Final report of the land-based testing of the Hyde-GuardianTM-system 1

Texel, January 2009

Final report of the land-based 

testing of the Hyde-GuardianTM 

-System, for Type Approval 

according to the Regulation D-2 

and the relevant IMO Guideline 

(April – July 2008)

Marcel J.W. Veldhuis Ph-D

Royal Netherlands Institute for Sea Research

P.O.Box 59

1790 AB, Den Burg, TEXEL

the Netherlands

phone: +31-222-369512/300

email: veldhuis@nioz.nl
Final report of the land-based testing of the Hyde-GuardianTM-system 2

Final report of the land-based 

testing of the Hyde-GuardianTM 

-System, for Type Approval 

according to the Regulation D-2 

and the relevant IMO Guideline 

(April – July 2008)

Marcel J.W. Veldhuis Ph-D

Frank Fuhr Dipl.-Biol.

Peter-Paul Stehouwer MSc.

Royal Netherlands Institute for Sea Research

P.O.Box 59

1790 AB, Den Burg, TEXEL

the Netherlands

phone: +31-222-369512/300

email: veldhuis@nioz.nl, ffuhr@nioz.nl, pstehouwer@nioz.nl
Final report of the land-based testing of the Hyde-GuardianTM-system 3

Final report of the land-based 

testing of the Hyde-GuardianTM 

-System, for Type Approval 

according to the Regulation D-2 

and the relevant IMO Guideline 

(April – July 2008)

Royal Netherlands Institute for Sea Research

P.O.Box 59

1790 AB, Den Burg, TEXEL

the Netherlands

phone: +31-222-369512/300

email: veldhuis@nioz.nl or ffuhr@nioz.nl
Final report of the land-based testing of the Hyde-GuardianTM-system 4

Content page

1 Executive summary 5

2 Zusammenfassung 6

3 Summary table 7

4 Acknowledgements 8

5 Introduction 8

6 Description of the test facility 9

6.1 NIOZ Royal Netherlands Institute for Sea Research 9

6.2 Portrait of Hyde Marine; producer of the Hyde-GuardianTM-system11

6.3 The test facility 11

6.4 Technical description of the Hyde-GuardianTM-system 13

7 Requirements to meet the D2 Standard 15

7.1 Requirements to meet: guideline G8 15

7.2 Experimental design 18

8 Results 24

8.1 Physical and chemical parameters 24

8.2 Biology 28

9 Environmental acceptability 35

10 Discussion and evaluation of results 37

11 References 39

Appendix 1: species list of phytoplankton and zooplankton 41

present during the various test runs.
Final report of the land-based testing of the Hyde-GuardianTM-system 5

1 Executive summary

The Hyde-GuardianTM-system, a ballast water management system was tested according to 

the standard as set out in regulation D-2 of the “International Convention for the Control 

and Management of Ships’ Ballast Water and Sediments,” hereafter referred to as the “D2-

Standard” and consistent with the IMO Guidelines for Type Approval testing (G8). The tests 

were conducted in the spring and early summer of 2008 in the harbour of the Netherlands 

Institute for Sea Research. 

In general most of the requirements for testing which concern water characteristics and 

abundance of organisms were met and in most test runs environmental conditions were far 

more challenging than strictly needed. The Hyde-GuardianTM-system is designed as a UV-light 

disinfection system (UV-reactor) with a mechanical filter as a pre-treatment step. The water 

is treated at intake and at discharge, i.e a two-fold disinfection of the ballast water. In total 

10 test runs in a row were successful for all relevant parameters and the BWT system 

performed on average much better than stated in Regulation-D2 by achieving values for 

organisms well below the requirements of the D2 Standard. For many size classes of 

organisms the residual number of organisms was often one order of magnitude lower than 

the number in D2-Standard. This system should therefore be regarded as an effective way of 

treating ballast water in ships, thereby minimizing the risk of new invasions originating from 

the ships ballast water. 

The sediment load was reduced in a pre-treatment step (self-cleaning disk filters) and the 

organisms were deactivated by ultraviolet light disinfection. 

Although numbers of phytoplankton and bacteria were also largely reduced by the 

ultraviolet light disinfection step not all cells were fully disintegrated. Complementary tests 

indicated that the remaining organisms possessed a reduced viability and were (partly) 

deactivated.

Treated water, which was transferred to favourable conditions, showed no regrowth within 

a period of 10 days. Environmental acceptability tests however showed that the growth of 

organisms, mainly plankton, was not limited by the discharge water indicating that the 

discharged water was still vital.
Final report of the land-based testing of the Hyde-GuardianTM-system 6

2 Zusammenfassung 

Das Hyde-GuardianTM-system zur Behandlung von Ballastwasser wurde im Frühjahr und 

Sommer 2008 gemäß der Regularien-D2 (D2-Standard), sowie der IMO Richtlinien über Tests 

für eine Typzulassung (G8) im Hafen des Königlich Niederländischen 

Meeresforschungsinstitut (NIOZ) getestet. Generell wurden die allermeisten Anforderungen 

bezüglich der abiotischen Parameter des Testwassers und zur Organismendichte erfüllt. In 

den meisten Tests waren die Bedingungen sogar schwieriger, als in den Richtlinien verlangt. 

Das Hyde-GuardianTM-System arbeitet mit UV-Licht als Desinfektionseinheit (UV-Reaktor). 

Zusätzlich verfügt es über einen mechanischen Filter als Vorabreinigungsstufe. Das Waser 

wird bei Aufnahme und Abgabe behandelt, d.h. es findet eine Doppelbehandlung des 

Ballastwassers statt. Es wurden 10 voneinander unabhängige Tests nacheinander, 

erfolgreich durchgeführt. Das System erfüllte alle Anforderungen der Regularien-D2 und der 

Richtlinie G8. Die Organismenanzahlen nach der Behandlung lagen deutlich unter den 

Anforderungen des D2-Standards, für einige Gruppen im Durchschnitt sogar um eine 

Größenordnung. Das System sollte demzufolge als effektive und sichere Möglichkeit zur 

Behandlung von Ballastwasser betrachtet werden, die dazu beitragen kann, weitere 

biologische Invasionen zu verhindern.

Die Sedimentfracht wurde in einem Vorbehandlungsschritt reduziert (selbstreinigende 

Scheibenfilter) und die Organismen durch UV-Bestrahlung inaktiviert.

Obwohl die Anzahl von Phytoplanktern und Bakterien durch die UV-Behandlung stark 

reduziert wurden, wurden nicht alle Zellen vollständig zerstört. Ergänzende Versuche 

ergaben, dass ein Teil dieser Zellen noch eine eingeschränkte Vitalität zeigte und nur 

teilweise inaktiviert war. Dennoch wurde in Inkubationsexperimenten über Zeiträume von 

mehr als 10 Tagen unter vorteilhaften Umweltbedingungen kein Planktonwachstum im 

behandelten Wasser festgestellt. Gleichzeitig durchgeführte Umweltverträglichkeitstests 

(Zugabe von Zeigeorganismen und Planktonkulturen, Verdünnungsreihen) mit dem 

behandelten Wasser ergaben jedoch keine negativen Auswirkungen auf das 

Planktonwachstum und zeigten somit, dass das behandelte Wasser selbst das Wachstum 

gesunder Organismen nicht beeinflusst.
Final report of the land-based testing of the Hyde-GuardianTM-system 7

3 Summary table with results for the Type Approval Certificate of the Hyde-GuardianTM-BWT-System

Land-based tests NIOZ Reference & Treated Reference Treated

salinity 22.1 PSU Intake Discharge Discharge Discharge*

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

total bacteria [counts/mL] 3.3+E6 1.3+E6 6.6+E6 2.1+E6 0.73+E6 3.6+E6 2.4+E6 0.38+E6 5.2+E6 0.31+E6

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

Enterococci [cfu/mL] <1 < 1 <1 < 1 < 0.1 1 < 0.1 < 0.1 < 0.1 < 1

plankton <10 μm [counts/mL] 5446 235 13646 589 62 1939 <10 <10 <10 12

plankton 10-50 μm [counts/mL] 1028 304 1784 147 140 156 <10 <10 <10 2.3

plankton >50 μm [counts/m3] 25.5+E5 10.1+E5 48.3+E5 3.95+E3 617 10.8+E3 2.9 n.d 7.3 -

Land-based tests NIOZ Reference & Treated Reference Treated

salinity 31.9 PSU Intake Discharge Discharge Discharge*

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

total bacteria [counts/mL] 4.2+E6 0.72+E6 6.3+E6 2.1+E6 0.33+E6 5.4+E6 0.88+E6 0.24+E6 1.2+E6 0.64+E6

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

Enterococci [cfu/mL] 8.3 < 1 17 < 0.1 < 0.1 1 < 1 < 1 < 1 < 1

plankton <10 μm [counts/mL] 2366 1194 3754 290 173 632 <10 <10 <10 1.9

plankton 10-50 μm [counts/mL] 1378 514 2090 140 121 151 <10 <10 <10 1.0

plankton >50 μm [counts/m3] 1.58+E5 0.52+E5 4.11+E5 1.54+E4 0.25+E4 0.84+E4 n.d. n.d. n.d. -

Summary table (average and minimum and maximum) of collected data covering the major groups of organisms at two series of 5 test-runs at a low and high 

salinity range, respectively. n.d.: not detectable in sample; -: no data; * numbers as determined in bottle incubations of discharge water.
Final report of the land-based testing of the Hyde-GuardianTM-System 8

4 Acknowledgements

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

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

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

project. 

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

Wadden Sea.

We also thank Mr. Rolf von Ostrowski, Mrs. Karin Sigler and Dr. Kai Trümpler of the 

Bundesambt für Seeshifffahrt und Hydrographie, acting on behalf of the German 

Administration, for their excellent collaboration during this whole certification process.

5 Introduction

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

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

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

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

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

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

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

end of a ship’s voyage. 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 chance of non-indigenous organisms to have large 

enough numbers for settling and expanding. The problem of invasive species is considered as 

one of the 4 major threats of the world’s oceans next to land-based marine pollution, 

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

To minimize these risks for the future, the International Maritime Organization (IMO) of the 

United Nations has adopted the Ballast Water Convention in 2004 (Anonymous 2005). The 

Convention states that finally ALL ships (>50,000 in number) should install proper ballast 

water treatment (BWT) equipment on board between 2009 and 2016. As a temporary and 

intermediate solution for the time being ship may reduce the risk of invasive species by 

performing ballast water exchange during their voyage when passing deep water (>200 m 

depth and 200 NM from the coast. Ballast water exchange faces many problems as to 

feasibility, safety and efficacy For a large part of ships’ voyages the required depth and/or 

distance to shore requirements are never met; BW exchange can affect the ships 

construction stability and in rough seas exchange is not possible because of the risk to ship 

and crew. Treatment of ballast water is therefore considered to be the best solution of 

reducing the risk of invasive species.

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 & Taylor 2001). However, next to a high efficacy there is more needed for a 

BWT system to be a good 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 Hyde-GuardianTM-System 9

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 & 

Taylor 2001; MEPC 49/2/13, 2003). Besides reducing the load of organisms the sediment 

load should be reduced as well. There are also considerable differences in ships operation, 

types of ships, the amount of space available for a ballast water treatment system on board 

and the way ships are operated. Ballast water treatment will develop into a new field of 

technology of its own with a commercial market estimated for the next 10 years in the order 

of 8 billion Euro (Haskoning 2001).

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

as to study 

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

treatment with the Hyde-GuardianTM- Ballast Water Treatment system (efficacy of the 

BWT system), 

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

3) to assess the environmental acceptability of discharging ultraviolet light 

disinfected ballast water by measuring residual effluent toxicity in order to determine 

latent effects, since ballast water treated with this specific UV-system does not need a 

complete G9 procedure (environmental impact).

This research strategy allows for more in depth testing, while it includes ALL organisms and 

not only the size classes as specified in the Convention D2-standard. 

6 Description of the treatment facility 

6.1 NIOZ Royal Netherlands Institute for Sea Research

NIOZ Royal Netherlands Institute for Sea Research is the National Oceanographic Institute of 

the Netherlands. NIOZ is part of the Netherlands Organization for Scientific Research (NWO). 

The institute employs around 200 people and the annual budget is approximately €20 

million.

The mission of NIOZ is to gain and communicate scientific knowledge on seas and oceans for 

the understanding and sustainability of our planet. The institute also facilitates and supports 

marine research and education in the Netherlands and in Europe.

In order to fulfil its mission, the institute performs tasks in three specific fields.

Research: The emphasis is on innovative and independent fundamental research in 

continental seas and open oceans. The institute also carries out research based on societal 

questions when this merges well with its fundamental work. The senior scientists at NIOZ all 

participate in international research projects.

Education: The institute educates PhD and other students of universities and schools for 

professional education. Together with universities NIOZ also organises courses for PhD 

students and master students in the marine sciences. A number of our senior scientists of 

NIOZ is also appointed as professor at Dutch and foreign universities.

Facilitary services: NIOZ invites marine scientists from Dutch and foreign institutes and 

universities to write scientific proposals involving the institute’s research vessels, 

laboratories, and the large research equipment, which is often designed and built by the 

institute’s own technical department.

The basic oceanographic disciplines studied at NIOZ are physics, chemistry, biology and 

geology. Multidisciplinary research is regarded as one of the main strengths of NIOZ.

More information on www.nioz.nl
Final report of the land-based testing of the Hyde-GuardianTM-System 10

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

TESO ferry (top).
Final report of the land-based testing of the Hyde-GuardianTM-System 11

6.2 Portrait of HYDE-MARINE/LAMOR (producer of the 

HYDE-GUARDIANTM-ballast water management system)

More information on: www.hydemarine.com

Ballast water treatment - The Hyde-GuardianTM-system

Hyde Marine, Inc. traces its origins back to the Hyde Windlass Company, established in Bath, 

Maine in 1865. Hyde Windlass was the largest supplier of anchor windlasses, winches, 

capstans, steering gear and specialty deck machinery in the United States before, during and 

after the First and Second World Wars. 

Hyde Marine has been involved in ballast water treatment (BWT) since 1996, when it 

teamed with the Univ. of Michigan to study potential technologies for BWT, particularly for 

ships operating in the Great Lakes St. Lawrence Seaway System. This led to Hyde's 

participation, beginning in 1997, as the engineering contractor for the Great Lakes Ballast 

Technology Demonstration Project (GLBTDP). The GLBTDP was one of the first BWT research 

programs in North America and conducted tests of filtration technology for BWT aboard a 

Canadian Great Lakes bulk carrier, the Algonorth. After a season aboard the Algonorth, the 

test equipment was transferred to a barge in Duluth Superior Harbor (Lake Michigan), where 

additional testing of the filtration system and other BWT technologies was conducted, also 

under the GLBTDP. 

Hyde first became involved in its own BWT equipment in 2000, when it installed its initial full 

scale first generation system aboard the US based cruise ship Regal Princess. In 2001 Hyde 

installed four additional systems, two on cruise ships and one each on a container ship and 

chemical tanker. In 2003, after the requirements were better defined, Hyde installed a 

state-of-the-art filtration and UV disinfection system aboard the Coral Princess. This system, 

named the Hyde-GuardianTM-system, was tested extensively on land based installations and 

on board the Coral Princess in the fall of 2004. The on-board tests demonstrated the Hyde 

Guardian's capability to meet the IMO BWT Convention requirements. This system is now 

first in line for acceptance into the U.S. Coast Guard STEP program. In the fall of 2006 an 

essentially identical system was installed aboard RCCL's Celebrity Mercury. It was 

commissioned early in 2007. The Hyde-GuardianTM-systems aboard the Coral Princess and 

Mercury were granted interim approval for use in Washington State waters by the State of 

Washington in 2004 and 2007 respectively. All seven systems were commercial transactions 

and the newest version, the Hyde-GuardianTM-system, has been fully commercially available 

since early 2003.

6.3 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 2). 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.
Final report of the land-based testing of the Hyde-GuardianTM-System 12

Figure 2: 

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 μmol quanta. 

m-2

.s-1)

The Hyde-GuardianTM-system is connected to a typical ballast water pump (capacity of 250 

m3

/h) 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 3.

Figure 3: P&I diagram of the Ecochlor and Hyde-GuardianTM-system installation at NIOZ with 

points of sampling 
Final report of the land-based testing of the Hyde-GuardianTM-System 13

6.4 Technical description of the Hyde-GuardianTM-System

The Hyde-GuardianTM-system operates in-line during the uptake of the ballast water. It is 

based on a modular concept which includes a physical separation as well as disinfection with 

a UV-light source resulting in permanent deactivation of organisms.

The Hyde-GuardianTM-system used during the tests at NIOZ has been build to the same 

standards as the double 250 m3/h containerised Hyde-GuardianTM-system that is installed on 

the test ship (fig. 4).

 

Figure 4: The Hyde-GuardianTM

skid mounted unit

The system consists of a UV-reactor (BersonInLine), a medium-pressure ultraviolet light 

disinfection system with a typical output of UV-C (Fig 5; 200 – 300 nm wavelength) combined 

with a pre-filter. The advantage of a pre-filter (Spin Klin® discs) is to reduce the number of 

larger organisms of which is known that UV is not a very effective way of deactivation 

(Raikow et al. 2007). In addition filtration reduces the load of (larger) particles and increases 

therefore the transparency of the water, hence the effectiveness of the UV irradiation.

Figure 5: spectral 

composition of the 

medium pressure (MP) 

lamp of the 

BersonInLine UVsystem used in the 

Hyde-GuardianTM-

system.

Unlike active substances the working of ultraviolet radiation (UV) is based on transferring 

electromagnetic energy to molecular components of organisms, resulting e.g. in DNA 

damage. This will prevent cell replication (growth and reproduction) but also causes 

(immediate) disintegration of cells, morphological changes, discoloration, cellular membrane 

leakage and cell damage. 
Final report of the land-based testing of the Hyde-GuardianTM-System 14

To disinfect municipal water UV is commonly used because in general it does not 

significantly contribute to Disinfection By-Products (DBP; (Malley et al. 1995; Chin & Bérubé 

2005).

The UV lamps in the Hyde-GuardianTM-system are of the medium pressure type and run at a 

nominal of 100 W per centimeter in length. This produces an average intensity of 140

mW/cm2

. The end of lamp life is not when a lamp fails to light but rather when its output has 

decreased by 30%. The physics inside a UV lamp does not change over time, i.e. at the heart 

of the lamp the produced UV remains constant. As the amount of UV that is emitted 

decreases it is necessary to increase the power applied to the lamp to keep the UV intensity

above the minimum levels. The Hyde-GuardianTM-system uses a UV sensor to continuously 

monitor the output of the lamps. Besides monitoring the output of UV-C (200 – 280 nm) the 

UV sensor can also monitor the cleanliness of the sleeves and indirectly the water quality.

Because the power delivered to the UV lamp is not infinitely adjustable the majority of the 

time the system will be overdosing the UV. This is probably the only disinfection technology 

that can safely be overdosed. The control system will keep the amount of energy used to a

minimum but given the discrete steps available for power control a new lamp will be running 

about 130% of the required UV. 

The Hyde-GuardianTM-system differs from other BWT-systems as the system is not only 

treating during intake (disc filter and UV-reactor) but also at discharge (only UV-reactor). 

Assessment of the efficacy of the BWT system was therefore immediately at intake (T0), at

discharge after a holding period of 5 days (T5) and 5 days after discharge (T10)

In addition to sampling the large holding basins for a period of 5 days, subsamples (with a 

volume of 10 L) of the reference and treated tank were taken at intake and discharge and 

incubated in a climate room under optimal growth conditions for the plankton community 

present in the test water. In case the treatment is insufficient and residual viable organisms 

remain present, or there is germination of resting stages or cysts, plankton growth will be 

stimulated under these favourable conditions. This outgrow experiment also allows studies 

on the effect of the treatment over a period longer than 5 days (up to 20 days). A sufficiently 

high level of nutrients will be maintained, favouring phytoplankton growth and possible cyst 

germination throughout the incubation experiments.

The applied test protocols were communicated with the Maritime Coastguard Agency (MCA) 

as well as with the German Administration (Federal Maritime and Hydrographic Agency of 

Germany; BSH) and a brief description of the various methods is included in the next section. 

During the certification process the whole practical procedure of intake and discharge has 

been witnessed on 4 occasions (intake and discharge) by a classification society (Lloyds 

Register, London and Rotterdam) as well as national and international agencies. 

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

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

made public at the moment of awarding the certificate.
Final report of the land-based testing of the Hyde-GuardianTM-System 15

7 Requirements to meet the D2-Standard

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

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

performance standard must discharge:

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

Furthermore, with exception of some indicator microbes (point 3) organisms < 10 μm are 

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

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

Nevertheless, the standard is clear with respect to the maximum number of organisms 

remaining present. On the other hand a proper definition of the dimensions of organisms is 

still subject of (academic) discussion. Moreover, as an operational definition for viable 

organisms the IMO is using: “organisms and any life stages thereof that are living”, but a 

more adequate (scientific) definition is: an organism that is able to complete it’s life-cycle, 

including reproduction (DNA replication). In the case of UV it is known that for certain 

organisms cell may be (genetically) damaged but remain active for a considerable period.

The organisms however, lack the ability to reproduce.

In addition to the (basic) requirements for the D2-Standard we have adopted a variety of 

methods and techniques to determine abundance, sizes and the viability status of different 

types of organisms. This includes also plankton < 10 μm other than bacteria (i.e. 

phytoplankton and viruses). Moreover, we extended our research effort to examine not only 

the fate of the organisms in the large-scale holding tanks but also took subsamples for 

incubation under optimal growth conditions in our to study the growth potential of 

remaining (viable) organisms and/or survival stages such as eggs, cysts or dormant cells over 

a longer period than the recommended 5 days. These later experiments allowed us also to 

study potential latent toxicity effects (environmental acceptability).

7.1 Requirements to meet: guideline G8

Next to the D2-Standard two guidelines were developed by the IMO/as a framework for 

approval of ballast water treatment systems (G8) and approval of the use of active 

substances in ballast water treatment systems (G9). For land-based testing MEPC 53/Annex 

3 Anonymous 2005; and the revision of MEPC 58) 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 

1) Less than 10 viable organisms per cubic metre greater than or equal to 50 

micrometers in minimum dimension;

2) Less than 10 viable organisms less than 50 micrometers in minimum dimension 

and greater than or equal to 10 micrometers in minimum dimension and

3) Less than the following concentrations of indicator microbes, as a human health 

standard:

1. Toxicogenic Vibrio chlorerae (serotypes O1 and O139) with less than 1 

colony forming unit (cfu) per 100 milliliters or less than 1 cfu per 1 

gramme (wet weight) of zooplankton samples:

2. Escherichia coli less than 250 cfu 100 milliliters;

3. intestinal Enterococci less than 100 cfu per 100 milliliters.
Final report of the land-based testing of the Hyde-GuardianTM-System 16

conditions. This is intended to ensure that the efficacy of the equipment is consistent and 

can be shown repeatedly. The test set-up should therefore be representative of the 

characteristics of the arrangements used and the type of environment the BWT system was 

designed for.

One of the main criteria in the G8 test requirements is the salinity range and related to this 

the differences in Total Suspended Solids (TSS), Particulate Organic Carbon (POC) and 

Dissolved Organic Carbon (DOC). This resulted in three main categories of test conditions 

(Table 1). 

Table 1: Three different salinity ranges and minimum concentrations of TSS, POC and 

DOC in the water.

Salinity

Parameter > 32 PSU 3 – 32 PSU < 3 PSU unit

Total Suspended 

Solids > 1 > 50 > 50 mg/L

Particulate Organic 

Carbon > 1 > 5 > 5 mg/L

Dissolved Organic 

Carbon > 1 > 5 > 5 mg/L

In general UV-reactors are not sensitive to changes in the salinity of the water except that 

water with a high alkalinity, iron and/or manganese will increase fouling of the UV-light 

source (Gundry 2007). Historically UV-reactors are widely used in treating (fresh) drinking 

water and sewage water and its use as a tool for disinfection is well studied. 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 

were identical (Table 1).

A further requirement is that the difference between the two salinity regimes should be at 

least 10 PSU. The test water, originating from the Wadden Sea, and the actual sampling did 

vary with the tide and as a result salinity was subject to variations. To assure the 10 PSU 

salinity difference it was decided to have the possibility of adding fresh water and upgrade 

coastal water of the North 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 Hyde-GuardianTM-System 17

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

and groups of organisms.

Influent test water

Parameter unit Remarks

organisms ≥ 50 micron > 105/ m3 at least 5 species from at least 3 

different phyla/divisions

10 ≤ organism size ≤ 50 

micron

> 103

/ mL at least 5 species from at least 3 

different phyla/divisions

heterotrophic bacteria > 104/ mL not further defined

The test water should contain minimum densities of plankton which are typical densities 

encountered in the Wadden Sea during the annual spring bloom in April/May. With respect 

to the species diversity, the Wadden Sea is known for its natural richness in organisms and 

during the test period (April – July) indeed a large diversity in organisms, adults, juveniles, 

eggs, etc. was encountered.

An important aspect, so far not recognized in the guidelines (G8), when dealing only with 

natural populations of organisms in the influent of the test water is the natural seasonality 

of species and blooms. The actual onset of the spring bloom is characterized by a dominance

in phytoplankton, but usually lacks high zooplankton abundance. Only at a later stage 

zooplankton starts to increase in abundance, subsequently due to predation it will diminish 

the numerical abundance of (smaller size) phytoplankton component. 

Furthermore, for the high salinity range, the composition of the organisms in the water 

resembles that of a typical oceanic environment. This implies an increase of smaller sized 

cells, down to the micrometer scale, and also a dramatic decline in the number of larger (>10 

μm) organisms. So far this shift in community structure has not been accounted for when 

using natural plankton for testing. 

Human pathogens

Table 3: Maximum allowed numbers of 3 groups of indicator microbes in the effluent test water 

on discharge. cfu: colony forming units

Effluent test water

Parameter unit Remarks

Toxicogenic 

Vibrio chlorerae

< 1 cfu/100 mL or < 1 cfu/ g wet 

weight of zooplankton serotypes O1 and O139

Escherichia coli <250 cfu/ 100 mL

intestinal 

Enterococci <100 cfu/ 100 mL

Within the group of prokaryotic microbes only bacteria and more specifically the 

heterotrophic group (Table 2) has been defined by the standard but for completeness this 

should include all bacteria and presently also Archeae. While these microbes are part of the 

natural community in the aquatic environment the indicator microbes (Table 3), i.e. the 

human pathogens, are introduced as part of human activity and often associated with 
Final report of the land-based testing of the Hyde-GuardianTM-System 18

sewage discharge. In the present research all microbes have been included as a bulk 

parameter, the number of heterotrophs as a viable component as well as the viability of the 

whole microbial community has been determined. 

Within the whole microbial community the number of heterotrophic bacteria was 

determined as well as E. coli and total enterococci. The test area of the institute is part of a 

tidal estuary of the Wadden Sea, which is essentially a pristine environment. Moreover, 

waste water treatment is highly developed in the Netherlands. Therefore, numbers of these 

human pathogens during the tests were to be expected to be low for most of the sampling 

period. On the other hand during the different treatment steps a significant amount of 

particulate organic material is transferred into dissolved organic carbon (DOC) which acts as 

an excellent substrate stimulating growth of (heterotrophic) bacteria. 

7.2 Experimental design

A variety of methods was applied to examine the biological efficacy of the Hyde-GuardianTM-

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 HydeGuardianTM-system (Anonymous 2008). 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, e.g. in the case of 

sampling and incubation of samples at discharge. Subsamples were taken at randomly or 

throughout the whole filling procedure of the tanks. 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 short description of each parameter and how it has been analysed is given 

below.

Physical and chemical properties of test water

Temperature

The water temperature was measured using a calibrated thermometer. 

pH

The pH-level is measured using a calibrated pH-meter.

Salinity

For salinity ca. 250 - 500 mL water is sampled and stored at room temperature (glass bottles) 

until analysis by direct measurement in the laboratory at NIOZ. Salinity of the water was 

measured after each test cycle using a refractometer (Atago) calibrated against 0 and 33 PSU 

standard (sea)water. The accuracy of the salinity measurement is 0.5 PSU. 

Dissolved Oxygen

The spectrophotometric method of the Winkler method (Winkler, 1888; Pai et al. 1993;

Reinthaler 2006) was used to determine the oxygen concentration in the water. Samples 

were taken using gastight tubing which was specially fitted to the sampling tubing that was 

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

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

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

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

H2SO4 is added prior to measuring the OD at 456 nm with a Hitachi U-3010 
Final report of the land-based testing of the Hyde-GuardianTM-System 19

Spectrophotometer. The oxygen concentration was calculated using standards and 

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

DOC

The concentration of dissolved organic carbon (DOC) was measured according to Reintaler & 

Herndl (Reinthaler & Herndl 2005). Samples for DOC (15 mL) were filtered through GF/C 

filters and sealed in pre-combusted glass ampoules after adding 50 μl of phosphoric acid 

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

laboratory by the high temperature combustion method using a Shimadzu TOC-5000 

analyzer. Standards were prepared with potassium hydrogen phthalate (Nacalao Tesque, Inc, 

Kioto, Japan). The mean concentration of triplicate injections of each sample (three in total) 

is calculated. The average analytical precision of the instrument is < 3 %.

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

For TSS/POC pre-weighted glass fibre filters (GF/C) are used. Each filter was coded and 

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

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

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

Practical volumes were between 100 and 1000 mL per sample. 

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

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

amount of suspended solids was calculated from the weight increase of the filter and 

averaged for the three replicates (mg/L).

Next, the filter is combusted at 500°C (overnight) and allowed to cool in a vacuum exicator 

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

measurement and the TSS weight.

 Figure 6: 

 Sampling point at tank 3

Biology

The majority of the large size fraction (>50 μm) consists of zooplankton, while the majority 

of the small size fraction (10-50 μm) consists of phytoplankton. Organisms > 50 μm are 

retained as recommended in MEPC 54/Inf.3 (using a Hydrobios net).

Samples for the 10 - 50 μm fraction were collected as whole undisturbed samples. These 

samples were then filtered over a 50 and a 10 μm sieve and fixated.

A second set of samples for this size class was taken and not separated from the organisms < 

10 μm in order to include the fate of the smaller sized (phyto)plankton community as well 

and to avoid further damage of the plankton. The results of these samples were compared 
Final report of the land-based testing of the Hyde-GuardianTM-System 20

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 7: 

1000 L container with a 50 μm Hydrobios sampling net 

Organisms > 50 μm

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

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

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

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

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

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

staining or during sample analysis.

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

detailed inspection of each intact individual is needed to assess viability. This includes the 

staining as well as the detection of internal (heart, gills) movement. Organisms which were 

not intact are assumed to be dead. 

Neutral Red is a reliable staining method for all major groups of organisms but inconsistent 

staining was found for bivalves. For this latter group movement (including internal such as 

heart and gills for juvenile mussels) has to be used obligatorily to determine viability. This is 

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

analyzed all samples.
Final report of the land-based testing of the Hyde-GuardianTM-System 21

Figure 8: (top) live copepod (left) and nauplia (right) from a 

Neutral Red stained sample. (bottom) dead copepod (left) and 

nauplia (right) from a NR stained sample. Pictures made with 

the FlowCam.

The samples are analyzed manually using a binocular with a 20x magnification for counting 

and up to 50x for species identification and measurements when necessary. For inter 

comparison a subset of samples was also analyzed using a semi-automated tool (FlowCam, 

Fluid Imaging Technologies; (Anonymous 2001)). Organisms need to be counted according to 

their size. Here organisms of 50 μm in minimum dimension are relevant. Several tests have 

shown that a single size bar is not efficient as viable organisms move in the counting 

chamber. Better results are achieved when the entire field of view is equipped with a size 

grid. The minimum dimension to measure will be adjusted to the specific organism groups. 

Organisms < 50 μm

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

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

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

using filtered seawater and fixed with Lugols solution. Sample analysis was conducted by 

microscopic count with an inverted microscope at 200x magnification (method by 

Utermöhl). Since the Utermöhl is not suitable to asses viability counting was restricted to the 

structural integrity of organisms and therefore the presence of intact cells (Paerl 1978). This 

method works for both zoo- and phytoplankton. This size fraction was also covered by flow 

cytometry on basis of a single cell measurement (Veldhuis & 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 
Final report of the land-based testing of the Hyde-GuardianTM-System 22

viability (Veldhuis et al. 2001; Veldhuis & 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) were analyzed using a calibrated flow cytometer. This yields the 

total number of particles (dead and live organisms as well as detritus) as well as their size 

range and the presence or absence of chlorophyll. For the counts exactly 1 mL was analyzed.

The size of the plankton was determined by comparison to standardized beads (10 and 50 

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

cytometer.

For organism viability testing, on the level of the individual cell, SYTOX Green was added to 1 

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

analyzed using the flow cytometer for the presence of dead and/or life organisms (cf. 

Veldhuis et al, 2001).

nucleus

A B

Figure: 9(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 PulseAmplitude Modulated fluorometer (PAM-fluorometry) WALTZ- water PAM (Schreiber et al. 

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

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

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

population of phytoplankton.

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

fate of the whole phytoplankton community (Jeffrey et al. 1997; Kraay et al. 1992). For this 

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

until further analysis. 

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

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

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

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

Bacteria

The classical method for counting bacteria in many applications is based on plating on 

selective media. Unfortunately, for studies in the aquatic environment this approach is by far 

insufficient for various reasons (Gasol & Giorgio 2000). As a result total bacteria were now 

determined by flow cytometry, using DNA-specific stains to get a more accurate bacteria 
Final report of the land-based testing of the Hyde-GuardianTM-System 23

number. In addition samples were taken at discharge for specific human pathogens and 

heterotrophic bacteria using a plate method. 

A 1.5 mL water sample was taken and pipetted in a Cryovial (in triplicate) and formaldehyde 

was added as a preservative. Samples were frozen and stored at -50 oC until further analysis. 

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

diluted with a TE-buffer, and the nucleic acid dye PicoGreen (MP) was added. Within 5 to 15 

minutes after the addition of the stain the sample is analyzed using a flow cytometer (cf. 

Gasol & Del Giorgio 2000; Veldhuis et al. 1997). A known bacterial standard is used for 

calibration and counting.

The number of total heterotrophic bacteria was determined using a plate method as the 

number of colony forming units (cfu’s) after incubation of the water at intake and discharge 

according to an international standard (NEN-EN-ISO 6222:1999).

Human pathogens

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

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

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

carried out according to NEN-EN-ISO standards.

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

transport container (4 °C). The analysis is carried out according NEN-EN-ISO 7899-2 for 

intestinal enterococci and NEN-EN-ISO 9308-1 for E. coli and related bacteria of the coli 

group as adopted for surface and waste water analysis in the Netherlands.
Final report of the land-based testing of the Hyde-GuardianTM-System 24

8 Results

The present section is a compilation of all relevant information needed for Type Approval 

Certification tests according to the Guidelines (G8), but also includes some relevant results 

of experiments conducted to assess the environmental acceptability of the applied UVreactor and/or its potential by-products in the environment upon discharge. Data are 

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

detailed species list of observed organisms is presented.

Figure 10: overview of the Hyde-GuardianTM- Ballast Water Treatment System in the NIOZ harbour. 

In front of the container are the three IBCs with the 50 μm inline sampling nets.

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

referred to as low and high salinity test series.

8.1 Physical and chemical parameters

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

of control and treated tanks for test runs 1 – 5.

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

1 - 5 22.1 0.92 8.5 - 10.4

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

low tide and fresh water was added to a maximum of 15% (v/v). 
Final report of the land-based testing of the Hyde-GuardianTM-System 25

Table 5: Average salinity and temperature of water at intake during the high salinity tests of 

control and treated tanks for test runs 6 – 10.

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

6 - 10 31.9 1.3 14.3 - 17.5

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

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

In terms of difference in salinity the variation between both salinity regimes is slightly less 

than 10 PSU. However, given the standard variation and the fact that tidal effects and 

prevailing winds showed occasionally disturbed the ideal situation the minor deviation from 

the ideal situation is considered to be of minor importance. Moreover, the present 

configuration of the Hyde-Guardian-BWT-system is not affected by changes in the salinity.

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.

Table 6: average or range of oxygen, pH, inorganic nutrients, TSS, POC and DOC concentration of 5 test 

series run at low salinity at intake and discharge of the reference and treated tank; 

1

: range of values

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

O2 reference1 9.3-12.9 7.9-10.7 mg/L

O2 treated1 9.4-13.0 8.1-11.5 mg/L

pH reference 8.17 0.10 8.19 0.12 μM

pH-treated 8.12 0.12 8.09 0.08 μM

NO3 reference 42.5 10.3 41.5 11.1 μM

NO3 treated 41.5 8.00 36.8 10.7 μM

NO2 reference 0.48 0.07 0.51 0.07 μM

NO2 treated 2.99 1.13 4.62 0.52 μM

NH4 reference 2.8 1.03 7.63 1.15 μM

NH4 treated 2.9 1.24 5.38 1.63 μM

PO4 reference 0.16 0.03 0.22 0.07 μM

PO4 treated 0.24 0.03 0.17 0.05 μM

TSS-reference 42.5 21.90 9.60 2.30 mg/L

TSS-treated 29.9 11.80 11.10 5.70 mg/L

POC -reference 16.1 6.80 5.40 1.30 mg/L

POC-treated 10.5 3.21 5.80 2.63 mg/L

DOC -reference 5.82 3.67 3.03 0.27 mg-C/L

DOC-treated 4.87 2.97 3.36 0.37 mg-C/L
Final report of the land-based testing of the Hyde-GuardianTM-System 26

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 as much as 20% compared to intake values. In contrast oxygen concentration in 

the treated tank was less affected, due to reduced bacterial activity, resulting in higher 

values (14% decline relative to intake) as compared to the reference tank. 

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

sufficiently high at intake but these values varied considerably among the different test runs. 

At discharge sediment concentration declined largely, mainly because of sedimentation in 

the storage tank during the holding period of 5 days. The applied filtration step effectively 

reduced the amount of TSS at intake and partly also the POC, on average by 30 and 35%, 

respectively. The remaining suspended material remained in suspension at discharge and 

half of its weight consisted of organic material. 

In contrast to the particulate fraction the amount of dissolved organic carbon (DOC) was 

hardly affected by the ballast water treatment system. Mainly due to microbial 

mineralisation of this water for 5 days resulted in a reduction of the DOC concentration in 

the reference and treated tank by as much as 48% and 40%, respectively. 

With respect to the concentration of inorganic nutrients (NO3, NO2, NH4 and PO4) there were 

with a few exceptions, only minor differences between the reference and treated ballast 

water tank. At the low salinity test series only the nitrite (NO2) concentration increased by a 

factor 6.2 after the UV-reactor. It is known that in presence of nitrate UV-photolysis 

produces nitrite (Buchanan et al. 2006). The observed elevated concentration was 

nevertheless within the range typical for the challenge water used and therefore not 

excessively high. For comparison the observed concentrations of nitrite were lower than the 

US (EPA) and EU maximum allowed concentration for drinking water of 71.4 and 7.9 μM 

(1000 μg-N/L and 110 μg-N/L), respectively (Sharpless et al. 2003).

In this respect it should be noted that the prolonged dark incubation also increased the 

nitrite as well as the ammonia concentration, in both the reference and treated tank.
Final report of the land-based testing of the Hyde-GuardianTM-System 27

High salinity 

Table 7: average or range of oxygen, pH, inorganic nutrients, TSS, POC and DOC concentrations 

of 5 test series run at high salinity at intake and discharge; 1

: range of values 

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

O2 reference1 4.7-9.2 2.6-6.1 mg/L

O2 treated1 4.2-8.8 2.9-6.7 mg/L

pH reference 8.07 0.05 8.12 0.09 μM

pH-treated 8.05 0.09 8.10 0.12 μM

NO3 reference 0.46 0.12 0.45 0.12 μM

NO3 treated 0.84 0.40 1.33 0.63 μM

NO2 reference 6.87 4.55 6.28 4.63 μM

NO2 treated 6.85 4.09 6.00 3.96 μM

NH4 reference 7.33 4.67 6.74 4.75 μM

NH4 treated 7.69 4.48 7.32 4.59 μM

PO4 reference 0.41 0.10 0.56 0.29 μM

PO4 treated 0.48 0.18 0.82 0.25 μM

TSS-reference 33.9 37.10 9.70 3.10 mg/L

TSS-treated 14 3.60 10.00 5.10 mg/L

POC -reference 10.2 5.30 4.80 1.30 mg/L

POC-treated 7.2 1.58 4.50 1.29 mg/L

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

DOC-treated 4.3 0.83 4.1 0.93 mg-C/L

The results of the basic parameters at the high salinity range were, with a few exceptions, on 

the whole the same as for the low salinity range (Table 7). At intake the oxygen 

concentrations were lower than observed for the low salinity test runs. This was mainly due 

to the temperature increase. Nevertheless, the ambient water was nearly 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 lower as compared to the low salinity test series but still 

considerably higher than the minimum requirements according to the G8 guidelines. Again 

the filtration step turned out to be an effective manner of reducing the particle load. Both 

the TSS content and the POC content declined by a 59% and 30%, respectively. 

Other differences were in the concentrations of the inorganic nutrients and in particular 

those containing nitrogen. While the nitrate concentration was very low, the level of nitrite 

and ammonia were elevated already in the water at intake. In fact these natural 

concentrations were much higher than the enhanced nitrite concentration caused by the 

UV-reactor. In contrast to the observations at the low salinity test runs the ammonia and 

nitrite concentration remained virtually constant during the 5 days of dark incubation.
Final report of the land-based testing of the Hyde-GuardianTM-System 28

8.2 Biology

Organisms > 50 μm

For the land based tests natural plankton was used and the required diversity of organisms 

(5 species of at least 3 different phyla) was easily fulfilled for all tests (Table 8). On average 

at least 10 different species of 4 to 5 different phyla were present in each sample (full details 

on species present see Annex 1).

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

the low salinity series (test run 1 -5) were all well above the minimum requirements. On 

average numbers in the >50 μm size fraction were 25 times higher than the minimum 

required number of 100,000 m-3

. At the high salinity range the water at intake was on 

average for the 5 test runs above the minimum numbers of organism but contained in two 

cases not sufficient numbers (test run 9 and 10). Test run 8 was slightly less than the 

required minimum of 100,000 organisms per m-3

.

 As explained earlier this is mainly due to 

the fact that natural seawater was used without any addition of (surrogate) 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 declined dramatically in the treated tank, to an 

average value for the low and high salinity test series of 2.9 and 2.4 organisms m-3

, 

respectively. It should however, be noted that the observed organisms, although still 

seemingly alive did receive prior to analysis a second UV-treatment. Since the applied BWT 

technology is known to have a delayed effect the organisms may be dying in the following 

day(s). Due to the very low concentration of organisms this could not be tested with 

sufficient statistical accuracy.

In fact during most test series at direct inspection immediately after intake already virtually 

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

discharge at day 5 well below 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 μm) of individual test runs and averages for

the salinity range at intake (T0) and on discharge at day 5 (T5) of the reference

(C) and treated (HG) tank. For test run 4 the same control was used, since this 

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

. 

total plankton > 50 micron numbers/m3

low salinity intake reference treated

C-T0 C-T5 HG-T5

I 1.24E+06 1.08E+04 3.0

II 4.83E+06 6.17E+02 1.7

III 1.01E+06 2.18E+03 0.0

IV 3.12E+06 2.18E+03 2.7

V 7.3

average 2.55E+06 3.95E+03 2.9

s.d. 1.79E+06 4.65E+03 2.7
Final report of the land-based testing of the Hyde-GuardianTM-System 29

Table 8: continued

total plankton > 50 micron

high salinity intake reference treated

C-T0 C-T5 HG-T5

VI 4.11E+05 1.31E+04 2.7

VII 2.75E+05 1.67E+04 0.3

VIII 8.57E+04 2.45E+03 1.7

IX 5.15E+04 1.73E+04 1.7

X 6.85E+04 2.76E+04 5.7

average 1.78E+05 1.54E+04 2.4

s.d. 1.58E+05 9.05E+03 2.0

Organisms 10 – 50 μm

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

ciliates and flagellates were occasionally present. 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 for the treated water also after the second UV-irradiance disinfection at discharge and 

incubated under optimal growth conditions. Results of plankton numbers in the 10 – 50 μm 

of both reference and treated incubations are included in the table for comparison with the 

actual measurement of the tanks (Table 9).

Table 9 shows that regarding the number of organisms at intake for both salinity regimes the 

test water contained sufficient plankton numbers, on average 50% higher numbers than 

required according to the Guidelines G8. Prolonged incubation of the plankton community in 

the dark (5 days) resulted in a considerable reduction in the number of plankton in the 10 –

50 μm size range, on average by a factor 10. Nevertheless, the plankton community of the 

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

requirements for discharge of the reference tank. Moreover, the bottle incubations, which 

were ran parallel to the tank incubations, showed, with two exceptions (series 3 and 4) a 

considerable increase in numerical abundance of the plankton within 5 days, i.e. clearly 

indicative of the presence of viable population of mainly phytoplankton. 

Figure 11: internal view of Hyde-GuardianTM-

system showing the UV-reactor. The UV-light 

is clearly visible in the open sampling line 

(yellow/greenish). 
Final report of the land-based testing of the Hyde-GuardianTM-System 30

In contrast, treatment resulted in an efficient removal of plankton although in terms of 

numerical abundance numbers remained fairly high, certainly in the test series at the high 

salinity range, after the first treatment. After 5 days in the tanks residual plankton cells were 

still detected flow cytometrically. However, analysis of the viability using SYTOX Green and 

the photosynthetic efficiency measurements clearly demonstrated that these cells, although 

intact, were no longer viable. The incubated experiments confirmed the low abundance of 

intact cells and on average the number of viable cells after the first treatment step was < 1 

cell/mL. Because the Hyde-GuardianTM-system also included a second UV-irradiance 

treatment at discharge this water was incubated for another period of 5 to 7 days to 

determine the efficacy of the second passing of the UV-reactor over a longer period. 

Measurement showed that there was indeed a delayed effect. The original numbers in the 

tank at discharge (HG-T5) were further reduced to average values of 2.3 and 1.0 (HG-Dis-Tx) 

for the low and high salinity ranges, respectively. Also compared to the bottle incubations of 

the intake samples the second UV-irradiance disinfection resulted in a further decline in 

numbers of organisms.

Table 9: number of total plankton (10 – 50 μm size range) of each test run and average for the salinity 

range at intake (T0), reference (C) and treated tank (HG), reference and treated incubated samples of 

the intake (Inc.) at day 5 (T5) during discharge and of the incubated discharge samples (Dis-Tx). Test 

run 4 and 5 share the same reference since this was a combined test run. All numbers are presented 

per mL as total counts or of viable cells. Tx= varying sampling day (5 to 7 days).

10- 50 μm 

cell numbers Reference Treated cells/mL

low salinity total viable total viable

x=5-7 

days

test run C-T0 C-T5 C-inc-T5 HG-T0 HG-T5 HG-T5 HG-inc-T5 HG-inc-T5 HG-Dis-Tx

I 1103 140 11184 677 242 <10 31.8 <1 0.0

 II 1719 156 18706 836 21.5 <10 1.5 <1 10.9

III 1530 144 1149 483 396 <10 28.9 <1 0.0

IV 1784 147 765 304 103 <10 4.4 <1 0.0

V 309 169 <10 6.7 <1 0.7

average 1534 147 7951 522 186 <10 15 <1 2.3

high salinity

VI 1657 121 9391 1091 53.3 <10 11.1 <1 0.0

VII 1657 149 6007 1453 68.8 <10 3.7 <1 2.2

VIII 2090 149 7774 2111 106 <10 6.7 <1 2.2

IX 1008 130 5754 514 7.4 <10 5.2 <1 0.7

X 1148 151 5941 1056 44.4 <10 3.7 <1 0.7

average 1512 140 6973 1245 56 <10 6.1 <1 1.0

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

fraction < 10 μm in size diameter, a single species of phytoplankton in the size range of ca. 6 

μm (Phaeocystis globosa) was also 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 HydeGuardianTM-system was also effective in reducing organisms in this size range. In general the 

response of Phaeocystis in terms of numbers and viable cells showed great similarity with 

the plankton in the 10 to 50 μm size class. Flow cytometry showed the presence of intact 

plankton in relatively high numbers in the treated tanks even after 5 days. This was 

confirmed by classical analysis of the chlorophyll and associated pigment content (data not 

shown). Viability tests indicated that the remaining cells were non-viable. This was also 
Final report of the land-based testing of the Hyde-GuardianTM-System 31

demonstrated in the incubation experiments. These bottle experiments showed that with 

exception of the first test run (204 cells/mL) numbers were already very low after the first 

treatment series. As with the 10 to 50 μm size range (Table 9), numbers of Phaeocystis cells 

also declined further after applying a second UV-irradiance disinfection step. 

Table 10: number of a dominant phytoplankton species present (Phaeocystis globosa; ca. 6 μm in size) 

at each test run and average for the salinity range at intake (T0), reference (C) and treated tank (HG), 

reference and treated incubated samples of the intake (Inc.) at day 5 (T5) during discharge and of the 

incubated discharge samples (Dis-Tx). Test run 4 and 5 share the same reference since this was a 

combined test run. All numbers are presented per mL as total counts or of viable cells. Tx= varying 

sampling day (5 to 7 days).

Phaeocystis 

~ 6 μm Reference Treated cells/mL

low salinity total viable total viable

x=5-7 

days

test run C-T0 C-T5 C-inc-T5 HG-T0 HG-T5 HG-T5 HG-inc-T5 HG-inc-T5 HG-Dis-Tx

I 7807 179 10170 13646 5865 <10 204 <1 0

II 2661 62 17175 235 88 <10 0.7 <1 57.7

III 5460 178 3314 6850 1559 <10 0 <1 0

IV 7738 1939 4491 3832 332 <10 0 <1 0

V 322 425 <10 9.6 <1 2.2

average 5916 589 8788 4977 1654 <10 43 <1 12

high salinity

VI 3754 632 9458 3475 493 <10 8.1 <1 0.7

VII 2227 204 4364 2611 463 <10 6.7 <1 3.7

VIII 2038 204 6650 3015 957 <10 3.0 <1 3.7

IX 1252 236 21473 1194 158 <10 1.5 <1 1.5

X 1395 173 14440 2705 665 <10 4.4 <1 0.1

average 2133 290 11277 2600 547 <10 4.7 <1 1.9

Photosynthetic efficiency

Another, fast and reliable approach to gain insight in the physiology and growth response of 

the whole phytoplankton community (2 to 50 μm in cells size) is by measuring the 

photosynthetic efficiency (Fv/Fm) of the phytoplankton cells. This can be done for the whole 

community or after filtration for different size classes when needed. For a clear 

interpretation 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 of the Wadden Sea 

(low salinity range) and coastal waters of the North Sea (high salinity range) was 

physiologically in a healthy condition, i.e. containing mostly photosynthetic active and 

therefore viable cells. Only at test run 7 the phytoplankton population showed some 

indication of stress. Prolonged incubation in the dark (5 days of dark stress) of phytoplankton 

in the reference tanks resulted in a severe reduction in the photosynthetic efficiency. 

Although the phytoplankton community did not enter the decay phase the majority of cells 

were far from healthy. In contrast the photosynthetic efficiency of the phytoplankton from 

the same water mass but incubated under optimal growth conditions showed that the cells 
Final report of the land-based testing of the Hyde-GuardianTM-System 32

possessed a healthy status (C-inc-T5). This is not surprising since phytoplankton also 

increased in numerical abundance (Tables 9 and 10). 

After the first treatment, at intake, most of the phytoplankton community was immediately

dead resulting in Fv/Fm values of 0 (HG-T). A holding time of 5 days in the tank did not 

change this pattern. Also in the incubated sample no recovery of the phytoplankton 

population was observed. This results did not change after the second UV-irradiance

disinfection step at discharge and a second incubation period of 5 to 7 days (HG-Dis-Tx).

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

range) at each test run and average values for the salinity range at intake (T0), reference (C) and 

treated tank (HG), reference and treated incubated samples of the intake (Inc.) at day 5 (T5) during 

discharge and of the incubated discharge samples (Dis-Tx). Test run 4 and 5 share the same reference 

since this was a combined test run. All numbers are presented per mL as total counts or of viable cells. 

Tx= varying sampling day (5 to 7 days). nd: not determined

low salinity Reference Treated

Fv/Fm C-T0 C-T5 C-inc-T5 HG-T0 HG-T5 HG-inc-T5 HG-Dis-Tx

I 0.65 0.16 0.69 0.00 0.01 0.00 nd

II 0.51 0.19 0.62 0.00 0.00 0.00 0.00

III 0.58 0.10 0.70 0.00 0.00 0.16 0.00

IV 0.51 0.12 0.81 0.00 0.00 0.00 0.00

V 0.01 0.01 0.00 0.00

average 0.56 0.14 0.71 0.00 0.00 0.03 0.00

s.d. 0.07 0.04 0.08 0.00 0.00 0.07 0.00

high salinity

VI 0.59 0.13 0.63 0.00 0.02 0.00 0.00

VII 0.44 0.11 0.65 0.00 0.04 0.00 0.00

VIII 0.67 0.16 0.60 0.00 0.00 0.01 0.05

IX 0.62 0.28 0.72 0.00 0.00 0.00 0.00

X 0.66 0.13 0.64 0.03 0.01 0.00 0.00

average 0.60 0.16 0.65 0.01 0.01 0.00 0.01

s.d. 0.09 0.07 0.04 0.01 0.01 0.01 0.02

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 three cases the numbers of 

human pathogens were above the detection limit (test runs 7, 8 and 9). 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 Hyde-GuardianTM-System 33

Table 12: plate counts of the human pathogens E. coli and total enterococci of each test run and 

averages for the salinity range at intake (T0), reference (C) and treated tank (HG), reference and 

treated incubated samples of the intake (Inc.) at day 5 (T5) during discharge and of the incubated 

discharge samples (Dis-Tx). Test run 4 and 5 share the same reference since this was a combined test 

run. All numbers are presented per mL as total counts or of viable cells. Tx= varying sampling day (5 to 

7 days).

human pathogens Reference Treated counts/mL

C-T0 C-T0 C-T5 C-T5 HG-T5 HG-T5 HG-Dis-Tx HG-Dis-Tx

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

I <0.1 <1 <0.1 <1 <0.1 <1 <0.1 <1

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

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

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

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

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

high 

salinity

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

VII 1 <1 <1 <0.1 <1

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

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

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

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

In contrast to the almost complete absence of human pathogens, the typical 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 ranging from 0.72 to 6.6 106 per mL (variation of 

factor 9.1) are typically observed in spring and early summer (Table 13). Of this total 

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

plate assay method (<10 to 60 per mL). Therefore, if only plating would be used as criteria

for heterotrophic bacteria numbers this would result in a severe underestimation of the 

actual bacteria numbers. After 5 days of incubation total bacteria numbers on average 

declined in the control tank but there was considerable variation between the different test 

runs (C-T5). 

As opposed to the tank total bacterial numbers increased, with exception of test run 2, in 

the incubated water of the reference tank (C-inc-T5) for both salinity regimes. This indicates 

that a significant fraction of the bacteria was viable.

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 1.9. In a similar 

manner the numbers of total bacteria of the incubated sample were comparable with those

of the holding tank samples. A clear reduction in bacteria number was observed after the 

second UV-disinfection step during discharge (HG-Dis-Tx) resulting in a decline in number by 

one order of magnitude relative to the number measured in the tank at day 5.
Final report of the land-based testing of the Hyde-GuardianTM-System 34

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 increased on average by a factor 5 after 5 days of 

incubation but the second UV-irradiance disinfection step reduced the number of cfu’s to 

below the detection limit. 

At all test runs at the high salinity range there the number of colony forming units was 

roughly identical to the water at intake. In contrast to the low salinity test runs the number 

of cfu’s increased in 3 out of 5 test runs at the high salinity tests after the second UVirradiance disinfection step. 

Table 13: Total bacteria number (numbers per mL) and colony forming heterotrophs (cfu/mL) of each 

test run and average for the salinity range at intake (T0), reference (C) and treated tank (HG), 

reference and treated incubated samples of the intake (Inc.) at day 5 (T5) during discharge and of the 

incubated discharge samples (Dis-Tx). Test run 4 and 5 share the same reference since this was a 

combined test run. All numbers are presented per mL as total counts or of viable cells. Tx= varying 

sampling day (5 to 7 days). nd: not determined

total bacteria numbers/mL

heterotrophic bacteria Reference Treated cfu/mL

low 

salinity C-T0 C-T0 C-T5 C-inc-5 C-T5 HG-T0 HG-T5

HG-incT5 HG-T5HG-DisTx HG-Dis-Tx

test run total colony total total colony total total total colony total colony

I 4.5E+06 20 2.3E+06 8.3E+06 80 4.1E+06 4.8E+06 7.4E+05 250 nd 10

II 6.6E+06 60 3.6E+06 2.4E+06 20 3.4E+06 5.2E+06 3.9E+06 20 2.1E+05 <10

III 3.0E+06 30 1.8E+06 4.8E+06 60 3.8E+06 9.6E+05 2.1E+06 300 1.3E+05 <10

IV 1.3E+06 30 0.7E+06 4.9E+06 <10 1.4E+06 3.8E+05 8.8E+05 375 9.4E+04 <10

V 20 1.0E+06 7.4E+05 4.0E+05 225 7.5E+05 <10

average 3.9E+06 32 2.1E+06 5.1E+06 43.0 2.8E+06 2.4E+06 1.6E+06 234 3.0E+05 <10

s.d. 2.3E+06 16.4 1.2E+06 2.4E+06 30.6 1.4E+06 2.4E+06 1.5E+06 132.6 3.1E+05

high salinity

VI 5.6E+06 20 0.9E+06 6.1E+06 10 6.3E+06 1.0E+06 3.6E+05 10 4.3E+05 <10

VII 0.7E+06 50 5.4E+06 8.2E+06 20 5.2E+06 7.1E+05 3.8E+05 70 3.1E+05 20

VIII 4.9E+06 20 1.7E+06 5.1E+06 30 4.3E+06 1.2E+06 2.5E+06 50 1.2E+05 120

IX 2.4E+06 30 0.3E+06 3.0E+06 <10 3.4E+06 2.4E+05 3.8E+06 10 1.2E+06 170

X 5.3E+06 10 2.4E+06 6.3E+06 60 4.2E+06 1.3E+06 3.3E+06 <10 1.2E+06 >1000

average 3.8E+06 26 2.1E+06 5.7E+06 30 4.7E+06 8.8E+05 2.1E+06 35 6.4E+05 <10->1000

s.d. 2.1E+06 15.2 2.0E+06 1.9E+06 21.6 1.1E+06 4.2E+05 1.6E+06 30.0 5.0E+05
Final report of the land-based testing of the Hyde-GuardianTM-System 35

9 Environmental acceptability

Ballast water treatment systems applying active substances, but also systems that do not use 

active substances should demonstrate according to the revised guidelines G8 (MEPC58, 

October 10th) that the treated water upon discharge is not harmful to the environment and 

organisms.

Although officially not obligatory for the Hyde-GuardianTM-system a series of studies were 

conducted to examine long-term (20 days) regrowth and vitality. In total 2 long-term 

incubation experiments, one at each salinity regime, were conducted with treated water 

which was collected in a clean container (10 L) and incubated under optimal growth 

conditions in a climate room. The experiments were done for treated water collected at 

intake (pre-filtration and UV-reactor) and at discharge (second UV-irradiance disinfection 

step). The second series of (discharge) samples were stored in the dark tank for 5 days prior 

to the incubation.

To stimulate regrowth of planktonic organism extra nutrients were added (nitrate; 30 μM, 

phosphate; 2 μM, silicate; 20 μM). In particular at the peak of the spring bloom and 

thereafter the ambient nutrient concentration (mainly silicate and phosphate) can be 

extremely low, which may prevent growth of phytoplankton and also bacteria. 

time [days]

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

phytoplankton 10-50 

μm [cells/mL]

1e-2

1e-1

1e+0

1e+1

1e+2

1e+3

1e+4

1e+5

control

Hyde-Guardian intake

Hyde-Guardian discharge

Figure 12: time course of phytoplankton numbers (10 – 50 μm size range) during a 25 day incubation 

period, control and treated water incubated at intake and treated water also incubated at discharge. 

Test run X; values are mean of three replicates. Line marks the D2-Standard value of 10 cells/mL.

Figure 12 shows that when incubated under optimal growth conditions the total cell 

numbers of phytoplankton in the 10-50 μm declined in the first three days. The same was 

observed for the smaller phytoplankton (data not shown). Regrowth of phytoplankton only 

occurred after 7 days of incubation. This observation indicates that the treatment did not 

deactivate the entire plankton community. Some cells survived as resting stages, cysts or did 

recover from the UV-irradiance disinfection step. Although figure 12 only shows the results 

for the larger fraction a similar trend was observed for the smaller phytoplankton as well 

(data not shown). Nevertheless, even under favourable conditions recovery, resulting in cell 

number exceeding the minimum number of 10/mL, was observed after 7 days.

For this reason a second UV-irradiance disinfection step was applied at discharge. This 

resulted in a further reduction of the total number of particles by an order of magnitude. 
Final report of the land-based testing of the Hyde-GuardianTM-System 36

Similar to the first UV- disinfection treatment there was a delayed effect of ca. 3 days (figure 

12). After this period the plankton community stabilized and regrowth started 14 days after 

the initial intake of the water. The second UV- irradiance disinfection step was certainly 

effective in this respect but the this water was still not entirely free of living plankton. 

In terms of vitality it should be noted that regrowth of phytoplankton was observed and the 

final numbers achieved in these experiments, exceeded even the numbers of the control 

bottles (Figure 9). Therefore, potential by- or end-products generated by the UV- reactor did 

not affect the water quality in such a manner that growth of phytoplankton in the receiving 

environment would be inhibited.

time [days]

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

bacteria [cells/mL]

1e+5

1e+6

1e+7

control

Hyde-Guardian intake

Hyde-Guardian discharge

Figure 13: time course of bacteria numbers during a 25 day incubation period, control and treated 

water incubated at intake and treated water also incubated at discharge. Test run X; values are 

mean of three replicates.

With respect to total bacteria numbers the control and the two treated water samples 

showed a trend over time which was basically similar to that of development of the 

phytoplankton community over time (fig. 13). In the control bottle bacteria increased on the 

first day, remaining at a constant level for 8 days and declining gradually till the last day of 

sampling (day 20). The final numbers at the end were one order of magnitude lower than 

the original values at intake.

In contrast bacteria numbers in the treated water at intake declined rapidly by a factor 10 in 

the first 5 days of incubation. From day 7 onwards there was rapid growth and at the day 20 

numbers had increased to a factor 2 higher than the initial numbers. 

Similar to the phytoplankton (figure 9) the second treatment with the UV-reactor at 

discharge resulted in a further reduction of the total amount of bacteria present. A recovery, 

in terms of bacterial growth was delayed until day 11. The bacterial growth rate was 

reduced, compared to that of the bacteria at intake, and final numbers after 25 days of 

incubation were only 50% of the original bacteria numbers at intake.
Final report of the land-based testing of the Hyde-GuardianTM-System 37

10 Discussion and evaluation of results

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

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

Hyde-GuardianTM-system under semi-in situ conditions. The initial idea of a treatment 

systems based on the (permanent) deactivation of organisms through ultraviolet light 

disinfection, both at intake and discharge turned out to be effective even in surface water 

with an extremely high density of mucilage containing organisms and a high turbidity. As a 

first step to reduce the particle load the coarse filter effectively removed the larger fraction 

including organisms. 

With only some minor deviations the present 10 test runs were conducted according to the 

guidelines G8 of the IMO. The basic chemistry and physical properties of the water were not 

altered by the treatment and no factors could be detected effecting the vitality of the 

treated water upon discharge. 

The treated water remained stable for most of the parameters over the whole holding 

period of 5 days. Concentrations of dissolved oxygen (DO) and dissolved organic carbon 

(DOC) hardly varied. Only the concentration of total suspended solids and the particulate 

organic carbon declined slightly mainly as a result of sedimentation. In this respect it should 

be noted that the application of the self-cleaning filter effectively removed larger particles 

and the load of sediment in the treated tanks was only minimal compared to that in the 

control tank.

With respect to the water chemistry a slight increase in nitrite concentration was observed 

during the low salinity test runs in the treated water (cf. Sharpless & Linden 2001). This 

apparent increase was still 50% of the natural nitrite concentrations observed during the 

second test series at high salinity and should therefore be considered as non hazardous. 

Moreover, the values were still lower than the maximum allowed concentration for drinking 

water (Sharpless et al. 2003).

The stable DO and DOC over time was indicative of a reduced biological activity during the 

holding period of 5 days. Numbers of organisms were in compliance with the D2-Standard at 

discharge but the second UV-disinfection effectively reduced the number of viable organism 

further. The treated water did not contain toxic or growth inhibiting substances. Neither 

were chemicals produced as by-products. On the contrary this water was rich in growth 

stimulating nutrients (nitrogen, phosphorus and silicate) for phytoplankton and bacteria. 

With apparently some living cells present and ample growth stimulating resources present,

phytoplankton and bacteria could start blooming in the presence of light. Still even under 

favourable conditions growth was delayed by a much as 10 days.

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

testing and legislation.

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

50 μm size range, cannot (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 μm since 

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

2. The long-term incubations (ca. 20 days) and the environmental acceptability studies 

provide good insight in the response of the whole community to the growth 

potential/limitation of the treated water when released into environment. These 

experiments should be conducted next to the standard set of toxicology and residual 

chemistry tests of the treated ballast water. 

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 Hyde-GuardianTM-System 38

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

were effectively deactivated. 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. Although UV systems, including the one applied in the present BWTsystem, in general have been proven to effective in the disinfection of (drinking) water 

no data test are available.

4. The Guidelines G8 and the update version as adopted at MEPC 58 (10 October 2008) 

are incomplete with respect to treatment systems including a second treatment at 

discharge. In line of the tank and sample volume for treatment at intake a similar 

protocol should be developed for treatment at discharge. However, considering the 

expected number of organisms at discharge and statistical errors associated with the 

actual counts a second sample tank to collect and store the discharge would be 

required with a volume two orders of magnitude larger than the present tank. i.e. 

20,000 m3

. As an alternative for these large volume incubations, but restricted to the 

smaller sized plankton, the bottle (10 L) incubations either in the dark or in a more 

favourable condition could be applied. 

In conclusion, the present configuration of the Hyde-GuardianTM-system offers a reliable and 

environmentally safe cleaning of the ballast water resulting in organism numbers well below 

the Standard of the IMO Regulation-D2.
Final report of the land-based testing of the Hyde-GuardianTM-System 39

11 References 

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

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

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

(G8). Annex3 Resolution MEPC.125(53) Annex:Parts 1,2,3 and 4

Anonymous (2008) Test protocol protocol for the biological efficacy testing of the 

Hyde-Guardian-Ballast water treatnebt system (ECOCHLOR, Inc) as part 

of the type approval process under the resolution MEPC 125.53

Buchanan W, Roddick F, Porter N (2006) Formation of hazardous by-products 

resulting from the irradiation of natural organic matter: comparison 

between UV and VUV irradiation. Chemosphere 63:1130 - 1141

Carlton JT, Geller JB (1993) Ecological roulette: the global transport of 

nonindiginous marine organisms. Science 261:78 - 82

Chin A, Bérubé P (2005) Removal of disinfection by-production precursors with 

ozone-UV advanced oxidation process. Water Res. 39:2136 - 2144

Crippen RW, & J.J. Perrier (1974) The use of neutral red and evans blue for livedead determinations of marine plankton. Stain Techn. 40:97 - 104

Drake LA, Choi K-H, Ruiz GM, Dobbs FC (2001) Global redistribution of 

bacterioplankton and virioplankton communities. Biological Invasions 

3:193 - 1999

Endresen Ø, Behrens HL, Brynestad S, Andersen AB, Skjong R (2004) 

Challenges in global ballast water management. Mar. Pollut. Bull. 48:615-

623

Fleming JM, Coughlan J (1978) Preservation of vitally stained zooplankton for 

live/dead sorting. Estuaries 1:135 - 137

Gasol JM, Del Giorgio PA (2000) Using flow cytometry for counting natural 

panktonic bacteria and understanding the structure of planktonic bacterial 

communities. Sci. Mar. 64:197 - 224

Gollasch S, Dammer M, Lenz J, Andres H-G (1998) Non-indigenous organisms 

introduced via ships into German waters. In: Carlton e.d., Ballast water: 

Ecological and fisheries implications ICES Cooperative Research Report 

No 224.:50 - 64

Gundry M (2007) Ultraviolet disinfection-practical aspects. Water Supply 

June/July:33 - 36

Hallegraeff GM, Valentine JP, Marshall J-A, Bolch CJ (1997) Temperature 

tolerances of toxic dinoflagellate cysts: application to the treatment of 

ship's ballast water. Aquatic Ecology 31:47 - 52

Hamer JP, McCollin TA, Lucas IAN (2000) Dinoflagellate cysts in ballast tank 

sediments: between tank variability. Mar. Pollut. Bull. 9:731 - 733

Haskoning R (2001) Global market analysis of ballast water treatment 

technology. Report committed by Northeast-Midwest Institute. 

42810/001R/HSK/SKO.

Hoagland P, Anderson DM, Kaoru Y, White AW (2002) The economic effects of 

Harmful Algal Blooms in the United States: estimates, assessment issues, 

and information need. Estuaries 25:819 - 837

Jeffrey SW, Mantoura RFC, Wright SW (1997) Phytoplankton pigments in 

oceanography. In: S.W. Jeffrey, R.F.C. Mantroura and S.W. Wright (eds) 

Phytoplankton pigments in oceanography: guidelines to modern methods 

SCOR-UNESCO, Paris pp. 661.

Kraay G, Zapata M, Veldhuis MJW (1992) Separation of chlorophylls c1, c2 and 

c3 of marine phytoplankton by reversed-phase-C18-high-perfomance 

liquid chromatography. J. Phycol. 28:708 - 712
Final report of the land-based testing of the Hyde-GuardianTM-System 40

Malley JJ, Shaw J, Ropp J (1995) Evaluation of by-products produced by 

treatment of groundwater with ultraviolet irradiation. AWWA Research 

Foundation and American Water Works Association

Paerl HW (1978) Effectiveness of various counting methods in detecting viable 

phytoplankton. N.Z Journal of Marine and Freshwater Research 12:67 -

72

Pai S-C, Gong G-C, Liu K-K (1993) Determination of dissolved oxygen in 

seawater by direct spectrophotometry of total Iodine. Mar. Chem. 41:343 -

351

Raikow D, Reid D, Blatchley E, Jacobs G, Landrum P (2007) effects of proposed 

physical ballast water treatments on aquatic invertebrates resting eggs. 

Environmental Toxicology and Chemistry 26:707 - 725

Reinthaler T (2006) Prokaryotic respiration and production in the open ocean. PhD thesis University of Groningen:pp. 128

Reinthaler T, Herndl GJ (2005) Seasonal dynamics of bacterial growth 

efficiencies in relation to phytoplankton in the southern North Sea. Aquat. 

Microb. Ecol. 39:7 - 16

Rigby G, Taylor AH (2001) Ballast water treatment to minimise the risks of 

introducing nonindigenous marine organisms into Australian ports. Astrl. 

Gov, BaWa research Ser. Rpt 13.

Ruiz GM, Rawlings TK, Dobbs FC, Drake LA, Mullady T, Huq A, Colwell RR 

(2000) Global spread of microorganisms by ships. Nature 408:49 - 50

Schreiber U, Neubauer C, Schliwa U (1993) PAM fluorometer based on mediumfrequency pulsed Xe-flash measuring light: A highly sensitive new tool in 

basic and applied photosynthesis. Photosynth. Res. 36:65 - 72

Sharpless C, Linden K (2001) UV photolysis of nitrate: effect on natural organic 

matter and dissolved organic carbon and implication for UV water 

disinfection. Envir. Sci. Techn. 35:2949 - 2955

Sharpless C, Page M, Linden K (2003) Impact of hydrogen peroxide on nitrite 

formation during UV disinfection Water Res. 37:4730 - 4736

Veldhuis MJW, Brussaard CPD (2006) Harmful algae and cell death. Ecological 

Studies; Springer-Verlag Berlin Heidelberg Vol. 189 E. Graneli & J.T. 

Turner (eds) Ecology of Harmful Algae 

Veldhuis MJW, Cucci TL, Sieracki ME (1997) Cellular DNA content of marine 

phytoplankton using two new fluorochromes: taxonomic and ecological 

implications. J. Phycol. 33:527 - 541

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

phytoplankton research: current applications and future perspectives. Sci. 

Mar. 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. Eur. J. Phycol. 36:167 - 177

Williams RJ, Griffiths FB, VanderWal EJ, Kelly J (1988) Cargo vessel ballast 

water as a vector for the transportation of non-indigenous marine species. 

Estuarine, Coastal and Shelf Science 26:409 - 420
Final report of the land-based testing of the Hyde-GuardianTM-System 41

Appendix 1

Phytoplankton Marsdiep AprilJuly 2008 +: present but rare, ++: present; +++: dominant;

data by Jolanda van Iperen ++++: very abundant; +++++: massively present

group species name relative 

dominance

autotrophic flagellate Phaeocystis globosa colony cell +++++

Phaeocystis globosa colonies < 50 μm ++++

Phaeocystis globosa colonies > 50 μm +++++

autotrophic flagellate Phaeocystis globosa flagellate cell +++++

heterotrophic flagellate parasite cyst of Ochromonas group ++++

diatom pennate Pseudonitzschia delicatissima group ++++

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

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

diatom centricate Thalassiosira spp. 10 μm ++++

diatom centricate Chaetoceros socialis ++++

autotrophic flagellate Hemiselmis group +++

diatom centricate Skeletonema "costatum" group +++

autotrophic flagellate Plagioselmis group +++

heterotrophic flagellate Paulinella spp. +++

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

autotrophic flagellate Pyramimonas spp. <10 μm ++

heterotrophic flagellate Choanoflagellatea indet. ++

hetero/auto flagellate Cryptophyceae "light" group ++

autotrophic flagellate Teleaulax acuta group ++

heterotrophic dinoflagellate Oxyrrhis marina ++

diatom centricate Leptocylindrus minimus ++

diatom centricate Minutocellus group ++

diatom centricate Thalassiosira spp. 10-30 μm ++

heterotrophic dinoflagellate Katodinium glaucum ++

diatom centricate Guinardia delicatula ++

freshwater green alga Pediastrum spp. ++

heterotrophic flagellate Ciliophrys group ++

autotrophic flagellate Prasinophyceae indet. <10 μm ++

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

diatom centricate Various species ++

autotrophic flagellate autotrophic flagellate indet. <10 μm ++

diatom centricate Chaetoceros spp. <10 μm solitairy cells +

autotrophic flagellate Chlorophyceae, Telonema spp. +

heterotrophic flagellate Chrysophyceae indet. +

freshwater green alga Crucigenia spp., Chlorophyta +

diatom centricate Skeletonema "costatum" lenses +

freshwater green alga Oocystis spp. +

diatom pennate Asterionellopsis glacialis +

autotrophic flagellate Rhodomonas group +

heterotrophic flagellate Bodo group +

 
Final report of the land-based testing of the Hyde-GuardianTM-System 42

Species list zooplankton and some larger (atypical) plankton

Appendix 1 continued

Phylum Class Subclass, 

Order, etc.

species 

no.

Identified 

genera

most likely 

present

Sarcomastigophora Dinoflagellida 2

Noctiluca, 

Protoperidinium

Bacillariophyceae 3+

Bidulphia, 

Coscinodiscus

Cnidaria Hydrozoa 2+ Obelia

Scyphozoa 2 Aurelia, Cyanea

Ctenophora 2+ Pleurobrachia, 

Beroe,

Mnemiopsis

Nemathelminthes Rotatoria 1+ Asplanchna

Nematoda 1+

Annelida Polychaeta 2+

Arthropoda Crustacea Order 

Calanoida

4+ Temora, 

Acartia, 

Centropages, 

Calanus

and/or 

Pseudocalanus

Oithona

Order 

Harpacticoida 2+ Tigriopus

Subclass 

Cirripedia 1+ Semibalanus

Suborder 

Cladocera 2 Podon, Evadne

Subclass 

Malacostraca 2+

Carcinus (zoea 

larvae)

Mollusca Gastropoda 1+ Littorina

Lamellibranchia 2+ Cerastoderma Mya

Echinodermata

Ophiuroidea 

and/or 2+

Ophiothrix, 

Echinocardium

Echinoidea

Urochordata Larvacea 1 Oikopleura

Minimum number of species encountered (10 phyla): 32