with the present 

land-based are documented in a separate report (G9 documents for Final Approval). 

Nevertheless, in the present study the vitality of the water was also determined as part of 

the environmental acceptability procedure. This was done by incubating treated water with 

freshly collected water from the Wadden Sea, containing the whole range of organisms 
Final report of the land-based testing of the ECOCHLOR®

-System 35

present in nature and not by means of a set of standard test organism, as typically is done in 

toxicology studies. 

time [days]

0 2 4 6 8 10 12 14 16 18 20 22 24 26

phytoplankton 10-50 

μm [cells/mL]

0.01

0.1

1

10

100

1000

10000

control

Ecochlor WS. nuts

Ecochlor WS

Ecochlor

Figure 10: time course of phytoplankton numbers (10 – 50 μm size range) during a 26 day 

incubation period, control and treated water (Ecochlor). At day 11 a 10% (v/v) of Wadden Sea 

water was added to the treated water and incubated in separate bottle with (Ecochlor WS nuts) 

and without additional nutrients (Ecochlor WS). Arrow indicates the number of phytoplankton 

cells in the original sample prior to addition to the treated water.

Test run X; values are mean of three replicates

Figure 10 shows that immediately after the addition of freshly collected Wadden Sea water 

numbers of phytoplankton increased rapidly, irrespective of the nutrient conditions of the 

water. This was observed for all other phytoplankton fractions as well (data not shown). 

Within 7 days, phytoplankton numbers increased by two orders of magnitude reaching the 

same values as measured in the control bottle.
Final report of the land-based testing of the ECOCHLOR®

-System 36

time [days]

0 2 4 6 8 10 12 14 16 18 20 22

bacteria [cells/mL]

0

2e+6

4e+6

6e+6

8e+6

control

Ecochlor

Figure 11: time course of bacteria numbers during a 20 day incubation period, control and 

treated water (Ecochlor). Test run X; values are mean of three replicates

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

pattern (fig. 11). In the control bottle bacteria first declined by a factor two but this was 

followed by a period of regrowth in the subsequent 5 days. After day 8 numbers declined 

again to a final number which was less than at intake. In contrast bacteria numbers in the 

treated water declined throughout the whole period at an almost constant rate. At day 20 

final numbers for both the control and treated water were nearly identical.

This gradual decline in the treated water implies that the active substance is actively 

reducing the number of bacteria like for the other organisms present. However, in contrast 

to phytoplankton which is immediately disintegrated after treatment bacteria remain 

present as intact cells and the degradation process takes far more time. Remarkable enough 

the regrowth of phytoplankton measured in test run 10 (Fig 9), starting at day 15, was not 

immediately followed by a growth response in bacteria. 
Final report of the land-based testing of the ECOCHLOR®

-System 37

10 Discussion and evaluation of results

The presented data show that the experimental design, type of test protocols used and 

additional experiments provide a solid data base of information on the performance of the 

ECOCHLOR®

-BWT-System under semi-in situ conditions. The initial idea of a treatment 

system based on the use of the active substance chlorine dioxide alone turned out to be 

ineffective in waters with an extremely high density of hard-shell organisms like barnacles. 

Despite the fact that a high percentage of these organisms were killed, sufficiently high 

numbers survived the 5 day incubation period as viable organisms. To some extent this was 

a surprise since the numerous onboard tests conducted by the University of Rhode Island

revealed that the system performed perfectly well without a coarse primary filter. 

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 

water discharge 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 longterm incubations also indicated that there was no regrowth of phyto- or zooplankton. 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, the treated water is far from ‘dead’ water. 

When a mixed plankton community from the Wadden Sea was added to the treated water 

the plankton community showed a rapid and unaffected growth response. 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. Nevertheless, the present growth experiments, as part of the environmental 

acceptability aspects, clearly demonstrate that upon discharge there is 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 > 50 μm fraction, and to some extent also in the 10 –

50 μm size range, cannot (easily) be met in the salinity range > 32 PSU. Further, the 

addition of cultured organisms is very difficult and poses a variety of problems. With 

increasing salinity the seawater will have the characteristics of typical open ocean 

water. This implies low to extremely low numbers of the organisms in the above 

indicated size ranges. For that reason we have extended our focus to the organisms < 

10 μm since there is no biologically relevant reasoning why tests should exclude this 

size fraction. 

2. The long-term incubations (ca. 20 days) and the 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.
Final report of the land-based testing of the ECOCHLOR®

-System 38

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 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 landbased 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 ECOCHLOR® -system its active 

compound (chlorine dioxide) has been evaluated for total and faecal coliforms (E. 

coli:; (Oviatt et al., 2002; URI unpublished on-going study) as well as with V. 

cholerea (Oviatt et al., 2002; URI unpublished on-going study) and proven to be an

effective method of disinfection.

In conclusion, the present configuration of the ECOCHLOR® -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 ECOCHLOR®

-System 39

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

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Appendix 1

Phytoplankton Marsdiep AprilJuly 2008 +: 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 +++++

heterotrophic flagellate parasite cyst of Ochromonas group ++++

diatom pennate Pseudonitzschia delicatissima group ++++

heterotrophic flagellate heterotrophic flagellate indet. <10 μm ++++

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 +++

heterotrophic flagellate Paulinella spp. +++

heterotrophic flagellate heterotrophic flagellate indet. 10-30 μm ++

autotrophic flagellate Pyramimonas spp. <10 μm ++

heterotrophic flagellate Choanoflagellatea indet. ++

hetero/auto flagellate Cryptophyceae "light" group ++

autotrophic flagellate Teleaulax acuta group ++

heterotrophic dinoflagellate Oxyrrhis marina ++

diatom centricate Leptocylindrus minimus ++

diatom centricate Minutocellus group ++

diatom centricate Thalassiosira spp. 10-30 μm ++

heterotrophic dinoflagellate Katodinium glaucum ++

diatom centricate Guinardia delicatula ++

freshwater green alga Pediastrum spp. ++

heterotrophic flagellate Ciliophrys group ++

autotrophic flagellate Prasinophyceae indet. <10 μm ++

hetero/auto dinoflagellate Gymnodiniaceae indet. 10-30 μm ++

diatom centricate Various species ++

autotrophic flagellate autotrophic flagellate indet. <10 μm ++

diatom centricate Chaetoceros spp. <10 μm solitairy cells +

autotrophic flagellate Chlorophyceae, Telonema spp. +

heterotrophic flagellate Chrysophyceae indet. +

freshwater green alga Crucigenia spp., Chlorophyta +

diatom centricate Skeletonema "costatum" lenses +

freshwater green alga Oocystis spp. +

diatom pennate Asterionellopsis glacialis +

autotrophic flagellate Rhodomonas group +

heterotrophic flagellate Bodo group +

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

-System 42

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