NIOZ Royal Netherlands Institute for Sea Research

2008 2

Final report of the land-based and 

shipboard testing of the SEDNA®-system

Marcel J.W. Veldhuis PhD, Frank Fuhr Dipl. Biol.
Final report of the land‐based testing of the SEDNA®

‐System     1

BSH / M5101             Texel March 2008

Final report of the land­based testing of the

SEDNA®­System, for Type Approval according to

Regulation­D2 and the relevant IMO Guideline  

(April – July 2007)

Final report of the shipboard testing of the SEDNA®­

System, for Type Approval according to Regulation   

D­2 and the relevant IMO Guideline  

(June – December 2007)

><((((°> 

Marcel J.W. Veldhuis Ph‐D   

Frank Fuhr Dipl.‐Biol.

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 or ffuhr@nioz.nl

S. Gollasch  Ph‐D  (GoConsult)

Grosse Brunnenstr. 61

22763 Hamburg

Germany

phone: +49 40 390 546 0

email: sgollasch@aol.com
Final report of the land‐based testing of the SEDNA®

‐System     2

BSH / M5101/ 

Final report of the land­based testing of the

SEDNA®­System, for Type Approval according to

Regulation­D2 and the relevant IMO Guideline  

(April – July 2007)

Marcel J.W. Veldhuis Ph‐D    

Frank Fuhr Dipl.‐Biol.

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 or ffuhr@nioz.nl
Final report of the land‐based testing of the SEDNA®

‐System     3

BSH / M5101             Texel January 2008

Final report of the land­based testing of the

SEDNA®­System, for Type Approval according to

Regulation­D2 and the relevant IMO Guideline  

(April – July 2007)

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 or ffuhr@nioz.nl
Final report of the land‐based testing of the SEDNA®

‐System     4

Content                  page 

Executive summary                5 

Zusammenfassung                5 

      

Acknowledgements                6   

Introduction                  6 

Description of the test facility            7 

NIOZ Royal Netherlands Institute for Sea Research        7 

Portrait of HAMANN AG  (producer of the SEDNA®‐system)      8   

Portrait of Evonik Degussa GmbH  (producer of PERACLEAN® Ocean)  8 

The test facility                9 

Technical description of the SEDNA system         10 

Requirements to meet the Regulation‐D2          12 

Requirements to meet: guideline G8           12 

Test procedures                15 

Results                  21 

Discussion of results                33 

Evaluation of results                34 

References                  35 

Appendix 1: species list of phytoplankton and zooplankton     37 

present during the various test runs. 
Final report of the land‐based testing of the SEDNA®

‐System     5

Executive summary

The SEDNA®‐System, a ballast water treatment system was tested according to 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

2007 in the harbour of the Netherlands Institute for Sea Research. Tests were successful and

the BWT system performed on average much better than Regulation‐D2 by achieving values

for organisms well below the requirements of the D2 Standard. This system should therefore

be regarded as an effective and safe way of cleaning ballast water in ships, thereby

minimizing the risk of new invasions originating from the ships ballast water.  

Sediment was removed by the hydrocyclone and filters and organisms were killed by the

active substance PERACLEAN® Ocean. In general the organisms disintegrated completely and

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

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

could be noted. The active substance was decomposed usually within 24 h and the

bacteriostatic agent (H2O2) was degraded within 3 days after addition. The by‐product, acetic

acid, reduced the pH of the water temporarily but was effectively and completely

mineralized and respired by heterotrophic bacteria. Although this caused a temporary

reduction in the ambient oxygen concentration no anoxia was observed. Upon discharge

after 5 days only in the pure treated water and the lowest dilution (1:1) with ambient

seawater tests showed a slight, but insignificant, effect of remaining acetic acid.   

Zusammenfassung

Das SEDNA®‐System zur Behandlung von Ballastwasser wurde im Frühjahr und Sommer 2007

gemäß Regulation‐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. Die Tests waren erfolgreich und das System zeigte eine bessere Leistung, als

gefordert. Die Organismenanzahlen nach der Behandlung lagen deutlich unter den

Anforderungen des D2‐Standards. Das System sollte demzufolge als effektive und sichere

Möglichkeit zur Behandlung von Ballastwasser betrachtet werden, die dazu beitragen kann,

weitere biologische Invasionen zu verhindern.

Sedimente wurden durch die Hydrozyklone und den Filter entfernt. Organismen wurden

durch die aktive Substanz PERACLEAN® Ocean abgetötet. In der Regel wurden die

Organismen vollständig aufgelöst und die Überreste konnten nur als amorphe Strukturen

oder in Form von gelöstem, organischem Kohlenstoff nachgewiesen werden. Beim Ablassen

des behandelten Wassers wurden keine Residualeffekte der chemischen Behandlung auf die

Umwelt festgestellt. Die aktive Substanz wurde normalerweise binnen 24 h zersetzt und die

bakteriostatische Komponente (H2O2) wurde innerhalb von 3 Tagen nach der Dossierung

abgebaut. Das Nebenprodukt des Abbaus, Essigsäure; senkte den pH‐Wert des Wasser

zeitweise, wurde aber effektiv und vollständig von heterotrophen Bakterien zersetzt.

Obwohl dadurch eine temporäre Abnahme der Sauerstoffkonzentration ausgelöst wurde,

wurden keine anoxischen Bedingungen festgestellt. Geringfügige Effekte der Essigsäure

konnten experimentell nach den 5 Tagen Aufbewahrungszeit nur in unverdünntem,

behandeltem und 1:1 mit Seewasser verdünntem, behandeltem Wasser nachgewiesen

werden.
Final report of the land‐based testing of the SEDNA®

‐System     6

Acknowledgements

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

Oosterhuis, Robert Lakeman, Santiago Gonzalez for making these tests possible. Without

their help and devotion the report would never have the standard it has now. Special tanks

also to Yolanda van Iperen for providing the species list of plankton in the Wadden Sea and

Lothar Reinecke, Matthias Welle and Andreas Meinhardt for running the SEDNA® system

without any technical problem and Bernd Hopf and Michael Volkmer for assisting us with the

PERACLEAN® Ocean measurements. We also would like to thank the commander and the

unit of the Royal Dutch Corp of Marines at Texel for their assistance.

Introduction

Ships transport 5‐10 billion tons of ballast water annually all over the globe (Endresen et al.

2004). The ballast water is loaded with particulate sediment and an enormous variety of

(living) organisms, which ranges from juvenile stages, larvae and eggs of fish and larger

zooplankton (Williams et al. 1988; Carlton and Geller 1993) to macroalgae, phytoplankton

(Hamer et al. 2000; Hallegraeff et al. 1997), bacteria and viruses (Gollasch et al. 1998). In

general these organisms belong to the natural ecosystem in and around the port of origin

but they might not be occurring naturally in the coastal waters and port of destination at the

end of a ship’s journey. In hundreds of cases around the world, this has resulted in severe

damage to the receiving ecosystem and to human health, because these non‐native

organisms developed into a plague. This often has a high impact on the ecosystem and can

cause economical damage (Hoagland et al. 2002), as it results in a decrease of stocks of

commercially valuable fish and shellfish species and occasionally outbreaks of diseases such

as cholera (Ruiz et al. 2000; Drake et al. 2001). If action is not taken the problem of invasive

species will increase in an exponential manner for several reasons. Ships are getting larger,

faster and the amount of traffic across the oceans is expected to increase rapidly during the

coming decades, and therefore also the change of non‐indigenous organisms to have large

enough number of 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 invasieve 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 fro 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.

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.  
Final report of the land‐based testing of the SEDNA®

‐System     7

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 and

Taylor 2001; MEPC 49/2/13, 2003). Besides reducing the load of organisms also 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 abundance and biodiversity of organisms prior, during and after a treatment

with the SEDNA®‐system (efficacy of the BWT system),  

2) to determine the viability status of the remaining organisms during discharge,  

3) to determine the environmental performance of the active substance and/or its

degradation products after discharge (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.  

Description of the treatment facility  

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.

More information on www.nioz.nl  
Final report of the land‐based testing of the SEDNA®

‐System     8

Portrait of HAMANN AG (producer of the SEDNA®­system)

HAMANN AG has been founded in 1972 by Mr. Knud Hamann under the name of Hamann

Wassertechnik GmbH. Concentrating on physio‐chemical waste water treatment plants for

all kinds of ships, the company constantly grew in both, turn over and staff. Over the years

the product range of Hamann Wassertechnik increased continuously. As a consequence,

different branches were established for the handling of spare parts, for emission control and

for ballast water treatment. All of these branches were joint together under the umbrella of

HAMANN AG in 2004, with the addition of freshwater production and in‐house workshop

facilities completing the picture. HAMANN AG is certified according to ISO 9001, which

guarantees the up most possible quality and reliability in the products and the workmanship.

With 70 people working at the head office in Hollenstedt and with a constantly growing

number of agents and service stations around the globe (currently, 31 agents are under

contract), HAMANN AG offers custom made products as well as a complete after sales

service.

More information on:  www.hamannag.com

Ballast water treatment ‐ The SEDNA®

‐System

The SEDNA®

‐System operates in‐line during the uptake of the ballast water. It is based on a

modular concept which includes a two‐step physical separation (hydrocyclone and self‐

cleaning filter) as well as a secondary treatment with an oxidising agent (PERACLEAN®

Ocean). The system is able to handle ballast water pump capacities from 250m3/h up to

1000 m3/h in a single module. A combination of modules is able to serve larger pump

capacities. For Navy vessels and Mega Yachts smaller plants are in the engineering phase.

Various configurations are feasible but the following are the three basic designs:

• Containerized system

• Skid mounted system

• Modular system

A full scale plant is in continuous operation aboard a newly build container ship since mid

2006.  

 

Portrait of Evonik Degussa GmbH (producer of PERACLEAN®

Ocean)

Evonik is a newly formed international industrial group with a workforce of about 43,000

and activities in more than 100 countries all over the world. The business areas are

Chemicals, Energy and Real Estate. The operations are bundled within 14 business units

which report directly to the Group board of management. The Corporate Center in Essen,

Germany, performs the Group’s strategic management functions. The business units and

Corporate Center are supported by Shared Services which provide cross‐locational services

such as procurement and payroll accounting.

Evonik Degussa GmbH is the producer of PERACLEAN® Ocean, a chlorine‐free disinfectant

especially designed for treating ballast water. The product contains as active substance

peracetic acid (PAA, 15 %) and H2O2 (14.3 %, effectively preventing re‐growth). During the

reaction PAA is converted into acetic acid (HAc), which is an organic carbon source normally

also present in natural aquatic environments.  

PERACLEAN® Ocean has been granted Basic Approval by IMO’s Marine Environment

Protection Committee on the 24th of March 2006; additional testing according to the

appropriate IMO Guidelines has been carried out in order to provide comprehensive

information needed to apply for Final Approval (G9 documentation).  

More information on:  www.evonik.com
Final report of the land‐based testing of the SEDNA®

‐System     9

The test facility 

The land‐based tests were carried out at the Royal Netherlands Institute for Sea Research

(NIOZ), Landsdiep 4, 1797 SZ 't Horntje, Texel, the Netherlands,  from March till July 2007

(www.nioz.nl).  

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 1). The tanks were (steam)cleaned after

each run. Water samples can be 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 1:  

Inside view of one of three

subterranean water tanks

According to the requirements of the Guidelines G8, sampling points are fitted before the

treatment system and directly after the system. Samples varying in volume from 500 mL up

to 1 m3 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. Different 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 oC; a light;dark regime of 16:8 h and 100 mol quanta.

m‐2

.s‐1)

The SEDNA®

‐System is 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, provision 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 2.
Final report of the land‐based testing of the SEDNA®

‐System     10

Figure 2: P&I diagram of SEDNA®‐System installation at NIOZ with points of sampling  

Technical description of the SEDNA®‐System

The SEDNA®

‐System operates in‐line during the uptake of the ballast water. It is based on a

modular concept which includes a two‐step physical separation as well as a secondary

treatment with an oxidizing agent.

The 1st step of the physical separation is a newly developed hydrocyclone that was specially

designed for ballast water applications. The number of hydrocyclones needed depends on

the flow rate of the ballast water pump. The 2nd step of the physical separation is a compact,

self‐cleaning filter with 50 μm meshes arranged in separate elements. The back‐flushing of

the filter is triggered by the differential pressure (max. 100 mbar), and the filter elements

are cleaned one by one with seawater without addition of any cleaning substances. Any

concentrates from the hydrocyclones (mainly sediments) and any rejects from the fine filter

remain at their point of origin and no new waste lines are produced, when the system is

operated on a ship.

The SEDNA®

‐System is self‐monitoring (PLC controlled) with automated bypass according to

the class requirements of the Germanischer Lloyd (GL). All materials and specifications are

selected in order to meet the class requirements of the Germanischer Lloyd for ballast water

systems and electric installations.

The SEDNA®‐System used during the tests at NIOZ has been build to the same standards as

the double 250 m3/h containerised SEDNA®

‐System that is installed on the test ship (certified

by Germanischer Lloyd).

The secondary treatment is the chlorine‐free disinfection with PERACLEAN® Ocean (150 ppm

= 150 mg/L), which is dosed automatically to the ballast water after the physical treatment.

PERACLEAN® Ocean has been granted Basic Approval from the MEPC at the 54th meeting in

March 2006 (MEPC 54/21). This compound contains as active substance PerAcetic Acid (PAA,

15%) and H2O2 (14.3%). Inert ingredients are water (43.5 %), acetic acid (26.5 %) and a

stabilizer (<1 %). At the time of injecting the product into the ballast water the

concentrations are 22.5 mg/L PAA and 21.5 mg/L H2O2. Afterwards, peracetic acid is
Final report of the land‐based testing of the SEDNA®

‐System     11

converted into acetic acid and together with the acetic acid already present in the original

formulation this results in a final acetic acid concentration in the treated ballast water of >50

mg/L. Acetic acid is easily biodegradable. Hydrogen peroxide is completely degrading

resulting in water and oxygen.

During a pilot study at the NIOZ harbour in 2004, no well‐described test methods were

available. Thus we had to develop these methods from the requirements laid‐out in

Guidelines for uniform interpretation of the IMO ballast water convention and the methods

developed for our fundamental scientific research on plankton. In practice sampling devices

and sample volumes had to be adapted taking into account the extremely low thresholds of

maximum allowed numbers of organisms per volume. For the large organisms (>50 m) a

new type of sampling net was designed by Hydrobios (Kiel, Germany) to concentrate the

organisms from several m3 of ballast water after it had been in the tanks. For the small

organisms (10 ‐ 50 m) the required sample sizes fitted well with the volumes used for semi‐

automated analysis by flow cytometry (Dubelaar et al. 1999; Veldhuis and Kraay 2000). In

addition to size class and maximum number of organisms per unit volume, the IMO D2‐

Standard refers to the presence of viable organisms rather than total organisms. To

differentiate between life and dead plankton, a newly developed staining method using the

dye SYTOX Green was successfully applied (Brussaard et al. 2001; Veldhuis et al. 2001;

Casotti et al. 2005). This method is based on the integrity of the cell membrane which is

intact in life cells but is disrupted when cells are exposed to stress, e.g. caused by physical

damage or chemical treatment.

The physical components of the tested BWT system (hydrocyclone and filters) turned out to

be effective in reducing the numerical abundance of the larger organisms but the chemical

treatment was still necessary to reduce the number of living organisms in all size classes to

below the maximal acceptable number. Moreover, the bacteriostatic compound of the

chemical (H2O2) delayed the regrowth of total bacteria by approximately 10 days.  

Currently our test protocols are communicated with various national and international

agencies. Upon agreement by national administrations and in collaboration with specialized

classification societies for ships, it is our goal to develop these protocols further to official

Guidelines used for Certification together with our international partners in Europe, the

United States and Asia.

NIOZ has the ambition to become the European test centre for the certification of BWT

equipment and related research. To achieve this, we have to develop or adapt automated

analyzing methods for phytoplankton, zooplankton and bacteria. We have had the opportunity to

collaborate with the German firm Hamann AG to optimize our test methodologies and at the

same time work on the certification of their SEDNA®

‐System. The NIOZ harbour was used as the

land‐based testing site for brackish water and high saline water under regulation G8 of the IMO

ballast water convention and testing was done according to a test protocol approved by the

Federal Maritime and Hydrographic Agency of Germany (BSH)  
Final report of the land‐based testing of the SEDNA®

‐System     12

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:

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.

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 m are

completely excluded. This is certainly an omission since this size class in particular includes

numerous phytoplankton species characterized as Harmful Algal Blooms (HABs).  

Nevertheless, 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 it’s life‐cycle,

including reproduction (DNA replication).

In addition to the 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. 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.

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.

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
Final report of the land‐based testing of the SEDNA®

‐System     13

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 

Additional experiments and previous tests (Veldhuis et al. 2006) showed that the SEDNA®

‐

System was not affected by differences in salinity, sediment or organisms load. The physical

steps, hydrocyclone and filtration, are unaffected by salinity but also the reactivity of the

active compound (PERACLEAN® Ocean) is not 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 (sea)salt. All other minimum requirements for TSS, POC and DOC

for these two water types are 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 difference it was decided to have the possibility of adding fresh surface water (this

water was freshly taken from Lake IJssel, a freshwater basin with an outflow into the

Wadden Sea and close to the institute) and upgrade Wadden Sea water 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 SEDNA®

‐System     14

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

micron) 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
Final report of the land‐based testing of the SEDNA®

‐System     15

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

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 the end product of the active substance is acetic acid, which is a

typical (natural) substrate found in the aquatic environment that is known to be consumed

by bacteria. The present quantities of acetic acid are high enough to boost the activity of in

particular heterotrophic bacteria. Therefore special attention was given to the degradation

rate of the elevated acetic acid concentration and its effect on the bacteria community.

Test procedures

A variety of methods was applied to examine the biological efficacy of the SEDNA®

‐System

for the different categories of organisms during the two test series. For detailed description

we refer to read the outline of the official test protocols for the SEDNA®‐System (Anonymous

2007). 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 at randomly and when

needed from multiple bottles or containers. As indicated previously there was great

emphasis on analysing of the freshly taken 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 brief 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
Final report of the land‐based testing of the SEDNA®

‐System     16

used to sample the ballast simulating tanks. The coded glass bottles are flushed at least

three times their volume (ca. 120 mL) with water.  

The sample bottles were stored in a dark container filled with water of the same

temperature as the samples until further analysis at the laboratory. In the laboratory 1 mL

H2SO4 is added prior to measuring the OD at 456 nm with a Hitachi U‐3010

Spectrophotometer. The oxygen concentration was calculated using standards and

expressed as M O2/L (or mg O2/L; = M 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 l 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 PERACLEAN® Ocean

Two water samples were taken during each filling of the treated tank to get the initial

concentrations during the actual filling of the tank. Sampling point was just behind the

PERACLEAN® Ocean injection of the SEDNA®

‐System.

After filling of the tank and in the following days, samples were taken from the sampling

point at tank 3 in the basement until the concentrations fell below the detection level.  

At least three samples per day were taken in order to get sufficient data, evenly distributed

over the day, to construct a degradation curve of the chemicals.  

  Figure 4:  

  Sampling point at tank 3
Final report of the land‐based testing of the SEDNA®

‐System     17

The amount of peracetic acid (PAA) and hydrogen peroxide (H2O2) was measured by

photometric determination according to Pinkernell et al (Pinkernell et al. 1997) and Wagner

& Ruck (Wagner and Ruck 1984), respectively. For each individual sample three

measurements were done, and the average value was calculated. The detection limits for

these methods are 0.5 mg/L PAA and 0.5 mg/L H2O2.

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 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:  

500 L container with 50 m

Hydrobios net using a modified lid

Organisms > 50 μm

The samples are pre‐concentrated over a Hydrobios 50 m net resulting in an end volume of

approximately 100 to 200 mL. The samples were transferred to the lab directly after

sampling and Neutral Red was added in a ratio that yields an end concentration of 1:50,000.

Staining time is 2+ hours. Neutral Red stains living organisms (Crippen 1974; Fleming and

Coughlan 1978 distinctively and quite rapidly (less than one hour, figure 6). Therefore the

viability assessment remains unaffected by the possible death of organisms during the

staining or during sample analysis.

It is assumed that dead but physically intact organisms will also be found. Consequently a

detailed inspection of each intact individual is needed to assess viability. This includes the
Final report of the land‐based testing of the SEDNA®

‐System     18

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

copepods from a

Neutral Red stained

sample.  

(bottom)  dead

copepods from a

NR stained sample.

FlowCam images

kindly provided by

N. Poulton  Fluid

Imaging

Technologies.

  

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 m 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.

For minimum dimension measurements the "body" of the organism has been measured.

Some examples are presented in figure 7.
Final report of the land‐based testing of the SEDNA®

‐System     19

Figure 7: Minimum dimension measurements (red line) in selected organism types: A =

bivalve larvae, B = gastropod larvae, C = polychaete, D = echinodermata larvae, E and F =

crustacean larvae and G = copepod.

Organisms < 50 m

Samples for visual inspection of species number and diversity were pre‐concentrated using a

sieve made of a Hydrobios 10 m mesh net using the 50 m prefiltered sample (effective

size range is 10 – 50 m). 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). Live‐dead‐separation in these samples was based on structural integrity of

organisms. This method works for both zoo‐ and phytoplankton.

This size fraction is 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 and Brussaard 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) are 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

m). 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 life organisms (cf.

Veldhuis et al, 2001).

A B

C D

E 

F 

G 
Final report of the land‐based testing of the SEDNA®

‐System     20

nucleus

A B

Figure: 8(A) Epifluorescence microscopic picture of a life 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 Pulse‐

Amplitude Modulated fluorometer (PAM‐fluorometry) WALTZ‐ water PAM (Schreiber et al.

1993). For this 3 mL of unfiltered sample water (control and treated, each in triplicate) are

filled into a glass cuvette and analysed using the Pulse‐Amplitude Modulated fluorometer.

The instrument was calibrated against filtered seawater and a healthy fast‐growing

population of phytoplankton.

Next to cell specific analysis chlorophyll a and its degradation products were determined to

assess the fate of the whole phytoplankton community (Jeffrey et al. 1997). For this purpose

water samples of 0.2 to 1.5 L (GF/C filters) were taken. The samples were frozen until further

analysis. Filters are extracted with acetone (90%) for at least 24 hours and chlorophyll a

concentration is determined spectrofluorometrically (standard procedures).

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 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 l 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 and Giorgio 2000; Veldhuis et al. 1997). A known bacterial standard is used for

calibration and counting.

The number of 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.
Final report of the land‐based testing of the SEDNA®

‐System     21

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.

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 a

species list of organisms observed is presented.

The tests were carried out at two different salinity regimes (Tables 4 and 5) hereafter

referred to as low and high salinity test series.

Salinity

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

control and treated tanks for test runs 1 – 6.

Test run Salinity [PSU] s.d. Temperature °C 

1 - 6 21.4 2.3 13 - 17 

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 7 – 12.

Test run Salinity [PSU] s.d. Temperature °C 

7 - 12 33.5 1.4 18 - 20 

To the high salinity test cycles coastal water from the North Sea was collected during high

tide and a brine solution made from natural seasalt was added to a maximum of 4 % (v/v).

Low salinity

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

DOC) is presented in table 6 for the reference and treated water sample at intake and

discharge.
Final report of the land‐based testing of the SEDNA®

‐System     22

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

runs in total) at intake and discharge; 1

: range of values; 2

: average of reference and treated tank at intake (pump samples).

Parameter Intake s.d. Discharge 

(day 5) s.d. unit 

O2 reference1 7.4-12.7 4.6-9.1 mg O2/L 

O2 treated 1 23.3-28.3 15.5-26.8 mg O2/L 

pH reference 8.31 0.16 8.27 0.16 

pH treated 6.42 0.18 

TSS 2 121.3 32.6 mg/L 

POC2 38.8 12.5 7.6 2.8 mg/L 

DOC reference 5.0 1.39 4.1 0.68 mg/L 

DOC treated 20.9 0.96 20.8 0.96 mg/L 

Table 6 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. 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. 30% compared to intake values. In contrast oxygen concentration in the

treated tank was increased by as much as 300% as compared to the reference tank. This

apparent super‐saturation is partly an artefact caused by the applied Winkler method for O2

measurements resulting in the dissociation of the H2O2 of PERACLEAN® Ocean (equation 1).

Based on the initial concentration of added H2O2 of ca. 25 mg/L the theoretical increase in

oxygen concentration according to equation 1 would be ca. 11.8 mg/L. In practice the

measured values in the tank are 20 – 30% less than the theoretical concentration. Although

not tested in detail this could be due to the simultaneous drop in pH altering the coloration

of the Winkler reagents, out‐gassing and oxygen respiration. After the required incubation

period of 5 days the oxygen concentration dropped by as much as 40% but at discharge the

oxygen level was still well above saturation.

Eq. 1. 

     2H2O2              2H2O   +  O2

The addition of PERACLEAN® Ocean caused a significant drop of the pH from an initial pH

value of 8.31 of the seawater to 6.52 on average in the treated tanks after addition of the

chemical (Table 6). This lowering of the pH is caused by the acetic acid present. At the end of

the incubation the pH in reference tanks remained nearly constant but a slight increase was

observed in the treated tank. As will be shown later the acidification of the water was

reduced as a result of microbial degradation of the acetic acid or by diluting the discharge

water with ambient seawater.

The total suspended solids (TSS) and particulate organic carbon (POC) concentrations were

sufficiently high at intake but reduced at discharge (data not shown). For the reference tank

this was mainly due to sedimentation but in the treated tank the mechanical treatment

(hydrocyclone and filter) reduced the particle load significantly at intake.  

Another striking difference between reference and treated tank was the concentration of

dissolved organic carbon (DOC). At intake the natural (background) concentration of DOC of

the Wadden Sea was ca. 5 mg DOC /L but this increased by 4 fold in the treated tank (Table
Final report of the land‐based testing of the SEDNA®

‐System     23

6). As can be derived from equation 2 this was entirely due to the amount of organic

compounds in the PERACLEAN® Ocean added.

Eq. 2 

15 %    PAA   22.5   mg/L   7.1   mg DOC /L     

(1 mole PAA = 76.05 g   including 24.02 g Carbon)) 

26.5 % HAc   39.75 mg/L    15.8 mg DOC /L      

(1 mole HAC = 60.05 g including 24.02 g Carbon)) 

‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 

  in total  as dissolved carbon  22.9 mg DOC/L  

DOC  estimation  on  addition  of  150  mg/L  PERACLEAN®

Ocean immediately after injection, containing as potential 

carbon  source  15  %  Peracetic  Acid  (PAA)  and  26.5  % 

Acetic acid (HAc). 

In practice the theoretical value of 22.9 mg DOC /L originating from the active substance was

not observed. At the end of the 5 days incubations the DOC content in the control tank

dropped by ca. 20% but remained virtually constant in the treated tank. This implies that the

discharge water contained a significant amount of acetic acid (HAc). The effect of this extra

organic carbon source was therefore subject of additional studies (see section Microbial

degradation of PERACLEAN® Ocean).  

High salinity  

Table 7: Oxygen, pH, TSS, POC and DOC concentrations of test series run at high salinity

(6 test runs in total) at intake and discharge;   1

: range of values;   2

: average of reference and treated at intake (pump samples).

Parameter Intake s.d. Discharge 

(day 5) s.d. unit 

O2 reference1 7.1-8.2 6.2-8.1 mg O2/L 

O2 treated 1 23.5-28.8 6.4-21.8 mg O2/L 

pH reference 8.13 0.05 8.08 0.04 

pH treated 6.27 0.07 6.29 0.06 

TSS 2 11.7 4.4 mg/L 

POC2 5.7 1.3 mg/L 

DOC reference 3.0 0.18 2.8 0.25 mg/L 

DOC treated 19.3 0.23 15.3 5.62 mg/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 7). At intake the ambient water was

saturated with oxygen and at discharge oxygen concentrations declined but they were still

far from depleted. Partly because of the increase in seawater from the open North Sea

sediment, POC and DOC values were much lower as compared to the low salinity test series.

The TSS content increased at discharge which was mainly due to an increase in POC, which

would be indicative of a potential increase in organisms since this was only observed in the

treated tank and not in the reference tank (POC control at day 5 declined to a value of 4.3

mg/L). The DOC concentration increased with a value of 16.3 mg/L, which was comparable

to the value of 15.9 mg/L observed at low salinity. This value was again lower than the

expected number based on the theoretical composition of the active substance. Unlike the
Final report of the land‐based testing of the SEDNA®

‐System     24

previous test series the DOC declined, by ca. 20% during the 5 days of incubation, which is

indicative of active (bacterial) degradation of the HAc starting already prior to discharge.

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).

Regarding the required minimal numbers of organisms per volume (Table 2), the values for

the low salinity series (test run 1  ‐6) were, with exception of run 6, well above the

requirements. On average numbers in the >50 m size fraction were 50 times higher than

the minimum required number of 100,000 m‐3

. At the high salinity range the water at intake

contained in two cases not sufficient numbers (test run 8 and 10). 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 are much lower in high saline water

than brackish water.

On discharge the number of organisms had dropped dramatically in the treated tank, to an

average value for the low and high salinity test series of 0.12 and 0.05 organisms m‐3

,

respectively. In fact during most test series at direct inspection immediately after intake

already no living organisms were observed. On average numbers in the treated tank were on

discharge at day 5 were a factor 100 lower than the D2‐standard of 10 organisms m‐3

. In

contrast numbers of organisms in the reference tank were still significantly above the G8

requirement and visual inspection and viability measurement indicated that these organisms

were both intact and viable.

Table 8: Number of organisms > 50 m at intake and on discharge at day 5 of the

reference and treated tank. For test run 11 and 12 the same control was used, since this

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

.  

total plankton > 50 micron counts /m3

low 

salinity Intake Reference Treated 

Test run day 0 day 5 day 5 

1 2300 E3 9.5 E3 0.7 

2 15200 E3 15 E3 0 

3 4600 E3 7.1 E3 0 

4 4900 E3 7.1 E3 0 

5 1400 E3 4.4 E3 0 

6 77.5 E3 12.7 E3 0 

average 9.3 E3 ± 3.9 E3 0.12 

high 

salinity 

7 103 E3 6.8 E3 0 

8 60.0 E3 2.5 E3 0 

9 113.2 E3 3.0 E3 0 

10 57.2 E3 600 0.3 

11 168 E3 1.5 E3 0 

12 168 E3 1.5 E3 0 

average 2.7 E3 ± 2.2 E3 0.05 
Final report of the land‐based testing of the SEDNA®

‐System     25

Additional analysis of some of the incubation experiments (see below for details) for

organisms larger than 50 μm showed higher levels of survival (up to 4 times) in the

incubated reference water as compared to the reference tank. However, in compliance with

the results from the treated tanks no living organisms larger than 50 μm were found in the

incubated treated water.

Organisms 10 – 50 μm

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

ciliates and flagellates were occasionally present in high numbers. 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 more specifically, on this group of

organisms. Finally, a 10 L subsample of both the reference and treated water was collected

at intake and incubated under optimal growth conditions. Results of plankton numbers in

the 10 – 50 m 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 in the reference tank a incubation of 5 days, in the dark, resulted in a

considerable reduction in the number of plankton in the 10 – 50 μm size range. At the low

salinity test series this decline was on average 55%, as compared with intake, but during the

high salinity test series this was much higher (81%). Nevertheless, the plankton community

of the reference tanks still contained sufficient numbers of viable organisms to meet the G8

requirements. Moreover, the bottle incubations showed, with two exceptions (series 3 and

11/12) a considerable increase in numerical abundance within 5 days, i.e. presence of viable

population of mainly phytoplankton. In contrast, treatment resulted in an efficient removal

of plankton. Although after 5 days in the tanks some residual plankton cells were detected

flow cytometrically, analysis of the viability using SYTOX Green, but also microscopic

observation showed no viable cells anymore. In the incubated bottles of the treated ballast

water even the detrimental particles were disintegrated after 5 days since virtually no

particles within the given size range could be observed.
Final report of the land‐based testing of the SEDNA®

‐System     26

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

and treated tank, reference and treated incubated samples (Inc.) at day 5 during discharge.

Runs 1 to 6 at low salinity range and 7 to 12 at high salinity range. Test run 11 and 12 share the

same reference since this was a combined test run. All numbers are presented per mL as total

counts or of viable cells (*).

total plankton 10 - 50 μm counts/mL 

low 

salinity Intake Reference Treated * viable cells 

Test run day 0 day 5 Inc. T5 day 5 total day 

5* 

Inc. 

T5 Inc. T5* 

1 1025 900 6070 <1 <0.1 0 <0.01 

2 2300 110 4720 18 <0.1 1 <0.01 

3 2200 1310 222 6 <0.1 0 <0.01 

4 1560 985 5060 12 <0.1 3 <0.01 

5 600 321 4180 8 <0.1 1 <0.01 

6 1100 350 7880 16 <0.1 0 <0.01 

average 1464 663 4689 12 <0.1 1 <0.01 

high 

salinity 

7 1242 810 2670 116 <0.1 7 <0.01 

8 2500 570 4432 35 <0.1 1 <0.01 

9 2079 114 3580 83 <0.1 4 <0.01 

10 1000 180 2517 90 <0.1 2 <0.01 

11 1841 201 933 48 <0.1 1 <0.01 

12 1841 201 933 196 <0.1 3 <0.01 

average 1751 346 2511 95 <0.1 3 <0.01 

In order to have a more complete insight of the fate of all organisms, i.e. also fraction the

planktonic fraction < 10 m in size diameter, a group of phytoplankton in the size range of

ca. 6 m 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 SEDNA®

‐System was also

effective in reducing organisms in this size range. Flow cytometry showed the presence of

intact particles in the treated tanks even after 5 days. This was confirmed by classical

analysis of the chlorophyll and associated pigment content (data not shown). Cells did loose

their pigmentation and therefore the signal is rapidly masked by the detrital particles also

present. With the high load of sediment, a clear differentiation of phytoplankton and other

particles is less pronounced. For this size group the incubation is a far better approach to

examine the residual growth potential of the plankton. Apparently the residual chlorophyll

signal is disappearing completely since no plankton was observed in the target group after

the incubation.
Final report of the land‐based testing of the SEDNA®

‐System     27

  

Table 10: Phytoplankton number of the <10 m size range at intake, reference and treated

tank, reference and treated incubated samples (Inc.) at day 5 during discharge. Runs 1 to 6

were test runs at low salinity range and 7 to 12 at high salinity range. Test run 11 and 12 share

the same reference since this was a combined test run. All numbers are presented per mL as

phytoplankton counts or of viable cells (*).

phytoplankton <10 μm (group of ca 6 μm) 

low 

salinity Intake Reference Treated 

Test run day 0 day 5 

Inc. 

day 5 day 5 day 5* Inc. T5 

Inc. 

day 5* 

1 8800 3100 60150 14400 <0.1 0 <0.01 

2 53000 31800 65200 29000 <0.1 6 <0.01 

3 13600 34600 31900 12500 <0.1 3 <0.01 

4 23500 336 22750 2080 <0.1 2 <0.01 

5 1400 76 15000 1720 <0.1 8 <0.01 

6 1160 810 8400 330 <0.1 3 <0.01 

average 16910 11787 33900 10005 3.7 <0.01 

high 

salinity 

7 1900 2650 8315 448 <0.1 3 <0.01 

8 8517 2179 13989 810 <0.1 2 <0.01 

9 5865 2352 14680 680 <0.1 3 <0.01 

10 1668 689 22291 771 <0.1 0 <0.01 

11 15619 671 5687 67 <0.1 1 <0.01 

12 15619 671 5687 391 <0.1 1 <0.01 

average 8198 1535 11775 528 1.7 <0.01 

Photosynthetic efficiency

Another approach to gain insight in the physiological and growth response of the

phytoplankton community is by measuring the photosynthetic efficiency (Fv/Fm) of the

plankton cells. This can be done for the whole community or after filtration for different size

classes when needed. For a clear understanding it must be noted that values of Fv/Fm > 0.4

are indicative of a healthy phytoplankton population; a Fv/Fm < 0.4 indicates that the

phytoplankton community is experiencing severe stress and a value < 0.1 is typically

observed in decaying phytoplankton populations.  

Table 11 shows that during intake the whole phytoplankton community in the water, at the

low as well as high salinity range was physiologically in a healthy condition, i.e. containing

mostly photosynthetic active and therefore viable cells. Prolonged incubation in the dark (5

days of dark stress) of phytoplankton in the reference tanks resulted in a reduction in the

photosynthetic efficiency, although some large differences in the magnitude of reduction

was observed for the different test runs. Nevertheless, with exception of test run 5 the

phytoplankton showed signs of stress but was certainly not entering the decaying stage. In

contrast the photosynthetic efficiency of the phytoplankton in the samples incubated under

optimal growth conditions showed a response varying from a mild stress to a healthy status

(Reference Inc. day 5). This is not surprising since phytoplankton also increased in numerical

abundance (Tables 9 and 10).  

After passing the treatment system most of the phytoplankton community was immediately

dead and as a result during discharge the Fv/Fm was typical of a decaying algal community.

Also in the incubated sample there was no recovery of the phytoplankton population
Final report of the land‐based testing of the SEDNA®

‐System     28

observed. It should be noted that the data as presented in Table 11 include the whole

phytoplankton community and not exclusively the fraction > 10 m.

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

m 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 1 to 6 were

test runs at low salinity range and 7 to 12 at high salinity range.

Fv/Fm Intake Reference Treated 

low salinity 

Test run day 0 day 5 Inc. day 5 day 5 Inc. day5 

1 0.5 0.21 0.41 0 0 

2 0.52 0.50 0.34 0 0 

3 0.44 0.22 0.32 0 0 

4 0.47 0.14 0.50 0 0 

5 0.5 0.09 0.29 0 0 

6 0.71 0.56 0.57 0 0 

high 

salinity 

7 0.63 0.18 0.56 0 0 

8 0.61 0.33 0.51 0 

9 0.58 0.12 0.43 0.002 0 

10 0.46 0.13 0.54 0 0 

11 0.65 0.22 0.58 0 0 

12 0.65 0.22 0.58 0.004 0 

Bacteria

For the microbial community the presence/absence of two types of human pathogens 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 the number of the target microorganisms was well

below the standard as indicated in the D2‐Standard. Only in two cases the numbers of

human pathogens were above the detection limit (test run 7 and 8). 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 SEDNA®

‐System     29

Table 12: Counts of the human pathogens E. coli and total enterococci at intake and during

discharge of the reference and treated tank.

Counts/mL Intake (day 0) Reference (day 5) Treated (day 5) 

low 

salinity 

Test run E. coli enterococci E. coli enterococci E. coli enterococci 

1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 

2 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 

3 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 

4 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 

5 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 

6 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 

high 

salinity 

7 0.34 <0.1 <0.1 <0.1 <0.1 <0.1 

8 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 

9 <0.1 0.33 <0.1 <0.1 <0.1 <0.1 

10 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 

11 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 

12 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 

In contrast to the nearly absence of human pathogens, the ‘normal’ marine microbial

community was abundantly present. The total bacteria 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 (Table 13) of numbers ranging from 3.4 to 7.6 106 per mL

are numbers typically present in spring and early summer. Of this total bacterial population

only a very small fraction could be identified as heterotrophic ones using the plate assay

method (10 to 140 per mL). Therefore, if only plating would be used as criteria for bacterial

numbers this would result in a severe underestimation of the actual bacteria numbers. After

5 days of incubation total bacteria numbers declined in most, but not in all test runs in both

the reference tank and in the incubated reference bottle. In general the incubation

contained a slightly higher number of bacteria than the control tank. In a same manner the

number of plated heterotrophs remained in the same order of magnitude at the low salinity

range but declined to below the detection limit in the high salinity test series.  

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. In nearly half of the test runs there was an actual increase whereas in the others

there was a decrease from the numbers at intake. These variations were nevertheless

moderate and never exceeded the original values by more than a factor of 2.2. In terms of

cultivable heterotrophic bacteria the treated water showed some remarkable differences

with the water at intake and the reference. During the low salinity test runs the numbers of

colony‐forming heterotrophs remained unaltered in the first two test runs but increased

significantly in the last 4 test series resulting in an increase of > 340 fold (test run 6). At all

test runs at the high salinity range there was a very high increase in colony forming units, up

to > 1000 fold increase, except for test run 12. As will be shown later in more detail this is

caused by a fast growing population of heterotrophic bacteria using the residual acetic acid

as their primary carbon and energy source. Growth of this bacterial group usually starts

around day 5 with some variation in the original onset of the growth period.  
Final report of the land‐based testing of the SEDNA®

‐System     30

Table 13: Total bacteria number and colony forming heterotrophs (numbers per mL) at intake

and during discharge of the reference and treated tank as well as the total bacteria numbers in

the incubated water samples (Inc.). n.d.: not determined.

Intake Reference Treated 

low 

salinity total bact colony total bact colony total bact total bact colony total bact 

Test run day 0 day 0 day 5 day 5 Inc. day5 day 5 day 5 Inc. day5 

1 3.6E+06 10 n.d. 50 n.d. n.d. 20 n.d. 

2 3.4E+06 10 1.3E+06 20 3.6E+06 4.5E+06 <10 4.9E+06 

3 5.8E+06 20 7.1E+06 30 2.7E+06 8.2E+06 200 5.2E+06 

4 7.6E+06 60 1.6E+06 50 3.0E+06 6.6E+06 190 7.2E+06 

5 5.8E+06 20 1.7E+06 10 2.3E+06 6.9E+06 275 6.1E+06 

6 5.2E+06 <10 3.0E+06 <10 3.1E+06 6.1E+06 3400 7.2E+06 

high 

salinity 

7 3.4E+06 20 2.6E+06 <10 3.0E+06 6.1E+06 >10000 4.0E+06 

8 4.7E+06 10 1.4E+06 <10 2.0E+06 4.6E+06 >10000 3.8E+06 

9 6.3E+06 140 0.9E+06 10 1.9E+06 2.8E+06 >10000 3.4E+06 

10 3.8E+06 10 1.2E+06 <10 3.9E+06 1.5E+06 6800 3.0E+06 

11 4.4E+06 10 9.5E+05 <10 2.6E+06 4.1E+06 2500 4.2E+06 

12 4.4E+06 10 9.5E+05 <10 n.d. 5.8E+06 50 5.0E+06 

Environmental effects after discharge

Microbial degradation of PERACLEAN® Ocean

Besides testing the SEDNA®

‐System for its efficiency to reduce the numbers of living

organisms the system was also examined for it impact on the (marine) environment since it

uses an active substance to kill organism in the second treatment step. The degradation of

PAA was very rapid, usually within 24 h after addition (Table 14). PAA was transformed in the

less reactive product acetic acid (Eq. 3). Together with the acetic acid already present in the

formulation this resulted in an acidification of the treated water (Tables 6, 7). Moreover, the

added amount of acetic acid generates a large pool of labile organic carbon which could

easily be decomposed by bacteria.  

DOC caused by 150 mg/L PERACLEAN® Ocean can be calculated as follows:  

Eq. 3   

150 ppm PERACLEAN® Ocean with 

   15% PAA     22.5 mg PAA /L  18.75 mg HAc /L  

26.5 % HAc                          39.75 mg HAc /L  

     ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 

  in total           58.5  mg HAc /L  

  as dissolved carbon:        23.4  mg DOC /L  

(1 mole of HAc = 60, 05 g and includes 24.02 g Carbon) 

The PAA (peracetic acid), which is the reactive substance is transformed into HAc (hydrogen

acetate= acetic acid). This dissolved organic pool of carbon can initially not be utilized by

(heterotrophic) bacteria community because the additional presence of the bacteriostatic
Final report of the land‐based testing of the SEDNA®

‐System     31

compound H2O2. H2O2 remains present for several days (table 14) but a major fraction of the

bacteria is also inactivated by the added PAA. Because of these two factors the regrowth of

heterotrophic bacteria shows a significant lag phase of several days and starts effectively

around day 5. It should be noted that mineralization of the weak organic acid (HAc) by

bacteria also results in an increase in pH.

Table 14: Temperature in the ballast tanks at intake and day 5 and degradation time of the

active ingredients PAA and H2O2 (h) after addition to detection limit (0.5 mg/L). n.d. not

determined.

 temperature PAA H2O2

low salinity degradation degradation 

Test run day 0 day 5 rate h rate h 

1 n.d n.d. 

2 13 14 16 114 

3 15 15 18 74 

4 16 16 18 90 

5 18 16 20 100 

6 16 17 18 106 

average 18 97 

high salinity 

7 18 18 16 90 

8 18 18 18 96 

9 20 20 18 68 

10 21 20 8 70 

11 21 19 8 54 

12 21 19 16 90 

average 14 78 
Final report of the land‐based testing of the SEDNA®

‐System     32

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

O2 [mg/l]

0

2

4

6

8

10

12

Oxygen 1:1 mg/l 

Oxygen 1:2 mg/l 

Oxygen 1:10 mg/l 

24.9

6.4

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

DOC [mg/l]

2

4

6

8

10

12

14

16

18

20

22

bacteria number [counts/ml]

0.0

5.0e+6

1.0e+7

1.5e+7

2.0e+7

2.5e+7

3.0e+7

3.5e+7

DOC mg/l 

bact. reference

bact treated

3.1 2.6

time [day]

-6 -4 -2 0 2 4 6 8 10 12 14

bacteria numbers [counts/ml]

0.0

2.0e+6

4.0e+6

6.0e+6

8.0e+6

1.0e+7

Bacteria 1:1 

Bacteria 1:2 

Bacteria 1:10 

Figure 9: A) Total DOC concentration and total bacteria numbers in the reference and treated

ballast water tank at intake (day  ‐5), discharge (day 0) and after storage in a large tank

(undiluted sample). Black bar is DOC concentration in reference tank at intake and discharge.

B) oxygen concentration at intake and discharger (bars) in treated tanks and after diluting the

discharge water in three concentrations with ambient seawater.  The 100% saturation value of

oxygen for the given temperature and salinity was 8.3 mg O2/L. C) bacteria numbers in the

same containers (for first 12 days only).
Final report of the land‐based testing of the SEDNA®

‐System     33

The degradation of the HAc was monitored as the residual DOC concentration in a large

volume tank over a period of 17 days (Fig. 9). The degradation during the initial 5 days

incubation was usually low to moderate, on average no more than 10% of the HAc was

mineralized during that period. The main component of the DOC content on discharge is

therefore HAc. The degradation of this compound has been studied in a variety of

experiments. Fig 9A shows that the complete bacterial degradation of HAc in a subsample

(700 L) down to the ambient DOC concentration of the seawater takes about 17 days. In that

period the bacteria numbers increase rapidly before declining again slightly.  

In a second set of experiments the treated water was diluted during discharge, with freshly

collected seawater in three dilution steps (1:1, 1:2 and 1:10 v/v) and oxygen and bacteria

numbers were measured during a 23 and 12 days period, respectively (Fig. 9B,C). The

dilution of the treated water with the water from the NIOZ harbour resulted in an initial

increase in the oxygen concentration to values above the saturation value for the given

temperature and salinity. In the following 4 days the oxygen concentration declined rapidly,

but this decline varied with the initial dilution step. In the least diluted tank (1:1) the oxygen

value dropped to a value of 15% of the normal saturation level and remained at that level for

another 6 days.   In the less diluted tanks oxygen levels did not drop that much and in all

tanks a recovery was observed after day 10 to values above 100% saturation values. This was

basically due to photosynthetic activity of the phytoplankton community (data not shown).

In terms of bacteria numbers only the least diluted tanks showed a significant increase in

numbers, thereby explaining the concurrent drop in oxygen concentration.  In the other two

tanks there was only a moderate increase in bacteria numbers and therefore also a

moderate drop in oxygen concentration.  

Discussion 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

SEDNA®

‐System BWT installation under semi in situ conditions. The outcome was in a way

not surprising since prior to testing according to the guidelines (Anonymous, 2005) of the

IMO some experience had been gained with testing the SEDNA®‐system for its efficacy

(Veldhuis et al. 2006). During the testing period we encountered three major issues which

need urgent attention.

1. The number of organisms in the > 50 m fraction, and to some extend also in the 10 –

50 m size range, can not (easily) be met in the salinity range > 32 PSU. 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 m since

there is no biologically relevant reasoning why tests should exclude this size fraction.

As demonstrated during these tests the proper tools and technologies are available to

test this size fraction for its presence and viability status.  

2. Short‐  and long‐term incubations should be an integrated part of land‐base testing.

While the large tank trials offer a more realistic scenario, sampling, sample handling

and analysis is still time‐consuming and labour‐intensive. In particular the microscopic

observations are a tedious task and the high load of sediments can be hampering

proper inspection of the samples. To some extend semi‐automated tools like flow

cytometry and flow cam reduce this ‘human’ error but do not completely solve it. An

elegant approach to test the efficacy of the ballast water system is to examine the

remaining viability of organisms after treatment. The best way to do so is to transfer a

sufficiently large volume of water under optimal growth conditions for plankton and

to monitor for abundance and viability for a considerable period.  

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 safety reasons accurate testing is therefore not possible.
Final report of the land‐based testing of the SEDNA®

‐System     34

Moreover, viability tests of the total bacteria community showed that not all bacteria

were effectively killed. Therefore, at least in theory the human pathogens would

remain a potential risk. This is a factor of concern for the land‐based tests as well as

for the ship‐board trials when the ship remains in fairly clean ports for intake and

discharge. In the case of PERACLEAN Ocean its active compound (PAA) has been

evaluated for total and fecal coliforms (E. coli: Antonelli et al. 2006 ) as well as with V.

cholerea (Baldry et al. 1995) and in both studies proven to be a effective method of

disinfection.

Evaluation of results

The present land‐based tests with the SEDNA®

‐System show that the BWT installation and

the management plan performs in accordance with the IMO Convention as stated in the

Standard‐D2 and according to the criteria for testing as indicated in the G8. The system has

been thoroughly tested for a considerable period (March till July) covering a full spring

season with a huge biodiversity and organism load. This period also included a large bloom

of the mucus containing phytoplankter Phaeocystis globosa, an organisms which is known to

clog nets of all sorts of sizes and certainly also the 50 m size used in the present filter. In

fact the system performed even better than the D2‐standard. For both size classes of

organisms the observed residual numbers of viable organisms on discharge were at least 100

fold lower than the Standard‐D2 allows. In addition also the plankton fraction < 10 m was

effectively killed by the system and no recovery was observed for up to 18 days. In general

the organism decompose completely and contribute to the pool DOC but can mainly be

traced as a amorphous fluffy substance free‐floating in the water or precipitating to the

bottom of the tanks.  

The fact that this system used an active substance, an organic acid combined with active

oxygen, resulted in a detailed examination of the (residual) effects of these compounds on

the organisms at intake but also after discharge. The life time of the active ingredients was

rather short, less than 24 h for PAA and a few days for H2O2, but this period increases at very

low temperatures (Lafontaine and Despatie 2006). Although this may result in a discharge of

low amounts of active compounds in the environment, these ingredients are not uncommon

in the aquatic environment, although natural concentrations are usually lower. The

degradation product of the active ingredient of PAA is HAc, also a common organic acid in

the aquatic environment although concentrations are usually several orders of magnitude

lower. Because of its acidic nature the pH was reduced, not fully buffered by the buffering

capacity of the seawater. In addition during microbial degradation of HAc oxygen was

utilized resulting in a reduced oxygen concentration of the water. These effects were only

temporarily and there was a full recovery within a period of a few days. The bacterial

degradation of the organic acid was completed within several days, resulting in a temporal

increase in microbial biomass. True oxygen depletion (anoxia) was never observed in the

treated tank. In contrast signs of true oxygen depletion were observed in the reference tanks

as could be derived from the typical smell of H2S during cleaning of the tanks.  

In conclusion, the present configuration of the SEDNA®

‐System and its active substance

PERACLEAN® Ocean 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 SEDNA®

‐System     35

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implications. Journal of Phycology 33: 527 ‐ 541

Veldhuis MJW, Fuhr F, Boon JP, Hallers‐Tjabbes CCt (2006) Treatment of ballast water;

how to test a system with a modular concept? Environmental technology 27:

909 ‐ 921

Veldhuis MJW, Kraay GW (2000) Application of flow cytometry in marine phytoplankton

research: current applications and future perspectives. Scientia Marina 64: 121 ‐ 

134

Veldhuis MJW, Kraay GW, Timmermans KR (2001) Cell death in phytoplankton:

correlation between changes in membrane permeability, photosynthetic

activity, pigmentation and growth. European Journal of Phycology 36: 167 ‐ 177

Wagner R, Ruck W (1984) Die Bestimmung von Wasserstoffperoxid und andere

Peroxyverbindungen. Z. Wasser‐ Abwasser‐Forsch. 17: 262 ‐ 267

Williams RJ, Griffiths FB, Van der Wal EJ, Kelly J (1988) Cargo vessel ballast water as a

vector for the transportation of non‐indigenous marine species. Estuar., Coast.

and Shelf Sci. 26: 409 ‐ 420
Final report of the land‐based testing of the SEDNA®

‐System     37

Appendix 1 

Phytoplankton Marsdiep April‐   July 2007 +: 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 Leptocylindrus danicus ++

diatom centricate Brockmanniella brockmannii ++

diatom centricate Cerataulina pelagica ++

autotrophic flagellate autotrophic flagellate indet. <10 μm   ++

diatom centricate Chaetoceros spp. <10 μm solitairy cells +

autotrophic flagellate Chlorophyceae indet.    +

heterotrophic flagellate Chrysophyceae indet.   +

freshwater green alga Crucigenia spp. +

heterotrophic flagellate Telonema spp. +

freshwater green alga Chlorophyta fresh indet.    +

diatom centricate Skeletonema "costatum" lenses +

freshwater green alga Oocystis spp. +

diatom pennate Asterionellopsis glacialis +

autotrophic flagellate Rhodomonas group +

diatom centricate Odontella aurita +

heterotrophic flagellate Bodo group +

diatom centricate Chaetoceros spp. >10 μm no bristle chloroplast +
Final report of the land‐based testing of the SEDNA®

‐System     38

Species list zooplankton and some larger (atypical) plankton 

Appendix 1 cont.

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       
Final report of the ship board testing of the SEDNA®

‐System 1

   

BSH / M5101                 Texel March 2008

Final report of the shipboard testing of the  

SEDNA®­System, for Type Approval according to  

Regulation D­2 and the relevant IMO Guideline

  

(June – December 2007)

Shipboard testing on board OOCL Finland

><((((°>

S. Gollasch  Ph‐D  (GoConsult)

Grosse Brunnenstr. 61

22763 Hamburg

Germany

phone: +49 40 390 546 0

email: sgollasch@aol.com

Marcel J.W. Veldhuis Ph‐D    

Frank Fuhr Dipl.‐Biol.

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 or ffuhr@nioz.nl
Final report of the ship board testing of the SEDNA®

‐System 2

   

BSH / M5101        

Final report of the shipboard testing of the  

SEDNA®­System, for Type Approval according to  

Regulation D­2 and the relevant IMO Guideline  

(June – December 2007)

Shipboard testing on board OOCL Finland

Dr. Stephan Gollasch 

 
Final report of the ship board testing of the SEDNA®

‐System 3

   

Content                   page

General                    4

Description of the ships ballast water system and sampling regime     4

Results                     5

Discussion of the results               8

References                   9
Final report of the ship board testing of the SEDNA®

‐System 4

   

General

The shipboard tests for final approval took place on three voyages of the container vessel OOCL Finland in

2007. The details of the vessel are outlined in the following tables 1 ‐ 3.

Table 1: Main dimensions of the test vessel

Length overall Loa 134.44 m 

Length b. perpendiculars Lpp 124.41 m 

Breadth (mld.) B 22.50 m 

Depth to main deck (mld.) D 11.30m / 14.00 m 

Scantling draft (mld.) dmax 8.712 m 

Table 2: Ballast water capacity of the test vessel

Total ballast water capacity 3932 m3 

Extra ballast water capacity 0 

Number of segregated ballast water tanks 21 

Number of other tanks used for ballast water 0 

Number of ballast water pumps 2 

Capacity of ballast water pumps (each) 225 m3/h 

Table 3: Tonnage measurement of the test vessel

Deadweight tdw abt. 11250 t 

Gross Tonnage 9981 GT 

Net Tonnage 6006 NT 

Description of the ships ballast water system and the sampling regime

The test ship is trading in short sea shipping, sailing in the Baltic and North Seas region. Due to the nature

of the trade (containers), it was difficult to identify a suitable set of ballast water tanks for the G8

certification tests. According to the requirements of the G8 guideline for shipboard testing, both ballast

water tanks, the control tank and the treated tank, must be filled and emptied in total, either in parallel or

in sequence. However, the stability of small container ships like the test ship do allow only for ballast

water operations in accordance with the load conditions. This normally includes the partly filling and/or

emptying of ballast water tanks. The double bottom tanks no. 2 P and no. 2 Stb were identified as the only

suitable tanks for the tests. Both tanks had a volume of 70m3. Because of this small volume, the samples

could be collected over each entire filling and emptying of the tanks, respectively. This resulted in a much

more accurate measurement of organism densities than just sub‐samples from parts of the ballast water

operation.

The installed SEDNA®‐System had a capacity to match the capacity of the ballast water pumps. Both

systems were installed on deck in a special 40‐foot container. The SEDNA®‐System was fully implemented

and integrated into the ballast system of the ship. A ballast water management plan was developed

especially for this ship, with the ballast water treatment being the preferred ballast water management

option. The Ballast water management plan as a whole was certified by Germanischer Lloyd (GL).
Final report of the ship board testing of the SEDNA®

‐System 5

   

Tests were conducted by Dr. S. Gollasch (GoConsult, Hamburg, Germany). In total five consecutive, valid

test runs were undertaken during the test period from June 2007 to December 2007. It should be noted

that during the fifth test run carried out from 05.12.2007 to 06.12.2007, the treated ballast water also

complied with the water quality standard D‐2.

Since no elevated level of mortality compared to the other test runs was observed in the control tank of

the fifth test run, the results were also taken into account for the evaluation of the systems performance.

During each of the test runs samples were collected by using multiple Hydrobios nets in parallel. The total

volume of each of the samples in summarized in table 4.

Table 4: Details of the test runs. The volumes given for the samples represent single samples for the control tank and

the treated tank upon uptake, as well as triplicate samples for the treated tank upon discharge

BWOperation Test-cycle no. 1 Test-cycle no. 2 Test-cycle no. 3 Test-cycle no. 4 Test-cycle no. 5 

Date BWuptake 16.06.07 29.08.07 02.09.07 28.11.07 05.12.07 

Location 

region leaving Gävle east of Bornholm Kotka near Texel west of St. Petersburg 

control treated control treated control treated control treated control treated 

Sample vol. 

[l] 1.232 1.534 1.290 1.429 1.141 1.207 1.010 1.012 1.438 1.672 

Retention 

time 32 hrs 58 hrs 42 hrs 31 hrs 30 hrs 

Date BWdischarge 18.06.07 01.09.07 04.09.07 30.11.07 06.12.07 

Location 

region 

before Kiel 

Canal Kotka St. Petersburg west of Bornholm 

before Kiel 

Canal 

control treated control treated control treated control treated control treated 

Sample vol. 2.117 

2.561 

2.914 

2.416 

3.071 

2.628 

2.542 

1.238 

1.859 

1.898 

1.887 

1.658 

10.17 

1.070 

1.004 

1.017 

2.220 

2.387 

2.383 

2.563 

Plankton organisms larger than 50 μm in minimum dimension were analysed on board directly after

sampling. Human pathogens were prepared onboard for later analysis by Vitens BV in Leeuwarden, The

Netherlands and all other samples were processed at NIOZ, Texel, The Netherlands. Procedures were

followed according to the test protocol. Like in the North Sea region the human pathogen Vibrio cholera

(O1 and O139) was not present in the Baltic Sea (e.g. Hökeberg 2007). Due to the reliable information

about the absence of V. cholera, in combination with the problems associated with testing for V. cholera

on a routine basis, it was decided not to perform extra tests for V. cholera. Furthermore the efficacy of

the active components used, peracetic acid and hydrogen peroxide, against V. cholera has been shown

before (Baldry et al 1995, Sagripanti et al 1997).

Results

The basic set of abiotic parameters analysed during the different tests runs are summarized in table 5.

Table 5: Physical parameters of the source water (temperature, salinity, total suspended solids and particulate

organic carbon) according to the G8 Guidelines. The values represent single samples for the control tank and the

treated tank upon uptake, as well as  the average of the triplicate samples for the treated tank upon discharge. b.d.l.:

below detection limit ( about 2 to 5 mg/l due to transport and handling)
Final report of the ship board testing of the SEDNA®

‐System 6

   

BWOperation Test-cycle no. 1 Test-cycle no. 2 Test-cycle no. 3 Test-cycle no. 4 Test-cycle no. 5 

Uptake 16.06.07 29.08.07 02.09.07 28.11.07 05.12.07 

Location 

region leaving Gävle east of 

Bornholm Kotka near Texel west of St. 

Petersburg 

control treated control treated control treated control treated control treated 

T°C 

Sal. 

TSS [mg/L] 

POC [mg/L] 

11,0 

9,11 

~ 30 

~ 10 

10,6 

8,92 

~ 30 

~ 10 

15,9 

5,2 

~ 40 

~ 15 

15,6 

5,2 

~ 40 

~ 15 

15,8 

5,3 

~ 50 

~ 30 

15,9 

5,3 

~ 40 

~ 20 

9,9 

20,5 

~ 20 

b.d.l. 

9,9 

20,5 

~ 20 

b.d.l. 

3,9 

5,2 

~ 40 

~ 25 

3,8 

5,1 

~ 40 

~ 25 

Discharge 18.06.07 01.09.07 04.09.07 30.11.07 06.12.07 

Location 

region 

before Kiel 

Canal Kotka St. Petersburg west of 

Bornholm 

before Kiel 

Canal 

 control treated control treated control treated control treated control treated 

T°C 

Sal. 

TSS [mg/L] 

POC [mg/L] 

14,6 

8,93 

~ 10 

b.d.l. 

14,4 

8,88 

~ 10 

b.d.l. 

14,3 

5,3 

~ 20 

~ 8 

14,1 

5,1 

~ 10 

b.d.l. 

14,4 

5,4 

~ 10 

b.d.l. 

14,6 

5,2 

~ 10 

b.d.l. 

8,4 

22,0 

~ 20 

~ 10 

7,4 

23,0 

~ 8 

b.d.l. 

7,1 

4,8 

~ 20 

~ 15 

7,0 

5,3 

~ 10 

b.d.l. 

The biological results of the shipboard tests are summarized in the tables 6 to 10. The results show that

the SEDNA®

‐ballast water treatment system complied with the standards in Regulation D‐2 for all tested

groups during all tests. This is consistent with the results of the land based tests. There are two remarks to

be made related to the results. Firstly, the low number of organisms in control tank at discharge during

the fifth test run (see above). Secondly, due to technical problems no results on the human pathogens are

available for the second test cycle (Table 7).  

Table 6: Results first test cycle from 16.06. (Gävle) to 18.06.2007 (Kiel Canal). Uptake: Number of organisms during

BW‐uptake, Discharge: Number of organisms during BW‐discharge, n.d.: not determined

Group Control Treated 

 uptake discharge uptake discharge 

> 50μm / m3 1.623 387 1.245 1,1 

10μm-50μm / mL 576 185 495 <0.1 

bacteria * 10^4/ mL* 143 168 148 231 

E. coli [cfu / mL] <0.1 0,16 <0.1 <0.1 

Enterococcus [cfu / mL] <0.1 <0.1 <0.1 <0.1 

Vibrio cholera 

[cfu / mL] n.d. n.d. n.d. n.d. 

total heterotrophic bacteria / 

mL 300 190 120 5 

* these are total bacteria counts 
Final report of the ship board testing of the SEDNA®

‐System 7

   

Table 7: Results second test cycle from 29.08. (Bornholm) to 01.09.2007 (Kotka). Uptake: Number of organisms

during BW‐uptake, Discharge: Number of organisms during BW‐discharge, n.d.: Not determined

Group Control Treated 

 uptake discharge uptake discharge 

> 50μm / m3 11.757 1.791 6.786 0,0 

10μm-50μm / mL 1.784 1123 1.254 <0.1 

bacteria * 10^4/ mL* 188 172 174 202 

E. coli [cfu / mL] n.d. n.d. n.d. n.d. 

Enterococcus [cfu / mL] n.d. n.d. n.d. n.d. 

Vibrio cholera 

[cfu / mL] n.d. n.d. n.d. n.d. 

total heterotrophic bacteria / 

mL n.d. n.d. n.d. n.d. 

* these are total bacteria counts

Table 8: Results third test cycle from 02.09. (Kotka) to 03.09.2007 (St. Petersburg). Uptake: Number of organisms

during BW‐uptake, Discharge: Number of organisms during BW‐discharge, n.d.: Not determined

Group Control Treated 

 uptake discharge uptake discharge 

> 50μm / m3 4.981 395 2.845 0,0 

10μm-50μm / mL 785 382 764 <0.1 

bacteria * 10^4/ mL* 201 236 245 211 

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

Enterococcus [cfu / mL] <0.1 <0.1 <0.1 <0.1 

Vibrio cholera 

[cfu / mL] n.d. n.d. n.d. n.d. 

total heterotrophic bacteria / 

mL >10000 >10000 30 <10 

* these are total bacteria counts
Final report of the ship board testing of the SEDNA®

‐System 8

   

Table 9: Results fourth test cycle from 28.11. to 30.11.2007. Uptake (To): Number of organisms during BW‐uptake,

Discharge (Ta): Number of organisms during BW‐discharge, n.d.: Not determined

Group Control Treated 

 uptake (To) discharge (Ta) uptake (To) discharge (Ta) 

> 50μm / m3 624 324 919 0,0 

10μm-50μm / mL 184 166 179 <0.1 

bacteria * 10^4/ mL* 385 295 315 198 

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

Enterococcus [cfu / mL] <0.1 <0.1 <0.1 <0.1 

Vibrio cholera 

[cfu / mL] n.d. n.d. n.d. n.d. 

total heterotrophic bacteria / 

mL 1300 <10 700 15 

* these are total bacteria counts

Table 10: Results fifth test cycle from 05.12. to 06.12.2007. Uptake (To): Number of organisms during BW‐uptake,

Discharge (Ta): Number of organisms during BW‐discharge, n.d.: Not determined

Group Control Treated 

 uptake (To) discharge (Ta) uptake (To) discharge (Ta) 

> 50μm / m3 209 20 224 0,0 

10μm-50μm / mL 41 39 39 <0.1 

bacteria * 10^4/ mL* 346 248 327 211 

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

Enterococcus [cfu / mL] <0.1 <0.1 <0.1 <0.1 

Vibrio cholera 

[cfu / mL] n.d. n.d. n.d. n.d. 

total heterotrophic bacteria / 

mL 140 >10000 <10 <10 

* these are total bacteria counts

Discussion of results

The ship board tests with the SEDNA®‐System showed that the ballast water treatment (BWT) installation

and the management plan performed in accordance with the standards as stated in Regulation D‐2 of the

IMO Ballast Water Management Convention and further meets the test criteria as indicated in the IMO

Guideline G8 for type approval tests of BWTS. The system has been thoroughly tested for a considerable

period (June until December) in different environmental conditions and during different seasons. During

these tests the SEDNA®

‐system has proven to be seaworthy and highly effective. The performance

onboard the OOCL Finland was consistent with results obtained during land based tests. The slightly

higher number of surviving organisms found in comparison to the land based tests can be attributed to
Final report of the ship board testing of the SEDNA®

‐System 9

   

residual, untreated water remaining present in the ballast pipes of the ship. This is due to the fact that for

shipboard testing not all ballast tanks were treated nor that the BWT system is used continuously. This is

most likely also the reason for the observed variations of numbers at intake, and especially for bacteria at

discharge of the control tank during the third test run. Unless the ballast water of the whole ship is

treated this will remain a problem and therefore a factor to consider.

Like for the land based test the shipboard tests also included inspection for all size ranges of plankton and

not only the size classes relevant to Regulation D‐2.  Although not presented in this report also in the size

class <10 m no living (phyto)plankton was observed in the treated water upon discharge. Also the

number of both total and heterotrophic bacteria dropped significantly in the treated water as compared

with the intake and control water. In this respect the results differed slightly with those of the land‐based

tests showing occasionally an increase in total and heterotrophic bacteria in the treated water. This was

mainly due to the fact that the remaining acetic acid of the PERACLEAN Ocean acted as a substrate for

growth of bacteria which apparently did not occurred during the ship tests during the given ballasting and

deballasting periods.  

  

References 

Baldry MGC, Cavadore A, French MS, Massa G, Rodrigues LM, Schirch PFT, Threadgold TL (1995) Effluent

disinfection in warm climates with peracetic acid Wat. Sci. Tech. 31: 161 ‐ 164

Sagripnati, J‐S, Eklund, CA, Trost, PA, Jinnemans, KC, Abeyta, C, Kaysner, CA, Hill, WE. 1997 Comparative

sensitivity of 13 species of pathogenic bacteria to seven chemical germicides. AJIC 335 – 339.

Hökeber, I, 2007. Swimmers catch severe infections in the Baltic Sea. EpiNorth

(www.epinorth.org./artikler/?id=45868)
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(NWO).

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