Shipboard Trials of Hyde ‘Guardian’ system in Caribbean 

Sea and Western Pacific Ocean, April 5th - October 7th, 2008. 

Final Report to Hyde Marine and Lamor Corp. 

by 

David A. Wright, University of Maryland Center for Environmental Science, 

Chesapeake Biological Laboratory, Solomons, Maryland 20688 

January 2009 
1 

Table of Contents

 Page . 

INTRODUCTION AND PROJECT DESCRIPTION 3 

Background. 3 

MATERIALS AND METHODS 6 

Disposition of treated and untreated ballast water. 6 

Sample collection. 6 

Examination of ‘challenge water’ immediately following ballasting/treatment 7 

Examination of treated and untreated water following residence time in the tanks. 7 

Sample Preparation and Counting. 9 

Zooplankton. 9 

Phytoplankton. 11 

Bacteria. 12 

Water Quality Measurement. 13 

Data Handling and Analysis. 13 

Whole Effluent testing. 13 

Trial 1, April 5th-18th, 2008. 14 

Sampling Timetable. 14 

Results and Discussion. 14 

Zooplankton. 14 

Phytoplankton. 15 

Bacteria. 21 

Water Chemistry. 24 

GENERAL CONCLUSIONS (Trial 1). 24 

Trial 2. June 30th – July 13th, 2008. 26 

Sampling Timetable. 26 

Results and Discussion. 27 

Zooplankton. 27 

Phytoplankton. 29 

Bacteria. 38 

Water Chemistry. 41 

GENERAL CONCLUSIONS (Trial 2) 41 

Trial 3. September 17th-October 6th, 2008 42 

Sampling Timetable. 42 

Sample collection 42 

Results and Discussion. 43 
2 

Zooplankton. 43 

Phytoplankton. 45 

Bacteria. 54 

Water Chemistry. 56 

GENERAL CONCLUSIONS (Trial 3) 57

Whole Effluent Toxicity Testing 59 

Rationale 59 

Sample Collection and Storage 60 

Results of Whole Effluent Toxicity Tests. 61 

WET Test Summary Data. 62 

Zooplankton. 62 

Invertebrate Assay 62 

Vertebrate Assay 66 

Phytoplankton Assay 69 

SUMMARY CONCLUSIONS 71 

LITERATURE CITED 78 

Appendix A. Raw zooplankton counts. Coral Princess. 80 

 Trial 1 80 

 Trial 2 84 

 Trial 3 90 

Appendix B. Bacterial Sampling Protocol for Ballast Water testing (Trial 3) 

showing mold counts not included in culturable heterotrophic bacteria counts. 96 

Appendix C. Maryland DNR, HAB Laboratory, Statement of Receipt. 103 

Appendix D. QA/QC Protocol related to shipboard trials 104 

Appendix E. Standard Operating Procedures for UMCES Analytical Services Division. 121 
3 

Shipboard Trials of Hyde ‘Guardian’ system in Caribbean Sea and Western 

Pacific Ocean, April 5th - October 6th, 2008. 

INTRODUCTION AND PROJECT DESCRIPTION 

Three trials were conducted aboard the Princess Cruise Lines ship M/V Coral Princess in 

2008 to test the efficacy of the Hyde Marine Inc. ‘Guardian’ Ballast Water Treatment system 

under normal working conditions. The system consists of a primary disc filter manufactured by 

Arkal Inc., Tel Aviv, Israel mounted in series with a medium pressure UV irradiation system 

rated by the manufacturer/vendor at 200 mJ cm2 for treatment of ballast water at flow rates up to 

250 m3 h

-1. Trials took place during the vessel’s regular spring schedule in the Caribbean Sea, the 

summer schedule in the N.W. Pacific Ocean between Whittier, Alaska and Vancouver, Canada, 

and during the repositioning cruise from the western Pacific to the vessel’s winter base in Fort 

Lauderdale, Florida. Trials consisted of determination of water quality parameters and a 

comparison of biological endpoints in treated and untreated ballast water samples, with reference 

to both IMO G8 and the U.S. Coast Guard Shipboard Technology Evaluation Program (STEP). 

Sampling procedures and endpoint determinations followed IMO G8 guidelines for shipboard 

trials and the exercise was designed to supplement land-based trials being conducted 

concomitantly at NIOZ, Texel, Netherlands to determine the efficacy of the BWT system under 

varying water quality conditions. Shipboard trials were designed to document system 

performance under normal seagoing conditions and under different geographical and seasonal 

conditions, with the objective of determining the degree of compliance with IMO and STEP 

requirements. Whole Effluent Toxicity (WET) tests were also conducted as part of the third and 

last trial to determine whether any significant chemical changes in ballast water after exposure to 

UV irradiation, which resulted in subsequent residual toxicity 

Background. 

It is now widely accepted that ships’ ballast water is the primary vector responsible for the 

introduction of non-indigenous aquatic species into coastal habitats (Carlton and Geller 1993, Cohen and 

Carlton 1998, Ruiz et al. 1997, 2000a, b). Pimental (2003) has estimated the total annual economic 

cost from invasive species to the U.S. is $123 billion, with $9 billion attributable to unwanted 

introductions of aquatic nuisance species (ANS) (Pimental et al. 2005). World-wide economic 
4 

costs associated with ANS are incomplete, but have been estimated at least in the tens of billions 

of dollars annually Raaymakers (2002). 

The 2004 IMO Convention for the Management of Ballast water and Sediment in Ships is 

pending ratification by port states representing 30% of global shipping. Criteria for ‘successful’ 

management or treatment have been published as Regulation D-1, relating to Open Ocean Ballast 

Water Exchange and Regulation D-2, described as the Ballast Water Performance Standard 

pertinent to the efficacy of ballast water treatment. The criterion for successful exchange was 

defined as a better than 95% volumetric replacement of water either through an empty-refill 

procedure or a pass-through procedure involving 3x the volume of the tank (or less if the 95% 

exchange is satisfactorily met). ‘Successful treatment’ was defined as the discharge of less than 

10 viable organisms/m3 greater than or equal to 50 μm in minimum dimension and less than 10 

viable organisms/ml less than 50 μm in minimum dimension and greater than or equal to 10 μm 

in minimum dimension. Specific bacteria are included as ‘indicator microbes’, namely serotypes 

O1 and O139 of Vibrio cholerae (standard: <1 cfu/100 ml or <1 cfu/gm wet weight 

zooplankton); Escherichia coli (standard: <250 cfu/100 ml); intestinal Enterococci (<100

cfu/100 ml). 

The April 2008 U.S. Coast Guard authorization Act (HR 2830, Section 503, sub-section 

1101; Ballast Water Management) contains standards that are up to 100x stricter than the IMO 

standards, and even more stringent standards were adopted in January 2008 by the state of 

California, essentially representing the complete elimination of plankton in the >50 μm 

(minimum dimension) size class and a live density of 0.01organisms/ml. in the >10 - <50 μm 

(minimum dimension) size class. California ballast water legislation also includes standards for 

total live bacteria post treatment, (less than 1,000 bacteria per 100 ml.) and viruses (less than 

10,000 viruses per 100 ml.) as well as more rigorous standards relating to indicator bacteria, i.e. 

concentrations of microbes that are less than 126 colony forming units/100 ml. of Escherichia 

coli; 33 colony forming units per 100 ml. of intestinal enterococci and 1 colony forming unit per 

100 ml. or 1 colony forming unit per gram of wet weight of zoological samples of toxicogenic 

Vibrio cholerae (serotypes 01 and 0139). 

In Washington State the interim ballast water discharge treatment standard is 95% 

zooplankton and 99% phytoplankton/bacteria elimination, with the stipulation that “Vessels that 

have not adequately exchanged their ballast water must treat their ballast to meet or exceed the 
5 

Washington State interim ballast water discharge standard prior to discharging in Washington 

waters”. Washington State legislation further states that only approved technologies may be used 

on specified vessels to discharge treated ballast in Washington waters. For approval, technologies 

must meet one of the following criteria: 

 Previously approved by Washington Department of Fisheries and Wildlife 

for use in WA waters 

 Approved by U.S. Coast Guard for use in national waters 

 Enrolled in U.S. Coast Guard Shipboard Technology Evaluation Program 

(STEP). 

 Approved by the State of California for use in California waters 

 Approved by the International Maritime Organization (IMO) and 

authorized by U. S. State Department and U.S. Coast Guard for use in 

national waters. 

 Vessel is enrolled in IMO approval process and is authorized by the U.S. 

State Department and U.S. Coast Guard for use in national waters. 

Table 1 provides a summary of current legislation, both national and international, 

relating to ballast water treatment standards. 

Table 1. 2008 Ballast Water Treatment Standards. 

IMO Regulation 

D-2 and 

Transport 

Canada 

2008 Ballast Water 

Management Act 

Section 1101 (f)i

2008 California 

Standard

Washington 

Administrative Code 

222-170 

Management 

approach 

Exchange moving 

towards treatment 

only 

Exchange moving 

towards treatment 

only 

Exchange moving towards 

treatment only 

Exchange or treatment 

Standard: Proposed Proposed Recommended Interim Adopted Interim: 

1) Organisms 

greater than 50 

microns in 

minimum 

dimension: 

 

2) Organisms 10-

50 microns in 

minimum 

dimension: 

 

3) Organisms less 

than 10 microns in 

minimum 

<10 viable 

organisms per 

cubic meter 

 

 

<10 viable 

organisms per ml 

No standards 

 

 

< 0.1 living 

organisms per cubic 

meter 

 

< 0.1 living 

organisms per ml 

 

 

No standard 

 

 

No detectable living 

organisms 

 

 

<10

-2 

living organisms per 

ml 

 

 

< 10

3

 cfu bacteria/100 ml 

 

 

Technology to 

inactivate or remove 

95% zooplankton 

 

99% bacteria & 

phytoplankton 
6 

MATERIALS AND METHODS. 

Disposition of treated and untreated ballast water.

 A matched pair of ballast tanks (5P, 5S) was identified for each of these trials. One tank 

(5S) was used for treated ballast water and the other for untreated water. Tank 5S was initially 

filled with treated water, followed by the filling of tank 5P with untreated water, as per normal 

ballasting procedure. The ‘treated first’ protocol was designed to eliminate any possible false 

‘positives’ through carry-over of untreated organisms in the ballasting system downstream from 

the BWT unit. For untreated samples, water followed the same path as the treated samples, 

except that the filter was by-passed and the UV unit was deactivated during the ballasting of the 

untreated tank. 

Sample collection.

 The sampling regime adopted for the Hyde Guardian BWT system aboard the M/V Coral 

Princess essentially followed 3 ‘in tank’ replicate x 3 time period (during the de-ballasting 

operation) x 2 treatment (i.e. treated/untreated) x 3 trial matrix design. It should be noted that, for 

the Guardian system, a ‘treatment’ consists of [filtration + UV irradiation] during the ballasting 

cycle PLUS [UV irradiation] during the de-ballasting cycle. Therefore, a treatment is not deemed 

complete until after the water passes through the UV system during de-ballasting. Therefore the 

full sampling matrix (below) only applies to treated and untreated water during the de-ballasting 

dimension: 

 

4) Escherichia coli 

 

5) Intestinal 

Enterococci 

 

6) Toxicogenic 

Vibrio cholerae 

(O1& O139) 

 

 

 

< 250 cfu/100 ml 

 

<100 cfu/100 ml 

 

 

<1 cfu/100 ml 

 

<1 cfu/gram of 

wet zooplankton 

samples 

<126 cfu/100 ml 

 

< 33 cfu/100 ml 

 

 

<1 cfu/100 ml 

 

<1 cfu/gram of wet 

weight of zoological 

samples; 

 

 

 

 

 

<126 cfu/100 ml 

 

<33 cfu/100 ml 

 

 

<1 cfu/100 ml 

 

< 1 cfu/gram of wet 

zoological samples 

 

<10

4

 viruses/100 ml 

 

Final standards – no 

discharge of living 

organisms 
7 

cycle, collected downstream from the BWT system following a residence time (4 days in Trial 1, 

5 days in Trial 2 and 10 days in Trial 3) in the ballast tanks. A sampling port, downstream from 

the Guardian system, established in the machinery space of the vessel for this purpose, and was 

used to process replicate samples of water for biological examination. 

Examination of ‘challenge water’ immediately following ballasting/treatment: 

In order to obtain information on the effect of tank residence time on biota, a series of 

samples (5 in Trial 1, 9 in Trials 2 and 3) were obtained from treated and untreated (control) 

tanks immediately following the initial ballasting/treatment cycle (T=0). The condition/numbers 

of biota in these samples was compared with treated and untreated samples collected later 

following a period of residence in the ballast tanks. 

Examination of treated and untreated water following residence time in the tanks.

Following the residence time in the tank, samples of treated and untreated water were collected 

according to the following sequence: 

1. Treated tank; replicate T1a filtered/sampled at the start of the de-ballasting cycle. 

2. Treated tank; replicate T1b filtered/sampled at the start of the de-ballasting cycle. 

3. Treated tank; replicate T1c filtered/sampled at the start of the de-ballasting cycle. 

4. Treated tank; replicate T2a filtered/sampled in the middle of the de-ballasting 

cycle. 

5. Treated tank; replicate T2b filtered/sampled in the middle of the de-ballasting 

cycle. 

6. Treated tank; replicate T2c filtered/sampled in the middle of the de-ballasting 

cycle. 

7. Treated tank; replicate T3a filtered/sampled at the end of the de-ballasting cycle. 

8. Treated tank; replicate T3b filtered/sampled at the end of the de-ballasting cycle. 

9. Treated tank; replicate T3c filtered/sampled at the end of the de-ballasting cycle. 
8 

10. Untreated (Control) tank; replicate C1a filtered/sampled at the start of the deballasting cycle. 

11. Untreated (Control) tank; replicate C1b filtered/sampled at the start of the deballasting cycle. 

12. Untreated (Control) tank; replicate C1c filtered/sampled at the start of the deballasting cycle. 

13. Untreated (Control) tank; replicate C2a filtered/sampled in the middle of the deballasting cycle. 

14. Untreated (Control) tank; replicate C2b filtered/sampled in the middle of the deballasting cycle. 

15. Untreated (Control) tank; replicate C2c filtered/sampled in the middle of the deballasting cycle. 

16. Untreated (Control) tank; replicate C3a filtered/sampled at the end of the deballasting cycle. 

17. Untreated (Control) tank; replicate C3b filtered/sampled at the end of the deballasting cycle. 

18. Untreated (Control) tank; replicate C3c filtered/sampled at the end of the deballasting cycle. 

All samples were prepared for biological examination within 1 h of collection. This 

involved some pre-filtration of samples within the ship’s machinery space, using 30 cm, 20 μm 

nets for zooplankton. Samples for phytoplankton and bacterial counts were unfiltered, although 

phytoplankton samples were further concentrated by filtration through 10 μm filters before growout and examination. These procedures are described below and a summary of the sampling 

scheme is shown in Figure 1. 
9 

Sample Preparation and Counting. 

Zooplankton. 

Samples for zooplankton counting were filtered through 30cm. nylon plankton nets 

consisting of 20μm mesh (nominal 50μm mesh nets can allow the passage of some >50μm 

organisms). During filtration, nets were submerged under the surface of a 125L plastic tub to 

soften the impact of the filtration procedure on the planktonic organisms. The 1L plastic bottle 

that forms the `cod-end' of the net also had 20μm mesh `windows' to facilitate the filtration 

process. Separate nets were used to sample from treated and untreated samples, and nets were 

rinsed with hot tap-water between sampling cycles. They were also examined for tears, leaks and 

imperfections and any repairs made. In the machinery space the contents of each mesocosm were 

filtered and concentrated to a volume suitable for manual transport up to the cabin space where 

samples were turned over to microscopists for examination. On receiving samples from the 

collection team, microscopists further concentrated samples through 10μm mesh filters to produce 

volumes suitable for microscopy, usually 10-20ml. Concentrated samples were transferred via 

Stempel pipets to counting wheels mounted on compound microscope stages for microscopical 

examination. For each sample, the appropriate multiplier was applied to each count for 

zooplankton densities to be expressed in terms of numbers of organisms per metric ton (m3). 

Records were kept of both alive and dead organisms in each sample, which were identified to the 

extent possible to the lowest taxonomic group (see Appendix A for taxonomic breakdown of 

zooplankton from each cruise), and records kept of the dimensions of each group of organisms, 

including maximum and minimum dimensions (μm). Dimensions of organisms were measures 

using reticule eye-pieces calibrated against National Institute of Standards and Technology 

certifies beads from 10 - 200μm in diameter. Sizing was also facilitated by seeding certified beads 

of known diameter into counting wheels. Live-dead status was assessed as movement of the 

organism, either as motility, heartbeat or movement of flagellum, velum or gut, following 

stimulation if necessary. This examination was supplemented by the use of the vital stain Neutral 

Red. 
10 

Figure 1. Schematic of Shipboard Trial sampling scheme design for Coral Princess Trials, 

2008 

>4day residence 

time 

UV UV UV 

UV 

Sea Chest filter 

400L 

Bacteria 

10-50/

200um

Zooplankton 

Phytoplankton 

500L 500L 500L 500L 

Bacteria 

10-50um 

200um

Zooplankton 

Phytoplankton 

Samples 

taken (t=0) 

24hr grow-out 

period samples 

Bacteria Phytoplankton Bacteria Phytoplankton 

48hr grow-out 

period samples 

Bacteria Bacteria 

T1 T2 T3 U1 U2 U3 

Filter 

Mesocosm 

Sample 

Ballast Tank 

(count ASAP) 

500L 500L 

72hr grow-out 

Bacteria period samples Bacteria 

(count ASAP) 
11 

Phytoplankton. 

 Live-dead status of phytoplankton for the determination of ballast water treatment 

efficacy remains problematic to the extent that, while some taxonomic groups, such as 

dinoflagellates, are clearly motile, many have vegetative stages that are immobile. The growth 

potential of non-motile forms can only be assessed by a variety of methods, including 

microscopic examination of chloroplast integrity, use of vital stain(s) and cell counts (of at least 

dominant groups) following a grow-out period under optimal growth conditions. The grow-out 

period for phytoplankton usually consists of a 24h (or 48h) period of irradiation under 

fluorescent lighting and non-limiting nutrient conditions through the addition of f/2 growth 

medium. Phytoplankton growth may be conveniently assessed by measuring in vivo chlorophyll 

a concentration before and after the grow-out period. While this represents a useful integrative 

determination of the status of the phytoplankton community as a whole, it does not provide 

information on individual taxonomic groups of phytoplankton that might have quite different 

characteristics in terms of size, shape, doubling time (growth rate) etc. Also, chlorophyll a data 

cannot be interpreted in terms of published standards. 

 Unfiltered 1L samples for phytoplankton analyses were taken from the same sample 

stream as that used for zooplankton sampling. Each sample was concentrated to a volume <10ml. 

for examination/grow-out, using a 10μm Nitex screen. Microscopic examination of 

cell/chloroplast integrity was supplemented by the use of Neutral Red as a vital stain. Unstained 

samples examined soon after collection were compared with ‘splits’ of the same sample that 

were stained with Neutral Red. Samples following a grow-out period were similarly examined, 

with and without Neutral Red. For these trials study in vivo chlorophyll a analyses was 

supplemented by individual cell counts of dominant phytoplankton taxa before and after a 24h 

grow-out period under fluorescent lights in ambient seawater supplemented with f/2 growth 

medium. Following initial examination of phytoplankton (before and after grow-out) to 

determine their general appearance, observations of chloroplast integrity and the activity of 

motile forms, samples were preserved in Lugol's Solution for more intensive taxonomy and 

determination of cell sizes. Typically counts were made of >200 squares of a 1000 square 

counting grid,. Determination of living phytoplankton was be made on the basis of (a) 

chlorophyll a analysis, (b) vital staining techniques and (c) cell counts before and after grow-out. 
12 

Bacteria. 

 Samples for bacterial analyses were be taken directly from the unfiltered discharge from 

the sampling port, and stored at temperatures just above freezing (1-4OC) prior to and during 

transport to the University of Maryland Center for Environmental Science, Chesapeake 

Biological Laboratory, although in Trial 3 some culturing/counting was carried out in cabin space 

aboard the vessel. Bacterial culture formed the basis of bacterial endpoints that were used to test 

the bactericidal effectiveness of the BWT system aboard this vessel. The following bactericidal 

endpoints were employed: 

• Plate counts of cultural heterotrophic bacteria. It is emphasized that only 

approximately 1% of marine bacteria will culture successfully, and that this figure 

may vary according to geographical area. This, therefore, represents only an 

approximate measure of the efficacy of BWT technology, and is somewhat 

variable according to geographical location. 

• Primary focus was on taxonomic groups specified in recently published IMO 

standards: colony-forming units of E.Coli, Enterococcus and Vibrio Cholera (with 

specific emphasis on virulent serotypes). Fluorescence-based techniques (IDEXX 

Laboatories.) were employed to quantify coliforms, E. Coli and Enterococcus in 

treated and untreated ballast water. 

Samples were diluted an order of magnitude with sterile deionized water for the IDEXX 

protocols. The established detection range for this technique is 10 – 24,190 cfu / 100 mL of sea 

water sample. 

Fluorescence and photometric counting of Coliform/E.coli was determined using the 

IDEXX Laboratories Inc. (ME) Colisure Quantitray counts following 18h incubation at 35OC. 

Fluorescence counting of Enterococcus was determined by IDEXX Laboratories Inc. 

(ME) Enterolert Quantitray 2000 counts following 24h incubation at 41OC. 

 Further details of microbiological protocols are found in Appendix B. 

Quantification of viable Vibrio cells was facilitated by the use of Polymerase Chain 

Reaction amplification techniques on refrigerated samples transported to the University of 
13 

Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Molecular 

Biology Laboratory, directed by Dr. Carys Mitchelmore. 

Water Quality Measurement.

 The following water quality parameters were analyzed: temperature, salinity, pH, 

dissolved oxygen, total suspended solids, total particulate carbon, dissolved organic carbon 

phosphate, nitrate, nitrite (Trial 3) and UV transmittance. 

Data Handling and Analysis. 

Data were entered into a SAS (software) file and a header file created. This included a 

factor for the calculation of volume of filtered water, enabling raw counts to be appropriately 

multiplied to compute numbers of organisms per ton of water. This file is included in this report 

as Appendix A. Results from biological analyses from these trials were compared with published 

standards. Additionally, zooplankton mortalities in treated samples will expressed in terms of 

percentage removal relative to untreated controls. An assessment of the effect of ballast tank 

residence time was also made. 

Whole Effluent Toxicity (WET) Testing. 

In order to comply with IMO G-8 requirements for ‘environmental acceptability of water 

treated by this technology, WET tests were conducted on standard test organisms exposed to 

treated and untreated water from Trial 3 to provide empirical information on possible residual 

toxicity on ballast discharge, resulting from chemical changes induced by UV irradiation during 

treatment. Details of these tests are provided on page 59. 
14 

TRIAL 1, April 5th-18th, 2008

Sampling Timetable

Ballasting took place close to the port of Aruba on April 8th, 2008, day 4 of a ten day 

cruise. Collection of samples 1-18 (see Materials and Methods) commenced immediately after the 

ballast pump began retrieving water from each of the respective tanks, following a residence time 

in the tanks of 96h. Ballast water was be taken on/treated after the vessel left Aruba on April 9th, 

2008) and was discharged/sampled/analyzed four days later, after the ship departed Ocho Rios, 

Jamaica on April 13th. Samples for bacterial examination were kept on ice until their return to the 

Chesapeake Biological Laboratory on April 15th incubation and grow-out of these samples 

continued until April 18th, 2008. 

Results and Discussion.

Zooplankton.

Results of zooplankton counts are summarized in table 2. These indicated a 98.6% 

mortality of zooplankton (>50μm minimum dimension) immediately following treatment on 

ballasting relative to untreated samples collected during the same sampling event, and a 100% 

mortality of zooplankton (>50μm minimum dimension) after a period of 4 days in the ballast 

tank followed by UV irradiation on de-ballasting. In contrast, untreated control samples 

demonstrated good survival following the 96h residence time in the tank. Overall there was no 

statistical decline in control numbers relative to those recorded from the intake water, although 

largevariations in organism numbers were apparent throughout the de-ballasting cycle, reflecting 

probable differences in plankton densities throughout the water column in the tanks. 
15 

Table 2. Summary of zooplankton results (>50μm minimum dimension) for Trial 1 of Hyde 

‘Guardian’ BWT system aboard M/V Coral Princess, April 5th-18th, 2008. 

Zooplankton Alive (>50μm). 

Density/m3

Dead (>50μm) 

Density/m3

Control (untreated) N=5 453±269 66±66 

Treated (UV + filter 

during ballasting) N=5 

6.4±6.7 1.6±3.6 

 

Control (untreated) 

Start de-ballast (N=3) 

432±345 232±236 

Control (untreated) Mid 

de-ballast (N=3) 

296±343 536±385 

Control (untreated) End 

de-ballast (N=3) 

755±164 1,195±352 

Treated (UV + filter 

during ballasting) →

(UV at de-ballasting) 

Start de-ballast (N=3) 

0 125±21 

Treated (UV + filter 

during ballasting) →

(UV at de-ballasting) 

Start de-ballast (N=3) 

0 131±99 

Treated (UV + filter 

during ballasting) →

(UV at de-ballasting) 

Start de-ballast (N=3) 

0 75±72 

Phytoplankton.

Phytoplankton counts from treated and untreated (control) samples from T=0 and T=96h are 

shown in tables 3-5. Based on microscopic examination, cells were scored as ‘live’ based on 

morphological characteristics such as chloroplast integrity and the ability to concentrate the 
16 

Table 3. Summary of phytoplankton results (>10<50μm minimum dimension) for Trial 1 of 

Hyde ‘Guardian’ BWT system aboard M/V Coral Princess, April 5th-18th, 2008. T=0 data 

including grow-out cell concentrations. 

Phytoplankton 

(>10μm-<50μm) 

‘Live’

Dinoflagellates. 

Density/m3

‘Live’

Diatoms. 

Density/m3 

Total ‘Live’

phytoplankton. 

Density/m3

Dead 

Dinoflagellates. 

Density/m3

Dead 

Diatoms. 

Density/m3 

Total Dead

phytoplankton. 

Density/m3

Untreated Controls, T=0

Rep. C1, T=0 9,821 173,661 183,182 0 0 0

Rep. C1, T=0 + 48h 

growout 

16,111 31,944 48,105 556 556 1,112

Rep. C2 T=0 4,018 62,946 66,964 0 0 0

Rep. C2 T=0 + 48h 

growout 

4,722 15,277 20,000 0 833 833

Rep. C3 T=0 23,661 127,679 151,340 0 0 0

Rep. C3 T=0 + 48h 

growout 

2,500 63,333 65,833 278 556 834

Mean ‘Live’ 

Phytoplankton. 

Control samples, T=0 

(after grow-out)

133,829±60,055

(44,646±23,111) 

Treated, T=0

Rep. T1, T=0 9,583 71,667 81,250 0 0 0

Rep. T1, T=0 + 48h 

growout 

4,444 5,000 9,444 833 278 1,111

Rep. T2 T=0 7,917 75,833 83,800 2,083 417 2,500

Rep. T2 T=0 + 48h 

growout 

3,056 6,111 9,167 0 0 0

Rep. T3 T=0 3,333 69,583 72,916 417 0 417

Rep. T3 T=0 + 48h 

growout 

3,056 5,278 8,334 0 0 0

Mean ‘Live’ 

Phytoplankton. 

Treated samples, T=0 

(after grow-out)

79,322±5,692

(8,982±578) 
17 

Table 4. Summary of phytoplankton results (>10<50μm minimum dimension) for Trial 1 of 

Hyde ‘Guardian’ BWT system aboard M/V Coral Princess, April 5th-18th, 2008. T=96h 

untreated (control) data. 

Phytoplankton. 

(>10μm-

<50μm) 

‘Live’

Dinoflagellates. 

Density/m3

‘Live’

Diatoms. 

Density/m3

Total ‘Live’

phytoplankton. 

Density/m3

Dead 

Dinoflagellates. 

Density/m3

Dead 

Diatoms. 

Density/m3

Total Dead 

phytoplankton. 

Density/m3

Untreated Controls, T=96h 

Start Deballast. Rep. 

C1a, T=96 

278 5,278 5,556 0 0 0 

Rep. C1b T=96 278 5,000 5,278 0 556 556 

Rep. C1c T=96 0 2,500 2,500 0 0 0 

Mean C1, T = 

96h samples 

 4,445±1,690 

Mid Deballast. Rep. 

C2a, T=96 

278 18,889 19,167 0 3,333 3,333 

Rep. C2b T=96 833 14,444 15,277 0 2,500 2,500 

Rep. C2c T=0 1,111 8,889 10,000 0 3,889 3,889 

Mean C2, T = 

96h samples 

14,815±4,601 

End Deballast. Rep. 

C3a, T=96 

1,111 14,722 15,883 556 5,278 5,834 

Rep. C3b T=96 833 16,389 17,222 278 3,889 4,167 

Rep. C3c T=96 556 20,556 21,112 0 3,889 3,889 

Mean C3, T = 

96h samples 

 18,072±2,716 

Mean Live 

Phytoplankton 

12,444±6,770 

(n=9) 
18 

Table 5 Summary of phytoplankton results (>10<50μm minimum dimension) for Trial 1 of Hyde 

‘Guardian’ BWT system aboard M/V Coral Princess, April 5th-18th, 2008. T=96h data 

from treated samples, including 45-47h grow-out cell concentrations. 

Phytoplankton. 

(>10μm-

<50μm) 

‘Live’

Dinoflagellates. 

Density/m3

‘Live’

Diatoms. 

Density/m3

Total ‘Live’ 

phytoplankton. 

Density/m3

Dead 

Dinoflagellates. 

Density/m3

Dead 

Diatoms. 

Density/m3

Total Dead 

phytoplankton 

Density/m3

Treated, T=96h 

Start Deballast. Rep. 

T1a, T=96 

556 3,611 4,167 278 0 278 

Rep. T1a, T=96 

+ 48h growout 

0 1,667 1,667 0 556 556 

Rep. T1b T=96 278 6,944 7,222 0 556 556 

Rep. T1b T=96 

+ 48h growout 

0 833 833 0 278 278 

Rep. T1c T=96 0 6,111 6,111 556 0 556 

Rep. T1c T=96 

+ 48h growout 

0 1,389 1,389 0 0 0 

Mean T1, 

T=96h samples 

(after growout)

 5,833±1,546 

(1,296±425) 

 

Mid Deballast. Rep. 

T2a, T=96 

278 7,222 7,500 278 278 556 

Rep. T2a, T=96 

+ 48h growout 

0 1,389 1,389 0 0 0 

Rep. T2b T=96 0 6,389 6,389 0 0 0 

Rep. T2b T=96 

+ 48h growout 

0 4,444 4,444 0 278 278 

Rep. T2c T=96 278 5,278 5,556 278 0 278 

Rep. T2c T=96 

+ 48h growout 

556 556 1,112 0 3,056 3,056 

Mean T2, T = 

96h samples 

(after growout)

6,482±975 

(2,315±1,849) 

End De- 556 3,611 4,167 0 2,778 2,778 
19 

ballast. Rep. 

T3a, T=96 

Rep. T3a, T=96 

+ 48h growout 

0 2,222 2,222 0 1,944 1,944 

Rep. T3b T=96 278 6,944 7,222 0 1,389 1,389 

Rep. T3b T=96 

+ 48h growout 

278 4,722 5,000 0 556 556 

Rep. T3c T=96 556 8,056 8,612 0 0 0 

Rep. T3c T=96 

+ 48h growout 

556 1,389 1,945 0 278 278 

Mean T3, T = 

96h samples 

(after growout)

 6,667±2,274 

(3,055±1,690) 

 

Mean Live 

Phytoplankton

(after growout)

6,327±1,421 

(n=9) 

(2,222±1,483) 

(n=9) 

vital stain Neutral Red. Based solely on these criteria, initial treatment (filter + UV during 

ballasting) resulted in an immediate 41% reduction in live cell numbers relative to untreated 

samples at T=0. However, following a 96h residence time in the tank, untreated ‘live’ cell 

numbers had fallen to 7% of the initial, untreated T=0 density, and treated ‘live’ cell numbers had 

fallen to 4.7% of that initial concentration (i.e. 95.3% removal). Under such circumstances a 

comparison between treated and control ‘live’ densities at 96h probably has little meaning as it is 

clear that the ballast tank provides an inhospitable environment for treated and untreated cells 

alike. If it is assumed that viability is best described by growth potential this assumption is 

further reinforced by cell counts following grow-out. Concentrations of treated cells following 

grow-out, shown in red in table 5, clearly indicate a failure to grow, based on the fact that they 

represent a mean reduction in cell numbers of 65% relative to the corresponding samples before 

grow-out. Based on growth potential, treated phytoplankton at 96h could reasonably be described 

as non-viable. (Grow-out data were not available for untreated (control) samples after 96h in the 

tank). This assessment is reinforced by measurement of chlorophyll a concentrations before and 

after grow-out (table 6). While some positive growth was seen in untreated control samples at 
20 

T=0, 2/3 control samples exhibited a small amount of growth after 96h, while no growth relative 

to intake water was recorded from treated samples at either T=0 or T=96h. 

 If ‘live’ cell counts based on morphological examination are used as the basis for 

regulatory compliance, the ‘live’ phytoplankton count of 6,327 recorded for treated samples at 

96h (table 5) would comply with both the current IMO standard of 107 live cells per m3 (10 live 

cells/mL) the U.S. Coast Guard Standard standard of 105 live cells per m3 (0.1 live cells/mL), 

and the much more rigorous standard of 104 live cells per m3 (0.01 live cells/mL) currently 

employed by the state of California. We conclude that cell numbers based on counts made after 

grow-out are even lower and probably best represent the criterion defining the term “live”, i.e. 

having potential for growth. 

Overall phytoplankton cell numbers were exceptionally low in challenge water samples, 

and we observe that, in this trial, even untreated samples would comply with IMO and U.S. 

Coast Guard standards at discharge, but not the more rigorous California standard. 

Table 6. Chlorophyll a concentrations from measurement of in vivo fluorescence. (all values are means 

of 3 determinations) 

T=0 T=0, Pre grow-out 

chlorophyll a conc. (μg 

L

-1)

chlorophyll a conc. (μg L-1) 

after 24h grow-out (T=0 + 24)

chlorophyll a conc. (μg 

L

-1) after 48h grow-out 

(T=0 + 48)

Control 1 start 

ballasting 

2.75 4.33 4.3

Control 2 mid 

ballasting 

1.81 1.95 1.98

Control 3 end 

ballasting 

7.78 8.58 8.76

Treated 1 start 

ballasting 

1.31 0.92 0.88

Treated 2 mid 

ballasting 

1 0.98 0.82

Treated 3 end 

ballasting 

1.13 1 0.94

T=96h T=96h, Pre grow-out 

Chlorophyll a conc. (μg 

L

-1)

Chlorophyll a conc. (μg L-1) 

after 24h grow-out (T=96 + 

24)

Chlorophyll a conc. (μg 

L

-1) after 48h grow-out 

(T=96 + 48)

Control 1 start deballasting 

1.2 1.29 1.4

Control 2 mid deballasting 

1.43 1.86 1.77
21 

Control 3 end deballasting 

1.26 1.45 1.22

Treated 1 start deballasting 

1.34 1.26 1.22

Treated 2 mid deballasting 

1.31 1.23 1.15

Treated 3 end deballasting 

1.22 1.22 1.07

Bacteria.

Based on raw counts of intestinal Enterococci, and coliforms (table 7), no viable cfus of 

these indicator microbes were detected in treated ballast water samples from this trial and only 

one each cfu of coliforms and Enterococci were recorded from control samples. No Vibrio 

cholerae cells, live or dead, were found in any of the samples examined. 

Low densities of cultural bacteria were detected in both control and treated samples (12.2 

± 12.9cfu per 100ml. and 19.9 ± 10.5 per 100ml. respectively). By 96h mean cfu per 100ml. had 

fallen to 5 and 1 respectively (table 8).

Table 7. Trial 1. Enterococci, Vibrio cholerae and colifom counts from treated and untreated 

(control) samples at T=0 and T=96h. 

 Coliforms E. Coli Enterrococci Vibrio 

Cholerae

Treatment Time Replicate Large Small Large Small Large Small

Control T=0 C1 1 0 0 0 0 0 0 

Control T=0 C2 0 0 0 0 0 0 0 

Control T=0 C3 0 0 0 0 0 0 0 

Control T=0 C4 0 0 0 0 0 0 0 

Control T=0 C5 0 0 0 0 0 1 0 

Treated T=0 T1 0 0 0 0 0 0 0 

Treated T=0 T2 0 0 0 0 0 0 0 

Treated T=0 T3 0 0 0 0 0 0 0 

Treated T=0 T4 0 0 0 0 0 0 0 

Treated T=0 T5 0 0 0 0 0 0 0 

Control T=96h C1a 0 0 0 0 0 0 0 

Control T=96h C1b 0 0 0 0 0 0 0 

Control T=96h C1c 0 0 0 0 0 0 0 

Control T=96h C2a 0 0 0 0 0 0 0 

Control T=96h C2b 0 0 0 0 0 0 0 

Control T=96h C2c 0 0 0 0 0 0 0 
22 

Control T=96h C3a 0 0 0 0 0 0 0 

Control T=96h C3b 0 0 0 0 0 0 0 

Control T=96h C3c 0 0 0 0 0 0 0 

Treated T=96h T1a 0 0 0 0 0 0 0 

Treated T=96h T1b 0 0 0 0 0 0 0 

Treated T=96h T1c 0 0 0 0 0 0 0 

Treated T=96h T2a 0 0 0 0 0 0 0 

Treated T=96h T2b 0 0 0 0 0 0 0 

Treated T=96h T2c 0 0 0 0 0 0 0 

Treated T=96h T3a 0 0 0 0 0 0 0 

Treated T=96h T3b 0 0 0 0 0 0 0 

Treated T=96h T3c 0 0 0 0 0 0 0 
23 

Table 8. Trial 1. Cultural bacteria in treated and untreated (control) samples at T=0 and 

T=96h. (10ml. samples were filtered through 0.45μm. Plates were read after 72h incubation 

at 25o 

 Colony 

forming 

units 

Colony 

forming 

units 

Colony 

forming 

units 

100% 10% 1%

Treatment Time Replicate 

Control T=0 C1 8 2 0 

Control T=0 C2 34 2 2 

Control T=0 C3 4 0 0 

Control T=0 C4 2 0 0 

Control T=0 C5 13 1 0 

Treated T=0 T1 33 1 0 

Treated T=0 T2 27 3 0 

Treated T=0 T3 18 0 0 

Treated T=0 T4 11 1 0 

Treated T=0 T5 8 0 0 

Control T=96h C1a 2 0 0 

Control T=96h C1b 3 3 1 

Control T=96h C1c 8 0 0 

Control T=96h C2a 19 2 0 

Control T=96h C2b 3 3 0 

Control T=96h C2c 3 0 0 

Control T=96h C3a 0 0 0 

Control T=96h C3b 3 1 0 

Control T=96h C3c 4 0 0 

Treated T=96h T1a 2 0 0 

Treated T=96h T1b 0 0 0 

Treated T=96h T1c 0 0 0 

Treated T=96h T2a 0 0 0 

Treated T=96h T2b 5 0 0 

Treated T=96h T2c 0 0 0 

Treated T=96h T3a 0 0 0 

Treated T=96h T3b 0 0 0 

Treated T=96h T3c 2 0 0 
24 

Water Chemistry. 

Ballast water during trial 1 was taken from near Aruba Harbor on April 8th, 2008. The salinity of 

challenge water (untreated water at T=0) varied between 36.4 – 37.1 PSU (mean 36.8), water 

temperatures ranged from 25.2 – 29.0o C (mean 26.8) and pH from 7.3-7.4. Dissolved oxygen 

(DO) concentrations ranged from 5.4mg L-1 to 6.6mg L-1 (mean 6.0mg L-1). DO and pH 

measured in ballast water immediately after treatment (T=0) showed no noticeable change from 

untreated water (table 9). UV transmittance was determined to be 95% in untreated water at T=0 

Nutrient levels in challenge water extremely low, ranging between 0.6 – 1.2 μg L-1 for nitrate and 

2.2 – 4.0 μg L-1 for phosphate. A higher nitrate level (9.72μg L-1) was recorded from treated 

samples at T=0. 

Table 9. Trial 1. Water Chemistry in untreated challenge water and treated ballast water at time of 

ballasting (T=0) near Aruba, April 8th, 2008. (DO = Dissolved O2; TSS = Total Suspended Solids; 

POC – Particulate Organic Carbon; DOC = Dissolved Organic Carbon; NO3 = Nitrate; PO4 = 

Phosphate) 

 pH DO 

(mg L-1) 

Salinity 

(PSU) 

Temp(

OC) 

TSS 

(mg L-1) 

POC 

(mg L-1) 

DOC 

(mg L-1) 

NO3

(μgN L-1)

PO4

(μgP L-1) 

C1, T=0 

(begin 

ballast) 

7.3 6.47 36.8 26.8 4.12±0.09 3.24±0.38 1.37±0.11 1.1±0.4 2.8±0.4 

C2, T=0 (mid 

ballast) 

 4.51±0.20 2.58±0.62 0.85±0.05 0.6±0.2 4.0±0.5 

C3, T=0 (end 

ballast) 

 4.46±0.38 2.56±1.16 0.92±0.07 1.2±0.8 2.2±0.4 

T1, T=0 

(begin 

ballast) 

7.4 5.41 4.50±0.23 3.00±0.90 1.75±0.36 9.7±0.37 4.4±0.7 

GENERAL CONCLUSIONS (Trial 1).

 Plankton densities in challenge water reflect oligotrophic conditions typified by low 

zooplankton and phytoplankton densities. However, zooplankton densities in untreated challenge 

water did exceed the value of 10 x D-2 required by IMO G8 guidelines. Based on results 

obtained from the first trial, the Hyde ‘Guardian’ system would comply with those portions of 

IMO Regulation D-2 and current California regulations relating to plankton. Viable bacteria, 

measured as colony forming units (cfu) of named indicator bacteria were within the prescribed 

limits published in current IMO Regulation D-2, U.S.C.G. and California regulations. Only 1 cfu 
25 

each of coliforms intestinal Enterococci were detected in untreated samples and none were seen 

in any treated samples. Culturable heterotrophic bacterial numbers were low in both treated 

samples, and actually decreased in both treated and untreated samples between T=0 and deballasting at T=96h. Cfus in treated samples declined more rapidly than in untreated samples 

over this time. At the time of de-ballasting treated and untreated samples contained 10 cfu/100 

ml. and 50 cfu/100 ml. respectively. No determination was made of performance of the BWT 

system against viruses, as per California regulations. 
26 

TRIAL 2. June 30th – July 13th, 2008

Sampling Timetable.

In Trial 2 the same matched pair of ballast tanks (5P, 5S) was employed as in Trial 1, i.e. 

tank (5S) was used for treated ballast water and the other (5P) for untreated water. Ballasting 

took place southeast of the port of Whittier, Alaska on July 1st, 2008, day 2 of an eight day 

cruise. As with Trial 1, tanks 5S and 5P were filled with treated and untreated water respectively, 

as per normal ballasting procedure, and for untreated samples, water followed the same path as 

the treated samples, except that the filter was by-passed and the UV unit was deactivated during 

the ballasting of the untreated tank. 

Sample collection.

 The same sampling regime was adopted as for Trial 1, i.e. 3 ‘in tank’ replicates x 3 time 

period (during the de-ballasting operation) x 2 treatment (i.e. treated/untreated), except that nine 

untreated samples and seven treated samples were obtained from the respective tanks 

immediately following the initial ballasting/treatment cycle. In the second trial a residence time 

of 114h elapsed before samples were retrieved from the tanks according to the sequence 

previously described. Ballast water was taken up/treated the day after the vessel left Whittier on 

July 1st, 2008, and was discharged/sampled/analyzed 4.5days later, after the ship departed 

Ketchekan on July 6th. Samples for bacteriological analysis were kept on ice following collection 

and accompanied the scientific team to Dr. Mitchelmore’s laboratory at the University of 

Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons, 

Maryland. Culture and analysis of these samples continued until July 13th 2008. 

Preparation of samples for biological examination followed procedures described for 

Trial 1 (see Figure 1.) Methods for sample examination and analysis were as previously 

described.
27 

Results and Discussion 

Zooplankton.

Results of zooplankton counts are summarized in table 10. Results indicated a 99.99% 

mortality/removal of zooplankton (>50μm minimum dimension) immediately following 

treatment on ballasting relative to untreated samples collected during the same sampling event. 

Samples were characterized by a dramatic difference in biomass between the treated and 

untreated samples. In treated samples a mean of 1.14 live organisms >50μm in minimum 

dimension per ton were found at T=0. No dead organisms were found in this size range in treated 

samples, indicating that the large majority were removed by the filter. In control samples at T=0, 

15,373±6118 live organisms >50μm and 141±117 dead organisms >50μm per ton were found. It 

is of interest to note that the live density of organisms >50μm in the challenge water showed a 

35-fold increase relative to the much more oligotrophic conditions encountered in Caribbean 

waters during the first, April 2008, trial. 

Unlike trial 1, there was a dramatic (98.4%) decline in overall numbers of live organisms 

in the untreated (control) tank after the 114h residence period. Both treated and untreated 

samples retrieved from the tanks after 114h were quite different in character from the samples 

examined at T=0. In treated samples, after 114h in the tank, and following UV irradiation on deballasting, numerous zooplankters in the >50μm minimum dimension size range were found in 

treated samples, although these all appeared to be dead, as judged by motility and many had 

begun to disintegrate. Many of these organisms were not seen in the T=0 untreated samples, and 

we conclude that many were living in the tanks before the start of the trial. 
28 

Table 10. Summary of zooplankton results (>50μm minimum dimension) for Trial 2 of 

Hyde ‘Guardian’ BWT system aboard M/V Coral Princess, June 30th – July 8th, 2008. 

Zooplankton 

Alive (>50μm). 

Density/m3

Dead (>50μm) 

Density/m3

Control (untreated) N=9 15,373±6118 141±117 

Treated (UV + filter 

during ballasting) N=7 

1.14±2.8 0 

Control (untreated) 

Start de-ballast (N=3) 

504±291 0 

Control (untreated) Mid 

de-ballast (N=3) 

437±624 1,123±572 

Control (untreated) End 

de-ballast (N=3) 

43±74 971±654 

332±431 698±683 

Treated (UV + filter 

during ballasting) →

(UV at de-ballasting) 

Start de-ballast (N=3) 

0 75±16 

Treated (UV + filter 

during ballasting) →

(UV at de-ballasting) 

Mid de-ballast (N=3) 

0 144±194 

Treated (UV + filter 

during ballasting) →

(UV at de-ballasting) 

End de-ballast (N=3) 

0 224±89 

Counts indicated live residuals of smaller zooplankton, the great majority of which were 

marine nematodes, in both treated and untreated samples taken at 114h, although these did not 

fall into the >50μm size category. Live/dead nematode numbers are shown in table 11. 

Nematode numbers were almost completely absent from the T=0 samples, and were seen to 

increase in T=114h samples from the beginning to the end of the de-ballasting cycle, reflecting a 

probable difference in plankton densities throughout the water column in the tanks. The most 

likely explanation for this is that, as the tank approaches empty, increasing numbers of organisms 

living in or on the surface of the residual sediment are stirred up and appear in the later samples. 
29 

In trial 1 it was noted that overall zooplankton densities at the end of the de-ballasting cycle were 

nearly double those at the beginning of the de-ballasting cycle, perhaps reflecting a similar 

phenomenon, although it was noteworthy that no nematodes were found in trial 1 samples. 

 

Table 11. Densities of living nematode worms in treated and untreated samples.

Trial 2. June 30th-July 11th 2008.

 Nematode densities (concentrations per m3

) 

Live Dead 

T=0h 

Control 0 0 

Treated 0 0 

T=114h 

Control (untreated). Start deballast (N=3) 

0 0 

Control (untreated) Mid deballast (N=3) 

0 0 

Control (untreated) End deballast (N=3) 

0 11±15 

Treated (UV + filter during 

ballasting) → (UV at deballasting). Start de-ballast (N=3) 

0 0 

Treated (UV + filter during 

ballasting) → (UV at deballasting) Mid de-ballast (N=3) 

27±27 69±98 

Treated (UV + filter during 

ballasting) → (UV at deballasting) End de-ballast (N=3) 

80±39 160±48 

Phytoplankton.

Phytoplankton counts from treated and untreated (control) samples from T=0 and T=114h are 

shown in tables 12-15. Based on microscopic examination, cells were scored as ‘live’ based on 

morphological characteristics such as chloroplast integrity and the ability to concentrate the vital 

stain Neutral Red. Unlike trial 1, initial treatment (filter + UV during ballasting) resulted in a 

dramatic (97%) reduction in live cell numbers based solely on morphological characteristics. 
30 

Following a 114h residence time in the tank, untreated ‘live’ cell numbers had fallen to 30% of 

the initial, untreated T=0 density (compared with 7% in trial 1). This, despite the presence of 

exceptionally high numbers of apparently viable diatoms in the first sample of this series (table 

14), perhaps reflecting the release of a ‘pulse’ of ‘live’ cells at the beginning of that sampling 

sequence. After 114h treated ‘live’ cell numbers had fallen to 1.9% of the initial (T=0) 

concentration (i.e. 98.1% removal). As with trial 1, it is clear that the ballast tank provides a poor 

environment for treated and untreated cells alike. Concentrations of treated cells following growout, shown in red in tables 12 and 13, indicate some growth capacity in 3/9 T=0 controls and 5/9 

T=0 treated samples, although taken overall cell numbers after grow-out show reductions in 

control cell densities in control and treated samples of 70% and 58% respectively. This is 

reinforced by measurement of chlorophyll a concentrations before and after grow-out (table 16). 

While some positive growth was seen in untreated control samples at T=0, treated samples at 

T=0, no growth relative to intake water was recorded from treated samples at either T=0 or 

T=114h. 

 If ‘live’ cell counts based on morphological examination are used as the basis for 

regulatory compliance, the ‘live’ phytoplankton count of 189,500 per m3 (0.189 live cells/mL) 

recorded for treated samples at 114h (table 15) would comply with current IMO standard of 107

live cells per m3, (10 live cells/mL) but not the 2008 U.S. Coast Guard regulations, 105 live cells 

per m3 (0.1 live cells/mL) or the much more rigorous standard of 104 live cells per m3 (0.01 live 

cells/mL)currently employed by the state of California. However, if cell counts after grow-out 

are taken into account, this number falls to 75,667 per m3 (0.076 live cells/mL; table 15 - total 

phytoplankton cells after grow-out), which does comply with U.S. Coast Guard requirements of 

0.1 live cells/mL). Again, California standards would not be met at this level, based on 

morphological characteristics alone. However, cell counts after grow-out indicated a 60% 

decrease in treated cell numbers compared with concentrations before grow-out, indicating that 

the treated phytoplankton population could not sustain growth. In contrast, untreated samples 

showed a small increase in numbers following grow-out (table 14), which correlated closely with 

chlorophyll a determinations made on the same samples (table 16). 
31 

Table 12. Summary of phytoplankton results (>10<50μm minimum dimension) for Trial 2 of Hyde 

‘Guardian’ BWT system aboard M/V Coral Princess, June 30th-July 8th, 2008. Untreated (control) 

T=0 data, including live grow-out cell concentrations (in red). Due to time constraints, dead cell 

numbers were not recorded from C2 orC3 samples following grow-out. 

Phytoplankton 

(10μm-50μm) 

Untreated 

Controls, T=0 

‘Live’

Dinoflagellates. 

Density/m3

‘Live’

Diatoms. 

Density/m3 

Total Live’

phytoplankton. 

Density/m3

Dead 

Dinoflagellates. 

Density/m3

Dead 

Diatoms. 

Density/m3 

Dead

Rep. C1a, T=0 259,700 12,045,500 12,305,200 0 0 0

Rep. C1a, T=0 

+ 42h growout 

 

774,200 

 

6,483,900 

 7,258,100 0 

354,800 

 

354,800 

Rep. C1b, T=0 449,400 19,887,600 20,337,000 0 0 0

Rep. C1b T=0 

+ 42h growout 

 

483.800 

 

11,838,700 

12,322,500 

32,300 

 

32300 

 

64600 

Rep. C1c, T=0 193,500 17,451,600 17,645,100 0 0 0

Rep. C1c T=0 + 

42h growout 

 

354.800 

 

12,709,700 

13,064,500 

96,800 

 

129,000 

 

225,800 

Mean C1 

samples (after 

grow-out)

16,762,433±4,088,004

10,881,700±3,159,984 

Rep. C2a T=0 392.200 19,779,400 20,171,600 0 0 0

Rep. C2a T=0 

+ 42h growout 

451.400 29,236,100 29,687,500 0 34,700 34,700

Rep. C2b, T=0 64,500 3,548,400 3,612,900 0 64,500 64,500

Rep. C2b T=0 

+ 42h growout 

290,300 6,838,700 7,129,000 0 129,000 129,000

Rep. C2c, T=0 741,900 4,838,700 5,580,600 0 0 0

Rep. C2c T=0 + 

42h growout 

129,000 3,709,700 3,838,700 96,700 129,000 225,800

Mean C2 

samples (after 

grow-out)

9,788,367±9,045,806

13,548,733±14,065,331

Rep. C3a, T=0 96,800 2,549,700 2,646,600 0 0 0

Rep. C3a T=0 

+ 42h growout 

220,600 7,389,700 7,610,300 0 404,400 404,400

Rep. C3b, T=0 122,500 2,279,400 2,402,000 0 0 0

Rep. C3b T=0 

+ 42h growout 

206,600 867,800 1,074,400 0 62,000 62,000

Rep. C3c, T=0 140,700 2,696,100 2,836,800 0 24,500 24,500

Rep. C3c T=0 + 

42h growout 

165,600 2,450,300 2,615,900 66,200 99,300 165,500

Mean C3 

samples (after 

grow-out)

2,628,467±217,966

3,766,867±3,416,583 
32 

Table 13. Summary of phytoplankton results (>10<50μm minimum dimension) for Trial 2 of Hyde 

‘Guardian’ BWT system aboard M/V Coral Princess, June 30th-July 8th, 2008. Treated (control) T=0 

samples, including live grow-out cell concentrations (in red). 

Mean ‘Live’ 

Phytoplankton 

(Nos. in red are 

after grow-out) 

9,726,400±7,880,600

2,936,800±6,608,000 

Phytoplankton 

(10μm-50μm) 

Treated, T=0 

‘Live’

Dinoflagellates. 

Density/m3

‘Live’

Diatoms. 

Density/m3 

Total ‘Live’

phytoplankton. 

Density/m3

Dead 

Dinoflagellates. 

Density/m3

Dead 

Diatoms. 

Density/m3 

Total Dead 

phytoplankton. 

Density/m3

Rep. T1a, T=0 129,000 64,500 193,500 0 0 0

Rep. T1a, 

T=0+42hr 

32,300 96,800 129,100 64,500 64,500 129,000

Rep. T1b, T=0 24,500 49,000 73,500 0 0 0

Rep. T1b, 

T=0+42hr 

96,700 32,200 129,000 32,200 0 32,200

Rep. T1c, T=0 147,100 0 147,100 0 0 0

Rep. T1c, 

T=0+42hr 

57,500 459,,800 517,200 0 57,400 57,400

138,033 ± 60,512

258,433±224,099 

Rep. T2a, T=0 135,100 216,200 351,300 0 0 0

Rep. T2a, 

T=0+42hr 

287,400 229,900 517,300 143,700 0 143,700

Rep. T2b, T=0 82,000 218,600 300,600 27,300 82,000 109,300

Rep. T2b, 

T=0+42hr 

322,600 451,600 774,200 32,300 96,800 129,100

Rep. T2c, T=0 129,000 258,100 387,100 0 0 0

Rep. T2c, 

T=0+42hr 

85,500 128,200 213,700 0 0 0

346,333±43,463

501,733±280,574 

Rep. T3a, T=0 193,500 64,500 258,000 0 0 0

Rep. T3a, 

T=0+42hr 

155,400 207,200 362,700 129,500 25,900 155,400

Rep. T3b, T=0 318,600 318,600 637,300 0 49,000 49,000

Rep. T3b, 

T=0+42hr 

188,700 141,500 330,200 141,500 94,300 235,800

Rep. T3c, T=0 73,500 612,700 686,300 24,500 98,000 122,500

Rep. T3c, 

T=0+42hr 

96,800 32,300 129,100 32,300 32,300 64,600

527,200±234,418

274,000±126,535 
33 

Table 14. Summary of phytoplankton results (>10<50μm minimum dimension) for Trial 2 of Hyde 

‘Guardian’ BWT system aboard M/V Coral Princess, June 30th-July 8th, 2008. T=114h untreated 

(control) samples. Due to time constraints, dead cell numbers were not recorded from C3 samples 

following grow-out. 

Phytoplankton. 

(>10μm-

<50μm). 

Untreated, 

T=114h 

‘Live’

Dinoflagellates. 

Density/m3

‘Live’

Diatoms. 

Density/m3

Total ‘Live’

phytoplankton. 

Density/m3

Dead 

Dinoflagellates. 

Density/m3

Dead 

Diatoms. 

Density/m3

Total Dead 

phytoplankton. 

Density/m3

Start De-ballast. 

Rep. C1a, 

T=114h 

1,200,500 18,117,300 19,317,800 81,800 381,900 463,800

Start De-ballast. 

Rep. C1a, 

T=114h + 42h 

988,500 17,823,400 18,811,900 101,200 299,600 400,800

Rep. C1b 

T=114h 

25,000 650,400 675,400 0 25,000 25,000

Rep. C1b 

T=114h + 42h 

0 349,900 349,900 46,000 54,600 100,600

Rep. C1c 

T=114h 

77,800 596,600 674,400 0 25,900 25,900

Rep. C1c 

T=114h + 42h 

36,800 602,700 639,500 0 36,400 36,400

Mean C1, T=

114h samples 

After grow-out 

6,889,200±10,763,500

6,600,433±8,635,620

Mid De-ballast. 

Rep. C2a, 

T=114h 

52,300 890,600 942,900 0 78,500 78,500

Mid De-ballast. 

Rep. C2a, 

T=114h + 42h 

54,600 1,237,900 1,292,500 0 36,800 36,800

Rep. C2b 

T=114h 

0 623,900 623,900 0 23,100 23,100

Mean ‘Live’ 

Phytoplankton 

(Nos. in red are 

after grow-out) 

 

298,400±229,700 

125,600±143,800 
34 

Rep. C2b 

T=114h + 42h 

0 789,100 789,100 0 37,000 37,000

Rep. C2c 

T=114h 

0 654,800 654,800 0 78,500 78,500

Rep. C2c 

T=114h + 42h 

0 1,046,700 1,046,700 0 46,000 46,000

Mean C2, T=

114h samples 

After grow-out 

740,600±176,000

1,042,767±205,531 

End De-ballast. 

Rep. C3a, 

T=114h 

125,000 800,500 925,500 0 125,000 125,000

End De-ballast. 

Rep. C3a + 42h 

87,900 1,238,600 1,326,500

Rep. C3b 

T=114h 

168,700 1,097,000 1,265,700 0 22,000 225,000

Rep. C3b 

T=114h +42h 

137,700 1,260,800 1,398,500

Rep. C3c 

T=114h 

96,200 722,000 818,200 0 120300 120300

Rep. C3c 

T=114h +42h 

102,200 902,300 1,004,500

Mean C3, T= 

114h samples 

After grow-out

 1,003,200±233,700

1,243,167±171,304 

 

Mean Live 

Phytoplankton 

After grow-out

2,877,600±6,168,400

2,962,122±5,613,127 
35 

Table 15 Summary of phytoplankton results (>10<50μm minimum dimension) for Trial 2 of Hyde 

‘Guardian’ BWT system aboard M/V Coral Princess, June 30th-July 8th, 2008. T=114h data from 

treated samples, including grow-out cell concentrations. 

Phytoplankton. 

(>10μm-<50μm) 

Treated, T=114h 

‘Live’

Dinoflagellates. 

Density/m3

‘Live’

Diatoms. 

Density/m3

Total ‘Live’

phytoplankton. 

Density/m3

Dead 

Dinoflagellat

es Density/m3

Dead 

Diatoms. 

Density/m3

Total Dead 

phytoplankton. 

Density/m3

Start De-ballast. 

Rep. T1a, 

T=114h 

49,000 98,000 147,000 0 49,000 49,000

Start De-ballast. 

Rep. T1a, 

T=114h + 42h 

19,900 37,000 56,900 0 56,800 56,800

Rep. T1b, 

T=114h 

25,900 207,500 233,400 0 51,800 51,800

Rep. T1b, 

T=114h + 42h 

20,500 28,000 48,500 22,000 67,600 89,600

Rep. T1c, 

T=114h 

50,000 200,100 250,100 0 0 0

Rep. T1c, 

T=114h + 42h 

0 40,500 40,500 0 22,000 22,000

Mean T1, T=

114h samples 

After grow-out

210,200±55,300

48,633±6,696 

Mid De-ballast. 

Rep. T2a, 

T=114h 

23,600 236,800 260,500 0 23,600 23,600

Mid De-ballast. 

Rep. T2a, 

T=114h + 42h 

0 23.600 23,600 0 23,600 23,600

Rep. T2b, 

T=114h 

0 156,800 156,800 0 0 0

Rep. T2b, 

T=114h + 42h 

0 92,000 92,000 0 0 0

Rep. T2c 

T=114h 

0 94,700 94,700 0 47,300 47,300

Rep. T2c 

T=114h + 42h 

0 56,900 56,900 0 64,900 64,900

Mean T1, T= 

114h samples 

After grow-out

170,700±83,800

57,500±27,927
36 

End De-ballast. 

Rep. T3a, 

T=114h 

32,900 65,800 98,700 0 0 0

End De-ballast. 

Rep. T3a, 

T=114h + 42h 

30,600 44,600 75,200 0 0 0

Rep. T3b, 

T=114h 

0 184,400 184,400 0 0 0

Rep. T3b, 

T=114h + 42h 

0 123,000 123,000 0 0 0

Rep. T3c, 

T=114h 

0 280,100 280,100 0 0 0

Rep. T3c, 

T=114h + 42h 

0 164,400 164,400 0 0 0

Mean T3, T= 

114h samples 

(After grow-out)

187,700±90,700

(120,867±36,447)

Mean Live 

Phytoplankton 

(After grow-out)

189,500±69,800

(75,667±41,861) 
37 

Table 16. Trial 2. Chlorophyll a concentrations from measurement of in vivo fluorescence. 

(all values are means of 3 determinations) 

T=0 T=0, Pre grow-out 

chlorophyll a conc. 

(μg L-1)

chlorophyll a conc. (μg 

L

-1) after 24h grow-out 

(T=0 + 24)

chlorophyll a conc. (μg L1

) after 42h grow-out 

(T=0 + 42)

Control 1 

start ballasting 

1.32 1.36 1.38 

Control 2 

mid ballasting 

2.2 2.54 2.9

Control 3 

end ballasting 

1.43 1.65 1.84

Treated 1 

start ballasting 

0.38 0.24 0.1

Treated 2 

mid ballasting 

0.47 0.32 0.14

Treated 3 

end ballasting 

0.42 0.27 0.09

T=114h Pre grow-out 

chlorophyll a conc. 

(μg L-1)

chlorophyll a conc. (μg 

L

-1) after 24h grow-out 

(T=114 + 24)

chlorophyll a conc. (μg L1

) after 42h grow-out 

(T=114 + 42)

Control 1 

start de-ballasting 

0.46 0.48 0.49 

Control 2 

mid de-ballasting 

0.56 0.65 0.71 

Control 3 

end de-ballasting 

0.57 0.63 0.72 

Treated 1 

start de-ballasting 

0.021 ND ND 

Treated 2 

mid de-ballasting 

0.017 ND ND

Treated 3 

end de-ballasting 

0.016 ND ND
38 

Bacteria.

No coliform bacteria, including E. Coli, were detected in any treated or untreated samples 

in this trial. Treated and untreated samples collected immediately following ballasting/treatment 

were all negative for intestinal enterococci, although both large and small cfus of Enterococci 

were detected in both untreated and treated samples retrieved from ballast tanks following a 

residence time of 114h (table 17). No Vibrio cholerae cells were detected in any of the samples 

examined. 

 No determination was made of performance of the BWT system against viruses, as per 

California regulations. 

Table 17. Trial 2. Enterococci, colifoms and Vibrio cholerae from treated and untreated 

(control) samples at T=0 and T=114h. 

 Coliforms E. Coli Enterrococci Vibrio 

cholerae

Treatment Time Replicate Large Small Large Small Large Small 

Control T=0 C1a 0 0 0 0 0 0 0 

Control T=0 C1b 0 0 0 0 0 0 0 

Control T=0 C1c 0 0 0 0 0 0 0 

Control T=0 C2a 0 0 0 0 0 0 0 

Control T=0 C2b 0 0 0 0 0 0 0 

Control T=0 C2c 0 0 0 0 0 0 0 

Control T=0 C3a 0 0 0 0 0 0 0 

Control T=0 C3b 0 0 0 0 0 0 0 

Control T=0 C3c 0 0 0 0 0 0 0 

Treated T=0 T1a 0 0 0 0 0 0 0 

Treated T=0 T1b 0 0 0 0 0 0 0 

Treated T=0 T1c 0 0 0 0 0 0 0 

Treated T=0 T2a 0 0 0 0 0 0 0 

Treated T=0 T2b 0 0 0 0 0 0 0 

Treated T=0 T2c 0 0 0 0 0 0 0 

Treated T=0 T3a 0 0 0 0 0 0 0 

Treated T=0 T3b 0 0 0 0 0 0 0 

Treated T=0 T3c 0 0 0 0 0 0 0 

Control T=114h C1a 0 0 0 0 2 0 0 

Control T=114h C1b 0 0 0 0 1 3 0 

Control T=114h C1c 0 0 0 0 1 0 0 

Control T=114h C2a 0 0 0 0 0 3 0 

Control T=114h C2b 0 0 0 0 2 0 0 

Control T=114h C2c 0 0 0 0 0 2 0 

Control T=114h C3a 0 0 0 0 0 0 0 

Control T=114h C3b 0 0 0 0 0 0 0 

Control T=114h C3c 0 0 0 0 0 0 0 
39 

Treated T=114h T1a 0 0 0 0 2 0 0 

Treated T=114h T1b 0 0 0 0 1 0 0 

Treated T=114h T1c 0 0 0 0 1 0 0 

Treated T=114h T2a 0 0 0 0 6 1 0 

Treated T=114h T2b 0 0 0 0 0 0 0 

Treated T=114h T2c 0 0 0 0 5 2 0 

Treated T=114h T3a 0 0 0 0 3 1 0 

Treated T=114h T3b 0 0 0 0 2 2 0 

Treated T=114h T3c 0 0 0 0 4 1 0 

Culturable heterotrophic bacteria showed markedly different characteristics in the second trial, 

compared with Trial 1. Numbers of cfus varied from 2->200 in T=0 controls, which did not differ 

significantly from treated samples examined at T=0. After a 144h residence time in the ballast 

tanks, both treated and untreated samples showed elevated counts, although this was particularly 

evident in treated samples where, in all but one (the last) sample investigators had to estimate the 

cfus as either >100 or >200 (table 18). 

Table 18. Trial 2. Culturable bacteria counts from treated and untreated (control) samples 

at T=0 and T=114h. 

 Colonyforming Units Colony-forming 

Units 

Colonyforming Units 

100% 10% 1%

Trial 2 Treatment Time Replicate 

Control T=0 C1a 3 0 0 

Control T=0 C1b 5 7 1 

Control T=0 C1c 18 4 0 

Control T=0 C2a >200 23 2 

Control T=0 C2b 8 3 2 

Control T=0 C2c 2 1 0 

Control T=0 C3a 45 4 0 

Control T=0 C3b 74 5 0 

Control T=0 C3c 4 0 0 

Treated T=0 T1a 23 2 0 

Treated T=0 T1b 4 3 0 

Treated T=0 T1c 4 0 0 

Treated T=0 T2a 84 8 0 

Treated T=0 T2b 9 0 0 

Treated T=0 T2c 1 0 0 

Treated T=0 T3a 8 3 0 

Treated T=0 T3b 56 3 1 

Treated T=0 T3c 5 0 0 
40 

Control T=114h C1a >200 28 6 

Control T=114h C1b 64 6 0 

Control T=114h C1c 81 0 0 

Control T=114h C2a >200 34 3 

Control T=114h C2b 86 13 1 

Control T=114h C2c 94 9 1 

Control T=114h C3a 81 11 1 

Control T=114h C3b 8 0 0 

Control T=114h C3c 15 3 0 

Treated T=114h T1a >200 25 2 

Treated T=114h T1b >200 24 3 

Treated T=114h T1c >100 10 2 

Treated T=114h T2a >200 17 5 

Treated T=114h T2b >200 23 1 

Treated T=114h T2c >100 17 5 

Treated T=114h T3a >200 38 4 

Treated T=114h T3b >100 11 0 

Treated T=114h T3c 35 13 1 

Water Chemistry. 

Ballast water during trial 2 was taken south of Whittier, Alaska on July 1st, 2008. The salinity of 

challenge water was 30.8 – 31.9 PSU, water temperatures ranged from 12.1 = 14.0o C and pH 

from 8.25 – 8.36. Dissolved oxygen (DO) concentrations ranged from 6.85 mg L-1 to 10.46 mg 

L

-1 (table 19). Total Suspended Solids (TSS) ranged from 5.37 – 9.95 in challenge water, with a 

tendency to the higher value early in the ballasting cycle. A similar pattern was seen in 

Particulate Organic Carbon (POC) levels, which ranged from 3.62 – 7.28 mg L-1. UV 

Transmittance was high (94%). 

 Nutrient levels in challenge water were significantly higher in the first trial, reflecting the 

overall greater productivity at this ballasting location. 
41 

Table 19. Trial 2. Water Chemistry in untreated challenge water and treated ballast water at time 

of ballasting (T=0) near Whittier AK, July 1st 2008. (DO = Dissolved O2; TSS = Total Suspended 

Solids; POC – Particulate Organic Carbon; DOC = Dissolved Organic Carbon; NO3 = Nitrate; PO4

= Phosphate) 

 pH DO 

(mg L-1) 

Salinity 

(PSU) 

Temp 

(

OC) 

TSS 

(mg L-1) 

POC 

 (mg L-1) 

DOC 

 (mg L-1) 

NO3

(μgN L-1) 

PO4

(μgP L-1) 

C1a, T=0 

(begin ballast) 

8.36 6.85 30.8 14.0 9.95±0.05 7.28±0.34 1.34±0.03 21.3±6.7 22.4±8.7 

C2a, T=0 (mid 

ballast) 

8.29 5.51±0.20 3.62±0.29 1.33±0.07 24.7±5.5 12.5±0.8 

C3a, T=0 (end 

ballast 

8.25 5.37±0.18 4.0±0.15 1.72±0.73 11.0±3.6 13.2±4.2 

T1a, T=0 

(begin ballast) 

8.25 10.46 31.9 12.1 

T2a, T=0 (mid 

ballast) 

8.32 

T3a, T=0 (end 

ballast 

8.35 

GENERAL CONCLUSIONS (Trial 2)

 Plankton concentrations in challenge water reflected higher densities of organisms 

compared with trial 1. However, zooplankton densities in untreated challenge water did exceed 

the value of 10 x D-2 required by IMO G8 guidelines. Based these results, the Hyde Guardian 

system would comply with those portions of IMO Regulation D-2 and current California 

regulations relating to plankton. 

 The appearance of intestinal Enterococci in samples collected at de-ballasting although 

none were detected in any T=0 samples poses a problem of interpretation. Clearly, no live 

enterococci were introduced into the tanks, i.e. none were detected in T=0 samples. We therefore 

interpret the appearance of these bacteria at de-ballasting as the result of their association with 

planktonic organisms present in the tanks before the trial took place. As with the culturable 

heterotrophic bacteria, treatment was actually associated with an increase in bacterial flora 

relative to controls. Mean numbers of Enterococci were 10 cfus per 100ml. and 36 cfus per 

100ml. for untreated and treated samples respectively. These data are consistent with the unusual 

nature of the samples collected during de-ballasting. Samples were characterized by a large 

amount of flocculated material, particularly in the treated tank, including several specimens of 

decaying zooplankton, some >>1000μm in the smallest dimension. 
42 

TRIAL 3. September 17th-October 6th, 2008

Sampling Timetable.

In Trial 3 the same matched pair of ballast tanks (5P, 5S) was employed as in Trials 1 and 2, i.e. 

tank (5S) was used for treated ballast water and the other (5P) for untreated water. The scientific 

team joined the vessel in Long Beach, CA on September 17

th. Ballasting took place on 

September 19th, 2008, day 3 of a seventeen day cruise. As with Trials 1 and 2, tanks 5S and 5P 

were filled with treated and untreated water respectively, as per normal ballasting procedure, and 

for untreated samples, water followed the same path as the treated samples, except that the filter 

was by-passed and the UV unit was deactivated during the ballasting of the untreated tank. 

Sample collection.

 The same sampling regime was adopted as for Trial 2, i.e. 3 ‘in tank’ replicates x 3 time 

period (during the de-ballasting operation) x 2 treatment (i.e. treated/untreated), except that nine 

samples were obtained from both untreated and treated tanks immediately following the initial 

ballasting/treatment cycle. In the third trial a residence time of 10 days elapsed before samples 

were retrieved from the tanks in Aruba Harbor on September 29th, according to the sequence 

previously described. For trial 3, Dr. Carys Mitchelmore and graduate student Jon Bearr joined 

the scientific team and performed all coliform and Enterococcus assays, and culturable 

heterotrophic bacterial assays for T=0 and T=10 Day samples in an air-conditioned passenger 

cabin. Other samples were carried over ice to the University of Maryland Center for 

Environmental Science, Chesapeake Biological Laboratory, Solomons, Maryland for further 

analysis. Culture and analysis of these samples continued until October 6th, 2008. 

Otherwise, preparation of samples for biological examination followed procedures 

described for previous trials (see Figure 1.) Methods for sample examination and analysis were as 

previously described.
43 

Results and Discussion 

Zooplankton.

Results of zooplankton counts are summarized in table 20. These indicated 100% 

mortality of zooplankton (>50μm minimum dimension) immediately following treatment on 

ballasting relative to untreated samples collected during the same sampling event (T=0). Unlike 

Trial 1 and similar to Trial 2 a massive die-off (99.7%) of zooplankton in the >50μm size 

category was recorded in the untreated tank at the time of de-ballasting (T=10 days) and no 

survivors in this size class appeared in the treated tank. While some smaller zooplankton, largely 

nematodes, survived, these fell into the >10 - <50μm size category. These organisms appeared 

only in the last sample taken and may have reflected some re-suspension from sediment present 

in the bottom of the tank. Such a conclusion is supported by the fact that nematodes appeared 

only at the end of the de-ballasting process, when the ballast tank was nearly empty (Table 21). 

Table 20. Summary of zooplankton results (>50μm minimum dimension) for Trial 3 of 

Hyde ‘Guardian’ BWT system aboard M/V Coral Princess, September 17th-October 6th, 

2008. 

Zooplankton 

Alive (>50μm). 

Density/m3

Dead (>50μm) 

Density/m3

Control (untreated) N=9 1,391±918 192±233 

Treated (UV + filter 

during ballasting) N=7 

0 11±4 

Control (untreated) 

Start de-ballast (N=3) 

24±0 32±13 

Control (untreated) Mid 

de-ballast (N=3) 

8±7 13±19 

Control (untreated) End 

de-ballast (N=3) 

16±17 13±10 

Treated (UV + filter 

during ballasting) →

(UV at de-ballasting) 

Start de-ballast (N=3) 

0 0 
44 

Treated (UV + filter 

during ballasting) →

(UV at de-ballasting) 

Mid de-ballast (N=3) 

0 3±4 

Treated (UV + filter 

during ballasting) →

(UV at de-ballasting) 

End de-ballast (N=3) 

0 3±4 

Table 21. Densities of living nematode worms in treated and untreated samples.

Trial 3. September 17th-October 6th, 2008.

 Nematode densities (concentrations per m3

) 

Live Dead 

T=0h 

Control 6±5 0 

Treated 0 0 

T= 10 Days 

Control (untreated). Start deballast (N=3) 

 0 0 

Control (untreated) Mid deballast (N=3) 

 0 0 

Control (untreated) End deballast (N=3) 

 0 0 

Treated (UV + filter during 

ballasting) → (UV at deballasting). Start de-ballast (N=3) 

 0 0 

Treated (UV + filter during 

ballasting) → (UV at deballasting) Mid de-ballast (N=3) 

 0 0 

Treated (UV + filter during 

ballasting) → (UV at deballasting) End de-ballast (N=3) 

3±4 13±14 
45 

Phytoplankton.

 Phytoplankton counts from trial 3 are shown in tables 22 – 25. Due to time constraints a 

full suite of grow-out data was only available for untreated samples collected at ballasting (table 

22). Only three treated samples at T=0 were subjected to grow-out (table 23). At de-ballasting 

(T=10 Days), grow-out information was obtained from four untreated and four untreated samples 

each (tables 24 and 25). 

Based on morphological characteristics treated samples at T=0 treated samples show a 

77% reduction in live cell counts relative to untreated samples. ‘Live’ cell numbers following 

grow-out had fallen to 2,469,400/m3 (2.4 cells/mL) and 1,181,737/m3 (1.2 cells/mL) in control 

and treated samples respectively. Following a 10 day residence period in the tank both treated 

and untreated samples showed a >50% drop in live cell numbers based on morphological 

characteristics, relative to corresponding T=0 samples. Following grow-out, further reductions of 

55% and 83% in ‘live’ cell numbers were reported from untreated and treated samples 

respectively. Based on grow-out data the number of viable cells in T= 10 Day treated samples 

after grow-out exceeded by 3% the 2008 standard published by the U.G. Coast Guard and the 

state of California, 103,000 live cells/m3 (0.103 cells/mL) vs. 100,000 live cells/m3 (0.100 

cells/mL). However, this number represents an 83% decrease in cell numbers relative to pregrow-out concentrations, indicating a predominantly non-viable phytoplankton population in 

treated samples. 

Chlorophyll a concentrations (table 26) indicate a small degree of growth potential in 

untreated samples at T=0, although treated samples appear non-viable as demonstrated by a 75% 

decrease in chlorophyll a following a 48h grow-out period. After 10 days residence in the ballast 

tank, untreated samples demonstrated no growth potential (90% decrease in chlorophyll a

following a 48h grow-out period), while treated samples indicated negligible chlorophyll a after 

10 days in the tank. 
46 

Table 22. Summary of phytoplankton results (>10<50μm minimum dimension) for Trial 3 

of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess, September 15th-October 6th, 

2008. Untreated (control) T=0 data. 

Phytoplankton. 

(>10μm-

<50μm). 

Untreated, T=0 

‘Live’

Dinoflagellates. 

Density/m3

‘Live’

Diatoms. 

Density/m3

Dead Dead 

Dinoflagellates. 

Density/m3

Dead 

Diatoms. 

Density/m3

Dead

Start De-ballast. 

Rep. C1a, T=0 

546,720 5,323,400 5,870,120 100,800 551,400 652,200

Rep. C1a, T=0 + 

48h 

250,500 123,500 374,000 186,000 448,300 634,300

Rep. C1b T=0 489,600 7,833,600 8,323,200 0 816,000 816,000

Rep. C1b T=0 + 

48h 

184,600 848,700 1,033,300 0 93,200 93,200

Rep. C1c T=0 488,889 3,422,222 3,911,111 244,444 244,444 488,888

Rep. C1c T=0 + 

48h 

384,500 336.800 721,300 0 36,800 36,800

Mean C1, T=0 

samples 

(After grow-out)

508,403 4,499,741 6,034,810±

2,210,650 

(709,533± 

329,807) 

115,096 537,297 652,393

Mid De-ballast. 

Rep. C2a, T=0 

408,000 8,976,000 9,384,000 0 408,000 408,000

Mid De-ballast. 

Rep. C2a, T=0 

+ 48h 

296,700 1,968,000 2,264,700 0 212,100 212,100

Rep. C2b T=0 2,760,000 10,304,000 13,064,000 368,000 552,000 920,000

Rep. C2b T=0 + 

48h 

1,176,300 2,349,600 3,525,900 102,000 464,000 566,000

Rep. C2c T=0 633,333 3,800,000 4,433,333 211,111 422,222 633,333

Rep. C2c T=0 + 

48h 

720,900 4,444,000 5,164,999 118,000 446,000 564,000

Mean C2, T=0 

samples 

(After grow-out)

1,267,111 7,693,333 8,960,444±

4,330,895 

(3,651,866± 

1,454,247)

193,037 460,741 653,778

End De-ballast. 

Rep. C3a, T=0 

998,045 500,122 1,498,167 97,618 384,067 481,685

Rep. C3a, T=0 + 

48h 

1,021,400 686,700 1,708,100 59,000 302,300 361,300
47 

Rep. C3b T=0 1,339,100 3,252,100 4,591,300 0 382,600 382,600

Rep. C3b T=0 + 

48h 

989,000 960,800 1,949,800 0 188,000 188,000

Rep. C3c T=0 785,700 6,678,500 7,464,200 196,400 392,800 589,200

Rep. C3c T=0 + 

48h 

484,600 4,998,000 5,482,600 202,400 447,600 650,000

Mean C3, T=0 

samples 

(After grow-out)

1,062,422 3,476,956 4,517,889±

2,983,694 

(3,046,833± 

2,112,895)

98,016 386,511 484,527

Mean Live 

Phytoplankton 

(After grow-out)

938,845 4,910,804 6,504,381

±3,458,482 

(2,469,400 

±1,866,202(n=9))

135,383 460,811 596,899
48 

Table 23. Summary of phytoplankton results (>10<50μm minimum dimension) for Trial 3 

of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess, September 15th-October 6th, 

2008. Treated T=0 data. Grow-outs were recorded for one sample each from T1, T2 and T3. 

Phytoplankton 

(10μm-50μm) 

Treated, T=0 

‘Live’

Dinoflagellates. 

Density/m3

‘Live’

Diatoms. 

Density/m3

Total ‘Live’ 

phytoplankton. 

Density/m3

Dead 

Dinoflagellates. 

Density/m3

Dead 

Diatoms. 

Density/m3

Total Dead 

phytoplankton. 

Density/m3

Rep. T1a, T=0 1,804,152 115,805 1,919,957 0 82,459 82,459

Rep. T1a, 

T=0+48hr 

ND

Rep. T1b, T=0 1,323,529 234,118 1,557,647 0 0 0

Rep. T1b, 

T=0+48hr 

2,479,339 0 2,479,339 0 165,289 165,289

Rep. T1c, T=0 2,476,546 0 2,476,546 0 164,300 164,300

Rep. T1c, 

T=0+48hr 

ND

Mean T1 T=0 

samples 

1,984,717±462,860

Rep. T2a, T=0 807,259 256,780 1,064,039 0 0 0

Rep. T2a, 

T=0+48hr 

ND

Rep. T2b, T=0 1,614,350 179,372 1,793,722 0 0 0

Rep. T2b, 

T=0+48hr 

0 330,579 330,579 0 0 0

Rep. T2c, T=0 1,735,296 234,118 1,969,414

Rep. T2c, 

T=0+48hr 

ND

1,609,058±480,106

Rep. T3a, T=0 1,438.090 0 1,438,090 0 0 0

Rep. T3a, 

T=0+48hr 

ND

Rep. T3b, T=0 2,152,466 0 2,152,466 0 0 0

Rep. T3b, 

T=0+48hr 

735,294 0 735,294 0 0 0

Rep. T3c, T=0 848,400 0 848,400

Rep. T3c, 

T=0+48hr 

ND

1,479,652±653,026

Mean ‘Live’ 

Phytoplankton 

(After growout)

1,372,720 124,073 1,511,770

±719,434 (n=9) 

(1,181,737± 

1,141,830 (n=3))

0 27,528 27,528
49 

Table 24. Summary of phytoplankton results (>10<50μm minimum dimension) for Trial 3 

of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess, September 15th-October 6th, 

2008. Untreated (control) T=10 Days data, including live grow-out cell concentrations (in 

red). Due to time constraints, no record was made of dead cell numbers following grow-out.

Phytoplankton 

(10μm-50μm) 

Untreated 

Controls, T=0 

‘Live’

Dinoflagellates. 

Density/m3

‘Live’

Diatoms. 

Density/m3

Dead Dead 

Dinoflagellates. 

Density/m3

Dead 

Diatoms. 

Density/m3

Dead

Rep. C1a, T=10 

Days 

170,940 1,538,462 1,709,002 0 683,761 683,761 

Rep. C1a, T=10 

Days + 48h 

392,157 2,156,863 2,549,020 

Rep. C1b, 

T=10 Days 

735,294 1,470,588 2,205,882 196,078 0 196,078 

Rep. C1b, 

T=10 Days + 

48h 

661,157 495,868 1,157,025 

Rep. C1c, T= 

10 Days 

540,300 4,898,270 5,438,570 147,059 0 147,059 

Rep. C1c, T= 

10 Days 

ND 

Mean C1, T=10 

Days 

 3,117,818± 

2,025,127

 

Rep. C2a T= 10 

Days 

1,661,157 2,495,868 3,157,025 0 165,289 165,289 

Rep. C2a T= 10 

Days + 48h 

ND 

Rep. C2b, T= 

10 Days 

441,177 1,294,118 1,735,295 0 0 0 

Rep. C2b, T= 

10 Days + 48h 

826,446 0 826,446 

Rep. C2c, T= 

10 Days 

826,446 3,878,460 4,704,906 0 165,289 165,289 

Rep. C2c, T= 

10 Days + 48h 

ND 

Mean C2, T=10 

Days 

 3,199,075± 

1,485,252

 

Rep. C3a, T= 400,969 2,180,200 2,581,169 0 211,007 211,007 
50 

10 Days 

Rep. C3a, T= 

10 Days + 48h 

ND 

Rep. C3b, T= 

10 Days 

0 661,157 661,157 0 0 0 

Rep. C3b, T= 

10 Days + 48h 

0 330,579 330,579 

Rep. C3c, T= 

10 Days 

0 3,150,787 3,150,787 0 330,579 330,579 

Rep. C3c, T= 

10 Days + 48h 

ND 

Mean C3, T=10 

Days 

668,115 1,997,381 2,220,086± 

1,358,517

 

Mean ‘Live’ 

Phytoplankton 

(After grow-out)

170,940 2,396,445 2,815,977± 

1,505,752 (n=9)

(1,271,768± 

951,515(n=4)) 

42,892 172,881 211,007 
51 

Table 25. Summary of phytoplankton results (>10<50μm minimum dimension) for Trial 3 

of Hyde ‘Guardian’ BWT system aboard M/V Coral Princess, September 15th-October 6th, 

2008. Treated T=10 Days data, including live grow-out cell concentrations (in red). 

Phytoplankton. 

(>10μm-<50μm) 

Treated, T=10 

Days 

‘Live’

Dinoflagellates. 

Density/m3

‘Live’

Viable

Diatoms. 

Density/m3

Total ‘Live’

phytoplankton. 

Density/m3

Dead 

Dinoflagellates 

Density/m3

Dead 

Diatoms. 

Density/m3

Total Dead 

phytoplankton. 

Density/m3

Start De-ballast. 

Rep. T1a, T=10 

Days 

431,304 646,957 1,078,261 215,600 215,600 431,200

Start De-ballast. 

Rep. T1a, T=10 

Days + 48h 

98,000 64,900 162,900 199,500 170,000 369,500

Rep. T1b, T=10 

Days 

0 0 0 2,160,000 540,000 2,700,000

Rep. T1b, T=10 

Days + 48h 

0 0 0 1,700,000 226,000 1,926,000

Rep. T1c, T=10 

Days 

652,174 434,783 1,086,957 0 652,174 652,174

Rep. T1c, T=10 

Days + 48h 

144,300 102,400 246,700 0 459,000 459,000

Mean T1, T=10 

Day samples 

(After grow-out)

361,159 360,580 721,739±625,060

(136,533 

±125,446) 

791,884 469,275 1,261,159

Mid De-ballast. 

Rep. T2a, T=10 

Days 

173,333 0 173,333 173,333 866,667 1,040,000

Mid De-ballast. 

Rep. T2a, T=10 

Days + 48h 

58,000 0 58,000 160,000 430,000 590,000

Rep. T2b, T=10 

Days 

891,429 1,089,524 1,980,953 198,095 0 198,095

Rep. T2b, T=10 

Days + 48h 

152,800 106,600 259,400 122,200 0 122,200

Rep. T2c T=10 

Days 

0 0 0 0 417,331 417,331

Rep. T2c T=10 

Days + 48h 

0 0 0 0 326,000 326,000

Mean T2, T=10 354,921 363,175 718,095± 123,809 427,999 551,809
52 

Day samples

(After grow-out)

1,097,095

(105,800

±136,146) 

End De-ballast. 

Rep. T3a, T=10 

Days 

0 842,857 842,857 168,571 0 168,571

End De-ballast. 

Rep. T3a, T=10 

Days + 48h 

0 102,000 102,000

Rep. T3b, T=10 

Days 

0 238,597 238,597 146,996 0 146,996

Rep. T3b, T=10 

Days + 48h 

0 100,700 100,700

Rep. T3c, T=10 

Days 

0 137,500 137,500 137,500 0 137,500

Rep. T3c, T=10 

Days + 48h 

0 0

Mean T3, T=10

Day samples 

(After grow-out)

0 490,179 406,318±381,418

(67,567±58,518)

153,036 0 153,036

Mean Live 

Phytoplankton 

(After grow-out)

238,582 376,690 614,273±677,711

(103,300 

±101,585) 

355,239 299,092 654,663
53 

Table 26. Trial 3. Chlorophyll a concentrations from measurement of in vivo fluorescence. 

(all values are means of 3 determinations) 

T=0 T=0, Pre grow-out 

chlorophyll a conc. 

(μg L-1)

chlorophyll a conc. (μg 

L

-1) after 24h grow-out 

(T=0 + 24)

chlorophyll a conc. (μg L1

) after 42h grow-out 

(T=0 + 42)

Control 1 

start ballasting 

0.257 0.260 0.2 

Control 2 

mid ballasting 

0.180 0.221 0.220 

Control 3 

end ballasting 

0.171 0.231 0.24 

Treated 1 

start ballasting 

0.141 0.051 0.044 

Treated 2 

mid ballasting 

0.142 0.049 0.034 

Treated 3 

end ballasting 

0.106 0.069 0.022 

T=10 Days Pre grow-out 

chlorophyll a conc. 

(μg L-1)

chlorophyll a conc. (μg 

L

-1) after 24h grow-out 

(T=10D + 24h)

chlorophyll a conc. (μg L1

) after 42h grow-out 

(T=10D + 42h)

Control 1 

start de-ballasting 

0.284 0.065 0.028 

Control 2 

mid de-ballasting 

0.282 0.087 0.044 

Control 3 

end de-ballasting 

0.287 0.039 0.02 

Treated 1 

start de-ballasting 

0.147 0.05 0.02 

Treated 2 

mid de-ballasting 

0.167 0.043 ND 

Treated 3 

end de-ballasting 

0.125 0.029 ND 
54 

Bacteria.

No coliform bacteria or, E. Coli were found in either treated or untreated samples 

immediately following ballasting/treatment in Trial 3, although a small number of cfus were 

detected in untreated samples following de-ballasting 10 days later (Table 27). No coliforms or 

E. Coli were found in any treated samples at de-ballasting, and no intestinal enterococci appeared 

in any samples examined during this trial. No Vibrio cholerae cells were found in any samples, 

treated or untreated. 

 Cfus of aerobic cultural bacteria were found in approximately equal numbers in both 

treated and untreated samples at T=0 (Table 28). However, both showed marked declines after 10 

days in the tanks. Of the treated samples, only one showed a positive response in terms of 

cultural bacteria. All indicator bacteria were within national and international standards. No 

determination was made of performance of the BWT system against viruses, as per California 

regulations. 

Table 27 . Trial 3. Enterococci, colifoms and Vibrio cholerae from treated and untreated 

(control) samples at T=0 and T=10 Days 

Coliforms E. Coli Enterrococci Vibrio 

cholerae 

Treatment Time Replicate Large Small Large Small Large Small

Control T=0 C1a 0 0 0 0 0 0 0 

Control T =0 C1b 0 0 0 0 0 0 0 

Control T =0 C1c 0 0 0 0 0 0 0 

Control T =0 C2a 0 0 0 0 0 0 0 

Control T =0 C2b 0 0 0 0 0 0 0 

Control T =0 C2c 0 0 0 0 0 0 0 

Control T =0 C3a 0 0 0 0 0 0 0 

Control T =0 C3b 0 0 0 0 0 0 0 

Control T =0 C3c 0 0 0 0 0 0 0 

Treated T =0 T1a 0 0 0 0 0 0 0 

Treated T =0 T1b 0 0 0 0 0 0 0 

Treated T =0 T1c 0 0 0 0 0 0 0 

Treated T =0 T2a 0 0 0 0 0 0 0 

Treated T =0 T2b 0 0 0 0 0 0 0 
55 

Treated T =0 T2c 0 0 0 0 0 0 0 

Treated T =0 T3a 0 0 0 0 0 0 0 

Treated T =0 T3b 0 0 0 0 0 0 0 

Treated T =0 T3c 0 0 0 0 0 0 0 

Control T=10 Days C1a 0 1 0 1 0 0 0 

Control T=10 Days C1b 0 1 0 1 0 0 0 

Control T=10 Days C1c 0 1 0 0 0 0 0 

Control T=10 Days C2a 0 0 0 0 0 0 0 

Control T=10 Days C2b 0 1 0 0 0 0 0 

Control T=10 Days C2c 0 0 0 0 0 0 0 

Control T=10 Days C3a 0 0 0 0 0 0 0 

Control T=10 Days C3b 0 0 0 0 0 0 0 

Control T=10 Days C3c 0 1 0 1 0 0 0 

Treated T=10 Days T1a 0 0 0 0 0 0 0 

Treated T=10 Days T1b 0 0 0 0 0 0 0 

Treated T=10 Days T1c 0 0 0 0 0 0 0 

Treated T=10 Days T2a 0 0 0 0 0 0 0 

Treated T=10 Days T2b 0 0 0 0 0 0 0 

Treated T=10 Days T2c 0 0 0 0 0 0 0 

Treated T=10 Days T3a 0 0 0 0 0 0 0 

Treated T=10 Days T3b 0 0 0 0 0 0 0 

Treated T=10 Days T3c 0 0 0 0 0 0 0 

Table 28. Trial 3. Culturable heterotrophic bacteria counts from treated and untreated 

(control) samples at T=0 and T=10 Days. 

 Colonyforming Units Colony-forming 

Units 

Colonyforming Units 

25% 10% 1%

 Treatment Time Replicate 

Control t=0 C1a 51 28 5 

Control t=0 C1b >100 17 4 

Control t=0 C1c 30 17 3 

Control t=0 C2a >100 22 1 

Control t=0 C2b 9 27 7 

Control t=0 C2c >100 88 15 

Control t=0 C3a 2 2 0 

Control t=0 C3b 0 97 24 

Control t=0 C3c 6 9 1 

Treated t=0 T1a >100 10 2 

Treated t=0 T1b 9 5 1 
56 

Treated t=0 T1c >100 9 6 

Treated t=0 T2a 4 2 1 

Treated t=0 T2b >100 22 4 

Treated t=0 T2c 3 4 0 

Treated t=0 T3a 3 0 0 

Treated t=0 T3b 23 10 1 

Treated t=0 T3c >100 2 0 

Control T=10 Days C1a 8 0 0 

Control T=10 Days C1b 2 0 0 

Control T=10 Days C1c 4 0 0 

Control T=10 Days C2a 0 0 0 

Control T=10 Days C2b 8 0 0 

Control T=10 Days C2c 23 4 0 

Control T=10 Days C3a 12 4 0 

Control T=10 Days C3b 4 1 1 

Control T=10 Days C3c 0 0 0 

Treated T=10 Days T1a 0 0 0 

Treated T=10 Days T1b 0 0 0 

Treated T=10 Days T1c 0 0 0 

Treated T=10 Days T2a 0 0 0 

Treated T=10 Days T2b 0 0 0 

Treated T=10 Days T2c 0 0 0 

Treated T=10 Days T3a 18 0 0 

Treated T=10 Days T3b 0 0 0 

Treated T=10 Days T3c 0 0 0 

Water Chemistry. 

Ballast water during trial 3 was taken south of Long Beach, S. California on September 

19th, 2008. The salinity of challenge water was 33.3 PSU, water temperatures ranged from 23.7 – 

24.3o C and pH from 7.5 - 7.82. Dissolved oxygen (DO) concentrations ranged from 7.5 mg L-1

to 8.83 mg L-1 (table 28). Total Suspended Solids (TSS) ranged from 3.59 – 7.3 in challenge 

water, will a tendency to the higher value early in the ballasting cycle. A similar pattern was seen 

in Particulate Organic Carbon (POC) levels, which ranged from 2.04 – 4.37 mg L-1. As in earlier 

trials percentage UV Transmittance was high (95%). 

 Nitrate levels in challenge water declined sharply throughout the initial ballasting 

sequence, from an initial concentration of 71.4μg L-1 to 5.2 ± 3.7 μg L-1. In treated samples at 

T=0 nitrate levels ranged from 2.1 – 6.4 μg L-1. At de-ballasting, ten days later, nitrate 

concentrations in untreated, control samples varied between 1.2 and 18.7 μg L-1. However, 
57 

treated samples at T=10 days contained much higher nitrate levels, possibly due to an increase in 

nutrient release from dying/dead organisms in these samples (table 29). Nitrite levels, although 

much lower, also showed a 3-4 fold increase in treated samples after 10 days (table 29). 

Phosphate concentrations in treated samples at T= 10 days also indicated higher concentrations 

than untreated (control) samples. DOC levels were slightly higher in both treated and untreated 

samples at T=0 than at T= 10 Days and showed no apparent relationship to treatment (table 29). 

Table 29. Trial 3. Water Chemistry in untreated challenge water and treated ballast water at time 

of ballasting (T=0) near Long Beach CA, September 19th, 2008. (DO = Dissolved O2; TSS = Total 

Suspended Solids; POC – Particulate Organic Carbon; DOC = Dissolved Organic Carbon; NO3 = 

Nitrate; NO2 = Nitrite; PO4 = Phosphate) 

 pH DO 

(mg L-1) 

Salinity 

(PSU) 

Temp 

(

OC) 

TSS 

(mg L-1) 

POC 

 (mg L-1) 

DOC 

 (mg L-1) 

NO3

(μgN L-1) 

NO2

(μgN L-1) 

PO4

(μgP L-1) 

C1, T=0 (begin 

ballast) 

7.5 8.83 33.3 23.7 7.3±0.176 4.37±0.11 4.08±1.43 71.4±17.2 0.8±0.2 11.6±2.3 

C2, T=0 (mid 

ballast) 

7.78 3.75±0.073 2.04±0.026 3.16±1.21 25.1±18.5 0.63±0.06 10.8±2.1 

C3, T=0 (end 

ballast 

7.73 3.59±0.066 2.11±0.06 1.87±0.88 5.2±3.7 0.6±0.0 10.8±1.0 

T1, T=0 (begin 

ballast) 

7.82 7.5 33.3 24.3 2.45±0.89 2.4±0.5 0.6±0.0 12.1±0.3 

T2, T=0 (mid 

ballast) 

7.78 3.01±1.27 2.1±0.8 0.6±0.0 13.4±0.06 

T3, T=0 (end 

ballast 

7.73 2.05±0.42 6.4±6.8 0.63±0.06 9.5±0.9 

C1, T= 10 Days 

(begin deballast) 

 1.5±0.4 1.2±0.8 0.6±0 1.76±0.4 

C2, T= 10 Days 

(mid deballast) 

 1.5±0.8 8.1±1.1 0.7±0.1 4.2±0.5 

C3, T= 10 Days 

(end deballast) 

 1.38±0.4 18.7±3.2 0.77±0.15 5.2±0.9 

T1, T=10 Days 

(begin deballast) 

 1.3±0.2 41.6±13.8 1.75±1.62 11.15±5.6 

T2, T=10 Days 

(mid deballast) 

 1.3±0.2 51.8±10.3 2.33±1.36 14.7±2.9 

T3, T=10 Days 

(end deballast) 

 1.5±0.3 52.6±11.8 2.73±1.34 15.65±1.6 

GENERAL CONCLUSIONS (Trial 3)

Zooplankton densities >50 μm in size range in trial 3 were initially only about 15% of 

concentrations seen in trial 2, but still exceed G-8 D2 regulations for challenge water. As in trial 

2, zooplankton mortalities (>50 μm) were 100% in treated samples at T=0 in trial 3, and numbers 
58 

of untreated organisms in this size category declined precipitously over the period of residence in 

the ballast tanks. 

 Phytoplankton cell numbers in trial 3 challenge water, 6.5 x 106 cells m3 (6.5 cells/mL) 

were approximately 2/3 of those seen in trial 2, 9.7 x 106 cells m3 (9.7 cells/mL). However, 

somewhat surprisingly, they fell less precipitously than in the earlier trial despite a residence 

time in the tank approximately twice as long. Based on morphological characteristics, live cell 

numbers at trial 3 de-ballasting, 2.81 x 106 cells m3 (2.8 cells/mL) were comparable to the 

numbers seen in trial 2 after 114h residence time 2.88 x 106

 cells m3 (2.9 cells/mL). Based on 

morphological characteristics, cell numbers in treated vs. untreated samples at de-ballasting had 

further decreased by respectively 93.4% and 78.2% in trials 2 and 3. If grow-out is considered as 

a criterion for phytoplankton cell viability, the viable cell count in treated samples is further 

reduced to 103,000 cells per m3 (0.103 cells/mL), just exceeding the U.S. Coast Guard standard 

of 0.100 cells/mL) for cells in the >10 - <50μm size category. The IMO D-@ standard is easily 

met for this phytoplankton population. 

All indicator bacteria were within national and international standards following 

treatment. 
59 

Test for Environmental Acceptence.

Whole Effluent Toxicity Testing.

Rationale.

Whole effluent toxicity tests were conducted on treated and untreated water samples collected 

during the shipboard ballasting and de-ballasting procedures described in Trial 3. The objective 

of these bioassays was to identify any residual chemical toxicity that could have resulted from 

UV irradiation of ballast water. A new (October 10, 2008) G8 resolution adopted by the Marine 

Environmental Protection Committee (MEPC 58) states: 

“ -- if it can reasonably be concluded that the treatment process could result in changes to 

the chemical composition of the treated water such that adverse impacts to receiving 

waters might occur upon discharge, the documentation should include results of toxicity 

tests of treated water. The toxicity tests should include assessments of the effects of hold 

time following treatment, and dilution, on the toxicity. Toxicity tests of the treated water 

should be conducted in accordance with paragraphs 5.2.3 to 5.2.7 of the Procedure for

approval of ballast water management systems that make use of Active Substances (G9), as 

revised, (resolution MEPC.169(57))” 

While, to date, no potentially toxic chemical changes resulting from UV irradiation have 

been identified, this resolution adopts a approach requiring tests for residual toxicity that 

essentially follow IMO G-9 guidelines, even for systems not involving the addition of active 

substances. The resolution was adopted too late for such testing to be incorporated into landbased testing of the system, which ended in July, 2008. Hence, three tests, one chronic (growthbased) and two acute toxicity bioassays, were incorporated into the third trial in order to provide 

empirical toxicological evidence on this point. 

 Using the convention adopted for sample collection and examination in this trial. Four 

‘treatments’ were tested: 
60 

Untreated, T=0 

Treated, T=0 

Untreated, T=10 Days 

Treated, T=10 Days 

As the prescribed treatment by the Hyde ‘Guardian’ system consists of two passes 

through the UV irradiation unit (on both ballasting and de-ballasting) the definitive comparison is 

regarded as that between treated and untreated water on final discharge (T=10 Days). 

Nevertheless, from an experimental standpoint, T=0 samples, untreated vs. treated, also represent 

a valid comparison, and provide a useful means of controlling for any possible toxic agents that 

might be introduced into the water as a result of prolonged storage in the ballast tanks. 

Sample collection and storage.

For WET tests 20L of each water sample to be tested was collected in the middle of each 

ballasting/deballasting operation. Thus, treated and untreated T=0 samples were collected during 

sequence T2a-T2c for treated samples and between sequence C2a-C2c for untreated samples. 

The logistics of being at sea during ballasting and de-ballasting demanded a deviation from 

standard U.S. EPA protocols for dealing with storage/shipment of whole effluent test samples. 

Standard practice stipulates that samples should be shipped over ice, with the temperature not to 

exceed 6OC. In order to preserve the integrity of the samples to the greatest degree possible it 

was therefore decided to freeze the samples as soon as possible after collection, pending toxicity 

bioassays. On landing at the destination port of Fort Lauderdale on October 2nd, 2008 frozen 

water samples in plastic ‘Cubitainers’ were transported overnight to the respective bioassay 

laboratories, where samples were thawed and testing commenced (October 3rd,2008 for larval 

fish and mysid shrimp assays; October 4th, 2008 for phytoplankton assay – Appendix C). Tests 

were accompanied by standard water quality measurements as well as nitrate, nitrite and 

phosphate analyses. 
61 

The following three bioassays were performed according to standardized U.S.EPA 

procedures for Acute (EPA 821-R-02-012) and Chronic (EPA 821-R-02-014) bioassays: 

Invertebrate. 

48-hour Acute Static Renewal Definitive Test using larval mysid shrimp, Americamysis 

bahia (Method 2007.0) 

Vertebrate.

96-hour Acute Static Renewal Definitive Test using larval topsmelt, Atherinops affinis 

 (Method 2006.0) 

Phytoplankton.

96-hour Chronic Static Non-renewal Definitive Test using the marine brown alga 

(diatom) Isochrysis galbana, strain T. Iso. (adapted from Method 1003.0) 

 The invertebrate and vertebrate tests were carried out at the University of Maryland 

Center for Environmental Science, Chesapeake Biological Laboratory, and the phytoplankton 

assay was conducted at the Maryland Department of Natural Resources, Harmful Algal Bloom 

(HAB) Laboratory, Annapolis, MD. 

Raw treatment water was subjected to a series of dilution steps with clean, reconstituted 

seawater matched to the salinity of the treatment water, in order to create a dilution series 

representing 100 %, 50 %, 25 %, 12.5 %, 6.25 % of the original raw treatment water including a 

negative control (reconstituted seawater) Static renewal 48 h and 96 h LC50 assays were 

conducted on 4-day-old mysid shrimp (Americamysis bahia) and 14-day-old topsmelt 

(Antherinops affinis) larvae, respectively. These organisms were obtained from Aquatic Research 

Organisms (ARO) Ltd., Maine. A culture of marine acclimated (30PSU) Isochrysis galbana

(Tahitian strain T. Iso) was obtained from the Provasoli-Guillard National Center for the Culture 

of Marine Phytoplankton (CCMP), Bigelow Laboratory, New Hampshire. Prior to the test the 

culture was grown to log phase in the test laboratory at 33PSU and 21OC. Tests were conducted 

at 21OC on a 14:10 light/dark cycle 
62 

Results of Whole Effluent Toxicity Tests.

Summary data from invertebrate Americamysis bahia and vertebrate Atherinops affinis assays are 

given below. Neither treated nor untreated samples resulted in any significant toxicity to either 

species. > 97% mysid shrimp survival was recorded from all samples, both treated and untreated, 

retrieved from the tanks at the time of de-ballasting. Topsmelt survival in undiluted treated and 

untreated water at time of discharge was 100% and 95.2% respectively with no noticeable 

toxicity associated with other treatments in the dilution series. 

WET Test Summary Data.

Zooplankton. 

Invertebrate Assay

(1) TEST SAMPLE ID: 10.03.08/CO/S

48 hour mysid test for C=0 control water

Initial Water Quality tests and characterization 

Criteria Test Water: CO water Dilution water

Final pH (SU) 8.00 8.15

Salinity (ppt) 33 33

Total ammonia (mg / L) 0.003 <0.003

Temperature: 19.6 21.6

Dissolved oxygen (DO mg /L): 5.71 7.27

SU = standard units 

ppt = parts per thousand 

Ranges of water quality tests for all treatments 

Criteria Treatment Waters

pH (SU) 7.93 - 8.18

Salinity (ppt) 31 - 33

Total ammonia (mg / L) 0.003-0.006

Temperature: 20.0 - 22.3

Dissolved oxygen (DO mg /L): 6.40 – 7.62

Survival of Americamysis bahia at test termination (48 hours) 

% test water % survival 48-h Statistics

0 97.5 LC50: N/A

6.25 100 LOEL:N/A
63 

12.5 95 NOEL:N/A

25 100

50 97.5

100 97.5

1: LC50 = median-lethal concentration (95% confidence interval) as measured by the trimmed 

spearman- Karber method. 

2: LOEL = lowest observable effect level as measured by steel many-one rank method 

3: NOEL = no observable effect level as measured by steel many-one rank method 

Protocol Deviations / problems: None. 

1) TEST SAMPLE ID: 10.03.08/TO/S

48 hour mysid test for T=0 treated water

Initial Water Quality tests and characterization 

Criteria Test Water: CO water Dilution water

Final pH (SU) 8.09 8.15

Salinity (ppt) 33 33

Total ammonia (mg / L) <0.003 <0.003

Temperature: 21.0 21.6

Dissolved oxygen (DO mg /L): 5.85 7.27

SU = standard units 

ppt = parts per thousand 

Ranges of water quality tests for all treatments 

Criteria Treatment Waters

pH (SU) 7.93 – 8.27

Salinity (ppt) 32 - 33

Total ammonia (mg / L) <0.003

Temperature: 21.1 - 22.3

Dissolved oxygen (DO mg /L): 6.05 – 7.62

Survival of Americamysis bahia at test termination (48 hours) 

% test water % survival 48-h Statistics

0 100 LC50:N/A

6.25 97.5 LOEL:N/A

12.5 100 NOEL:N/A

25 100

50 97.5

100 100

1: LC50 = median-lethal concentration (95% confidence interval) as measured by the trimmed 

spearman- Karber method. 
64 

2: LOEL = lowest observable effect level as measured by steel many-one rank method 

3: NOEL = no observable effect level as measured by steel many-one rank method 

Protocol Deviations / problems: None. 

(1) TEST SAMPLE ID: 10.03.08/C12/S

48 hour mysid test for C=10 Day control water

Initial Water Quality tests and characterization 

Criteria Test Water: CO water Dilution water

Final pH (SU) 8.11 8.15

Salinity (ppt) 30 33

Total ammonia (mg / L) 0.004 <0.003

Temperature: 20.6 21.6

Dissolved oxygen (DO mg /L): 6.08 7.27

SU = standard units 

ppt = parts per thousand 

Ranges of water quality tests for all treatments 

Criteria Treatment Waters

pH (SU) 7.93 - 8.25

Salinity (ppt) 29 - 33

Total ammonia (mg / L) 0.003-0.014

Temperature: 21.1 - 22.3

Dissolved oxygen (DO mg /L): 6.30 – 7.62

Survival of Americamysis bahia at test termination (48 hours) 

% test water % survival 48-h Statistics

0 100 LC50:N/A

6.25 97.5 LOEL:N/A

12.5 100 NOEL:N/A

25 100

50 97.5

100 100

1: LC50 = median-lethal concentration (95% confidence interval) as measured by the trimmed 

spearman- Karber method. 

2: LOEL = lowest observable effect level as measured by steel many-one rank method 

3: NOEL = no observable effect level as measured by steel many-one rank method 

Protocol Deviations / problems: None. 

(1) TEST SAMPLE ID: 10.03.08/T12/S

48 hour mysid test for T=10 Day treated water
65 

Initial Water Quality tests and characterization 

Criteria Test Water: CO water Dilution water

Final pH (SU) 7.92 8.15

Salinity (ppt) 33 33

Total ammonia (mg / L) <0.003 <0.003

Temperature: 21.0 21.6

Dissolved oxygen (DO mg /L): 6.41 7.27

SU = standard units 

ppt = parts per thousand 

Ranges of water quality tests for all treatments 

Criteria Treatment Waters

pH (SU) 7.93 - 8.22

Salinity (ppt) 31 - 34

Total ammonia (mg / L) <0.003

Temperature: 21.4 - 22.3

Dissolved oxygen (DO mg /L): 6.11 – 7.62

Survival of Americamysis bahia at test termination (48 hours) 

% test water % survival 48-h Statistics

0 100 LC50:N/A

6.25 97.5 LOEL:N/A

12.5 100 NOEL:N/A

25 100

50 97.5

100 100

1: LC50 = median-lethal concentration (95% confidence interval) as measured by the trimmed 

spearman- Karber method. 

2: LOEL = lowest observable effect level as measured by steel many-one rank method 

3: NOEL = no observable effect level as measured by steel many-one rank method 

Protocol Deviations / problems: None. 
66 

Vertebrate Assay. 

(1) TEST SAMPLE ID: 10.03.08/CO/F

96 hour larval fish test for T=0 control water

Initial Water Quality tests and characterization 

Criteria Test Water: CO water Dilution water

Final pH (SU) 8.00 8.15

Salinity (ppt) 33 33

Total ammonia (mg / L) 0.003 <0.003

Temperature: 19.6 21.6

Dissolved oxygen (DO mg /L): 5.71 7.27

SU = standard units 

ppt = parts per thousand 

Ranges of water quality tests for all treatments 

Criteria Treatment Waters

pH (SU) 7.93 - 8.18

Salinity (ppt) 31 - 33

Total ammonia (mg / L) 0.003-0.006

Temperature: 20.0 - 22.3

Dissolved oxygen (DO mg /L): 6.40 – 7.62

Survival of Atherinops affinis at test termination (96 hours) 

% test water % survival 48-h Statistics

0 90 LC50:N/A

6.25 95 LOEL:N/A

12.5 90 NOEL:N/A

25 100

50 95

100 90

1: LC50 = median-lethal concentration (95% confidence interval) as measured by the trimmed 

spearman- Karber method. 

2: LOEL = lowest observable effect level as measured by steel many-one rank method 

3: NOEL = no observable effect level as measured by steel many-one rank method 

Protocol Deviations / problems: None. 

 

(1) TEST SAMPLE ID: 10.03.08/TO/F

96 hour larval fish test for T=0 treated water
67 

Initial Water Quality tests and characterization 

Criteria Test Water: CO water Dilution water

Final pH (SU) 8.00 8.15

Salinity (ppt) 33 33

Total ammonia (mg / L) <0.003 <0.003

Temperature: 19.6 21.6

Dissolved oxygen (DO mg /L): 5.71 7.27

SU = standard units 

ppt = parts per thousand 

Ranges of water quality tests for all treatments 

Criteria Treatment Waters

pH (SU) 7.93 - 8.18

Salinity (ppt) 31 - 33

Total ammonia (mg / L) <0.003

Temperature: 20.0 - 22.3

Dissolved oxygen (DO mg /L): 6.40 – 7.62

Survival of Atherinops affinis at test termination (96 hours) 

% test water % survival 48-h Statistics

0 95 LC50:N/A

6.25 100 LOEL:N/A

12.5 90 NOEL:N/A

25 80

50 75

100 100

1: LC50 = median-lethal concentration (95% confidence interval) as measured by the trimmed 

spearman- Karber method. 

2: LOEL = lowest observable effect level as measured by steel many-one rank method 

3: NOEL = no observable effect level as measured by steel many-one rank method 

Protocol Deviations / problems: None. 

 

(1) TEST SAMPLE ID: 10.03.08/C12/F

96 hour larval fish test for T=10 Day control water

Initial Water Quality tests and characterization 

Criteria Test Water: CO water Dilution water

Final pH (SU) 8.11 8.15

Salinity (ppt) 30 33

Total ammonia (mg / L) 0.004 <0.003
68 

Temperature: 20.6 21.6

Dissolved oxygen (DO mg /L): 6.08 7.27

SU = standard units 

ppt = parts per thousand 

Ranges of water quality tests for all treatments 

Criteria Treatment Waters

pH (SU) 7.93 - 8.25

Salinity (ppt) 29 - 33

Total ammonia (mg / L) 0.003-0.014

Temperature: 21.1 - 22.3

Dissolved oxygen (DO mg /L): 6.30 – 7.62

Survival of Atherinops affinis at test termination (96 hours) 

% test water % survival 48-h Statistics

0 100 LC50:N/A

6.25 100 LOEL:N/A

12.5 100 NOEL:N/A

25 95.2

50 85.7

100 95.2

1: LC50 = median-lethal concentration (95% confidence interval) as measured by the trimmed 

spearman- Karber method. 

2: LOEL = lowest observable effect level as measured by steel many-one rank method 

3: NOEL = no observable effect level as measured by steel many-one rank method 

Protocol Deviations / problems: None. 

 

(1) TEST SAMPLE ID: 10.03.08/T12/F

96 hour larval fish test for T=10 Day treated water

Initial Water Quality tests and characterization 

Criteria Test Water: CO water Dilution water

Final pH (SU) 7.92 8.15

Salinity (ppt) 33 33

Total ammonia (mg / L) <0.003 <0.003

Temperature: 21.0 21.6

Dissolved oxygen (DO mg /L): 6.41 7.27

SU = standard units 

ppt = parts per thousand 

Ranges of water quality tests for all treatments 
69 

Criteria Treatment Waters

pH (SU) 7.93 - 8.22

Salinity (ppt) 31 - 34

Total ammonia (mg / L) <0.003

Temperature: 21.4 - 22.3

Dissolved oxygen (DO mg /L): 6.11 – 7.62

Survival of Atherinops affinis at test termination (96 hours) 

% test water % survival 48-h Statistics

0 100 LC50:

6.25 95 LOEL:

12.5 100 NOEL:

25 90

50 95

100 100

1: LC50 = median-lethal concentration (95% confidence interval) as measured by the trimmed 

spearman- Karber method. 

2: LOEL = lowest observable effect level as measured by steel many-one rank method 

3: NOEL = no observable effect level as measured by steel many-one rank method 

Protocol Deviations / problems: +None. 

Phytoplankton Assay.

Results from the phytoplankton whole effluent test are summarized in table 30. Results 

indicate variable growth in T=0 control (untreated) samples, with no clear relationship to the 

dilution series, and a slight positive relationship between degree of dilution and growth rate in 

treated T=0 samples. Definitive samples taken during de-ballasting indicate some growth 

inhibition, although this appears not to be related to UV irradiation. 
70 

Table 30. Summary data from Phytoplankton WET bioassay. T=0 samples collected 

September 19th, 2008. T=10 Days samples collected September 29th, 2008. Test begum 

October 4th, 2008. 

Untreated, 

T=0h Phytoplankton Cell Counts 

Before 

Growout 72h After 96h Grow-out 

Mean ± S.D. 5 reps 

% T. Iso 

@ 96h/0h

% growth 

relative to 

controls @ 96h

6.25% 251,697 1066547 1,227,844±315,040 488 72.21 

12.50% 249,546 1195623 1,597,287±366,670 640 93.94 

25% 255,761 1369875 1,511,954±379,578 591 88.92 

50% 249,546 1647627 2,207,671±349,221 885 129.84 

100% 270,820 1015395 1,486,712±243,092 549 87.44 

 

Treated, 

T=0h 

 

6.25% 266,517 1698904 2,112,251±253,418 793 124.22 

12.50% 268,908 1726984 2,171,482±267,665 808 127.71 

25% 259,107 1497038 1,631,086±230,473 630 95.93 

50% 248,112 953725 1,173,489±235,826 473 69.01 

100% 245,722 1026390 1,127,643±176,926 459 66.32 

 

Untreated, 

T=10 Days 

 

6.25% 244,527 1111246 2,057,322±324,840 841 120.99 

12.50% 256,956 1164310 1,961,950±144,565 764 115.38 

25% 253,371 1318245 1,908,216±301,344 753 112.22 

50% 248,351 1135627 1,429,680±470,361 576 84.08 

100% 266,995 924086 1,028,255±84,855 385 60.47 

 

Treated, 

T=10 Days 

 

6.25% 261,498 1377285 1,861,175±337,341 712 109.46 

12.50% 248,829 1357684 1,973,662±128,668 793 116.07 

25% 268,430 1109333 1,596,570±290,540 595 93.9 

50% 263,171 1105270 1,553,019±223,125 590 91.33 

100% 247,634 998663 1,363,134±55,263 550 80.17 

 

Lab 

Controls 

256,275 

1214745 1,700,356 675 
71 

SUMMARY CONCLUSIONS.

 In conducting these trials investigators were aware of several different 

requirements and interests that guided the testing timetable and other procedures. For example, 

under IMO G-8 guidelines there is a requirement to perform three tests, the first and last of 

which should be separated by a period of not less than six months, in order to provide a seasonal 

dimension to the testing regime. Under U.S. Coast Guard Shipboard Technology Evaluation 

Program (STEP) requirements, two tests should be conducted in two different water bodies in 

which the vessel normally operates. These requirements were met by conducting these three 

trials between April 5th and October 6th, and by testing water picked up respectively in the 

Caribbean Sea, the North West Pacific off Alaska, and the Western Pacific off Long Beach. The 

distributors of the system, Hyde Marine Inc. expressed an interesting in varying the retention 

time of ballast water in the tanks in order to investigate any possible latent effects of UV 

irradiation. This was accommodated by varying the retention times by as much as 6 days: 96h in 

Trial 1, 114h in Trial 2 and 10 days in Trial 3. Actual ballasting times were at the discretion of 

the officers and crew of the vessel, according to their schedule and other logistics. 

In all three trials the numbers of live zooplankton >50μm (narrowest dimension) 

complied with all published ballast water treatment regulations, in that no live zooplankton in 

this size class were seen at the time of ballast water discharge. While some smaller taxa survived 

treatment, e.g. marine nematodes, these were very much narrower than 50μm. In making 

live/dead assessment investigators relied on movement of some part of the organism to confirm 

viability, although microscopic examination of this group was aided by the vital stain neutral red, 

which greatly facilitated the location of potentially live organisms in the microscope field of 

view. Trial 2 was characterized by a large amount of detritus appearing in samples collected 

during the de-ballasting operation close to the end of the cruise. Several large organisms were 

present in samples from both treated and untreated tanks, although these were dead in treated 

samples. These are discussed later in the context of bacterial counts. 

Planktonic organisms in the >10μm - <50μm represent, in several respects, the most 

problematic of all the endpoints measured. This size range primarily, but not exclusively, consists 

of phytoplankton, and is subject to standards that differ by as much as three orders of magnitude 

among jurisdictions (table 1). Natural populations of phytoplankton may have an even greater 

concentration range among different water bodies, and cell densities encountered during these 
72 

trials demonstrated 100-fold differences between Caribbean waters and Alaskan waters. Alaskan 

water (trial 2) showed the highest densities encountered throughout these trials (ca. 108 cells/m3), 

although very much higher cell concentrations would not be unusual in Pacific waters. We, 

therefore, categorize, phytoplankton densities throughout this sequence of trials as moderately 

low (trial 2) to extremely low (trial 1), with trial 3 intermediate. In view of the fact that most of 

the groups comprising this size class are non-motile, the primary problem confronting scientific 

investigation is that of viability. Several discussions of this issue exist (e.g. Wright 2007, 

Veldhuis et al. 2001, Veldhuis and Brussaard 2006). Further problems concern the fact that some 

phytoplankton taxa are actually larger than 50μm (Wright 2007) and in many cases a significant 

proportion of phytoplankton fall into the <10 μm size category as was the case in the current 

trials. 

In all of these trials phytoplankton numbers following treatment (at de-ballasting) were 

well below the standard of 107 live cells/m3 set by IMO under G-8 guidelines. When compared to 

the U.S. Coast Guard standard of 105 live cells/m3 (=100,000 live cells/m3), the grand mean from 

all three trials (2,222, 75,667, 103,300/3 = 60,398 live cells/m3) complies with the standard, 

although the individual value from trial 3 (103,300 live cells/m3) is marginally higher than the 

100,000 live cells/m3 U.S. Coast Guard standard. Another way of looking at these data takes note 

of the fact that cell densities following grow-out represent reductions in cell densities of 65%, 

60% and 83% for trials 1-3, compared with cell numbers before grow-out. A case can, therefore, 

be made for assuming that all these natural populations of phytoplankton are incapable of 

growth, and therefore non-viable. A similar result was seen in 2004 trials of the Hyde BWT 

system aboard Coral Princess (Wright et al. 2007), where a comprehensive analysis was made of 

individual genera/species of phytoplankton in treated and untreated ballast water. In untreated 

samples 27 out of 43 genera for which data were available showed positive growth rate (average 

increase after grow-out 1.25), whereas no treated samples exhibited positive growth of any 

phytoplankton genus (average decline of 75% in cell numbers after grow-out). Several analytical 

techniques are currently available to differentiate between dead and live, but non-motile cells 

(Bruussard et al 2001, Veldhuis et al. 2001, Veldhuis and Bruussard 2006), although 

quantification is difficult within the context of current published standards 

Bacterial counts illustrate some of the difficulties involved with conducting trials aboard 

working ships where, unlike land-based trials, there is limited ability to control some test 
73 

parameters. A summary of bacterial counts from all three trials is shown in table 31. 

As judged by indicator bacteria, water examined from trials 1 and 3 indicate relatively 

pristine conditions. Trial 1 showed very low densities of coliforms and Enterococci in control 

(untreated) samples at T=0, but there was no evidence of these groups in any other samples from 

this trial. Counts were made of heterotrophic cultural bacteria in all samples. Although they are 

not regulated by IMO or the U.S. Coast Guard, January 2008 California regulations set a 

standard of 100cfu/100ml for this group (Table 1). While cfus for cultural bacteria exceed this 

standard in trial 1 at T=0, both treated and untreated numbers declined at the time of discharge to 

levels below the California standard. At de-ballasting, cfus in treated samples were as low as one 

Table 31 . Summary of Bacterial endpoints for Trial 1 (April), Trial 2 (July) and Trial 3 

(September). Numbers are reported as cfu/100ml. 

Total 

Culturable 

Coliforms E. Coli Enterrococci Vibrio 

cholerae

Treatment Time Trial

Control t=0 1 122±129 0.2±0 ND 0.2±0 0

Treated t=0 1 194±104 ND ND ND 0

Control t=96h 1 50±57 ND ND ND 0

Treated t=96h 1 10±17 ND ND ND 0

Control t=0 2 522±707 ND ND ND 0

Treated t=0 2 211±262 ND ND ND 0

Control t=114h 2 1156±1201 ND ND 2±1 0

Treated t=114h 2 1978±881 ND ND 36±27 0

Control t=0 3 341±342 ND ND ND 0

Treated t=0 3 71±67 ND ND ND 0

Control t=10 days 3 27±29 6±5 3±5 ND 0

Treated t=10 days 3 8±23 ND ND ND 0

tenth of that standard. 

Samples from trial 3 were similar to trial 1. No indicator bacteria were reported in either 

treated or untreated samples at T=0, although low numbers of coliforms, including E. Coli, 

appeared in untreated samples at the time of discharge. No indicator bacteria were reported from 

any treated samples from trial 3. Cultural bacteria were present in both treated and untreated 

samples at T=0 in trial 3 (Table 31) although, as in trial 1, numbers at the time of discharge were 

lower than the California standard (table 30), particularly in treated samples. 
74 

Bacterial counts from trial 2 differed markedly from the other two trials, and were 

characterized by: 

1. High counts of cultural bacteria at the time of discharge (de-ballasting) 

2. Appearance of intestinal enterococci in both treated and untreated samples at time of 

de-ballasting, despite the fact that no cfus from this taxa were recorded in the 

respective T=0 samples. 

While IMO and the U.S. Coast Guard have no standards applicable to culturable 

heterotrophic bacteria, the numbers of cfu in these samples clearly exceeded the California 

standards. No coliforms were recorded from any samples in this trial. With respect to 

Enterococci, numbers of cfus in treated samples at de-ballasting, 36±27cfu/100ml (table 31), 

were noticeably higher than in untreated samples (2±1cfu/100 ml.). The figure of 

36±27cfu/100ml is below the standard of 100cfu/100ml set for this group by IMO and, therefore, 

complies with IMO regulations. However it marginally fails the corresponding standard of 33 

cfu/100ml set jointly by U.S. Coast Guard and the state of California (table 1). These anomalous 

figures should be viewed, however, within the context of the zooplankton samples, which had 

high detritus loads and large zooplankton specimens that were not seen in T=0 samples. These 

included several harpacticoid copepod adults >1000μm (some >2000μm), and indicated a quite 

different population that that sampled at T=0. Despite the presence of these organisms at the time 

of de-ballasting, all specimens in the treated samples were dead; some recently, others in varying 

states of decay. It must be borne in mind that the filtered was turned off at de-ballasting as per 

normal de-ballasting protocol, and would not have filtered out large organisms during the 

discharge cycle. 

It seems reasonable to conclude that the appearance of Enterococci in samples 

withdrawn from the tank at discharge would result from the decay/disintegration of these 

planktonic organisms with the concomitant release of endogenous bacteria that had been shielded 

from the effect of UV irradiation. In contrast, the corresponding T=0 samples were free from 

Enterococci and were remarkably “clean”, indicating that the filter appeared to be working 

correctly. Two explanations could explain the presence of large planktonic organisms identified 

at discharge but not previously seen: 
75 

1. An earlier ballasting operation (before trial 2) may not have properly employed 

the filter, or 

2. Small eggs and/or juvenile stages (<50μm) may have passed through a correctly 

functioning filter rated for a 50μm cut-off, then subsequently grew and formed a 

live population within the tank. 

A similar situation has been observed by this author in previous shipboard trials (Wright et al. 

2007 and unpublished). A drawback of conducting shipboard tests, therefore, relates to the fact 

that it is impossible to know if residual flora/fauna inhabit the tank prior to the onset of a trial. If 

such a situation exists, there may be significant, extraneous, qualitative and quantitative 

differences between the discharged water and that characterized by T=0 sampling. Such 

differences are avoided in land-based testing, where the collection/storage tanks can be 

vigorously rinsed between trials. In shipboard trials a similar problem relates to the flushing of 

the, often lengthy, piping and associated dead-space that constitutes the ballasting system. 

 Current IMO shipboard testing protocols simply require a comparison between treated 

and untreated water in shipboard trials following a certain defined residence time in the tanks, 

although there is an additional requirement to characterize the challenge (untreated) water at 

ballasting (T=0). For a system such as the one tested here, where treatment is not deemed 

complete until the second pass through the UV system at de-ballasting, it is clear that, from a 

regulatory standpoint, that the definitive samples should be those collected at discharge. 

Nevertheless, problems such as those described above illustrate the importance of inline 

sampling and analysis of a representative number of treated samples as well as untreated samples 

at T=0, particularly where a filter is involved. 

Results of Whole Effluent Toxicity (WET) bioassays conducted during trial 3 indicated 

no significant differences between the toxicity of treated vs. untreated water samples at the time 

of discharge from the vessel. In invertebrate and vertebrate larval assays undiluted water from 

both treated and untreated tanks appeared not to show any significant toxicity relative to 

laboratory controls. In the case of phytoplankton there appeared to be a small degree of toxicity 

associated with water retrieved from ballast waters following the (10 day) residence time, 

although the toxicity did not differ significantly between treated and untreated water. It might be 

concluded from those data that any toxic element present in discharged water did not result from 

UV irradiation. Such results have been supported by an extensive literature search that has 
76 

revealed no evidence of residual chemical toxicity in water resulting from prior UV irradiation of 

that water. While some changes in nutrient levels appeared to be associated with water treatment 

it was not possible to definitively conclude that these changes directly resulted from UV 

irradiation. For example, both phosphate and nitrate levels at de-ballasting were higher in treated 

samples relative to untreated samples at the time of discharge from the ship. It is possible that 

part of this difference could be attributed to an increase in the degradation of planktonic 

organisms resulting from the treatment process, rather than UV irradiation directly. Nitrite levels 

were also higher in treated samples 10 days after treatment. While UV photolysis of nitrate to 

nitrite has been demonstrated in UV-irradiated drinking water (e.g. Sharpless and Linden 2001) 

the levels involved are very small relative to the U.S. drinking water standard of 1000μg L-1, or 

the European standard of 100μg L-1. Nitrate levels reported in this trial were negligible relative 

to these standards and pose no toxicological threat associated with discharge. 

Table 32 provides a summary of compliance/non-compliance of the Hyde ‘Guardian’ 

BWT system with current published national and international standards. Under the conditions 

encountered during these trials, the system is seen to comply with all IMO G-8 standards relating 

to the elimination of biota and with respect the issue of residual toxicity of treated water related 

to chemicals generated during treatment. A bacterial (Enterococcus) excursion seen in Trial 2 

(see table 32 footnote) and minimally exceeding U.S. Coast Guard standards, was interpreted as 

an artifact related to the presence of a population of large zooplankton living in the tank prior to 

the test. Several of these organisms were probably alive until UV irradiation on de-ballasting. In 

view of the fact that no Enterococci were detected in T=0 samples, endogenous bacteria were 

probably released from dead plankton at de-ballasting. 

Problems such as this illustrate one of the drawbacks of shipboard trials, i.e. the problem 

of not being able to rigorously control all conditions related to the test. Another concerns the 

relative lack of flexibility in certain cases in determining the exact timing of a ballasting/deballasting event. Biological productivity in such cases may be less than optimal. Within these 

constraints, shipboard trials nevertheless represent a useful and informative exercise providing 

critical information regarding the performance of a ballast water treatment system under ‘real 

world conditions’. In this case, under the ambient conditions and for the flow rates encountered 

throughout these trials, the Hyde Marine ‘Guardian’ system appears to be a highly effective 

means of treating ballast water to remove potentially invasive species from the ballast stream. 
77 

 

Table 32. A summary of compliance (√) / non-compliance (X) with current published 

national and international ballast water management standards. NT = Not Tested 

 

IMO 

Regulation 

D-2 and 

Transport 

Canada

Ballast Water 

Management 

Act Section 

1101 (f) i

2008 

California 

Standard

IMO G-9 

Discharge 

Standard for 

residual 

Toxicity 

Trial 1 

Zooplankton √ √ √

NT 

Trial 1 

Phytoplankton √ √ √

Trial 1 

Bacteria √ √ √

Trial 2 

Zooplankton √ √ √

Trial 2 NT 

Phytoplankton √ √ X

3

Trial 2 

Bacteria √ X

1 X 

Trial 3 

Zooplankton √ √ √

Trial 3 √

Phytoplankton √ X

2 X3

Trial 3 

Bacteria √ √ √

1

 – Intestinal enterococci appeared at de-ballasting despite not being seen in T=0 samples, 

thought to be associated with zooplankton present in the treated tank prior to the conduct of the 

test. 

2

 – ‘Live’ phytoplankton present in treated samples after 10 days in tank + UV irradiation on deballasting. Residuals exceed U.S, C.G. standard by 3% following 83% reduction in cell 

concentration after grow-out. Such a reduction in cell count following a grow-out period could 

be interpreted as non-viable (Wright et al. 2007; Perrins et al. 2006). 

3

 – While phytoplankton cells can be scored as ‘live’ based on morphological characteristics their 

lack of growth potential determined through cell counts before and after grow-out can be 

interpreted as ‘non-viable’, in which case California regulations would be met.
78 

LITERATURE CITED.

Brussaard C.P.D., Marie, D, Thyrhaug R. and Bratbak, G. (2001) Flow cytometric 

analysis of phytoplankton viability following viral infection. Aquatic Microbial Ecology 

26, 157 – 166. 

Carlton, J.T. and Geller, J.B. (1993). Ecological roulette: The global transport of 

nonindigenous marine organisms. Science 261, 78-82. 

Cohen, A.N. and Carlton, J.T. (1998). Accelerated invasion rate in a highly invaded 

estuary. 279, 555-558. 

Perrins, J.C. Cooper, W. J., van Leeuwen, J. and Herwig, R.P. (2006). Ozonation of seawater 

from different locations: formation and decay of total residual oxidant – implications for ballast 

water treatment. Marine Pollution Bulletin 52, 1023 – 1033. 

Pimentel, D., Zuniga, R. and Morrison, D. (2005). Update on the environmental and 

economic costs associated with alien-invasive species in the United States. Ecological 

Economics 52, 273-288. 

Pimentel, D. (2003). Economic and Ecological Costs Associated with Aquatic Invasive 

Species. Proceedings of the Aquatic Invaders of the Delaware Estuary Symposium, 

Malvern, Pennsylvania, May 20, 2003, pp. 3-5. 

 

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25-38. 

 

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80 

Appendix A. Raw zooplankton counts. Coral Princess. 

Trial 1

 CORAL PRINCESS BALLAST WATER TRIAL #1 APR 5-15 2008 (updated 10/08/2008) 

 CONSOLIDATED SPECIES LIST 

 GROUP SPECNAME LIFESTAGE 

 ACTINULA ACTINULA 

 BIVALVE BIVALVE D-HINGE 

 CHAETOGNATH CHAETOGNATH 

 CLADOCERAN CLADOCERAN 

 COPEPOD ACARTIA HELGOLANDICA ADULT 

 COPEPOD CALANOID ADULT 

 COPEPOD CALANOID COPEPODITE 

 COPEPOD CANDACEA ADULT 

 COPEPOD CENTROPAGES HAMATUS ADULT 

 COPEPOD CLAUSOCALANUS ADULT 

 COPEPOD COPEPOD ADULT 

 COPEPOD COPEPOD COPEPODITE 

 COPEPOD CORYCAEUS 

 COPEPOD CORYCAEUS ADULT 

 COPEPOD CORYCAEUS COPEPODITE 

 COPEPOD CYCLOPOID COPEPODITE 

 COPEPOD FARANULA CARINATA ADULT 

 COPEPOD HALICYCLOPS ADULT 

 COPEPOD HALICYCLOPS COPEPODITE 

 COPEPOD HARPACTICOID 

 COPEPOD HARPACTICOID ADULT 

 COPEPOD HARPACTICOID UNDETERMINED 

 COPEPOD LABIDOCERA ADULT 

 COPEPOD MACROSETELLA 

 COPEPOD MACROSETELLA ADULT 

 COPEPOD MICROSETELLA ADULT 

 COPEPOD OITHONA ADULT 

 COPEPOD OITHONA COPEPODITE 

 COPEPOD ONCAEA 

 COPEPOD ONCAEA ADULT 

 COPEPOD ONCAEA-POPEYE ADULT 

 COPEPOD PARACALANUS ADULT 

 COPEPOD SAPHIRELLA COPEPODITE 

 COPEPOD TEMORA UNDETERMINED 

 COPEPOD NAUPLII COPEPOD NAUPLIUS 

 EGGS EGGS 

 FORAMINIFERA GLOBIGERINA 

 GASTROPOD GASTROPOD 

 NEMATODE NEMATODE 

 NOZOO NOZOOPLANKTON 

 POLYCHAETE POLYCHAETE 

 POLYCHAETE POLYCHAETE LARVAE 

 PROTOZOAN PROTOZOAN 

 TINTINNID TINTINNID 
81 

CORAL PRINCESS BALLAST WATER TRIAL #1 (CARIBBEAN SEA ) APR 5-15 2008 (updated 10/08/2008) 

 Num. Num. Total Total Grand 

Event Treatment Rep Group Alive Dead Total alive dead total 

---------------------------------------------------------------------------- 

T0 

 CONTROL 1 COPEPOD 328 136 464 

 1 COPEPOD NAUP 72 0 72 

 1 EGGS 312 0 312 

 1 NEMATODE 8 0 8 

 1 POLYCHAETE 48 0 48 768 136 904 

 2 CLADOCERAN 16 0 16 

 2 COPEPOD 128 24 152 

 2 COPEPOD NAUP 168 8 176 

 2 EGGS 224 104 328 

 2 POLYCHAETE 80 0 80 616 136 752 

 3 BIVALVE 16 0 16 

 3 COPEPOD 104 40 144 

 3 COPEPOD NAUP 24 0 24 

 3 TINTINNID 96 0 96 240 40 280 

 4 COPEPOD 32 0 32 

 4 COPEPOD NAUP 64 0 64 

 4 EGGS 88 0 88 

 4 POLYCHAETE 16 0 16 

 4 TINTINNID 112 0 112 312 0 312 

 5 BIVALVE 32 8 40 

 5 COPEPOD 40 0 40 

 5 COPEPOD NAUP 80 8 88 

 5 EGGS 368 0 368 

 5 NEMATODE 8 0 8 

 5 POLYCHAETE 24 0 24 552 16 568 

 TREATED 1 COPEPOD 16 0 16 

 1 TINTINNID 0 8 8 16 8 24 

 2 COPEPOD 8 0 8 

 2 POLYCHAETE 0 8 8 8 8 16 

 3 NOZOO 0 0 0 0 0 0 

 4 NOZOO 0 0 0 0 0 0 

 5 COPEPOD 16 0 16 16 0 16 

T96 

 CONTROL 1 BIVALVE 8 0 8 

 1 COPEPOD 40 104 144 

 1 COPEPOD NAUP 24 8 32 

 1 EGGS 384 0 384 

 1 POLYCHAETE 8 0 8 464 112 576 

 2 BIVALVE 8 8 16 

 2 COPEPOD 48 72 120 

 2 EGGS 16 0 16 72 80 152 

 3 BIVALVE 16 8 24 
82 

CORAL PRINCESS BALLAST WATER TRIAL #1 (CARIBBEAN SEA ) APR 5-15 2008 (updated 10/08/2008) 

 Num. Num. Total Total Grand 

Event Treatment Rep Group Alive Dead Total alive dead total 

---------------------------------------------------------------------------- 

T96 3 COPEPOD 24 272 296 

 3 COPEPOD NAUP 24 0 24 

 3 EGGS 624 240 864 

 3 POLYCHAETE 64 0 64 

 3 PROTOZOAN 8 0 8 

 3 TINTINNID 16 0 16 776 520 1296 

 4 BIVALVE 8 8 16 

 4 COPEPOD 8 88 96 

 4 EGGS 32 0 32 48 96 144 

 5 BIVALVE 16 8 24 

 5 COPEPOD 128 80 208 

 5 COPEPOD NAUP 0 8 8 

 5 POLYCHAETE 8 0 8 

 5 TINTINNID 8 0 8 160 96 256 

 6 CHAETOGNATH 0 8 8 

 6 COPEPOD 0 328 328 

 6 COPEPOD NAUP 680 344 1024 

 6 FORAMINIFERA 8 0 8 

 6 POLYCHAETE 24 0 24 

 6 TINTINNID 0 24 24 712 704 1416 

 7 CHAETOGNATH 16 8 24 

 7 COPEPOD 0 344 344 

 7 COPEPOD NAUP 584 440 1024 

 7 EGGS 0 8 8 

 7 NEMATODE 24 0 24 

 7 POLYCHAETE 16 0 16 

 7 TINTINNID 0 8 8 640 808 1448 

 8 BIVALVE 8 8 16 

 8 CHAETOGNATH 24 8 32 

 8 COPEPOD 80 648 728 

 8 COPEPOD NAUP 552 752 1304 

 8 EGGS 0 40 40 

 8 FORAMINIFERA 16 0 16 

 8 POLYCHAETE 32 8 40 

 8 TINTINNID 0 32 32 712 1496 2208 

 9 CHAETOGNATH 8 0 8 

 9 COPEPOD 8 672 680 

 9 COPEPOD NAUP 856 568 1424 

 9 EGGS 0 8 8 

 9 FORAMINIFERA 16 0 16 

 9 POLYCHAETE 48 16 64 

 9 TINTINNID 0 16 16 936 1280 2216 
83 

CORAL PRINCESS BALLAST WATER TRIAL #1 (CARIBBEAN SEA ) APR 5-15 2008 (updated 10/08/2008) 

 Num. Num. Total Total Grand 

Event Treatment Rep Group Alive Dead Total alive dead total 

---------------------------------------------------------------------------- 

 T96 TREATED 1 ACTINULA 0 16 16 

 1 BIVALVE 0 8 8 

 1 COPEPOD 0 104 104 

 1 GASTROPOD 0 8 8 

 1 NEMATODE 0 16 16 0 152 152 

 2 ACTINULA 0 16 16 

 2 COPEPOD 0 80 80 0 96 96 

 3 COPEPOD 0 128 128 

 3 COPEPOD NAUP 0 8 8 

 3 TINTINNID 0 8 8 0 144 144 

 4 COPEPOD 0 176 176 

 4 COPEPOD NAUP 0 8 8 

 4 FORAMINIFERA 0 8 8 0 192 192 

 5 COPEPOD 0 184 184 0 184 184 

 6 COPEPOD 0 16 16 0 16 16 

 7 COPEPOD 0 64 64 

 7 COPEPOD NAUP 0 16 16 0 80 80 

 8 COPEPOD 0 144 144 0 144 144 

 9 NOZOO 0 0 0 0 0 0 
84 

Trial 2 

CORAL PRINCESS BALLAST WATER TRIAL #2 JUN 30-JUL 7 2008 (updated 10/08/2008) 

 CONSOLIDATED SPECIES LIST 

 GROUP SPECNAME LIFESTAGE 

 AMPHIPOD HYPERIA SP ADULT 

 B? B? 

 BIVALVE BIVALVE LARVA 

 BIVALVE BIVALVE UMBO 

 COPEPOD CALANOID 

 COPEPOD CALANOID ADULT 

 COPEPOD CALANOID COPEPODITE 

 COPEPOD CALANOID2 ADULT 

 COPEPOD CALANOID3 ADULT 

 COPEPOD COPEPOD EGG 

 COPEPOD OITHONA ADULT 

 COPEPOD OITHONA COPEPODITE 

 COPEPOD ONCAEA ADULT 

 COPEPOD NAUPLIUS COPEPOD NAUPLIUS 

 DECAPOD SHRIMP POSTLARVA 

 EGGS COPEPOD EGG 

 EGGS EGG DARK 

 EGGS EGG GREEN 

 EGGS EGG MASS 

 EGGS FISH EGGS 

 FORAMINIFERA GLOBEGERINA 

 GASTROPOD LAMACINA 

 GASTROPOD SNAIL 

 NEMATODE NEMATODE 

 NOZOO NOZOOPLANKTON 

 POLYCHAETE POLYCHAETE LARVA 

 PROTOZOAN PROTOZOAN 

 ROTIFER ROTIFER 

 TINTINNID TINNINNID V-SHAPE 
85 

 CORAL PRINCESS BALLAST WATER TRIAL #2 ALASKAN COAST JUN 30-JUL 7 2008 (as of 10/08/2008) 

 Sample Sample Sample 

 Num* Num grand total total 

Event Treatment Rep Group Total* Alive* Dead* total* alive* dead* 

------------------------------------------------------------------------------------------- 

T0 CONTROL 1 B? 16 16 0 

 COPE NAUPLII 2992 2944 48 

 COPEPOD 4136 3992 144 

 EGGS 48 48 0 

 POLYCHAETE 8 8 0 

 ---------------------------- 7200 7008 192 

 2 BIVALVE 40 40 0 

 COPE NAUPLII 7100 6880 220 

 COPEPOD 13220 12840 380 

 DECAPOD 40 40 0 

 ---------------------------- 20400 19800 600 

 3 B? 48 48 0 

 COPE NAUPLII 2064 2064 0 

 COPEPOD 2928 2928 0 

 EGGS 208 208 0 

 PROTOZOA 32 32 0 

 ---------------------------- 5280 5280 0 

 4 COPE NAUPLII 11504 11472 32 

 COPEPOD 7808 7760 48 

 ---------------------------- 19312 19232 80 

 5 COPE NAUPLII 13600 13536 64 

 COPEPOD 9600 9536 64 

 DECAPOD 32 32 0 

 EGGS 320 320 0 

 ---------------------------- 23552 23424 128 

 6 AMPHIPOD 64 64 0 

 BIVALVE 32 32 0 

 COPE NAUPLII 10976 10976 0 

 COPEPOD 6912 6720 192 

 DECAPOD 32 32 0 

 EGGS 576 576 0 

 GASTROPOD 32 32 0 

 ---------------------------- 18624 18432 192 

 7 BIVALVE 32 32 0 

 COPE NAUPLII 8144 8144 0 

 COPEPOD 11712 11712 0 

 EGGS 176 176 0 

 ---------------------------- 20064 20064 0 

 8 COPE NAUPLII 11584 11584 0 

 COPEPOD 3952 3872 80 

 EGGS 128 128 0 

 GASTROPOD 16 16 0 

 ---------------------------- 15680 15600 80 

 9 COPE NAUPLII 4464 4464 0 

 COPEPOD 4656 4656 0 

 EGGS 368 368 0 

 PROTOZOA 32 32 0 

 ---------------------------- 9520 9520 0 

T0 TREATED 1 ---------------------------- 0 0 0 

 2 ---------------------------- 0 0 0 

 3 ---------------------------- 0 0 0 
86 

 CORAL PRINCESS BALLAST WATER TRIAL #2 ALASKAN COAST JUN 30-JUL 7 2008 (as of 10/08/2008) 

 Sample Sample Sample 

 Num* Num grand total total 

Event Treatment Rep Group Total* Alive* Dead* total* alive* dead* 

------------------------------------------------------------------------------------------- 

 4 ---------------------------- 0 0 0 

 5 ---------------------------- 0 0 0 

 6 COPEPOD 8 8 0 

 ---------------------------- 8 8 0 

 7 ---------------------------- 0 0 0 

T114 CONTROL 1 COPE NAUPLII 8 8 0 

 COPEPOD 192 192 0 

 ---------------------------- 200 200 0 

 2 COPE NAUPLII 160 160 0 

 COPEPOD 240 240 0 

 EGGS 16 16 0 

 ---------------------------- 416 416 0 

 3 COPE NAUPLII 256 256 0 

 COPEPOD 624 624 0 

 EGGS 16 16 0 

 ---------------------------- 896 896 0 

 4 COPE NAUPLII 504 336 168 

 COPEPOD 1224 808 416 

 EGGS 40 40 0 

 POLYCHAETE 16 8 8 

 PROTOZOA 48 24 24 

 ROTIFERA 32 16 16 

 ---------------------------- 1864 1232 632 

 5 COPE NAUPLII 704 32 672 

 COPEPOD 1120 96 1024 

 GASTROPOD 32 32 0 

 ---------------------------- 1856 160 1696 

 6 COPE NAUPLII 544 0 544 

 COPEPOD 544 0 544 

 EGGS 32 0 32 

 ---------------------------- 1120 0 1120 

 7 COPE NAUPLII 416 0 416 

 COPEPOD 896 0 896 

 EGGS 128 0 128 

 NEMATODE 32 0 32 

 ---------------------------- 1472 0 1472 

 8 COPE NAUPLII 512 0 512 

 COPEPOD 704 64 640 

 EGGS 64 0 64 

 GASTROPOD 96 64 32 

 ---------------------------- 1376 128 1248 

 9 COPE NAUPLII 32 0 32 

 COPEPOD 192 0 192 

 ---------------------------- 224 0 224 
87 

 CORAL PRINCESS BALLAST WATER TRIAL #2 ALASKAN COAST JUN 30-JUL 7 2008 (as of 10/08/2008) 

 Sample Sample Sample 

 Num* Num grand total total 

Event Treatment Rep Group Total* Alive* Dead* total* alive* dead* 

------------------------------------------------------------------------------------------- 

T114 TREATED 1 COPEPOD 56 0 56 

 ---------------------------- 56 0 56 

 2 COPEPOD 96 0 96 

 ---------------------------- 96 0 96 

 3 COPE NAUPLII 8 0 8 

 COPEPOD 64 0 64 

 ---------------------------- 72 0 72 

 4 COPE NAUPLII 16 0 16 

 COPEPOD 24 0 24 

 ---------------------------- 40 0 40 

 5 COPE NAUPLII 8 0 8 

 COPEPOD 32 0 32 

 NEMATODE 16 16 0 

 ---------------------------- 56 16 40 

 6 BIVALVE 48 0 48 

 COPE NAUPLII 80 0 80 

 COPEPOD 240 0 240 

 EGGS 64 0 64 

 FORAMINIFERA 16 0 16 

 NEMATODE 272 64 208 

 PROTOZOA 16 16 0 

 TINTINNID 32 0 32 

 ---------------------------- 768 80 688 

 7 COPE NAUPLII 80 0 80 

 COPEPOD 320 0 320 

 FORAMINIFERA 16 0 16 

 NEMATODE 192 80 112 

 ---------------------------- 608 80 528 

 8 COPE NAUPLII 32 0 32 

 COPEPOD 224 0 224 

 GASTROPOD 80 0 80 

 NEMATODE 336 128 208 

 ---------------------------- 672 128 544 

 9 COPEPOD 144 0 144 

 EGGS 32 0 32 

 NEMATODE 384 32 352 

 ---------------------------- 560 32 528 

------------------------------------------------------------------------------------------- 

*Density expressed as numbers per ton of water. 
88 

 CORAL PRINCESS BALLAST WATER TRIAL #2 ALASKAN COAST JUN 30-JUL 7 2008 (as of (as of 

10/08/2008) 

 ANALYST=MARCIA. NUM DEAD=DEAD PINK + DEAD CLEAR 

 Num Num Dead Dead 

Event Treatment Rep Group Alive* Dead* pink clear 

-------------------------------------------------------------------------------- 

T0 CONTROL 4 COPE NAUPLII 11472 32 . . 

 COPEPOD 7760 48 . . 

 5 COPE NAUPLII 13536 64 . . 

 COPEPOD 9536 64 . . 

 DECAPOD 32 0 . . 

 EGGS 320 0 . . 

 6 AMPHIPOD 64 0 . . 

 BIVALVE 32 0 . . 

 COPE NAUPLII 10976 0 . . 

 COPEPOD 6720 192 . . 

 DECAPOD 32 0 . . 

 EGGS 576 0 . . 

 GASTROPOD 32 0 . . 

 8 COPE NAUPLII 11584 0 . . 

 COPEPOD 3872 80 . . 

 EGGS 128 0 . . 

 GASTROPOD 16 0 . . 

T0 TREATED 3 

 4 

 5 

 6 COPEPOD 8 0 . . 

T114 CONTROL 5 COPE NAUPLII 32 672 672 0 

 COPEPOD 96 1024 928 96 

 GASTROPOD 32 0 0 0 

 6 COPE NAUPLII 0 544 544 0 

 COPEPOD 0 544 480 64 

 EGGS 0 32 0 32 

 7 COPE NAUPLII 0 416 416 0 

 COPEPOD 0 896 896 0 

 EGGS 0 128 0 128 

 NEMATODE 0 32 0 32 

 8 COPE NAUPLII 0 512 512 0 

 COPEPOD 64 640 640 0 

 EGGS 0 64 0 64 

 GASTROPOD 64 32 32 0 

 9 COPE NAUPLII 0 32 32 0 

 COPEPOD 0 192 192 0 

T114 TREATED 6 BIVALVE 0 48 0 48 

 COPE NAUPLII 0 80 80 0 

 COPEPOD 0 240 240 0 

 EGGS 0 64 0 64 

 FORAMINIFERA 0 16 0 16 

 NEMATODE 64 208 64 144 

 PROTOZOA 16 0 0 0 

 TINTINNID 0 32 0 32 

 7 COPE NAUPLII 0 80 80 0 

 COPEPOD 0 320 48 272 

 FORAMINIFERA 0 16 0 16 

 NEMATODE 80 112 32 80 
89 

 CORAL PRINCESS BALLAST WATER TRIAL #2 ALASKAN COAST JUN 30-JUL 7 2008 (as of (as of 

10/08/2008) 

 ANALYST=MARCIA. NUM DEAD=DEAD PINK + DEAD CLEAR 

 Num Num Dead Dead 

Event Treatment Rep Group Alive* Dead* pink clear 

-------------------------------------------------------------------------------- 

 8 COPE NAUPLII 0 32 0 32 

 COPEPOD 0 224 96 176 

 GASTROPOD 0 80 0 80 

 NEMATODE 128 208 32 176 

 9 COPEPOD 0 144 0 144 

 EGGS 0 32 0 32 

 NEMATODE 32 352 288 64 

------------------------------------------------------- ----------------------- 

*Density expressed as numbers per ton of water. 
90 

Trial 3

CORAL PRINCESS BALLAST WATER TRIAL #3 SEP 19-29 2008 (updated 10/08/2008) 

 CONSOLIDATED SPECIES LIST 

 GROUP SPECNAME LIFESTAGE 

 BARNACLE BARNACLE CYPRIS STAGE 

 BARNACLE BARNACLE NAUPLIUS 

 BIVALVE BIVALVE D-HINGE 

 COPEPOD CALANOID COPEPODITE 

 COPEPOD CALANOID4 ADULT 

 COPEPOD CALANOID5 ADULT 

 COPEPOD CALANOID5 COPEPODITE 

 COPEPOD COPEPOD ADULT 

 COPEPOD COPEPOD COPEPODITE 

 COPEPOD CORYCEUS ADULT 

 COPEPOD CYCLOPOID ADULT 

 COPEPOD CYCLOPOID1 ADULT 

 COPEPOD CYCLOPOID1 COPEPODITE 

 COPEPOD CYCLOPOID2 COPEPODITE 

 COPEPOD HARPACTICOID ADULT 

 COPEPOD HARPACTICOID COPEPODITE 

 COPEPOD SAPHIRELLA COPEPODITE 

 COPEPOD NAUPLIUS COPEPOD NAUPLIUS 

 DECAPOD SHRIMP POST LARVA 

 ECHINODERMATA STARFISH LARVA 

 NEMATODE NEMATODE 

 NOZOO NOZOOPLANKTON 

 POLYCHAETE POLYCHAETE 

 POLYCHAETE POLYCHAETE ADULT 

 POLYCHAETE POLYCHAETE LARVA 

 POLYCHAETE POLYCHAETE TROCHOPHORE 

 ROTIFER ROTIFER 
91 

 CORAL PRINCESS BALLAST WATER TRIAL #3 PACIFIC COAST SEP 19-29 2008 (as of 10/08/2008) 

 Sample Sample Sample 

 Num* Num grand total total 

Event Treatment Rep Group Total* Alive* Dead* total* alive* dead* 

------------------------------------------------------------------------------------------- 

T0 CONTROL 1 BIVALVE 16 16 0 

 COPEPOD 200 160 40 

 COPEPOD NAUPLII 3696 3216 480 

 NEMATODE 8 8 0 

 POLYCHAETE 24 24 0 

 ---------------------------- 3944 3424 520 

 2 COPEPOD 32 32 0 

 COPEPOD NAUPLII 1832 1776 56 

 NEMATODE 8 8 0 

 ---------------------------- 1872 1816 56 

 3 COPEPOD 32 32 0 

 COPEPOD NAUPLII 1608 1600 8 

 NEMATODE 8 8 0 

 ---------------------------- 1648 1640 8 

 4 COPEPOD 72 64 8 

 COPEPOD NAUPLII 1096 1024 72 

 POLYCHAETE 24 24 0 

 ---------------------------- 1192 1112 80 

 5 COPEPOD 16 16 0 

 COPEPOD NAUPLII 736 736 0 

 ROTIFERA 16 16 0 

 ---------------------------- 768 768 0 

 6 COPEPOD 184 184 0 

 COPEPOD NAUPLII 1064 1016 48 

 NEMATODE 8 8 0 

 POLYCHAETE 8 8 0 

 ---------------------------- 1264 1216 48 

 7 COPEPOD 232 232 0 

 COPEPOD NAUPLII 1448 1440 8 

 NEMATODE 8 8 0 

 ---------------------------- 1688 1680 8 

 8 BIVALVE 8 8 0 

 COPEPOD 72 72 0 

 COPEPOD NAUPLII 224 184 40 

 POLYCHAETE 8 8 0 

 ---------------------------- 312 272 40 

 9 COPEPOD 138 138 0 

 COPEPOD NAUPLII 492 492 0 

 ECHINODERMATA 15 15 0 

 NEMATODE 15 15 0 

 ---------------------------- 662 662 0 

T0 TREATED 1 COPEPOD 8 0 8 

 COPEPOD NAUPLII 8 0 8 

 ---------------------------- 16 0 16 

 2 ---------------------------- 0 0 0 

 3 ---------------------------- 0 0 0 

 4 COPEPOD NAUPLII 16 0 16 

 ---------------------------- 16 0 16 

 5 BIVALVE 8 0 8 
92 

 CORAL PRINCESS BALLAST WATER TRIAL #3 PACIFIC COAST SEP 19-29 2008 (as of 10/08/2008) 

 Sample Sample Sample 

 Num* Num grand total total 

Event Treatment Rep Group Total* Alive* Dead* total* alive* dead* 

------------------------------------------------------------------------------------------- 

 COPEPOD NAUPLII 40 0 40 

 ---------------------------- 48 0 48 

 6 ---------------------------- 0 0 0 

 7 ---------------------------- 0 0 0 

 8 ---------------------------- 0 0 0 

 9 ---------------------------- 0 0 0 

T240 CONTROL 1 COPEPOD 32 16 16 

 POLYCHAETE 8 8 0 

 ---------------------------- 40 24 16 

 2 COPEPOD 48 16 32 

 POLYCHAETE 8 8 0 

 ---------------------------- 56 24 32 

 3 COPEPOD 72 24 48 

 ---------------------------- 72 24 48 

 4 BARNACLE 8 8 0 

 DECAPOD 8 8 0 

 ---------------------------- 16 16 0 

 5 COPEPOD 48 8 40 

 ---------------------------- 48 8 40 

 6 ---------------------------- 0 0 0 

 7 BIVALVE 8 0 8 

 COPEPOD 8 0 8 

 COPEPOD NAUPLII 8 8 0 

 ---------------------------- 24 8 16 

 8 BARNACLE 8 8 0 

 COPEPOD 24 16 8 

 COPEPOD NAUPLII 16 16 0 

 ECHINODERMATA 8 0 8 

 POLYCHAETE 8 0 8 

 ---------------------------- 64 40 24 

 9 ---------------------------- 0 0 0 

T240 TREATED 1 ---------------------------- 0 0 0 

 2 ---------------------------- 0 0 0 

 3 ---------------------------- 0 0 0 

 4 BIVALVE 8 0 8 

 ---------------------------- 8 0 8 

 5 ---------------------------- 0 0 0 

 6 ---------------------------- 0 0 0 

 7 BARNACLE 8 0 8 
93 

 CORAL PRINCESS BALLAST WATER TRIAL #3 CARIBBEAN SEP 19-29 2008 (as of 10/08/2008) 

 Sample Sample Sample 

 Num* Num grand total total 

Event Treatment Rep Group Total* Alive* Dead* total* alive* dead* 

------------------------------------------------------------------------------------------- 

 NEMATODE 32 0 32 

 ---------------------------- 40 0 40 

 8 NEMATODE 8 8 0 

 ---------------------------- 8 8 0 

 9 NEMATODE 8 0 8 

 ---------------------------- 8 0 8 

------------------------------------------------------------------------------------------- 

*Density expressed as numbers per ton of water. 
94 

 CORAL PRINCESS BALLAST WATER TRIAL #3 PACIFIC COAST SEP 19-29 2008 (as of 10/08/2008) 

 ANALYST=MARCIA. NUM DEAD=DEAD PINK + DEAD CLEAR 

 Num Num Dead Dead 

Event Treatment Rep Group Alive* Dead* pink clear 

-------------------------------------------------------------------------------- 

T0 CONTROL 1 BIVALVE 16 0 . . 

 COPEPOD 160 40 24 16 

 COPEPOD NAUPLII 3216 480 280 200 

 NEMATODE 8 0 . . 

 POLYCHAETE 24 0 . . 

 2 COPEPOD 32 0 . . 

 COPEPOD NAUPLII 1776 56 0 56 

 NEMATODE 8 0 . . 

 3 COPEPOD 32 0 . . 

 COPEPOD NAUPLII 1600 8 0 8 

 NEMATODE 8 0 . . 

T0 TREATED 1 COPEPOD 0 8 0 8 

 COPEPOD NAUPLII 0 8 0 8 

 2 

 3 

 4 COPEPOD NAUPLII 0 16 0 16 

 5 BIVALVE 0 8 . . 

 COPEPOD NAUPLII 0 40 0 40 

 6 

T240 CONTROL 7 BIVALVE 0 8 0 8 

 COPEPOD 0 8 0 8 

 COPEPOD NAUPLII 8 0 . . 

 8 BARNACLE 8 0 . . 

 COPEPOD 16 8 8 0 

 COPEPOD NAUPLII 16 0 . . 

 ECHINODERMATA 0 8 0 8 

 POLYCHAETE 0 8 8 0 

 9 

T240 TREATED 3 

 4 BIVALVE 0 8 . 8 

 5 

 6 

 7 BARNACLE 0 8 . 8 

 NEMATODE 0 32 24 8 

 8 NEMATODE 8 0 . . 

 9 NEMATODE 0 8 8 0 

------------------------------------------------------- ----------------------- 

*Density expressed as numbers per ton of water. 
95 

Appendix B. 

Bacterial Sampling Protocol for Ballast Water testing (Trial 3) showing mold counts not 

included in culturable bacteria counts. 

(1) General sample details:

Name : Carys Mitchelmore 

Date of test : 19th September 2008 

Test ID : Princess Cruise/Hyde Maine – trial 3, T=0 

Type of sample : Ballasting of treated and control tanks; 3 samples each tank; early, mid and end 

samples (n=3 each time point; n=9 total each tank). 

Time and details of sample collection : 

Three sets (A-C) of 55ml sterile PP tubes filled from water outlet for collection, stored in 

cold cooler once collected. 

(1) Treated tank; : Start of collection: 11.40am (in cold cooler) 

 : Number of samples: 9 

 : Id of samples: T1, T2, T3....through T9 (start-end time in order) 

 : End of collection: 12.00pm 

(2) Control tank: : Start of collection: 12.05pm (in cold cooler) 

 : Number of samples: 9 

 : Id of samples: C1, C2, C3....through C9 (start-end time in order) 

 : End of collection: 12.25pm 

Sample processing/storage: 

 

Samples processed immediately after sampling for Idexx : Coliform/E.coli/Enterococci (Tube set 

A), tube set A placed in fridge after processing. Other samples (Tubes set B and C) stored in 

fridge until processing <24hr (see specific tests). 
96 

(2) Idexx Tests:

(1) Coliform / E.coli (colilert 18) 

Sample ID: Trial 3, T=0 

Date: 19th September 2008 samples collected 

Storage: N/A; samples processed immediately 

Time: 15.40-18.10; processed Treated first (9 samples T1-T9), then control samples (9 

samples C1-C9). Distilled water used as control. 

Protocol: As per standard Idexx method. 

Specific details: 90ml of DI water placed in sterile Idexx bottles. 10ml of test water (at RT) 

added (one bottle per sample. Vial opened and added to water, mixed till dissolved. Samples 

placed in trays and tray sealer used. Specific times made noted. Treated samples processed first 

then control samples. Samples read at 18 hours and later (up to 22 hours), scored for coliform 

(yellow wells) and E.coli (blue fluorescence). Positive wells scored as (large; L or small; S). 

After 22 hours tests are invalid. 

Incubation times: Treated samples placed in incubator at 17.15 at 35OC (19th Sept 2008) 

 Control samples placed in incubator at 18.10pm at 35OC (19th Sept 2008) 

Read times: Treated samples read at 11.15 (20th Sept 2008) – 18 hours 

 Control samples read at 12.00 (20th Sept 2008) ~ 18 hours 

Sample ID Coliform counts MPN Coliform E.coli counts MPN E.coli 

 Large Small Large Small 

T1 0 0 0 0 0 0 

T2 0 0 0 0 0 0 

T3 0 0 0 0 0 0 

T4 0 0 0 0 0 0 

T5 0 0 0 0 0 0 

T6 0 0 0 0 0 0 

T7 0 0 0 0 0 0 

T8 0 0 0 0 0 0 

T9 0 0 0 0 0 0 

C1 0 0 0 0 0 0 

C2 0 0 0 0 0 0 

C3 0 0 0 0 0 0 

C4 0 0 0 0 0 0 

C5 0 0 0 0 0 0 

C6 0 0 0 0 0 0 

C7 0 0 0 0 0 0 

C8 0 0 0 0 0 0 

C9 0 0 0 0 0 0 

DI water 0 0 0 0 0 0 

Additional notes: 

Samples also scored at 23.20; C3 positive coliform (11B, 14S) (but this was at 28 hours!) 
97 

(2) Idexx Tests:

(2) Enterobacteriacae (Enterolert) 

Sample ID: Trial 3, T=0 

Date: 19th September 2008; sample collection 

Storage: N/A; processed immediately 

Time: 17.40-18.50; processed Treated first (9 samples T1-T9), then control samples (9 

samples C1-C9). Distilled water used as control. 

Protocol: As per standard Idexx method. 

Specific details: 90ml of DI water placed in sterile Idexx bottles. 10ml of test water (at RT) 

added (one bottle per sample). Vial opened and added to water, mixed till dissolved. Samples 

placed in trays and tray sealer used. Specific times made noted. Treated samples processed first 

then control samples. Samples read at 24 hours and later (up to 28 hours), scored for 

Enterobacteriacea (blue fluorescence). Positive wells scored as (large; L or small; S). After 28 

hours tests are invalid. 

Incubation times: Treated samples placed in incubator at 18.30pm at 41C (19th Sept 2008) 

 Control samples placed in incubator at 18.55pm at 41C (19th Sept 2008) 

Read times: Treated samples read at 20.10pm (20th Sept 2008) ~ 25 hours 

 Control samples read at 20.30pm (20th Sept 2008) ~ 25 hours 

Sample ID Entero. counts MPN Entero. 

 Large Small 

T1 0 0 0 

T2 0 0 0 

T3 0 0 0 

T4 0 0 0 

T5 0 0 0 

T6 0 0 0 

T7 0 0 0 

T8 0 0 0 

T9 0 0 0 

C1 0 0 0 

C2 0 0 0 

C3 0 0 0 

C4 0 0 0 

C5 0 0 0 

C6 0 0 0 

C7 0 0 0 

C8 0 0 0 

C9 0 0 0 

DI water 0 0 0 

Additional notes: 

Samples also scored at 23.30pm; no positives (but this was 29 hours).
98 

(4) Vibrio cholera sample tests: 

Sample ID: Trial 3, T=0 

Date: 19th September 2008 (sample collection) 

Storage: Samples stored at 4C until processing (on September 20th 2008). 

Time: 16.00-17.40; processed Treated first (9 samples T1-T9), then control samples (9 

samples C1-C9). Distilled water used as control. 

Protocol: As per standard methods. 

Specific details: 100ml of each sample filtered onto 0.2um filters. Filters folded onto themselves 

in half, then half again. Placed in foil and frozen. Stored at -20OC until processing. Filters at 4OC 

until freezing on September 21st 2008.
99 

(4) Aerobic (culturable bacteria) sample tests: 

Sample ID: Trial 3, T=0 

Date: 19th September 2008 (sample collection) 

Storage: Samples stored at 4OC until processing (on September 20th 2008). 

Time: 11.40-14.40; processed Treated first (9 samples T1-T9), then control samples (9 

samples C1-C9). Distilled water used as control. 

Protocol: As per standard plate counting methods. Four plates used per sample. Four dilutions / 

volumes used; 25ml (A), 10ml (B), 1ml (C) and 0.1ml (D) of sample per plate. Dilutions 

prepared as follows ; for 1ml volume (C), 1.1ml of sample and 9.9ml of DI. For 0.1ml sample 

(D) use 1ml of sample (C) and 9ml of DI (1:10 dilution). Blackfield 0.45um filters placed on 

vacuum manifold and 25ml or 10ml samples slowly filtered onto filters. Filters placed in 47mm 

sterile plates, with pads containing (soaked in) 2ml of sterile marine broth. Plates placed upside 

down in humid incubator at 35C. Plates read at 48 hours (or longer times also). Scored for 

colonies; I note the colored versus clear colonies, also make note of fungi. Score mid-large 

colonies very tiny ones (which do not grow at longer incubations) do not score, but make note. 

Incubation times: Treated samples placed in incubator at 13.00 at 35OC (20th Sept 2008) 

 Control samples placed in incubator at 14.40 at 35OC (20th Sept 2008) 

Read times: Treated samples read at 14.05 (22nd Sept 2008) ~ 49 hours 

 Control samples read at 14.50pm (22nd Sept 2008) ~ 49 hours 

Sample 

Id 

counts counts Notes 

 25ml 10ml 1ml 0.1ml 

T1 ? (m) 10 (m) 2 (m) 0 mold 

T2 9 (m) 5 (m) 1 1 mold 

T3 ? (m) 9 (m) 6 0 mold 

T4 4 (m) 2 (m) 1 0 mold 

T5 ? 22 (m) 4 0 mold 

T6 3 (m) 4 (m) 0 0 mold 

T7 3 (m) 0 (m) 0 0 mold 

T8 23 (m) 10 1 0 mold 

T9 ? 2 0 0 

 7.11±6.31 

C1 51 28 5 0 

C2 ? (m) 17 4 0 

C3 30 17 (m) 3 0 mold 

C4 ? (m) 22 1 1 

C5 9 27 7 0 V.small25ml 

C6 tntc 88 15 1 

C7 ? 2 2 ? 0 0 

C8 97 24 2 
100 

C9 6 (m) 9 1 0 mold 

 34.1±32.2 

DI 

water 

0 0 0 0 

2

nd Read times: Treated samples read at 23.00 (22nd Sept 2008) ~ 58 hours 

 Control samples read at 23.30pm (22nd Sept 2008) ~ 58 hours 

Sample 

ID 

counts counts Notes 

 25ml 10ml 1ml 0.1ml 

T1 15 (m) 11 (m) 2 (m) 0 mold 

T2 11 (m) 6 (m) 1 1 mold 

T3 5 (m) 7 5 0 mold 

T4 6 (m) 3 (m) 1 0 mold 

T5 30 (m) 37 (m) 3 0 mold 

T6 5 (m) 4 (m) 0 0 mold 

T7 3 (m) 2 (m) 0 0 mold 

T8 44 (m) 12 1 0 mold 

T9 ? 3 (m) 0 0 (m) 

Mean 8.74±10.6 

C1 86 23 4 0 

C2 61 39 (m) 6 0 

C3 28 (m) 32 3 0 mold 

C4 26 ? 32 1 1 v.small-25/10ml 

C5 39 ? 38 10 0 v.small- 25/ml 

C6 tntc 87 17 1 

C7 ? 14 54 0 0 v.small-25ml 

C8 164 98 22 2 

C9 15 ? (m) 14 5 0 mold 

 46.3±26.9 

DI 

water 

0 0 0 0 
101 

3

rd Read times: Treated samples read at 09.30 (23rd Sept 2008) ~ 67 hours 

 Control samples read at 09.30 (23rd Sept 2008) ~ 67 hours 

Sample 

ID 

counts counts Notes 

 25ml 10ml 1ml 0.1ml 

T1 15 (12m) 11 (5m) 2 (1m) 0 mold 

T2 14 (8m) 4 (6m) 1 1 mold 

T3 ? (m) 9 (m) 6 0 mold 

T4 4 (m) 2 (m) 1 0 mold 

T5 28 (1m) 46 (3m) 4 (2m) mold 

T6 4 (1m) 3 (4m) 0 0 mold 

T7 3 (12m) 0 (5m) 0 0 mold 

T8 34 (3m) 12 (2m) 1 0 mold 

T9 ? 2 (2m) 0 0 (1m) 

C1 46? 28 4 (1m) 0 

C2 49 (4m) 18 4 0 

C3 31 (2m) 17 (1m) 3 0 mold 

C4 23 (4m) 17 (1m) 2 1 

C5 33 28 12 0 V.small- 25ml 

C6 tntc 80 (1m) 19 2 

C7 ? 43 46 ? 0 0 

C8 152 (1m) 99 25 2 

C9 13 (1m) 9 9 1 mold 

DI 

water 

0 0 0 0 
102 

4

th

 Read times: Treated samples read at 17.20 (23rd Sept 2008) ~ 75 hours 

 Control samples read at 17.50 (23rd Sept 2008) ~ 75 hours 

 (NB not score prior negative plates) 

Sample 

ID 

counts counts Notes 

 25ml 10ml 1ml 0.1ml 

T1 16 (9m) 12 (5m) 2 (1m) 0 mold 

T2 9 (5m) 5 (5m) 1 1 mold 

T3 4 (3m) 7 4 mold 

T4 6 (3m) 2 (2m) 1 0 mold 

T5 24 (1m) 25 (3m) 4 mold 

T6 5 (1m) 3 (4m) 0 0 mold 

T7 4 (13m) 0 (6m) 0 mold 

T8 36 (3m) 12 (2m) 2 mold 

T9 ? 2 (2m) 0 0 (1m) 

C1 44? 19 4 0 

C2 ? (4m) 24 4 0 

C3 33 (2m) 19 (1m) 3 0 mold 

C4 20 (4m) 20 (1m) 2 3 

C5 33 28 12 0 V.small- 25ml 

C6 tntc 81 (1m) 14 (5m) 2 

C7 ? 27 12 ? 0 0 

C8 152 (1m) 99 25 2 

C9 9 8 (1m) 11 1 mold 

DI 

water 

0 0 0 0 
103 
104 

Appendix D. 

QA/QC Protocols Related to Shipboard Trials of the Hyde Marine ‘Guardian’ 

BWTS, April – October 2008.

The testing and quality assurance procedures described in this document are based on 

Standard Operating Procedures (SOP) of the University of Maryland Center for Environmental 

Science, Guidelines of the U.S. Environmental Protection Agency (USEPA) and the American 

Society for Testing and Materials (ASTM). 

Shipboard testing of the Hyde Marine Guardian Ballast Water Treatment System 

(BWTS) was conducted by a team led by Dr. David Wright, Professor of Environmental 

Toxicology at the University of Maryland Center for Environmental Science (UMCES), 

Chesapeake Biological Laboratory, Solomons Maryland 20688, U.S.A. Dr. Wright has more 

than 20 years research experience with Aquatic Nuisance Species and has conducted more than 

15 shipboard trials of a variety of BWTS. The test team and associates for IMO G-8 trials of the 

Hyde Marine ‘Guardian’ BWTS is summarized in table 1, below: 

Table 1. Shipboard testing team and associates for G-8 shipboard trial of the Hyde Marine 

‘Guardian’ BWTS, April October 2008. 

Personnel/Title Role 

David.A. Wright, Professor UMCES. Project Director, 

Dr. Rodger. Dawson, UMCES (ret.). Environmental 

Research Services. 

Consultant Marine Chemist, water 

quality, nutrient analysis, logistics 

Dr. Carys Mitchelmore. Associate Professor, 

UMCES 

Microbiologist, Toxicologist 

responsible for WET testing 

(zooplankton) 

Jonathon Bearr, PhD student, UMCES Assistant for microbiology and 

WET testing (phytoplankton 

Marcia Olson MS. Morgan State University, 

Environmental Research Laboratory, Lusby, MD. 

Plankton microscopy, data entry, 

QA/QC 

Celia Orano-Dawson MS. MD Department of 

Natural Resources, Harmful Algal Bloom 

Laboratory 

Plankton microscopy, data entry. 

WET testing (phytoplankton) 

UMCES Analytical Services Division. Dr. Walter 

R. Boynton, Professor and Principal Investigator; 

Carl F. Zimmerman, Advanced Faculty Research 

Assistant; Carolyn Keefe, Advanced Faculty 

Research Assistant; Kathy Wood, Advanced 

Faculty Research Assistant; Nancy Kaumeyer, 

Advanced Faculty Research Assistant; Adriene 

Capers, Senior Faculty Research Assistant; Maggie 

Weir, Advanced Faculty Research Assistant 

Nutrient analyses, QA/QC 
105 

Objectives and Background. 

 The objective of the shipboard trials was to measure the biological effectiveness of 

shipboard filtration/separation and ultraviolet radiation equipment as a ballast water treatment 

method. The sampling protocol was designed to satisfy IMO G8 requirements, with the objective 

of comparing biological endpoints to those published under regulation D-2 of the IMO Ballast 

Water Convention (IMO 2004). Investigators were also aware of other standards set by U.S. 

federal and state regulatory bodies described below (table 2). 

In order to comply with IMO G-8 guidelines for shipboard testing, three trials were 

conducted aboard the Princess Cruise Lines ship M/V Coral Princess between April and October 

2008. Samples of treated and untreated ballast water were taken from a customized sampling 

port mounted downstream of the BWTS in the machinery space of the vessel. These samples 

were compared in order to test the efficacy of the system to IMO and other standards (table 2) 

standards under normal working conditions. The BWTS system consists of a primary disc filter 

manufactured by Arkal Inc., Tel Aviv, Israel mounted in series with a medium pressure UV 

irradiation system rated by the manufacturer/vendor at 200 mJ cm2 for treatment of ballast water 

at flow rates up to 250 m3 h

-1. Trials took place during the vessel’s regular spring schedule in the 

Caribbean Sea, the summer schedule in the N.W. Pacific Ocean between Whittier, Alaska and 

Vancouver, Canada, and during the repositioning cruise from the western Pacific to the vessel’s 

winter base in Fort Lauderdale, Florida. Trials consisted of determination of water quality 

parameters and a comparison of biological endpoints in treated and untreated ballast water 

samples, with reference to both IMO G8 and the U.S. Coast Guard Shipboard Technology 

Evaluation Program (STEP). Sampling procedures and endpoint determinations followed IMO 

G8 guidelines for shipboard trials and the exercise was designed to supplement land-based trials 

being conducted concomitantly at NIOZ, Texel, Netherlands to determine the efficacy of the 

BWT system under varying water quality conditions. Shipboard trials were designed to 

document system performance under normal seagoing conditions and under different 

geographical and seasonal conditions, with the objective of determining the degree of 

compliance with IMO and STEP requirements. Whole Effluent Toxicity (WET) tests were also 

conducted as part of the third and last trial to determine whether any significant chemical 

changes in ballast water after exposure to UV irradiation, which resulted in subsequent residual 

toxicity. 

Table 2. . 2008 IMO and U.S. Ballast Water Treatment Standards.

IMO Regulation 

D-2 and 

Transport 

Canada 

2008 Ballast Water 

Management Act 

Section 1101 (f)i

2008 California 

Standard

Washington 

Administrative Code 

222-170 

Management 

approach 

Exchange moving

towards treatment 

only 

Exchange moving 

towards treatment 

only 

Exchange moving towards 

treatment only 

Exchange or treatment 

Standard: Proposed Proposed Recommended Interim Adopted Interim: 

1) Organisms 

greater than 50 

<10 viable 

organisms per 

< 0.1 living 

organisms per cubic 

No detectable living 

organisms 

Technology to 

inactivate or remove 
106 

Endpoints Measured. 

The primary measurements in 2008 shipboard trials were: 

• Plankton Viability/Growth Potential (Plankton Abundance) 

• Microbial activity as determined by plate counts and MPN 

• Water Quality Measurements, e.g. Salinity, Temperature, DO, TSS, POC, 

Nitrate 

 To assess biological effectiveness, the following protocols were employed in order to 

comply with IMO G-8 guidelines and produce results that were compatible with IMO D-2 

regulations (IMO 2004). Procedures were modified in certain cases in order to account for 

problems encountered aboard a working vessel. For example, logistics dictated that it was not 

always possible to ballast the vessel in port, and that the challenge water was not ‘optimal’ in 

terms of organism densities, bacterial flora and TSS. The following procedures describe both 

sampling strategies and analytical techniques employed during shipboard trials. Most biological 

endpoint analyses began aboard the vessel, an essential requirement where rapid assessment of 

live plankton was required. However, in two out of three trials, samples for bacterial analysis 

microns in 

minimum 

dimension: 

 

2) Organisms 10-

50 microns in 

minimum 

dimension: 

 

3) Organisms less 

than 10 microns in 

minimum 

dimension: 

 

4) Escherichia coli 

 

5) Intestinal 

Enterococci 

 

6) Toxicogenic 

Vibrio cholerae 

(O1& O139) 

 

 

 

cubic meter 

 

 

<10 viable 

organisms per ml 

No standards 

 

 

< 250 cfu/100 ml 

 

<100 cfu/100 ml 

 

 

<1 cfu/100 ml 

 

<1 cfu/gram of 

wet zooplankton 

samples 

meter 

 

< 0.1 living 

organisms per ml 

 

 

No standard 

 

 

 

<126 cfu/100 ml 

 

< 33 cfu/100 ml 

 

 

<1 cfu/100 ml 

 

<1 cfu/gram of wet 

weight of zoological 

samples; 

 

 

 

 

 

 

<10

-2 

living organisms per 

ml 

 

 

< 10

3

 cfu bacteria/100 ml 

 

 

<126 cfu/100 ml 

 

<33 cfu/100 ml 

 

 

<1 cfu/100 ml 

 

< 1 cfu/gram of wet 

zoological samples 

 

<10

4

 viruses/100 ml 

 

Final standards – no 

discharge of living 

organisms 

95% zooplankton 

 

99% bacteria & 

phytoplankton 
107 

were preserved pending analysis using internationally recognized standards/chain of custody 

procedures. While standard water quality parameters were measured aboard ship, nutrient 

analyses were conducted by the University of Maryland Analytical Services Division, which has 

been an independent, nationally and internationally recognized analytical resource for over thirty 

years. 

Biological Endpoints

Figure 1 depicts the basic sampling strategy employed in the 2008 shipboard trials. As a 

measure of accuracy a 3 x 3 sampling matrix was employed, with U1, U2 and U3 representing 

untreated ballast water sampled in triplicate at the beginning, middle and end of a de-ballasting 

cycle, and T1, T2 and T3 representing treated samples taken at the beginning, middle and end of 

a de-ballasting cycle. 

 

>4day residence 

time 

UV UV UV 

UV 

Sea Chest filter 

400L 

Bacteria 

10-50/

200um

Zooplankton 

Phytoplankton 

500L 500L 500L 500L 

Bacteria 

10-50um 

200um

Zooplankton 

Phytoplankton 

Samples 

taken (t=0) 

24hr grow-out 

period samples 

Bacteria Phytoplankton Bacteria Phytoplankton 

48hr grow-out 

period samples 

Bacteria Bacteria 

T1 T2 T3 U1 U2 U3 

Filter 

Mesocosm 

Sample 

Ballast Tank 

(count ASAP) 

500L 500L 

72hr grow-out 

Bacteria period samples Bacteria 

(count ASAP) 
108 

Sampling Strategy.

 Triplicate samples were taken at the beginning, middle and end of a collection cycle. It 

must be emphasized that treatment by the Hyde ‘Guardian’ system is only complete following a 

second ‘pass’ through the UV system at the time on discharge from the ship. Therefore, 

definitively treated samples can only be collected and examined during de-ballasting. Related to 

this is the fact that shipboard testing cannot be subject to the same degree of control as landbased testing, e.g. the test team cannot start with clean ‘rinsed’ tanks. It is, therefore of particular 

importance to define the nature of the intake (challenge) water as well as discharged water, in 

order to identify important differences between the biology and chemistry of water at the time of 

uptake, and that discharged from the vessel. 

Biological Endpoints.

Zooplankton.

Samples for zooplankton counting were taken from a sampling port located downstream 

from the Ballast Water Treatment System (BWTS) in the machinery space of the vessel. At that 

location samples were filtered through 30cm. nylon plankton nets consisting of 20μm mesh 

(nominal 50μm mesh nets can allow the passage of >50μm organisms). During filtration, each 

net was submerged under the surface of a 125L plastic tub to soften the impact of the filtration 

procedure on the planktonic organisms. The 1L plastic bottle that forms the `cod-end' of the net 

also had 20μm mesh `windows' to facilitate the filtration process. Separate nets were used to 

sample from treated and untreated samples, and nets were rinsed with hot tap-water between 

sampling cycles. They were also examined for tears, leaks and imperfections and any repairs 

made. In the machinery space the contents of each mesocosm were filtered and concentrated to a 

volume suitable for manual transport up to the cabin space where samples were turned over to 

microscopists for examination. On receiving samples from the collection team, microscopists 

further concentrated samples through 20μm mesh filters to produce volumes suitable for 

microscopy, usually 10-20ml. Concentrated samples were transferred via Stempel pipets to 

counting wheels mounted on compound microscope stages for microscopical examination. Using 

these procedures, it was possible to begin microscopic examination for plankton density/activity 

within one hour of collection. As a measure of precision, a majority of plankton samples are split 

and processed as duplicates to indicate the amount of variability sample processing may add to 

the results. Counts are made of at least 100 individuals from at least five taxonomic groups. 

Sample-sharing between microscopists was practiced throughout these trials. Organism 

sizes were recorded as the minimum dimension of the cell/organism, using calibrated reticule 

eyepieces mounted in the eyepiece lens of each microscope. However, a particularly useful 

means of cross-calibration involved the ‘seeding’ of counting slides with glass beads of specific 

sizes (10 μm, 20 μm, 50 μm, 100 μm, 200 μm). These glass beads are certified by the U.S. 

National Institute for Standards as calibration standards for particle sizing technology. Their use 

as a supplement to microscopy provides an excellent cross-calibration tool among microscopists 

and has been referenced in recent shipboard studies (Wright et al. 2009). In order to address 

precision associated with counting a majority of plankton samples are split and processed as 

duplicates to indicate the amount of variability sample processing may add to the results. 
109 

Precision of nutrient analyses is addressed through regular running of standards between samples 

batches to ensure consistent equipment operation and calibration. 

 

 QA/QC considerations for shipboard sampling include rigorous washing of the sampling 

nets with freshwater and examination of nets and other sampling devices containers for 

flaws/leaks between sampling runs. Sampling aboard a working vessel involves pumping water 

through numerous pipes/tank segments, which may involve dead spaces. Unlike land-based 

operations, extensive rinsing of tanks/pipes is not possible between sampling episodes. 

Therefore, to the extent possible under the constraints of the sampling regime, sufficient time 

should be given to flushing water through the ballasting system of the shipping before sampling 

commences. An important consideration in this respect is that the collection of treated samples 

always precedes the collection of untreated samples. This minimizes the potential of carry-over 

of live individuals (i.e. Type 1 error) that might have been trapped in dead-spaces. Conversely, 

carry-over of dead organisms (Type 2 error) through a ‘treated first’ protocol is likely to 

insignificant in the context of current published standards. Given the rigor of current standards, 

type 1 errors should be avoided at all costs. These and other aspects of shipboard sampling and 

testing are discussed by Wright (2007). 

Phytoplankton.

Samples for phytoplankton counting/assessment consisted of 1L of unfiltered water 

collected from the sampling port. These samples were passed through a 200μm mesh net to 

remove any large predatory zooplankton. Experience with shipboard trials has indicated that 

grazing can be a factor, even within the context of ballast tank residence time and sample holding 

time prior to examination. Each sample was concentrated to a volume <10ml. for 

examination/grow-out, using a 10μm Nitex screen. This enabled unusually low densities of 

phytoplankton to be counted with greater precision, as it resulted in greater cell densities on the 

counting slide. Typically counts were made of >200 squares of a 1000 square counting grid,. 

Determination of living phytoplankton was be made on the basis of (a) chlorophyll a analysis, (b) 

vital staining techniques and (c) cell counts before and after grow-out. 

Use of grow-out technique.

Live-dead status of phytoplankton for the determination of ballast water treatment 

efficacy remains problematic to the extent that, while some taxonomic groups, such as 

dinoflagellates, are clearly motile, many have vegetative stages that are immobile. The growth 

potential of non-motile forms can only be assessed by a variety of methods, including 

microscopic examination of chloroplast integrity, use of vital stain(s) and cell counts (of at least 

dominant groups) following a grow-out period under optimal growth conditions. Other, more 

sophisticated techniques, such as flow cytometry are also available, but do not present a realistic 

option for shipboard trials. For shipboard trials, it was determined that growth potential 

represents a robust indicator of phytoplankton viability within the constraints of shipboard 

analyses, although this endpoint remains difficult to quantify in terms of regulatory endpoints. 

The grow-out period for phytoplankton usually consisted of a 24h (or 48h) period of irradiation 

under fluorescent lighting and non-limiting nutrient conditions through the addition of f/2 growth 

medium. This medium had the following constituents: FeCl3.6H2O, 1.45 mg L-1; KNO3 200 mg 

L

-1 ; NaH2PO4.2H20, 34.8 mg L-1; H3BO3, 34.2 mg L-1; EDTA.2H20, 30 mg L-1; MnCl2.4H20, 4.3 

mg L-1; CoCl2.6H20, 0.13 mg L-1; ZnSO4

.

7H20, 0.364 mg L-1; H2SeO3, 0.00173 mg L-1; 
110 

Thiamine HCl, 0.1 mg L-1; Vit B12, 0.001 mg L-1; Biotin, 0.002 mg L-1. 

An effort was made to identify and count as many taxonomic groups as possible, with 

particular focus on dominant taxa, although individual cell counts before and after grow-out are 

still subject to different interpretation. While ‘live’ counts depended on such parameters as 

chloroplast integrity, motility and neutral red staining (useful for several but not all taxonomic 

groups), numbers of viable cells determined in this way often declined rather than increased 

following ‘grow-out’. Under such circumstances it may be concluded that the population as a 

whole was non-viable, i.e. incapable of growth. 

Other determinants of phytoplankton viability.

Microscopic examination of cell/chloroplast integrity was supplemented by the use of 

Neutral Red as a vital stain, which proved a useful stain for many, but not all taxonomic groups.. 

Unstained samples examined soon after collection were compared with ‘splits’ of the same 

sample that were stained with Neutral Red. Samples following a grow-out period were similarly 

examined, with and without Neutral Red. Following initial examination of phytoplankton (before 

and after grow-out) to determine their general appearance, observations of chloroplast integrity 

and the activity of motile forms, samples were preserved in Lugol's Solution for more intensive 

taxonomy and determination of cell sizes. 

 Chlorophyll a 

Phytoplankton growth may be conveniently assessed by measuring in vivo chlorophyll a

concentration before and after the grow-out period. While this represents a useful integrative 

determination of the status of the phytoplankton community as a whole, it does not provide 

information on individual taxonomic groups of phytoplankton that might have quite different 

characteristics in terms of size, shape, doubling time (growth rate) etc. Also, chlorophyll a data 

cannot be interpreted in terms of published standards, which are based on cell numbers. While 

good quantitative relations can be drawn between chlorophyll a and cell numbers in 

monocultures (Wright et al. 2006), natural phytoplankton communities may include taxa which 

differ in volume by more than two orders of magnitude. Under such circumstances chlorophyll a 

levels would be biased towards larger cells. We, nevertheless find that useful information can be 

obtained from chlorophyll a analyses, particularly with respect to growth capacity. While two 

analytical methodologies are described in UMCES standard operating procedures (see Appendix 

E), the method described by Welschmeyer was used for these trials. For this study, chlorophyll a

analyses were made aboard the vessel, using a Turner Designs Aquafluor fluorimeter, Model # 

8000-001 calibrated aboard the vessel using a solid standard, which in turn was calibrated 

against a chlorophyll a standard at the UMCES Analytical Services Division. 

Bacteria. 

 250ml. samples for bacterial analyses were be taken directly from the unfiltered discharge 

from the sampling port, and stored at temperatures just above freezing (1-4OC) prior to and 

during transport to the University of Maryland Center for Environmental Science, Chesapeake 

Biological Laboratory, although in Trial 3 some culturing/counting was carried out in cabin space 

aboard the vessel. Bacterial culture formed the basis of bacterial endpoints that were used to test 

the bactericidal effectiveness of the BWT system aboard this vessel. The following bactericidal 

endpoints were employed: 

• Cultural bacteria. For total culturable heterotrophic bacteria, samples were 
111 

serially diluted three to four orders of magnitude. 20 ml. full strength and diluted 

samples were filtered through 0.45 um black field filters to isolate the bacteria on 

the filter. The filter was then transferred to a Petri dish that contained a sterile 

absorbent pad soaked in 2ml of autoclaved marine broth (DifcoTM Marine Broth 

2216). The filter paper was placed so as to expose the side inoculated with 

bacteria. The Petri dishes were then closed and placed upside down in a 25 ±0.5 

oC incubator. After 72 hours, the dishes were removed and colony forming units 

were enumerated using a colony counter. 

It is emphasized that only approximately 1% of marine bacteria will culture successfully, 

and that this figure may vary according to geographical area. This, therefore, represents only an 

approximate measure of the efficacy of BWT technology, and is somewhat variable according to 

geographical location. 

Primary focus was on taxonomic groups specified in published IMO (2004) D-2 regulations: 

colony-forming units of E.Coli, Enterococcus and Vibrio Cholera (with specific emphasis on 

virulent serotypes). Fluorescence-based techniques (IDEXX Laboatories.) were employed to 

quantify coliforms, E. Coli and Enterococcus in treated and untreated ballast water. 

Samples were diluted an order of magnitude with sterile deionized water for the IDEXX 

protocols. The established detection range for this technique is 10 – 24,190 cfu / 100 mL of sea 

water sample. 

• Total coliform and E.coli levels were enumerated using IDEXX Colilert-18 

Quantitray/2000 test kits (IDEXX Laboratories Inc., ME), which are US EPA 

approved standard methods for E.coli analysis. Samples were diluted an order of 

magnitude with sterile deionized water , added with the detection media into the 

Quantitray and sealed and incubated at 37 oC for 18 hours. The analytical method 

is a multi-well Most Probable Number (MPN) method involving exposure to a 

mixture of o-nitrophenol-β-D-galactopyranoside (ONPG) and 4-methylumbelliferyl-β-D-gluronide (MUG). Coliform bacteria produce a yellow color 

resulting from the formation of β-galactosidase. In addition to the yellow color, 

E.coli also produce fluorescence (under a 6 watt, 365nm UV light) as a result of 

the action of β-glucuronidase. Yellow and fluorescent squares were counted and 

converted to cfu/100ml. 

• Counting of a variety of Enterococci sps. including faecalis, faecium, avium and 

gallinarum is achieved using a MUG-based nutrient-indicator and a fluorescent 

end-product. Like the coliform method, the IDEXX Laboratories Inc. Enterolert 

Quantitray 2000 (ASTM method # D6503-99) relies on a MPN endpoint,

following 24h incubation at 41oC. A blue fluorescence signifies a positive result 

for Enterococci. 

Quantification of viable Vibrio cells was facilitated by the use of Polymerase Chain 

Reaction amplification techniques on refrigerated samples transported to the University of 

Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Molecular 

Biology Laboratory, directed by Dr. Carys Mitchelmore. 
112 

Water Quality and Nutrient Analyses.

Only chlorophyll a and standard water quality criteria were determined aboard the vessel. 

As previously reported chlorophyll a analyses were cross-calibrated via chlorophyll a standard 

measurements conducted both aboard ship and at the UMCES Analytical Services Division, 

Solomons MD. pH, dissolved oxygen (DO), salinity, temperature were all measured using a 

Yellow Springs Instruments ‘Multimeter’, which was calibrated against laboratory-based 

instrumentation before and after each trial, with pH/salinity re-calibrations against known 

standards between sampling batches. Spare membranes for the DO meter were available ‘in the 

field’. 

 Apart from chlorophyll a other nutrient analyses were performed by the UMCES 

Analytical Services Division, Chesapeake Biological Laboratory, Solomons, MD, U.S.A. 

Standard Operating Procedures from this Laboratory have been published as comprehensive 

document, available online at http://cbl.umces.edu.nasl/Methods (Appendix E), and only those 

analyses relevant to the 2008 shipboards trials are summarized below 

Total Suspended Solids (TSS).

 Total Suspended Solids (TSS) and Particulate Organic Carbon (POC) were measured 

using GF/C filters that were initially dried to (102-105OC), pre-weighed (gm.) to 4 decimal 

places and brought aboard the vessel in individual sealed Petri dishes. Filter volumes varied 

between 500ml. -1-5 L. Filters were rinsed with freshwater to remove salt and brought back to 

the laboratory over ice. Each filter was then dried at 60OC for 24h and re-weighed (APHA 1975, 

USEPA 1979a). Total Suspended Solids (TSS) were calculated from the difference in between 

this weight and that of the pre-weighed filter. 

For POC measurement filters were then combusted in a 500OC oven overnight, stored in a 

desiccators and then re-weighed. POC was calculated as the loss in weight relative to the TSS 

weight. 

Both TSS and POC are expressed as mg L-1. 

Dissolved Organic Carbon (DOC).

The Shimadzu TOC-5000 uses a high temperature combustion method to analyze aqueous 

samples for TIC, TOC and non-purgeable organic carbon. Samples for DOC analysis are treated 

with hydrochloric acid and sparged with ultra pure carrier grade air to drive off inorganic carbon. 

High temperature combustion (680 °C) on a catalyst bed of platinum-coated alumina balls breaks 

down organic carbon into carbon dioxide (CO2). The CO2 is carried by ultra pure air to a nondispersive infrared detector (NDIR) where CO2 is detected (Sugimura and Suzuki 1988). 

Nitrite + Nitrate

Filtered samples are passed through a granulated copper-cadmium column to reduce nitrate to 

nitrite. The nitrite (originally present plus reduced nitrate) then is determined by diazotizing with 

sulfanilamide and coupling with N-1- naphthylethylenediamine dihydrochloride to from a 

colored azo dye. Nitrate concentration is obtained by subtracting the corresponding nitrite value 

from the nitrite + nitrate concentration. (Technicon 1977, USEPA 1979b). 
113 

Orthophosphate

Ammonium molybdate and antimony potassium tartrate react in an acid medium with dilute 

solutions of phosphorus to form an antimony-phospho-molybdate complex which is reduced to 

an intensely blue-colored complex by ascorbic acid. Color is proportional to phosphorus 

concentration. (Technicon 1973, USEPA 1979c). 

Ammonium (analyzed) in conjunction with Whole Effluent Testing (WET) only (Trial 3). 

Determination of ammonium is by the Berthelot Reaction in which a blue-colored 

compound similar to indophenol forms when a solution of ammonium salt is added to sodium 

phenoxide, followed by the addition of sodium hypochlorite. The addition of a potassium 

sodium tartrate and sodium citrate solution prevents precipitation of hydroxides of calcium and 

magnesium (Technicon 1986, Kerouel and Aminot 1987). 

University of Maryland Analytical Services Division - Statement of Quality 

Assurance/Quality Control Procedures.

The analytical services division of the University of Maryland Center for Environmental 

Science (UMCES) is a nationally and internationally recognized resource for aquatic chemical 

analyses, which for over thirty years has provided this service to numerous State and Federal 

agencies and other clients. Standard Operating Procedures (SOP) used by this group are available 

online at http://cbl.umces.edu.nasl/Methods and are reproduced here as Appendix E. A 

representative summary of QA/QC components practices by this laboratory is given below. 

A constant consideration of the UMCES Analytical Services laboratory is assuring the 

quality of data generated by the procedures presented in this manual. Further, indication of data 

quality is accomplished by analyzing duplicates, spikes, standards-as-samples, standard reference 

materials and participating in cross-calibration exercises. 

Laboratory Duplicates 

Approximately 5% of the total number of samples analyzed consist of laboratory 

duplicates. For dissolved analytes, after a sample is analyzed, the same sample container is 

placed farther along in the automatic sampler and re-analyzed. The mean of the two values is 

reported as the concentration for that sample. If a difference of >10% is observed between 

replicates, then all of the replicates for that particular analytical run are carefully reviewed. If 

only one of the duplicate pairs is in question, then only that sample is re-analyzed. If all show a 

similar trend, then instrumentation/reagent problems are suspected and the analytical run is 

halted until such time as the problem is resolved. This procedure is practiced for all dissolved 

analytes that are not consumed completely in the analytical procedure. For those that are 

completely consumed and for particulate analytes, duplicate samples constitute actual duplicate 

samples collected in the field and analyzed in the same analytical run. 

Values for each duplicate analyzed are recorded in a separate QA/QC data file along with 

the sample number, sample collection date and analysis date. The mean concentration and 

standard deviation of the replicates are calculated in this data file. In the case of particulate 

carbon and nitrogen, total suspended solids and chlorophyll a, 10 percent of the total number of 
114 

samples are analyzed as duplicates. This generates sufficient quality assurance data to 

compensate for the omission of laboratory spikes for these non-aqueous samples. 

Laboratory duplicates serve as an indicator of instrument stability, consistency in 

laboratory sample preparation and analysis, as well as an estimate of field proficiency. 

Cross Calibration Exercises

UMCES Analytical Services Division has participated in many cross calibration exercises. 

Participation in such programs is an excellent means of determining accuracy of results. 

Examples of such cross calibration exercises include the Chesapeake Bay Quarterly Split Sample 

Program, US EPA Method Validation Studies and International Council for the Exploration of 

the Sea Intercomparison Exercise for Nutrients in Sea Water. 

Sample Custody

Upon arrival at the laboratory, samples are counted, observed for potential problems 

(melting, broken containers, etc.) and placed in a freezer until analysis. Sample information and 

date of arrival are recorded on a log sheet. 

Instrument Maintenance

Analytical instruments are maintained on a regular basis and records are kept of hours of 

operation, scheduled maintenance, pump tube changes, etc. A critical spare parts inventory is 

maintained for each instrument. Instrument down-time is minimized by troubleshooting 

instrument problems telephonically with manufacturers and service representatives. Spare parts 

can be received within 24 hours via next-day air service.

Statement on Instrument Comparability

The Nutrient Analytical Services Laboratory develops a data quality maintenance 

program for each analyte whenever new instrumentation is acquired. It is the policy of the 

Nutrient Analytical Services Laboratory to report any data from new instrumentation only after 

thorough and satisfactory side-by-side comparisons with existing instrumentation are preformed. 

No predetermined number of data pairs are used to make the assessment on data comparability 

between new and existing methodology. Even in the case of instrumentation with similar 

methods of detection (i.e., automated colorimetric), no specific number of data pairs is used. 

Comparability at low and high concentrations, salinity and other possible matrix interferences, 

sensitivity and precision are all factors in determining the number of pairs that must be addressed 

before bringing an instrument on-line and in determining instrument comparability. 

The analyst who performs these comparisons should be experienced, open-minded and 

impartial. This person can give an evaluation of ease of instrument operation and a very 

important general statement of comparability. This statement on comparability must then be 

substantiated via statistical analysis of the data. As previously mentioned, these data must 

encompass the entire concentration range, matrix interferences, percent recovery, results of 

standard reference material analyses, etc. The data interpretation must support comparability. 

The analyst and laboratory QA/QC officer must concur and finally, some sort of presentation 
115 

(written or verbal) must be given to the contractor explaining what procedure was followed and 

the results that were obtained to bring this instrument on-line. 

Temperature Logs.

Temperature logs of freezers, refrigerators and drying ovens are kept on a monthly basis. 

Thermometers used in this equipment are calibrated against a certified NBS thermometer. 

Laboratory Spikes 

Approximately 5% of the total number of samples analyzed consist of laboratory spikes. 

A spike is prepared by adding a known volume of standard to a known volume of pre-analyzed 

sample. We routinely add enough concentrated standard to provide a significant response on our 

instruments that is distinguishable from the original concentration of the sample. This 

concentrated standard is used to minimize any possible change in sample matrix by the addition 

of spike. The spiked sample is analyzed and its expected concentration calculated as the sum of 

the original concentration and the spike concentration, normalized for the constituent volumes. A 

comparison is made between the actual value and the expected value. These concentrations 

(original, expected and actual) are recorded in a separate QA/QC data file along with sample 

number, sample collection date, analysis date and the amount of spike added. In the case of 

particulate phosphorus, the volume filtered is not used in the calculation to determine percentage 

recovery. If a value of >115% or <85% is observed for percentage recovery of the spike, then all 

of the spikes for that particular analytical run are carefully reviewed. If only one of the spikes is 

in question, then only that sample is re-analyzed. If all show poor recovery, then 

instrumentation/reagent problems are suspected and the analytical run is halted until such time 

that the problem is resolved. This procedure is adhered to for all dissolved analytes and for 

particulate phosphorus and biogenic silica. 

Documentation of Slopes

A running record of the slopes of the standard curves (the so-called "F," "S" and "K" 

factors) is maintained for each analysis. Random up and down movement within a predetermined 

range as a function of time indicates the analysis is under control. Consistent upward or 

downward movement of these factors indicates the analysis is out of control and requires 

immediate attention. 

Standard as Sample

Standards are analyzed as samples throughout the analytical run. This is an excellent 

means of evaluating instrument performance during the course of an analytical run. Standards are 

analyzed every 12 - 20 samples, depending on the instrument and analyte. 

Limits of Detection.

Limits of detection, the lowest concentration of an analyte that the analytical procedure 

can reliably detect, have been established for all parameters routinely measured by Nutrient 

Analytical Services. The limit of detection is 3 times the standard deviation of a minimum of 7 

replicates of a single low concentration sample. 
116 

Standard Reference Materials

Particulate Carbon, Nitrogen and Phosphorus: BCSS-1 is a marine sediment reference 

material prepared by the National Research Council of Canada. It is certified by the Council for 

carbon content, gives a non-certified range of results for phosphorus, but no information for 

nitrogen. We have analyzed this sediment for many years and maintain a substantial database for 

nitrogen and phosphorus, as well as carbon values. We analyze this sediment quarterly and 

compare these results to the certified value, non-certified range of values and our historical 

values. Dissolved Analytes and Hardness: Standard reference materials for ammonium, nitrite + 

nitrate, nitrite, orthophosphate, dissolved nitrogen, dissolved phosphorus, dissolved organic 

carbon, sulfate and chloride and hardness are supplied by SPEX, a US EPA certified company. 

The samples arrive in ampules and we prepare final concentrations to approximate typical 

estuarine concentrations. The samples are then placed in pre-cleaned poly bottles, frozen and 

analyzed on a quarterly basis.The analysis of these frozen standard reference materials as a 

function of time also provides data on the effect of our preservation technique (freezing) on the 

integrity of the concentration of samples. The US EPA recommends a holding time of 28 days 

for many of the parameters we routinely analyze. 

Organic compounds are included with each dissolved nitrogen and dissolved phosphorus 

digestion to determine the completeness of the digestion procedure. Glutamic acid and 

glycerophosphate are used as the N and P sources, respectively. 

 

Data Management.

In general, Standard Methods (APHA, 1998), section 9020B.10-11, guided the 

procedures for documentation, recordkeeping and data handling. Data were analyzed using 

desktop and software such as Microsoft Excel, QuattroPro and system software such as Statistics 

and Analysis System (SAS). 

 The project director was present throughout every aspect of shipboard trials, a total of 

thirty-one days at sea over three trials, and was an integral member of the microscopy team. The 

responsibility for maintenance of quality for a project lies with every member of the project. All 

project personnel aid in identifying perceived problems that may affect quality and report such 

problems to the project director. A team approach is adopted in order to correct observed 

deviations for which there is an obvious solution during the routine implementation of the 

sampling procedures. During data validation and assessment, deviations in the project data are 

corrected as appropriate. If data are determined to be invalid, they are denoted as such and not 

included in the data set. The project director is responsible for assigning/conducting QA 

assessments and is responsible for correcting any deviations observed when accuracy, precision, 

completeness, and comparability are not to specifications, and for inspection of the final reports 

to confirm that the methods, procedures, and observations are accurately and completely 

described, and that the reported results accurately and completely reflect the raw data of the 

studies. 

 Responsible persons for zooplankton, phytoplankton and microbial assessments provide 

preliminary reports on data derived from the first day of testing to the project director to allow 

the test team to correct any readily apparent problems. They also prepare a report to the project 

manager within one month of the period of ship trials. The report includes but is not limited to 

the following: 
117 

1. An introduction stating an overview of the procedures performed; 

2. Methods/SOPs followed for the analysis; 

3. Brief analytical assessment of the results highlighting pertinent information about the 

data (i.e. outliers, deviations, unexpected results etc); 

4. Compliance with SOPs and chain-of-custody procedures (see following sample form) 
118 

UMCES, Chesapeake Biological Laboratory 

 

FIELD CHAIN-OF-CUSTODY - Sample Custodian

Project Title: Coral Princess BW Testing Trial, _________2008 #:________ Time_________ 

Sample Type: 

1 = Preserved Zooplankton 

2 = Live Phytoplankton 

3 = Preserved Phytoplankton 

4 = Microbial (Macrophage) 

5 = Microbial (TPC) 

1. Collected by:___________________________ 

 Date:_____________ Time:_______________ 

 Relinquished by:_______________________ 

 Date:_____________ Time:_______________ 

2. Received by:_________________________ 

 Date:_____________ Time:_____________ 

 Relinquished by:_____________________ 

 Date:_____________ Time:_______________ 

3. Received by:_________________________ 

 Date:_____________ Time:_______________ 

 Relinquished by:______________________ 

 Date:_____________ Time:_______________ 
119 

Literature Cited. 

American Public Health Association APHA. 1975. Method 208D. Total Nonfilterable Residue 

Dried at 103-105 °C (Total Suspended Matter) in Standard Methods for the Examination of 

Water and Wastewater, 14th Edition. American Public Health Association. Washington, D.C. 

1193pp. 

American Public Health Association (APHA), American Water Works Association, Water 

Environment Federation. 1998. Standard Methods for the examination of water and wastewater 

(20th edition). Clesceri, LS., A.E. Greenberg and A.D. Eaton, eds. American Public Health 

Association.Washington DC. 

Froelich, P.N. and M.E.Q. Pilson. 1978. Systematic absorbance error with Technicon 

AutoAnalyzer II colorimeter. Water Res. 12:599-603. 

IMO (International Maritime Organization) 2004. International convention for the control and 

management of ships’ ballast water and sediment. Available at 

www.imo.org/Conventions/Mainframe.asp?topic_id=867. 

Kerouel, R. and A. Aminot. 1987. Procédure optimisée hors-contaminations pour l'analyze des 

éléments nutritifs dissous dans l'eau de mer. Mar. Environ. Res. 22:19-32. 

Standard Methods for the Examination of Water and Wastewater, 20th Edition (1998) under 

sections 10-20 and 10-21, “Biological Examination” 

Sugimura, Y. and Y. Suzuki. 1988. A high temperature catalytic oxidation method for the 

determination of non-volatile dissolved organic carbon in seawater by direct injection of a liquid 

sample. Mar. Chem. 24:105-131. 

Technicon Industrial Method No. 155-71W/Tentative. 1973. Technicon Industrial Systems. 

Tarrytown, New York, 10591. 

Technicon Industrial Method No. 158-71 W/A† Tentative. 1977. Technical Industrial Systems. 

Tarrytown, New York, 10591. 

Technicon Industrial Method No. 804-86T. August 1986. Technicon Industrial Systems. 

Tarrytown, New York, 10591. 

United State Environmental Protection Agency 1979a. Method No. 160.2 (with slight 

modification) in Methods for chemical analysis of water and wastes. United States 

Environmental Protection Agency, Office of Research and Development. Cincinnati, Ohio. 

Report No. EPA-600/4-79-020 March 1979. 460pp. 

United State Environmental Protection Agency 1979b. Method No. 365.2 in Methods for 

chemical analysis of water and wastes. United States Environmental Protection Agency, Office of 

Research and Development. Cincinnati, Ohio. Report No. EPA-600/4-79-020 March 1979. 

460pp. 
120 

United State Environmental Protection Agency 1979c. Method No. 365.1 in Methods for 

chemical analysis of water and wastes. United States Environmental Protection Agency, Office of 

Research and Development. Cincinnati, Ohio. Report No. EPA-600/4-79-020 March 1979. 

460pp. 

United State Environmental Protection Agency. Office of Research and Development. 1996. 

ICR Microbial Laboratory Manual. EPA/600/R-95/178. 

Welschmeyer, N.A. 1994. Fluorometric analysis of chlorophyll a in the presence of chlorophyll b

and phaeopigments. Limnol. Oceanogr., 39: 1985-1992. 

Wright, D.A., R. Dawson, C.E.F. Orano-Dawson, G.R. Morgan and J. Coogan. (2006) The 

development of ultraviolet irradiation as a method for the treatment of ballast water in ships. 

Proc. Inst. Mar. Eng. Sci. Technol. J. Mar. Sci. Environ. (C4) 3-12. 

Wright, D.A. (2007) Problems associated with performance compliance testing for ballast water 

treatment. Proc. Inst. Mar. Eng. Sci. Technol. J. Mar. Design Ops. (B12) 25-38. 

 

Wright, D.A., Dawson, R., Caceres, V., Orano-Dawson, C.E., Kananen, G., Cutler, S.J. 

and Cutler, H.G. (2009). Shipboard testing of the efficacy of SeaKleen® as a ballast 

water treatment to eliminate non-indigenous species aboard a working tanker in Pacific 

waters. Environ. Technol. (in review) 
121 

Appendix E. 

Standard Operating Procedures for UMCES Analytical Services Division. 
NUTRIENT ANALYTICAL SERVICES LABORATORY

STANDARD OPERATING PROCEDURES

Carolyn W. Keefe

Kimberly L. Blodnikar

Walter R. Boynton

Cheryl A. Clark

Jerome M. Frank

Nancy L. Kaumeyer

Margaret M. Weir

Kathryn V. Wood

Carl F. Zimmermann

Special Publication Series No. SS-80-04-CBL 

 Center for Environmental and Estuarine Studies

The University of Maryland System

Chesapeake Biological Laboratory

 P.O. Box 38

Solomons, Maryland 20688-0038

(410) 326-7252

www.cbl.umces.edu/nasl

February, 2004

TABLE OF CONTENTS

INTRODUCTION ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 1

DISSOLVED INORGANIC ANALYTES ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 3

Orthophosphate ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 7

Ammonium ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 11

Nitrite ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 14

Nitrite + Nitrate.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 17

Silicate ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 21

Sulfate, Chloride and Nitrate ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 24

ORGANIC ANALYTES ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . 27

Total Dissolved Nitrogen and Phosphorus ... ... ... ... ... ... ... ... ... ... ... ... ... . . 29

Total and Dissolved Organic Carbon ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 35

PARTICULATE ANALYTES ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 38

Particulate Carbon and Nitrogen ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 39

Particulate Phosphorus and Particulate Inorganic Phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Particulate Biogenic Silica ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 48

Total Suspended Solids ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 52

Total Volatile Solids ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 54

Chlorophyll a and Phaeopigments ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 55

APPENDIX A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 59

APPENDIX B ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 60

ACKNOWLEDGEMENTS ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 65
LIST OF TABLES

Table 1. Parameters routinely analyzed at the Nutrient Analytical Services Laboratory at CBL . . . . . 1

Table 2. Sampling and storage for organic analytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Table 3. Minimum limits of detection of CBL ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 62

LIST OF FIGURES

Figure 1. Manifold assembly for orthophosphate method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Figure 2. Manifold assembly for ammonium method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Figure 3. Manifold assembly for nitrite method ... ... ... ... ... ... ... ... ... ... ... ... ... ... . 14

Figure 4. Manifold assembly for nitrite + nitrate method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Figure 5. Manifold assembly for silicate method ... ... ... ... ... ... ... ... ... ... ... ... ... ... 21

Figure 6. Flow diagram of nitrogen analysis ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 27

Figure 7. Flow diagram of phosphorus analysis ... ... ... ... ... ... ... ... ... ... ... ... ... ... 28

Figure 8. Manifold assembly for total dissolved phosphorus method . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Figure 9. Manifold assembly for total dissolved nitrogen method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Figure 10. Schematic diagram of the Exeter Analytical, Inc. (EAI) CE-440 Elemental Analyzer . . . . 39

Figure 11. Manifold assembly for particulate phosphorus method . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Figure 12. Manifold assembly for particulate biogenic silica method . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Figure 13. Percentage recovery of glutamic acid as an internal standard for total dissolved

nitrogen digestion/analysis ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 64

Figure 14. Percentage recovery of glycerophosphate as an internal standard for total dissolved

phosphorus digestion/analysis ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... . . 64
1

INTRODUCTION

The following pages document the analytical methodologies performed by the Nutrient Analytical

Services Laboratory at the University of Maryland Chesapeake Biological Laboratory (CBL). This

manual includes sections on dissolved inorganic nutrients, dissolved organic nutrients and particulate

nutrients. Within each of these sections, sample collection, storage, preparation and analysis are

discussed. A final section addresses data management and quality assurance/quality control (QA/QC).

Many of the procedures described are used for the Maryland Mainstem portion of the Chesapeake Bay

Program.

Instrumentation includes:

C Technicon AutoAnalyzer II,

C Two channel Technicon TrAAcs-800 Nutrient Analyzer,

C Shimadzu TOC-5000 and 5000 A Total Organic Carbon Analyzers,

C Turner Designs TD-700 Fluorometer,

C Shimadzu UV-120-02 Spectrophotometer,

C Exeter Analytical, Inc. CE-440 Elemental Analyzer, and

C Dionex DX-120 Ion Chromatograph.

PC Computers with complete spreadsheet packages are used for data reduction and management.

High quality (18.3 megohm-cm) deionized water is provided via 2 Barnstead NANOpure II systems. The

Barnstead system produces Type 1 Reagent Grade water equal to or exceeding standards established by

the American Society for Testing and Materials. First, water is filtered through a reverse osmosis

membrane. Final product water then passes through a series of five filters: one organic colloid, two

mixed bed, one organic free, and one final 0.2 :m filter. Throughout this manual, the term "deionized

water" refers to 18.3 megohm-cm water and "frozen" refers to temperature < -20°C.

Table 1. Parameters routinely analyzed at the Nutrient Analytical Services Laboratory at CBL

DISSOLVED INORGANIC ORGANIC PARTICULATE

Ammonium-Nitrogen Persulfate Nitrogen Carbon

Nitrite-Nitrogen Persulfate Phosphorus Nitrogen

Nitrite+Nitrate-Nitrogen Dissolved Carbon Phosphorus

Phosphate-Phosphorus Total Suspended Solids

Silicate Total Volatile Solids

Sulfate Biogenic Silica

Chloride Chlorophyll a

Nitrate-Nitrogen Phaeopigments
2
3

DISSOLVED INORGANIC ANALYTES

Instrumentation

All of the dissolved inorganic nutrient procedures, particulate phosphorus and biogenic silica procedures

require the use of a segmented continuous flow analyzer such as the Technicon AutoAnalyzer II or

TrAAcs-800. Samples and reagents are continuously added in a specific sequence along a path of glass

tubing and mixing coils. Within the system of tubes, air bubbles injected at precise intervals sweep the

walls of the tubing and prevent diffusion between successive samples. Reactions in the AutoAnalyzer or

TrAAcs-800 do not develop to completion as in manual methods, but reach identical stages of

development within each sample since every sample follows the same path, timing and exposure to

specific reagents.

The basic function of each component of the segmented continuous flow analyzer is discussed briefly

below. This explanation is similar to that of Sanborn and Larrance (1972).

Automatic Sampler

At a timed interval, the sampler probe alternately draws fluid from a tray of discrete samples and a wash

fluid receptacle. After each sample is drawn, the sample tray advances to the next sample position. A

bubble of air, which acts as a diffusion barrier, is aspirated into the sample stream between sample and

wash. The ratio of sample-to-wash time and the number of samples analyzed per hour are controlled by

the TrAAcs software, or by a cam or timer assembly located in the top well of the AutoAnalyzer II

sampler. Cams and timer assemblies are adjusted easily to provide various sampling rates.

The wash solution separates successive samples in the sample stream as indicated on the graphical record

by alternating minima (wash) and maxima (sample). The sample probe is connected to a stream divider

that delivers identical samples simultaneously to each manifold via the pump.

Proportioning Pump 

The proportioning pump is a peristaltic-type pump that continuously delivers air, reagents and samples to

the manifold. Plastic pump tubes of various diameters are pressed between a series of moving rollers and

a platen. The motion of the rollers along the tubes delivers a continuous flow of samples and reagents. 

The delivery rate is determined by the inside diameter of the tubes since the rollers move at a constant

rate. These pump tubes are available in a large assortment of delivery rates. The pump holds a

maximum of 28 tubes and has an air bar that mechanically measures and injects identically sized air

bubbles into the analytical stream. The pump tubes delivering reagents, air and samples are connected to

appropriate manifolds.

Manifold

Each analysis requires a manifold specifically designed for the chemical determination employed. The

manifolds are composed of a series of glass coils, injection fittings and heating baths arranged for the

proper sequence of reactions leading to color development. The glass coils permit mixing of the sample

and the reagents; as two solutions with different densities travel around each turn of the mixing coil, the

denser solution falls through the less dense one, causing mixing and creation of a homogeneous mixture
4

of the two solutions. The length of the coil determines the amount of time allowed for chemical reaction

between the addition of successive reagents. Injection fittings for each of the reagents are placed

between mixing coils; thus, a sample enters one end of the manifold, a reagent is added, and mixed; and

then another reagent is added and mixed. After the addition of all reagents, and an adequate reaction

time, the solution flows into a colorimeter.

Colorimeter

The colorimeter measures the absorption of monochromatic light by the solution in the flow cell. Light

from a single source passes through two separate but identical interference filters that pass light within a

narrow spectral band. The light then passes through the appropriate flow cell and is projected onto a

phototube. The phototube generates an electrical signal in response to the intensity of the impinging

light. The output from each phototube is a measure of transmittance and is converted electronically by

the colorimeter to a signal proportional to absorbance. The relationship between transmittance and

absorbance is given by the equation A = log 1/T; where A = absorbance and T = transmittance. The

resulting signal is linear in absorbance and directly proportional to concentration. As each sample passes

through the flow cell, the signals are sent to a recorder.

Recorder

Results of the analyses are continuously recorded by strip chart recorders and/or by computer using an

IBM compatible DP500 software system by Labtronics Inc., or AACE software system by Bran and

Luebbe. Each recorder can simultaneously monitor two separate analyses. The DP500 and AACE

systems can each collect and analyze data from up to four different detectors simultaneously. The output

of the colorimeter is proportional to absorbance and standards of known concentration must be analyzed

to relate absorbance to concentration. The analog signals can be converted to absorbance values by

referring to the Technicon reference curve and the standard calibration control.

Sampling and Storage 

 Collected water samples are filtered through Whatman GF/F filters (nominal pore size 0.7 :m), placed

in either polypropylene bottles or directly into 4 ml AutoAnalyzer cups and frozen. Samples for silicate

are treated in the same manner but are refrigerated at 4°C. All samples are routinely analyzed within 28

days.

Operating Procedures

The following describes step-by-step operating procedures for the AutoAnalyzer II system.

1. Colorimeter - Turn power on and allow 10 minutes for warm-up. Set standard calibration setting for desired

determination.

2. Recorder (or Computer) - Turn power on and allow 10 minutes for recorder warm-up. Check recorder paper supply. If

using computer for data collection, load software and select appropriate sample method and sample table. Refer to

Labtronics Inc. DP500 and Bran and Luebbe AACE users’ manuals for descriptions of system operation.

3. Sampler Water Reservoirs - Check and fill the deionized water reservoirs.

4. Pump - Connect pump tubes and attach platen to pump. Start pump with deionized water flowing through the system. 

Check for leaks in tubes at connections and for a regular bubble pattern in the manifold.
5

5. Recorder - Start recorder. Paper should begin to move.

6. Colorimeter - Check ZERO and FULL SCALE knob. ZERO simulates a zero output so that ZERO adjustment

(screwdriver) of the recorder can be made. Set knob to NORMAL and establish a baseline with deionized water using the

BASELINE CONTROL adjustment knob and a standard calibration (STD CAL) setting of 1.0; i.e., wherein the full scale

absorbance is 1.00.

7. Allow reagents to pump through the system and note any rise in baseline and readjust the baseline to zero. Refer to this

rise as the REAGENT BLANK (at a STD CAL of 1.0).

8. An extremely wide range of nutrient concentrations found in Chesapeake Bay waters, both temporally and spatially,

requires use of a standard curve covering a large range and that covers a few STD CAL control settings.

9. Reset zero baseline at the STD CAL control setting normally used for that determination (e.g., particulate phosphorus STD

CAL of 4.0). Next, switch the STD CAL setting to 1.0. There should be no deflection of the pen at zero baseline. Note

peak heights of standards at the various STD CAL settings along with the STD CAL settings. This allows the operator to

use STD CAL settings in the range of 1 to 4 (for this example) in analyzing standards and samples that otherwise would

have gone off scale. Intersperse standards in the run after approximately every 20 samples, including a standard analyzed

at each STD CAL setting employed during the preceding 40 samples. A visual comparison with the day's initial standard

curve should indicate no greater variance than 5% of the peak height (e.g., initial standard peak height 60.0; subsequent

standards acceptable in the range of 57.0 to 63.0). If the variance exceeds 5%, identify the source of the problem, correct

and re-analyze affected samples. Adjust baseline after approximately every 20 samples. If an adjustment of more than 1

unit is required, identify the source of the problem, correct and re-analyze affected samples.

10. At completion of the run, remove lines from reagents and place tubes in deionized water.

11. Shut-down - Turn off recorder. Wash system with 1 N hydrochloric acid for 15 minutes, followed by a 15 minute wash

with deionized water. Turn off pump, release proportioning platen and loosen pump tubes. Turn off colorimeter.

Glassware

Glassware are cleaned appropriately for the determination. Most are acid-washed with 10% hydrochloric

acid followed by numerous rinses with deionized water. Other cleaning protocols are listed in their

specific sections.

Calibration and Standardization

Please refer to each specific determination for the appropriate STD CAL control setting and for the

standard concentrations used.

The STD CAL control setting located on the colorimeter allows the operator to adjust the electrical

output to the concentration range of the samples. Extremely low values (:g/L) require high STD CAL

settings (high sensitivity) whereas high values (mg/L) require lower STD CAL settings (lower

sensitivity).

Concentrations of nutrients are calculated from the linear regression of the standard concentration

(independent variable) against the corresponding peak height (dependent variable). All standards

analyzed at a particular STD CAL setting are included in the regression for that set of calculations. Only

samples whose peak heights were measured at that individual STD CAL setting are calculated from that

regression. If a broad range of sample concentrations requires that more than one STD CAL setting be

used throughout the course of a run, then a separate regression must be employed for each STD CAL

setting. For example, peak heights obtained from standards read at STD CAL 9.0 are used to obtain the

linear regression for calculating only the concentrations of samples whose peak heights were read at
6

STD CAL 9.0. Likewise, peak heights obtained from standards read at STD CAL 2.0 are used to obtain

the linear regression for calculating only the concentrations of samples whose peak heights were read at

STD CAL 2.0.

Peak heights are read manually from the strip chart or automatically by the DP500 or AACE software

systems. Operator vigilance is necessary throughout the run to ensure that all peaks indicate steady state

conditions for the reaction for each sample. If steady state conditions are not obtained, the samples are

re-analyzed.

Stock standards are prepared with primary standard grade chemicals of each nutrient in deionized water. 

As a general rule, stock solutions are made every 6 months and the preparation date logged. Secondary

standards, where appropriate, are prepared with deionized water. Working standards are prepared daily

with deionized water or the appropriate matrix as described by the specific determination method and

should encompass the range of the samples.

All analysis documents are kept electronically and in notebooks. A copy is also given to the investigator

or granting agency. Information provided includes:

C name of the method;

C collection date;

C source of samples;

C analyst;

C analysis date;

C sample number;

C sample concentration;

C results of duplicate analyses;

C results of spike analyses; and.

C results of analyses of commercially prepared standard reference materials.

Literature Cited

Sanborn, H. and J. Larrance. 1972. An operations manual of the AutoAnalyzer for seawater nutrient

analysis. NOAA/NMFS. Seattle, Washington. 44pp.
7

ORTHOPHOSPHATE Revised 2/2004

Ammonium molybdate and antimony potassium tartrate react in an acid medium with dilute solutions of

phosphorus to form an antimony-phospho-molybdate complex which is reduced to an intensely bluecolored complex by ascorbic acid. Color is proportional to phosphorus concentration.

Methodology

Technicon Industrial Method No. 155-71W/Tentative. 1973. Technicon Industrial Systems. Tarrytown,

New York, 10591.

USEPA. 1979. Method No. 365.1 in Methods for chemical analysis of water and wastes. United States

Environmental Protection Agency, Office of Research and Development. Cincinnati, Ohio. Report

No. EPA-600/4-79-020 March 1979. 460pp.

Froelich, P.N. and M.E.Q. Pilson. 1978. Systematic absorbance error with Technicon AutoAnalyzer II

colorimeter. Water Res. 12:599-603.

Instrumentation

Technicon AutoAnalyzer II

Manifold Assembly

Figure 1. Manifold assembly for orthophosphate method.
8

Specifications

Standard Calibration Settings: 9.0, 6.0, 2.0, 1.0

Damp: Normal

Sampling Rate: 40/hour, 9:1 sample/wash ratio

Filter: 880 nm

Phototube: 199-B021-04

Flowcell: 50 mm

Relative Absorbance ( 18 μM P): ~.85

Interferences: Silicon at a level of 100 :M Si causes an interference equivalent to approximately 0.04

:M P. High iron may cause precipitation (i.e., loss) of phosphorus. High sample turbidity and color may

interfere.

Reagents

Deionized Water Diluent

Sod 3 2 11 3ium dodecyl sulfate, (SDS)[CH (CH ) OSO Na;

m.w.=288.38; phosphate #0.0001 %] 0.05 g

Deionized water up to 500 mL

Add 0.05g of sodium dodecyl sulfate to approximately 500 mL of deionized water. Mix well.

Sulfuric Acid Solution, 4.9 N

Su 2 4 lfuric acid (H SO ), concentrated (sp. gr. 1.84) 136 mL

Deionized water up to 1000 mL

To a 1000 mL volumetric flask, add 136 mL of concentrated sulfuric acid to approximately 800 mL of deionized water while

cooling (cold water bath). After the solution is cooled, dilute to 1000 mL with deionized water.

Ammonium Molybdate Solution

Ammon 4 6 7 24 2ium molybdate [(NH ) Mo O @4 H O] 40 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 40 g of ammonium molybdate in 800 mL of deionized water. Dilute to 1000 mL with

deionized water. Store in plastic bottle away from direct sunlight.

Ascorbic Acid Solution

Asco 686 rbic acid (C H O ) 18 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 18 g of ascorbic acid in 800 mL of deionized water. Dilute to 1000 mL with deionized

water and dispense -40 mL aliquots into clean polybottles and freeze. Thaw overnight in the refrigerator before use.

Antimony Potassium Tartrate Solution

Ant 446 2imony potassium tartrate [K(SbO)C H O @1⁄2 H O] 3 g

Deionized water up to 1000 mL
9

In a 1000 mL volumetric flask, dissolve 3 g of antimony potassium tartrate in 800 mL deionized water. Dilute to 1000 mL with

deionized water.

Working Reagents

Reagent A

Sulfuric Acid Solution, 4.9 N 50 mL

Ammonium Molybdate Solution 15 mL

Antimony Potassium Tartrate Solution 5 mL

Sod 3 2 11 3ium dodecyl sulfate, (SDS) [CH (CH ) OSO Na;

m.w. 288.38; phosphate #0.0001%] 0.15 g

Reagent B

Ascorbic Acid Solution 30 mL

SDS 0.05 g

Standards

Stock Orthophosphate Standard, 12,000 :M

Potassium phosphate, monobasic

2 4 (KH PO ), dried at 45°C 1.632 g

Deionized water up to 1000 mL

Ch 3loroform (CHCl ) 1 mL

In a 1000 mL volumetric flask, dissolve 1.632 g of potassium phosphate in -800 mL deionized water. Dilute to 1000 mL with

deionized water then add 1 mL of chloroform as a preservative (1 mL contains 12 :moles P).

Secondary Orthophosphate Standard

Stock Orthophosphate Standard 1 mL

Deionized water up to 100 mL

In a 100 mL volumetric flask, dilute 1 mL of Stock Orthophosphate Standard to 100 mL with deionized water (1 mL contains

0.12 :moles P).

Working Orthophosphate Standards

Dilute 0.1, 0.25, 0.5, 1.0, 1.5, 2.5, 5 and 10 mL of Secondary Orthophosphate Standard to 100 mL with deionized water to yield

concentrations of 0.12 :M P (0.00372 mg/L), 0.3 :M P (0.0093 mg/L), 0.6 :M P (0.0186 mg/L), 1.2 :M P (0.0372 mg/L), 1.8

:M P (0.0558 mg/L), 3.0 :M P (0.093 mg/L), 6.0 :M P (0.186 mg/L) and 12.0 :M P (0.372 mg/L), respectively.

Refractive Index Correction

Working Reagents

Refractive Reagent A

Sulfuric Acid Solution, 4.9 N 50 mL

Deionized water 20 mL

SDS 0.15 g
10

Refractive Reagent B

Ascorbic Acid Solution 30 mL

SDS 0.05 g

Procedure

1. Obtain peak heights for all samples and standards with Refractive Reagent A and Reagent B in place of Reagent A and

Reagent B of the orthophosphate determination. Peak heights for the refractive index correction must be obtained at the

same STD CAL setting and on the same colorimeter as the corresponding samples and standards (Froelich and Pilson

1978).

2. Subtract the refractive index peak heights from the heights obtained for the orthophosphate determination.

3. Calculate the regression equation using the corrected standard peak heights.

4. Calculate the concentration of samples from the regression equation using the corrected sample peak heights.

When a large data set has been amassed in which each sample's salinity is known, a regression for the

refractive index correction on the colorimeter can be calculated. For each sample, the apparent

orthophosphate concentration due to refractive index is calculated from its peak height obtained with

Refractive Reagent A and Reagent B, and the regression of orthophosphate standards obtained with

orthophosphate Reagent A and Reagent B. The salinity is entered as the independent variable and its

apparent orthophosphate concentration due to its refractive index in that colorimeter is entered as the

dependent variable. The resulting regression allows the operator to subtract an apparent orthophosphate

concentration when the salinity is known, as long as other matrix effects are not present; thus, the

operator is not required to obtain the refractive index peak heights for all samples when a large data set

has been found to yield consistent orthophosphate concentrations due to salinity.
11

AMMONIUM Revised 2/2004

Determination of ammonium is by the Berthelot Reaction in which a blue-colored compound similar to

indophenol forms when a solution of ammonium salt is added to sodium phenoxide, followed by the

addition of sodium hypochlorite. The addition of a potassium sodium tartrate and sodium citrate

solution prevents precipitation of hydroxides of calcium and magnesium.

Methodology

Technicon Industrial Method No. 804-86T. August 1986. Technicon Industrial Systems. Tarrytown,

New York, 10591.

Kerouel, R. and A. Aminot. 1987. Procédure optimisée hors-contaminations pour l’analyze des éléments

nutritifs dissous dans l’eau de mer. Mar. Environ. Res. 22:19-32.

Instrumentation

Technicon TrAAcs-800 Nutrient Analyzer

Manifold Assembly

Figure 2. Manifold assembly for ammonium method.
12

Specifications

Gain: 40 - 45

Baseline: 32 - 38

Sampling Rate: 60/hour

Filter: 630 nm

Flowcell: 50 mm

Relative Absorbance (12 :M N): 0.22 - 0.24 

Contamination: Ammonium determination is highly susceptible to skin and atmospheric contamination

(Kerouel and Aminot 1987). 

 Reagents

Complexing Reagent

Sod 446 2ium potassium tartrate (KNaC H O @4 H O) 39 g

Sod 2 2 2ium citrate [HOC(COONa)(CH OONa) @2 H O] 28 g

Sulfuric acid (H2 4 SO ), concentrated (sp. gr. 1.84) as required

Deionized water up to 1000 mL

Brij-35, 30% 1 mL

In a 1000 mL beaker, dissolve 39 g of potassium sodium tartrate and 28 g of sodium citrate in 950 mL of deionized water. 

Adjust the pH of the solution to 5.0 using concentrated sulfuric acid. Dilute to 1000 mL with deionized water in a volumetric

flask. Add 1 mL of 30% Brij-35 and mix thoroughly. Store in a light resistant container and refrigerate. Make fresh weekly.

Alkaline Phenol Reagent

Pheno 6 5 l (C H OH), 88% 23.6 mL

Sodium hydroxide (NaOH), 50% (w/w) 18 g

Deionized water up to 250 mL

In a 250 mL volumetric flask, slowly add 23.6 mL of 88% phenol to -150 mL of deionized water. Weigh out exactly 18 g of

50% (w/w) sodium hydroxide, then while in an ice bath, slowly add the sodium hydroxide to the phenol/water solution. Dilute to

250 mL with deionized water. Store in a light resistant glass container and refrigerate. Make fresh weekly.

WARNING: Phenol is extremely dangerous and should be handled accordingly. Prepare reagent fresh weekly under a fume

hood and wearing protective gloves.

Sodium Hypochlorite, 1%

Sodium hypochlorite (CLOROX Ultra - 6.0%) 44 mL

Deionized water up to 200 mL

In a 250 mL volumetric flask, dilute 44 mL of any commercially available bleach containing 6.0% sodium hypochlorite to 250

mL with deionized water. Make fresh every other day.

Sodium Nitroprusside (Sodium Nitroferricyanide), 0.05%

Sodium 2 5 2 nitroprusside [Na Fe(CN) NO@2 H O] 0.5 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 0.5 g of sodium nitroprusside in 900 mL of deionized water. Dilute to 1000 mL with

deionized water. Store in a light resistant container.
13

Standards

Stock Ammonium Standard, 1,500 :M

Ammonium 4 2 4 sulfate [ (NH ) SO ], dried at 45°C 0.100 g

Deionized water up to 1000 mL

Ch 3loroform (CHCl ) 1 mL

In a 1000 mL volumetric flask, dissolve 0.1 g of ammonium sulfate in -900 mL of deionized water. Dilute to 1000 mL with

deionized water. Add 1 mL chloroform to act as a preservative (1 mL contains 1.5 :moles N).

Working Ammonium Standards

Dilute 0.1, 0.2, 0.4, 0.6 and 0.8 mL of Stock Ammonium Standard to 100 mL with deionized water to yield concentrations of 1.5

:M N (0.021 mg/L), 3 :M N (0.042 mg/L), 6 :M N (0.084 mg/L), 9 :M N (0.126 mg/L) and 12 :M N (0.168 mg/L),

respectively.

Procedure

1. Check the level of all reagents at start-up to ensure adequate supply.

2. Place all lines into their respective containers and start the proportioning pump. Allow -10 minutes equilibration time.

3. Place a saline primer (artificial seawater) before the actual samples to avoid an abnormal peak for the first sample. Place a

deionized water cup before an intersample calibrant.

A typical tray protocol appears as follows:

P, 5C, H@2, 2L@6, A@119, 20S>7, W@120, 2I@3, S@119, 20S>26,....

where: P = primer (100% FS standard),

C = calibrant,

H = 100% FS standard,

L = 20% FS standard,

A = artificial seawater,

S = samples,

W = deionized water, and

I = intersample calibrant.

4. At shut-down, remove all reagent lines from their respective containers excluding the sampler solution line and place them

in deionized water containing 1 mL/L of 30% Brij-35.

5. After 5 minutes, allow all lines to pump dry. Stop the pump and release the platen. 

NOTES:

1. The gain setting of 40 - 45 should yield peaks of -70 - 80 lines with a 12 :M N standard.

2. Reagent blanks appear to be quite variable. If the reagent blank is >10 lines with the appropriate gain setting (40 - 45) and

baseline setting (32 - 38), then prepare fresh reagent.

3. Close attention should be paid to prevent contamination of samples. Smoking and the use of ammonium-containing

cleaning solutions should be prohibited within the air-handling limits surrounding the laboratory. Sample bottles should

be stored in a clean environment and only opened when necessary. Samples should never be pipetted by mouth. Samples

should be analyzed immediately upon opening.
14

NITRITE Revised 2/2004

Nitrite reacts under acidic conditions with sulfanilamide to form a diazo compound that couples with N1-naphthylethylenediamine dihydrochloride to form a reddish-purple azo dye measured at 520 nm..

Methodology

Technicon Industrial Method No. 818-87T. February 1987. Technicon Industrial Systems. Tarrytown,

New York, 10591.

Instrumentation

Technicon TrAAcs-800 Nutrient Analyzer

Manifold Assembly

Figure 3. Manifold assembly for nitrite method.

Specifications

Gain: 53

Baseline: 36 - 37

Sampling Rate: 60/hour

Filter: 520 nm

Flowcell: 50 mm

Relative Absorbance (3 :M N): -0.165
15

Reagents

Sulfanilamide Solution

Sulfan 2 6 4 2 2ilamide (4 NH C H SO NH ) 2.5 g

Hydrochloric acid (HCl), concentrated 25 mL

Deionized water up to 500 mL

Brij-35, 30% few drops

In a 500 mL volumetric flask, add 2.5 g of sulfanilamide and 25 mL of concentrated hydrochloric acid to -300 mL of deionized

water. Swirl to dissolve and dilute to 500 mL with deionized water. Add a few drops of Brij-35, 30% and mix thoroughly. 

Store in light resistant container. Refrigerate when not in use.

N-1-Naphthylethylenediamine Dihydrochloride Solution

N-1-naphthylethylenediamine dihydrochloride

12 14 2

(CHN @2HCl) 0.25 g

Deionized water up to 500 mL

Brij-35, 30% few drops

In a 500 mL volumetric flask, dissolve 0.25 g of N-1-naphthylethylenediamine dihydrochloride in -300 mL of deionized water. 

Dilute to 500 mL with deionized water. Add a few drops of Brij-35, 30% and mix thoroughly. Store in a light resistant

container. Refrigerate when not in use.

Standards

Stock Nitrite Standard, 5,000 :M 

Sod 2ium nitrite (NaNO ), dried at 45°C 0.345 g

Deionized water up to 1000 mL

Ch 3loroform (CHCl ) 1 mL

In a 1000 mL volumetric flask, dissolve 0.345 g of sodium nitrite in -600 mL of deionized water. Dilute to 1000 mL with

deionized water. Add 1 mL of chloroform as a preservative (1 mL contains 5 :moles N).

Secondary Nitrite Standard

Stock Nitrite Standard 0.8 mL

Deionized water up to 100 mL

In a 100 mL volumetric flask, dilute 0.8 mL of Stock Nitrite Standard to 100 mL with deionized water to yield a concentration of

40 :M N.

Working Nitrite Standards

Dilute 0.5, 1, 2.5, 5 and 7.5 mL of Secondary Nitrite Standard to 100 mL with deionized water to yield concentrations of 0.2 :M

N (0.0028 mg/L), 0.4 :M N (0.0056 mg/L), 1 :M N (0.014 mg/L), 2 :M N (0.028 mg/L) and 3 :M N (0.042 mg/L),

respectively.
16

Procedure

Run this analysis concurrently with ammonium, using a synthetic seawater cup prior to a sample set and a deionized water

cup prior to an intersample calibrant (See Ammonium methodology). The 3-way stream divider (PT 2 connector, P/N 116-B000-

01) used to split the sample is the appropriate size for the pump tubes used. Stream dividers with smaller ports create gas

bubbles with the rapid movement of sample through the fitting.

NOTES:

1. A gain setting of 53 should yield peaks of -80 lines using a 3 :M N standard.

2. This is a very steady analysis with a smooth baseline, well defined peaks, minimal baseline correction and minimal carryover correction.
17

NITRITE + NITRATE Revised 2/2004

Filtered samples are passed through a granulated copper-cadmium column to reduce nitrate to nitrite.

The nitrite (originally present plus reduced nitrate) then is determined by diazotizing with sulfanilamide

and coupling with N-1-naphthylethylenediamine dihydrochloride to form a colored azo dye. Nitrate

concentration is obtained by subtracting the corresponding nitrite value from the nitrite + nitrate

concentration.

Methodology

Technicon Industrial Method No. 158-71 W/A† Tentative. 1977. Technicon Industrial Systems. 

Tarrytown, New York, 10591.

USEPA. 1979. Method No. 353.2 in Methods for chemical analysis of water and wastes. United States

Environmental Protection Agency, Office of Research and Development. Cincinnati, Ohio. Report

No. EPA-600/4-79-020 March 1979. 460pp.

Instrumentation

Technicon AutoAnalyzer II

Manifold Assembly

Figure 4. Manifold assembly for nitrite + nitrate method.
18

Specifications

Standard Calibration Settings: 

ORN/GRN sample tube: 3.0, 1.0

ORN/YEL sample tube: 2.0, 1.0

BLK/BLK sample tube: 9.0, 6.0, 2.0, 1.0

Dilution loop: 3.0, 1.0

Damp: Normal

Sampling Rate: 40/hour, 9:1 sample/wash ratio

Filter: 550 nm

Phototube: 199-B021-01

Flowcell: 50 mm

Relat 3ive Absorbance (20 μM NO -N ; BLK/BLK sample tube): ~.55 - 

Interferences: Metal ions may interfere if present in sufficient concentrations. The presence of large

concentrations of sulfide and/or sulfate greatly reduce the sensitivity of the copper-cadmium column.

Reagents

Alkaline Water

Sodium hydroxide (NaOH, pellets) 0.20 ± 0.02 g

Deionized water up to 1000 mL

Add 0.20 g of sodium hydroxide pellets to 1000 mL of deionized water.

Copper Sulfate Reagent, 2%

Copper su 4 2 lfate (CuSO @5 H O) 2 g

Deionized water up to 100 mL

In a 100 mL volumetric flask, dissolve 2 g of copper sulfate in -80 mL of deionized water. Dilute to 100 mL with deionized

water.

Ammonium Chloride Reagent

Ammonium 4 chloride (NH Cl) 10 g

Alkaline Water up to 1000 mL

Copper Sulfate Reagent, 2% 6 drops

In a 1000 mL volumetric flask, dissolve 10 g of ammonium chloride in -800 mL of Alkaline Water and dilute to 1000 mL with

Alkaline Water. Attain a pH balance of 8.5. Add 6 drops of 2% Copper Sulfate Reagent.

Color Reagent

Sulfan 6822 ilamide (C H N O S) 20 g

Pho 3 4 sphoric acid (H PO ), concentrated (80%) 200 mL

N-1-naphthylethylenediamine dihydrochloride

12 14 2

(CHN @2HCl) 1 g

Deionized water up to 2000 mL

Brij-35, 30% 1 mL

In a 2000 mL volumetric flask, add 200 mL concentrated phosphoric acid and 20 g of sulfanilamide to -1500 mL deionized
19

water. Dissolve completely. Add 1 g of N-1-naphthylethylenediamine dihydrochloride and dissolve. Dilute to 2000 mL with

deionized water and add 1 mL of 30% Brij-35. Store in refrigerator. Prepare every 6 weeks.

Preparation of Copper-Cadmium Column

1. Use good quality cadmium filings (25 - 60 mesh size).

2. Clean 10 g of cadmium with 20 mL of acetone. Rinse twice with 20 mL of deionized water. Clean cadmium next with 50

mL of 1 N hydrochloric acid for 1 minute. Cadmium turns silver in color. Decant the hydrochloric acid and wash the

cadmium with another 50 mL of 1 N hydrochloric acid for 1 minute.

3. Decant the hydrochloric acid and wash the cadmium several times with deionized water.

4. Decant the deionized water 4 2 and add 20 mL of 2% (w/v) copper sulfate (CuSO @5H O). Wash the cadmium until no blue

color remains in the solution.

5. Add another 20 mL of 2% (w/v) copper sulfate and wash the cadmium until no blue color remains in the solution. The

cadmium will be dark brown in color.

6. Decant and wash thoroughly (-10 times) with deionized water.

7. Fill the reductor column (22 cm length of 0.110-inch ID tubing) with Ammonium Chloride Reagent and transfer the

prepared cadmium particles to the column using a Pasteur pipette or some other method that prevents contact of the

cadmium pellets with air. Do not allow any air bubbles to be trapped in the column. Pack the entire column uniformly

with filings such that, visually, the packed filings have separation gaps #-1 mm.

8. When the entire column is well-packed with granules, insert glass wool plugs at the outlet end of the column. With

reagents running through the system, attach the column. Remember to have no air bubbles in the valve and to attach the

column to the intake side of the valve first.

9. Check for good flow characteristics (good bubble pattern) after the addition of air bubbles beyond the column. If the

column is packed too tightly, an inconsistent flow pattern will result. Allow Ammonium Chloride Reagent to flow through

the column for one hour.

10. Prior to sample analysis, condition the column with approximately 100 mg nitrate/L for 5 minutes followed by 100 mg

nitrite/L for 10 minutes.

11. Analyze 3 2 Working Standard A (40 :M NO -N) and Working Nitrite Standard (40 :M NO -N). If the peak height of - -

Working Standard A is <90% of the peak height of Working Nitrite Standard, prepare a new cadmium reduction column.

Standards

Stock Nitrate Standard, 5,000 :M

Potass 3ium nitrate (KNO ), dried at 45°C 0.5055 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 0.5055 g of potassium nitrate in -800 mL of deionized water. Dilute to 1000 mL with

deionized water (1 mL contains 5 :moles N).

Working Standard A

Stock Nitrate Standard 0.8 mL

Deionized water up to 100 mL

In a 100 mL volumetric flask, dilute 0.8 mL of Stock Nitrate Standard to 100 mL with deionized water to yield a concentration

of 40 :M N (0.56 mg N/L).
20

Working Nitrate Standards

Dilute 2.5, 5, 10, 15 and 25 mL of Working Standard A to 100 mL with deionized water to yield concentrations of 1 :M N

(0.014 mg N/L), 2 :M N (0.028 mg N/L), 4 :M N (0.056 mg N/L), 6 :M N (0.084 mg N/L) and 10 :M N (0.140 mg N/L),

respectively.

Dilute 0.8 mL of Stock Nitrate Standard to 200 mL with deionized water to yield concentrations of 20 :M N (0.28 mg N/L).

Dilute 1.0 mL and 1.5 mL of Stock Nitrate Standard to 100 mL with deionized water to yield concentrations of 50 and 75 :M N

4

(0.70 and 1.05 mg N/L), respectively for use with the ORN/YEL sample tube, YEL/BLU NH Cl tube and sample concentrations

of 0.50 - 1.00 mg N/L (nitrite + nitrate).

Dilute 2.0 mL of Stock Nitrate Standard to 100 mL with deionized water to yield concentrations of 100 :M N (1.40 mg N/L) for

use 4 with the ORN/GRN sample tube, YEL/BLU NH Cl tube and sample concentrations of 1.00 - 1.40 mg N/L (nitrite + nitrate).

Dilute 3.0, 5.0, 6.0 and 8.0 mL of Stock Nitrate Standard to 100 mL with deionized water to yield concentrations of 150, 250,

300 and 400 :M N (2.1, 3.5, 4.2 and 5.6 mg N/L) for use with the dilution loop and sample concentrations of 1.40 - 5.60 mg

N/L (nitrite + nitrate). The nitrate sample is sampled originally with an ORN/WHT sample tube and YEL/YEL deionized water

diluent tube which 4 is then "resampled" to an ORN/YEL resample tube and YEL/BLU NH Cl tube.

Stock Nitrite Standard, 5,000 :M

Sod 2ium nitrite (NaNO ), dried at 45°C 0.345 g

Deionized water up to 1000 mL

Ch 3loroform (CHCl ) 1 mL

In a 1000 mL volumetric flask, dissolve 0.345 g of sodium nitrite in -600 mL of deionized water. Dilute to 1000 mL with

deionized water. Add 1 mL of chloroform as a preservative (1 mL contains 5 :moles N).

Working Nitrite Standard

Dilute 0.8 mL of Stock Nitrite Standard to 100 mL with deionized water to yield a concentration of 40 :M N (0.56 mg N/L).
21

SILICATE Revised 2/2004

This reaction is based on the reduction of silicomolybdate in acidic solution to "molybdenum blue" by

ascorbic acid. Oxalic acid is added to minimize interference from phosphates.

Methodology

Technicon Industrial Method No. 811-86T. November 1986. Technicon Industrial Systems. Tarrytown,

New York, 10591.

Instrumentation

Technicon TrAAcs-800 Nutrient Analyzer

Manifold Assembly

Figure 5. Manifold assembly for silicate method.

Specifications

Gain: 8

Baseline: 39 - 42

Sampling Rate: 60/hour

Filter: 800 nm

Flowcell: 50 mm

Relative Absorbance (75 :M Si): -1.10

Interferences: Salinity may interfere.
22

Reagents

Oxalic Acid Solution

Oxa 2 2 4 2lic acid (H C O @2H O)100 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 100 g of oxalic acid in -900 mL deionized water and dilute to 1000 mL with deionized

water.

Ascorbic Acid Solution

Oxa 2 2 4 2lic acid (H C O @2 H O) 5 g

Asco 686 rbic acid (C H O ), U.S.P. quality 100 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 5 g of oxalic acid in -800 mL of deionized water. Add 100 g of ascorbic acid and mix

until dissolved. Dilute to 1000 mL with deionized water.

Ammonium Molybdate Solution

Ammon 4 6 7 24 2ium molybdate [(NH ) Mo O @4 H O] 7.5 g

Deionized water up to 250 mL

In a 250 mL volumetric flask, dissolve 7.5 g of ammonium molybdate in -200 mL of deionized water. Dilute to 250 mL with

deionized water.

Stock Phosphate Solution (Baseline Spike)

Potass 2 4 ium phosphate (KH PO ), dried at 45°C 0.4394 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 0.4394 g of potassium phosphate in -600 mL of deionized water. Dilute to 1000 mL

with deionized water. Prepare fresh when making 0.7 N Sulfuric Acid Solution.

Sulfuric Acid Solution, 0.7 N

Su 2 4 lfuric acid (H SO ), concentrated (sp. gr. 1.84) 4.06 mL

Stock Phosphate Solution 21.4 mL

Sod 3 2 11 3ium dodecyl sulfate, (SDS) [CH (CH ) OSO Na;

m.w. = 288.38; phosphate #0.0001 %] -0.25 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, add 4.06 mL of concentrated sulfuric acid and 21.4 mL of Stock Phosphate Solution to -600 mL

of deionized water. Dilute to 1000 mL with deionized water. Add -0.25 g of SDS.

Sodium Hydroxide Wash Solution

Sodium hydroxide (NaOH) 4 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 4 g of sodium hydroxide in -800 mL of deionized water. Dilute to 1000 mL with

deionized water.
23

Sampler (Baseline) Solution

3 2 Isopropanol [(CH ) CHOH] 100 mL

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dilute 100 mL of isopropanol to 1000 mL with deionized water.

Standards

Stock Silicate Standard, 10,000 :M

Sod 2 6 ium silicofluoride (Na SiF ), dried at 45°C 1.88 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 1.88 g of sodium silicofluoride in -900 mL of deionized water. Dilute to 1000 mL with

deionized water (1 mL contains 10 :moles Si). Store in a plastic container.

Working Silicate Standards

Dilute 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.75 mL of Stock Silicate Standard to 100 mL with deionized water to yield

concentrations of 5 :M Si (0.14 mg/L), 10 :M Si (0.281 mg/L), 20 :M Si (0.562 mg/L), 30 :M Si (0.843 mg/L), 40 :M Si

(1.124 mg/L), 50 :M Si (1.41 mg/L) and 75 :M Si (2.10 mg/L), respectively.

NOTE: 

1. The use of glassware with this method should be avoided because it may be a source of silica contamination. All

chemicals used for reagents and the deionized water should be of good quality and low in silica.

2. Sampler (Baseline) Solution of isopropanol is used instead of deionized water as baseline solution to counter the effect of

salt water on the system due to its high refractive index relative to deionized water.

Procedure

1. At start-up, check the level of all reagents to ensure an adequate supply.

2. With the molybdate line pumping air, place all other lines into their respective containers and start the proportioning pump.

3. After 2 - 3 minutes, place the molybdate line into its container. Allow -10 minutes equilibration.

4. A typical tray protocol appears as follows:

P, 5C, H, 2L, nS, I,....

where: P = primer (100% FS standard),

C = calibrant,

H = 100% FS standard,

L = 20% FS standard,

S = samples, and

I = intersample calibrant.

5. At shut-down, remove all reagent lines and place into the Sodium Hydroxide Wash Solution container. Allow to pump 5

minutes.

6. Remove all lines from the wash container and place into deionized water with SDS. Allow to pump 10 minutes.
24

SULFATE, CHLORIDE AND NITRATE Revised 2/2004

A small volume of filtered sample is introduced into an ion chromatograph (IC). The sample is pumped

through a pre-column, separator column, suppressor column and conductivity detector. Anions are

separated in the pre-column and separator column based on their affinity for resin exchange sites in the

columns. The suppressor column converts the sample anions to their acid form. The separated anions are

measured by the conductivity detector. The concentration of anions is determined by comparing peak

areas of unknowns to a calibration curve generated from known standards.

Methodology

Pfaff, J.D., C.A. Brockhoff and J.W. O’Dell. August 1991. USEPA Test Method No. 300.0. The

determination of inorganic anions in water by ion chromatography. Environmental Monitoring and

Systems Laboratory, Cincinnati, OH 45268.

USEPA. 1987. Section 11.0. Determination of chloride, nitrate and sulfate by ion chromatography in

Handbook of methods for acid deposition studies: Laboratory analyses for surface water chemistry.

United States Environmental Protection Agency, Office of Research and Development. Washington,

D.C.. Report # EPA 600/4-87/026 September 1987. 336pp.

Instrumentation

Dionex-120 Ion Chromatograph with PeakNet software (version 5.21) was brought on-line in January

2003. A Dionex AS-40 AutoSampler was added to the system in January 2004.

Reagents

Eluent, 2 3 3 3.5M Na CO /1.0M NaHCO Solution

2 3 Sodium carbonate (Na CO ) 0.742 g

3

 Sodium bicarbonate (NaHCO ) 0.164 g

 Deionized water up to 2000 mL

 

In a 2000 mL volumetric flask, dissolve 0.742 g of sodium carbonate and 0.164 g of sodium bicarbonate in approximately 1500 mL

of deionized water. Dilute to 2000 mL with deionized water.

Regenerant

2 4 Sulfuric acid (H SO ), concentrated (sp.gr. 1.84) 2.8 mL

 Deionized water up to 2000 mL

 

2 4 In a 2000 mL volumetric flask, add 2.8 mL of concentrated H SO to approximately 1500 mL of deionized water. Dilute to 2000 mL

with deionized water. The regenerant reservoir must be filled to the absolute top of the container. CBL’s system holds six liters of

acid.

This eluent/regenerant system is called displacement chemical regeneration. As the regenerant is used up, the used eluent takes its

place in the reservoir. It is crucial that every time that the eluent is changed, that the regenerant is also changed.
25

Standards

Stock Sodium Chloride Standard

Sodium Chloride (NaCl), dried at 45° C 1.6485 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 1.6485 g of sodium chloride in ~ 800 mL deionized water. Dilute to 1000 mL with deionized

water (1mL contains 1 mg Cl ). -

Stock Sodium Sulfate Standard

Sod 2 4 ium sulfate (Na SO ), dried at 45° C 1.8141 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 1.8141 g of sodium sulfate in ~ 800 mL deionized water. Dilute to 1000 mL with deionized

water 4 (1mL contains 1 mg SO ). -2

Stock Sodium Nitrate Standard

Sod 3ium nitrate (NaNO ), dried at 45° C 6.0679 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 6.0679 g of sodium nitrate in ~ 800 mL deionized water. Dilute to 1000 mL with deionized

water 3 (1mL contains 1 mg NO -N). -

Working 5:5:0.5 Chloride/Sulfate/Nitrate Standard (mg/L)

Stock Sodium Chloride Standard 0.5 mL

Stock Sodium Sulfate Standard 0.5 mL

Stock Sodium Nitrate Standard 0.05 mL

Deionized water up to 100 mL

In a 100 mL volumetric flask, dilute 0.5 mL of stock sodium chloride standard, 0.5 mL stock sodium sulfate standard and 0.05

mL stock sodium nitrate standard to 100 mL with deionized water. The final concentration of this standard is 5 mg Cl /L, 5 mg -

SO4 3 /L and 0.5 mg NO -N/L. 

-2 -

A comparable procedure may be used to make different working standards based on the

concentrations of the samples to be analyzed.

Procedure

Each sample to be analyzed must be pre-treated to remove organic particles that may bind to the column beads, reducing column

efficiency, reliability and also column life. Organic particles are removed by a BakerBond SPE C-18 column and filtration

through a Gelman or Whatman 0.2 μm Acrodisk designed for use with an IC system.

1. To prepare the C-18 columns, pass one column volume of HPLC grade methanol through each column. Then pass three

column volumes of deionized water through each column.

2. To prepare a sample, first remove all the deionized water from the column and then immediately place 1 mL of sample into the

column.

3. Place an acrodisk at the end of the column containing the sample.

4. Pass the sample through the column using positive pressure (pushing air through a syringe). Discard the first five drops.
26

Capture the next 11 drops in a 0.5 Ml Dionex sample vial, place a Dionex sample cap on top of the vial and refrigerate until

analysis.
27

ORGANIC ANALYTES

Rationale

Methods for measuring dissolved organic carbon, nitrogen and phosphorus are described in the following

sections. Dissolved organic carbon concentrations are determined by high temperature combustion. For

total dissolved nitrogen and phosphorus, potassium persulfate is added to a sample, which, when under

heat and pressure, breaks down the organic constituents to inorganic forms. Inorganic fractions then are

subtracted from the total dissolved sample to yield the dissolved organic concentration. See Figures 6

and 7.

Sampling and Storage

 Collected water samples are filtered through Whatman GF/F filters (nominal pore size 0.7 :m) and

placed in appropriate containers and preserved. Table 2 presents sampling and storage practices for

organic analytes. Containers for dissolved organic carbon are acid washed in 10% HCl and rinsed

thoroughly with deionized water. Containers for nitrogen and phosphorus are autoclaved with potassium

persulfate solution before use, then rinsed thoroughly with deionized water only.

Table 2. Sampling and storage for organic analytes

ANALYTE VOLUME (mL) STORAGE CONTAINER

Dissolved organic carbon -30 -20°C Teflon or Borosilicate Glass

Vial1

Dissolved nitrogen/phosphorus 10 -20°C Borosilicate Glass Screw-cap

Tube

 Teflon vials can be used for all samples. Wheaton-33 low extractable borosilicate glass vials 1 ®

(recommended by EPA) are used for saltwater samples.

Figure 6. Flow diagram of nitrogen analysis (TN = total nitrogen, PN = particulate nitrogen, TDN =

total dissolved nitrogen, DIN = dissolved inorganic nitrogen, DON = dissolved organic nitrogen).
28

Figure 7. Flow diagram of phosphorus analysis (TP = total phosphorus, PP = particulate phosphorus,

TDP = total dissolved phosphorus, DIP = dissolved inorganic phosphorus, DOP = dissolved organic

phosphorus).
29

TOTAL DISSOLVED NITROGEN Revised 2/2004

AND PHOSPHORUS

This method is a persulfate oxidation technique for nitrogen and phosphorus where, under initially

alkaline conditions, nitrate is the sole nitrogen product. Phosphate is the sole phosphorus product after

acidic conditions are achieved following further autodecomposition of the persulfate in the heated

oxidation tubes..

Digested samples are passed through a granulated copper-cadmium column to reduce nitrate to nitrite.

The nitrite then is determined by diazotizing with sulfanilamide and coupling with N-1-

naphthylethylenediamine dihydrochloride to form a colored azo dye. Color is proportional to nitrogen

concentration.

Ammonium molybdate and antimony potassium tartrate react in an acid medium with dilute solutions of

phosphorus to form an antimony-phospho-molybdate complex which is reduced to an intensely bluecolored complex by ascorbic acid. Color is proportional to phosphorus concentration.

Methodology

D'Elia, C.F., P.A. Steudler and N. Corwin. 1977. Determination of total nitrogen in aqueous samples

using persulfate digestion. Limnol. Oceanogr. 22:760-764.

Valderrama, J.C. 1981. The simultaneous analysis of total nitrogen and total phosphorus in natural

waters. Mar. Chem. 10:109-122.

Instrumentation

Technicon AutoAnalyzer II

Manifold Assembly

Figure 8. Manifold assembly for total dissolved phosphorus method.
30

Figure 9. Manifold assembly for total dissolved nitrogen method.

Specifications

Damp: Normal

Sampling Rate: 40/hour, 9:1 sample/wash ratio

Filters: 550 nm for nitrate and 880 nm for phosphate

Phototubes: 199-B021-01 for nitrate and 199-B021-04 for phosphate

Flowcell: 50 mm

Relat 3ive Absorbance (40 μM NO -N; ORN/YEL sample tube): ~.59 -

Relative Absorbance (18 μM P): ~.85

Interferences: Metal ions may interfere if present in sufficient concentrations. The presence of high

concentrations of sulfate will reduce the sensitivity of the copper-cadmium column. Silicon at a

concentration of 100 :M Si causes interferences equivalent to -0.04 :M P.

Reagents

Oxidizing Reagent - Low, Moderate and High N and P Concentrations

Sodium hydroxide (NaOH) 3 g

Potass 2 2 8ium persulfate (K S O ), low N (<0.001 %) 20.1 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 3 g of sodium hydroxide and 20.1 g of potassium persulfate in -800 mL of deionized

water. Dilute to 1000 mL with deionized water. Make fresh daily.
31

Oxidizing Reagent - Very High N and P Concentrations

Sodium hydroxide (NaOH) 3 g

Potass 2 2 8ium persulfate (K S O ), low N (<0.001 %) 20.1 g

Deionized water up to 3000 mL

In a 3000 mL volumetric flask, dissolve 3 g of sodium hydroxide and 20.1 g of potassium persulfate in -800 mL of deionized

water. Dilute to 3000 mL with deionized water. Make fresh daily.

Buffer Solution

Boric acid (H3 3 BO ) 61.8 g

Sodium hydroxide (NaOH) 8 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 61.8 g of boric acid in -300 mL deionized water. Add 8 g of sodium hydroxide and

dilute to 1000 mL with deionized water. The solution is stable for several weeks.

Internal Standards

Stock Glutamic Acid Standard, 5,040 :M

Glutam 3 2 2 2ic acid [C H NH (COOH) ], dried at 45°C 0.3705 g

Deionized water up to 500 mL

Ch 3loroform (CHCl ) 0.5 mL

In a 500 mL volumetric flask, dissolve 0.3705 g of glutamic acid in -400 mL of deionized water and dilute to 500 mL with

deionized water. Add 0.5 ml of chloroform as a preservative (1 mL contains 5.04 :moles N).

Working Glutamic Acid Standard - Low, Moderate and High N and P Concentrations

Stock Glutamic Acid Standard 1 mL

Deionized water up to 100 mL

In a 100 mL volumetric flask, dilute 1 mL of Stock Glutamic Acid Standard to 100 ml with deionized water to yield a

concentration of 50.4 :M N (0.7056 mg N/L).

Working Glutamic Acid Standards - Very High N and P Concentrations

Stock Glutamic Acid Standard 6 mL

Deionized water up to 100 mL

In a 100 mL volumetric flask, dilute 6 mL of Stock Glutamic Acid Standard to 100 mL with deionized water to yield a

concentration of 302.4 :M N (4.23 mg N/L).

Stock Glycerophosphate Standard, 309 :M

$-Glycerophosphoric acid, disodium salt, 5-hydrate

2 2 3 2 2

[(HOCH ) CHOPO Na @5 H O] 0.0473 g

Deionized water up to 500 mL

Ch 3loroform (CHCl ) 0.5 mL

In a 500 mL volumetric flask, dissolve 0.0473 g of $-Glycerophosphoric acid, disodium salt, 5-hydrate in -400 mL of deionized

water. Dilute to 500 mL with deionized water. Add 0.5 mL of chloroform as a preservative ( 1 mL contains 0.309 :moles P).
32

Working Glycerophosphate Standard - Low, Moderate and High N and P Concentrations

Stock Glycerophosphate Standard 1 mL

Deionized water up to 100 mL

In a 100 mL volumetric flask, dilute 1 mL of Stock Glycerophosphate Standard to 100 mL with deionized water to yield a

concentration of 3.09 :M P (0.096 mg P/L).

Working Glycerophosphate Standard - Very High N and P Concentrations

Stock Glycerophosphate Standard 2 mL

Deionized water up to 100 mL

In a 100 mL volumetric flask, dilute 2 mL of Stock Glycerophosphate Standard to 100 mL with deionized water to yield a

concentration of 6.18 :M P (0.192 mg P/L).

Working Inorganic Standards

Stock Nitrate Standard, 5,000 :M

Potass 3ium nitrate (KNO ), dried at 45°C 0.5055 g

Deionized water up to 1000 mL

Ch 3loroform (CHCl ) 1 mL

In a 1000 mL volumetric flask, dissolve 0.5055 g of potassium nitrate in 1000 mL of deionized water. Add 1 mL of chloroform

as a preservative (1 mL contains 5 :moles N).

Working Nitrate Standards - Low N and P Concentrations

Dilute 0.5, 1.0 and 1.5 mL of Stock Nitrate Standard to 100 mL with deionized water to yield concentrations of 25 :M N (0.35

mg N/L), 50 :M N (0.70 mg N/L) and 75 :M N (1.05 mg N/L), respectively.

Working Nitrate Standards - Moderate N and P Concentrations

Dilute 0.5, 1.0 and 2.0 mL of Stock Nitrate Standard to 100 mL with deionized water to yield concentrations of 25 :M N (0.35

mg N/L), 50 :M N (0.70 mg N/L) and 100 :M N (1.40 mg N/L), respectively.

Working Nitrate Standards - High N and P Concentrations

Dilute 1.0, 2.0 and 4.0 mL of Stock Nitrate Standard to 100 mL with deionized water to yield concentrations of 50 :M N (0.70

mg N/L), 100 :M N (1.40 mg N/L) and 200 :M N (2.80 mg N/L), respectively.

Working Nitrate Standards - Very High N and P Concentrations

Dilute 3.0, 6.0 and 8.0 mL of Stock Nitrate Standard to 100 mL with deionized water to yield concentrations of 150 :M N (2.1

mg N/L), 300 :M N (4.2 mg N/L) and 400 :M N (5.6 mg N/L), respectively.

Stock Orthophosphate Standard, 12,000 :M

Potassium phosphate, monobasic

2 4 (KH PO ), dried at 45°C 1.632 g

Deionized water up to 1000 mL

Ch 3loroform (CHCl ) 1 mL
33

In a 1000 mL volumetric flask, dissolve 1.632 g of potassium phosphate in - 800 mL of deionized water. Dilute to 1000 mL with

deionized water. Add 1 mL of chloroform as a preservative (1 mL contains 12 :moles P).

Secondary Orthophosphate Standard

Stock Orthophosphate Standard 1 mL

Deionized water up to 100 ml

In a 100 mL volumetric flask, dilute 1 mL of Stock Orthophosphate Standard to 100 mL with deionized water (1 mL contains

0.12 :moles P).

Working Orthophosphate Standards - Low, Moderate and High N and P Concentrations

Dilute 0.5, 2.0 and 4.0 mL of Secondary Orthophosphate Standard to 100 mL with deionized water to yield concentrations of 0.6

:M P (0.0186 mg/L), 2.4 :M P (0.0744 mg/L) and 4.8 :M P (0.1488 mg/L), respectively.

Working Orthophosphate Standards - Very High N and P Concentrations

Dilute 2.0, 5.0 and 10.0 mL of Secondary Orthophosphate Standard to 100 mL with deionized water to yield concentrations of

2.4 :M P (0.0744 mg/L), 6.0 :M P (0.186 mg/L) and 12.0 :M P (0.372 mg/L), respectively.

Procedure

1. Select appropriate standards and oxidizing reagent for samples to be analyzed. For 0 - 200 :M N and 0 - 5.0 :M P, use

10 mL samples and 5 mL of Oxidizing Reagent for Low, Moderate and High N and P Concentrations, following steps 2 -

9. For 150 - 400 :M N and 2.4 - 12.0 :M P, use 5 mL samples and 15 mL of Oxidizing Reagent for Very High N and P

Concentrations, following steps 2VH - 9VH.

2. Place 10 mL of filtered water (Whatman GF/F filter, 0.7 :m) in a 30 mL screw cap test tube and freeze.

2VH. Place 5 mL of filtered water (Whatman GF/F filter, 0.7 :m) in a 30 mL screw cap test tube and freeze.

3. Place 10 mL of each standard (3 replicates of each) in 30 mL screw cap test tubes and treat exactly as samples.

3VH. Place 5 mL of each standard (3 replicates of each) in 30 mL screw cap test tubes and treat exactly as samples.

4. When ready to analyze, thaw samples at room temperature.

5. Add 5 mL of Oxidizing Reagent. A precipitate will form with seawater samples. Cap test tubes fairly tightly. Invert

twice.

5VH. Add 15 mL of Oxidizing Reagent. A precipitate will form with seawater samples. Cap test tubes fairly tightly. Invert

twice.

6. Place test tubes in a pressure cooker at 100 - 110°C and 3 - 4 psi for 60 minutes. Bring back to atmospheric pressure over

1 hour.

7. Remove test tubes, tighten caps and cool to room temperature. Samples can be stored at this point.

8. Add 1 mL of Buffer Solution to each tube and shake. The pH of the sample should be 4 - 4.5 after the addition of the

buffer solution. Transfer an aliquot of each sample to AutoAnalyzer cups using a Pasteur pipette.

9. Analyze for nitrite + nitrate and orthophosphate as described in the Dissolved Inorganic Analytes section. Use the pump

3 4 tubes for low sample concentrations of 0.00 - 1.00 mg N/L; ORN/YEL NO sample tube and YEL/BLU NH Cl tube, for -

modera 3 4 te sample concentrations of 0.35 - 1.4 mg N/L; ORN/GRN NO sample tube and YEL/BLU NH Cl tube, for high -

3

sample concentrations of 0.70 - 2.80 mg N/L; ORN/WHT NO sample tube and YEL/YEL deionzed water diluent on the -
34

dilu 4tion loop "resampled" to a BLK/BLK resample tube and YEL/YEL NH Cl tube.

9VH. Analyze for nitrite + nitrate and orthophosphate as described in the Dissolved Inorganic Analytes section. Use a

BLK/BLK NO3 sample tube and YEL/YEL deionzed water diluent on the dilution loop "resampled" to a BLK/BLK -

4

resample tube and YEL/YEL NH Cl tube.

NOTES:

1. The use of internal organic standards (glutamic acid and glycerophosphate) allows the check for percentage recovery and is

incorporated into each digestion batch.

2. The procedure includes an internal dilution factor of samples and standards due to addition of reagents of 1:1.6 for low N

and P concentration samples and 1:4.2 for very high N and P concentration samples.

3. Reagent Blanks: Oxidizing reagent only (3 replicates) is digested in 30 mL test tubes, and buffered. The analyzed peak

heights of the nitrate and phosphate reagent blanks are normalized to the sample + reagent volume by adding 10 mL of

deionized water immediately before analyzing for nitrite + nitrate and phosphate. The resultant reagent blank peak height

then is subtracted from the sample peak heights before calculating the concentrations based on the regression from the peak

heights of the standards. Reagent blank for N should be #8% of the peak height of the 75:M N standard. Reagent blank

for P should be #6% of the 4.8 :M P standard.

4. Deionized Water Blanks: 10 mL of deionized water (3 replicates) are digested, and analyzed with the samples and

standards. Their peak heights are included with the peak height of the digested inorganic working standards in the

regression to obtain the slope used to calculate concentrations of the samples. The value of the reagent blank is generally

about 0 - 5 chart lines less than the value of the y intercept obtained in the regression of inorganic working standards and

deionized water blanks.
35

TOTAL AND DISSOLVED ORGANIC Revised 02/2004

CARBON

The Shimadzu TOC 5000/5000A utilizes high temperature combustion in determining organic carbon,

and is used to analyze all ranges of salinity. 

SHIMADZU TOC-5000 TOTAL ORGANIC CARBON ANALYZER METHOD

Methodology

Sugimura, Y. and Y. Suzuki. 1988. A high temperature catalytic oxidation method for the determination

of non-volatile dissolved organic carbon in seawater by direct injection of a liquid sample. Mar.

Chem. 24:105-131.

Instrumentation

The Shimadzu TOC-5000/5000A uses a high temperature combustion method to analyze aqueous samples

for TIC, TOC and non-purgeable organic carbon (NPOC). Samples are treated with hydrochloric acid

and sparged with ultra pure carrier grade air to drive off inorganic carbon. High temperature combustion

(680°C) on a catalyst bed of platinum-coated alumina balls breaks down organic carbon into carbon

diox 2 2 ide (CO ). The CO is carried by ultra pure air to a non-dispersive infrared detector (NDIR) where

CO2 is detected.

Specifications

Analytical Range: 100 ppb - 4000 ppm using 250 :L syringe and 4 - 100 :L injection volume, using

regular sensitivity catalyst.

Calibration: Up to 4 concentrations may be used to calibrate the instrument

Reagents

Hydrochloric Acid, 2 N

Hydrochloric acid (HCl), concentrated 172 mL

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, add 172 mL of concentrated hydrochloric acid to -600 mL of deionized water. Dilute to 1000 mL

with deionized water.

Standard

Stock Potassium Hydrogen Phthalate (KHP) Standard, 1000 mg/L

Potas 6 4 sium hydrogen phthalate (HOCOC H COOK),

dried at 45°C 2.125 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 2.125 g of potassium hydrogen phthalate in -800 mL of deionized water. Dilute to 1000
36

mL with deionized water. Make fresh every 4 - 6 months. Store at 4°C.

Procedure

1. Samples are stored frozen at -20°C. When ready to analyze, thaw samples at room temperature and transfer to

autosampler vials.

2. Make 3 working standards (generally <20 mg/L) from the Stock KHP Standard for the standard curve, and load into

autosampler. Use deionized water as the zero concentration. Numerous standard concentrations may be analyzed as

samples.

3. Acidify each sample with 100 :L of 2 N Hydrochloric Acid and add 250 :L of 2 N Hydrochloric Acid to each standard

vial. It is possible to program the instrument to do this automatically.

4. The standards and samples are sparged with ultra pure carrier (UPC) grade Air for 6 minutes at 60 mL/minute, driving

off any inorganic carbon in the sample.

5. Standards and samples are injected onto a catalyst bed of platinum-coated alumina balls and combusted at 680°C. The

2

resulting CO gas is carried to the NDIR detector by the UPC grade Air.

6. For the standard curve, use the area of the deionized water as the water blank in calculating the 4 standard

concentrations.

7. Calculate a linear regression of the 4 standard concentrations, first subtracting the water blank from each standard.

8. The absolute value of the y-intercept is an indicator of the instrument blank and is subtracted from the area of the

samples. The water blank includes the instrument blank, so only one value is subtracted from the standards.

Calculation of TOC

TOC concentration is calculated using the following equation:

whe sre: A = area of the sample,

b = y-intercept, and

m = slope of the regression line.

Dissolved inorganic carbon, carbonate alkalinity, and hardness:

Dissolved inorganic carbon also can be determined with direct reaction with 25% phosphoric acid. Once

the inorganic concentration is determined, carbonate alkalinity can be calculated using the following

equation:
37

NOTE: This method can also be used on saline waters of < 10 ppt salinity.

Titrimetric hardness determination is appropriate for freshwater samples (APHA, 1975).

Methodology

APHA. 1975. Method 309B. EDTA Titrimetric Method in Standard Methods for the Examination of

Water and Wastewater, 14 Edition. American Public Health Association. Washington, D.C. th

1193pp.
38

PARTICULATE ANALYTES

Rationale

The direct measurement of particulate carbon, particulate nitrogen and particulate phosphorus is the

preferred method used by the Nutrient Analytical Services Laboratory. A large volume can be filtered

onto the pad, yielding a representative sample. The alternative, subtraction of the dissolved concentration

from the total sample concentration to determine the particulate carbon, nitrogen or phosphorus

concentration, is imprecise, sometimes yielding negative concentrations. Direct measurement is rapid,

sensitive and more precise.

Instrumentation

Particulate phosphorus and biogenic silica procedures require the use of a segmented continuous flow

analyzer such as the AutoAnalyzer II, previously described in the section Dissolved Inorganic Analytes. 

Particulate carbon and particulate nitrogen procedures require the use of an elemental analyzer.

Sampling and Storage

 A known volume of the collected water is filtered through Whatman GF/F filters (25 mm for particulate

carbon and nitrogen, and 47mm for particulate phosphorus, nominal pore size 0.7 :m). The filter is

folded, placed in an aluminum foil pouch and frozen until analysis. For biogenic silica, water is filtered

through a 0.4 μm Nuclepore polycarbonate filter. The filter is placed in a 50 mL plastic centrifuge tube

and stored in a refrigerator. 

Sediment samples are collected, dried and ground with a mortar and pestle to thoroughly blend the

sample.
39

PARTICULATE CARBON AND NITROGEN Revised 2/2004

Samples a 2re combusted in pure oxygen (O ) under static conditions. Products of combustion are passed

over suitable reagents in the combustion tube where complete oxidation occurs. In the reduction tube,

ox 2ides of nitrogen (N) are converted to molecular N. The carbon dioxide (CO ), water vapor and N are

mixed and released into the thermal conductivity detector where the concentrations of the sample gases

are measured.

Instrumentation

Exeter Analytical, Inc. (EAI) CE-440 Elemental Analyzer

Operating Principles

2 2 2

C Carbon (as CO ), hydrogen (as H O) and nitrogen (N ) content in organic and inorganic compounds

can be determined.

2

C Combustion of the weighed or filtered sample occurs in pure O under static conditions (Figure 13).

Figure 10. Schematic diagram of the Exeter Analytical, Inc. (EAI) CE-440 Elemental Analyzer.
40

C Helium (He, ultra-pure carrier grade) carries the combustion products through the analytical system

to the atmosphere. Helium also purges the instrument.

C Solenoids A - G control the gas flow through the system. Valves H and I are used for automatic leak

testing.

C Products of combustion pass over suitable reagents in the combustion tube where complete oxidation

occurs.

2

C In the reduction tube, oxides of N are converted to molecular N. The CO , water vapor and N are

then flushed into a mixing volume where they are thoroughly homogenized at a precise volume,

temperature and pressure. The mixture is released through the sample volume into the thermal

conductivity detector.

C Between the first of three pairs of thermal conductivity cells, an absorption trap removes water

2

(H O) from the sample gas. The differential signal read before and after the trap reflects the amount

of 2 hydrogen (as H O) in the original sample. A similar measure is made of the signal output of a

2

second pair of thermal conductivity cells between which a trap removes CO . The remaining gas

consists only 2 of N and He. This gas passes through a thermal conductivity cell and the output

2

signal is compared to a reference cell through which pure He flows. This gives the N concentration.

Standard

Standard is acetanilide 3 6 5 (CH CONHC H ) stored at 45°C. Composition of acetanilide is 71.09% C, 6.71%

H and 10.36% N.

Procedure

1. Filter a known volume of sample water through a Whatman GF/F filter pad (25 mm; 0.7 :m pore size; pre-combusted in

oven set at 500°C for 90 minutes and cooled to room temperature in a dessicator. This setting on CBL’s oven has been

determined to be 550°C.) Filter samples in duplicate for back-up and 10% QA/QC.

2. Fold filter pads in half inward, and place in an aluminum foil pouch. Label and freeze for later analysis.

3. When ready for analysis, remove aluminum foil pouches from freezer and place in a drying oven at 45°C overnight.

4. Load nickel sleeves (pre-combusted at 875°C for 90 minutes and cooled to room temperature) into the filter ram. Load

filter pads into the nickle sleeves. Place loaded nickel sleeves into the sample wheel.

5. Dry sediment samples at 45°C overnight and weigh into aluminum capsules (pre-combusted at 550°C for 90 minutes and

cooled to room temperature). Load nickel sleeves into the sample wheel and place aluminum capsules containing the

sediment samples into the nickel sleeves.

6. Load standards (-1500 :g acetanilide) and blanks (empty capsule for standards and weighed samples; filter pad for filtered

samples) into the sample wheel. Approximately 50 samples can be run per day. 

7. Place 3 standards at the beginning of each run to determine the K-Factor.

8. Insert standard checks between every 15 - 20 samples and at the end of each run.
41

The Run Cycle

1. At the start of each run, the entire system is flushed with helium (He) at a high flow rate while the sample is in the cool

zone.

2. The injection box is automatically purged using the P valve.

3. The combust 2ion train is filled with O and the sample is injected.

4. Shortly after sample injection, D valve closes to seal off the combustion train from the rest of the analytical system that is

still being flushed with He.

5. Combust 2 ion occurs under static conditions in an excess of O at about 975°C. During this time, the mixing volume is being

purged with E and F valves open.

6. Valve F closes to allow the pressure in the mixing volume to reach atmospheric pressure.

7. Close to the end of the combustion period, a high-temperature heating coil around the combustion tube vaporizes any

condensates at the entrance of the combustion tube that may have been produced by diffusion of the sample during initial

stages of combustion.

8. To a 2ssure complete combustion, the ladle is retracted -1 inch, a small amount of O is added and the ladle is fully injected.

9. During high heat, valve E closes, A and D re-open and the combustion products are completely flushed from the

combustion train into the mixing volume.

10. When a pressure of 1500 mm Hg is reached, valve D closes, trapping the sample gas in the mixing volume. The time

required to reach this pressure is called "fill time" and is usually 20 - 50 seconds.

11. The combustion train remains under positive pressure until the end of the complete cycle.

12. While the sampling gases are mixing, pure He flows from valve C through the sample volume and through the detectors.

13. The signal from each detector bridge is read and stored in memory to provide a baseline reading with no sample gas in the

detector.

14. After mixing is complete and baseline reading is set, F and G open allowing the sample gas captured in the mixing volume

to expand through the sample volume to the atmosphere. During this time, valve C is closed and there is low flow through

the detector.

15. When 2 2 2 sample gases are near atmospheric pressure, valves F and G close and C opens. The H O, CO and N concentrations

of the sample are measured by displacing the sample gas through the detectors to the atmosphere.

16. The volume of sample gas in the system is large enough so that the He flow allows measurement of the contents of each

detector in sequence, under steady-state conditions.

17. The sample gas passes through the detectors at a constant flow, pressure and temperature. This eliminates any variation in

water vapor concentration due to changes in water adsorption on the walls of the pneumatic system.

18. While the sample gas is displaced through the detectors, the output signals are recorded.

19. The difference in microvolts (:V) between each "read" signal and the baseline ("zero") level for the same detector is

directly proportional to the concentration of the sample gas measured.

20. At the end of a cycle, the exhaust valves open to allow the sample gases to escape to the atmosphere.

21. The computer then prints out the calculated results, places the instrument in standby with C valve open and waits for the

next command.
42

22. With the HA automatic injector, the results are printed after each run. The run cycle continues until the pre-selected

number of runs have been completed.

Definition of Terms

Blank: Blank value = blank read (see Read Signal below) minus blank zero (see Zero Value below).

Capsule: Pre-combusted aluminum or nickel container. Used for sealing samples with an accurate

weight and maintains integrity prior to combustion.

Combustion 2 Time: Time for sample to fully combust in O environment.

Combustion Tube: Quartz tube used for packing reagents and for sample combustion.

Conditioner: Coats the walls of the system surfaces, particularly the mixing and sample volumes, with

H2 2 O 2 vapor, CO and N that simulate actual sample running conditions. The conditioner we use is

acetanilide as recommended by the manufacturer. A sample is considered conditioner when placed

before a blank or a standard in a run. Blanks should be run immediately after a conditioner.

Detector: The heart of the analyzer consisting of three bridges. Determines the percentages of C, H and

N in the sample via thermal conductivity.

Detector Oven: Keeps the temperature of the detector, pressure transducer, mixing volume and sample

volume constant.

Double Drop: On HA automation, two samples are dropped for one run used for >25 mm filters. 

Sample ID requires a "+" prefix.

Fill Time: Time required to build up the pressure in the mixing volume to 1500 mm Hg.

Furnace: Heats the reduction and combustion tubes to operating temperature.

Injection: Moving the ladle into the combustion furnace.

Injection Box: For the HA automation, the box assembly that houses the sample wheel.

K-Factor: Instrument sensitivity factor in :V/:g, calibrated using a chemical standard.

Ladle: Transports the boat or capsule containing the sample into the combustion furnace.

Mixing Volume: Spherical bottle in which sample gases become homogenous.

Mother Board: The main circuit board. Where all CE-440 power supplies are located.

Read Signal: Steady-state signal produced by the detector when sample gases are present in stable

concentration.
43

Reduction 2 Tube: Quartz tube with reduced copper that removes excess O from the sample gas and

2

reduces oxides of N to free N.

Run: One sample analysis from start to finish, including print-out.

Run Cycle: The entire analytical sequence of runs from the first run to the last run in a day, including

the transfer of the run cycle data to the disk.

Sample Volume: Tube where sample gas is exhausted from the mixing volume prior to entering the

detector.

Scrubber 2s: Removes water and CO from gas supplies.

Traps: 2 Used for removing water and CO from the sample gas.

Zero Value: Bridge signal with only pure He flowing through the detector.

Calibration

The following formula is used to calculate K-Factors, as well as C, N and H concentrations in unknown

samples:

where: K = calibration factor of the instrument,

W = sample weight,

R = read signal of sample gas,

Z = zero reading or instrument baseline, and

B = blank signal (instrument, ladle and capsules).

Always run a conditioner before a standard and before and after a blank.

K-Factors vary greatly among instruments but should be within the following ranges:

C

 K = 18 - 25

H

 K = 55 - 76

KN = 7 - 10
44

PARTICULATE PHOSPHORUS Revised 2/2004

AND

PARTICULATE INORGANIC PHOSPHORUS

Ammonium molybdate and antimony potassium tartrate react in an acid medium with dilute solutions of

phosphorus to form an antimony-phospho-molybdate complex which is reduced to an intensely bluecolored complex by ascorbic acid. Color is proportional to phosphorus concentration.

Methodology and Related Reference

Aspila, I., H. Agemian and A.S.Y. Chau. 1976. A semi-automated method for the determination of

inorganic, organic and total phosphate in sediments. Analyst 101:187-197.

Keefe, C.W. 1994. The Contribution of Inorganic Compounds to the Particulate Carbon, Nitrogen, and

Phosphorus in Suspended Matter and Surface Sediments of Chesapeake Bay. Estuaries 17:122-130.

Instrumentation

Technicon AutoAnalyzer II with IBM compatible Bran and Luebbe AACE ver. 5.22 Software data

collection system.

Manifold Assembly

Figure 11. Manifold assembly for particulate phosphorus method.
45

Specifications

Standard Calibration Setting: 4.0

Damp: Normal

Sample Rate: 40/hour, 9:1 sample/wash ratio

Filter: 880 nm

Phototube: 199-B021-04

Flowcell: 50 mm

Relative Absorbance (48 μM P): ~.27

Interferences: Silicon (Si) at analysis temperature >40°C and/or <2.2 N sulfuric acid in the mixed

reagent solution causes interference in the concentration range of >0.05 mg/mL Si in the extract. These

conditions are avoided by maintaining an acid concentration of 2.45 N sulfuric acid in the reagents and

analysis at 37°C.

Reagents

Hydrochloric Acid, 1 N

Hydrochloric acid (HCl), concentrated (sp. gr. 1.9) 86 mL

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, add 86 mL of concentrated hydrochloric acid to -800 mL of deionized water. After cooled, dilute

to 1000 mL with deionized water.

Deionized Water Diluent

Sod 3 2 11 3ium dodecyl sulfate, (SDS) [CH (CH ) OSO Na]

m.w. = 288.38; phosphate #0.0001 %] 0.05 g

Deionized water up to 500 mL

Add 0.05 g of sodium dodecyl sulfate to 500 mL deionized water. Mix well.

Sulfuric Acid Solution , 4.9 N

Su 2 4 lfuric acid (H SO ), concentrated (sp. gr. 1.84) 136 mL

Deionized water up to 1000 mL

To a 1000 mL volumetric flask, add 136 mL of concentrated sulfuric acid to approximately 800 mL of deionized water while

cooling (cold water bath). After the solution is cooled, dilute to 1000 mL with deionized water.

Ammonium Molybdate Solution

Ammon 4 6 7 24 2ium molybdate [(NH ) Mo O @ 4 H O] 40 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 40 g of ammonium molybdate in 800 mL of deionized water. Dilute to 1000 mL with

deionized water. Store in plastic bottle away from direct sunlight.

Ascorbic Acid Solution

Asco 686 rbic acid (C H O ) 18 g

Deionized water up to 1000 mL

Sod 3 2 11 3ium dodecyl sulfate, (SDS) [CH (CH ) OSO Na] 1.66 g
46

In a 1000 mL volumetric flask, dissolve 18 g of ascorbic acid and 1.66 g of SDS in 800 mL of deionized water. Dilute to 1000

mL with deionized water and dispense -40 mL into clean polybottles and freeze. Thaw overnight in refrigerator before use.

Antimony Potassium Tartrate Solution

Ant 446 2imony potassium tartrate [K(SbO)C H O @ 1⁄2 H O] 3 g

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, dissolve 3 g of antimony potassium tartrate in 800 mL deionized water. Dilute to 1000 mL with

deionized water.

Working Reagents

Reagent A

Sulfuric Acid Solution, 4.9 N 50 mL

Ammonium Molybdate Solution 15 mL

Antimony Potassium Tartrate Solution 5 mL

SDS 0.15 g

Reagent B

Ascorbic Acid Solution 30 mL

SDS 0.05 g

Standards

Stock Phosphorus Standard, 12,000 :M

Potassium phosphate, monobasic

2 4 (KH PO ), dried at 45°C 1.632 g

Deionized water up to 1000 mL

Ch 3loroform (CHCl ) 1 mL

In a 1000 mL volumetric flask, dissolve 1.632 g of potassium phosphate in -800 mL of deionized water. Dilute to 1000 mL with

deionized water. Add 1 mL of chloroform to act as a preservative (1 mL contains 12 :moles P).

Working Phosphorus Standards

Dilute 0.05, 0.1, 0.2, 0.3 and 0.4 mL of Stock Phosphorus Standard to 100 mL with 1 N Hydrochloric Acid to yield

concentrations of 6 :M P (0.185 mg P/L), 12 :M P (0.37 mg P/L), 24 :M P (0.74 mg P/L), 36 :M P (1.11 mg P/L) and 48 :M P

(1.48 mg P/L), respectively.

Particulate Phosphorus Procedure

1. Filter a known volume of water through a Whatman GF/F filter (47 mm; 0.7 :m pore size, pre-dried at 103 - 105°C for 24

hours, if also using pad for total suspended solids). Using forceps, fold filter pad in half, sample inside, and place in an

aluminum foil pouch. Collect sediment samples in a clean container.

2. Freeze aluminum foil pouches and/or sediments.

3. If filters are used also for Total Suspended Solids, dry overnight at 103-105 C. Dry sediments at 50°C overnight or until

 o 

dry. Grind sediment samples to blend thoroughly.

4. Fold filters in quarters using forceps and place in numbered crucibles with lids. Weigh sediment into crucibles. In our

laboratory, generally about 25 mg of sediment is used. Cover crucibles. Record sample numbers and corresponding
47

crucible numbers. Prepare 5% of samples in duplicate.

5. Combust samples in crucibles in an oven set at 500 ° C for 90 minutes. This setting on CBL’s oven has been determined

to be 550 ° C.

6. When crucibles are cool, remove from muffle furnace. Place filter or sediment in a labelled 50 mL plastic screw cap

centrifuge tube.

7. Add 10 mL of 1 N Hydrochloric Acid to each tube containing a filter. For sediments, add 20 mL to each tube.

8. Cap tubes and let stand for a minimum of 24 hours. Shake tubes several times during the 24-hour period.

9. Transfer supernatant to an AutoAnalyzer cup with a Pasteur pipette.

10. Analyze for phosphate using an AutoAnalyzer II system equipped with a Teflon sample probe. Spike 5% of samples 1:1

with 1.11 mg P/L standard solution and analyze.

11. Blank filter pads should be carried through the procedure above; approximately one blank per -20 samples.

Calculation of Phosphorus Concentration

Phosphorus concentration is calculated using Bran and Luebbe AACE ver.5.22 software. The calculation

is based on the following equation:

where: B = mean reading of blanks,

F = inverse of the regression slope of the standards,

VE = volume of hydrochloric acid used for extraction (L)

 ( i.e., 0.01 for filters and 0.02 for sediments), and

VF = volume of sample filtered (L).

Technicon AutoAnalyzer II with 50 mm flowcell and STD CAL setting of 4.0 typically gives the

following results:

slope 43.63

r 0.9999 2

Particulate Inorganic Phosphorus Procedure

1. Follow procedure for Particulate Phosphorus Steps 1 through 3. Do not combust the samples. Place filter or weighed

sediment sample directly in labeled 50 mL screw cap centrifuge tube. Follow procedure for Steps 7 through 11. 

2. Calculate Particulate Inorganic Phosphorus concentration.
48

PARTICULATE BIOGENIC SILICA Revised 2/2004

Silicomolybdate is reduced in acid solution to "molybdenum blue" by ascorbic acid. Oxalic acid is added

to eliminate interference from phosphates. Detection of the silicomolybdate complex is by colorimetry.

Methodology

Technicon Industrial Method No. 186-72W/B . Technicon Industrial Systems. Tarrytown, New York, †

10591.

Paasche, E. 1973. Silicon and the ecology of marine plankton diatoms. I. Thalassiosira pseudonana

(Cyclotella nana) grown in a chemostat with silicate as limiting nutrient. Mar. Biol. 19:117-126.

Instrumentation

Technicon AutoAnalyzer II with IBM compatible Bran and Luebbe AACE vers. 5.22 Software data

collection system

Manifold Assembly

Figure 12. Manifold assembly for particulate biogenic silica method.
49

Specifications

Standard Calibration Setting: 5.0

Damp: Normal

Sampling Rate: 40/hour, 9:1 sample/wash ratio

Filter: 660 nm

Phototube: 199-B021-04

Flowcell: 50 mm

Relative Absorbance (42 μM Si): ~.27

Interferences: Tannin, large quantities of iron and sulfide, strong coloration and turbidity may interfere.

Digestion Equipment

2 digital timers

100°C water bath

ice bath

centrifuge tube racks

2 polypropylene re-pipettes

2 1000 mL polypropylene volumetric flasks

Digestion Reagents

Sodium Hydroxide , 0.2 N

Sodium hydroxide (NaOH) 8 g

Deionized water up to 1000 mL

In a 1000 mL polypropylene volumetric flask, dissolve 8 g of sodium hydroxide pellets in -800 mL of deionized water. Dilute to

1000 mL with deionized water.

Sulfuric Acid Solution, 1 N

Su 2 4 lfuric acid (H SO ), concentrated (sp. gr. 1.84) 28 mL

Deionized water up to 1000 mL

In a 1000 mL volumetric flask, carefully add 28 mL of concentrated sulfuric acid to -800 mL of deionized water. When cool,

dilute to 1000 mL with deionized water.

Sample Collection

1. Filter a known volume (volume dependent on water source) of water through a 0.4 :m Nuclepore polycarbonate filter.

®

2. Fold filter in half, sample inside, and place in a labelled 50 mL polypropylene centrifuge tube, cap and refrigerate.

Procedure

1. Fill water bath to depth that will cover liquid in tubes and heat to 100°C.

2. Prepare ice bath.

3. To the sample pad in the centrifuge tube, add 10 mL of 0.2 N Sodium Hydroxide from a polypropylene re-pipette. Make
50

sure that the filter pad is covered by the Sodium Hydroxide. Cap the tube leaving it loosened 1⁄4 turn.

4. Place the centrifuge tube in the 100°C water bath for exactly 20 minutes.

5. After exactly 20 minutes, remove the tube from the hot water bath and place in ice bath for exactly 4 minutes.

6. Add 2.5 mL of 1 N Sulfuric Acid Solution to tube to neutralize. Cap and shake. The samples can be stored now, until

analyzed.

7. Transfer extract to an AutoAnalyzer cup using a polyethylene Pasteur pipette. Avoid particulate pieces. Samples may need

to be centrifuged at 3000 rpm for 10 minutes.

8. Analyze for silicon on an AutoAnalyzer II system. 

9. Digest blanks and standards interspersed with samples. Digest 5% of samples in duplicate and spike 5% of sample

duplicates with 0.4 mL of Secondary Silica Standard (2.5 :M Si/mL). Analyze blanks and standards before samples to

establish standard curve.

Analysis Reagents

Oxalic Acid Solution

Oxa 2 2 4 2lic acid (H C O @2H O) 50 g

Deionized water up to 1000 mL

In a 1000 mL polypropylene volumetric flask, dissolve 50 g of oxalic acid in -900 mL deionized water and dilute to 1000 mL

with deionized water.

Ascorbic Acid Solution

Asco 686 rbic acid (C H O ), U.S.P. quality 17.6 g

Ace 3 2 tone [(CH ) CO] 50 mL

Sod 3 2 11 3ium dodecyl sulfate, (SDS) [CH (CH ) OSO Na],

m.w. = 288.38; phosphate #0.0001 %] 0.3 g

Deionized water up to 1000 mL

In a 1000 mL polypropylene volumetric flask, dissolve 17.6 g of ascorbic acid in 500 mL of deionized water and 50 mL of

acetone. Mix and dilute to 1000 mL with deionized water. Add 0.3 g of sodium dodecyl sulfate. Dispense 200 mL aliquots into

poly bottles and freeze.

Sulfuric Acid Solution, 0.07 N

Su 2 4 lfuric acid (H SO ), concentrated (sp. gr. 1.84) 1.96 mL

Deionized water up to 1000 mL

In a 1000 mL polypropylene volumetric flask, add 1.96 mL of concentrated sulfuric acid to -800 mL of deionized water. Dilute

to 1000 mL with deionized water.

Ammonium Molybdate Solution

Ammon 4 6 7 24 2ium molybdate [(NH ) Mo O @4 H O] 7 g

Sulfuric Acid Solution, 0.07 N up to 1000 mL

Dissolve 7 g of ammonium molybdate in 1000 mL of 0.07 N Sulfuric Acid Solution. Store in an amber plastic container in the

dark. Prepare fresh every few days.
51

Standards

Stock Silica Standard, 10,000 :M

Sod 2 6 ium silicofluoride (Na SiF ), dried at 45°C 1.88 g

Deionized water up to 1000 mL

In a 1000 mL polypropylene volumetric flask, dissolve 1.88 g of sodium silicofluoride in -800 mL of deionized water. Dilute to

1000 mL with deionized water (1 mL contains 10 :moles Si).

Secondary Silica Standard

Stock Silica Standard 25 mL

Deionized water up to 100 mL

In a 100 mL polypropylene volumetric flask, dilute 25 mL of Stock Silica Standard to 100 mL with deionized water (1 mL

contains 2.5 :moles Si).

Working Silica Standards

To labelled 50 mL polypropylene screw cap centrifuge tubes, add 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mL of Secondary Silica

Standard. Digest standards as samples. After digestion, standard tubes will contain 0, 7.0, 14.0, 21.1. 28.1, 35.1 and 42.2 :g Si,

respectively.

NOTE: The use of glassware with this method should be avoided because it may be a source of Si contamination. All chemicals

used for reagents and the deionized water should be of good quality and low in Si concentration.

Calculation of Silica Concentration

Silica concentration is calculated using Bran and Luebbe AACE ver. 5.22 software. The calculation is

based on the following equation:

where: B = mean reading of the blanks,

F = inverse of the regression slope of the standards, and 

V = volume filtered (L)

Technicon AutoAnalyzer II with 50 mm flowcell and STD CAL setting of 5.0 typically gives the

following results:

slope 1.60

r 0.9998 2
52

TOTAL SUSPENDED SOLIDS Revised 2/2004

Total suspended solids (TSS) is the retained material on a standard glass filter pad after filtration of a

well-mixed sample of water. Results are expressed in mg/L.

Methodology

APHA. 1975. Method 208 D. Total Nonfilterable Residue Dried at 103 - 105 C (Total Suspended

Matter) in Standard Methods for the Examination of Water and Wastewater, 14 Edition. American th

Public Health Association. Washington, D.C. 1193pp. 

USEPA. 1979. Method No. 160.2 (with slight modification) in Methods for chemical analysis of water

and wastes. United States Environmental Protection Agency, Office of Research and Development. 

Cincinnati, Ohio. Report No. EPA-600/4-79-020 March 1979. 460pp.

Specifications

Interferences: Excessive residue may form a water-trapping crust. Sample size should be limited to

yield <200 mg of residue.

Sample Collection and Handling

1. Sequentially number Whatman 47 mm GF/F filters (0.7 :m pore size) along the outside edge, where the sample will not

pass through, using a fine-tipped permanent marker.

2. Dry filters at 103 - 105°C overnight.

3. Weigh filters (in grams) to 4 decimal places to obtain pre-weight. Pre-weighed filters are stored in sealed containers until

ready for use.

4. Filter a known volume of water through the filter pad (conducted in the field). Rinse filter with deionized water to remove

salts. Fold pad in half, sample inside, and place in aluminum foil pouch.

5. Freeze filter pads for storage.

Procedure

1. Dry filters at 103 - 105°C overnight. Allow samples to cool to room temperature in dessicator.

2. Weigh filters and record weights (in grams).

3. Subsample a portion of the filters and replace them in the drying oven for a minimum of 1 hour.

4. Re-weigh subsampled filters. If there is >0.5 mg weight loss between the first and second weight of the subsampled filter

pads, then all filter pads should be re-dried and re-weighed.

5. Repeat steps 3 and 4 as necessary.
53

Calculation of TSS

TSS concentration is calculated using the following equation:

whe post re: W = dry weight of filter pad after filtering (g),

Wpre = dry weight of filter pad before filtering (g), and

V L = volume of water filtered (L).
54

TOTAL VOLATILE SUSPENDED SOLIDS Revised 2/2004

Total volatile solids (TVS) is the volatilized material that is lost on ignition from Total Suspended Solids

(TSS). It is calculated from the measurement of a total suspended solid (TSS) sample minus the

measurement of the quantity remaining after combustion. Results are expressed in mg/L.

 

 Methodology

APHA. 1975. Method 208 E (with modification). Total volatile and fixed residue at 550° C. in Standard

Methods for the Examination of Water and Wastewater, 14 th Edition. American Public Health

Association. Washington, D.C. 1193pp.

 Sample Collection and Handling

1. Combust Whatman 47 mm GF/F filters (0.7 :m pore size) in an oven set at 500° C for 90 minutes. This setting on

CBL’s oven has been determined to be 550° C.

2. Weigh filters (g) to 4 decimal places to obtain pre-weight and store individual pads in a numbered petri dish.

3. Follow sample filtration procedure for total suspended solid sample. Make note of pad number on foil pouch.

 Procedure

1. Dry filters overnight at 103°-105° C. Allow to cool to room temperature in dessicator.

2. Weigh filters and record weights (in g). Calculate TSS.

3. Transfer filter to a porcelain crucible and combust at 500° C for 90 minutes. Allow to cool to room temperature in

dessicator.

4. Weigh filter and record weights (in g). Calculate TVS.

 Calculation of TVS

 

combust where: W = dry weight of filter pad and sample after combustion (in grams).

post W = dry weight of filter pad after filtering (in grams).

L

 V = volume of water filtered (L)
55

CHLOROPHYLL a AND Revised 2/2004

PHAEOPIGMENTS

Two fluorometric methods are available for analysis of Chlorophyll a. Chlorophyll, in a measured

volume of water, is concentrated by filtering through a glass fiber filter, and the pigments on the filter are

extracted in 90% acetone. Fluorescence is proportional to chlorophyll concentration. In one method,

fluorescence of the extract is measured before and after acidification using a fluorometer. In the second

method, the fluorometer is equipped with specific wavelength filters which read only emissions from

chlorophyll a, without interferences from chlorophyll b and phaeopigments. Therefore, the sample is not

acidified.

Methodology

Strickland, J.D.H. and T.R. Parsons. 1972. A practical handbook of seawater analysis. Bulletin 167 (2nd

ed.). Fisheries Research Board of Canada, Ottawa, Canada.

Parsons, T.R., Y. Maita and C.M. Lalli. 1984. Determination of chlorophylls and total carotenoids: 

Spectrophotometric method. pp. 101 - 112 in Parsons, T.R., Y. Maita and C.M. Lalli. A manual of

chemical and biological methods for seawater analysis. Pergamon Press, Oxford.

Welschmeyer, N.A. 1994. Fluorometric analysis of chlorophyll a in the presence of chlorophyll b and

phaeopigments. Limnol. Oceanogr., 39: 1985-1992.

Instrumentation

Acidification Method:

Fluorometer: Turner Designs Model TD700 calibrated against a spectrophotometer using pure

chlorophyll a from spinach (Sigma Chemical Company, C 5753), or liquid standards from Turner

Designs, #10-850.

Excitation filter: Turner Designs 10-050R, 340-500 nm

Emission filter: Turner Designs 10-051R, > 665 nm

Lamp: Daylight White Lamp, Turner Designs 10-045

Welschmeyer Technique:

Fluorometer: Turner Designs Model TD700 calibrated against a spectrophotometer using pure

chlorophyll a from spinach (Sigma Chemical Company, C 5753), or liquid standards from Turner

Designs, #10-850.

Excitation Filter: 436 nm

Emission Filter: 680 nm

Lamp: blue, F4T4.5B2 equivalent

Reagents

Acetone, 90%

Ace 3 2 tone [(CH ) CO] 900 mL

Deionized water 100 mL

In a 1000 mL volumetric flask, add 900 mL of acetone and add 100 mL deionized water.
56

Hydrochloric Acid, 1N

Hydrochloric acid (HCl), concentrated 8.6 mL

Deionized water up to 100 mL

In a 100 mL volumetric flask, add 8.6 mL of concentrated hydrochloric acid to -70 mL of deionized water. Dilute to 100 mL

with deionized water.

Procedure

1. Filter a known volume of water through a Whatman GF/F filter pad (nominal pore size 0.7 :m). Only a faint color is

needed on the pad.

2. Fold pad in half, sample inside, wrap in aluminum foil, label and freeze for analysis within 4 weeks.

3. Before analysis, briefly thaw pads, then place in a 15 ml centrifuge tube. Add 10 mL of 90% Acetone.

4. Using a Teflon pestle, grind the filter against the side of the tube until the filter is well ground. Allow the sample to extract

for 2 - 24 hours in the dark under refrigeration. Overnight is recommended.

5. Remove tubes from refrigerator and allow to warm to room temperature.

6. Shake tubes, centrifuge at -2300 rpm for 5-10 minutes and pipette liquid into a 5 mL round cuvette for reading on the

fluorometer. If a refrigerated centrifuge is used, a longer spin time may be used, but is not necessary. 

7. If phaeopigments are to be measured, add 2 drops of 1N Hydrochloric Acid and read on the fluorometer again. If using the

Welschmeyer technique, do not acidify the sample.

Calculation of Chlorophyll a and Phaeopigment Concentration

Chlorophyll a, phaeopigment, and active chlorophyll a concentrations are calculated using the following

equations:

and
57

whe bre: R = fluorometric reading before adding acid,

Ra = fluorometric reading after adding acid,

Fs b = calibrating std conc./reading of std, (C/R ),

b a r = R /R determined on a calibrating extract, and

V = volume filtered (mL).

10 = extract volume.

Calibration of the TD700: Determine the concentration of the chlorophyll a stock solution on the

spectrophotometer, or use liquid standards from Turner Designs. Follow instructions in the manual to

calibrate for raw fluorescence or concentration.

Calculation for the Welschmeyer Technique:

The instrument can be calibrated to read in concentration. To correct for volume filtered, the above

calculation is used. 

Chlorophyll a Calibration of the Fluorometer:

1. Empty 1 mg of dried chlorophyll a into a 100 mL volumetric flask and dilute to 100 mL with 90%

Acetone (10 mg/L).

2. Make serial dilutions of 1.0 mg/L, 0.1 mg/L, and 0.01 mg/L from the 10 mg/L solution (10 mL of

each solution diluted to 100 mL with 90% Acetone).

3. Read the 10 mg/L and 1 mg/L solutions on a spectrophotometer with a 10 cm cell path length at 750

nm, 665 nm, 664 nm, 647 nm and 630 nm. Add 2 drops of 1N Hydrochloric Acid and read again. 

The reading at 750 nm is a turbidity blank. If its value is greater than .005, centrifuge the sample at

2300 rpm for 5 minutes and read it again. The reading at 665 nm is used in the determination of

phaeopigments.

4. Read the 10, 1, 0.1 and 0.01 mg/L solutions on the fluorometer, using many dilutions.

5. The calculations for the Spec. numbers are as follows:

664 647 630 mg Chl a /L = 11.85(R ) - 1.54(R ) - 0.08(R )
58

whe nre: R = Reading at n nm

Determination of the calibration factors:

Multiply the 1 mg/L value by 10, and average this value and the 10 mg/L value. By using this

average and the fluorescence readings from each standard, including dilutions, calibration factors

can be calculated. 

 C = concentration of standard. 

Rb = fluorescence reading before adding acid. 

Determination of r:

Determ b a ine the ratio of R /R for all of the readings. Average.

Use a rolling average of the last three calibrations as the working factor and r.
59

APPENDIX A

SAMPLE CUSTODY

Upon arrival at the laboratory, samples are counted, observed for potential problems (melting, broken

containers, etc.) and placed in a freezer until analysis. Sample information and date of arrival are

recorded on a log sheet, and entered in an ACCESS file for internal tracking.

INSTRUMENT MAINTENANCE

Analytical instruments are maintained on a regular basis and records are kept of hours of operation,

scheduled maintenance, pump tube changes, etc.

A critical spare parts inventory is maintained for each instrument. Instrument down-time is minimized by

troubleshooting instrument problems by telephone with manufacturers and service representatives. Spare

parts can be received within 24 hours via next-day air service.

TEMPERATURE LOGS

The temperatures in freezers and refrigerators used for sample storage are monitored with NIST certified

and calibrated thermometers. Temperatures in drying ovens are monitored with NIST certified and

calibrated thermometers.

DATA HANDLING

Data produced by computer driven instruments are transferred electronically via LIM system to the main

laboratory data base. Other data are manually entered in Lotus 123 © or Microsoft Excel © spreadsheets

by data management personnel. A hard copy of manually entered data is produced and undergoes a

manual point-by-point verification process. Any necessary corrections are made. Subsequently, all data

are further checked by a series of programs to ensure the following:

324 C NO + NO + NH < Total Dissolved Nitrogen, --+

4

C PO < Total Dissolved Phosphorus, -3

C DOC < TOC, and

223 C NO < NO + NO .

---

Any data indicating errors are given specific error codes. Final printed data files are created and sent

along with an electronic copy to the client.
60

APPENDIX B

STATEMENT ON INSTRUMENT COMPARABILITY

The Nutrient Analytical Services Laboratory develops a data quality maintenance program for each

analyte whenever new instrumentation is acquired. It is the policy of the Nutrient Analytical Services

Laboratory to report any data from new instrumentation only after thorough and satisfactory side-by-side

comparisons with existing instrumentation are preformed.

No predetermined number of data pairs are used to make the assessment on data comparability between

new and existing methodology. Even in the case of instrumentation with similar methods of detection

(i.e., automated colorimetric), although no specific number of data pairs is used, there are at least 100

data pairs. Comparability at low and high concentrations, salinity and other possible matrix interferences,

sensitivity and precision are all factors in determining the number of pairs that must be addressed before

bringing an instrument on-line and in determining instrument comparability.

The analyst who performs these comparisons should be experienced, open-minded and impartial. This

person can give an evaluation of ease of instrument operation and a very important general statement of

comparability. This statement on comparability must then be substantiated via statistical analysis of the

data. As previously mentioned, these data must encompass the entire concentration range, matrix

interferences, percent recovery, results of standard reference material analyses, etc. The data

interpretation must support comparability. The analyst and laboratory QA/QC officer must concur and

finally, some sort of presentation (written or verbal) must be given to the contractor explaining what

procedure was followed and the results that were obtained to bring this instrument on-line.

STATEMENT OF QUALITY ASSURANCE/QUALITY CONTROL PROCEDURES

A constant consideration of Nutrient Analytical Services is assuring the quality of data generated by the

procedures presented in this manual. Further, indication of data quality is accomplished by analyzing

duplicates, spikes, standards-as-samples, standard reference materials; and participating in crosscalibration exercises.

Laboratory Duplicates

Approximately 5% of the total number of samples analyzed consist of laboratory duplicates. For

dissolved analytes, after a sample is analyzed, the same sample container is placed farther along in the

automatic sampler tray and re-analyzed. The mean of the two values is reported as the concentration for

that sample. If a difference of >10% is observed between replicates, then all of the replicates for that

particular analytical run are carefully reviewed. If only one of the duplicate pairs is in question, then only

that sample is re-analyzed. If all show a similar trend, then instrumentation/reagent problems are

suspected and the analytical run is halted until such time as the problem is resolved. This procedure is

practiced for all dissolved analytes that are not consumed completely in the analytical procedure. For

those that are completely consumed and for particulate analytes, duplicate analyses are actually duplicate

samples collected in the field and analyzed in the same analytical run.

Values for each duplicate analyzed are recorded in a separate QA/QC data file along with the sample

number, sample collection date and analysis date. The mean concentration and standard deviation of the
61

replicates are calculated in this data file.

In the case of particulate carbon and nitrogen, total suspended solids and chlorophyll a, 10% of the total

number of samples are analyzed as duplicates. This generates sufficient quality assurance data to

compensate for the omission of laboratory spikes for these non-aqueous samples.

Laboratory duplicates serve as an indicator of instrument stability, consistency in laboratory sample

preparation and analysis, as well as an estimate of field proficiency.

Laboratory Spikes

Approximately 5% of the total number of samples analyzed consist of laboratory spikes. A spike is

prepared by adding a known volume of standard to a known volume of pre-analyzed sample. We

routinely add enough concentrated standard to provide a significant response on our instruments that is

distinguishable from the original concentration of the sample. This concentrated standard is used to

minimize any possible change in sample matrix by the addition of spike.

The spiked sample is analyzed and its expected concentration calculated as the sum of the original

concentration and the spike concentration, normalized for the constituent volumes. A comparison is

made between the actual value and the expected value. These concentrations (original, expected and

actual) are recorded in a separate QA/QC data file along with sample number, sample collection date,

analysis date and the amount of spike added. In the case of particulate phosphorus, the volume filtered is

not used in the calculation to determine percentage recovery.

If a value of >115% or <85% is observed for percentage recovery of the spike, then all of the spikes for

that particular analytical run are carefully reviewed. If only one of the spikes is in question, then only

that sample is re-analyzed. If all show poor recovery, then instrumentation/reagent problems are

suspected and the analytical run is halted until such time that the problem is resolved. This procedure is

adhered to for all dissolved analytes and for the extracts of particulate phosphorus and biogenic silica.

Documentation of Slopes, etc.

A running record of the slopes of the standard curves (the so-called "F," "S" and "K" factors) is

maintained for each analysis. Random up and down movement within a predetermined range as a

function of time indicates the analysis is under control. Consistent upward or downward trend of these

factors indicates the analysis is out of control and requires immediate attention.

Limits of Detection

Limits of detection, the lowest concentration of an analyte that the analytical procedure can reliably

detect, have been established for all parameters routinely measured by Nutrient Analytical Services. The

limit of detection is 3 times the standard deviation of a minimum of 7 replicates of a single low

concentration sample.

Table 3 presents the current minimum limits of detection. These values are reviewed and revised

periodically.
62

Table 3. Minimum limits of detection of CBL, October/November 1987, March 1994,

February/December 1998, February 1999, January 2004 a

MEAN CONC. STANDARD DETECTION LIMIT

NUTRIENT (mg/L) DEVIATION (mg/L) (:M)

Ammonium 0.0007 0.001 0.0030 0.21

Nitrite 0.0002 0.0001 0.0003 0.02

Nitrite + Nitrate 0.0011 0.00023 0.0007 0.05

Phosphate 0.0027 0.00025 0.0007 0.02

Dissolved Organic Carbon 3.5800 0.050 0.1500 12.50

Total Suspended Solids 13.4000 0.800 2.4000

Total Volatile Solids 3.95 0.67 1.98

Particulate Phosphorus 0.0187 0.0008 0.0024 0.08

Particulate Inorganic Phosphorus 0.0027 0.0002 0.0006 0.02

Total Dissolved Nitrogen 0.39 0.0096 0.0300 2.06

Total Dissolved Phosphorus 0.0057 0.0005 0.0015 0.05

Silicate 0.2500 0.003 0.0100 0.32

Particulate Nitrogen 0.3170 0.0041 0.0123 0.88

Particulate Carbon 2.2600 0.0253 0.0759 6.32

Particulate Biogenic Silica 0.1630 0.003 0.0090 0.32 

Sediment C (10 mg) 2.1830% 0.044 0.1300%

Sediment N (10 mg) 0.1950% 0.003 0.0084%

Sediment P (34.8 mg) 0.0304% 0.003 0.0087%

Chlorophyll a 24.26 0.22 0.65 (:g/L)

FRESHWATER DETECTION LIMITS

Chloride 5.84 0.08 0.23 6.57

Sulfate 4.90 0.03 0.09 2.80

 Results based on a minimum of seven replicates collected from one cubitainer and analyzed randomly a

on a typical day of analyses. 

Standard as Sample

Standards are analyzed as samples throughout the analytical run. This is an excellent means of evaluating

instrument performance during the course of an analytical run. Standards as samples are analyzed every

12 - 20 samples, depending on the instrument and analyte.

Standard Reference Materials

Particulate Carbon, Nitrogen and Phosphorus: BCSS-1 is a marine sediment reference material prepared

by the National Research Council of Canada. It is certified by the Council for carbon content, gives a

non-certified range of results for phosphorus, but no information for nitrogen. We have analyzed this
63

sediment for many years and maintain a substantial database for nitrogen and phosphorus, as well as

carbon values. We analyze this sediment at least quarterly and compare these results to the certified

value, non-certified range of values and our historical values.

Dissolved Analytes and Hardness: Standard reference materials for ammonium, nitrite + nitrate, nitrite,

orthophosphate, dissolved nitrogen, dissolved phosphorus, dissolved organic carbon, sulfate and chloride

and hardness are supplied by SPEX, a US EPA certified company. The samples arrive in ampules and we

prepare final concentrations to approximate typical estuarine concentrations. The samples are then placed

in pre-cleaned Teflon or poly bottles, frozen and analyzed on at least a quarterly basis.

The analysis of these frozen standard reference materials as a function of time also provides data on the

effect of our preservation technique (freezing) on the integrity of the concentration of samples. The US

EPA recommends a holding time of 28 days for many of the parameters we routinely analyze.

Dissolved organic carbon results provide a good example. SPEX standards (2 and 5 mg C/L) were

prepared in deionized water on 31 May 1995. Several of each concentration were placed in pre-cleaned

Teflon bottles and frozen for subsequent analysis. Results for the Shimadzu TOC-5000 Carbon Analyzer

are presented below.

SHIMADZU TOC-5000 TOTAL ORGANIC CARBON ANALYZER

(Concentrations in mg C/L)

ANALYSIS DATE

Standard 6/14/95 7/5/95 8/16/95 11/6/95

(Frozen) (Frozen) (Frozen) (Frozen)

5.00 5.30 5.23 5.32 5.42

2.00 2.11 2.13 2.15 2.13

Organic compounds are included with each dissolved nitrogen and dissolved phosphorus digestion to 

determine the completeness of the digestion procedure. Glutamic acid and glycerophosphate are used as

the N and P sources, respectively. Figures 14 and 15 illustrate the percentage recoveries of these internal

standards for a specific project in which we participated in 1994 - 1995.

Cross Calibration Exercises

Nutrient Analytical Services has participated in many cross calibration exercises. Participation in such

programs is an excellent means of determining accuracy of results. Examples of such cross calibration

exercises include the Chesapeake Bay Program Quarterly Split Samples, Chesapeake Bay Program Blind

Audits, USGS Standard Reference Sample Project, US EPA Method Validation Studies and International

Council for the Exploration of the Sea Intercomparison Exercise for Nutrients in Sea Water.
64

Figure 13. Percentage recovery of glutamic acid as an internal standard for total dissolved nitrogen

digestion/analysis. (1995)

Figure 14. Percentage recovery of glycerophosphate as an internal standard for total dissolved

phosphorus digestion/analysis. (1995)
65

ACKNOWLEDGEMENTS

We appreciate Dr. Christopher D’Elia’s help in assembling the earlier versions of this manual. In 1978 he

started acquiring equipment and hiring personnel to establish NASL to support research at CBL and,

later, “outside” programs. This manual reflects his insistence on appropriate methods for the sample

matrix to ensure data accuracy.

Erin Connor authored part of the 1997 manual, and assembled all the sections and diagrams in a

consistent format. Adriene Capers helped assemble this manual and, also, the previous versions. 

We thank Dr. Theodore Loder and Dr. Leonard Haas for their editorial suggestions and review of the

1997 version of this document. 
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

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Final report of the land-based testing of the Hyde-GuardianTM-System 40

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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
1 (24)

Environmental acceptability evaluation of the Hyde GUARDIANTM

Ballast Water Treatment System as part of the Type Approval Process

Version 3

Non-Confidential

20 April 2009

Authors

Dr. Cato ten Hallers, CaTO Marine Ecosystems & Royal Netherlands Institute for Sea Research (NIOZ),

Netherlands

Professor Emeritus Erkki Leppäkoski, MeriEko Ltd and Åbo Akademi University, Finland

Mr. Jukka Sassi, VTT Technical Research Centre of Finland, Finland

Mr. Matt Granitto, Hyde Marine Inc., US

Mr. Tom Mackey, Hyde Marine Inc., US

Mr. Mats Björkendahl, Hyde Marine Inc., a Lamor Group Company, Finland
2 (24)

20 April 2009

Table of Contents

Glossary

1 Introduction ................................................................................................................................ 5

2 UV light and disinfecting action.................................................................................................. 5

3 Identification of Preparations, Active Substances and Relevant Chemicals ................................. 8

3.1 Preparations (G9: 2.1.3; GESAMP-BWWG 6, Annex IV: 1.1.3)............................................. 8

3.2 Active Substances (G9: 2.1.1; GESAMP-BWWG 6, Annex IV: 1.1.4) ................................... 9

3.3 Relevant Chemicals (G9: 2.1.4; GESAMP-BWWG 6, Annex IV: 1.1.6)................................. 9

3.4 Other components (GESAMP-BWWG 6, Annex IV: 1.1.5).................................................. 10

4 Data on Effects on Aquatic Plants, Invertebrates and Fish, and Other Biota, Including Sensitive

and Representative Organisms (G9: 4.2.1.1; GESAMP-BWWG 6, Annex IV: 3.3) .............................. 10

4.1 Environmental effects ........................................................................................................... 10

4.2 Aquatic toxicity .................................................................................................................... 12

4.2.1 Acute Aquatic Toxicity (GESAMP-BWWG 6, Annex IV: 3.3.2) .................................. 12

4.2.2 Chronic aquatic toxicity (GESAMP-BWWG 6, Annex IV: 3.3.3) ................................. 13

4.2.3 Endocrine disruption (GESAMP-BWWG 6, Annex IV: 3.3.4) ...................................... 14

4.2.4 Sediment toxicity (GESAMP-BWWG 6, Annex IV: 3.3.5) ........................................... 14

4.3 Bioavailability/Biomagnification/Bioconcentration/Food web/Population effects (GESAMPBWWG 6, Annex IV: 3.3.6, 3.3.7) ................................................................................................... 14

5 Data on Mammalian Toxicity (G9: 4.2.1.2; GESAMP-BWWG 6, Annex IV: 3.4) .................... 14

5.1 Data on mutagenicity and genotoxicity (GESAMP-BWWG 6, Annex IV: 3.4.8) .................. 14

6 Data on Environmental Fate and Effect under Aerobic and Anaerobic Conditions (G9: 4.2.1.3;

GESAMP-BWWG 6, Annex IV: 3.5)................................................................................................... 15

7 Physical and Chemical Properties for the Preparations, Active Substance and Treated Ballast

Water (G9: 4.2.1.4; GESAMP-BWWG 6, Annex IV: 3.6).................................................................... 15

8 Analytical Methods at Environmentally Relevant Concentrations (G9: 4.2.1.5; (GESAMPBWWG 6, Annex IV: 3.7).................................................................................................................... 15

9 Use of Active Substance or the Preparation (GESAMP-BWWG 6, Annex IV: 4)...................... 15

10 Material Safety Data Sheets (GESAMP-BWWG 6, Annex IV: 5)............................................. 15

11 Risk Characterization................................................................................................................ 15

11.1 Evaluation of the treated ballast water (G9: 5.2; GESAMP-BWWG 6, Annex IV: 6.2) ......... 15

11.1.1 Results from the Land Based Tests................................................................................ 15

11.1.2 Results from the shipboard tests.................................................................................... 16

11.2 Prediction of discharge and environmental concentrations (GESAMP-BWWG 6, Annex IV:

6.3.3) 17

11.3 Effects on sediment (G9: 5.1; GESAMP-BWWG 6, Annex IV: 6.3.7) .................................. 17

12 Quality Control/Quality Assurance (G9: 4.2.4) ......................................................................... 17

13 Use of the Active Substance and Preparations........................................................................... 18

14 Risk Assessment (G9: 6, 7)....................................................................................................... 18

14.1 Risk to safety of ship (GESAMP-BWWG, Annex IV: 7.1) ................................................... 18

14.1.1 Ship Integrity and the BWTS ........................................................................................ 18

14.1.2 Hazardous Area............................................................................................................. 19

14.1.3 Corrosion ...................................................................................................................... 19

14.1.4 Fire and Explosion ........................................................................................................ 20

14.1.5 Chemical Storage.......................................................................................................... 20

14.1.6 Accidental Chemical Release ........................................................................................ 20

14.2 Risk to technicians and/or ship’s crew .................................................................................. 20

14.3 Risks to human health (GESAMP-BWWG, Annex IV: 7.2).................................................. 21

14.4 Risks to the aquatic environment (GESAMP-BWWG, Annex IV: 7.3) ................................. 21
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14.5 Noise .................................................................................................................................... 21

15 Assessment Report (G9: 4.3; GESAMP-BWWG, Annex IV: 8)................................................ 21

16 Appendices............................................................................................................................... 23

List of Figures

Figure 2-1. Formation of thymine – thymine dimer from adjacent thymine residues. .............................. 6

Figure 2-2. Graph depicting the germicidal curve and typical lamp technology outputs.......................... 8
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20 April 2009

Glossary

BWM Ballast Water Management

BWTS Ballast Water Management System

CAS Chemical Abstracts Service

DBP Disinfection By-products

DOC Dissolved Organic Carbon

FAO Food and Agriculture Organization

GESAMP-BWWG Group of Experts on the Scientific Aspects of Marine Protection - Ballast

Water Working Group

G2 Resolution MEPC.173(58) Sampling for Compliance Control (G2)

G8 Resolution MEPC.174(58) Guidelines for Approval of Ballast Water Management Systems (G8)

G9 Resolution MEPC.169(57) Guidelines for Approval of Ballast Water Management Systems that Make Use of Active Substances (G9)

HAA Haloacetic Acids

IMO International Maritime Organization

IMO D-2 Regulation D-2 Ballast Water Performance Standard

LC50 Lethal Concentration, 50%

LPHO Low Pressure High Output (lamps)

MEPC Marine Environment Protection Committee (of the IMO)

MSDS Material Safety Data Sheet

NIOZ The Royal Netherlands Institute for Sea Research

NOEC No Observed Effect Concentration

NOM Natural Organic Matter

Pa Pascals

PAH Polycyclic Aromatic Hydrocarbon

PSU Practical Salinity Units

STEP U.S. Coast Guard Shipboard Technology Evaluation Program

THM Trihalomethane

TOC Total Organic Carbon

TOX Total Organic Halides

US EPA U.S. Environmental Protection Agency

UV-A Ultraviolet Light, wavelength 315-400 nm

UV-B Ultraviolet Light, wavelength 280-315 nm

UV-C Ultraviolet Light, wavelength 200-280 nm

UV-Vac Ultraviolet Light, wavelength 10-200 nm (UV-V)

VOC Volatile Organic Compound

WET Whole Effluent Toxicity

WHO World Health Organization
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20 April 2009

1 Introduction

With this document, accompanying the type approval test reports (land based and shipboard tests) Hyde

Marine Inc. whose registered office is at 28045 Ranney Parkway, Cleveland, OH 44145 USA, applies

for approval of environmental acceptability according to International Ballast Water Management Convention Guideline G8 (Guidelines for Approval of Ballast Water Management Systems) and in agreement with G9 (Procedure for Approval of Ballast Water Management Systems that Make Use of Active

Substances),where appropriate. The document is structured to support evidence that no active substances

are used or generated for ballast treatment and that relevant chemicals and other compounds (DBPs) are

either not produced or not produced at unacceptable levels.

The document first argues the case for absence of active substances by the treatment method (filtration

and UV irradiation) and then presents information on potential by- and end products, their toxicological

profiles and the actual presence or absence of such products in the treatment process under the different

circumstances the system might meet.

Reference is made to the relevant paragraphs of BWM Guideline G9: Resolution MEPC.169(57) –

Procedure for approval of ballast water management systems that make use of Active Substances (G9)

and the GESAMP-BWWG Methodology as described in MEPC/58/2/7, 6th GESAMP-BWWG Meeting

report, Annex IV.

The environmental performance of the Hyde GUARDIANTM system is being evaluated according to the

definitions in G9, the guideline for approval of ballast water management systems that make use of active substances, to which G8 (Resolution MEPC.174 (58) - Approval of ballast water management systems) refers in the paragraphs on environmental acceptability, and according to the methodology developed by the GESAMP-BWWG.

This review is based on the existing literature and on the results from land based and shipboard toxicity

tests in the application of Hyde GUARDIANTM in fully marine and brackish water. It is known from e.g.

oxidation processes that the formation of disinfection by-products (DBPs) in marine waters may differ

considerably from those in the more frequently studied drinking-, process- and wastewater applications1.

Therefore it was important to document whether similar problems as those that occur in some of the

fresh-water applications may also arise from UV ballast water treatment saline waters.

2 UV light and disinfecting action

Ultraviolet light (UV) is an electromagnetic radiation of shorter wavelengths (Ȝ) than visible light, with

wavelengths ranging from 10 – 400 nm and energies from 3 eV to 124 eV. The ultraviolet spectre consists of frequencies (f) above those of the visible colour violet.

Ultraviolet (UV) wavelengths 10-400 nm lay outside the visible light spectre. UV is classified according

to spectral bands (expressed in nanometres – nm):

x UV-A – 315-400 nm

x UV-B – 280-315 nm

x UV-C – 200-280 nm

x UV-Vac – 10-200 nm (UV-V).

1 Kornmüller A (2007) Review of fundamentals and specific aspects of oxidation technologies in marine waters. Water Science and Technology 55, 1-6
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20 April 2009

Natural solar UV includes the full spectrum of UV; artificially UV is made by specific devices, including a UV lamp and a generator, which, depending on construction, can be specialised to generate UV of

a specific spectral band or of one single wavelength. Artificial UV is used for several purposes in and

outside water treatment. This paper will focus on UV as used in water treatment, where UV is mainly

used for disinfection and for photolysis of chemical bonds. For optimal action both processes require

specific spectral bands that differ from each other. UV-C is optimal for disinfection, while UV-V results

in photolysis of chemical bonds. Wavelengths below 200 nm can also result in unwanted by-products

due to photolysis, such as ozone.

Physical process

UV disinfection is a physical form of disinfection, as opposed to the chemical form of chlorine and other

chemicals. Some molecules, when subjected to UV light, will absorb its energy. Once absorbed, the

electronic energy is sufficient to break bonds and promote the formation of new bonds within the molecule, leaving it damaged. For this reason, UV-C light is called phototoxic (toxic light). The most important molecules of living cells, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are very sensitive to phototoxicity. The most common effect of UV-C is the formation of a cyclobutyl ring between

two adjacent thymine nucleic acids located on the same strand of DNA/RNA, as shown on Figure 2-1.

Figure 2-1. Formation of thymine – thymine dimer from adjacent thymine residues2.

The resulting structure, called a thymine dimer, locally distorts the helical structure of the DNA/RNA

molecule preventing the proper attachment of transcriptional and replicating enzyme complexes. This

damage most commonly results in inhibition of the transcription and replication of the genetic molecules

within the affected cell, which results in death of that single cell. 3

Structural damage to DNA material, rendering the DNA unable to replicate, inhibits reproduction of the

organism. For disinfection and germicidal purposes UV-C penetrates through cell membranes and cyto2 New York State Energy Research and Development Authority (NYSERD) (2004) Evaluation of Ultraviolet (UV) radiation disinfection technologies for wastewater treatment plant effluent.).

Final Report 04-7. December 2004.

3 New York State Energy Research and Development Authority (NYSERDA) (2004) Evaluation of Ultraviolet (UV) radiation disinfection technologies for wastewater treatment plant effluent.).

Final Report 04-7. December 2004.
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20 April 2009

plasm and can enter the cell nucleus; in both nucleus and cytoplasm UV-C damages or destroys genetic

material, rendering the DNA unable to reproduce. The optimal wavelength for disinfection is 265 nm.

The treatment method is nonintrusive and does not alter the chemistry or physical property of the water

to be treated. No organisms have been demonstrated to be resistant to UV.

UV-B, of larger wavelengths, penetrates less deep in fluids and is hence less fit for disinfection. UV-V

risks the formation of by-products due to ionisation and dissociation of chemical compounds by photolysis.

Artificial UV-C is generated by purpose-constructed devices that generate UV-C through different process, pressures and temperature regimes.

Low pressure UV lamps consist of a quartz lamp with attached electrodes. The lamp is filled with inert

gas and a minor amount of mercury. For disinfection purposes the quartz should be natural, as artificial

quartz causes the generation of not only UV at 254 nm, but also UV-V of 185 nm. The latter bears the

risk of generation of ozone as a by-product. A low pressure UV generator has typically a pressure of 1.3

– 0.13 Pa. The temperature in a low pressure UV lamp is 40.5 – 43oC. The lamp functions optimally in

water of around 21oC, else the UV is less effective. The lamp functions at 120-140 V, with a current of <

500 mA and a power output of 40 - >100 W. The mercury in the lamp is partly vaporised. The lamp

typically emits light of one specific wavelength.

Medium pressure UV-C lamps function at much higher pressures (13-1300 kPa). Temperatures inside

the UV lamp rise to 500-600 oC, at which temperature the mercury is completely vaporised. The lamp is

stable in all temperature conditions and has a broad spectral output. The high temperature and pressure

creates a plasma excitation of the mercury, which collapses emitting characteristic absorption lines. The

principle of the construction of the medium pressure lamp that operates with a reactor is similar as that

of the low pressure lamps. Here too the quartz of the lamp should be natural, so as to prevent the formation of potentially harmful by-products.

UV irradiation for disinfection is most effective at germicidal wavelengths (240-280 nm, Figure 2-2).

The effectiveness of the UV treatment (adequate destruction of the genetic material) requires that a sufficient energy dose is absorbed by the organism. The energy dose is measured as the product of the

lamps intensity (rate at which photons are delivered to the target) and the time of exposure. UV lamps at

low pressure and low intensity are most common in disinfection, however for shipboard use, medium

pressure lamps (>3 kW) are better suited for the application due to compact size and lower power consumption.4

Basically, low pressure lamps have a mercury vapour pressure of between 0.1 and 10 Pa, whereas medium pressure lamps have a mercury vapour pressure (when hot) of between 50 and 300 kPa.

Low intensity (normal) low pressure lamps have only mercury present (plus an argon buffer gas),

whereas high intensity low pressure (usually called low pressure high output or LPHO lamps) either

have an amalgam spot (a compound with mercury and another metal such as indium) or a special long

electrode section with a 'cold spot' (Bolton, James R., Bolton Photosciences Inc. 2009. Personal communication).

The low pressure low intensity lamps are typically about 65 W each. The low pressure high intensity

lamps can go as high as 300 W. Regardless of the intensity low pressure lamps are monochromatic

(Granitto, Matt, Hyde Marine Inc. 2009. Personal communication)

4 Techneau (2007) UV disinfection and UV/H2O2 oxidation: by-product formation and control. Techneau D2.4.1.1, May 2007.
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20 April 2009

Figure 2-2. Graph depicting the germicidal curve and typical lamp technology outputs.

All organisms have different responses to UV irradiation. The way in which UV is measured is called

dose. The UV dose is simply a multiplication of the intensity of the UV and contact time. An organism

exposed to 140 mW/cm2 radiation (intensity) for 0.5 s would receive a UV dose of 70 mWs/cm2or 70

mJ/cm2. As an example of appropriate amounts of UV, the US EPA’s standard for using UV to disinfect

drinking water requires a dose of 40 mJ/cm2.

The Hyde GUARDIANTM system is designed to provide a minimum average dose of 200 mJ/cm2. This

average minimum dose to be effective for disinfection is calculated using the expected end of lamp life

intensity of the UV lamp and at a water transmission of T10 = 92%.

BWT through the use of UV is repeatedly proven to be effective against zooplankton, phytoplankton,

bacteria and viruses (Malmlook et al 20075). In addition, ”UV irradiation is an attractive, rapid means of

disinfection, as it leaves no harmful residues6”.

3 Identification of Preparations, Active Substances and Relevant Chemicals

3.1 Preparations (G9: 2.1.3; GESAMP-BWWG 6, Annex IV: 1.1.3)

“Preparation” means any commercial formulation containing one or more Active Substances including

any additives. This term also includes any Active Substances generated on board for purposes of ballast

water management and any relevant chemicals formed in the in the ballast water management system

that makes use of Active Substances to comply with the Convention.

5 Malmlook R, Badran O, Abu-Khader MM, Holdo A, Dales J (2008) Fuzzy sets analysis for ballast water treatment systems: best available control technology. Clean Technologies and Environmental Policy 10, 397-407

.

6 Oemcke DJ, Van Leeuwen J (2007) Chemical and physical characterization of ballast water. Part 1: Effects on ballast water treatment processes. Journal of Marine Environmental Engineering

7, 47-64
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20 April 2009

Following this definition the Hyde GUARDIANTM system is not using any preparations other than UV

light.

3.2 Active Substances (G9: 2.1.1; GESAMP-BWWG 6, Annex IV: 1.1.4)

”Active Substance” means a substance or organism, including a virus or a fungus that has a general or

specific action on or against harmful aquatic organisms and pathogens.

Hyde GUARDIANTM system does not use or generate Active Substances, but only uses MP UV-light for

disinfection; the photons emitted by the UV light are energy packages and therefore not substances or

organisms. The process of disinfection is demonstrated by the description of the system in Chapter 2.2.

3.3 Relevant Chemicals (G9: 2.1.4; GESAMP-BWWG 6, Annex IV: 1.1.6)

”Relevant Chemicals” means transformation or reaction products that are produced during and after employment of the ballast water management system in the ballast water or in the receiving environment

and that may be of concern to the ship’s safety, aquatic environment and/or human health.

UV from solar radiation produces short-lived OH radicals that can act as an intermediate to reaction byproducts. In that sense OH radicals may be considered relevant chemicals. The potential to form OH

radicals by the Hyde GUARDIANTM system is discussed below. Ozone is only formed at wave lengths

below the spectre of the Hyde GUARDIANTM system (below 200 nm) and should therefore not be considered a relevant chemical for the Hyde GUARDIANTM system.

Formation of OH radicals is enhanced by the presence of chlorine radicals in the water, a situation particular for drinking water purification by UV when chlorine is present in the upstream water to be purified7

. Little is known about the fundamental photochemistry of free chlorine. Following photolysis of

aqueous free chlorine to HOCl, the HOCl may absorb a photon (Ȝ< 511 nm), leading to an OH radical

and a Cl radical. Although in seawater chlorine is present in NaCl, the situation as described for drinking

water is not to be expected to play an important role in seawater. For fresh water shipping areas, NaCl is

not present or only at very low levels. The other situation where OH formation is enhanced is in the

presence of added hydrogen peroxide (H2O2)

8

 or added ozone (O3). This situation is not to be expected

in an aquatic environment. Solar UV OH formation might be promoted by the ozone when that is present

in natural circumstances. As neither H2O2, not excess chlorine is present in the conditions that the Hyde

GUARDIANTM system operates, the formation of OH radicals is not expected to be at a level that may

cause adverse effects; levels may even be too low to be detectable at all, even when measured at the

nanosecond life span of OH radicals. By using UV of Ȝ >200 nm only, the potential to form by-products,

such as OH and O3, through photolysis is prevented. Such by-products may be formed at lower wave

length, typically at Ȝ 185 nm, a wave length that is emitted when the lamp is made of synthetic (artificial) quartz. Pure quartz (quartz of natural origin) does not emit wavelengths below Ȝ 200 nm. The

lamps used in the Hyde GUARDIANTM system are all made of pure quartz. Furthermore, the UV source

in the Hyde GUARDIANTM system is shielded by double pure quartz layer, since also the quartz sleeve

that covers the UV lamp is made of pure quartz.

7 Watts MJ, Linden KG (2007) Chlorine photolysis and subsequent OH radical production during UV treatment of chlorinated water. Water Research 41, 2871-2878

8 Techneau (2007) UV disinfection and UV/H

2

O

2

 oxidation: by-product formation and control. Techneau D2.4.1.1, May
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3.4 Other components (GESAMP-BWWG 6, Annex IV: 1.1.5)

“Other components” of a preparation means any other substances in a preparation, other than the Active

Substance(s) or Relevant Chemicals, produced during the treatment of ballast water.

For evaluating the presence of other components, the reaction by-products from the Hyde GUARDIANTM system, we investigate disinfection by- and end products (DBPs), i.e., chemical organic and inorganic substances that can form during a reaction of UV as disinfectant with the treated ballast water

and/or with organic matter in the ballast and receiving water9.

4 Data on Effects on Aquatic Plants, Invertebrates and Fish, and Other Biota, Including Sensitive and Representative Organisms (G9: 4.2.1.1; GESAMP-BWWG 6, Annex IV: 3.3)

Disinfection by UV is a physical process, where bonds are broken through photolysis. Such bonds can

either be DNA and other cell material in living organisms or bonds of hazardous organic chemicals, such

as may be present in wastewater streams. The administered energy should be sufficient to break the

bonds. For disinfection this means sufficient energy to destroy the genetic material so as to render it incapable of reproduction (recombining DNA material). When the administered energy is too low DNA

material might be damaged only, but still able to recombine, which can result in mutations that might

prove harmful10. In the UV spectral band produced by the Hyde GUADRIANTM system (200-400 nm)

the energy delivered to the ballast water is 200 mJ/cm2per treated m3. This minimum dosing, which is

considerably higher than the US EPA’s standard for UV to disinfect drinking water (40 mJ/cm2), is set

sufficiently high to prevent the possibility that DNA can still recombine.

4.1 Environmental effects

Scientific and technical reports on UV disinfection and by- and end products are only partly applicable

to screening of environmental and health impacts of ballast water treated by UV. UV disinfection is

common in treatment of drinking water purification and wastewater treatment, reducing bacterial and

microorganism loads as well as breaking down organic contaminants. By-products formed by UV disinfection on waters containing hazardous chemical compounds, not present naturally in an aquatic environment (such as disinfection products of wastewaters containing PAHs ), are mentioned in this report

but are not considered relevant for the purpose of environmental evaluation of MP UV in ballast water

treatment.

By-products from UV treatment arise (1) directly through photochemical reactions, and (2) indirectly

through reactions with products of photochemical reactions. By-products formed at manufacturing of

drinking water are rather well documented. It was shown in several studies that UV disinfection at UV

doses < 400 mJ/cm2 does not affect formation of trihalomethanes (THMs), haloacetic acids (HAAs) and

Total Organic Halides (TOX)11.

UV treatment on its own was shown to have very little impact on the organic constituents in raw water.

At low irradiation intensities, UV treatment does not change pH, turbidity, dissolved OC level, colour,

9 www.lenntech.com/water-disinfection/disinfection-byproducts.htm

10 Kalisvaart BF (2001) Photobiological effects of polychromatic medium pressure UV lamps. Water Science and Technology 43, 191-197; Taghipour F, Sozzi A (2005) Modeling and design

of ultraviolet reactors for disinfection by-product precursor removal. Desalination 176, 71-80; Cooper WJ, Jones AC, Whitehead RF, Zika RG (2007) Sunlight-induced photochemical decay of

oxidants in natural waters: implication in ballast water treatment. Environmental Science and Technology 41, 3728-3733

11Malley J, Show J, Ropp J (1996) Evaluation of the by-products produced by the treatment of groundwaters with ultraviolet radiation. American Water Works Association Research Foundation, Denver, CO; Liu W, Andrews SA, Bolton JR, Linden KG, Sharpless C, Stefan M (2002) Comparison of disinfection byproduct (DBP) formation from different UV technologies at bench

scale. Water Science and Technology: Water Supply 2, 515-521; Kashinkunti RD, Linden KG, Shin G-A, Metz DH, Sobsey MD, Moran MC, Samuelson AM (2004) Investigating multibarrier

inactivation for Cincinnati-UV, by-products, and biostability. American Water Works Association Journal 96,114-127.
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20 April 2009

nitrite, nitrate, bromide, iron or manganese of the water being treated11. The maximum UV dosage used

(1.6 Ws/cm2) did not result in any significant reduction of TOC (total organic carbon), chloroform or

HAA (haloacetic acids) formation potentials12.

There is a plethora of applications, other than ballast water treatment, in which UV has been used for

water purification for years or even decades. Among these, commonly known uses in e.g. disinfection of

drinking water and municipal wastewater, and uses in pharmaceutical and cosmetic industries, laboratories, food and brewery industries etc, have been evaluated and accepted by administrations worldwide.

Many references focus on disinfection of drinking water (i.e. freshwater), the DBPs reported and discussed being chlorine and different daughter substances formed by chlorination. Often UV treatment of

drinking water is combined with ozonisation or chlorination13. Research has focused on drinking water

and on the impact of UV on the formation of halogenated DBPs after subsequent chlorination, and on

the transformation of organic material to more degradable components14. Addition of chlorine may result

in the formation of different DBPs, such as trihalomethanes (THM), haloacetic acids (HAAs), carboxylic

acids, aldehydes, and increase of TOX (Total Organic Halogen).15 Buchanan et al. (2006) reported on

the formation potential of THM, HAA, nitrite and peroxide resulting from UV photo-oxidation followed

by biological treatment for the removal of natural organic matter (NOM). Samples exposed to different

doses of UV were found to be non-cytotoxic and non-mutagenic16.

For groundwater and filtered drinking water, UV disinfection at typical doses did not impact the formation of THMs and HAAs. Several studies have shown the formation of low levels of (in the US nonregulated) DBPs, such as aldehydes. In filtered drinking water no significant change in aldehydes, carboxylic acids, or total organic halides was found (Kashinkunti et al. 2004).

UV-radiation is also used in seawater reverse osmosis (SWRO) desalination plants. Munshi et al. (1999)

noted that UV did not cause any phase change in water, which does not lead to any large-scale accumulation of toxic by-products in the water phase; in summary, UV-radiation was found to be a potential alternative to chlorination 17

.

At hospitals water is treated by UV to insure pure water for pathology laboratories and kidney dialysis

where bacteria-free water is of utmost importance18. For water used in the preparation of dialysis fluid,

chemical contaminant levels are set very low. The additional treatment ensures that the water meets the

more stringent requirements.

Application of UV disinfection for wastewater treatment is considered environmentally positive: (1) no

chemicals are added to the effluent stream, reducing the risk of detrimental effects to aquatic life; (2) no

trihalomethane (THM) are formed and (3) UV disinfection reduces safety, handling or explosion risks19.

12 Chin A, Berube PR (2005) Removal of disinfection by-product precursors with ozone-UV advanced oxidation process. Water Research 39, 2136-2144

13 E.g., Watts MJ, Linden KG (2007) Chlorine photolysis and subsequent OH radical production during UV treatment of chlorinated water. Water Research 41, 2871-2878

14 Swaim PD, Cotton CA, Jeyanayagam SS (2004) Ultraviolet disinfection. In: Water Treatment Plant Design. McGraw-Hill

15 Kashinkunti RD, Linden KG, Shin G-A, Metz DH, Sobsey MD, Moran MC, Samuelson AM (2004) Investigating multibarrier inactivation for Cincinnati-UV, by-products, and biostability.

American Water Works Association Journal 96, 114-127

16 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

17 Munshi HA, Sasikumar N, Jamaluddin AT, Mohammed K (1999) Evaluation of ultra-violet radiation disinfection on the bacterial growth in the Swro Pilot plant, Al-Jubail. Proceedings of

the 4th Gulf Water Conference, 1 Bahrain, Feb.13-17, 1999. 17 pp

18 E.g., Hoenich N, Levin R (2008) Water Treatment for Dialysis: Technology and Clinical Implications In: Ronco C, Cruz DN (eds): Hemodialysis - From Basic Research to Clinical Trials.

Contribution to Nephrology Basel, Karger, 161, 1-6

19 Lau PJ (1997) Applying disinfection alternatives to wastewater treatment. Pollution Engineering 29, 64-66
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4.2 Aquatic toxicity

The results from land- and ship-based toxicity tests used in this document were performed in full marine

(31.9 PSU) and in brackish water (22.1 PSU). It is known from, e.g., oxidation processes that the formation of disinfection by-products (DBPs) may be considerably different in marine waters from fresh water

applications20. Therefore it was important to document whether similar problems may arise also from

UV treatment for water of higher salinities.

4.2.1 Acute Aquatic Toxicity (GESAMP-BWWG 6, Annex IV: 3.3.2)

Some few examples only of acute (eco)toxicity of UV-treated were found during the literature search. In

an acute immobilisation test, UVC-treated freshwater appeared to be toxic to the water flea Daphnia

carinata. The observed toxicity was attributed to photo-oxidative degradation of metal-binding sites in

natural organic matter, which resulted in the release of bioavailable copper ions21.

Nitrite toxicity in seawater

Some changes in nutrient levels were recorded during the shipboard tests22. For example, both phosphate

and nitrate levels at deballasting were higher in treated samples relative to untreated samples at the time of

deballasting. It was not possible to definitively conclude that these changes directly resulted from UV irradiation: part of the differences could be attributed to an increase in the degradation of planktonic organisms resulting from the treatment process, rather than UV irradiation directly.

Photolysis of nitrate to nitrite has been demonstrated in UV-irradiated drinking water (e.g. Sharpless &

Linden 2001)23. In shipboard tests, the concentration of NO2 (μgN L-1 increased from an initial concentration of 0.6 up to 1.75 – 2.73 μgN L-1 in treated samples 10 days after treatment. In the land-based

tests24, at the low salinity test series only the nitrite concentration increased by a factor 6.2 after the UV

reactor, up to 4.62 μM at discharge (day 5). The observed elevated concentration was nevertheless

within the range typical for the test water used and therefore not excessively high. It is also possible that

the prolonged dark incubation also increased the nitrite concentration in both the reference and treated

tank.

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), respectively25. In seawater conditions these nitrite levels seem not to be of any toxicological significance. High concentrations of chloride ions effectively reduce the toxicity of nitrite. Seawater fish are usually less sensitive to

nitrite than freshwater fish26. For example, nitrite was found to be 55 times more toxic to juvenile milkfish (Chanos chanos) in fresh water than in brackish (16 psu) water. The 48-h median LC50 values were,

respectively, 12 and 675 mg NO2-N L-1 27. The 96-h LC50 of nitrite-N for European eel (Anguilla anguilla) can range from 84.0 mg L-1 in freshwater to 812.0mg L-1 in full strength seawater (salinity = 36.0

20 Kornmüller A (2007) Review of fundamentals and specific aspects of oxidation technologies in marine waters. Water Science and Technology 55, 1-6

21 Parkinson A, Barry MJ, Roddick FA, Hobday MD (2001) Preliminary toxicity assessment of water after treatment with uv-irradiation and UVC/H2O2. Water Research 35, 3656-3664

22 Wright DA (2009) Shipboard Trials of Hyde ‘Guardian’ system in Caribbean Sea and Western Pacific Ocean, April 5 th - October 7th, 2008. Final Report to Hyde Marine and Lamor Corp.

University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons Maryland, 122 pp

23 Sharpless CM, Linden KG (2001) UV photolysis of nitrate. Effects of natural organic matter and dissolved inorganic carbon and implications for UV water disinfection. Environmental

Science and Technology 29, 2949-2955

24 Veldhuis MJW, Fuhr F, Stehouwer P-P (2009) Final report of the land-based testing of the Hyde-GuardianTM -System, for Type Approval according to Regulation-D2 and the relevant IMO

Guideline (April – July 2008). Royal Netherlands Institute for Sea Research, Den Burg, Texel, the Netherlands, 42 pp

25 Sharpless C, Page M, Linden K (2003) Impact of hydrogen peroxide on nitrite formation during UV disinfection Water Research 37, 4730 - 4736

26 Avilez IM, de Aguiar LH, Altran AE, Moraes G (2004) Acute toxicity of nitrite to matrinxã, Brycon cephalus (Günther, 1869) (Teleostei-Characidae). Ciência Rural, Santa Maria 34,1753-

1756

27 Spotte SH (1992) Captive Seawater Fishes: Science and Technology. Wiley, 976 pp
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psu)28. Similar results were obtained when the relative toxicity of nitrite to chinook salmon fingerlings

(Oncorhynchus tshawytscha) was measured by static bioassay. In freshwater, the 48-h median lethal nitrite concentration was 19 mg L-1. In seawater, 1,070 mg L-1 nitrite caused only 10% mortality in 48 h29

.

Bromate formation in UV-treated seawater through ozone

It is known that (1) ozone can be produced by ultraviolet light and (2) ozonation or chlorination of bromide-containing water can facilitate the conversion of bromide into bromate (BrO3(-), which is a carcinogenic chemical30

.

No data were found on concentrations of ozone produced during short-term exposure of seawater to UVC nor the risk of production of bromate (and its derivates) in UV-treated seawater. In April 2006, a thorough assessment of environmental risks of bromate as a relevant chemical was performed by Japan

MEPC 55/2 in connection with basic approval of active substances used by Special Pipe Ballast Water

Management System (combined with ozone treatment) in accordance with the Procedure for approval of

Ballast Water Management Systems that make use of active substance (G9)31. The risk evaluation to the

environment focused on the bromate ion. It was considered that the short-term toxicity of ballast water

discharged after being treated in the system is basically insignificant: acute toxicity of the bromate ion in

seawater was low (NOEC 32 mg/L). Since the concentration of bromate ions (oxidant concentration) at

the time of discharge is lower than 0.16 mg/L, Long-term effects are unlikely to occur. Therefore the

bromate ion to be discharged from this system will not cause any long-term or short-term effects on

aquatic organisms. The Japanese system, submitted to MEPC 55 for approval, uses ozone as an active

substance whereas in Hyde GUARDIANTM system ozone is not likely to be produced since the Hyde

GUARDIANTM operates in a higher UV spectral bandwidth (above 200 nm).

4.2.2 Chronic aquatic toxicity (GESAMP-BWWG 6, Annex IV: 3.3.3)

The most applicable and relevant indirect evidence of zero NOEC (No Observed Effect Concentration)

of UV-treated saltwater originates from uses in marine aquariums and hatcheries, in which bacteria-free

water prevents disease agents (bacteria, fungi) from growing without producing any DBPs harmful to

marine organisms. In mussel and oyster culture, UV-treated water is used for rearing all stages of spat,

from fertilization to larvae and juveniles32, which are regarded as the most sensitive life-cycle stages.

Ultraviolet treatment is sometimes employed also in water purification systems for tropical freshwater,

marine and cool water aquaria and pond systems, not only to reduce microbial levels but also to aid in

oxidation of organics, phosphate and nitrogenous compounds through the collateral production of ozone

(O3)

33

.

In fish aquaculture, the applications include treatment of water in hatcheries, and fry rearing tanks34. UV

is ideally suited for these applications as it uses no chemicals and does not create by-products, which

would harm the fish stock, or other aquatic life, on discharge35 36. However, the results from a 5-month

experiment indicate that seawater recently disinfected with ultraviolet radiation (254 nm) can increase

28 Saroglia MG, Scarano G, Tibladi E (1981) Acute toxicity of nitrite to sea bass (Dicentrarchus labrax) and European eel (Anguilla anguilla). Journal of the World Aquaculture Society 12,

121-126 (quoted in Avilez et al. 2004)

29 Crawford RE, Allen GH (1977) Seawater inhibition of nitrite toxicity to chinook salmon. Transactions of the American Fisheries Society 106, 105-109.

30 Huang X, Gao N, Deng Y (2008) Bromate ion formation in dark chlorination and ultraviolet/chlorination processes for bromide-containing water. J Environ Sci (China) 20, 246-51

31 MEPC 55/2, submitted by Japan in 2006

32 Helm MM, Bourne N, Lovatelli A (eds) (2004) Hatchery culture of bivalves. A practical manual. FAO Fisheries Technical Paper. No. 471. Rome, FAO.177 pp.

www.fao.org/docrep/007/y5720e/y5720e06.htm

33 Saltwater Aquariums. http://saltaquarium.about.com

34 Shackley SE, King PE (2006) A sterile sea water circulating system for the jar culture of developing fish eggs. Journal of Fish Biology 12, 235-237

35 Hanovia News (2007) UV water disinfection in fish farms and hatcheries. http://halmapr.com/news/hanovia/2007/09/28/aquaculture/

36 Øye AK, Rimstad E (2001) Inactivation of infectious salmon anaemia virus, viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus in water using UVC irradiation.

Diseases of Aquatic Organisms 48, 1-5
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the frequency of cataract (cloudiness of the lens of the eye) in juvenile cod. It is hypothesized that ozone

or other photoproducts formed by UV-C radiation may cause cataract in fish37; however, other studies on

this topic found seem to deal with natural exposure to UV-A and UV-B38, not UV-C.

In 2002, chlorine was replaced by UV treatment for water purification in a dolphinarium in Edmonton,

Alberta, Canada39. A routine water-testing package consisted of a number of common water parameters

(sodium, potassium, calcium, magnesium, total nitrate, iron, manganese, sulphate, chloride, carbonate,

bicarbonate, fluoride, hardness, alkalinity, and total dissolved solids). The total nitrate level was the only

parameter that changed with the implementation of the UV system. Other parameters that were measured throughout the implementation process included dissolved oxygen, pH, suspended solids, and temperature. It was found that both prior to and after implementation the aforementioned parameters did not

undergo any change.

4.2.3 Endocrine disruption (GESAMP-BWWG 6, Annex IV: 3.3.4)

No evidence has been found that the by-products that could be formed or have been found to be present

at low levels (nitrite) can cause endocrine disruption.

4.2.4 Sediment toxicity (GESAMP-BWWG 6, Annex IV: 3.3.5)

No evidence has been found that by-products could result in sediment toxicity.

4.3 Bioavailability/Biomagnification/Bioconcentration/Food web/Population effects (GESAMPBWWG 6, Annex IV: 3.3.6, 3.3.7)

No evidence has been found that short lived nitrites as a DBP of UV-C treatment could result in biomagnification or bioconcentration.

5 Data on Mammalian Toxicity (G9: 4.2.1.2; GESAMP-BWWG 6, Annex IV: 3.4)

Mammalian toxicity studies have not been performed on DBP (nitrite) specifically for Hyde GUARDIANTM system. There are no scientific data available indicating that nitrite concentrations at the levels

found in the Hyde GUARDIANTM discharge can be expected to be toxic to mammalians.

40

5.1 Data on mutagenicity and genotoxicity (GESAMP-BWWG 6, Annex IV: 3.4.8)

An environmental concern, which has been assumed but not investigated in depth, is the risk of genetic

mutation. If the exposure to UV-C is insufficient to kill all of the organisms, the damage to the genetic

material might lead to viable mutations and reproduction might still be possible, and hence pose an environmental risk41. Even if most or all of the surviving organisms with damaged genetic material will fail

to reproduce, a possibility exists that genetically altered DNA will benefit the surviving (micro)organisms.

37 Björnsson B (2004) Can UV-treated seawater cause cataract in juvenile cod (Gadus morhua L.)? Aquaculture 240, 187-199

38 Cullen AP, Monteith-McMaster CA, Sivak JG (1994) Lenticular changes in rainbow trout following chronic exposure to UV radiation. Current Eye Research 13, 731 – 737; Sharma JG

Masuda R, Tanaka M (2005) Ultrastructural study of skin and eye of UVB irradiated ayu Plecoglossus altivelis. Journal of Fish Biology 67, 1646-1652; Formicki G, Stawarz R (2006) Ultraviolet influence on catalase activity and mineral content in eyeballs of gibel carp (Carassius auratus gibelio). Science of the Total Environment 369, 447-450

39 Dombrosky J (2002) The use of ultraviolet irradiation for sole disinfection of an inland marine mammal facility.www.trojanuv.com/en/images/IC/Dolphin%20Lagoon.pdf

40 FAO/WHO (2003): Nitrite (and potential endogenous formation of N-nitroso compounds). In: Safety evaluation of certain food additives and contaminants. Geneva, World Health Organization, Joint FAO/WHO Expert Committee on Food Additives. WHO Food Additives Series No. 50; http://www.inchem.org/documents/jecfa/jecmono/v50je05.htm

41 Buchholz K, Tanis D, Macomber S, Farris E (1998) Ballast Water Secondary Treatment Technology Review. Final Report to Northeast-Midwest Institute. www.nemw.org/Balsurv2_UV.htm
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The risk of thriving mutants is prevented by the high minimum dosing of UV (200 mJ/cm2 - 5 times the

dose required by the US EPA drinking water disinfection standard), which dose is sufficiently high to

ensure the adequate level of DNA damage. As an extra precaution the Hyde GUARDIANTM system exposes ballast water to UV-C disinfection once more to the same high dose during de-ballasting.

6 Data on Environmental Fate and Effect under Aerobic and Anaerobic Conditions (G9:

4.2.1.3; GESAMP-BWWG 6, Annex IV: 3.5)

Nitrite released with ballast water will enter the natural nitrogen cycle run by the marine ecosystem

where NO2 is present in low concentrations, produced by e.g. planktonic algae and through decomposition of fecal pellets and dead zooplankton organisms42. It is expected that NO2 from BWT will follow

the same reduction and oxidation pathways as NO2 from natural sources; NO2 will be oxydised to NO3

and reduced to NH4.

7 Physical and Chemical Properties for the Preparations, Active Substance and Treated Ballast

Water (G9: 4.2.1.4; GESAMP-BWWG 6, Annex IV: 3.6)

Nitrite is the univalent radical NO2 or a compound containing it, such as a salt or an ester of nitrous acid.

Its regulatory name is nitrite, molecular formula NO2-, and molecule weight is 46 g/mol 43. Nitrite CAS

number is 14797-65-0.44

8 Analytical Methods at Environmentally Relevant Concentrations (G9: 4.2.1.5; (GESAMPBWWG 6, Annex IV: 3.7)

DBPs (nitrite) had been analysed using standardised methods. The impact of by-products in the discharge was analysed in 20-days incubation studies in a climate room.

9 Use of Active Substance or the Preparation (GESAMP-BWWG 6, Annex IV: 4)

The Hyde GUARDIANTM system does not use or generate Active Substances.

10 Material Safety Data Sheets (GESAMP-BWWG 6, Annex IV: 5)

Material safety data sheet for nitrite is presented in Appendix 1.

11 Risk Characterization

Nitrite is not persistent and does not bioaccumulate. Nitrite is not toxic at the concentrations resulting

from the Hyde GUADRIANTM system.

11.1 Evaluation of the treated ballast water (G9: 5.2; GESAMP-BWWG 6, Annex IV: 6.2)

11.1.1 Results from the Land Based Tests

The land based test trials of the Hyde GUARDIANTM system were carried out at the Royal Netherlands

Institute for Sea Research (NIOZ), Texel, the Netherlands, from March till July 2008. The System was

tested according to the D-2 Standard and the IMO Guidelines for Type Approval testing (G8).

42 Carlucci AF, Hartwig EO, Bowes PM (1970) Biological production of nitrite in seawater. Marine Biology 7, 161-166

43 http://www.lenntech.com/hazardous-substances/nitrite.htm

44 http://www.epa.gov/OGWDW/dwh/t-ioc/nitrates.html
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The main findings from the test trials are summarised below45:

x 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;

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

during the low salinity test runs in the discharge. This is in agreement with findings by Sharpless

& Linden46. Yet the increase was only half of the natural nitrite concentrations observed during

the second test series at high salinity and should therefore be considered to be within the natural

range. The values were also below the maximum allowed concentration for drinking water47

x The treated water did not contain toxic or growth inhibiting substances, neither were other

chemicals than nitrite found that could have been formed as by-products from the treatment.

The complete report of Veldhuis et al. (2009) has been submitted to Lloyds Register and to Maritime

and Coastguard Agency in December 2008.

11.1.2 Results from the shipboard tests

The three shipboard test trials were conducted aboard the Princess Cruise Lines ship M/V Coral Princess in 2008 to test the efficacy of the Hyde GUARDIANTM system under normal working conditions.

The trials took place during the vessel’s regular spring schedule in the Caribbean Sea, the summer

schedule in the N.W. Pacific Ocean between Whittier, Alaska and Vancouver, Canada, and during the

repositioning cruise from the western Pacific to the vessel’s winter base in Fort Lauderdale, Florida. Trials consisted of determination of water quality parameters and a comparison of biological endpoints in

treated and untreated ballast water samples, with reference to both IMO G8 and the U.S. Coast Guard

Shipboard Technology Evaluation Program (STEP).

In the report from the shipboard test trials48 the following conclusions can be found:

x While, to date, no potentially toxic chemical changes resulting from UV irradiation have been

identified, this resolution adopts an approach requiring tests for residual toxicity that essentially

follow IMO G9 guidelines, even for systems not involving the addition of active substances. The

resolution was adopted too late for such testing to be incorporated into land based testing of the

system, which ended in July 2008. Hence, three tests, one chronic (growth based) and two acute

toxicity bioassays, were incorporated into the third trial in order to provide empirical toxicological evidence on this point.

x Results of Whole Effluent Toxicity (WET) bioassays conducted during trial 3 indicated no significant differences between the toxicity of treated vs. untreated water samples at the time of discharge from the vessel. In invertebrate and vertebrate larval assays undiluted water from both

treated and untreated tanks appeared not to show any significant toxicity relative to laboratory

controls. In the case of phytoplankton there appeared to be a small degree of toxicity associated

with water retrieved from ballast waters following the (10 day) residence time, although the toxicity did not differ significantly between treated and untreated water."

x It might be concluded from those data that any toxic element present in discharged water did not

45 Veldhuis MJW, Fuhr F, Stehouwer P-P (2009) Final report of the land-based testing of the Hyde-GuardianTM -System, for Type Approval according to Regulation-D2 and the relevant IMO

Guideline (April – July 2008). Royal Netherlands Institute for Sea Research, Den Burg, Texel, the Netherlands, 42 pp

46 Sharpless CM, Linden KG (2001) UV photolysis of nitrate. Effects of natural organic matter and dissolved inorganic carbon and implications for UV water disinfection. Environmental

Science and Technology 29, 2949-2955

47 Sharpless C, Page M, Linden K (2003) Impact of hydrogen peroxide on nitrite formation during UV disinfection Water Research 37, 4730 – 4736

48 Wright DA (2009) Shipboard Trials of Hyde ‘Guardian’ system in Caribbean Sea and Western Pacific Ocean, April 5 th - October 7th, 2008. Final Report to Hyde Marine and Lamor Corp.

University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons Maryland, 122 pp
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20 April 2009

result from UV irradiation. Such results have been supported by an extensive literature search

that has revealed no evidence of residual chemical toxicity in water resulting from prior UV irradiation of that water. While some changes in nutrient levels appeared to be associated with water

treatment it was not possible to definitively conclude that these changes directly resulted from

UV irradiation. For example, both phosphate and nitrate levels at de-ballasting were higher in

treated samples relative to untreated samples at the time of discharge from the ship. It is possible

that part of this difference could be attributed to an increase in the degradation of planktonic organisms resulting from the treatment process, rather than UV irradiation directly. Nitrite levels

were also higher in treated samples 10 days after treatment. While UV photolysis of nitrate to nitrite has been demonstrated in UV-irradiated drinking water49, the levels involved are very small

relative to the U.S. drinking water standard of 1000ȝg L-1, or the European standard of 100ȝg L1

. Nitrate levels reported in this trial were negligible relative to these standards and pose no toxicological threat associated with discharge.

The final report of Wright (2009) has been submitted to Lloyds Register and to Maritime and Coastguard Agency in January 2009.

11.2 Prediction of discharge and environmental concentrations (GESAMP-BWWG 6, Annex IV: 6.3.3)

The nitrite levels measured from the treated ballast water during the land based testing were 4.62 μM in

low salinity seawater. In water intake the nitrite concentration was 2.99 μM. The nitrite concentration in

the treated water, in high salinity water, was 6.00 μM. In water intake the concentration was 6.85 μM.

During the shipboard testing nitrite levels showed a 3-4 fold increase, from 0.8 to 2.73 μg/l in trial #3, in

treated samples after 10 days.

11.3 Effects on sediment (G9: 5.1; GESAMP-BWWG 6, Annex IV: 6.3.7)

Particles and organisms larger than 50 μm are removed by the filter. This means that small particles, that

may have a higher organic content, will accumulate inside the ballast tanks. Experience from the ships

that have had the Hyde GUADRIANTM system installed show little sediment accumulation inside the

ballast tank and only a thin layer of silt (< 2 mm) has been observed. The DBP (nitrite) is not expected

to have adverse effects on sediment.

12 Quality Control/Quality Assurance (G9: 4.2.4)

The Quality Control and Quality Assurance issues have been addressed in two separate reports. Concerning the shipboard trials of the Hyde GUADRIANTM system the QA/QC protocol has been presented

in the Appendix D and the Standard Operating Procedures (SOP) for UMCES Analytical Services Division in the Appendix E of the reference (Wright, 2009). 50

Regarding the land based test trials a description of the Test Protocol and Quality Assurance Project Plan

(QAPP) has been presented in the reference (NIOZ, 2009)51. According to the reference it has been approved by Maritime Coastguard Agency (UK) and Lloyds Register (LR) in April 2008.

The both reports have been submitted to LR in January 2009.

49 E.g. Sharpless CM, Linden KG (2001) UV photolysis of nitrate. Effects of natural organic matter and dissolved inorganic carbon and implications for UV water disinfection. Environmental

Science and Technology 29, 2949-2955

50 Wright, DA (2009). Shipboard Trials of Hyde ‘Guardian’ system in Caribbean Sea and Western Pacific Ocean, April 5th – October 7th, 2008. Final Report to Hyde Marine and Lamor Corp.

University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons, Maryland 20688.

51 Royal Netherlands Institute for Sea Research (NIOZ) (2009). Test protocol and Quality Assurance Project Plan (QAPP ) for the biological efficacy testing of the Hyde Marine Ballast Water

Treatment System (HBWTS, Hyde Marine Inc) as part of the Type Approval Process under Resolution MEPC 125.53.
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13 Use of the Active Substance and Preparations

The Hyde GUARDIANTM system does not use or generate Active Substances for its treatment.

14 Risk Assessment (G9: 6, 7)

14.1 Risk to safety of ship (GESAMP-BWWG, Annex IV: 7.1)

The potential risk to the safety of the ship and crew raised by the operation of the Hyde GUARDIANTM

system is assessed, in sections below.

14.1.1 Ship Integrity and the BWTS

The Hyde GUARDIANTM system is installed in parallel with the ship’s ballast piping system. The system is always installed somewhere down the ballast pipe line after the ballast pump. There are no specific requirements or restrictions as of distance from the ballast pump or any of the associated ballast

tanks. The Hyde GUARDIANTM filtration system can be installed separately from the disinfection system. The disinfection system (UV unit) can be installed either vertically or horizontally as this has no

affect on the operation or efficacy of the system. However the multi spine Galaxy filter system is recommended to be installed in a vertical position in order for the back-flush of the filter spines to be efficient. A small inclement (less than 30o) does not have affect but a horizontal (90o) installation position

could make the back-flushing to be more inefficient, as the spines would back-flush against each other.

This recommendation does not apply for the single spine Crystal filter system.

The Hyde GUARDIANTM system is normally isolated from the ballast piping system by inlet and outlet

pneumatically actuated butterfly valves which are normally closed when the Hyde GUARDIANTM system is not in operation. For emergency use the inlet and outlet valves can be operated via the fail safe

Supervisory Control System, but also mechanically at valve location. Aside from the in-built by pass

pipe which by-passes the filter system during de-ballasting but forces ballast water flow through the UV

disinfection unit, the entire system can be bypassed in case of emergency by operation of ships valves.

Operation of the system bypass valves should be restricted to emergency operation only as this will violate the ballast water treatment convention. Theses valves should remain locked and their use should require permission of the chief or captain. The bypassing of the treatment system will need to be recorded

in the ballast water log book.

The Hyde GUARDIANTM system is fully automatic and the system can be fully integrated to the ships

control. If any of the logged operating parameters of the BWMTS are not within the acceptable and predetermined tolerances, it will shut down the Hyde GUARDIANTM system and not allow untreated water

to pass.

Generally speaking the Hyde GUARDIANTM system after installed on the ship will be one of the ships

equipment that requires least attention by the ships crew in terms of operation of the system (especially

if fully integrated into ships control) but also in terms of maintenance. The ships crew can easily be

trained on site how to operate the system from the Supervisory Control Board (local control) LCD display and control panel. The control panel, as a standard Hyde GUARDIANTM option, features simple

function buttons and a simple and clear LCD text display. The ships crew can start and stop the BWTS

system pressing simple function buttons and also control and monitor the BWTS operating parameters,

simply by following the clear instructions on the LCD text display.
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If the Hyde GUARDIANTM system is chosen not to fully integrate into ships control, but installed as

stand alone equipment. The ships crew need to manually start the BWTS system locally after having

started the ships ballasting pump(s). After starting the Hyde GUARDIANTM at the Control Panel and

choosing either ballast or de-ballast mode, the system opens the inlet valve to the system and keeps the

delivery valve closed, and waits for the UV unit to heat up to its operational temperature. After the UV

unit has reached its operational temperature and is ready for treating the water, only then the delivery

valve of the system is opened and water flows through the system. The warm up time is slightly dependant on the ambient water temperature, but normally the operating temperature is reached within 2 min

from starting of the system. The system will continue to run automatically without input from the ships

crew.

When filling or emptying the ballasting tanks (i.e. ballasting procedure), is finished, the crew needs to

stop the BWTS system locally. The system will close the delivery valve and conduct a final back-flush

cycle of the filters (only if ballasting of tanks has taken place). After this the system will shut down and

the UV unit cools down.

If the Hyde GUARDIANTM system is fully integrated into ships control via a digital link, (such as Modbus and Profibus), the controls and monitoring of the BWTS are managed from the station where ballasting the procedure is taking place, without manual interface from the ships crew. In that case the starting

signal to the ballast pump and the selection of ballasting or de-ballasting mode, enabled by the ships

crew, will give the signal to the Hyde GUARDIANTM Control system to run the system without input of

the ships crew.

14.1.2 Hazardous Area

The Hyde GUARDIANTM system can be safely installed onboard ships having hazardous areas, e.g.

tankers. However there are limitations what concern the location of the individual system components.

The Hyde GUARDIANTM filter system can be installed safely inside a hazardous area since it’s completely pneumatically operated. The Hyde GUARDIANTM UV disinfection chamber can, with a small

modification, be made into an explosion proof system by simply purging the UV chamber. This is principally done by pressurizing the internal part of the UV chamber which features the electrical components. Hence no dangerous gases or vapours can enter inside the UV chamber which if penetrated could

get in touch with the electrical system and potentially cause an electrical arc and consequential

fire/explosion by igniting the gas and/or vapour. By requirement also the areas which are classified as

hazardous areas, e.g. a Cargo Pump room features also on-line constant gas and vapour monitoring

equipment which potentially would give an alarm even before gas and/or vapour would enter into the

UV chamber. However a fail safe pressure switch is installed to the UV chamber which constantly

measures the pressure inside the UV chamber. In case of pressure loss inside the chamber, the pressure

switch would give a signal disconnect all power to the UV system.

The Hyde GUARDIANTM Power Panel and Control Panel would be installed outside the hazardous area,

e.g. machinery room space. All the penetrations through the bulkhead into the hazardous area would

have to be done by type approved penetration gland and sealing for hazardous areas. The booster pump,

which is only used during the back-flush mode of the filters, would be supplied with an explosion proof

electric or hydraulic motor.

14.1.3 Corrosion

The Hyde GUARDIANTM system does not cause corrosion of the ships structure or the ships systems to

which the Hyde GUARDIANTM system is in contact with. The Hyde GUARDIANTM filtration stage is
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pure solid liquid separation system and the UV disinfection treatment does not change the treated water

by any means which could have an affect on corrosion. If a corrosion problem exist onboard the ship

where the Hyde GUARDIANTM system is installed, the system will not enhance nor will it reduce the

corrosion rate, since the Hyde GUARDIANTM system has no affect on corrosion.

14.1.4 Fire and Explosion

The Hyde GUARDIANTM system does not possess any risk for explosion. The filters are pressure rated

to 10.0 bar and under exceptional circumstances the water pressure of a ships ballast system rarely

reaches above 7.0 bar. Under normal circumstances the operating pressure of a ships ballast system is in

the range of 1.5 to 5.0 bars. In the case of the Hyde GUARDIANTM system, during the back-flushing

and when the delivery valve of the system is closed the pressure is temporarily raised to 5.0 bar pressure

by external booster pump to enable efficient back flushing. If the ships own ballast pump deliver 5.0 bar

pressure, the external booster pump is not required. The Hyde GUARDIANTM filter system is however

equipped with a simple pressure relief valve fitted on the centre manifold which in case of pressure build

up would let out excessive pressure and drain small amounts of the ballast water to ships bilge system.

There is no risk of explosion in the UV chamber.

The Hyde GUARDIANTM system features an electrically powered UV disinfection unit, power panel,

and control cabinet components. These Hyde GUARDIANTM components are encapsulated in steel

frames and ingress protected to min IP 54 standard in accordance with general industry standard and in

accordance with ships classification society rules. And hence does not possess any higher risk of fire

then any other electrical system onboard. In case of fire e.g. short circuited system caused by any of the

Hyde GUARDIANTM electrical components or in case of fire from external source which would damage

any of the Hyde GUARDIANTM electrical components, the Supervisory Control system will shut down

the BWTS.

14.1.5 Chemical Storage

There are no chemicals used in the Hyde GUARDIANTM system.

14.1.6 Accidental Chemical Release

There are no chemicals used in the Hyde GUARDIANTM system.

14.2 Risk to technicians and/or ship’s crew

The Hyde GUARDIANTM system does not possess any risk for the ships technicians and/or ships crew.

The system does not feature any mechanically rotating parts which could cause damage or injury to personnel operating, or conducting maintenance to, the Hyde GUARDIANTM system. The Hyde GUARDIANTM system which does not use any active substance nor produce any active substance during the

treatment process is completely safe for the crew to operate or inspect during operation and also when

the system is idle or when stopped.

As of ships crew conducting routine maintenance to the system, as the Hyde GUARDIANTM system features electrical components, proper safety procedures are to be adhered to. These safety procedures are

general and are not different from any other safety procedure for a ships electrical systems or components. All the safety procedures specific to the Hyde GUARDIANTM system are well described in English language in the Owners Operating and Maintenance Manual supplied with each system. Also all the

electrical components on the Hyde GUARDIANTM system are labelled with industry accepted WARNING signs for electrically powered components.
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20 April 2009

14.3 Risks to human health (GESAMP-BWWG, Annex IV: 7.2)

There is no direct human health risk associated with the Hyde GUARDIANTM system. The system is

very robust, totally isolated from the outside world; the routine maintenance is simple and robust and is

easy to adhere to. Only by violating the simple and easy to read safety procedures related to the routine

maintenance of the system there might be a risk for human health. Even in such case or in an emergency

direct eye contact with a powered up UV-C lamp should be prevented at all times. Under normal operation there is no possibility for a person to get in contact with UV-C as the UV is well confined inside the

UV chamber. And the small amount of UV light which might be visible through drain hose connected to

the UV chamber does not cause any harm to eye sight. The UV-lamps contain a small amount of mercury (< 200 mg/lamp) that that is well enclosed within the device under normal operation. If a lamp

breaks during lamp replacement work, the mercury can be rendered harmless by binding it with sulphur

powder. Contact with skin and eyes and inhaling vapours should be prevented

14.4 Risks to the aquatic environment (GESAMP-BWWG, Annex IV: 7.3)

With the Hyde GUARDIANTM system there are no currently known risks to the aquatic environment.

The Hyde GUARDIANTM system does not use an active substance nor is a active substance produced

during the treatment process so there is no risk of any active substance being released to the aquatic environment which could cause a risk for the same

14.5 Noise

The power cabinet of the Hyde GUARDIANTM does emit a slight hum, <40db from the transformers.

The cabinet also has cooling fans will contribute to the ambient noise level.

The treatment part of the Hyde GUARDIANTM system does not generate any sound or noise except of

the sound of water flowing through a standard ships ballast pipe system, which in the case of a ship installation will be absorbed or is neglected by the ambient noise level existing onboard a ship in a machinery -, a pump room or a cargo pump room space.

15 Assessment Report (G9: 4.3; GESAMP-BWWG, Annex IV: 8)

The Hyde GUARDIANTM system, using UV (Ȝ 200-400 nm) does not use or produce active substances

for its meant ballast water treatment.

The Hyde GUARDIANTM ballast water treatment system does not produce any DBPs that would fit into

either of the two distinct classes52 of undesirable DBPs - those that affect water’s aesthetic quality and

those that are harmful to the environment or human health. Based on literature that is currently available,

it was also found that UV treatment produces neither odour nor taste.

As the result from the land-based tests it can be concluded that the present configuration of the Hyde

GUARDIANTM system was found to offer a reliable and environmentally safe cleaning of the ballast water resulting in organism numbers well below the IMO Standard D2.

Additionally, the results from the ship-board tests prove that under the conditions encountered during

these trials, the system was found to comply with all IMO G-8 standards relating to the elimination of

biota and with respect the issue of residual toxicity of treated water related to chemicals generated dur52 Wolfe RL (1990) Ultraviolet disinfection of potable water. Environmental Science and Technology 24, 768-773
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20 April 2009

ing treatment.

When the shipboard and land-based tests with the Hyde GUARDIANTM system were agreed on in early

2008, the valid G8 Guidelines at that time (Resolution MEPC.125(53)) did not include the requirements

for the environmental acceptability of the discharged water, which are included in the revised G8 Guidelines as adopted in October 2008. The land-based tests were performed in the spring and early summer

of 2008. During those tests the discharge water was analysed for potential by- or end products of the UV

treatment, as a precautionary action. Only a slight increase in nitrite, formed by conversion of nitrate in

the water was found. As stated in this report, nitrite is a constituent of sea and fresh waters.

The revised Guidelines G8 (Resolution MEPC.174(58)) were adopted on 10 October 2008. The revised

Guidelines include a reference to environmental acceptability, as introduced in paragraph 1.8: "These

Guidelines contain recommendations regarding the design, installation, performance, testing, environmental acceptability and approval of ballast water management systems".

The Resolution also states in paragraph 5.1.5 that “if it can reasonably be concluded that the treatment

process could result in changes to the chemical composition of the treated water such that adverse impacts to receiving waters might occur upon discharge, the documentation should include results of toxicity tests of treated water. The toxicity tests should include assessments of the effects of hold time following treatment, and dilution, on the toxicity. Toxicity tests of the treated water should be conducted in accordance with paragraphs 5.2.3 to 5.2.7 of the Procedure for approval of ballast water management

systems that make use of Active Substances (G9), as revised, (Resolution MEPC.169(57))”. Resolution

MEPC.174(58) was adopted after all land-based tests with the Hyde GUARDIANTM system were completed in July 2008.

To accommodate the environmental acceptability requirements of the revised G8, all possible effort was

made by the testing bodies to provide toxicity data required in Resolution MEPC.174(58). During the

last shipboard test trial Whole Effluent Toxicity (WET) tests were conducted on treated and untreated

water samples. The objective was to identify any residual chemical toxicity that could have resulted

from the Hyde GUARDIANTM ballast water treatment system. Results of the WET bioassays indicated

no significant differences between the toxicity of treated vs. untreated water samples at the time of discharge from the vessel.

The reviewed literature did indicate that in the UV BW treatment process no relevant chemicals (i.e.,

metabolites, intermediates, residual substances) are produced at levels that are expected to cause adverse

impacts on aquatic life by the discharge of UV-treated ballast water. These findings are also supported

by the results of the Hyde GUARDIANTM land based and shipboard test trials.

Based on the practical operational experience from the first Hyde GUARDIANTM system installed on

board the Coral Princess in 2003, the system has proven its reliability and safety to the ship and its crew.

Based on the results from the land-based and ship-board tests, and on the findings from the literature, it

can reasonable be concluded that the Hyde GUARDIANTM Ballast Water Treatment System could not

result in changes to the chemical composition of the treated water such that adverse impacts to receiving

waters might occur upon discharge.
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20 April 2009

16 Appendices

Annex 1.

Technical Factsheet on: NITRATE/NITRITE 53

As part of the Drinking Water and Health pages, this fact sheet is part of a larger publication: National

Primary Drinking Water Regulations

Drinking Water Standards (in mg/L)

Nitrate- MCLG: 10; MCL: 10; 10-day HAL: 10

Nitrite- MCLG: 1; MCL: 1; 10-day HAL: 1

Total (Nitrate+Nitrite)- MCLG: 10; MCL: 10; 10-day HAL: 10

Health Effects Summary

Acute: Excessive levels of nitrate in drinking water have caused serious illness and sometimes death.

The serious illness in infants is due to the conversion of nitrate to nitrite by the body, which can interfere

with the oxygen-carrying capacity of the child's blood. This can be an acute condition in which health

deteriorates rapidly over a period of days. Symptoms include shortness of breath and blueness of the

skin.

Drinking water levels which are considered "safe" for short-term exposures: For a 10-kg (22 lb.) child

consuming 1 liter of water per day, a ten-day exposure to 10 mg/L total nitrate/nitrite.

Chronic: Effects of chronic exposure to high levels of nitrate/nitrite include diuresis, increased starchy

deposits and hemorrhaging of the spleen.

Cancer: There is inadequate evidence to state whether or not nitrates or nitrites have the potential to

cause cancer from lifetime exposures in drinking water.

Usage Patterns

Most nitrogenous materials in an aquatic environment tend to be converted to nitrate, so all sources of

combined nitrogen, particularly organic nitrogen and ammonia, should be considered as potential nitrate

sources. Primary sources of organic nitrates include human sewage and livestock manure, especially

from feedlots.

The primary inorganic nitrates which may contaminate drinking water are potassium nitrate and ammonium nitrate. Potassium nitrates are used mainly as fertilizers (85%), with the remainder in heat transfer

salts, glass and ceramics, and in matches and fireworks. Ammonium nitrates are used as fertilizers (84%)

and in explosives and blasting agents (16%).

Release Patterns

The major environmental releases of inorganic sources of nitrates are due to the use of fertilizers. According to the Toxics Release Inventory, releases to water and land totalled over 112 million pounds

from 1991 through 1993. The largest releases of inorganic nitrates occurred in Georgia and California.

53 http://www.epa.gov/OGWDW/dwh/t-ioc/nitrates.html
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Environmental Fate

Due to its high solubility and weak retention by soil, nitrates are very mobile in soil, moving at approximately the same rate as water, and has a high potential to migrate to ground water. Because it does not

volatilize, nitrate/nitrite is likely to remain in water until consumed by plants or other organisms. Ammonium nitrate will be taken up by bacteria. Nitrate is more persistent in water than the ammonium ion.

Nitrate degradation is fastest in anaerobic conditions.

Chemical/Physical Properties

CAS Number: Nitrate ion: 14797-55-8; Nitrite ion: 14797-65-0

Color/ Form/Odor: Domestic fertilizer grade ammonium or potassium nitrates are in prilled (beaded) or

crystalline forms, usually coated with an anti-caking agent and adsorbed fuel oil.

Solubilities: Nitrates and nitrites are highly soluble in water

Soil sorption coefficient: N/A

Bioconcentration Factor: N/A

Trade Names/Synonyms: Potassium salt: Potnit, Hitec, Niter, Nitrate of potash, Saltpeter.Ammonium

salt: German or Norway saltpeter, Varioform I, Merco or Herco prills, Nitram.

Other Regulatory Information

Monitoring:

-- For Ground Water Sources:

Initial Frequency-Nitrate: 1 sample annually

Nitrite: 1 sample during first 3-year compliance period

Repeat Frequency-Nitrate: 1 sample annually

Nitrite: determined by State

-- For Surface Water Sources:

Initial Frequency-Nitrate: 1 sample each quarter

Nitrite: 1 sample during first 3-year compliance period

Repeat Frequency-Nitrate: 1 sample annually

Nitrite: determined by State

--Triggers - If detect at > 5 mg/L nitrate, sample quarterly.

If detect at > 0.5 mg/L nitrite, sample quarterly.

If detect total nitrate + nitrite > 5 mg/L, sample quarterly

Analysis

Reference Source Method Number

EPA 600/4-79-020 353.1; 353.2; 353.3; 300.0; 354.1

Standard Methods 418C; 418F

ASTM D3867-85A; D3867-85B

Treatment/Best Available Technologies: Ion exchange; Reverse osmosis; Electrodialysis (nitrate only)

For Additional Information:

Sources of toxicological and environmental fate data include:

Toxic Substance Control Act Information Line - 202/554-1404

Toxics Release Inventory, National Library of Medicine - 301/496-6531

Agency for Toxic Substances and Disease Registry - 404/639-6000