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 

April 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 

Mean Control No. at deballast 494±284 654±324 

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 

Mean treated No. at deballast 110±64 

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/mL 

‘Live’

Diatoms. 

Density/ mL 

Total ‘Live’

phytoplankton. 

Density/ mL 

Dead 

Dinoflagellates. 

Density/ mL 

Dead 

Diatoms. 

Density/ mL 

Total Dead

phytoplankton. 

Density/ mL 

Untreated Controls, T=0

Rep. C1, T=0 0.01 0.17 0.18 0 0 0

Rep. C1, T=0 + 48h 

growout 

0.016 0.032 0.048 0.0006 0.0006 0.001

Rep. C2 T=0 0.004 0.062 0.07 0 0 0

Rep. C2 T=0 + 48h 

growout 

0.005 0.015 0.02 0 0.0008 0.0008

Rep. C3 T=0 0.024 0.13 0.15 0 0 0

Rep. C3 T=0 + 48h 

growout 

0.003 0.063 0.066 0.0003 0.0006 0.0008

Mean ‘Live’ 

Phytoplankton. 

Control samples, T=0 

(after grow-out)

0.133±0.061

(0.45±0.023) 

Treated, T=0

Rep. T1, T=0 0.01 0.072 0.081 0 0 0

Rep. T1, T=0 + 48h 

growout 

0.004 0.005 0.009 0.0008 0.0003 0.001

Rep. T2 T=0 0.008 0.076 0.084 0.002 0.0004 0.0025

Rep. T2 T=0 + 48h 

growout 

0.003 0.006 0.009 0 0 0

Rep. T3 T=0 0.003 0.070 0.072 0.0004 0 0.0004

Rep. T3 T=0 + 48h 

growout 

0.003 0.005 0.008 0 0 0

Mean ‘Live’ 

Phytoplankton. 

Treated samples, T=0 

(after grow-out)

0.080±0.006

(0.009±0.0006) 
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/mL 

‘Live’

Diatoms. 

Density/ 

mL 

Total ‘Live’

phytoplankton. 

Density/ mL 

Dead 

Dinoflagellates. 

Density/ mL 

Dead 

Diatoms. 

Density/ mL 

Total Dead 

phytoplankton. 

Density/ mL 

Untreated Controls, T=96h 

Start Deballast. Rep. 

C1a, T=96 

0.0003 0.005 0.006 0 0 0 

Rep. C1b T=96 0.0003 0.005 0.005 0 0.0006 0.0006 

Rep. C1c T=96 0 0.003 0.003 0 0 0 

Mean C1, T = 

96h samples 

 0.004±0.002 

Mid Deballast. Rep. 

C2a, T=96 

0.0003 0.019 0.019 0 0.003 0.003 

Rep. C2b T=96 0.0008 0.014 0.015 0 0.0025 0.0025 

Rep. C2c T=0 0.001 0.009 0.01 0 0.004 0.004 

Mean C2, T = 

96h samples 

0.015±0.005 

End Deballast. Rep. 

C3a, T=96 

0.001 0.015 0.016 0.0006 0.006 0.006 

Rep. C3b T=96 0.0008 0.016 0.017 0.0003 0.004 0.004 

Rep. C3c T=96 0.0006 0.021 0.021 0 0.004 0.004 

Mean C3, T = 

96h samples 

 0.018±0.003 

Mean Live 

Phytoplankton 

0.012±0.007 

(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/mL 

‘Live’

Diatoms. 

Density/ 

mL 

Total ‘Live’

phytoplankton. 

Density/ mL 

Dead 

Dinoflagellates. 

Density/ mL 

Dead 

Diatoms. 

Density/ 

mL 

Total Dead 

phytoplankton

. Density/ mL 

Treated, T=96h 

Start Deballast. Rep. 

T1a, T=96 

0.0006 0.004 0.004 0.0003 0 0.0003 

After 48h 

grow-out 

0 0.002 0.002 0 0.0006 0.0006 

Rep. T1b T=96 0.0003 0.007 0.007 0 0.0006 0.0006 

After 48h 

grow-out 

0 0.0008 0.0008 0 0.0003 0.0003 

Rep. T1c T=96 0 0.006 0.006 0.0006 0 0.0006 

After 48h 

grow-out 

0 0.001 0.001 0 0 0 

Mean T1, 

T=96h samples 

(after growout)

 0.006±0.002 

(0.001±0.0004) 

 

Mid Deballast. Rep. 

T2a, T=96 

0.0003 0.007 0.008 0.0003 0.0003 0.0006 

After 48h 

grow-out 

0 0.001 0.001 0 0 0 

Rep. T2b T=96 0 0.006 0.006 0 0 0 

After 48h 

grow-out 

0 0.004 0.004 0 0.0003 0.0003 

Rep. T2c T=96 0.0003 0.005 0.006 0.0003 0 0.0003 

After 48h 

grow-out 

0.0006 0.0006 0.001 0 0.003 0.003 

Mean T2, T = 

96h samples 

(after growout)

0.006±0.001 

(0.002±0.002) 
19 

End Deballast. Rep. 

T3a, T=96 

0.0006 0.004 0.004 0 0.003 0.003 

After 48h 

grow-out 

0 0.002 0.002 0 0.002 0.002 

Rep. T3b T=96 0.0003 0.007 0.007 0 0.001 0.001 

After 48h 

grow-out 

0.0003 0.005 0.005 0 0.0006 0.0006 

Rep. T3c T=96 0.0006 0.008 0.009 0 0 0 

After 48h 

grow-out 

0.0006 0.001 0.002 0 0.0003 0.0003 

Mean T3, T = 

96h samples 

(after growout)

 0.007±0.002 

(0.003±0.002) 

 

Mean Live 

Phytoplankton

(after growout)

0.006±0.001 

(n=9) 

(0.002±0.001) 

(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 
20 

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

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
21 

Control 2 mid deballasting 

1.43 1.86 1.77

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 
22 

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

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±356 0

Control (untreated) Mid 

de-ballast (N=3) 

464±670 1,123±572

Control (untreated) End 

de-ballast (N=3) 

43±74 971±654

Mean control (untreated) 

No. at de-ballast 

337±441 698±683

Treated (UV + filter 

during ballasting) → (UV 

at de-ballasting) Start deballast (N=3) 

0 75±20

Treated (UV + filter 

during ballasting) → (UV 

at de-ballasting) Mid deballast (N=3) 

0 256±374

Treated (UV + filter 

during ballasting) → (UV 

at de-ballasting) End deballast (N=3) 

0 533±9

Mean treated No. at deballasting 0 288±274

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 de-ballast 

(N=3) 

0 0 

Control (untreated) Mid de-ballast 

(N=3) 

0 0 

Control (untreated) End de-ballast 

(N=3) 

0 11±15 

Treated (UV + filter during 

ballasting) → (UV at de-ballasting). 

Start de-ballast (N=3) 

0 0 

Treated (UV + filter during 

ballasting) → (UV at de-ballasting) 

Mid de-ballast (N=3) 

27±27 69±98 

Treated (UV + filter during 

ballasting) → (UV at de-ballasting) 

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. 

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

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) 

‘Live’ 

Dinoflagellates. 

Density/mL 

‘Live’ 

Diatoms. 

Density/ 

mL 

Total ‘Live’ 

phytoplankton. 

Density/ mL 

Dead 

Dinoflagellates. 

Density/ mL 

Dead 

Diatoms. 

Density/ 

mL 

Total Dead 

phytoplankton. 

Density/ mL 

Rep. C1a, T=0 0.26 1.2 12.3 0 0 0

After42h 

growout 

0.77 6.5 7.3 0 0.30 0.30

Rep. C1b, T=0 0.45 19.9 20.3 0 0 0

After42h 

growout

0.48 11.8 12.3 0.03 0.03 0.06

Rep. C1c, T=0 0.19 17.4 17.6 0 0 0

After42h 

growout

0.36 12.7 13.1 0.1 0.13 0.23

Mean C1 

samples (after 

grow-out)

16.8±4.1

(10.9±3.2) 

Rep. C2a T=0 0.39 19.8 20.2 0 0 0

After42h 

growout

0.45 29.2 30.0 0 0.03 0.03

Rep. C2b, T=0 0.64 3.5 3.6 0 0.06 0.06

After42h 

growout

0.29 6.8 7.1 0 0.13 0.13

Rep. C2c, T=0 0.74 4.8 5.6 0 0 0

After42h 

growout

0.13 3.7 3.8 0.1 0.13 0.23

Mean C2 

samples (after 

grow-out)

9.8±9.0

13.5±14.1 

Rep. C3a, T=0 0.97 2.5 2.6 0 0 0

After42h 

growout

0.22 7.4 7.6 0 0.40 0.40

Rep. C3b, T=0 0.12 2.3 2.4 0 0 0

After42h 

growout

0.21 0.88 1.1 0 0.06 0.06

Rep. C3c, T=0 0.14 2.7 2.8 0 0.02 0.02

After42h 

growout

0.17 2.4 2.6 0.07 0.1 0.16

Mean C3 

samples (after 

grow-out)

2.6±0.22

3.8±3.4 
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 

( after growout)

9.7±7.9

2.9±6.6 

Phytoplankton 

(>10μm-

<50μm) 

‘Live’

Dinoflagellates. 

Density/mL 

‘Live’

Diatoms. 

Density/ 

mL 

Total ‘Live’

phytoplankton. 

Density/ mL 

Dead 

Dinoflagellates. 

Density/ mL 

Dead 

Diatoms. 

Density/ 

mL 

Total Dead 

phytoplankton

. Density/ mL 

Rep. T1a, T=0 0.13 0.06 0.19 0 0 0

After42h 

growout

0.03 0.10 0.13 0.06 64,500.060 0.13

Rep. T1b, T=0 0.02 0.05 0.07 0 0 0

After42h 

growout

0.10 0.03 0.13 0.03 0 0.03

Rep. T1c, T=0 0.15 0 0.15 0 0 0

After42h 

growout

0.06 0.46 0.51 0 0.06 0.06

Mean treated 

No. at T=0 

(after grow-out) 

0.14 ± 0.06

0.26±0.22 

Rep. T2a, T=0 0.13 0.22 0.35 0 0 0

After42h 

growout

0.29 0.23 0.52 0.14 0 0.14

Rep. T2b, T=0 0.08 0.22 0.30 0.03 0.08 0.11

After42h 

growout

0.32 0.45 0.77 0.03 0.10 0.13

Rep. T2c, T=0 0.13 0.26 0.39 0 0 0

After42h 

growout

0.09 0.13 0.21 0 0 0

Mean treated 

No. at T=0 

(after grow-out)

0.35±0.04

0.50±0.28 

Rep. T3a, T=0 0.19 0.06 0.26 0 0 0

After42h 

growout

0.16 0.21 0.36 0.13 0.03 0.16

Rep. T3b, T=0 0.32 0.32 0.64 0 0.05 0.05

After42h 

growout

0.19 0.14 0.33 0.14 0.09 0.23

Rep. T3c, T=0 0.07 0.61 0.61 0.02 0.10 0.12

After42h 

growout

0.10 0.03 0.13 0.03 0.03 0.06
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/mL 

‘Live’

Diatoms. 

Density/ 

mL 

Total ‘Live’

phytoplankton. 

Density/ mL 

Dead 

Dinoflagellates. 

Density/ mL 

Dead 

Diatoms. 

Density/ 

mL 

Total Dead 

phytoplankton. 

Density/ mL 

Start De-ballast. 

Rep. C1a, 

T=114h 

1.2 18.1 19.3 0.82 0.38 0.46

After 42h growout 

1.0 17.8 18.8 0.10 0.30 0.40

Rep. C1b 

T=114h 

0.025 0.65 0.68 0 0.025 0.025

After 42h growout

0 0.35 0.35 0.05 0.05 0.10

Rep. C1c 

T=114h 

0.08 0.06 0.67 0 0.03 0.03

After 42h growout

0.04 0.60 0.64 0 0.04 0.04

Mean C1, T=

114h samples 

After grow-out 

6.9±10.8

6.6±8.6

Mid De-ballast. 

Rep. C2a, 

T=114h 

0.05 0.90 0.90 0 0.08 0.08

After 42h growout

0.05 1.24 1.29 0 0.04 0.04

Rep. C2b 

T=114h 

0 0.62 0.62 0 .0.02 0.02

After 42h grow- 0 0.79 0.79 0 0.04 0.04

Mean treated 

No. at T=0 

(after grow-out)

0.53±0.23

0.27±0.13 

Mean ‘Live’ 

Phytoplankton 

(after grow-out) 

0.29±0.23

0.13±0.14 
34 

out

Rep. C2c 

T=114h 

0 0.65 0.65 0 0.08 0.08

After 42h growout

0 1.05 1.05 0 0.05 0.05

Mean C2, T=

114h samples 

After grow-out 

0.74±0.18

1.04±0.21 

End De-ballast. 

Rep. C3a, 

T=114h 

0.13 0.80 0.93 0 0.13 0.13

After 42h growout

0.88 1.24 1.33

Rep. C3b 

T=114h 

0.17 1.10 1.30 0 0.02 0.23

After 42h growout

0.14 1.26 1.40

Rep. C3c 

T=114h 

0.09 0.72 0.81 0 0.12 0.12

After 42h growout

0.10 0.90 1.0

Mean C3, T= 

114h samples 

After grow-out

 1.0±0.23

1.2±0.17 

 

Mean Live 

Phytoplankton 

After grow-out

2.9±6.2

3.0±5.6 
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/mL 

‘Live’

Diatoms. 

Density/ 

mL 

Total ‘Live’

phytoplankton. 

Density/ mL 

Dead 

Dinoflagellates. 

Density/ mL 

Dead 

Diatoms. 

Density/ 

mL 

Total Dead 

phytoplankton. 

Density/ mL 

Start De-ballast. 

Rep. T1a, 

T=114h 

0.05 0.98 0.15 0 0.05 0.05

After42h 

growout

0.02 0.04 0.06 0 0.06 0.06

Rep. T1b, 

T=114h 

0.03 0.21 0.23 0 0.05 0.05

After42h 

growout

0.02 0.03 0.05 0.02 0.07 0.09

Rep. T1c, 

T=114h 

0.05 0.20 0.25 0 0 0

After42h 

growout

0 0.04 0.04 0 0.02 0.02

Mean T1, T=

114h samples 

After grow-out

0.21±0.06

0.05±0.007 

Mid De-ballast. 

Rep. T2a, 

T=114h 

0.02 0.24 0.26 0 0.02 0.02

After42h 

growout

0 0.02 0.02 0 0.02 0.02

Rep. T2b, 

T=114h 

0 0.16 0.16 0 0 0

After42h 

growout

0 0.09 0.09 0 0 0

Rep. T2c 

T=114h 

0 0.09 0.09 0 0.05 0.05

After42h 

growout

0 0.06 0.06 0 0.06 0.06

Mean T1, T= 

114h samples 

After grow-out

0.17±0.08

0.06±0.03
36 

End De-ballast. 

Rep. T3a, 

T=114h 

0.03 0.07 1.0 0 0 0

After42h 

growout

0.03 0.04 0.07 0 0 0

Rep. T3b, 

T=114h 

0 0.18 0.18 0 0 0

After42h 

growout

0 0.12 0.12 0 0 0

Rep. T3c, 

T=114h 

0 0.28 0.28 0 0 0

After42h 

growout

0 0.16 0.16 0 0 0

Mean T3, T= 

114h samples 

(After grow-out)

0.19±0.09

(0.12±0.04)

Mean Live 

Phytoplankton 

(After grow-out)

0.19±0.07

(0.08±0.04) 
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) 

during ballasting 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±16 

Control (untreated) Mid 

de-ballast (N=3) 

8±7 13±19 

Control (untreated) End 

de-ballast (N=3) 

16±17 13±10 

Mean Control No. at deballasting 16±13 20±18 

Treated (UV + filter 

during ballasting) →

(UV at de-ballasting) 

0 0 
44 

Start de-ballast (N=3) 

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 

Mean Treated Nos. at 

de-ballasting 

0 1.8±3.5 

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/mL 

‘Live’

Diatoms. 

Density/ 

mL 

Total ‘Live’

phytoplankton. 

Density/ mL 

Dead 

Dinoflagellates. 

Density/ mL 

Dead 

Diatoms. 

Density/ 

mL 

Total Dead 

phytoplankton. 

Density/ mL 

Start Ballast. 

Rep. C1a, T=0 

0.55 5.32 5.87 0.10 0.55 0.65

After 48h growout 

0.25 0.12 0.37 0.19 0.04 0.63

Rep. C1b T=0 0.49 7.83 8.32 0 0.82 0.82

After 48h growout

0.18 0.85 1.03 0 0.09 0.09

Rep. C1c T=0 0.49 3.42 3.91 0.24 0.24 0.49

After 48h growout

0.38 0.34 0.72 0 0.04 0.04

Mean C1, T=0 

samples 

(After grow-out)

0.51 4.50 6.03±

2.21 

(0.71± 

0.33) 

0.12 0.54 0.65

Mid De-ballast. 

Rep. C2a, T=0 

0.04 9.0 9.4 0 0.4 0.4

After 48h growout

0.3 2.0 2.3 0 0.2 0.2

Rep. C2b T=0 2.8 10.3 13.1 0.4 0.5 0.9

After 48h growout

1.2 2.3 3.5 0.1 0.5 0.6

Rep. C2c T=0 0.6 3.8 4.4 0.21 0.42 0.63

After 48h growout

0.72 4.44 5.16 0.11 0.45 0.56

Mean C2, T=0 

samples 

(After grow-out)

1.27 7.70 8.96±4.33

(3,65±1.45)

193,037 460,741 653,778

End De-ballast. 

Rep. C3a, T=0 

1.0 0.5 1,5 1.0 0.4 1.4

After 48h growout

1.02 0.70 1.72 0.06 0.3 0.36

Rep. C3b T=0 1.34 3.25 4.59 0 0.38 0.38

After 48h grow- 0.99 0.96 1.95 0 0.19 0.19
47 

out

Rep. C3c T=0 0.79 6.69 7.46 0.19 0.39 0.60

After 48h growout

0.48 5.0 5.48 0.20 0.45 0.65

Mean C3, T=0 

samples 

(After grow-out)

1.0 3.5 4.5±2.98

(3.05±2.11)

0.98 0.39 0.48

Mean Live 

Phytoplankton 

(After grow-out)

0.94 4.91 6.5±3.46

(2.47±1.90(n=9))

0.14 0.46 0.60
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/mL 

‘Live’

Diatoms. 

Density/ 

mL 

Total ‘Live’

phytoplankton. 

Density/ mL 

Dead 

Dinoflagellates. 

Density/ mL 

Dead 

Diatoms. 

Density/ 

mL 

Total Dead 

phytoplankton. 

Density/ mL 

Rep. T1a, T=0 1.80 0.12 1.92 0 0.08 0.08

After 48h growout

ND

Rep. T1b, T=0 1.32 0.23 1.56 0 0 0

After 48h growout

2.48 0 2.48 0 0.16 0.16

Rep. T1c, T=0 2.48 0 2.48 0 0.16 0.16

After 48h growout

ND

Mean T1 T=0 

samples 

1.98±0.46

Rep. T2a, T=0 0.81 0.26 1.06 0 0 0

After 48h growout

ND

Rep. T2b, T=0 1.61 0.18 1.79 0 0 0

After 48h growout

0 0.33 0.33 0 0 0

Rep. T2c, T=0 1.73 0.23 1.97

After 48h growout

ND

1.61±0.48

Rep. T3a, T=0 1.44 0 1.44 0 0 0

After 48h growout

ND

Rep. T3b, T=0 2.15 0 2.15 0 0 0

After 48h growout

0.73 0 0.73 0 0 0

Rep. T3c, T=0 0.85 0 0.85

After 48h growout

ND

1.48±0.65

Mean ‘Live’ 

Phytoplankton 

(After growout)

1.37 0.12 1.51±0.72 (n=9)

(1.18±1.14 

(n=3))

0 0.03 0.03
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/mL 

‘Live’

Diatoms. 

Density/ 

mL 

Total ‘Live’

phytoplankton. 

Density/ mL 

Dead 

Dinoflagellates. 

Density/ mL 

Dead 

Diatoms. 

Density/ 

mL 

Total Dead 

phytoplankton. 

Density/ mL 

Rep. C1a, T=10 

Days 

0.17 1.54 1.71 0 0.68 0.68 

After 48h growout

0.39 2.16 2.55 

Rep. C1b, 

T=10 Days 

0.73 1.47 2.20 0.20 0 0.20 

After 48h growout

0.66 0.50 1.16 

Rep. C1c, T= 

10 Days 

0.54 4.90 5.44 0.15 0 0.15 

After 48h growout

ND 

Mean C1, T=10 

Days 

 3.12±2.03 

Rep. C2a T= 10 

Days 

1.66 2.50 3.16 0 0.16 0.16 

After 48h growout

ND 

Rep. C2b, T= 

10 Days 

0.44 1.29 1.73 0 0 0 

After 48h growout

0.83 0 0.83 

Rep. C2c, T= 

10 Days 

0.83 3.88 4.71 0 0.16 0.16 

After 48h growout

ND 

Mean C2, T=10 

Days 

 3.20±1.49 

Rep. C3a, T= 

10 Days 

0.4 2.18 2.58 0 0.21 0.21 

After 48h growout

ND 

Rep. C3b, T= 

10 Days 

0 0.66 0.66 0 0 0 
50 

After 48h growout

0 0.33 0.33 

Rep. C3c, T= 

10 Days 

0 3.15 3.15 0 0.33 0.33 

After 48h growout

ND 

Mean C3, T=10 

Days 

0.67 1.98 2.22±1.36 

Mean ‘Live’ 

Phytoplankton 

(After grow-out)

0.17 2.40 2.82±1.50 

(n=9) 

(1.27±0.95 

(n=4))

0.04 0.02 0.02 
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/mL 

‘Live’

Diatoms. 

Density/ 

mL 

Total ‘Live’

phytoplankton. 

Density/ mL 

Dead 

Dinoflagellates

. Density/ mL 

Dead 

Diatoms. 

Density/ 

mL 

Total Dead 

phytoplankton. 

Density/ mL 

Start De-ballast. 

Rep. T1a, T=10 

Days 

0.43 0.65 1.01 0.22 0.22 0.44

After 48h growout

0.10 0.06 0.16 0.20 0.17 0.37

Rep. T1b, T=10 

Days 

0 0 0 2.16 0.54 2.7

After 48h growout

0 0 0 1.70 0.22 1.92

Rep. T1c, T=10 

Days 

0.65 0.43 1.09 0 0.65 0.65

After 48h growout

0.14 0.10 0.25 0 0.50 0.50

Mean T1, T=10 

Day samples 

(After grow-out)

0.36 0.36 0.72±0.62

(0.14±0.12) 

0.79 0.50 1.26

Mid De-ballast. 

Rep. T2a, T=10 

Days 

0.17 0 0.17 0.17 0.87 1.04

After 48h growout

0.06 0 0.06 0.16 0.43 0.59

Rep. T2b, T=10 

Days 

0.89 1.09 1.98 0.19 0 0.19

After 48h growout

0.16 0.11 0.26 0.12 0 0.12

Rep. T2c T=10 

Days 

0 0 0 0 0.42 0.42

After 48h growout

0 0 0 0 0.33 0.33

Mean T2, T=10 

Day samples 

(After grow-out)

0.36 0.36 0.72±1.1 0.12 0.43 0.55
52 

(0.11±0.14)

End De-ballast. 

Rep. T3a, T=10 

Days 

0 0.84 0.84 0.17 0 0.17

After 48h growout

0 0.10 0.10

Rep. T3b, T=10 

Days 

0 0.24 0.24 0.15 0 0.15

After 48h growout

0 0.10 0.10

Rep. T3c, T=10 

Days 

0 0.14 0.14 0.14 0 0.14

After 48h growout

0 0

Mean T3, T=10 

Day samples 

(After grow-out)

0 0.49 0.41±0.38

(0.07±0.06)

0.15 0 0.15

Mean Live 

Phytoplankton 

(After grow-out)

0.24 0.38 0.61±0.68

(0.10±0.10) 

0.36 0.30 0.66
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 

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

Treated T =0 T3a 0 0 0 0 0 0 0 
55 

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 

25mL 10mL 1mL 

 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 

Treated t=0 T1c >100 9 6 

Treated t=0 T2a 4 2 1 
56 

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, 

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 
57 

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 

of untreated organisms in this size category declined precipitously over the period of residence in 

the ballast tanks. 
58 

 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-2 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 an 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. Growth was determined as percentage change in cell count before and after 

grow-out

Untreated, 

T=0h Phytoplankton Cell Counts 

Before 

Grow-out 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 

5

th 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 
72 

concentration range among different water bodies, and cell densities encountered during these 

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 
73 

working ships where, unlike land-based trials, there is limited ability to control some test 

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 1000cfu/100ml for this group (Table 1). Cfus for cultural heterotrophic bacteria 

nominally comply with this standard in all three trials, thereby illustrating the sparse bacterial 

flora in areas from which ballast water was obtained. Noticeably higher concentrations of 

culturable bacteria were recorded from treated samples at 114h in trial 2, although the mean 

figure of 148.3 must be regarded as approximate in view of the ‘>100cfu’ and ‘>200cfu’ 

designations given the most densely populated plates (Table 18). 

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

heterotrophic 

Coliforms E. Coli Enterrococci Vibrio 

cholerae

Treatment Time Trial

Control t=0 1 12.2±12.9 0.2±0 ND 0.2±0 0 

Treated t=0 1 19.4±10.5 ND ND ND 0 

Control t=96h 1 5.0±5.7 ND ND ND 0 

Treated t=96h 1 1.0±1.7 ND ND ND 0 

Control t=0 2 39.6±65.0 ND ND ND 0 

Treated t=0 2 21.6±29 ND ND ND 0 

Control t=114h 2 94.1±68.3 ND ND 1.6±1.4 0 

Treated t=114h 2 148.3±64.4 ND ND 3.4±2.6 0 

Control t=0 3 44.2±44.75 ND ND ND 0 

Treated t=0 3 49.1±48.1 ND ND ND 0 

Control t=10 

days 

3 6.8±7.3 0.6±0.53 0.3±0.5 ND 0 

Treated t=10 

days 

3 2.0±6.0 ND ND ND 0 

Samples from trial 3 were similar to trial 1. No indicator bacteria were reported in either 
74 

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. 

Bacterial counts from trial 2 differed from the other two trials, and were characterized by 

higher counts of cultural bacteria at the time of discharge (de-ballasting) and the 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. No coliforms were 

recorded from any samples in this trial. With respect to Enterococci, numbers of cfus in treated 

samples at de-ballasting, 3.4±2.6 cfu/100ml (table 31), were higher than in untreated samples 

(1.6±1.4cfu/100 ml.). The figure of 3.4±2.6 cfu/100ml is below the standard of 100cfu/100ml set 

for this group by IMO and also complies with the corresponding standard of 33 cfu/100ml 

proposed by the U.S. Coast Guard and adopted by the state of California (table 1). Appearance of 

intestinal enterococci in both treated and untreated samples at time of de-ballasting, despite no 

observation of cfus from this taxa in the respective T=0 samples should be viewed 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

2

Trial 2 

Bacteria √ √ √

Trial 3 

Zooplankton √ √ √

Trial 3 √

Phytoplankton √ X

1 X2

Trial 3 

Bacteria √ √ √

1

 – ‘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). 

2

 – 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. 

 

Raaymakers, S. (2002). The Ballast Water Problem:Global Ecological, Economic and 

Human Health Impacts Presentation at theRECSO / IMO Joint Seminar on Tanker 

Ballast Water Management & Technologies. Dubai, UAE 16-18 Dec 2002. 

Ruiz, G.M., Carlton, J.T., Grosholz, E.D. and Hines, A.H.. (1997). Global invasions of 

marine and estuarine habitats by non-indigenous species: mechanisms, extent and 

consequences. Am. Zool. 37, 621-632. 

Ruiz, G.M., Fofonoff, P.W., Carlton, J.T., Wonham, M.J.and Hines, A.H.. (2000a). 

Invasion of coastal marine communities in North America: apparent patterns, processes 

and biases. Ann. Rev. Ecol. Syst. 31, 481-531. 

Ruiz, G.M., Rawlings, T.K., Dobbs, F.C., Drake, L.A., Mullady, T., Huq, A. and 

Colwell, R.R. (2000b). Global spread of microorganisms by ships. Nature 408, 49-50. 

Sharpless, C.M. and Linden K.G. (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. 
79 

Veldhuis M.J.W. and Brussaard, C.P.D. (2006) Harmful algae and cell death. Ecological 

Studies; Vol. 189. Ecology of Harmful Algae E. Graneli & J.T. Turner (eds). SpringerVerlag, Berlin, Heidelberg. 

Veldhuis M.J.W., Kraay, G.W. and Timmermans, K.R. (2001) Cell death in 

phytoplankton: correlation between changes in membrane permeability, photosynthetic 

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

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., R. Dawson, S.M. Moesel and C.E.F. Orano-Dawson (2005). Shipboard trials of 

ballast water management options: Part 2. Efficacy of ballast water exchange. Proc. Inst. Mar. 

Eng. Sci. Technol.. (C3). J. Mar. Sci. Environ., 15-20. 

Wright, D.A., Dawson, R., Orano-Dawson, C.E.F. and Moesel, S.M. (2007). A test of the 

efficacy of a Ballast Water Treatment System aboard the Vessel Coral Princess. Marine 

Technology 44, 57-67. 

Zhang, F and M. Dickman (1999). Mid-ocean exchange of container vessel ballast 

water. 1. Seasonal factors affecting the transport of harmful diatoms and dinoflagellates. 

Marine Ecology Progress Series, 176, 243-251. 
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. 
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